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Photocatalytic modification of bioactive molecules incontinuous-flow microreactorsCitation for published version (APA):Bottecchia, C. (2019). Photocatalytic modification of bioactive molecules in continuous-flow microreactors.Eindhoven: Technische Universiteit Eindhoven.

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Photocatalytic modification of bioactive molecules in

continuous-flow microreactors

Cecilia Bottecchia

Cecilia Bottecchia

Photocatalytic modification of bioactive molecules in continuous-flow microreactors

The work described in this thesis has been carried out within the Micro Flow

Chemistry and Synthetic Methodology group, at the Eindhoven University of

Technology, The Netherlands. This research was financially supported by the

European Union’s Horizon 2020 research and innovation program via a Marie

Sklodowska-Curie grant (641861).

A catalogue record is available from the Eindhoven University of Technology

Library

ISBN: 978-90-386-4692-3

Copyright © 2019 by Cecilia Bottecchia

Printed by ProefschriftMaken, 2019

Cover design by Remco Wetzels, www.remcowetzels.nl

Photocatalytic modification of bioactive molecules in

continuous-flow microreactors

PROEFSCHRIFT

Ter verkrijging van de graad van doctor aan de Technische Universiteit Eindhoven,

op gezag van de rector magnificus prof.dr.ir. F.B.T. Baaijens, voor een commissie

aangewezen door het College voor Promoties, in het openbaar te verdedigen op

woensdag 20 februari 2019 om 11:00 uur

Door

Cecilia Bottecchia

geboren te Milaan, Italië

Dit proefschrift is goedgekeurd door de promotoren en de samenstelling van de

promotiecommissie is als volgt:

Voorzitter: prof.dr.ir. E.J.M. Hensen

Promotor: dr. T. Noël

Copromotor(en): prof.dr. F. Gallucci

Leden: prof.dr. A. Madder (Universiteit Gent)

prof.dr. J.G. Roelfes (RUG)

prof.dr. R.P. Sijbesma

prof.dr. J.H. van Maarseveen (UvA)

dr.ir. A.R.A. Palmans

Het onderzoek of ontwerp dat in dit proefschrift wordt beschreven is uitgevoerd

in overeenstemming met de TU/e Gedragscode Wetenschapsbeoefening

Table of Contents:

Chapter 1: Introduction to visible light photoredox catalysis

andcontinuous-flow chemistry

9

Chapter 2: Visible light-induced trifluoromethylation and

perfluoroalkylation of cysteine in batch and continuous

flow

53

Chapter 3: Batch and flow synthesis of disulfides by visible light

induced TiO2 photocatalysis

73

Chapter 4: Visible-light-mediated selective arylation of cysteine in

batch and flow

93

Chapter 5: Visible light induced trifluoromethylation of highly

functionalized arenes and heteroarenes in continuous

flow

117

Chapter 6: De novo design of organic photocatalysts: bithiophene

derivatives for the visible-light induced C-H

functionalization of heteroarenes

135

Chapter 7: A fully automated continuous-flow platform for

fluorescence quenching studies and Stern-Volmer

analysis

167

Conclusions and Summary 191

List of abbreviations 196

List of publications 199

Acknowledgements 201

About the author 203

“Pass on what you have learned.

Strength, mastery. But weakness, folly, failure also.

Yes, failure most of all. The greatest teacher, failure is.”

-Master Yoda

CHAPTER 1

Introduction to visible light photoredox catalysis and continuous-flow chemistry

This chapter is based on:

Bottecchia, C.; Noël, T. Photocatalytic modification of amino acids, peptides and proteins. Chem. – Eur. J. 2019, 25, 26-42.

Bottecchia, C.; Cambié, D.; Straathof, N. J. W.; Hessel, V.; Noël, T. Applications of continuous-flow photochemistry in organic synthesis, material science, and water treatment. Chem. Rev. 2016, 116, 10276-10341.

1

Introduction 11

PHOTOCHEMISTRY AND PHOTOCATALYSIS

Sunlight strikes our planet every hour with more energy than we consume in a

whole year.[1] Therefore, many researchers have explored ways to efficiently harvest

and use light energy for the activation of organic molecules.[2] Light activation of

molecules provides access to reaction pathways which are otherwise impossible

to reach with classical thermochemical activation.[3] Another fascinating aspect of

photochemistry is the use of photons as “traceless and green reagents”, hence

rendering photochemical processes green and sustainable.

However, up to date the use of sunlight to drive chemical reactions is still far from

reality. The main drawbacks preventing the widespread use of solar photochemistry

are the difficulty associated with the efficient harvesting of solar light and the

fluctuating nature of the radiation itself (e.g. its variability and irreproducibility

due to weather conditions or to the different geographical areas on the planet).[4] Nevertheless, significant advances have been recently reported thanks to the

implementation of continuous-flow reactors for solar photochemistry.[5] Thus, it is

expected that the technology necessary to enable solar photochemistry in organic

synthesis will soon be available.

A brief historical perspective of photochemistry

The first experimental account of a chemical modification induced by light dates

back to 1834, when Hermann Trommsdorff described the effects of sunlight

exposure on α-santonin crystals, a sesquiterpene lactone used as febrifuge and

isolated from Artemisia plants.[6] Remarkably, through the use of a prism, he

understood the dependence of the wavelength on the crystal decomposition

(“santonin is turned yellow not only by the undivided, but also by the blue and

violet rays… whereas… the yellow, green and red ones cause not even the slightest

changes”).[7] The photodegradation of santonin was further investigated by another

pioneer of photochemistry, Stanislao Cannizzaro.[8] Starting from his seminal

work, his disciples Paternó, Ciamician and Silber took the first steps towards

modern photochemistry. Paternó first discovered in 1909 the eponym [2+2]

photocycloaddition between alkenes and carbonyls, further expanded by Büchi.[9]

Around the same period, Giacomo Ciamician and Paolo Silber laid the foundation

of modern photochemistry through a systematic investigation of photochemical

reactions (photoreductions[10], photocycloadditions[11], α- and β-cleavage of

ketones[12]).[13]

1

12 Chapter 1 Introduction 13

Figure 1.1: Giacomo Ciamician surrounded by a number of sun-exposed flasks on his laboratory terrace at the University of Bologna. Beginning of 20th century. Reprinted with permission from Ref.[14] Copyright 2003 Royal Society of Chemistry.

The importance of Ciamician’s work lies also in the awareness that photochemistry

could be a more sustainable alternative to thermally-induced transformations,

which depend on non-renewable fossil sources.[15] In his 1912 visionary paper, the

following statement can be found:

“On the arid lands there will spring up industrial colonies without smoke and without

smokestacks; forests of glass tubes will extend over the plains and glass buildings

will rise everywhere; inside of these will take place the photochemical processes that

hitherto have been the guarded secret of the plants, but that will have been mastered

by human industry which will know how to make them bear even more abundant

fruit... And if in a distant future the supply of coal becomes completely exhausted,

civilization will not be checked by that, for life and civilization will continue as long

as the sun shines!”

Deriving from these earliest approaches, photochemistry continuously evolved.[16] In the late 20th century, fundamental applications emerged: among them the

synthesis of cubane[17], lycopodine[18], estrone[19] and ginkgolide B[20]. It did not take

long before the usefulness of photochemistry was implemented in the chemical

industry. For example, the industrial synthesis of vitamin D3 proceeds through a

photochemical step. More specifically, the 9,10 double bond in provitamin D is

cleaved via UV irradiation to yield the E and Z isomers of previtamin D.[21] Moreover,

since the installation of the first photochemical production unit in 1963, the

synthesis of ε-caprolactam through the photonitrosation of cyclohexane (Toray

process) has been an important step towards Nylon 6.[21]

Over the course of the last century, many other photochemical reactions have

been reported, too many to be discussed in detail in this dissertation. Among them,

photocycloadditions represent one of the most studied photochemical reactions.[22] Their popularity lies in the possibility to rapidly prepare highly complex organic

molecules, such as substituted cyclobutanes.[3b, 23] Another class of light induced

reactions that has been extensively studied over the years are photoisomerizations.[24] These transformations rely on the principle that excited states, reached through

light absorption, may induce reversible or irreversible isomerizations of organic

substrates. Consequently, relatively simple starting materials can be converted in

complex structures (e.g. cyclobutenone rings), which are often elusive to prepare

via other thermal or chemical pathways. Reversible photoisomerization reactions

are also key in the design of chemical switches and molecular motors.[24b, 25] Finally,

photodecarboxylations are an interesting variant of decarboxylation reactions,

valuable in organic synthesis as they allow the controlled extrusion of CO2 from

organic substrates.[26]

The advent of photoredox catalysis

In photochemical reactions, the substrate that undergoes the chemical

transformation is also the absorbing species capable of reaching an excited state

upon absorption of light. Typically, photochemical reactions are driven by high-

energy UV light. This is due to the fact that the vast majority of organic molecules

do not possess highly conjugated systems capable of absorbing light in the visible

range. Instead, the presence of C-heteroatom double bonds or of non-conjugated

π systems typically results in absorption bands in UV-C to UV-B region (λ= 100 to

315 nm). The high costs, poor efficiency and health risks associated with the use of

UV lamps render the implementation of photochemical reactions impractical and

expensive on the large scale. Moreover, photochemical reactions driven by UV light

often exhibit poor selectivity due to the fact that side reactions involving other

absorbing species are hard to prevent. As a consequence, chemists have focused

their attention on the possibility to employ visible light to drive photochemical

transformations. To solve the inherent disadvantage deriving from the fact that

only a small fraction of organic substrates can absorb light in the visible light

range, chemists devised the use of light-harvesting molecules, also known as

photocatalysts. The understanding of the photophysical and photochemical

aspects governing the interactions of photocatalysts with light has been of primary

interest in the photophysical community.[3a, 27] The translation of this knowledge to

the organic chemistry community has been one of the key points that boosted the

field of visible light photoredox catalysis.

1


In the late 70’s, seminal work from Kellogg and co-workers demonstrated how,

upon visible-light absorption, ruthenium metal-based complexes can efficiently

fulfil the role of photocatalyst by engaging in redox processes with the substrate

of interest.[28] Around the same time, other publications confirmed that similar Ru

species could participate in the visible-light induced reduction of a wide range of

organic substrates (e.g. benzylic and phenacyl halides, electron deficient olefins

and aromatic ketones).[29] The first researcher that appreciated the potential of the

Ru photocatalytic systems for reductive intermolecular and intramolecular bond

formation were Deronzier and Okada.[30] Despite these early findings, the field of

photoredox catalysis remained unexplored until the first decade of this century,

when the groups of Macmillan, Yoon and Stephenson showcased how varied the

redox reactivity of transition-metal based photocatalysts can be.[31]

Photoredox catalysis: basic principles

Owing to the generally mild reaction conditions (i.e. room temperature and

ambient pressure), the use of visible light as a perennial energy source and its broad

applicability, visible light photoredox catalysis has gained tremendous momentum

in the past decade, triggering a renewed interest towards photochemistry in

general.

In photoredox catalysis, the ability of transition metal-based or organic

photocatalysts to harvest visible light and convert it to an electrochemical potential

is exploited to activate organic substrates.[32] Specifically, upon absorption,

photocatalysts reach an excited state in which they are prone to engage in

single electron transfers (SETs) with organic substrates acting as either electron

donors or acceptors, thus de facto activating them and resulting in the formation

of radical intermediates (Scheme 1.4).[32-33] In order to best comprehend the

mechanisms involved in photoredox chemistry, an excursus on the photophysical

properties of commonly used photocatalysts appears necessary. For the purpose

of this discussion, the photophysical properties of the tris(bipyridine)ruthenium(II)

complex (i.e. Ru(bpy)32+) will be taken as example. Upon excitation by a photon

with energy corresponding to the band-gap of the photocatalyst (in the case

of Ru(bpy)32+ a photon with energy corresponding to blue light, λ ~ 450 nm), an

electron from the highest occupied molecular orbital (HOMO) is promoted to the

lowest unoccupied molecular orbital (LUMO) (Scheme 1.1).

Scheme 1.1: Simplified view of the Molecular Orbital involved in the photocatalytic activity of Ru(bpy)3

2+ MLCT = metal to ligand charge transfer, ISC = intersystem crossing.

Specifically, for Ru(bpy)32+ it has been demonstrated that an electron is transferred

from one metal-centred t2g orbital to a ligand-centred π* orbital.[34] For this reason,

this electronic transition is often referred to as metal to ligand charge transfer

(MLCT). As a consequence of the MLCT, the metal is oxidized to a Ru(III) species,

while the ligand framework becomes reduced (Scheme 1.1).[35]

At this stage, it is important to underline that when a ground state molecule is

excited to a higher energy level, the induced excited state can be a singlet or a

triplet. In a singlet state, the spin of the excited electron is still paired with the

ground state electron (i.e. they have opposite spins, see Scheme 1.2). On the

contrary, in a triplet state the excited electron is no longer paired with the ground

state electron; (i.e. they have same, parallel spins, see Scheme 1.2). In general, since

the formation of a triplet state requires an additional “forbidden” spin transition,

singlet states are more likely to be generated upon visible light absorption.

However, the initial singlet excited state can transition to a triplet state through

a non-radiative process known as intersystem crossing (ISC). This phenomena is

especially relevant for photoredox catalysts because ISC results in the formation

of a more stable (i.e. lower in energy) and long-lived triplet excited state (named

excited triplet state in Scheme 1.1 for Ru(bpy)32+ or *PCn(T1) in Scheme 1.4). Being

EXCITED STATE

eg*

*

t2g

eg*

*

t2g

eg*

*

t2g

eg*

*

t2g

MLCT+ ISC

N

N

RuII

NN

N

N

2+

N

N

RuIII

NN

N

N

*2+

Ru(bpy)32+ *Ru(bpy)32+

Ru(bpy)33+

Reductivequenching

Oxidativequenching

E1/2 *Ru(II)/Ru(I) = 0.77VE1/2 Ru(III)/Ru*(II) = 0.81VLifetime ( ): 1100 ns

max = 452 nm

GROUND STATE

OXIDIZED

E1/2 Ru(III)/Ru(II) = 1.29V

Ru(bpy)3+REDUCED

E1/2 Ru(II)/Ru(I) = -1.33V

(triplet)

1


spin-forbidden, the decay from the excited triplet state back to singlet ground

state is typically slower, which explains the long excited state lifetime of transition

metal-based photocatalysts.[32]

Scheme 1.2: : Singlet and triplet energy levels and spins of the electron populating them. HOMO = highest occupied molecular orbital LUMO = lowest unoccupied molecular orbital.

Importantly, this excited triplet state is able to engage in SET events with other

organic molecules, thus activating them. The excited triplet state can undergo

a reduction or an oxidation (respectively from *Ru(II) to Ru(I) or from *Ru(II) to

Ru(III))(Scheme 1.1).

Scheme 1.3: Energy levels of HOMO and LUMO orbitals for ground state and excited state. The effect of photoexcitation is the formation of an excited specie which is a better oxidant and a better reductant compared to ground state. HOMO = highest occupied molecular orbital, LUMO = lowest unoccupied molecular orbital.

Perhaps the most remarkable effect of photoexcitation is that the excited state is

a more powerful oxidant and a more powerful reductant compared to the ground

state. This concept can be clearly explained when looking at the energy levels

depicted in Scheme 1.3: In the excited state configuration, an electron is on a

higher energy level than it used to be, thus making its donation to an acceptor more

favourable than on the ground state. In other words, this means that the excited

state compound is a better reducing agent than the ground state counterpart. At

the same time, excitation causes the formation of an electron hole in the lower

energy level. Because this energy level is lower than the LUMO of the ground

state, the insertion of an electron from a donor will occur more easily compared to

ground state. Ultimately, this means that the excited state compound is a better

oxidizing agent than the ground state counterpart.

Scheme 1.4: Schematic overview of the single electron transfer events involved in a typical photocatalytic cycle. PC = photocatalyst, T1 = triplet excited state, S1 = singlet excited state, S0 = singlet ground state and ISC = intersystem crossing.

When quenching of the excited state of a photocatalyst results in the donation of

an electron to an acceptor species (Scheme 1.4), the corresponding catalytic cycle

is defined as an oxidative quenching cycle (where oxidative refers to the fact that

the photocatalyst is oxidized). The oxidized catalyst can then undergo a consecutive

SET (often referred to as reductive recombination) in which the electron missing

in order to return to the ground state configuration is taken from a donor species

(Scheme 1.4). In other words, this second SET event “closes” the photocatalytic

catalytic cycle yielding a ground state configuration ready for another excitation

event. This closed catalytic cycle also explains why the absorbing species can be

used in catalytic amounts in organic reactions. Alternatively, when quenching

of the excited state of a photocatalyst results in the photocatalyst accepting an

Energy

LUMO

HOMO

Oppositespins

SINGLETGROUND STATE

SINGLETEXCITED STATE

h ISC TRIPLETEXCITED STATE(lower energy)

Parallelspins

Energy

LUMO

HOMO

High energyelectron acceptor

Low energyelectron donor

High energyelectron donor

Low energyelectron acceptor

GROUND STATE EXCITED STATEh

*PCn (S1)

*PCn (T1)Acceptor

ISC

PCn (S0)

OXIDATIVEQUENCHING CYCLE

REDUCTIVEQUENCHING CYCLE

PCn-1PCn+1

Acceptor

Donor

Donor

Acceptor

Acceptor

Donor

Donor

Luminescence

1


electron from a donor species (Scheme 1.4), the corresponding catalytic cycle is

defined as a reductive quenching cycle (where reductive refers to the photocatalyst

being reduced). The reduced catalyst can then back-donate the extra electron in a

consecutive SET to an acceptor species (Scheme 1.4), an event also referred to as

oxidative recombination.

The driving force determining whether a SET can occur is described as redox

strength. Specifically, for each photocatalyst the ability to undergo a single

electron reduction or oxidation can be estimated by measuring (typically via

cyclic voltammetry) the electrochemical half potential of the corresponding

redox reactions. If the electrochemical half potential of a substrate of interest is

known, potential values can be useful in predicting whether the excited state half

potential of a certain photocatalyst will be able sufficient to reduce/oxidize the

substrate, thus activating it. The structures, optical properties and excited states

redox potentials for a series of commonly used transition metal-based and organic

photocatalyst are depicted in Scheme 1.5.

Scheme 1.5: Overview of the optical and redox properties of some commonly used transition metal-based and organic photocatalysts: λmax refers to the absorption maximum. All potential values are in volt and referred to a saturated calomel electrode (SCE).The lifetime of the excited states (τ) for Eosin Y is of the triplet state.

The lifetime of the excited states able to engage in SETs are presented as well. A

striking difference can be found between the lifetime values of transition metal-

based and organic photocatalysts, with the latter typically showing shorter excited

state lifetime (with at least one order of magnitude difference). Due to the fact

that long excited state lifetimes essentially mean that the probability for the

photocatalyst to engage in SETs is higher, this aspect might be responsible for

the often observed superior reactivity of transition metal-based photocatalysts

compared to the organic counterparts.

Obviously, the absorption of photons is crucial to carry out photochemical

reactions. An important parameter that can be used to describe the efficiency

of a photochemical reaction is the quantum yield (φ) Defined as the number of

molecules of product formed over the number of photons absorbed, the quantum

yield of a reaction can be determined by carrying out photon flux measurements

and subsequently performing the reaction in the same setup.[36] (Equation 1.1).

The photon flux is defined as the number of photons observed per unit time. It is

important to understand that :1) not every photon emitted by the light source will

end up in the photoreactor, and 2) not every photon reaching the reactor will give

rise to the conversion of one molecule of starting material; as excited states can

return to their ground state by radiative or non-radiative processes (e.g. heating).

Theoretically, the maximum value for φ is 1, meaning that for every photon

absorbed in the system one molecule of product is formed.[37] However, many

photocatalytic reactions exhibit quantum yields > 1. This means that, on average,

each excited state of the photocatalyst produces more than one molecule of

product. In other words, whenever a reaction’s quantum yield is > 1, other radical

mechanisms must occur concurrently to the photoredox cycle (e.g. radical chain

mechanism). [38] Therefore, quantum yields < 1 are consistent with a closed catalytic

cycle, while quantum yields >> 1 indicate a radical chain propagating mechanism

(Scheme 1.6).[38a]

Scheme 1.6: : Difference between closed and open photocatalytic cycles (respectively φ< 1

and φ> 1). The example shown refers to a reductive quenching cycle, but the same principle applies also to oxidative quenching cycles.

Ru(bpy)32+

- max = 452 nm- E1/2(PC*/PC-) = 0.77V- E1/2(PC+/PC*) = -0.81V- lifetime ( ): 1100 ns

N

N

RuN

N

N

N

N

IrN

N

2+

NMe

Me

MeMe

O

COO

Br

Br

OBr

O

Br

Ir(ppy)3

- max = 375 nm- E1/2(PC*/PC-) = 0.31V- E1/2(PC+/PC*) = -1.73- lifetime ( ): 1900 ns

Mes-Acr+

- max = 425 nm- E1/2(PC*/PC-) = 2.06V- E1/2(PC+/PC*) = n.a.- lifetime ( ): 6 ns

Eosin Y

- max = 520 nm- E1/2(PC*/PC-) = 0.83V- E1/2(PC+/PC*) = -1.15- lifetime ( ): 2 ns

𝜙𝜙𝜙𝜙 = number of molecules of product formednumber of photons absorbed by reactive medium

PCn

*PCn

PCn-1

Sub

Sub +

Prod +

Reductivequenching

Oxidativerecombination

h

Prod

Sub'

Prod'

ChainPropagation

(Open CatalyticCycle)

R

ClosedCatalyticCycle

< 1

>> 1

(1.1)

1


PHOTOREDOX CATALYSIS FOR BIOMOLECULE MODIFICATION

As mentioned earlier, compared to other catalytic approaches photoredox catalysis

offers the advantage of enabling the activation of organic substrates under mild

reaction conditions, while making use of visible-light irradiation as a sustainable

source of energy. Moreover, because of the inability of the majority of organic

substrates to absorb light in the visible spectrum, together with the fact that most

organic molecules possess an activation barrier that cannot be overcome at room

temperature, photoredox-based reactions typically exhibit high selectivities, with

little or no side products observed.[39]

Scheme 1.7: Overview of the advantages and challenges in the development of photoredox methodologies for biomolecule modification.

As a consequence of the growing interest in peptides as drug candidates, and due

to the undeniable importance of antibody-drug conjugates in current state-of-

the-art therapeutics, the need for novel bioconjugation strategies is constantly

on the rise.[40] In other words, selective chemical transformations aimed at the

modification of native or non-native amino acids as well as robust techniques

allowing the incorporation of exogenous entities (e.g. drugs, tracers or tools

for immobilization) in peptides and/or proteins are of fundamental importance

in chemical biology.[41] However, traditional organic chemistry approaches

are often inadequate solutions for bioconjugation, as their biocompatibility

is usually limited. Ideally, bioconjugation strategies should provide selective

transformations resulting in the formation of stable conjugates, while providing

mild and biocompatible reaction conditions (i.e. room temperature, atmospheric

pressure, physiological pH, aqueous buffered solutions as solvent).[42]

Despite the many advances in the field of bioconjugation, innovative strategies to

answer the remaining open challenges (e.g. modification of elusive amino acids,

general strategies for regioselective modification of exposed residues in proteins),

would greatly contribute to enlarge the toolbox of available methods for post-

translational modification methods.[43]

When looking at the intrinsic advantages offered by visible-light photoredox

catalysis, the reasons in favor of its application to the development of novel

methodologies for bioconjugation become apparent. Firstly, the use of visible light

to drive chemical transformations is advantageous both in terms of sustainability

(i.e. light is a green, traceless reagent) and in preserving the delicate nature of

bioactive molecules (as opposed to UV irradiation, often disruptive towards the

conformational integrity of proteins).[2a, 3a, 33d, 44] Secondly, photoredox-based

reactions can be conducted at room temperature and generally proceed smoothly

in buffers or in aqueous mixtures, thus offering biocompatible reaction conditions.[3f] Moreover, reaction kinetics of photocatalytic transformations can be easily

controlled owing to their strong dependence on the photon flux.[45] Consequently,

the vast majority of photoredox reactions can be easily quenched by simply

switching off the light. Such a straightforward on\off approach to control the

reaction progression is an attractive feature when it comes to bioconjugation

methods, as it allows to circumvent the need for quenchers and can simplify

subsequent purification methods. Keeping all these inherent advantages in mind,

the recent trend of applying photoredox catalysis to biomolecule modification

comes as no surprise.

Photocatalytic modification of amino acids: a brief overview

The idea to employ photocatalytic methods for bioconjugation purposes is

relatively new, and prior to the beginning of the research described in this thesis

only few examples on this matter were published. However, over the last few

years, the field of photocatalysis continued to attract attention in the organic

chemistry community, and the first concrete examples of its application to amino

acid modification were reported. A brief overview of the current methods for

photocatalytic amino acid modification will be described in the next section, while

the three strategies developed for cysteine modification in the context of this

dissertation are discussed in details in Chapter 2, 3 and 4.

Methodology requires:

- Physiological conditions

- High selectivity

- Non-toxicity

- Fast kinetics

ChallengesPhotocatalysis offers:

- Visible light as harmless,

traceless reagent

- Mild reaction conditions

- On-off approach

- Simplified purification

Advantages

NIr

N

N

O

COO-

-O

Br

Br Br

O

Br

H2N OHH2N OH

1


Among the 20 proteinogenic amino acids, only few residues represent viable

targets for the development of successful bioconjugation methods. Typically,

bioconjugation methodologies rely on the intrinsic reactivity of the different

amino acids to achieve chemoselectivity (e.g. nucleophilicity/electrophilicity

or acid-base behavior). Alternatively, the accessibility of one specific residue in

proteins or peptides (i.e. position in the sequence or spatial orientation in the

overall structure) can be exploited to achieve site-selectivity in presence of other

reactive amino acids. A straightforward approach to classify methods for amino

acid modification is to organize them based on the targeted residue.

Cysteine

Generally speaking, cysteine represents one of the most targeted residues in

bioconjugation methods.[46] This is mainly due to its relatively high nucleophilicity

and to its low natural abundance, which render this amino acid an ideal candidate

to achieve regio- and chemo-selective modification of peptides and proteins.

The so-called thiol-ene click approach, that is the reaction between a thiol and

an alkene, is a very efficient transformation to establish a C–S linkage through

reaction[47]. This reaction is typically initiated by a radical initiator or UV light.

However, due to the incompatibility of peptides and proteins with high energy

UV light,[44] several reports have appeared in recent years on the development

of visible-light thiol-ene transformations using photocatalysts, including

Ru(bpy)32+[48], Ru(bpz)3

2+[49], and 9-mesityl-10-methylacridium+.[50] These catalysts

are able to absorb visible light to reach an excited state which is reductively

quenched to generate a thiyl radical cation. Upon deprotonation, a thiyl radical is

formed which can subsequently add to an olefin in an anti-Markovnikov fashion.

The biocompatibility of this transformation has been demonstrated by Yoon et

al.[51] The authors developed a visible light photocatalytic protocol which allows to

couple glutathione with various olefins in aqueous media. Ru(bpz)32+ was used as a

photocatalyst and p-toluidine served as a redox mediator. A variety of biologically-

relevant olefinic coupling partners, containing azides, PEG oligomers, protected

sugars and biotin, were efficiently hydrothiolated under these conditions (Scheme

1.8a).

Guo et al. have developed an efficient visible-light photocatalytic protocol to

desulfurize cysteine moieties within peptides into alanine (Scheme 1.8b).[52]

Notably, the reaction can be carried out in aqueous phosphate buffer solution.

Ru(bpy)32+ was used as a photocatalyst to generate cysteine radicals which can

react subsequently with a water-soluble phosphine (i.e. 3,3’,3”-phosphinidynetris

(benzenesulfonic acid) trisodium salt, TPPTS). The corresponding phosphoranyl

radical converts via β scission into an alkyl radical which abstracts a proton from

cysteine to generate the alanine residue. Notably, a broad variety of different,

deprotected peptide sequences could be subjected to the reaction protocol

providing the target alanine-containing peptide in good to excellent yield.

Scheme 1.8: Overview of photocatalytic methods for cysteine modification.

An approach to cysteine arylation was developed by Molander et al. using a dual

Ni/photocatalytic catalytic cycle.[53] The reaction utilizes aryl bromides as coupling

partners and proceeds in the presence of ammonium bis(catechol) silicate as

a hydrogen atom transfer reagent, [Ni(dtbbpy)(H2O)4]Cl2 as the cross-coupling

catalyst, [Ru(bpy)3](PF6)2 as the photocatalyst in DMF (Scheme 1.8c). Glutathione

was used as a benchmark example showing excellent reactivity towards a diverse

set of aryl bromides bearing various functional groups. Interestingly, the method

was also applicable to a fully deprotected cysteine-containing 9-mer peptide

bearing other reactive residues such as Trp, His, Glu and Tyr using 20 equivalents

of aryl bromide.

Tyrosine

Tyrosine is another interesting residue to enable site-selective modification as it is

relatively rare on protein surfaces. Furthermore, the reactivity of individual tyrosyl

residues can be tuned by selective deprotonation since the pKa depends on the

microenvironment around the residue.

Properties:

HS OH

O

NH2

Low natural abundancy(1-2% in native proteins)Relatively highnucleophilicity (pKa 8.2)

Photoredox cysteinemodification:

a) thiol-ene reactionb) desulfurization(conversion to Ala)c) dual catalytic arylationd) trifluoromethylation (Ch. 2)e) disulfide formation (Ch. 3)f) arylation (Ch. 4)

Cysteine

b) Desulfurization: from Cys to Ala (Guo et al., 2016)

phosphate buffer

Ru(bpy)3Cl2TPPTS, TBM

NH

O

SH

NH

Me

O

c) Dual catalytic arylation (Molander et al., 2018)

a) Visible light-induced thiol-ene (Yoon et al., 2015)

DMF, 4-48 h

iBu[Si-]Ni catalyst

[Ru(bpy)3](PF6)2Biomolecule

Y

Br

R

Biomolecule

SY

R

R

Ru(bpz)3(PF6)2p-toluidineGlutathione

Glutathione

SH2O

SH

SH

R

1


Scheme 1.9: Overview of photocatalytic modification of tyrosine

Researchers at Merck have developed a photocatalytic approach to install

trifluoromethyl moieties on tyrosine sidechains.[54] The photocatalytic method

utilizes 20 equivalents NaSO2CF3 and 15 mol% of Ir[dF(CF3)ppy]2(dtbbpy)]PF6

in a CH3CN/10% aqueous AcOH mixture, with 20 hours of irradiation (Scheme

1.9a). The method displayed good selectivity towards tyrosine in the presence of

histidine and phenylalanine sidechains. However, in the presence of tryptophan

residues, a slightly higher selectivity was observed for tryptophan over tyrosine.

The photocatalytic method displayed greater yields for the trifluoromethylated

products than analogous non-photocatalytic radical trifluoromethylation

reactions. A diverse set of biologically-relevant peptides were modified using

this strategy, including deltorphin I, angiotensin I, angiotensin II, β-casomorphin,

dermorphin and even insulin.

Nakamura et al. developed a broadly applicable photocatalytic method to enable

protein modification using Ru(bpy)32+.[55] The photocatalyst generates tyrosyl

radicals upon light irradiation, which subsequently react with various radical

trapping reagents containing an N’-acyl-N,N- dimethyl-1,4-phenylenediamine

(Scheme 1.9b). The method was successfully applied to the modification of

peptides (e.g. angiotensin II) and proteins (e.g. Bovine serum albumin).[56] Notably, to

demonstrate the potential of this method to enable selective protein modification

via a local single electron transfer, the authors synthesized a benzenesulfonamide-

conjugate ruthenium-based photocatalyst. This benzenesulfonamide moiety

functions as a ligand for the protein carbonic anhydrase and upon protein-ligand

interaction a significantly higher modification efficiency of nearby tyrosine

residues was observed.

Methionine

Few examples of conjugation methodologies involving methionine can be found in

the literature.[57] This is largely due to the fact that, compared to the other sulfur

containing proteinogenic amino acid (i.e. Cys), methionine exhibits an intrinsic

lower nucleophilicity and high hydrophobicity.[40a, 41a] Thus, one of the biggest

challenges in the development of bioconjugation methods for methionine is to

achieve selectivity under pH-neutral conditions in presence of other competing,

and more nucleophilic residues (i.e. Lys, Cys, Tyr, Ser). However, due to the fact

that all competing nucleophilic residues exist in a protonated form at low pH,

selectivity towards methionine could be observed in methods employing acidic

conditions.[40a, 58] Nevertheless, no photocatalytic methodologies for methionine

bioconjugation have been currently reported. On the other hand, photocatalytic

methods for the chemical modification of the individual amino acid do exist,

often exploiting the fact that this amino acid is prone to oxidation. In biological

systems, the oxidation of methionine was found to be a relevant post-translational

modification controlling several signalling pathways at a cellular level.[59]

Scheme 1.10: Photocatalytic oxidation of methionine

Moreover, methionine sulfoxide, that is the oxidized form of methionine, has been

investigated for its role in the pathogenesis of neurodegenerative diseases, as

well as oxidative stress.[60] A photocatalytic oxidation of methionine was described

N

NRu

NN

NN

O

NH

2+

Ligand withphotocatalyst

Target proteinO

Neighboring Tyr residue

Local SETN NH

O

Labeled tyrosyl radicaltrapping agent (TRT)

MES buffer,pH 6, 5 min

HO

Distant Tyr residuesremain untouched

Target protein

HO

OH

NH

O

N

Labeled protein

OH

O

NH2HO

20h

NH

ONH

O

HO HOCF3

a) trifluoromethylationb) cross-linking of tyrosyl radical

Properties:

- Relatively low naturalabundancy- pKa depending onmicroenvironment

Ir photocatalystNa2SO2CF3

MeCN/ aq. AcOH1: 1 mixture

a) Trifluoromethylation (Merck, 2018)

b) Protein modification via cross-linking of tyrosyl radicals (Nakamura et al., 2013)

TyrosinePhotoredox tyrosine

modification:

Properties:

Photoredox methioninemodification:

Methioninea) Photosensitized oxidation (Dreesen et al., 2017)

OH

O

NH2

- Second rarest proteinogenicamino acid- high hydrophobicity-Eox varies depending onnieghboring residues

a) Photosensitized oxidation

SMe Rose bengal,

O2,

quantitative

H2O, 1.4 min

OHO

NH2

SMe

OHO

NH2

SMe

O

1


by Dreesen, Heinrichs, Monbaliu and co-workers (Scheme 1.10).[61] In this scalable

continuous-flow protocol, the oxidation of methionine via the formation of singlet

oxygen was obtained with rose Bengal as photosensitizer. The biphasic reaction

(i.e. liquid reagent and molecular oxygen) was performed in a glass mesoscale

commercially available photoreactor and afforded quantitative yields within 1.4

minutes of reaction time and a productivity up to 132 g/day.

Tryptophan

With a calculated abundance of ~1.3%, tryptophan is the rarest of all proteinogenic

amino acids.[62] However, it was estimated that approximately 90% of all proteins

contain at least one Trp residue.[63] In other words, many proteins contain only

one Trp in their sequence. Thus, methodologies affording chemoselective

modification of Trp would result in site-selective bioconjugation on a vast number

of endogenous substrates. Up to date, the majority of methods developed for

Trp conjugation showed that the C-2 position of indole is the most reactive, while

other positions on the aromatic ring or in β to the stereocenter proved harder to

modify.[41a, 64] Moreover, the free NH on the indole moiety of Trp has been found

incompatible with some transition metal-based methodologies, hence rendering

the use of protecting groups necessary.[65] Thus, in this context, the development

of novel photocatalytic methodologies selectively targeting Trp would represent

a positive advancement in the field of photoredox catalysis.

In a recent example, Shi and co-workers reported a photocatalytic and

chemoselective β-modification of Tryptophan (Scheme 1. 11).[66] Notably, the

selective transformation of the β-position was essentially unprecedented in the

literature, with only one report illustrating a similar modification, albeit through

an enzymatic approach.

Scheme 1. 11: Photocatalytic modification of tryptophan

The authors postulated that the observed selectivity at the β-position might

derive from the formation of a stabilized Trp-skatolyl radical. Specifically, a first

SET from the excited state of the photocatalyst (i.e. Ir[dF(CF3)ppy]2(dtbbpy)PF6) to

the N in the indole moiety of Tryptophan would result in the formation of a radical

cation, in which the benzylic proton (i.e. the β-proton) exhibits a higher acidity.

Thus, in presence of K2HPO4, extraction of the β-proton affords the stabilized

Trp-skatolyl radical. Finally, this radical can undergo conjugation with any Michael

acceptor, ultimately affording the desired bioconjugation product after back

electron transfer to the photocatalyst. A broad range of Michael acceptors proved

compatible with the transformation, and the selectivity and tolerance of the

methodology towards other amino acids was demonstrated with a small array

of synthetic and natural peptides (e.g. glucagon and GLP-1). Depending on the

conformation and accessibility of other amino acids, competitive reactions with Lys

and His residues were observed. Interestingly, when the reaction was performed

on human insulin, which does not contain any Trp residue, decarboxylation of the

C-term residue on the β-chain was observed. This result differed from what was

reported by MacMillan and co-workers, who observed selective decarboxylation

of the C-term in the α-chain of insulin under similar photoredox conditions (see

Scheme 1.13).[67]

Dehydroalanine

The incorporation of non-canonical amino acids in peptides and proteins offers

several advantages.[40b] For instance, these residues can be exploited to increase

the metabolic stability, affinity and physical properties of peptide drug candidates.[68] Moreover, including non-canonical amino acids in peptides or proteins

can provide strategies to achieve effective and selective post-translational

modifications methods. One prominent example of a non-canonical amino acid

enabling selective transformations is dehydroalanine (DHA). In nature, DHA is

found in peptides of microbial origin and has been observed in proteins as the

result of post-transcriptional modifications.[69] The incorporation of DHA residues

in peptides and proteins has been demonstrated both biosynthetically[70] and

through chemical approaches (e.g. activation/elimination of the thiol moiety

in Cys or starting from selenocysteine).[43b, 71] Unlike most proteinogenic amino

acids, DHA is a good electrophile, and is therefore interesting from a synthetic

perspective as it exhibits good reactivity towards nucleophiles. Therefore, several

post-translational modifications of DHA based on transition-metal catalysis or

Michael additions have been reported.[72]

Properties:

Photoredox tryptophanmodification:

Tryptophana) Ir-catalyzed alkylation at -position with Michael acceptors(Shi et al., 2018)

- Rarest proteinogenic amino acid- Most reactive position is C2 indole-Indole N-H might causeinterference when attemptingselective modification

a) Ir-catalyzed alkylation at-position with Michael acceptors

OH

O

NH2HN

Ir[dF(CF3)ppy]2(dtbbpy)PF6K2HPO4,

NH

O

NH

DMF, 16hNH

O

NH

OMe

O

O

OMe

1


In 2017, Jiu and co-workers reported the possibility to activate pyridyl halides via

a photoredox-induced single electron transfer and demonstrated the subsequent

addition of these radical species to dehydroalanine derivatives.[73] Optimized

reaction conditions comprised the use of [Ir(ppy)2(dtbbpy)]PF6 as photocatalyst,

excess of Hantzsch ester as reductant and a DMSO/H2O solvent mixture. The

developed protocol proved efficient for the preparation of a wide range of β–

heteroaryl α–amino acid derivatives (Scheme 1.12).

Scheme 1.12: Photocatalytic modification of dehydroalanine.

Notably, to obviate the fact that the use of DHA as electrophilic alkenes inevitably

results in the formation of products as racemic mixtures, the authors adapted the

procedure to use a chiral oxazolidinone as the alkene partner (Scheme 1.12).[74] The

product resulting from the transformation exhibited good diastereocontrol, and,

following concurrent carbamate cleavage and hemiaminal hydrolysis, gave access

to the desired β–heteroaryl α–amino acid derivatives in high stereochemical purity

(i.e. 97% ee).

More recently, de Bruijn and Roelfes reported the late-stage functionalization of

dehydroalanine-containing antimicrobial peptides (e.g. thiostrepton and nisin) by

means of photoredox catalysis.[75] Specifically, under irradiation with blue LEDs and

by employing Ir[dF(CF3)ppy]2(dtbbpy)PF6 as photoredox catalyst, the formation of

carbon-centered radicals starting from trifluoroborate salts was achieved. These

carbon-centered radicals were then trapped by the dehydrated amino acids present

in thiostrepton and nisin. Interestingly, in the case of thiostrepton chemoselective

modification of the DHA-16 residue was obtained, while in the case of nisin a triple-

modified product (corresponding to the modification of both DHA present in the

sequence and of one dehydrobutyrine residue) was observed.

Photocatalytic decarboxylation strategies: side chain or C-term modifications

Among the transformations enabled by photoredox catalysis, the decarboxylation

of carboxylic acids has been thoroughly investigated.[70] In this context, MacMillan

and co-workers described the photoredox decarboxylation of native amino

acids to achieve site- and chemoselective bioconjugation. In a first report, the Ir-

catalyzed decarboxylation of C-terminal carboxylates was exploited to achieve

intramolecular cyclization, thus affording a novel and mild strategy for the synthesis

of cyclic peptides.[71] Specifically, the excited state of Ir[dF(CF3)ppy]2(dtbbpy)PF6

can be reductively quenched by the C-terminal carboxylate, thus generating, after

CO2 extrusion, a nucleophilic C(sp3) radical. The intramolecular addition of the

nucleophilic α-amino radical to a pendant Michael acceptor placed at the N-term of

the sequence results in the formation of the desired macrocyclic peptide (Scheme

1.13). Notably, selective oxidation of the C-term carboxylate group over other acid-

containing side chains (i.e. aspartate or glutamate) was achieved owing to its lower

pKa and oxidation potential. A range of macrocyclic peptides, including challenging

medium-sized peptidic rings, were prepared following the methodology, and the

synthesis of a cyclic somatostatin receptor agonist was showcased.

In a later report, a similar methodology based on the visible-light induced

decarboxylation of the C-terminus position was applied to the alkylation of a set

of biologically active peptides and proteins (Scheme 1.13b).[52]

In order to render the methodology broadly applicable and compatible with

biologically-relevant reaction conditions, the authors identified riboflavin

tetrabutyrate as an efficient water-soluble photocatalyst. Selectivity and

compatibility towards all canonical amino-acids was demonstrated, with the sole

exception of Tyrosine, which underwent competitive oxidation even at lower

pH. However, the use of a less oxidizing flavin photocatalyst (i.e. lumiflavin)

partially solved the issue. Sequences incorporating other side-chain carboxylates

underwent selective decarboxylation at the C-term. As mentioned earlier, this

was rationalized taking into account that the C-term carboxylate is more readily

oxidized due to the stabilizing effect of the adjacent nitrogen atom on the

α-amino radical generated upon decarboxylation. Finally, the broad applicability

of the methodology was demonstrated for a series of tetramers and of longer

biologically active peptides (e.g. angiotensin II, bradykinin, bivalirudin). Notably,

human insulin was also successfully alkylated while maintaining its structural

Properties:

Photoredox tryptophanmodification:

Dehydroalaninea) Heteroaryl amino acid synthesis via SET pyridyl halide activation andsubsequent radical addition to DHA (Jiu et al., 2017)

- Electrophilic- Introduced via biosyntheticor chemical approaches- Does not contain a chiralcenter

a) Heteroaryl amino acid synthesisvia SET pyridyl halide activation andsubsequent radical addition to DHAb) Ir-catalyzed alkylation withorganoborates as radical precursors

OH

O

NH2

N

NO

tBu

O

CbzN

O

O

tBuCbz

With chiral oxazolidinoneas alkene:

68%

N

98%, (97% ee)

NH2

OH

O

Aq. HCl80 °C

Ir[(ppy)2(dtbbpy)]PF6Hantzsch ester

DMSO/H2O 5:1rt, 16h

N Br

1


integrity, with decarboxylation of the C-terminus happening selectively at chain A

of the dimer (Scheme 1.13b).

Scheme 1.13: Overview of photocatalytic strategies for the selective modification of peptides and proteins via visible light-induced decarboxylation

Despite all the aforementioned strategies for the photocatalytic modification of

amino acids, further improvement of the existing methodologies will be required to

render them broadly applicable in chemical biology. Current limitations observed

when implementing photocatalytic methods in bioconjugation strategies concern

the sensitivity of certain substrates to the photogenerated radical species, which

might result in oxidative damage. Moreover, in certain cases selective modification

of one amino acid in presence of other residues still remains an unsolved issue.

CONTINUOUS FLOW TECHNOLOGY: A BRIEF INTRODUCTION

Despite being the most commonly used tool for organic synthesis, traditional round-

bottom flasks are not appropriate vessels for large scale production. This is due to

the fact that a precise control over heating/cooling of the reaction mixture as well

as an efficient mixing are hard to obtain on the large scale.[76] As a matter of fact,

in the petrochemical sector traditional stirring tank reactors are often replaced by

tubes and pipes as adequate vessels for production scale. Such continuous mode

of operation often outperforms batch processing in terms of performance, cost,

safety and atom-efficiency.[77] On the other hand, in the pharmaceutical and fine

chemical industry, batch processes remain the main production mode. Mainly, this

derives from the fact that for the production scales required in such industries

(hundreds to thousands of Kg), multipurpose plants in which any type of chemistry

can be conducted are more economically viable than continuous production plants

dedicated to one process

Nevertheless, the last two decades have witnessed a renaissance in continuous-

flow technology, both in the academic community and in the pharmaceutical

sector.[39, 78] The intrinsic safety associated with flow reactors is perhaps the

main reason rendering this technology attractive to industrial process chemists.[79] Instead, researchers in academia turned their attention to flow chemistry as

enabling technology offering a precise control over key reaction parameters, and

thus granting access to otherwise “impossible” chemistries.[80] Within continuous

flow chemistry, microreactor technology is the branch concerned with the

development, characterization and implementation of microflow reactors. For

the purpose of this dissertation, the term microreactor should be intended as

a continuous-flow reactor with an inner diameter ≤ 1 mm. Flow reactors with a

diameter larger than 1 mm will be referred to as milli- or meso-reactors. Due to

their smaller dimensions, microreactors exhibit especially high surface-to-volume

ratios, which in turn result in very efficient mass- and heat-transfers. Overall,

this means that a fine control over many reaction parameters (e.g. temperature,

Photoredox decarboxylative strategies

a) Decarboxylative macrocylization (Macmillan et al., 2017)

Ir photocat.K2HPO4,

NHPhO

NH

O

HN

iPr

O

HNPh

O

OHN

COOH

NHPh

ONH

O

HN

iPr

O

HNPh

OO

HN

DMF, rt

86%

95:5formate buffer/glycerol

pH 3.5, 8h

Lumiflavin

aa

COO-

Glu aaNH

RO

O-

aa

COO-

Glu aaNH

RR'

R''

No functionalizationon internal Glu residuesR'

R''

Cys

Cys Thr Ser

Gly

Ile

Val

Glu

Gln

Ile

CysS SSer

Leu

Tyr

GlnLeu Glu

Asn

Tyr

Cys HN

O

O

O

NH2SS

CysValLeu

Gly

Glu

Arg

GlyPhe

PheTyr

ThrPro

LysThrHOOC

SS

Tyr

LeuAla

GluCys

ValLeuHisSer

Gly

LeuHis

Gln

Asn

Val

Phe

H2N

H2N

Human insulin: selective A chain functionalization, 49%

A chain

B chain

b) Photocatalytic selective C-term decarboxylation/alkylation (Macmillan et al., 2017)

1


pressure, mixing etc.) is straightforward in microreactors. From a safety point

of view, the accumulation of significant quantities of hazardous materials or

reaction intermediates is prevented due to the small volumes of microreactors.

Moreover, the scaling-up of continuous flow microreactors can be regarded a

rather straightforward procedure, often requiring only a minimal redesign of the

reactor/reaction conditions. Other advantages of microreactors are the improved

mixing, ease of processing multiphase reaction mixtures and potential to include

inline analytical technologies, allowing to develop fully automated and/or self-

optimizing processes. Specifically, inline spectroscopic tools, self-optimization

protocols and automation allow to further reduce human interaction (see Chapter

7).[81]

CONTINUOUS FLOW PHOTOCHEMISTRY

Over the last decade, the renewed interest for photocatalysis in organic synthesis

also brought some old and unsolved problems back to the surface. The majority of

the issues associated with the use of light to promote chemical transformations

derive from the complexity of photochemical processes. However, the main

practical issue preventing the widespread use of photochemical transformations in

the chemical industry is that scalability is hampered. This is due to the attenuation

effect of photon transport described by the Bouguer−Lambert−Beer law, which

prevents the use of a dimension-enlarging strategy for scale-up. If larger reactors

are used, over-irradiation of the reaction mixture can become an important issue as

the reaction times are substantially increased. This often results in the formation

of byproducts, complicating the purification process significantly.

Thus, the use of continuous-flow microreactors for photochemical applications

has received a lot of attention as it allows to overcome the issues associated with

batch photochemistry. The narrow channels of a typical microreactor provide the

means to ensure a uniform irradiation of the entire reaction mixture. Consequently,

photochemical reactions can be substantially accelerated (from hours/days in batch

to seconds/mins in flow) and lower photocatalyst loadings are often feasible. This

reduction in reaction time minimizes potential byproduct formation and increases

the productivity of the photochemical process.

PHOTOREDOX CATALYSIS IN CONTINUOUS FLOW: FUNDAMENTALS

In the following section, the fundamental aspects that render continuous flow

technology an ideal platform to carry out photochemical transformations will be

discussed in details, with an emphasis on the most relevant principles pertinent to

the research work described herein.

Good reasons why to “go with the flow”:

1. Improved irradiation of the reaction mixture

Photochemical reactions are driven by the absorption of photons. Consequently, a

homogeneous energy distribution inside the photoreactor is crucial to obtain high

yields and high selectivities. According to the well-known Bouguer-Lambert-Beer

law (Equation 1.2), the radiation distribution will not be uniform in a reactor due

to absorption effects.

(1.2)

This equation shows a clear correlation between the absorption and the molar

extinction coefficient (ε) of the light absorbing molecules, their concentration (c)

and the path length of light propagation (l). By plotting this relationship between

absorption and reactor radius, the importance of reactor size can be conclusively

shown (Figure 1.2). With strong absorbers, such as common photocatalysts and

organic dyes, the light intensity is rapidly decreasing towards the centre of the

reactor. In order to keep the radiation distribution uniform and thus maximize the

efficiency of the photochemical process, microreactor technology can be used.[82]

Figure 1.2: Transmission of light as a function of distance in a photocatalytic reaction using Ru(bpy)3Cl2 (c = 0.5, 1 and 2 mM, ε = 13,000 cm-1 M-1) utilizing the Bouguer-Lambert-Beer correlation (Eq. 1.1).

𝐴𝐴𝐴𝐴 = log10 𝑇𝑇𝑇𝑇 = log10𝐼𝐼𝐼𝐼0𝐼𝐼𝐼𝐼

= 𝜀𝜀𝜀𝜀𝜀𝜀𝜀𝜀𝜀𝜀𝜀𝜀

1


2. Improved reaction selectivity and increased reproducibility.

One of the key aspects in any chemical reaction is the product selectivity. It is

generally accepted that microreactors provide opportunities to increase the

reaction selectivity substantially. This is often attributed to the enhanced mass-,

heat- and photon-transport phenomena observed in microchannels, which offers a

high reproducibility of the reaction conditions. Such benefits can be related to the

increased surface-to-volume ratio in microchannels.

Scheme 1.14: Schematic representation of a photochemical microreactor setup including a mixing zone (top), a reaction zone (centre), and a quenching zone (bottom).

A typical flow setup can be schematically represented as depicted in Scheme 1.8

and consists of a mixing zone, a reaction zone and a quenching zone. In batch,

reaction time is defined by how long the reactants are residing in the vessel. In

contrast, in flow reactors, reaction time (also called residence time) is defined by

the average time that the reactants spend in the reactor. Consequently, reaction/

residence time is related to the flow rate. Due to the presence of a quencher,

reaction times can be precisely controlled and thus follow-up reactions (e.g.

degradation of the compound) can be avoided by minimizing the time spent in the

reaction zone.

3. Fast mixing

One of the key features of microreactors is their size. Generally speaking, the

reactor’s size is very important to explain the observed mixing phenomena

in continuous-flow photochemistry.[83] In microscale photomicroreactors, the

observed flow pattern is laminar flow (Scheme 1.9). In this flow regime, fluid

is flowing in parallel layers and mixing is governed by diffusion across parallel

lamellae. It is easy to understand that the smaller the diameter of the reactor, the

faster a uniform concentration of reactants across the channel will be obtained.

This insight led to the development of micromixers in which the diffusion distance

is minimized to obtain mixing times in the milliseconds range.[84]

Scheme 1.15: Laminar flow regime encountered in microchannels. Note that the velocity profile in the fully developed region (after t2n) is parabolic. The velocity at the wall is much lower than the velocity in the centre of the capillary.

Mixing is especially important to prevent the formation of by-products which

originate because of local concentration gradients. This is called disguised chemical

selectivity and occurs when the mixing time is much larger than the reaction time.[85] On a macroscale, such selectivity issues are overcome by lowering the reaction

temperature as this will slow down the reaction kinetics significantly. However, in

microscale reactors, the mixing efficiency can be substantially increased due to

the small size of the reactor. This provides opportunities to carry out reactions at

higher temperatures than in conventional batch equipment.

4. Multiphase chemistry

Multiphase reactions are very common in the chemical industry, e.g. oxidations,

hydrogenations, halogenations and phase-transfer catalysis reactions. They

involve the combination of two or more immiscible phases (e.g. liquid-liquid or

Reaction zone

Mixing zone

reagent A

Substrate

reagent B

Controlled reactionenvironment (T, P,irradiation, etc.)

micromixer

Reagents introduced bypumps (syringe pumps,HPLC, etc.)

Quenching zone

microreactor

additional modules (optional)

quench A

inline analysis (optional)

Controlled reaction quench

Products

Laminar flow:

tt0 tn t2n

1


gas-liquid reactions). When mass transfer from one phase to the other is the rate-

determining step, it is crucial to maximize the interfacial area between the phases.

In batch, the interfacial area is low and poorly defined, which leads to prolonged

reaction times. Due to the small characteristic dimensions of microreactors, large

and well-defined interfacial areas are observed, which can reach up to 9000 m2·m-

3. These large interfacial areas lead to efficient mass transfer between the two

phases. More specifically, the mass transfer between two phases can be described

with the overall volumetric mass transfer coefficient (kLa), which in microreactors

is one to two orders of magnitude higher than for classical multiphase reactors.

Several microreactor designs have been developed to carry out multiphase

reaction conditions (Scheme 1.10).[86]

Scheme 1.16: Overview of the most common microreactor types used for multiphase flows.

A first design involves single channel microreactors, e.g. transparent polymer-

based capillaries, which are commonly used in photochemical applications. Two

flow regimes are typically used in single channel microreactors, i.e. segmented

flow (also known as Taylor flow or slug flow) and annular flow (sometimes referred

to as pipe flow). Segmented flow is characterized by liquid slugs and elongated

bubbles of an immiscible phase (e.g. gas or liquid, see for example Chapter 2). In

both, toroidal vortices are established which cause a powerful mixing effect and

intensify the mass transfer from one phase to the other. The bubble is separated

from the reactor walls by a thin liquid film. In photochemical applications, this thin

film will receive the highest intensity of photons and it can be anticipated that

reaction rates are locally very high. The internal circulation patterns will result in

a thin-film renewal exposing different fluid to high photon fluxes. Annular flow is

obtained when one phase flows at a higher velocity in the centre of the capillary,

while the wetting phase forms a thin layer at the channel wall. This thin film will be

in close contact with the phase flowing in the centre of the reactor and will receive

the highest photon flux. A second design concerns the falling film microreactor in

which the liquid phase is directed over the channels by using gravitational forces.

The gas phase can be subsequently directed co- or counter-currently in front of

the liquid phase. A third design is the membrane reactor in which the two phases

are separated by a permeable membrane. Through the membrane, reactants can

diffuse from one phase to the other. A notable and popular example of membrane

reactors are the so-called tube-in-tube reactors.[87]

5. Immobilized catalyst

Immobilization of photocatalysts is an interesting way of recuperating and recycling

the catalyst and thus increasing the economic viability of the photocatalytic

process.[83b, 88] Heterogeneous photocatalysts, such as semiconductors (e.g.

TiO2 and ZnO), are extensively used in photochemical applications due to their

abundance, low cost, and robustness.[89] These catalysts can be introduced in the

reactor as a slurry. However, such an approach can lead rapidly to reactor clogging

when using microstructured photoreactors and a downstream separation step

is required to remove the catalyst from the products. An alternative approach is

the immobilization of this catalyst on the reactor walls or in a packed bed (see

Chapter 3).[90]. Alternatively, wall-coated photomicroreactors have been reported

in literature.[91] In these examples, a thin layer of photocatalyst is immobilized on

the reactor walls and the catalyst can be irradiated from the front or the backside

of the catalyst. Due to the short diffusion distances and the large surface-to-

volume ratios in the microchannels, enhanced performances are usually observed.

The illuminated catalyst surface area per unit of liquid in the reactor (κ) is high

for microreactors (κ = 11,667 m2·m-3), compared to slurry photoreactors (κ = 2,631

m2·m-3) or external type annular photoreactors (κ = 27 m2·m-3).[92] Photocatalysts

can also be immobilized on a membrane.[93]

Slug flow(Taylor regime):

tt0 tn t2n

immiscible phaseinternal

convection

(a) Diffusion into reaction bulk & thin film(b) High photon flux at thin film

(b)

(a)

Annular flow:

immiscible phase continuous thin-film

side view

LightsourceGravity assisted

flow direction

Gaseous flow

Back plate

gaseous bulk on-topof reaction mixture

Falling Film Microreactor:

Membrane Microreactor

liquid reaction phasegas permeablemembrane

side viewgaseous phase

(a)

Single channel microreactor:

1


6. Increased safety of operation

Increasing the operational safety of hazardous reactions is one of the main

arguments for the chemical industry to switch to a continuous-flow protocol.

Microreactors have small dimensions which reduces the total inventory of

hazardous chemicals and thus avoids any safety risks associated with its handling.

Furthermore, the impact of an explosion is directly related to the total amount

of explosive materials present in the reactor to the power of 1/3. Despite the

advantages of microreactors, one must still be careful as explosions can propagate

into the neighbouring storage vessels which might contain larger amounts of

explosive material.[94] This also demonstrates the need of suitable quenchers

at the reactor outlet, which can prevent explosion propagation. Due to the

increased safety, reaction conditions which are impossible to carry out in batch

can be accessible in flow microreactors. This is especially true for photochemical

reactions involving gases (see Chapter 2) or potentially hazardous intermediates

(e.g. diazonium salts, see Chapter 4).

7. Other reasons

Besides the aforementioned reasons, other advantages inherent to continuous-flow

technology might be beneficial when carrying out photochemical transformations.

However, as these advantages are not directly correlated with the experimental

work presented in this dissertation, they will only be briefly described herein.

Due to their high surface-to-volume ratio, microreactors provide an efficient

heat dissipation.[80a] This feature is especially relevant in the case of highly

exothermic reactions or to dissipate the heat generated by artificial light sources.

Consequently, hot spot formation can be largely avoided and microreactors can

often be considered as isothermal reactors. Continuous-flow chemistry is also an

attractive technology to facilitate multistep reaction sequences as it allows several

reaction and purification steps to be combined in one continuous streamlined flow

process.[95] Such flow networks require less manual handling for the practitioner

and result in substantial time and economic gains. As mentioned before, due

to the Bouguer−Lambert−Beer law limitations, scalability of photochemical

processes even on a laboratory scale has been a long-known problem. Microreactor

technology has been embraced by the scientific community as the ideal reactor to

scale photochemistry.[96] Essentially, two strategies can be distinguished to scale-up

photochemistry with photomicroreactors: 1) longer operation times and increased

flow rates (which in turn result in an increased throughput) and 2) numbering-up.

With the second strategy, two main different approaches can be distinguished:

external and internal numbering-up. The first one is simply achieved by placing

several microreactors along with their pumping system and process control in

parallel.[97] Therefore, the entire microreactor setup is copied several times until

the desired amount of product can be reached. On the other hand, internal

numbering is more economically feasible as only the reactor itself is numbered-

up while the process control and pumping system is shared. The essential part

in the design of efficient internal numbering up is the distributor section, which

equalizes and regulates the reaction streams over the different microreactors.[98]

Small differences in pressure drop will lead to significant maldistribution and thus

in non-equal reaction conditions in the different reactors.[99]

PHOTOCATALYTIC MODIFICATION OF BIOACTIVE MOLECULES IN CONTINUOUS-FLOW MICROREACTORS

AIM AND OBJECTIVES

Up to 2015, only few reports on the use of photocatalytic strategies for amino

acid or protein modification existed. Moreover, none of these examples focused

on the use of continuous-flow systems to achieve a homogeneous and efficient

irradiation of the reaction mixture. Thus, at the beginning of the research

described in this dissertation, the primary goal was to test the benefits deriving

from the combination of this mild catalytic approach with continuous-flow as

enabling technology. From a chemical point of view, a decision was made to

target cysteine as the amino acid to be modified. Among endogenous amino acids,

cysteine represents one of the most targeted residues for the post-translational

modification of peptides and proteins.[46] The reason behind the popularity of

bio-modification strategies targeting cysteine can be found in its low natural

abundancy (~ 1-2% in proteins), together with its relatively high nucleophilicity.

These characteristics combined offer an advantageous starting point when trying

to achieve the site-selective introduction of relevant moieties. Furthermore,

cysteine displays high nucleophilicity, particularly in its deprotonated thiolate

form (pKa ~ 8.2 depending on the conformation, neighboring residues and reaction

mixture), making it an ideal target for rapid and efficient modification.

Another important goal of this dissertation was to develop continuous-flow

systems specifically designed to accommodate the reaction conditions required

for the photocatalytic methodologies of interest. In other words, depending on the

chemical reactions developed, a concrete effort towards an optimal reactor design

was made. In certain instances, this resulted in reactors optimized to safely handle

1


gaseous reagents, while in other cases reactors for the handling of heterogeneous

catalysts were assembled (vide infra).

Parallel to these two main goals, extra efforts were dedicated to further explore

the chemical space enabled by the positive combination of photoredox catalysis

and continuous-flow technology. Despite not being centered on bioconjugation,

the results obtained in this other research endeavors lead to other applications

on the combination of photoredox catalysis and continuous-flow technology and,

thus, will be described herein.

THESIS OUTLINE

To facilitate the organization of the research conducted, the results presented in

this dissertation have been divided into two main research lines.

A first research line focused on the development of novel photoredox methodologies

for biomolecule modification (vide supra). Thus, Chapters 2 to 4 describe three

different approaches allowing cysteine modification, with an emphasis on the

application of this methods to the modification of peptides (Scheme 1.11). In

all three cases, the implementation of continuous-flow technology afforded a

homogeneous irradiation of the reaction mixture and resulted in an increased

reaction rate compared to batch methods. Moreover, these three methodologies

can serve as positive examples to demonstrate how different inherent advantages

of continuous-flow chemistry can improve photoredox reactions.

Scheme 1.17: Overview of the photocatalytic methods for cysteine modification described in this dissertation

For instance, in one case the assembled microflow reactor afforded safe and

efficient handling of a gaseous reagent (Chapter 2). In another instance the

preparation of a packed-bed reactor enabled an adequate use of a heterogeneous

catalyst while allowing to significantly accelerate S-S bond formation (Chapter 3).

Finally, in a third case the optimal mixing deriving from the formation of a slug-flow

regime afforded an incredible reduction in reaction time (30 seconds residence

time, Chapter 4).

However, despite all the benefits provided by microflow technology, it is important

to state that one of the main objectives of this research line was to demonstrate the

application of the developed photoredox methodologies to peptide substrates.

Thus, a parallel effort was made into rendering biocompatible the aforementioned

methodologies by employing buffered water solutions as solvents and neutral

pH. Moreover, in order to render these methods highly practical and appealing

for chemical biology labs that might not have the resources to assemble flow

reactors, reactions performed on peptides were carried out in standard vials.

Consequently, the methodologies developed can have dual applications. On the

one hand, flow reactors can be employed to rapidly obtain large quantities of

cysteine derivatives (or small modified peptides). Notably, such quantities are

compatible with the amounts required for standard solid phase peptide synthesis

(SPPS), thus rendering the developed methodologies useful for the synthesis

of typically expensive unnatural amino acids. On the other hand, another set of

optimal reaction conditions to afford peptide modification in batch mode were

described, resulting in easy protocols that can appeal to a broad community of

chemists and biologists. It is worth noting that in the majority of cases the reactions

were performed on crude, non-purified peptides, hence minimizing the need for

intermediate purification steps.

In the second part of this dissertation, projects designed to address some of the

limitations currently preventing the widespread use of photoredox catalysis in the

pharmaceutical industry are discussed. Specifically, in Chapter 5 the combination

of photoredox catalysis and microreactor technology allowed the development

of a trifluoromethylation strategy suitable for the late stage modification of drug

candidates.

In the context of structure-activity relationship (SAR) studies, the incorporation

of fluorinated moieties is of great interest as it improves the lipophilicity and

bioavailability of biologically active compounds (Scheme 1.12).[100] Moreover, the

replacement of methyl groups with their trifluoromethyl counterparts is of high

interest because, while being conservative in terms of steric hindrance, it can

H2N

OHO

SH

H2N

OHO

S

H2N

OHO

S

H2N

OHO

S

CF3 or

SR

Chapter 4:Arylation

Chapter 2:Fluorination

Chapter 3:Disulfideformation

Key features

- Mild reaction conditions- Selective in presence of other aa- Different modifications possible:a) arylationb) trifluoromethylationc) disulfide formation

Arylation, disulfide:-Labeling and tagging

Trifluoro- or perfluoroalkyaltion:- Precursors of PET tracers- Study of hydrophobic pocketsin enzymes

Possible Applications

CF2COOEt

1


represent a valuable strategy to block potential metabolic sites in drug candidates,

thus prolonging their half-life and metabolic stability.[100a]

Scheme 1.18: Relative size comparison between a trifluoromethyl group and other alkyl substituents; effects of the introduction of F in drug candidates and two top-selling drugs currently on the market containing a trifluoromethyl moiety.

Indeed, the introduction of fluorine substituents is often a good strategy in drug

development, as demonstrated by the fact that an estimated 20% of the drugs

currently approved on the market contain at least one fluorine moiety.[101] To

showcase the relevance of this method in the context of drug discovery processes,

the reaction scope was conducted on a series of highly-functionalized arenes

and heteroarenes, with a focus on N-containing heterocycles bearing halogen

substituents as convenient handles for further modification via standard cross-

coupling methods (see Chapter 5).

The most commonly used photocatalysts in photoredox catalysis are transition

metal-based complexes (i.e. Ir and Ru polypyridyl complexes). Despite their

excellent redox properties and the long lifetime of the photo-generated excited

species, the use of this strongly absorbing species is also associated with some

drawbacks (Scheme 1.13). Firstly, due to their limited availability in nature, Ir and

Ru polypyridyl complexes have prohibitive costs. Thus, the implementation of

photoredox methods employing these compounds for the large scale production

of fine chemicals is not economical at this stage.

Secondly, transition metal-based complexes can be prone to photobleaching,

which can represent an issue for catalyst stability and long-term usage. Hence,

in the last years significant efforts have been devoted by the photochemical

community into the use of organic dyes as valuable and economical alternatives to

transition metal-based complexes (Scheme 1.13). The development of new classes

of organic photoredox catalysts with good redox properties, largely available or

easily prepared in high yields would contribute to render photoredox methods an

economically viable catalytic approach. In Chapter 6, the de novo synthesis and

characterization of a series of substituted bis-thiophene derivatives as novel and

inexpensive organic photocatalysts was described. Based on the predictions on a

large series of bithiophene derivatives deriving from DFT calculations, a cluster

of six new molecules was synthesized via a one-step procedure starting from

commercially available precursors. Notably, for all compounds the calculated

absorption spectra and redox potentials matched closely with the collected

empirical data. The ability of this new series of organic photocatalysts to catalyse

photoredox reactions was then tested. In two distinct visible-light mediated

strategies for the C-H functionalization of heteroarenes excellent results were

obtained. Significantly, the develop photocatalyst compared positively with other

commonly used organic or transition-metal photocatalysts.

Scheme 1.19: Overview of the different properties of transition metal-based and organic photocatalysts.

Finally, the last project discussed in this dissertation demonstrates the potential

of continuous-flow systems in terms of automation of chemical synthesis. Recent

breakthroughs in the flow chemistry community sparked the idea that automated

platforms designed to execute all the routinely aspects of organic synthesis

might soon become a reality.[5a, 102] Inspired by these ideas, an automated platform

continuous-flow platform capable to perform fluorescence quenching studies and

Effects of F in drug candidates

pKa

permeability

potency

clearance ( by blockinglabile metabolic sites)

MeMe

Me

Relative size comparison of CF3 group with alkyl substitutents

>MeF

Me>

FF

F>

HH

Me>

HH

H

CF3

ONH

Me

CF3-containing drugs

Fluoxetine(Prozac)

OS

NN

Me

NH2O

F3C

Celecoxib(Celebrex)

Transition metal complexes

Advantages:- Long lifetime of excited states- Excellent redox potentialsDisadvantages:- Expensive- Not widely available- Contain metals

Organic photocatalystsRu(bpy)32+ fac-[Ir(ppy)3]

Mes-Acr+

N

N

RuN

N

N

N

N

IrN

N

2+

NMe

Me

MeMe

Advantages:- Widely available- Good redox potentials- AffordableDisadvantages:- Activity can be pH dipendent- Prone to photobleaching- Shorter lifetime of excited states

Eosin Y2-

O

COO

Br

Br

OBr

O

Br

1


Stern-Volmer analysis was conceived. These analysis are relevant in photoredox

catalysis as they can be used to probe the interaction of organic substrates with the

excited state of photocatalysts, thus revealing important information concerning

the rate and the nature of a photocatalytic cycle.

Scheme 1.20: Overview of the automated continuous-flow setup for fluorescent quenching studies and Stern-Volmer analysis. For a detailed description see Chapter 7.

As described in Chapter 7, this system has the potential to accelerate reaction

discovery in photoredox catalysis and to contribute to elucidate the underlying

mechanisms involved. Overall, compared to manually executed experiments,

the use of the automated system affords accurate and reproducible data while

minimizing human errors

LED light source

Autosampler

or Flow cuvette

SpectrophotometerComputer

Waste

Catalyst

Solvent Quencher

UV-Vis

Control interface

- Completely automated

- Flexible set-up

- High accuracy (R2 >0.98)

- High reproducibility

- For reaction discovery/

Stern-Volmer analysis

Key features

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CHAPTER 2

Visible light-induced trifluoromethylation and perfluoroalkylation of cysteine in batch and continuous flow


Bottecchia, C.; Wei, X.-J.; Kuijpers, K. P. L.; Hessel, V.; Noël, T., J. Org. Chem., 2016, 81, 7301-7307

2

54 Chapter 2 Visible light-induced trifluoromethylation and perfluoroalkylation of cysteine 55

ABSTRACT

In this chapter, a visible light-induced trifluoromethylation and perfluoroalkylation

strategy for cysteine conjugation is described. The methodology relies on the use

of Ru(bpy)32+ as photocatalyst and inexpensive perfluoroalkyl iodides as coupling

partners. The protocol allows the introduction of a variety of perfluoro alkyl groups

(C1-C10) and of a CF2COOEt moiety. The reaction is high yielding (56-94% yield) and

fast (2 hours in batch, 12 examples). Process intensification in a photomicroreactor

accelerated the reaction (5 minutes reaction time) and increased the yields.

Interestingly, quantum yield investigations support a radical chain mechanism.

INTRODUCTION

The incorporation of fluorinated moieties is of great interest as it improves the

metabolic stability, lipophilicity and bioavailability of biologically active compounds.[1] In addition, access to fluorinated biomolecules allows one to use 19F-NMR

spectroscopy to investigate complex biological interactions.[2] Recent advances in 18F-radiochemistry also provide access to 18F-labeled small molecules, peptides and

proteins thus rendering positron emission tomography (PET) techniques suitable

for in vivo monitoring of biochemical processes.[3] Several biosynthetic approaches

have been developed allowing the introduction of fluorinated amino acids through

protein engineering.[4] However, most of these methods fail to provide efficient

strategies for the incorporation of highly fluorinated amino acids, which are often

prepared through post-translational chemical modification of native sequences or

through insertion of fluorinated residues via standard solid phase peptide synthesis.[5] In this regard, examples from literature showed viable synthetic pathways towards

trifluoromethylated amino acids or peptides.[6] As part of our interest to develop

efficient synthetic tools for chemical biology purposes, we have been evaluating

the use of visible light photoredox catalysis for efficient cysteine conjugation.[7] A

cysteine residue provides a unique reactivity which allows it to engage in radical or

ionic reaction processes.[8] In general, the reactivity of aliphatic and aromatic thiols

towards perfluoroalkyl iodides under UV irradiation has been reported before

and it has been speculated that it mightollow a nucleophilic radical substitution

mechanism.[9] As example, Soloshonok et al. utilized CF3I to construct the cysteine

S–CF3 bond but the method suffered from unpractical reaction conditions (such

as, liquid ammonia, -50°C and UV irradiation, Scheme 2.1).[10] Also other radical

approaches were troubled with low yields and limited scope.[11] A more convenient

method was developed by Togni, Seebach et al. utilizing hypervalent iodine(III)

trifluoromethylating reagents but still required cryogenic reaction conditions

(Scheme 2.1).[6c, 12] Furthermore, introduction of other perfluoroalkylated analogues

requires the synthesis of unique hypervalent iodine(III) perfluorinating reagents,

which are either expensive, not commercially available, or difficult to synthesize.

In order to develop a general protocol for the perfluoroalkylation of cysteines,

we turned our attention to RF-I reagents (RF = perfluoroalkyl) which can generate

electrophilic perfluoroalkyl radicals under visible light photocatalytic reactions

conditions and are commercially available and cost efficient (Scheme 2.1).[13]

We anticipated that such electrophilic perfluoroalkyl radicals can be used to

functionalize cysteine residues, thereby rendering a more general and practical

approach to access perfluoroalkylated cysteines. We also demonstrate that the

2


use of continuous-flow photochemistry allows to accelerate this transformation,

which provides a convenient and scalable method able to efficiently handle

gaseous reactants (e.g. CF3I).

Scheme 2.1: Trifluoromethylation strategies for cysteine modification

RESULTS AND DISCUSSIONBased on our experience with the visible-light induced photocatalytic

trifluoromethylation of aromatic thiols, viable reaction conditions that might be

suitable for the trifluoromethylation were selected (i.e. photocatalyst, organic

base, CF3 source).[14]

Thus, we chose to initiate our investigations by trifluoromethylating cysteine

1 with gaseous CF3I in the presence of Ru(bpy)3Cl2 as a photocatalyst and

tetramethylethane-1,2-diamine (TMEDA) in acetonitrile (Table 2.1). Irradiation of

the reaction mixture was achieved by a 24 W white CFL (compact fluorescent light).

In the absence of any light or nitrogen base, no reaction product (2a) could be

obtained (Table 2.1, Entries 1 and 2). The formation of SCF3 product in the

absence of any photocatalyst occurs via homolytic cleavage of the CF3–I bond

upon irradiation (Bond Dissociation Energy [CF3–I] = 52.6 ± 1.1 kcal/mol which

corresponds to 544 nm photons) (Table 2.1, Entry 3).[15] However, a more efficient

and faster reaction was observed in the presence of Ru(bpy)3Cl2 (Table 2.1, Entry

4). When the reaction was conducted in methanol (MeOH), a lower product yield

This work: Batch/flow perfluoroalkylation of cysteine via photo-induced RF generation

RF-I

-Inexpensive-Commerciallyavailable

HN

O SH

MeO

R' HN

O

MeO

R'

SRF

Visible lightPhotoredox catalysis

room temperature5 min flow / 2 hours batch

MeOH/H2O -78°C

Togni and Seebach (2008)

I OF3C

HN

O SH

MeO

R' HN

O

MeO

R'

SCF3

12 h

Liquid NH3, -50°C

NH2

O S

HO

CF3

NH2

O SH

HO

Soloshonok (1992)

CF3-I

UV-irradiation

was obtained (Table 2.1, Entry 5), while water proved to be an incompatible solvent

which is mainly caused by solubility issues (i.e. poor solubility of both CF3I and the

Table 2.1: Optimization studies for the visible light-induced trifluoromethylation

of cysteine 1 with CF3I protected cysteine in water, Table 2.1, Entry 6). Further,

the amount of CF3I could be lowered (from a large excess of 10 equivalents to 4

equivalents) without any impact on the reaction yield (Table 2.1, Entries 4 and 7).

The use of an inorganic base proved to be ineffective for this transformation (Table

2.1, Entries 8 and 9). Satisfyingly, in contrast to our observations with aromatic and

other aliphatic thiols, no disulfide byproduct formation could be observed when

analyzing the reaction mixture in GC-MS.

Table 2.1: Optimization studies for the visible light-induced trifluoromethylation of cysteine 1 with CF3I

Entry Conditionsa Solvent Base Eq. of CF3Ib Yield (%)c

1 No light MeCN TMEDA 10 n.r.

2 CFL MeCN No base 10 n.r.

3 CFL, no catalyst MeCN TMEDA 10 / 4 43 / 23

4 CFL MeCN TMEDA 10 84

5 CFL MeOH TMEDA 10 49

6 CFL H2O TMEDA Sat. n.r.

7 CFL MeCN TMEDA 4 82

8 CFL MeCN KOAc 4 35

9 CFL MeCN Na3PO4 4 23

aStandard reaction conditions: N-Boc-L-Cys-OMe (1) (0.5 mmol), Ru(bpy)3Cl2•6H2O (3.75 mg, 1mol%)

TMEDA (1 mmol), and CF3I in 5 ml MeCN. bCF3I added directly to the reaction mixture or via a stock

solution in MeCN, visible light irradiation with a CFL (compact fluorescence light), 2 hours. cYield

determined by 19F-NMR with addition of an internal standard (α,α,α-trifluorotoluene, 0.5 mmol)

With optimal conditions in hand, we investigated the scope of this photo-

induced trifluoromethylation protocol (Scheme 2.2). Two L-cysteine derivatives

with a different amine protecting group were efficiently trifluoromethylated

in excellent isolated yield (compounds 2a and 2b). Notably, good to excellent

yields were also obtained for dipeptides Boc-Leu-Cys-OMe (4, 56%) and Boc-

Phe-Cys-OMe (5, 94%), thus showing the selectivity of our methodology in the

presence of these two amino acid residues. However, further investigations

to test the selectivity of the trifluoromethylation reaction in presence of other

MeCN, RT

1 mol % Ru(bpy)3Cl2CF3I

TMEDA

1 2a

HN

O S

MeO

OtBu

O

CF3

HN

O SH

MeO

OtBu

O

2


more reactive residues (e.g. lysine, serine or histidine) would be necessary in

order to demonstrate the overall general applicability of this methodology.

Scheme 2.2: Direct photo-induced trifluoromethylation of cysteine residues in batch.

Reaction conditions: Cysteine derivative (0.5 mmol), Ru(bpy)3Cl2•6H2O (1mol%), TMEDA (1

mmol), and CF3I (2 mmol) in 5 ml MeCN. 24 W white CFL, 2 hours.

Several studies have shown that the introduction of multiple highly fluorinated

amino acids can significantly alter the properties of proteins.[16]. Due to the high

electronegativity of fluorine, C-F bonds exhibit a strong and opposite dipole

moment compared to C-H bonds.[17] However, C-F bonds are also characterized by

a lower polarizability compared to C-H bonds (this is due to the high number of

tightly packed electrons in fluorine outer shell and to its small radius compared to

other halogens ).[18] These two features become more relevant in highly fluorinated

molecules, which are typically characterized by hydrophobic interactions with their

environment.[19] Therefore, a perfluoroalkylated cysteine residue could change the

overall acidity of the protein or could participate in hydrophobic interactions in a

biological environment. Specifically, the modified residue could be harbored within

hydrophobic pockets of proteins and enzymes, therefore providing an enabling tool

for the investigation of hydrophobic protein-protein or even protein-membrane

interactions.[5b] Moreover, the synthetic access to various perfluoroalkylated amino

acids would be of high interest to generate a small library of compounds, which

can be used for rapid screening of such interactions. In addition, long fluorous tags

can be used to recover peptides and proteins by enabling extraction techniques

with fluorinated solvents.[20]

2a, 90%

Ru(bpy)3Cl2, TMEDA

CH3CNCF3I

2b, 82% 4, 96% 5, 94%

HN

O S

MeOOtBu

O

CF3

HN

O S

MeOMe

O

CF3O

HN

iPrO

O

ONH

S

OMeCF3

tBu

Boc-Leu-Cys-OMe

OHN

BnO

O

ONH

S

OMeCF3

tBu

Boc-Phe-Cys-OMe

HN

O SH

MeO

R'HN

O

MeO

R'

SCF3

Scheme 2.3: Direct photo-induced perfluoroalkylation of cysteine in batch. Reaction conditions: N-Boc-L-Cys-OMe (0.5 mmol), Ru(bpy)3Cl2•6H2O (1mol%) TMEDA (1 mmol), and RF-I (1 mmol) in 5 ml MeCN. 24 W white CFL, 2 hours.

To showcase the utility of our methodology for the preparation of highly fluorinated

cysteine residues, the scope of the perfluoroalkyl coupling partner was further

expanded by using a wide variety of commercially available perfluoroalkylated

iodides (Scheme 2.3).A complete range of perfluoroalkyl-substituted cysteines,

bearing perfluoroalkylated chains of variable length (C3 to C10), was obtained in

good to excellent yields (60-90% isolated yield). Moreover, derivative 3h, bearing

an ethyl difluoroacetyl moiety, could be obtained in good yield (75% isolated

yield). This compound constitutes an intermediate of interest for the preparation

of difluoromethyl-substituted compounds or for the introduction of 18F via Ag-

catalyzed decarboxylative fluorination.[3a]

Next, we focused our research efforts to transfer our trifluoromethylation and

perfluoroalkylation protocol to a continuous-flow microreactor. In general,

such devices provide a more homogeneous irradiation/energy distribution and

an increased gas-liquid mass transfer.[21] The observed process intensification

3a, 60%

Ru(bpy)3Cl2, TMEDA

3

HN

O S

MeO

OtBu

O

RF

HN

O S

MeOOtBu

O

F F

FF

CF3

HN

O S

MeOOtBu

O

F F

FF

F F

CF3

HN

O S

MeOOtBu

O

F F

FF

F FCF3

F F

HN

O S

MeOOtBu

O

F F

FF

F F

F F

F F

CF3

HN

O S

MeOOtBu

O

F F

FF

F F

F F

F FCF3

F F

HN

O S

MeOOtBu

O

F F

FF

F F

F F

F F

F FCF3

F F

HN

O S

MeOOtBu

O

F F

FF

F F

F F

F F

F F

F F

CF3F F

F F

OEt

O

HN

O S

MeOOtBu

O

CH3CNRF-I

1

HN

O SH

MeO

OtBu

O

3b, 84% 3c, 71% 3d, 62%

3e, 90% 3f, 67% 3g, 60%

3h, 75%

F F

2


of photochemical transformations in microreactors often results in increased

yields, reduced reaction times and easy scale-up.[22] The microflow setup consists

of perfluoroalkoxyalkane microcapillary tubing (PFA, 760 μm ID, 2.0 m, 883 μL)

wrapped around a plastic holder, which is placed into a 3D-printed beaker (See

Scheme 2.4 and experimental section).[23]

Scheme 2.4: Schematic representation of the microflow setups for (A) gas-liquid trifluoromethylation and (B) perfluoroalkylation of cysteine. For more details on the procedure see experimental section.

The use of 3D-printing techniques to prepare such beakers represents a fast and

straightforward way to obtain reactor holders that match perfectly the reactor

size, thus allowing a more efficient disposition of the light source toward the

reactor. The reactor is subjected to irradiation generated by a blue LED strip

(1 m length, 78 Lumen, 3.12 Watt). The use of such miniaturized light sources,

instead of CFL light sources, allows to increase the overall photonic efficiency

and to minimize unproductive heat generation.[24] For the trifluoromethylation

protocol, CF3I gas was dosed into the liquid stream via a mass flow controller

(Scheme 2.4A). For the perfluoroalkylation protocol, the two liquid phases were

introduced in the reactor by means of syringe pumps (Scheme 2.4B). For both, the

PFA microreactor760 µm ID, 883 µL

MFC

CF3I

N-Boc-Cys-OMe,Ru(bpy)3Cl2TMEDA, MeCN


I-RF,TMEDA, MeCN

N-Boc-Cys-OMe,Ru(bpy)3Cl2, MeCN

RF

HN

O S

MeOOtBu

O

HN

O

MeO

R'

SCF3

A) Gas-liquid trifluoromethylation of cysteine in flow

B) Perfluoroalkylation of cysteine in flow

two streams were combined in a Tefzel T-micromixer (500 μm ID) upon entering

the photomicroreactor. Notably, because of the high solubility of CF3I gas in

acetonitrile, the formation of a slug flow regime was not observed. A significant

acceleration of the reaction rate was observed for both the trifluoromethylation

and perfluoroalkylation chemistry, thus affording the formation of the desired

products in comparable or slightly higher yields than in batch and within only 5

minutes residence time (Scheme 2.5, 62-92%).

Scheme 2.5: Direct photo-induced trifluoromethylation and perfluoroalkylation of cysteine in continuous flow. Reaction conditions: N-Boc-L-Cys-OMe (0.5 mmol), Ru(bpy)3Cl2•6H2O (1mol%) TMEDA (1 mmol), and RF-I (4 equiv. for CF3I, 2 equiv. for RF-I) in MeCN are mixed with a T-mixer and irradiated with an array of. 3.12 W blue LEDs, 5 minutes residence time.


2a, 79%

HN

O S

MeOOtBu

O

F F

FF

CF3

HN

O S

MeOOtBu

O

F F

FF

F F

CF3

HN

O S

MeOOtBu

O

F F

FF

F FCF3

F F

HN

O S

MeOOtBu

O

F F

FF

F F

F F

F F

CF3

F2C OEt

O

HN

O S

MeOOtBu

O

HN

O S

MeOOtBu

O

CF3

HN

O S

MeOMe

O

CF3O

HN

BnO

O

ONH

S

OMeCF3

tBu

Trifluoromethylations

Ru(bpy)3Cl2, TMEDA

HN

O SH

MeO

R' HN

O

MeO

R'

SRF

CF3IorRF-I

2b, 91% 5, 85%

Boc-Phe-Cys-OMe

Perfluoroalkylations

3a, 70% 3b, 92% 3c, 70%

3d, 62% 3h, 81%

2


Scheme 2.6: Proposed mechanism of the Ru(bpy)32+-catalyzed radical perfluoroalkylation of

cysteine residues.

A plausible mechanism for this process is outlined in Scheme 2.6. Upon absorption

of blue light, [Ru(bpy)3]2+ undergoes a metal-to-ligand charge transfer which

is subsequently reductively quenched by TMEDA. Stern-Volmer quenching

experiments indeed demonstrated that this step occurs under our reaction

conditions (see experimental section). Next, [Ru(bpy)3]+ is oxidized to its ground

state generating an electrophilic RF radical. This radical can subsequently react

with cysteine to establish the S–RF linkage. In order to generate a neutral species,

the radical anion needs to undergo another single electron transfer step (SET).

This can be either done with [TMEDA]●+ (chain-terminating SET) or with RFI

(chain propagating SET) to generate another RF radical. In order to elucidate this

step and to update our previously proposed mechanism on the photocatalytic

trifluoromethylation of aromatic thiols, we calibrated the quantum yield of this

transformation against the oxidation of 1,9-diphenylanthracene with singlet

oxygen.[14, 25]

The obtained quantum yield value (defined as the number of molecules of product

formed over the number of photons absorbed, see Eq. 1.1 in Chapter 1) was φ =

126 which demonstrates that indeed a chain propagating SET step is present in the

light-induced perfluoroalkylation of cysteine (see experimental section for details

on the procedure).[26]

CONCLUSIONIn summary, we have developed a visible light-induced photocatalytic route to

prepare a wide variety of trifluoromethylated and perfluoroalkylated cysteine

[Ru(bpy)3]2+

[Ru(bpy)3]2+*

[Ru(bpy)3]+

TMEDA

TMEDAReductivequenching

Oxidativerecombination

h

ChainPropagation

= 126

RFI

RF

HN

O S

MeO

R'

RFHN

O S

MeO

R'

RF

Product

HN

O S

MeO

R'

residues. The mild reaction conditions and the broad scope renders our

methodology amenable to the synthesis of perfluoroalkylated cysteines, which can

then be deprotected on the acid moiety and subsequently employed in standard

peptide synthesis protocols. Moreover, the implementation of a continuous-flow

photomicroreactor afforded similar or increased product yields (up to 10% more

product formation compared to batch) and reduced reaction times (5 minutes vs

2 hours in batch).

EXPERIMENTAL SECTIONGeneral procedure for the trifluoromethylation/perfluoroalkylation of cysteine residues in batch (GP1, for compounds 2a-b, 4, 5): In an oven-dried vial equipped with a magnetic stirrer and a PTFE septum, 3.75 mg (1 mol %) of Ru(bpy)3Cl2•6H2O was added to a mixture of N-Boc-L-Cys-OMe (117.65 mg, 0.5 mmol), N,N,N’,N’-tetramethylethylenediamine (TMEDA) (116.24 mg, 1.0 mmol) and α,α,α-Trifluorotoluene (73.1 mg, 0.5 mmol, internal standard) in MeCN. The fluorinating agent (2 mmol, 4 equiv. for CF3I or I-CF2R, 1 mmol, 2 equiv) was added dropwise to the reaction mixture. For the insertion of CF3I, stock solutions of known concentrations in MeCN were prepared and immediately used for the reaction. The vial was subjected to visible light irradiation with a 24W white CFL. The reaction was stirred at 1000 rpm for 2 hours. The reaction mixture was pre-adsorbed onto silica, dried in vacuo and purified by flash chromatography to yield the fluorinated product.

General procedure for the trifluoromethylation of cysteine residues in a continuous-flow microreactor (GP2, for compounds 2a-b, 5): A 10 mL syringe containing 7.5 mg (1 mol%) of Ru(bpy)3Cl2•6H2O, N-Boc-L-Cys-OMe (235.3 mg, 1 mmol, 0.1 M), α,α,α-Trifluorotoluene (146.1 mg, 1 mmol, internal standard) and TMEDA (232.4 mg, 2.0 mmol) in 10 mL MeCN was mounted on a syringe pump. The liquid flowrate was fixed at 176.6 μL/min. The liquid stream was merged with gaseous CF3I in a T-Mixer before entering the reactor. CF3I was added to the reaction mixture at a flowrate of 1.72 mL/min by means of a mass flow controller. After reaching steady state, a reaction sample was collected until 0.5 mmol of product was collected in a vial kept in the dark. The reaction mixture was pre-adsorbed onto silica, dried in vacuo and purified by flash chromatography to yield the trifluoromethylated product.

General procedure for the perfluoroalkylation of cysteine residues in a continuous-flow microreactor (GP3, for compounds 3a-d, 3h): A 5 mL syringe containing 7.5 mg (1 mol%) of Ru(bpy)3Cl2•6H2O, N-Boc-L-Cys-OMe (235.3 mg, 1.0 mmol, 0.2 M), α,α,α-Trifluorotoluene (146.1 mg, 1 mmol, internal standard) in 5 mL MeCN and a 5 mL syringe containing TMEDA (232.4 mg, 2.0 mmol) and the fluorinating agent (I-CF2R, 2 mmol,2 eq.) in 5 mL MeCN were mounted on a single syringe pump. The liquid flowrate (per syringe) was fixed at 88.3 μL/min. The two liquid streams were merged in a T-Mixer. After reaching steady state, a

2


reaction sample was collected until 0.5 mmol of product was collected in a vial kept in the dark. The reaction mixture was pre-adsorbed onto silica, dried in vacuo and purified by flash chromatography to yield the fluorinated product.

General procedure for the synthesis of dipeptides Boc-Leu-Cys-OMe and Boc-Phe-Cys-OMe: The dipeptides used as starting materials for the synthesis of derivatives 4 and 5 and were prepared through a two-step procedure adapted from literature.[27]

Step 1: Formation of the thioester derivative: L-Boc-Leu-OH or L-Boc-Phe-OH (1.0 equiv.) and DCM or ethyl acetate (0.5 mmol/mL) were added to an oven-dried flask and placed in an ice bath (0°C). Then, DCC (1 equiv.) and HOBt•H2O (1 equiv.) were added together with thiophenol (1 equiv.). The flask was closed with a PTFE septum and the reaction mixture was placed under argon atmosphere. The reaction was checked for completion by TLC (4 -24 hours). The reaction mixture was washed with HCl (1M), NaHCO3 (sat) and brine. The organic layer was dried with MgSO4. The crude was absorbed on silica gel and purified with PE:EtOAC 7:1.

Step 2: Native chemical ligation: L-Cysteine methyl ester HCl (1.0 equiv.) and the thioester derivative (1.0 equiv.) were added to MeOH (0.25 mmol/mL) in an oven-dried flask kept under argon atmosphere and closed with a PTFE septum. Next tributylphopshine (0.6 equiv.) was added by the use of a disposable syringe and the reaction mixture was stirred at r.t. until completion (24 hours). The crude was evaporated under vacuo, and re-dissolved in EtOAc. The organic layer was extracted with H2O (3 times) and with brine (3 times). The organic layer was then dried with MgSO4 and concentrated on silica gel under vacuo. The purification by column chromatography afforded the desired dipeptides Boc-Leu-Cys-OMe (45%) and Boc-Phe-Cys-OMe (24%) (DCM:MeOH 9:1 + 1% acetic acid).

General procedure for Stern-Volmer analysis: All samples used in the luminescence quenching studies were prepared under oxygen free conditions. The photocatalysts Ru(bpy)3

2+ was weighed into a vial, dissolved in acetonitrile and degassed by freeze-pump-thaw. For each measurement, the appropriate amount of the solution of photocatalyst and of a solution of quencher were added to a cuvette and diluted to 1 mL with degassed acetonitrile. The luminescence emission spectrum of Ru(bpy)3

2+ was measured multiple times (three different samples were prepared and measured each twice) and an average was taken as the standard reference spectrum. Solutions of increasing concentrations of quenchers (TMEDA or CF3I) were prepared in acetonitrile and tested. The samples containing the photocatalyst and different concentrations of quencher were each measured three times and an average was taken.

The Stern-Volmer plot obtained for the quenching of Ru(bpy)32+ with TMEDA is reported

below.

Figure 2. 1: Stern-Volmer plot on the quenching of photocatalyst Ru(bpy)32+ with TMEDA.

General procedure for the quantum yield determination for the photocatalytic trifluoromethylation of cysteine: The actinometry experiments were conducted with an AvaSpec- 2048L spectrophotometer from Avantes, equipped with an Avalight-DH-S-BAL light source. In order to quantify the mechanism involved in the trifluoromethylation of N-Boc-Cys-OMe (1), quantum yield measurements were performed. As shown by Scaiano et al.[25], these measurements can be done by the use of a Ru(bpy)3Cl2 actinometer, which in the case of our transformation is also the photocatalyst. The quantum yield measurements consist typically of three steps: First of all, the actinometer is measured. Secondly, the photon flux is determined. Then, the reaction of interest is performed with the same catalyst concentration of the actinometer experiment. Every other parameter is also kept under exactly the same conditions (e.g. reactor volume, light irradiation, residence time, temperature, pressure).

Finally, by comparison between the actinometer and the reaction of interest, the quantum yield for the transformation of interest can be calculated. It is worth nothing that the quantum yields experiments were performed in flow both for the trifluoromethylation reaction and the actinometer. We decided to use the calibrated oxidation of 1,9-diphenylanthracene (DPA) with singlet oxygen and Ru(bpy)3Cl2 as actinometer. The quantum yield of this transformation is known to be of ϕ =0.019, thus allowing to derive the photon flux (ϕ, Einstein/s) in our system. The photon flux obtained for the flow system can then be used to derive the quantum yield of the trifluoromethylation reaction with the following formula:

where ϕ = photon flux (Einstein/s), τ = residence time(s)

𝜙𝜙𝜙𝜙 = 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑚𝑚𝑚𝑚𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝ℎ𝑚𝑚𝑚𝑚𝑜𝑜𝑜𝑜𝑚𝑚𝑚𝑚𝑜𝑜𝑜𝑜 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑜𝑜𝑜𝑜𝑎𝑎𝑎𝑎 𝑚𝑚𝑚𝑚𝑝𝑝𝑝𝑝𝑚𝑚𝑚𝑚𝑠𝑠𝑠𝑠𝑎𝑎𝑎𝑎𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑠𝑠𝑠𝑠𝑚𝑚𝑚𝑚𝑜𝑜𝑜𝑜𝑐𝑐𝑐𝑐𝑚𝑚𝑚𝑚𝑎𝑎𝑎𝑎𝑜𝑜𝑜𝑜𝑎𝑎𝑎𝑎𝑜𝑜𝑜𝑜𝑎𝑎𝑎𝑎 𝑚𝑚𝑚𝑚𝑠𝑠𝑠𝑠𝑎𝑎𝑎𝑎𝑚𝑚𝑚𝑚𝑜𝑜𝑜𝑜𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑜𝑜𝑜𝑜𝑚𝑚𝑚𝑚

𝜑𝜑𝜑𝜑 ∙ 𝜏𝜏𝜏𝜏

2


COMPOUND CHARACTERIZATION

Methyl N-(tert-butoxycarbonyl)-S-(trifluoromethyl)-L-cysteinate (2a)[12] was made according to GP1 on a 0.5 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 100:0 to 90:10) yielding 124.4 mg (0.41 mmol, 82%) of derivative 2a as a white solid (Mp: 67.6-67.9 °C). The reaction according to GP2 on a 0.5 mmol scale afforded. 120 mg (0.39 mmol, 79 %) of product 2a after 5 minutes residence time. 1H NMR (399 MHz, Chloroform-d) δ 5.35 (s, 1H), 4.62 (s, 1H), 3.79 (s, 3H), 3.58 – 3.23 (m, 2H), 1.45 (s, 9H). 13C NMR (101 MHz, Chloroform-d) δ 170.1, 154.9, 130.5 (q, J = 306.4 Hz), 80.6, 53.0, 52.9, 32.1, 28.2. 19F NMR (376 MHz, Chloroform-d) δ -41.0 HRMS (ESI) calculated for C5H9F3NO2S [M-Boc+H]+: 204.0306; found: 204.0308. IR (ATR, cm-1): 3358, 3000, 1724, 1674, 1519, 1369, 1342, 1328, 1292, 1251, 1161, 1145.

Methyl N-acetyl-S-(trifluoromethyl)-L-cysteinate (2b)[11] was made according to GP1 on a 0.5 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl ether 1:1) yielding 110.3 mg (0.45 mmol, 90%) of derivative 2b as a white solid (Mp: 68.6-69.4 °C). The reaction according to GP2 on a 0.5 mmol scale afforded 111.5 mg (0.46 mmol, 91%) of product 2b. 1H NMR (400 MHz, Chloroform-d) δ 6.72 (s, 1H), 5.01 – 4.60 (m, 1H), 3.74 (s, 3H), 3.59 – 3.10 (m, 2H), 2.00 (s, 3H13C NMR (101 MHz, Chloroform-d) δ 170.3, 170.1, 130.5 (q, J = 306.4 Hz), 53.0, 51.9, 31.6, 22.8, 15.2.19F NMR (376 MHz, Chloroform-d) δ -41.1. HRMS (ESI) calculated for C7H11F3NO3S [M+H]+: 246.0412; found: 246.0401. IR (ATR, cm-1): 3317, 2958, 1741, 1734, 1641, 1537, 1340, 1255, 1105, 1039.

Methyl N-(tert-butoxycarbonyl)-S-(perfluoropropyl)-L-cysteinate (3a) was made according to GP1 on a 0.5 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 100:0 to 90:10) yielding 121 mg (0.3 mmol, 60 %) of derivative 3a as yellow oil. The reaction according to GP3 on a 0.5 mmol scale afforded 141.2 mg (0.35 mmol, 70 %) of compound 3a.1H NMR (399 MHz, Chloroform-d) δ 5.37 (s, 1H), 4.61 (s, 1H), 3.78 (s, 3H), 3.60 – 3.28 (m, 2H), 1.44 (s, 9H). 13C NMR (100 MHz, Chloroform-d) δ 170.2, 155.1, 128.0 – 120.8 (m), 117.4 (dt, J = 288.1, 33.3 Hz), 114.0 – 106.9 (m), 80.7, 53.2, 52.9, 30.9, 28.2. 19F NMR (376 MHz, Chloroform-d) δ -76.30 – -82.31 (m), -84.76 – -89.66 (m), -124.08. HRMS (ESI) calculated for C7H9F7NO2S+ [M-Boc+H]+: 304.0237; found: 304.0238: 254. IR (ATR, cm-1): 3367, 2982, 1724, 1518, 1336, 1180, 1161, 1112.

Methyl N-(tert-butoxycarbonyl)-S-(perfluorobutyl)-L-cysteinate (3b) was made according to GP1 on a 0.5 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 100:0 to 90:10) yielding 190.2 mg (0.42 mmol, 84%) of derivative 3b as yellow oil. The reaction according to GP3 on a 0.5 mmol scale afforded 209.3.mg (0.46 mmol, 92 %) of compound 3b.1H NMR (399 MHz, Chloroform-d) δ 5.49 (s, 1H), 4.58 (s, 1H), 3.74 (s, 3H), 3.56 – 3.25 (m, 2H), 1.40 (s, 9H). 13C NMR (100 MHz, Chloroform-d) δ 170.2,

155.1, 127.8 – 123.4 (m), 122.7 – 120.5 (m), 117.4 (dt, J = 288.1, 33.3 Hz), 114.6 – 105.3 (m), 80.7, 53.2, 52.9, 30.9, 28.2. 19F NMR (376 MHz, Chloroform-d) δ -81.39 (t, J = 9.8 Hz), -85.67 – -88.16 (m), -119.94 – -121.26 (m), -125.32 – -126.23 (m). HRMS (ESI) calculated for C8H9F9NO2S+ [M-Boc+H]+: 354.0205; found: 354.0206. IR (ATR, cm-1): 3370, 2997, 1724, 1681, 1518, 1348, 1225, 1198, 1163, 1136.

Methyl N-(tert-butoxycarbonyl)-S-(perfluoropentyl)-L-cysteinate (3c) was made according to GP1 on a 0.5 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 100:0 to 90:10) yielding 178.7 mg (0.36 mmol, 71%) of derivative 3c as yellow oil. The reaction according to GP3 on a 0.5 mmol scale afforded 176.1.mg (0.35 mmol, 70%) of compound 3c. 1H NMR (399 MHz, Chloroform-d) δ 5.37 (s, 1H), 4.63 (s, 1H), 3.79 (s, 3H), 3.61 – 3.27 (m, 2H), 1.44 (s, 9H). 13C NMR (100 MHz, Chloroform-d) δ 170.0, 154.8, 127.8 – 126.3 (m), 124.6 – 123.5 (m), 122.3 – 120.3 (m), 119.3 – 117.9 (m), 115.9 (d, J = 33.1 Hz), 80.6, 53.0, 52.9, 30.9, 28.1. 19F NMR (376 MHz, Chloroform-d) δ -80.47 – -81.14 (m), -85.48 – -87.66 (m), -119.18 – -120.38 (m), -121.60 – -122.76 (m), -125.63 – -127.13 (m). HRMS (ESI) calculated for C9H9F11NO2S+ [M-Boc+H]+: 404,0173; found: 404,0171. IR (ATR, cm-1): 3371, 2997, 2949, 1726, 1680, 1518, 1288, 1223, 1099.

Methyl N-(tert-butoxycarbonyl)-S-(perfluorohexyl)-L-cysteinate (3d) was made according to GP1 on a 0.5 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 100:0 to 90:10) yielding 171.4 mg (0.31 mmol, 62%) of derivative 3d as yellow oil. The reaction according to GP3 on a 0.5 mmol scale afforded 171.6 mg (0.31 mmol, 62%) of compound 3d. 1H NMR (399 MHz, Chloroform-d) δ 5.40 (s, 1H), 4.62 (s, 1H), 3.78 (s, 3H), 3.55 – 3.25 (m, 2H), 1.43 (s, 9H 13C NMR (100 MHz, Chloroform-d) δ 170.0, 154.8, 125.5 (dt, J = 292.5, 34.1 Hz), 121.6 – 120.8 (m), 117.1 (dt, J = 288.5, 33.2 Hz), 114.4 – 112.2 (m), 111.6 – 109.6 (m), 109.2 – 107.1 (m), 80.5, 53.0, 52.8, 30.8, 28.1. 19F NMR (376 MHz, Chloroform-d) δ -80.11 – -81.65 (m), -85.12 – -88.04 (m), -119.78, -121.51, -122.94, -125.67 – -127.15 (m). HRMS (ESI) calculated for C10H9F13NO2S+

[M-Boc+H]+: 454,0141; found: 454.0153. IR (ATR, cm-1): 3379, 2980, 2951, 1728, 1695, 1682, 1518, 1317, 1199, 1188, 1163, 1147.

Methyl N-(tert-butoxycarbonyl)-S-(perfluoroheptyl)-L-cysteinate (3e) was made according to GP1 on a 0.5 mmol scale. The crude product was purified by flash chromatography (petroleum ether : diethylether 16:1 to 8:1 )yielding 269.5 mg (0.44 mmol, 90 %) of derivative 3e as yellow oil. 1H NMR (399 MHz, Chloroform-d) δ 5.42 (s, 1H), 4.62 (s, 1H), 3.77 (s, 3H), 3.61 – 3.27 (m, 2H), 1.43 (s, 9H). 13C NMR (100 MHz, Chloroform-d) δ 170.2, 155.1, 127.8 – 123.6 (m), 122.2 – 120.3 (m), 117.3 (dt, J = 288.5, 33.0 Hz), 114.5 – 112.4 (m), 112.2 – 109.9 (m), 109.2 – 107.2 (m), 106.7 – 104.5 (m), 80.7, 53.2, 53.0, 31.0, 28.3. 19F NMR (376 MHz, Chloroform-d) δ -81.07 (t, J = 10.0 Hz), -84.97 – -88.54 (m), -119.78, -121.39, -122.18, -122.93, -126.00 – -126.69 (m). HRMS (ESI) calculated for C11H9F15NO2S+

2


[M-Boc+H]+: 504,0109; found: 504,0114. IR (ATR, cm-1): 3377, 2991, 1728, 1693, 1681, 1518, 1321, 1230, 1193, 1149.

Methyl N-(tert-butoxycarbonyl)-S-(perfluorooctyl)-L-cysteinate (3f) was made according to GP1 on a 0.5 mmol scale. The crude product was purified by flash chromatography (petroleum ether :ether 10:1 to 6:1) yielding 218.9 mg (0.34 mmol, 67%) of derivative 3f as yellow oil. 1H NMR (399 MHz, Chloroform-d) δ 5.40 (s, 1H), 4.63 (s, 1H), 3.78 (s, 3H), 3.56 – 3.30 (m, 2H), 1.43 (s, 9H). 13C NMR (100 MHz, Chloroform-d) δ 170.2, 155.0, 127.4 (d, J = 33.4 Hz), 124.3 (t, J = 33.9 Hz), 119.5 – 118.2 (m), 118.1 – 117.2 (m), 115.8 (t, J = 33.4 Hz), 112.0 – 109.8 (m), 109.5 – 107.0 (m), 80.8, 53.2, 53.1, 31.1, 28.3.19F NMR (376 MHz, Chloroform-d) δ -81.02 (t, J = 10.0 Hz), -84.55 – -89.24 (m), -119.59 – -119.88 (m), -121.20 – -121.46 (m), -121.80 – -122.27 (m), -122.74 – -123.14 (m), -126.25 – -126.46 (m). HRMS (ESI) calculated for C12H9F17NO2S+ [M-Boc+H]+: 554,0077; found: 554.0076. IR (ATR, cm-1): 3383, 2991, 1695, 1681, 1516, 1369, 1195, 1118, 1082.

Methyl N-(tert-butoxycarbonyl)-S-(perfluorodecyl)-L-cysteinate (3g) was made according to GP1 on a 0.5 mmol scale. The crude product was purified by flash chromatography (petroleum ether : ether 12:1 to 8:1) yielding 225.9 mg (0.3 mmol, 60 %) of derivative 3g as a white solid (Mp: 65.5-67.1 °C). 1H NMR (399 MHz, Chloroform-d) δ 5.38 (s, 1H), 4.64 (s, 1H), 3.79 (s, 3H), 3.65 – 3.22 (m, 2H), 1.44 (s, 9H). 13C NMR (100 MHz, Chloroform-d) δ 170.2, 155.0, 127.7 – 127.0 (m), 126.3 – 125.5 (m), 124.9 – 123.9 (m), 122.0 – 121.1 (m), 119.4 – 118.0 (m), 116.5 – 115.1 (m), 114.4 – 113.0 (m), 111.9 – 109.7 (m), 109.3 – 106.8 (m), 80.8, 53.2, 53.1, 31.1, 28.3. 19F NMR (376 MHz, Chloroform-d) δ -80.48 – -81.23 (m), -85.58 – -87.69 (m), -119.21 – -119.96 (m), -120.84 – -121.55 (m), -121.55 – -122.26 (m), -122.62 – -123.05 (m), -125.67 – -126.94 (m). HRMS (ESI) calculated for C14H9F21NO2S+ [M-Boc+H]+: 654,0013; found: 654,0019. IR (ATR, cm-1): 3383, 2982, 1728, 1695, 1684, 1516, 1198, 1141.

Methyl N-(tert-butoxycarbonyl)-S-(2-ethoxy-1,1-difluoro-2-oxoethyl)-L-cysteinate (3h) was made according to GP1 on a 0.5 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 100:0 to 90:10) yielding 134.2 mg (0.38 mmol, 75%) of derivative 3h as yellow oil. The reaction according to GP3 on a 0.5 mmol scale afforded 144.7.mg (0.40 mmol, 81%) of compound 3h. 1H NMR (400 MHz, Chloroform-d) δ 5.36 (s, 1H), 4.55 (s, 1H), 4.31 (q, J = 7.1 Hz, 2H), 3.73 (s, 3H), 3.48 – 3.18 (m, 2H), 1.40 (s, 9H), 1.32 (t, J = 7.1 Hz, 3H).13C NMR (101 MHz, Chloroform-d) δ = 170.4, 161.4 (t, J=32.6), 154.9, 120.0 (t, J=287.4), 80.4, 63.8, 53.0, 52.8, 30.9, 28.2, 13.8. 19F NMR (376 MHz, Chloroform-d) δ = -80.9 – -82.4 (m). HRMS (ESI) calculated for C8H14F2NO4S+ [M-Boc+H]+: 258,0612; found: 258,0616. IR (ATR, cm-1): 3387, 2980, 1755, 1714, 1504, 1247, 1161, 1010.

Methyl N-((tert-butoxycarbonyl)-L-leucyl)-S-(trifluoromethyl)-L-cysteinate (4) was made according to GP1 on a 0.50 mmol scale. The crude product was purified by flash

chromatography (petroleum ether: ether: 6:1 to 3:1) yielding 117 mg (0.28 mmol, 56%) of derivative 4 as a white solid (Mp: 78.8-79.3 °C). 1H NMR (400 MHz, Chloroform-d) δ 7.22 (s, 1H), 5.00 (s, 1H), 4.80 (q, J = 5.3 Hz, 1H), 4.14 (s, 1H), 3.75 (s, 3H), 3.52 – 3.24 (m, 2H), 1.74 – 1.58 (m, 2H), 1.51 – 1.44 (m, 1H), 1.42 (s, 9H), 0.91 (t, J = 6.8 Hz, 6H). 13C NMR (101 MHz, Chloroform-d) δ 172.8, 169.7, 155.6, 130.5 (q, J = 306.4 Hz), 80.2, 53.1, 52.9, 51.8, 40.8, 31.4, 28.2, 24.7, 22.8, 21.9. 19F NMR (376 MHz, Chloroform-d) δ -41.03. HRMS (ESI) calculated for C16H27F3N2NaO5S+ [M+Na]+: 439,1485; found: 439,1487. IR (ATR, cm-1): 3334, 2958, 1755, 1686, 1654, 1514, 1367, 1153, 1103.

Methyl N-((tert-butoxycarbonyl)-L-phenylalanyl)-S-(trifluoromethyl)-L-cysteinate (5)[6c] was made according to GP1 on a 0.50 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl ether 50:50 to 0:100) yielding 211.5 mg (0.47 mmol, 94%) of derivative 5 as a white solid (Mp: 112.2-112.9 °C). The reaction according to GP2 on a 0.5 mmol scale afforded 191.3.mg (0.43 mmol, 85%) of compound 5. 1H NMR (400 MHz, Chloroform-d) δ 7.41 – 7.16 (m, 5H), 6.83 (s, 1H), 4.94 (s, 1H), 4.82 (s, 1H), 4.42 (s, 1H), 3.79 (s, 3H), 3.58 – 3.27 (m, 2H), 3.20 – 3.04 (m, 2H), 1.45 (s, 9H). 13C NMR (101 MHz, Chloroform-d) δ 171.5, 169.5, 155.5, 141.8 – 129.7 (m), 129.4, 129.0, 128.9, 127.2, 80.7, 55.8, 53.1, 52.0, 38.0, 31.6 (d, J = 2.2 Hz), 28.4. 19F NMR (376 MHz, Chloroform-d) δ -40.9. HRMS (ESI) calculated for C19H25F3N2NaO5S+ [M+Na]+: 473,1329; found: 473,1323. IR (ATR, cm-1): 3325, 2972, 1741, 1681, 1666, 1516, 1439, 1298, 1220, 1153.

ASSOCIATED CONTENTThe Supporting Information for this article is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.6b01031

2


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CHAPTER 3

Batch and flow synthesis of disulfides by visible light induced TiO2 photocatalysis


Bottecchia, C.; Erdmann, N.; Tijssen, P.; Milroy, L. G.; Brunsveld, L.; Hessel, V.; Noël, T., ChemSusChem, 2016, 9, 1781-1785

3

74 Chapter 3 Batch and flow synthesis of disulfides by visible light induced TiO2 photocatalysis 75

ABSTRACT

In this chapter, a mild and practical method for the preparation of disulfides via

visible light induced photocatalytic aerobic oxidation of thiols is reported. The

method involves the use of TiO2 as a heterogeneous photocatalyst. The catalyst’s

high stability and recyclability makes this method highly practical. The reaction

can be substantially accelerated in a continuous-flow packed-bed reactor, which

enables a safe and reliable scale-up of the reaction conditions. The batch and

flow protocol described herein can be applied to a diverse set of thiol substrates

for the preparation of homo- and hetero-dimerized disulfides. Furthermore,

biocompatible reaction conditions (room temperature, visible light, neutral buffer

solution, no additional base) have been developed, which permits the rapid and

chemoselective modification of densely functionalized peptide substrates without

recourse to complex purification steps.

INTRODUCTION

Several synthetic approaches reported in the literature suggest viable strategies

for the formation of symmetrical and unsymmetrical disulfides.[1] In Nature,

disulfides play a pivotal role in protein folding and oligomerization.[2] Disulfides are

also fundamental to the production of many fine chemicals and pharmaceuticals, as

well as vulcanizing reagents for industrial-scale rubber production.[3] Nevertheless,

traditional synthetic methods might require the use of harsh reaction conditions,

such as stoichiometric amounts of metal oxides or peroxides. More recently, milder

and greener approaches have been developed, including aerobic oxidation of thiols

in presence of molecular oxygen or air, catalyzed by transition metal complexes.[4] Owing to the prevalence of disulfides in proteins, a number of biocompatible

and bio-orthogonal methodologies have also been developed.[5] In such cases, S–S

bond formation serves as a controlled trigger for oxidative folding or as a method

to enable bioconjugation.[6] The redox-sensitive nature of disulfide bonds also

renders them viable candidates for drug delivery systems.[7] Nevertheless, rapid,

straightforward and biocompatible strategies are still in demand to obviate the

need for long reaction times and activating agents (e.g. Ellmann’s reagent or

methanethiosulfonate reagents).[7c, 8]

We recently reported a photocatalytic approach to symmetrical disulfides catalyzed

by the organic dye Eosin Y.[9] Despite the mild reaction conditions and fast reaction

times of our approach, the use of Eosin Y as a homogeneous photocatalyst still

necessitates an additional purification step to recover catalyst and isolate the

target disulfide. In peptide and protein modification, purification steps are

undesired and often problematic, especially for delicate proteins that are prone

to denature. Furthermore, the use of a transition-metal complex as homogeneous

catalyst might lead to bio-incompatibility issues, since these catalysts tend to give

undesired binding interactions.[6a, 10]

In our efforts to develop efficient synthetic tools for chemical biology purposes, our

attention was drawn to the use of TiO2 as a cheap and recyclable photocatalyst of

aerobic S–S bond formation.[11] To date, the main application of TiO2 photocatalysis

has been in the water treatment industry,[12] while its use for organic synthesis

remains scarce.[11, 13] In light of the high energy band-gap of nanoscale TiO2 (3.2 eV

for the anatase form), its photocatalytic activity is typically associated with UV-

light irradiation.[14] However, given the incompatibility of UV light to peptides and

proteins, [15] we reasoned that visible-light photoredox catalysis would be better

suited to chemical biology applications owing to the milder reaction conditions

3


(e.g. room temperature and visible light). In this regard, several recent reports

have shown that surface interactions between nanoscale TiO2 and some organic

substrates, such as amines, can overcome the innate lack of visible-light absorption.[16] In particular, Chen and co-workers showed that the use of triethylamine (TEA)

allows for visible light TiO2 excitation, which in turn enables the selective aerobic

oxidation of sulfides to sulfoxides.[17] Here, we report the use of TiO2 as catalyst for

the visible light-mediated formation of symmetrical and unsymmetrical disulfide

bonds. The chemistry is amenable to intramolecular S–S bond formation in complex

peptides under biocompatible reaction conditions (buffer solution, no additional

base required, visible light, simple purification by filtration or centrifugation).

Furthermore, we demonstrate that TiO2 can be repeatedly recycled without

noticeable catalyst deactivation, which enabled the development of a highly

effective continuous-flow protocol with much reduced reaction times.

RESULTS AND DISCUSSION

We began our study with benzyl mercaptan 1, a relatively unreactive substrate

which proved challenging for our Eosin Y based-protocol (Table 3.1). In the absence

of any light or base, almost no reaction was observed (Table 3.1, Entry 1 and 2).

Upon light irradiation, 33% of the target disulfide product 2 was observed (Table

3.1, entries 3 and 4). It should be noted that photo-oxidative disulfide formation is

possible in the presence of a base under visible light irradiation in the absence of

any photocatalyst as shown by Yoon et al.[18]

However, the transformation has a very narrow scope and is limited to those thiols

of which the corresponding thiolate absorbs in the visible light region, e.g. p-NO2-

thiophenol. Improved results were obtained when the light source was switched

from CFL (compact fluorescent light) to white LEDs (Table 3.1, Entries 5, 6).

Interestingly, a 28% yield of 2 was attained when a carbonate buffer was used as

the base (Table 3.1, Entry 7). This result is significant as it provides opportunities

to perform this chemistry under biocompatible conditions. The lower yield can be

explained by the triple phase-transfer reaction conditions, i.e. oxygen to liquid,

reactants to TiO2 and base from the water to the organic phase. In all cases, no

over-oxidation to the corresponding thiosulfinate and thiosulfonate was observed

due to a careful management of the reaction time (Table 3.1).

To reduce the risks associated with the handling of molecular oxygen and to

overcome potential gas-liquid mass transfer limitations, we further concentrated

our efforts on transferring this transformation to a continuous-flow protocol (see

Supporting Information).[19] [20] [21] Initial attempts to introduce a slurry of TiO2 in

Table 3.1: Optimization table of solvents, light sources and basea

Entry Light source Solvent Base Yield (%)b

1 No light CH3CN TMEDA 2

2 CFL CH3CN No base 0.5

3 CFL CH3CN TMEDA 32

4 CFL EtOH TMEDA 33c

5 White LED EtOH TMEDA 43c

6 White LED EtOH TMEDA 75d

7 White LED H2O/CH3CN (1:3) Carbonate buffer 28c

aStandard reaction conditions: thiol 1 (1 mmol), TMEDA (1 mmol), TiO2 (Aeroxide® P25, 12.5 mol %),

EtOH (2 mL), O2, visible light irradiation, 1.5 hours. bYield determined with GC-MS and internal standard. cReaction time 5 hours. dReaction time 8 hours.

the liquid phase into a capillary microreactor immediately proved unpractical. We

found that the TiO2 nanoparticles tended to aggregate in the presence of TMEDA

leading to rapid clogging of the microreactor. Next, a packed-bed reactor strategy

was used in which the TiO2 nanoparticles were loaded in a glass packed-bed

reactor. A bed consisting purely of TiO2 resulted in an excessive drop in pressure,

which could be remediated by adding small glass beads to the bed (60 mg TiO2,

310 mg glass beads, 200 µL void for the combined gas-liquid phase, ID 3 mm, 4 cm

length) (see experimental section). To circumvent potential leaching of TiO2 from

the reactor, the packed-bed was flushed with a solution of TMEDA (1 M in EtOH)

prior to the reaction. This caused the TiO2 particles to aggregate and effectively

avoided any leaching of these small particles, which can cause clogging at the

reactor outlet. Substantial rate accelerations (from 8 h in batch to 5 min in flow)

were observed when performing this reaction in flow. This can be attributed to the

enhanced gas-liquid mass transfer characteristics and the improved irradiation of

the reaction mixture (Scheme 3.1).[19a, 22]

To verify whether the TMEDA-induced aggregation of TiO2 nanoparticles might be

influencing their catalytic properties, we analysed the size and the morphology of

the TiO2 nanoparticles by scanning electron microscopy (SEM) and X-ray diffraction

(XRD).[16f, 23] Samples of the catalyst were collected at the inlet and outlet of the

microreactor after reaction, and were dried and compared to a sample of fresh

EtOH, RT

cat. TiO2O2

TMEDA

1 2

SH SS

2

3


TiO2. By both SEM and XRD, we were unable to detect any substantial difference

between the different samples, suggesting that the composition of TiO2 remained

unchanged, and that the catalyst was stable under our reaction conditions.

However, further dynamic light scattering (DLS) experiments proved that large

TiO2 aggregates were formed in the presence of TMEDA.

Scheme 3.1: Comparison of flow and batch yields for symmetrical disulfide formation. Reaction conditions in batch: thiol (1 mmol), TMEDA (1 mmol), TiO2 (Aeroxide® P25, 12.5 mol %), EtOH (2 mL), O2, visible light irradiation. The yields reported are isolated. For the detailed reaction conditions in flow see experimental section.

Next, we evaluated the influence of the increased particle size of TiO2 in presence

of TMEDA with respect to potential deactivation and recyclability of the catalyst.

The conversion of thiophenol to disulfide 3 was continuously monitored in a

continuous-flow packed-bed reactor for >28 hours. No decrease in reaction yield

was observed, thus proving the stability of the catalyst and the reliability of the

continuous-flow set-up in terms of catalyst leaching. Furthermore, in batch, the TiO2

nanoparticles were separated by centrifugation and reused up to ten consecutive

times with no impact on the reaction yield. With the optimized batch and flow

conditions in hand, we examined the scope of the aimed transformation with an

array of (hetero)aromatic thiols (Scheme 3.1). Disulfides 2 to 7 were obtained in

good to excellent yields (72-96% in batch vs 60-99% in flow). The formation of

unsymmetrical disulfides via oxidative coupling is rarely described in literature,

most likely due to the selectivity issues (i.e. homo- or hetero-dimerization) arising

under the reaction conditions.[1a],[24] Previous reports of unsymmetrical disulfide

formation often rely on the use of reactive reagents.[25]

Scheme 3.2: Scope of unsymmetrical disulfides. Reaction conditions in batch: (hetero)aromatic thiol (1 mmol), aliphatic thiol (5 mmol) TMEDA (6 mmol), TiO2 (Aeroxide® P25, 12.5 mol %), EtOH (2 mL), O2, visible light irradiation. The yields reported are isolated. For the detailed reaction conditions in flow for compound 13 see experimental section.

Under our reaction conditions, the use of an excess of the less reactive thiol (i.e.

5 equiv. of the aliphatic thiol) overcame statistical formation of homo- and hetero

disulfides and allowed us to obtain a diverse range of unsymmetrical aryl-alkyl

disulfides 8-13 in good yields (23-86%) with good selectivity to the unsymmetrical

disulfide (< 5% of the symmetrical disulfide of the aromatic thiol; more symmetrical

disulfide is formed from the less reactive thiol due to the large excess) (Scheme

3.2). Encouraged by these promising results, we anticipated that our protocol

would be suitable for the formation of symmetrical and unsymmetrical disulfides

on cysteine-containing derivatives. Pleasingly, starting from Boc-L-Cys-OMe,

disulfide 14 was isolated in 90% yield, while the unsymmetrical disulfide 15 could

be isolated in 69% yield (Scheme 3.3).

These results demonstrate the potential of our methodology for the formation of

symmetrical and unsymmetrical disulfide derivatives as handles for bioconjugation.[26] However, larger peptide sequences are typically only stable at neutral or

slightly basic pH. A further evaluation of the reaction conditions revealed that

the chemistry could be carried out in aqueous phosphate buffer (pH= 7.4) in the

absence of TMEDA. These reaction conditions are practical in a chemical biology

2

cat. TiO2O2,TMEDA

EtOH

R1 SH2

SS S

S

F

F

SS

Br

Br

SS

MeO

OMe

S

NS

N

SS

batch: 8h - 75%flow: 5min - 60%

R1S

SR1

3


4



batch: 3h - 94% batch: 7h - 72%

5 3 4

8

cat. TiO2O2,TMEDA

EtOHR1 SH

batch: 5h - 80%

R1S

SR1

9

batch: 6h - 77%

10

batch: 7h - 78%

batch: 16h - 23% batch: 24h - 65% flow: 3min - 86%

11 12 13

R2HS

SS

MeO

SS

MeO

S

NS

S

NS

SS

HO

6 6 6

SS 6

OMe

3


Scheme 3.3: Symmetric and unsymmetrical disulfide formation on a cysteine residue. Reaction conditions for 14: Boc-Cys-OMe (1 mmol), TMEDA (1 mmol), TiO2 (Aeroxide® P25, 12.5 mol %), EtOH (2 mL), O2, visible light irradiation, 6 hours. Reaction conditions for 15: Boc-Cys-OMe (1 mmol), Thiophenol (5 mmol) TMEDA (6 mmol), TiO2 (Aeroxide® P25, 12.5 mol %), EtOH (2 mL), O2, visible light irradiation, 6 hours. The yields reported are isolated.

Scheme 3.4: Intramolecular disulfide formation yielding the native form of Oxytocin 17. Reaction conditions: crude of precursor 16 (10 mg, 9.91 μmol), TiO2 (Aeroxide® P25, 2 mg), 20 mM phosphate buffer (20.0 mL), O2, visible light irradiation, 30 minutes.

laboratory and suitable for working with sensitive and complex biomolecules, such

as peptides, proteins, and in principle, antibodies. Practically, only buffer solution,

TiO2, visible light irradiation and atmospheric conditions are required to carry out

the transformation. To probe these new reaction conditions, an intramolecular

disulfide bond formation reaction was carried out to yield the cyclic nonapeptide

Oxytocin starting from its reduced form 16. Oxytocin is a hormone peptide

produced in mammals and associated with the brain modulation of several social

and non-social behaviours.[27]

Scheme 3.5: Intramolecular disulfide formation yielding the native form of yeast-derived y1fatc peptide 18. Reaction conditions: precursor 18 (0.25 mg, 0.13 μmol), TiO2 (Aeroxide® P25, 1 mg), 20 mM phosphate buffer (0.25 mL), milliQ water (0.25) mL, O2, visible light irradiation, 1 hour.

The active form of Oxytocin 17 was obtained within only 30 minutes (Scheme 3.4).

Notably, the methodology also worked on a crude sample of the Oxytocin precursor

isolated by precipitation after cleavage from the solid-phase resin, emphasizing the

feasibility of the transformation on impure peptide mixtures. After the reaction,

the TiO2 nanoparticles were easily separated by centrifugation and Oxytocin was

purified by standard preparative scale LC-MS. Next, we examined the photocatalytic

intramolecular disulfide transformation on a more challenging peptide 18, the

reduced form of the yeast-derived C-terminal lipid-binding motif of the TOR1

(target of rapamycin) FATC domain (y1fatc).[28] Interestingly, the redox state of the

peptide regulates the membrane binding properties of the protein, with the oxidized

disulfide form binding to lipid membranes more tightly than the reduced form.

cat. TiO2O2,TMEDA

EtOH

HS+

SH

HN

OMe

O

Boc SS

HN

OMe

O

Boc

SH

HN

OMe

O

Boc

SS

HN

OMe

O

Boc

NH

OMe

O14

15

2

90%

69%

Boc

cat. TiO2O2,TMEDA

EtOH

phosphate buffer

cat. TiO2O2,TMEDA

Me

NH

NH2

ONH

OO

MeNH

O

NHOH

O

NH2

SH

NH

N

H2N

OO

O

SHO

NH

Me

Me

HN

O

NH2O

Me

HNNH2

O

HN O

O

Me

NH

O

NH

OH

O

NH2

SHN

N NH2OO

O

S

O

HNMe

HN O

H2N

O

16 17

Me

Oxytocin

cat. TiO2O2,TMEDA

phosphate buffer

HN

HOO

O

AcHN

NHO

HN

HN

NH2

HN

O

MeMe

HN

O

S

NH

O

ONH2

NHO

NH

N

NH

O

OHHN

O

Me

Me

NH

O

NH

O

HN

O

S

N

HN O

NH

OPh

HO O

NH

HN

OH

OO

NHAcNH

O

NH

NHH2N

HN

O

MeMe

NHO

HS

HNO

O

H2N

NHO

NH

N

HNO

HO

NHO Me

Me

NHO

HN

O

HN

ON

NHO

NH

O

Ph

HOOHN

18

SH

19

3


For our purposes, peptide 18 serves as an interesting model to test the selectivity

of our disulfide formation approach, because of the presence of other nucleophilic

and aromatic residues that might interfere with the transformation. Satisfyingly,

peptide 18 was also converted within 1 h to its native form 19 when subjected to

our methodology (Scheme 3.5).

CONCLUSION

In conclusion, we have developed a convenient protocol for the preparation of

disulfides via visible-light photocatalytic aerobic oxidation of thiols with TiO2

as a recyclable photocatalyst. Unsymmetrical disulfides were also prepared

in good yield using an excess of the less reactive coupling partner. The use of a

continuous-flow TiO2 packed-bed reactor substantially reduced the reaction times

(from hours in batch to minutes in flow). The stability of the TiO2 photocatalyst

was investigated and no noticeable degradation was observed in both batch and

flow. We note as well that the mild reaction conditions (room temperature, visible

light, neutral buffer solution) and the facile catalyst recuperation by filtration

makes this protocol particularly attractive for routine application in biomolecule

modification, as exemplified in the synthesis of Oxytocin and peptide 19.

EXPERIMENTAL SECTION

General batch procedure for the synthesis of symmetrical disulfides (GP1): In an oven-dried vial equipped with a magnetic stirrer and a PTFE septum, 10 mg (12.5 mol %) of TiO2 was added to a mixture of thiol (1.0 mmol) and N,N,N’,N’-tetramethylethylenediamine (TMEDA) (1.0 mmol) in EtOH (2.0 mL). The vial was subjected to visible light irradiation and oxygen was added to the reaction mixture through bubbling. The reaction was stirred at 1000 rpm and monitored by TLC. When full conversion was reached, the reaction mixture was adsorbed on silica and purified by flash chromatography to give the desired disulfide.

General batch procedure for the synthesis of unsymmetrical disulfides (GP2): In an oven-dried vial equipped with a magnetic stirrer and a PTFE septum, 10 mg of TiO2 was added to a mixture of aromatic thiol (0.5 mmol, 1 equiv.) and N,N,N’,N’-tetramethylethylenediamine (TMEDA) (3.0 mmol, 6 equiv.) in EtOH (2.0 mL). The aliphatic thiol (2.5 mmol, 5 equiv.) was then added to the reaction vial under stirring. The vial was subjected to visible light irradiation and oxygen was added to the reaction mixture through bubbling. The reaction was stirred at 1000 rpm. When full conversion was reached according to TLC, the reaction mixture was adsorbed on silica and purified by flash column chromatography.

General continuous-flow procedure for the synthesis of symmetrical disulfides in a packed-bed reactor (GP3): A 5 mL syringe containing 0.5 M substrate and a 5 mL syringe containing 0.5 M TMEDA were mounted on a single syringe pump. The liquid flowrate (per syringe) was

fixed at 8.3 μL/min. The two liquid streams were merged in a T-Mixer and to this stream O2 was added at a flowrate of 50 μL/min by means of a mass flow controller. After reaching steady state, a reaction sample was collected until 1 mmol of product was collected. This sample was purified by flash chromatography and analysed by NMR and IR.

General continuous-flow procedure for the synthesis of symmetrical disulfides in a packed-bed reactor (GP4): A 5 mL syringe with 0.375 M (1. eq.) of aromatic thiol in ethanol and a 5 mL syringe with 1.875 M (5 eq.) of aliphatic thiol in ethanol were inserted on a single syringe pump. The two solutions were mixed in a T-mixer at a flowrate of 5.5 μL/min. A 5 mL syringe containing 2.25 M (6 eq.) TMEDA in ethanol was inserted in another syringe pump and mixed with the substrate stream at a flowrate of 5.5 μL/min. The liquid streams were merged in a T-Mixer and to this stream O2 was added at a flowrate of 50 μL/min by means of a mass flow controller. After reaching steady state, a reaction sample was collected until 1 mmol of product was collected. This sample was purified by flash chromatography and analysed by NMR and IR.

General batch procedure for the synthesis of oxytocin (GP5): 2 mg of TiO2 was weighed in a 25 mL Falcon tube. 20 mL of 20 mM phosphate buffer and 10 mg (9.91 μmol) of crude precursor 16 were added consecutively. The Falcon tube was positioned in the batch set-up and closed with its cap. Prior to the reaction the cap was perforated with a hot cannula to allow the insertion of gas. Oxygen was then delicately bubbled through the reaction mixture with a balloon. An extra needle was inserted in the septum of the vial to allow ventilation. The reaction was not magnetically stirred, while the bubbling of oxygen ensured mixing. After 30 minutes, the reaction vial was centrifuged to separate TiO2 and the reaction mixture was analyzed by LC-MS. Purification of the crude reaction mixture was done with preparative scale LC-MS.

General batch procedure for the synthesis of peptide 19 (GP6): 1 mg of TiO2 was weighed in an Eppendorf vial (2.5 mL). 0.25 mL of 20 mM phosphate buffer and 0.25 mg (0.13 μmol) of crude precursor 18 were added consecutively. The Eppendorf vial was positioned in the batch set-up and closed with a layer of Parafilm. Oxygen was delicately bubbled through the reaction mixture with a balloon. An extra needle was inserted in the septum of the vial to allow ventilation. The reaction was not magnetically stirred, while the bubbling of oxygen ensured mixing. After 1 hour, the Eppendorf vial was centrifuged to separate TiO2 and the reaction mixture was analyzed by LC-MS.

3


Preparation of starting materials:

Synthesis of oxytocin precursor 16 and peptide 18

Both peptides were synthesized via automated solid-phase peptide synthesis (Intavis MultiPep RSi, INTAVIS Bioanalytical Instruments AG) on Rink amide resin (Novabiochem®, 100-200 mesh, 0.59 mmole/g loading).[3] Briefly, per reaction column, the resin (50 µmole scale) was pre-swollen in 1,7 mL of NMP for 5 x 10 min and the N-Fmoc protecting group cleaved by treating the resin with 1,5 mL of a stock solution of 20 % piperidine (v/v) in NMP for 2 x 8 min. Each amino acid coupling was performed by premixing 1052 µL of a 0.38 M stock solution of O-benzotriazole-N,N,N’,N’- tetramethyluronium-hexafluoro-phosphate (HBTU) in N-methyl-2-pyrrolidone (NMP) with 500 µL of a 0.5 M stock solution of the amino acid in NMP, followed by 840 µL of a 1.6 M stock solution of N,N-diisopropylethylamine (DIPEA) stock solution, also in NMP. The reaction mixture was immediately added to the resin and the reaction vessel vortexed intermittently at ambient temperature for 2 x 30 min (double coupling). After the final Fmoc deprotection step, the peptides were manually cleaved from the resin, with simultaneous deprotection of the side chain functional groups, by incubating the resin in 3 mL of a 92.5/2.5/2.5/2.5 (v/v) mixture of trifluoroacetic acid (TFA)/H2O/triisopropylsilane (TIS)/ ethanedithiol (EDT), and then precipitated through dropwise addition into ice-cold diethyl ether. Peptide 16 was then employed for the reaction without further purification. Peptide 18 was purified by reverse-phase HPLC using an Alltima HP C18 column (5 μm, length 125 mm, ID: 20 mm) and 0.1% trifluoroacetic acid (TFA) in H2O/MeCN as mobile phase. The H2O/MeCN solvents were removed to dryness by lyophilization to leave the pure peptide 18 as a white powder. Peptide analysis by LC-MS using a Shimadzu LC Controller V2.0, LCQ Deca XP Mass Spectrometer V2.0, Alltima C18- column 125 x 2.0 mm, Surveyor AS and PDA with solvent eluent conditions: CH3CN/H2O/1% TFA. Alternatively, the peptides were analyzed by LC-MS using a LCQ Fleet from Thermo Scientific on a C18 column, Surveyor AS and PDA with solvent eluent conditions: MeCN/H2O/0.1% formic acid.


Dibenzylsulfide (2) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 20:1) yielding 95.4 mg (0.39 mmol, 75 %) of disulfide 2 as a colorless solid (Mp: 68.6-69.4). The reaction according to GP3 on a 1.5 mmol scale afforded 110.9 mg (0.45 mmol, 60 %) of disulfide 2 after 5 minutes residence time. 1H NMR (399 MHz, Chloroform-d) δ 7.34 – 7.20 (m, 10H), 3.59 (s, 4H).13C NMR (100 MHz, Chloroform-d) δ 137.5, 129.5, 128.6, 127.5, 43.4. MS (GC-MS) m/z: 246. IR (ATR, cm-1): 2980, 2887, 1395, 1252, 1152, 1070, 966, 694.

Diphenyldisulfide (3) was made according to GP1 on a 1.1 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 40:1) yielding 110 mg (0.50 mmol, 95%) of disulfide 3 as a colorless solid (Mp: 58.9-59.3). The reaction according to GP3 on a 0.95 mmol scale afforded 103 mg (0.47 mmol, 99%) of disulfide 3. 1H NMR (399 MHz, Chloroform-d) δ 7.38 (d, J = 8.3 Hz, 4H), 6.81 (d, J = 8.3 Hz, 4H), 3.75 (s, 6H).13C NMR (100 MHz, Chloroform-d) δ 137.2, 129.2, 127.6, 127.3. MS (GC-MS) m/z: 218. IR (ATR, cm-1): 2980, 1574, 1474, 1437, 1260, 1072, 1020, 798, 735, 685.

Di-4-fluorophenyldisulfide (4) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 20:1) yielding 126.4 mg (0.5 mmol, 96 %) of disulfide 4 as a transparent oil. The reaction according to GP3 on a 1.45 mmol scale afforded 171.7 mg (0.68 mmol, 98 %) of disulfide 4. 1H NMR (400 MHz, CDCl3) δ: 7.49 – 7.41 (m, 4H), 7.06 – 6.98 (m, 4H) ppm. 13C NMR (100 MHz, CDCl3) δ: 163.98, 161.51, 132.35, 132.32, 131.46, 131.38, 116.51, 116.29 ppm. 19F NMR (376 MHz, CDCl3) δ: -113.46 ppm. MS (GC-MS) m/z: 254. IR (ATR, cm-1): 2980, 1587, 1485, 1396, 1224, 1153, 1092, 1076, 1026, 821, 621.

Di-4-methoxyphenyldisulfide (5) was made according to GP1 on a 1.2 mmol scale. The crude zproduct was purified by flash chromatography (petroleum ether: ethyl acetate 20:1) yielding 151.8 mg (0.55 mmol, 91 %) of disulfide 5 as a yellow solid (Mp: 35.9-36.4). The reaction according to GP3 on a 0.75 mmol scale afforded 74.7 mg (0.29 mmol, 78 %) of disulfide 5. 1H NMR (400 MHz, CDCl3) δ: 7.38 (d, J = 9.0 Hz, 4H), 6.81 (d, J = 9.2 Hz, 4H), 3.75 (s, 6H) ppm. 13C NMR (100 MHz, CDCl3) δ: 159.98, 132.67, 128.48, 114.69, 55.40 ppm. MS (GC-MS) m/z: 278. IR (ATR, cm-1): 2980, 2970, 1489, 1386, 1244, 1169, 1151, 1072, 1028, 966, 823.

Di-4-bromophenyldisulfide (6) was made according to GP1 on a 1.1 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 20:1) yielding 194 mg (0.52 mmol, 94 %) of disulfide 6 as a colorless solid (Mp: 92.8-93.4). 1H NMR (400 MHz, CDCl3) δ: 7.42 (d, J = 7.2 Hz, 4H), 7.34 (d, J = 8.8 Hz, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ: 135.85, 132.33, 129.52, 121.67 ppm. MS (GC-MS) m/z: 376. IR (ATR, cm-1): 2980, 1466, 1383, 1066, 1005, 810, 721.

Dipyridine-2-yldisulfide (7) was made according to GP1 on a 1.1 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 89.6 mg (0.41 mmol, 72 %) of disulfide 7 as a yellow solid (Mp: 54.7-55.4). 1H NMR (400 MHz, CDCl3) δ: 8.46 – 8.38 (m, 2H), 7.61 – 7.52 (m, 4H), 7.11 – 7.02 (m, 2H) ppm. 13C NMR (100 MHz, CDCl3) δ: 158.96, 149.59, 137.41, 121.15, 119.73 ppm. MS (GC-MS) m/z: 220. IR (ATR, cm-1): 3043, 2981, 1570, 1558, 1445, 1414, 1277, 1146, 1111, 1084, 1043, 986, 754, 717, 617.

3


S-(4-methoxyphenyl)-S’-(octyl)-disulfide (8) was made according to GP2 on a 0.53 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 40:1) yielding 120 mg (0.42 mmol, 80 %) of disulfide 8 as an oil. 1H NMR (400 MHz, CDCl3) δ: 7.49 (d, J = 9.1 Hz, 2H), 6.87 (d, J = 7.3 Hz, 2H), 3.81 (s, 3H), 2.74 (t, J = 7.3 Hz, 2H), 1.72 – 1.62 (m, 2H), 1.39 – 1.23 (m, 10H), 0.93 – 0.85 (m, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ: 159.58, 131.70, 128.70, 114.70, 55.45, 39.04, 31.91, 29.25, 28.83, 28.59, 22.75, 14.20 ppm. MS (GC-MS) m/z: 284. IR (ATR, cm-1): 2926, 1713, 1491, 1362, 1246, 1221, 910, 825, 731.

S-(pyridine)-S’-(octyl)-disulfide (9) was made according to GP2 on a 0.49 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 9:1 to 1:1) yielding 95.9 mg (0.38 mmol, 77 %) of disulfide 9 as an oil. 1H NMR (399 MHz, Chloroform-d) δ = 8.43 (s, 1H), 7.71 (d, J=7.4, 1H), 7.61 (t, J=7.7, 1H), 7.05 (s, 1H), 2.85 – 2.61 (m, 2H), 1.75 – 1.60 (m, 2H), 1.45 – 1.15 (m, 10H), 0.85 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 160.80, 149.61, 136.98, 120.49, 119.56, 39.10, 29.31, 29.28, 29.26, 29.21, 22.70, 14.17 ppm. MS (GC-MS) m/z: 255. IR (ATR, cm-1): 2924, 2853, 1574, 1558, 1445, 1418, 1117, 758, 717.

S-(phenol)-S’-(octyl)-disulfide (10) was made according to GP2 on a 0.72 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 20:1 to 9:1) yielding 151.9 mg (0.56 mmol, 77 %) of disulfide 10 as an oil. 1H NMR (400 MHz, CDCl3) δ: 7.43 (d, J = 8.1 Hz, 2H), 6.80 (d, J = 8.1 Hz, 2H), 2.73 (t, J = 7.3 Hz, 2H), 1.71 – 1.62 (m, 2H), 1.38 – 1.19 (m, 10H), 0.88 (t, J = 6.8 Hz, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ: 155.53, 131.90, 128.88, 116.23, 39.04, 31.89, 29.24, 28.83, 28.59, 22.75, 14.20 ppm. MS (GC-MS) m/z: 270. IR (ATR, cm-1): 2924, 1489, 1456, 1234, 1167, 824.

S-(pyridine)-S’-(propyl)-disulfide (11) was made according to GP2 on a 0.81 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 9:1) yielding 34.7 mg (0.19 mmol, 23 %) of disulfide 11 as an oil. 1H NMR (400 MHz, CDCl3) δ: 8.44 (d, J = 4.9 Hz, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.06 (t, J = 6.1 Hz, 1H), 2.81 – 2.61 (m, 2H), 1.78 – 1.65 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ: 160.81, 149.65, 137.04, 120.55, 119.59, 41.03, 22.41, 13.22 ppm. MS (GC-MS) m/z: 185. IR (ATR, cm-1): 1711, 1418, 1362, 1221, 910, 729.

S-(4-methoxyphenyl)-S’-(propyl)-disulfide (12) was made according to GP2 on a 0.50 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 40:1) yielding 70.5 mg (0.32 mmol, 65 %) of disulfide 12 as an oil. 1H NMR (400 MHz, CDCl3) δ: 7.48 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 8.9 Hz, 2H), 3.81 (s, 3H), 2.71 (t, J = 7.2 Hz, 2H), 1.78 – 1.65 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ: 159.59, 131.68,

128.67, 114.73, 55.49, 40.97, 22.21, 13.22 ppm. MS (GC-MS) m/z: 214. IR (ATR, cm-1): 2960, 1591, 1458, 1287, 1244, 1171, 1032, 824.

S-(2-methoxyphenyl)-S’-(octyl)-disulfide (13) was made according to GP4

on a 0.52 mmol scale. The crude product was purified by flash chromatography

(petroleum ether: ethyl acetate 40:1) yielding 122.1 mg (0.45 mmol, 86 %) of disulfide 13 as an oil. 1H NMR (400 MHz, CDCl3) δ: 7.72 (d, J = 6.0 Hz, 1H), 7.30 – 7.17 (m, 1H), 7.01 (t, J = 7.7 Hz, 1H), 6.86 (d, J = 8.2 Hz, 1H), 3.89 (s, 3H), 2.73 (t, J = 7.4 Hz, 2H), 1.74 – 1.64 (m, 2H), 1.43 – 1.21 (m, 10H), 0.90 (t, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ: 156.60, 127.70, 127.66, 125.61, 121.26, 121.15, 110.69, 55.92, 38.79, 31.89, 29.25, 29.03, 28.65, 22.74, 14.20 ppm. MS (GC-MS) m/z: 284. IR (ATR, cm-1): 2924, 1578, 1474, 1462, 1433, 1269, 1236, 1057, 1024, 744, 677.

N-Boc-cysteine-OMe disulfide (14) was made according to GP1 on a 1 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 3:1) yielding 212.8 mg (0.46 mmol, 90 %) of disulfide 14 as an oil. 1H NMR (400 MHz, Chloroform-d) δ = 5.38 (d, J=6.8, 2H), 4.59 (d, J=6.5, 2H), 3.75 (s, 6H), 3.15 (d, J=4.7, 4H), 1.44 (s, 18H) ppm. 13C NMR (101 MHz, Chloroform-d) δ = 171.2, 155.1, 80.3, 52.8, 52.6, 41.3, 28.3 ppm. MS (LC-MS) [M-2*Boc]+1: 269.17.

Methyl N-(tert-butoxycarbonyl)-S-(phenylthio)-L-cysteinate (15) was made according to GP2 on a 1 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 10:1) yielding 236.9 mg (0.69 mmol, 69 %) of disulfide 15 as yellow oil. 1H NMR (400 MHz, Chloroform-d) δ = 7.59 – 7.49 (m, 2H), 7.40 – 7.31 (m, 2H), 7.30 – 7.25 (m, 1H), 5.31 (s, 1H), 4.63 (s, 1H), 3.74 (s, 3H), 3.38 – 3.04 (m, 2H), 1.44 (s, 9H) ppm. 13C NMR (101 MHz, Chloroform-d) δ = 171.1, 155.0, 136.6, 129.2, 128.3, 127.4, 80.2, 52.9, 52.6, 40.9, 28.3 ppm.MS (LC-MS) [M-*Boc]+1: 244.08.

ASSOCIATED CONTENT

The Supporting Information for this article is available free of charge on the Wiley Publications website at DOI: 10.1002/cssc.201600602.

3


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90 Chapter 3

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CHAPTER 4

Visible Light-Mediated Selective Arylation of Cysteine in Batch and Flow


Bottecchia, C.; Rubens, M.; Gunnoo, S. B.; Madder, A.; Noël, T. Angew. Chem, Int. Ed. 2017, 56, 12702-12707

4

94 Chapter 4 Visible-Light-Mediated Selective Arylation of Cysteine in Batch and Flow 95

ABSTRACT

In this chapter, a mild visible light-mediated strategy for cysteine arylation is

presented. The method relies on the use of Eosin Y as a metal-free photocatalyst

and aryldiazonium salts as arylating agents. The reaction can be significantly

accelerated in a microflow reactor, whilst allowing the in situ formation of the

required diazonium salts. The batch and flow protocol described herein can be

applied to obtain a broad series of arylated cysteine derivatives and arylated

cysteine-containing dipeptides. Moreover, the method was applied to the

chemoselective arylation of a model peptide in biocompatible reaction conditions

(room temperature, PBS buffer) within a short reaction time.

INTRODUCTION

The formation of C–S bonds is of high interest in the fields of organic synthesis

and drug discovery.[1] However, due to the undesired coordination between

metal catalysts and sulfur atoms, traditional cross-coupling methods are often

inadequate strategies for C–S bond formation.[2] Despite this, some transition-

metal catalyzed cross-coupling methods for C–S bond formation have been

reported.[3] However, these methods often rely on high reaction temperatures

and/or require stoichiometric amounts of a strong base. A well-known strategy

largely applied in industry for C–S bond formation is the so-called Stadler-Ziegler

reaction, in which a diazonium salt is reacted with an aryl thiolate to afford the

desired thioether derivative.[4] Starting from the original conditions reported by

Stadler and by Ziegler, a plethora of methodologies have emerged, allowing milder

reaction conditions.[5] Among them, our group reported a mild one-pot procedure

for the synthesis of aryl sulfides facilitated by photoredox catalysis.[6]

In the interest of developing mild methodologies for chemical biology purposes[7],

we envisaged modifying our procedure to achieve a visible light-induced protocol

for cysteine arylation. Specifically, we directed our attention towards the

development of a biocompatible metal-free strategy involving inexpensive organic

dyes as photoredox catalysts. In addition, due to the incompatibility of UV light to

peptides and proteins, we reasoned that visible-light photoredox catalysis would

be perfectly suited to chemical biology applications owing to the milder reaction

conditions (e.g. room temperature and visible light).

Novel selective chemical modifications of peptides and proteins are of pivotal

importance for the study of protein-protein interactions and for the development

of novel bioconjugates and drug candidates.[8] Compared to other amino acids

commonly targeted for post-translational modifications, cysteine exhibits

low natural abundancy and a relatively high nucleophilicity.[9] Together, these

characteristics account for the generally higher selectivity and the broad reactivity

profile typical for post-translational chemical modifications involving cysteine

residues. Some of the most widespread strategies for cysteine bioconjugation

include disulfide formation,[10] thiol-maleimide reactions,[11] and alkylation with

haloalkyl reagents.[12] Other strategies use cysteine as precursor for the formation

of dehydroalanine[13] (Dha), or as a handle for nucleophilic aromatic substitution

allowing access to perfluorinated staples in peptides and proteins.[14] Moreover,

several methodologies relying on thiol-ene[11] (or thiol-yne[15]) reactions have

been reported, often requiring UV irradiation to generate the thiyl radical.

Fewer records in the literature describe the use of transition metals for cysteine

4


modification. Among them, recent developments illustrate methodologies for

cysteine arylation[16] as well as viable protocols for cysteine arylation in proteins.[17]

Inspired by these reports, we envisioned that photoredox catalysis could serve our

purpose to obtain a mild and straightforward methodology for cysteine arylation.

Moreover, we hypothesized that highly electrophilic benzenediazonium salts

would be suitable as arylating agents, able to easily generate aryl radicals (Ered as

high as 0.5V vs SCE) via a SET pathway.[18] The generated aryl radicals could then be

trapped by the nucleophilic thiol moiety of cysteine.


We commenced our investigation with the arylation of N-Ac-L-cysteine-OMe 1a

using 4-fluorobenzenediazonium tetrafluoroborate in acetonitrile (MeCN) under

batch conditions. In the absence of light and photocatalyst, a modest 26% of the

desired arylated product 3g was obtained within 2 hours reaction time (Table

4.1, Entry 1). When exposed to a 24W compact fluorescence light source (CFL),

a similar yield of 25% was observed, indicating that visible light alone does not

significantly increase aryl radical formation (Table 4.1, Entry 2). However, in the

presence of Ru(bpy)3Cl2•6H2O (1 mol %) as a benchmark photoredox catalyst, a

higher yield of 40% was obtained (Table 4.1, Entry 3). In order to minimize the

risks associated with the handling of potentially explosive diazo intermediates and

desiring to simplify our protocol into a one-pot procedure, we investigated the in

situ formation of the diazonium salt starting from readily available 4-fluoroaniline,

tert-butyl nitrite (t-BuONO, 2.0 equiv.) and catalytic amounts of tetrafluoroboric

acid (HBF4, 1.5 mol %). Within 2 hours, product 3g could be isolated in an improved

56% yield (Table 4.1 Entry 4). Tetrafluoroborate benzenediazonium salts are

easily isolated and exhibit higher stabilities as compared to diazonium salts

bearing other counterions. However, by implementing the in situ formation of

diazonium salts, the counterion choice appeared less restrictive (i.e. no need to

use BF4 counter-ion to afford shelf-stable diazonium salts). Instead, we chose to

use catalytic amounts of easy-to-handle para-toluenesulfonic acid (TsOH.H2O),

which gave similar results (59%, Table 4.1 Entry 5). To develop a biocompatible

strategy, we further tested the possibility to employ an organic dye, Eosin Y, as

photocatalyst for our transformation. Gratifyingly, in the presence of 1 mol% of

Eosin Y, the desired product was obtained in 59% (Table 4.1, Entry 6). This is in

line with recent reports on the ability of Eosin Y to be oxidatively quenched by

diazonium salts, thus generating aryl radicals.[19] Further increasing the amount of

tBuONO to 3 equiv. did not lead to any improvement in yield (Table 4.1, Entry 7).

Solvent screening revealed that the reaction afforded lower yields in DMSO (15%,

Table 4.1, Entry 8) but proceeded well in PBS buffer (pH = 8, 46% Table 4.1, Entry

9), a commonly used solvent for peptide and protein modifications.

One of the major limitations of photocatalytic reactions conducted in batch is

the inefficient irradiation of the reaction mixture, often resulting in sub-optimal

yields and difficulty of scale-up.[20] In order to circumvent these issues, we

translated our arylation protocol into a micro-flow procedure. We developed a

photomicroreactor assembly consisting of a 3D-printed holder equipped with 0.45

mL PFA microcapillary tubing (500 µm ID) and 3.12 W white LEDs (see experimental

section for more details).[21]

Table 4.1: Optimization of Reaction Conditions in Batch for Cysteine Arylationa

Entry Light source Catalyst Changes from optimized conditions Isolated Yield (%)

1 No light none Pre-made diazonium 26

2 CFL none Pre-made diazonium 25

3 CFL Ru(bpy)3Cl2 Pre-made diazonium 40

4 CFL Ru(bpy)3Cl2 In situ formation, HBF4 56

5 CFL Ru(bpy)3Cl2 In situ formation, PTSA 59

6 CFL Eosin Y None 59

7 CFL Eosin Y In situ formation, 3 eq. tBuONO 52b

8 CFL Eosin Y DMSO 15

9 CFL Eosin Y PBS 46

10 White LEDs Eosin Y Continuous flow f 79 (92b)aStandard reaction conditions: 0.5 mmol N-Ac-L-cysteine-OMe (1a), 4-fluoroaniline (1.3 equiv), t-BuONO

(2.0 equiv), 1.5 mol% TsOH∙H2O and 1 mol% Eosin Y in 5 ml MeCN (0.1 M), white CFL, 2 hours reaction

time. For pre-made diazonium salts: 4-fluorobenzenediazonium tetrafluoroborate was used in absence

of t-BuONO and TsOH∙H2O. bYield determined by GC-MS with n-decane as internal standard. fFor

detailed flow conditions, see Scheme 1 and ESI.

Remarkably, within only a 30 second residence time, 79% of compound 3g was

obtained (Table 4.1, Entry 10). Due to the evolution of nitrogen gas (consistent

with the reduction of diazonium salts), the formation of a slug flow was observed,

which ensured optimal mixing efficiency.[22] The significant acceleration of reaction

1a 3g

HN

O S

MeO

Me

O

HN

O SH

MeO

Me

O

F

photocatalyst (1 mol%)tBuONO, TsOH·H2O4-fluoroaniline

MeCNRT, 2 h

4


kinetics and increase in product yields can be attributed to the optimal irradiation

of the reaction mixture.[20a]

With optimized conditions in hand, we evaluated the scope of our protocol both

in batch and in continuous flow (Scheme 4.1). The arylation reaction tolerated a

wide variety of substituents on the aniline coupling partner. Anilines bearing alkyl

substituents reacted in modest yields in batch to give the corresponding arylated

cysteine derivatives (3a to 3c) and improved yields were obtained in flow for

compounds 3a and 3b. Notably, compound 3d (49% batch vs 61% flow) bearing

an alkyne moiety could be of use for further functionalization of biomolecules

through copper-catalyzed alkyne-azide cycloaddition methods (CuAAC).[23] Anilines

bearing both ortho and para substituents also reacted in modest to good yields to

give the desired arylated derivatives 3e and 3f (28% and 66% batch vs 73% in flow

for 3f). In general, we observed that electron-deficient anilines gave higher yields

as compared to the electron-rich anilines. This can be attributed to the difference

in reactivity of their corresponding diazonium salts. In fact, electron-deficient

aryldiazonium salts are less stable and therefore more prone to reduction via SET.[18b] Moreover, a series of fluorinated derivatives was obtained in good to excellent

yields (3g to 3m). Specifically, para- and ortho-fluoro (3g 59% batch, 82% flow, 3h

70% batch), para- and meta-trifluoromethyl- (3k 60% batch, 89% flow and 3l 81%

flow) and trifluoromethoxy- (3m 62% flow) arylated cysteine derivatives were

all prepared in good yields. Additionally, perfluoroarylated derivatives 3i (flow

40%) and 3j (batch 42%, flow 45%) were synthesized in satisfactory yields. Similar

perfluoroarylated cysteine derivatives have been reported by Pentelute and co-

workers as convenient intermediates for peptide stapling.[14a, 14b]

Next, we explored the potential of our methodology for Cl, Br and I containing

anilines, as all halogenated derivatives could represent useful synthetic handles

for further peptide functionalization. Both ortho- and para-Cl derivatives were

obtained in satisfactory yields (3o 78%, 3p 62%) as well as the ortho-Br derivative

3n (79%). Moreover, para- and meta-I derivatives were synthesized (3q 35%, 3r

41%) albeit in slightly diminished yields. The lower yields observed in the presence

of an iodine atom could be explained by considering that iodoarene moieties are

prone to iodine transfer to aryl radicals, thus affording 1,4-diiodobenzene, which

we did observe as a significant side product in our reaction (detected in GC-MS).[19e]

Additionally, we explored the possibility of employing keto- and ester-containing

anilines, thus obtaining para-methyl ketone and ortho-methoxy ester derivatives

3s (78%) and 3t (75%) in good yields. Finally, we probed the reactivity of the

heterocycle 3-amino-5-Cl pyridine towards our transformation. Gratifyingly, the

pyridine-containing cysteine derivative 3u was obtained in 69% yield in flow.

Scheme 4.1: Scope of cysteine arylation in batcha and flowb. aReaction conditions batch: 1.0 mmol N-Ac-Cys-OMe (1a), aniline (1.3 eq), t-BuONO (2 mmol), 1.5mol %TsOH∙H2O and 1mol% Eosin Y in 10 ml ACN (0.1 M), white CFL, 2 h reaction time; bReaction conditions flow: 2.0 mmol N-Ac-Cys-OMe (1a), aniline (1.3 eq), t-BuONO (2 mmol), 4 mol %TsOH∙H2O and 1mol% Eosin in 40 ml ACN (0.05 M), white LED light, 30 seconds residence time; Reported yields are isolated yields [average of two runs]; c60 seconds residence time, d150 seconds residence time. eGram scale experiment in continuous flow (5 mmol scale)

Owing to the ease of scalability, our flow protocol could be easily employed to

obtain arylated cysteine derivatives on gram scales. Consequently, this notable

feature allows one to prepare sufficient quantities for use in automated solid phase

peptide synthesis (SPPS). As an example, we performed a continuous-flow scale-up

experiment with N-Ac-L-cysteine-OMe 1a (5 mmol) and 3-trifluoromethylaniline.

Within approximately two hours of operation time, 1.16 g (72%) of derivative 3l

was obtained.

tBuONO, TsOH·H2OMeCN, RT

Batch (2 h) or continuous-flow (30 s)HN

O SH

MeO

Me

O

NH2

R

+HN

O S

MeO

Me

O

R

3aBatch: 50%Flow: 72%

3bBatch: 33%Flow:d 62%

tBu

3cBatch: 29%

Me

Me

Me

3dBatch: 49%Flow:c 61%

3eBatch: 28%

OMe

3fBatch: 66%Flow: 73%

NO2

Me Me

3g, 3hBatch: p-F 59%Flow: p-F 79%Batch: o-F 70%

3iFlow:d 40%

F

3jBatch: 42%Flow: 45%

3kBatch: 60%Flow: 89%

CF3

3lFlow: 81%

Gram scale:e 75%, 1.16g

F

3mFlow: 62%

3n,3o,3pBatch: o-Br 79%Batch: o-Cl 78%Batch: p-Cl 62%

OCF3

CF3

F

F

F

F

F

F

F

F

F

F

F

F

3q,3rBatch: p-I 35%Batch: m-I 41%

3sBatch: 78%

IN

3uFlow: 69%

Cl

F

COMe

3tBatch: 75%

COOMe

X

EY

4


Encouraged by the results obtained for the arylation of N-Ac-L-Cys-OMe, we

prepared a small array of cysteine-containing dipeptides to test the compatibility

of our methodology with simple model peptides. Therefore, four dipeptides (4

N-Boc-L-Ala-L-Cys-OMe, 5 N-Boc-L-Leu-L-Cys-OMe, 6 N-Boc-L-Trp-L-Cys-OMe and

7 N-Boc-L-Phe-L-Cys-OMe) were prepared in solution via native chemical ligation,

and were subjected to our arylation protocol (Scheme 4.2).[24] Satisfyingly, N-Boc-L-

Ala-L-Cys-OMe afforded the corresponding arylated dipeptides 8a (60%), 8b (56%)

and 8c (42%) in good yields. Similarly, good yields were obtained with N-Boc-L-

Leu-L-Cys-OMe for derivatives 9a to 9d. A remarkable acceleration and increase in

yield was observed when the arylation of N-Boc-L-Leu-L-Cys-OMe was conducted

in flow. When attempting the arylation of N-Boc-L-Trp-L-Cys-OMe, we found the

presence of indole to be incompatible with the in situ diazonium formation.[25]

However, when pre-formed diazonium salt was added to N-Boc-L-Trp-L-Cys-OMe,

the corresponding arylated derivative 10a was obtained in 39% yield. Finally,

N-Boc-L-Phe-L-Cys-OMe afforded the corresponding arylated derivative 11a in

55% yield.

Scheme 4.2: Arylation of cysteine-containing dipeptides in batcha and flowb: aReaction conditions for dipeptide arylation in batch are the same as for the arylation of N-Ac-L-cysteine-OMe but on a 0.25 mmol scale. bReaction conditions for dipeptide arylation in flow are the same as for the arylation of N-Ac-L-cysteine-OMe but on a 1 mmol scale. cFor Trp-Cys pre-made 4-tBu benzenediazonium tetrafluoroborate was used. d150 seconds residence time

In order to further demonstrate the utility of our methodology, we focused

our attention on performing our arylation strategy on more complex peptide

substrates. However, we anticipated that the in situ formation of diazonium salts

might be incompatible with the delicate nature of peptides and proteins. Keen

to adapt our protocol to biologically relevant reaction conditions, we tested the

possibility of employing pre-made diazonium salts and aqueous phosphate buffer

(pH = 8) for our cysteine arylation.

Scheme 4.3: Arylation of a cysteine-containing peptide: 1 eq of crude peptide 12 (0.47 μmol), 10 eq diazonium salt, 1 mol% Eosin Y in 1 mL PBS buffer (pH = 8), white CFL, 30 min reaction time.

Thus, we applied our arylation protocol under these mild reaction conditions on

peptide 12, which was used upon resin cleavage without further purification. In

the presence of para-F benzenediazonium salt tetrafluoroborate or para-OCF3

benzenediazonium tetrafluoroborate, full conversion to the desired products

13 and 14 was achieved within 30 minutes as detected by LC/MS (Scheme 4.3).

Notably, no selectivity issues were observed in presence of lysine and serine

residues, and no organic solvent was required, thus demonstrating the excellent

compatibility of our protocol with other common post translational modification

methods involving these residues.

HN

O SH

MeO

R'HN

O S

MeO

R'

R

HN

O S

MeO

8a, R = CF3 Batch: 60%8b, R = F Batch:56%8c, R = tBu Batch:42%

9a, R = CF3 Batch: 61%, Flow: 86%9b, R = F Batch:46%, Flow: 78%9c, R = penta-F Flow: 40%d

9d, R = tBu Batch: 32%, Flow: 61%

Ala-Cys Leu-Cys

O

NHMe

OtBuO

R

HN

O S

MeOO

NH

OtBuO

R

4-7 8-11

Me

Me

NH2

R

2

+

tBuONO, TsOH·H2OMeCN, RT

Batch (2 h) or continuous-flow (30 s)

HN

O S

MeO

10a, Batch:39%c 11a, Batch:55%

O

NH

OtBuO

tBu

HN

O S

MeOO

NH

OtBuO

CF3Trp-Cys Phe-Cys

HN

EY

12

13, R = F14, R = OCF3

N2+ BF4-

Rphosphate bufferroom temperature

30 min

HN

O SH

HN

O

NH

O

OH

HN

O

HO

NH

O

NH2

H2N

O

NH

O

HN

O S

HN

O

NH

O

OH

HN

O

HO

NH

O

NH2

H2N

O

NH

O

R

EY

4


CONCLUSION

In conclusion, we reported a one-pot protocol for cysteine arylation via visible light

photoredox generation of aryl radicals from their corresponding diazonium salts.

In situ formation of diazonium salts starting from readily available anilines reduces

the risks associated with the handling of potentially explosive intermediates.[20a, 22b] An array of arylated cysteine derivatives decorated with a broad range of

substituents was obtained in moderate to good yields (17 examples, 28-79%).

The implementation of a microflow reactor afforded faster reaction times and

increased yields (30 to 150 seconds residence time, 11 examples, 45-89% yield).

Moreover, a diverse set of cysteine containing dipeptides was arylated successfully

in batch and in flow (12 examples, 32-86% yield). The reaction was easily scaled-up,

affording more than one gram of the arylated cysteine derivative within 2 hours of

total operation time. Finally, in biologically relevant conditions, a model peptide

containing additional nucleophilic side chains was selectively converted to its Cys-

arylated derivative within 30 minutes. Taking into account the simplicity of our

reaction conditions (atmospheric conditions, visible light irradiation, short reaction

time), we believe that our procedure will be appealing to chemical biologists for

post translational chemical modification of cysteine.


General batch procedure (GP1): In an oven-dried glass vial equipped with a magnetic stirrer and a PTFE septum, photoredox catalyst Eosin Y (1 mol%, 0.010 mmol) was added to a mixture of N-Ac-L-cysteine-OMe (1a)/cysteine containing peptide (1.0 mmol), aniline (1.3 eq, 1.3 mmol) and p-toluenesulfonic acid monohydrate (1.5 mol%, 0.015 mmol) in MeCN (10 mL). The vial was purged with argon and the PTFE septum was punched with a disposable syringe needle (for venting N2). The vial was irradiated with a 24W CFL and tert-butylnitrite (2.0 mmol, 2 equiv.) was added dropwise to the mixture. The reaction was monitored by TLC, and stopped after 2 hours. After completion, water was added to the reaction mixture, and the organic layer was extracted with EtOAc (3x). The organic layer was then washed with brine, dried with MgSO4 and evaporated under reduced pressure. The resulting crude compound was absorbed on silica gel and purified via column chromatography. (EtOAc/Hexane, varying ratios). The isolated compound was analysed by GC-MS and NMR.

General continuous-flow procedure in a capillary microreactor (GP2): In an oven-dried glass vial equipped with a magnetic stirrer, photoredox catalyst Eosin Y (1 mol%,0.02 mmol) was added to a mixture of N-Ac-L-cysteine-OMe (1a)/cysteine containing peptide (2.0 mmol), aniline (1.3 eq, 2.6 mmol) and p-toluenesulfonic acid monohydrate (4 mol%, 0.08 mmol)

in MeCN (20 mL). The vial was fitted with a PTFE septum and purged with argon. A second oven-dried glass vial was fitted with a septum, purged with argon and tert-butylnitrite (459 μL, 4 mmol, 2 equiv.) was added through a syringe and diluted with 20 mL of MeCN. The two solutions were transferred into two 20 mL BD Discardit plastic syringes and introduced into the photo-microreactor through a syringe pump. The two liquid streams were merged with a T-Mixer (ID = 500 μm) before entering the reactor. The flow rate was set to 900 μL/min (450 μL/min per syringe), thus resulting in 30 seconds residence time (volume of reactor = 450 μL). After reaching steady state (approximately 4 residence times), a reaction sample was collected in a vial kept in the dark under argon atmosphere. The volume collected was measured and the sample was then diluted with water and extracted with EtOAc (3x). The organic layer was washed with brine, dried with MgSO4 and evaporated under reduced pressure. The resulting crude compound was absorbed on silica gel and purified via column chromatography (EtOAc/Hexane, varying ratios). The isolated compound was analysed by GC-MS and NMR. A schematic representation of the continuous-flow set-up used for the photocatalytic arylation of cysteine is represented below:

Scheme 4.4: Schematic representation of the microflow setups for the photocatalytic arylation of cysteine.

Photocatalytic synthesis of arylated peptide 13 and 14:

1/1.3 mg of para-F/ para-COF3 benzene diazonium salt (10 eq) was weighed in a 2.5 mL Eppendorf vial. To this vial, 150 μL (0.3 mg, 0.468 μmol, 1 eq) of a 3.15 μM solution of peptide 12 in PBS (2 mg of peptide in 1 ml PBS pH = 8) was added together with 30.3 μL (0.1 eq) of a solution Eosin Y in PBS (1 mg in 1 ml PBS). More PBS was added to reach a final volume of 1 mL. The Eppendorf vial then closed and positioned in the proximity of a 24W white CFL lamp. Prior to the reaction the lid of the vial was perforated with a needle to allow the evolution of gas. The reaction was magnetically stirred. After 30 minutes the reaction was analysed in LC-MS.


tBuONO, MeCN

N-Ac-Cys-OMe,Eosin Y, PTSA H2O, MeCN

HN

O S

MeOMe

O

Cysteine arylation in flow

R

4


Preparation of starting materials:

Synthesis of peptides 4, 5, 6 and 7 via native chemical ligation: the dipeptides used as starting materials for the synthesis of derivatives 8 to 11 were prepared through a two-step procedure adapted from literature.2

Step 1: Formation of the thioester derivative: L-Boc-Ala-OH or L-Boc-Leu-OH or L-Boc-Trp-OH or L-Boc-Phe-OH (1.0 equiv.) and DCM or ethyl acetate (0.5 mmol/mL) were added to an oven-dried flask and placed in an ice bath (0°C). Then, DCC (1 equiv.) and HOBt•H2O (1 equiv.) were added together with thiophenol (1 equiv.). The flask was closed with a PTFE septum and the reaction mixture was placed under argon atmosphere. The reaction was checked for completion by TLC (4 -24 hours). The reaction mixture was washed with HCl (1M), NaHCO3 (sat) and brine. The organic layer was dried with MgSO4.The crude was absorbed on silica gel and purified with PE:EtOAC 7:1. Purification by column chromatography (DCM:MeOH 9:1 + 1% acetic acid) afforded the desired thioester intermediates.

Step 2: Native chemical ligation: L-Cysteine methyl ester HCl (1.0 equiv.) and the thioester derivative obtained from the previous step (1.0 equiv.) were added to MeOH (0.25 mmol/mL) in an oven-dried flask kept under argon atmosphere and closed with a PTFE septum. Next tributylphopshine (0.6 equiv.) was added and the reaction mixture was stirred at r.t. until completion (24 hours). The crude was evaporated under vacuo, and re-dissolved in EtOAc. The organic layer was extracted with H2O (3 times) and with brine (3 times). The organic layer was then dried with MgSO4 and concentrated on silica gel under vacuo. Purification by column chromatography (DCM:MeOH 9:1 + 1% acetic acid) afforded the desired dipeptides Boc-Ala-Cys-OMe 4 (overall yield 37%), Boc-Leu-Cys-OMe 5 (overall yield 39%), Boc-Trp-Cys-OMe 6 (overall yield 77%) and Boc-Phe-Cys-OMe 7 (overall yield, 20%).

Synthesis of peptide 12 (starting material): Automated peptide synthesis were performed on a fully-automated SYRO Multiple Peptide Synthesizer robot, equipped with a vortexing unit for the 24-reactor block (MultiSynTech GmbH). Reactions were open to the atmosphere and executed at ambient temperature. Peptide 12 was synthesized using the Fmoc/tBu strategy with HBTU/DIPEA-mediated couplings. Peptide sequence synthesized ABA-CGSSK-CONH2. After the final Fmoc deprotection step, the peptide was manually cleaved from the resin. Cleavage cocktail (500 μL – 1 mL of 95% TFA, 2.5% TIPS and 2.5% H2O) was added to peptide resin, and the reaction was shaken at r.t. for 2 h. Cleavage cocktail containing peptide was precipitated into cold ether and centrifuged (10 mins, 10 kprm). Ether was poured off, and the pellet was re-suspended in fresh cold ether and centrifuged (10 mins, 10 kprm). The resulting pellet peptide was dried on an oil pump. Peptide 12 was then analysed by LC/MS and employed for the reaction without further purification.


Methyl N-acetyl-S-phenyl-L-cysteinate (3a) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 126.4 mg (0.50 mmol, 50 %) of 3a as a yellow oil. The reaction according to GP2 on a 1.825 mmol scale afforded 328.3 mg (1.30 mmol, 72%) of 3a within 30 seconds residence time. 1H NMR (400 MHz, Chloroform-d) δ 7.41 (d, J = 8.1 Hz, 2H), 7.32 – 7.27 (m, 2H), 7.23 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 6.6 Hz, 1H), 4.92 – 4.79 (m, 1H), 3.56 (s, 3H), 3.52 – 3.33 (m, 2H), 1.87 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 170.8, 169.9, 134.8, 131.2, 129.2, 127.3, 52.6, 52.5, 36.6, 23.0. HRMS (ESI) m/z [M+H]+ calculated for C12H16NO3S: 254.0846, found 254.0849.

Methyl N-acetyl-S-(4-tert-butylphenyl)-L-cysteinate (3b) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 101.4 (0.33 mmol, 33 %) of 3b as a yellow oil. The reaction according to GP2 on a 1.75 mmol scale afforded 335.6 mg (1.09 mmol, 62%) of 3b within 150 seconds residence time. 1H NMR (400 MHz, Chloroform-d) δ 7.38 – 7.29 (m, 4H), 6.18 (d, J = 7.0 Hz, 1H), 4.85 (dt, J = 7.6, 4.4 Hz, 1H), 3.54 (s, 3H), 3.47 – 3.29 (m, 2H), 1.84 (s, 3H), 1.29 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 170.8, 169.9, 150.7, 131.5, 131.1, 126.2, 52.6, 52.5, 36.9, 34.6, 31.3, 23.0. HRMS (ESI) m/z [M + H]+ calculated for C16H24NO3S: 310.1472, found 310.1479.

Methyl N-acetyl-S-mesityl-L-cysteinate (3c) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 159.9 mg (0.59 mmol, 59 %) of 3c as a yellow oil.1H NMR (400 MHz, Chloroform-d) δ 6.91 (s, 2H), 6.17 (d, J = 6.72 Hz, 1H), 4.82 – 4.73 (m, 1H), 3.59 (s, 3H), 3.29 – 3.07 (m, 2H), 2.47 (s, 6H), 2.24 (s, 3H), 1.91 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 171.1, 169.8, 142.5, 138.5, 129.4, 129.0, 52.8, 52.7, 36.6, 23.1, 21.9, 21.1. HRMS (ESI) m/z [M + H]+ calculated for C15H22NO3S: 296.1315, found 296.1317

Methyl N-acetyl-S-(4-ethynylphenyl)-L-cysteinate (3d) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 136.4 (0.49 mmol, 49 %) of 3d as a yellow oil. The reaction according to GP2 on a 1.75 mmol scale afforded 296.3 mg (1.07 mmol, 61%) of 3d within 60 seconds residence time. 1H NMR (399 MHz, Chloroform-d) δ 7.42 – 7.30 (m, 4H), 6.24 (d, J = 7.4 Hz, 1H), 4.87 (dt, J = 7.4, 4.5 Hz, 1H), 3.59 (s, 3H), 3.44 (ddd, J = 57.0, 14.3, 4.5 Hz, 2H), 3.10 (s, 1H), 1.90 (s, 3H). 13C NMR (100 MHz, Chloroform-d) δ 170.7, 169.9, 136.4, 132.7, 129.9, 120.6, 83.0, 78.2, 77.5, 77.2, 76.8, 52.8, 52.4, 35.9, 23.1. HRMS (ESI) m/z [M + H]+ calculated for C14H16NO3S: 278.0846, found 278.0853

4


Methyl N-acetyl-S-(4-methoxy-2-methylphenyl)-L-cysteinate (3e) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 83.4 mg (0.28 mmol, 28 %) of 3e as a yellow oil. 1H NMR (399 MHz, Chloroform-d) δ 7.37 (d, J = 8.54 Hz, 1H), 6.76 (d, J = 2.77 Hz, 1H), 6.69 (dd, J = 8.53, 2.84 Hz, 1H), 6.18 (d, J = 6.91 Hz, 1H), 4.79 (dt, J = 7.54, 4.58 Hz, 1H), 3.77 (s, 3H), 3.57 (s, 3H), 3.34 – 3.16 (m, 2H), 2.43 (s, 3H), 1.91 (s, 3H). 13C NMR (100 MHz, Chloroform-d) δ 171.0, 169.8, 159.7, 142.4, 135.4, 124.0, 116.2, 112.2, 55.4, 52.6, 52.5, 37.2, 23.1, 21.2. HRMS (ESI) m/z [M + H]+ calculated for C14H20NO4S: 298.1108, found 298.1113

Methyl N-acetyl-S-(2-methyl-4-nitrophenyl)-L-cysteinate (3f) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 206.2 (0.66 mmol, 66 %) of 3f as a yellow oil. The reaction according to GP2 on a 1.85 mmol scale afforded 421.4 mg (1.35 mmol, 73%) of 3f within 30 seconds residence time. 1H NMR (399 MHz, Chloroform-d) δ 7.98 (d, J = 11.02 Hz, 2H), 7.36 (d, J = 8.42 Hz, 1H), 6.45 (d, J = 6.02 Hz, 1H), 5.03 – 4.69 (m, 1H), 3.69 (s, 3H), 3.65 – 3.34 (m, 2H), 2.36 (s, 3H), 1.95 (s, 3H). 13C NMR (100 MHz, Chloroform-d) δ 170.6, 170.0, 145.4, 144.8, 137.7, 126.0, 124.7, 121.6, 53.1, 52.0, 34.2, 23.2, 20.5. HRMS (ESI) m/z [M + H]+ calculated for C13H17N2O5S: 313.0853, found 313.0860

Methyl N-acetyl-S-(4-fluorophenyl)-L-cysteinate (3g) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 159.9 mg (0.59 mmol, 59%) of 3g as a yellow oil. The reaction according to GP2 on a 1.85 mmol scale afforded 396.1 mg (1.46 mmol, 79%) of 3g within 30 seconds residence time. 1H NMR (400 MHz, Chloroform-d) δ 7.51 – 7.36 (m, 2H), 7.10 – 6.94 (m, 2H), 6.20 (s, 1H), 4.83 (dt, J = 7.50, 4.55 Hz, 1H), 3.58 (s, 3H), 3.43 (dd, J = 14.28, 4.56 Hz, 1H), 3.30 (dd, J = 14.28, 4.56 Hz, 1H), 1.92 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 170.8, 169.8, 162.3 (d, J = 248.00 Hz), 134.0 (d, J = 8.13 Hz), 129.8 (d, J = 3.37 Hz), 116.3 (d, J = 21.94 Hz), 52.6, 52.4, 37.7, 23.1. 19F NMR (376 MHz, Chloroform-d) δ -113.79 – -113.90 (m). HRMS (ESI) m/z [M+H]+ calculated for C12H15FNO3S: 272.0751, found 272.0755.

Methyl N-acetyl-S-(2-fluorophenyl)-L-cysteinate (3h) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 189.9 mg (0.70 mmol, 70%) of 3h as a yellow oil. 1H NMR (400 MHz, Chloroform-d) δ 7.45 (td, J = 7.84, 1.76 Hz, 1H), 7.31 – 7.19 (m, 1H), 7.13 – 7.01 (m, 2H), 6.28 (d, J = 6.26 Hz, 1H), 5.14 – 4.59 (m, 1H), 3.54 (s, 3H), 3.40 (dd, J = 4.45, 3.27 Hz, 2H), 1.92 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 170.7, 169.8, 162.3 (d, J = 246.57 Hz), 134.5, 129.9 (d, J = 8.02 Hz), 124.7 (d, J = 3.81 Hz), 121.4 (d, J = 17.47 Hz), 116.1 (d, J = 22.76 Hz), 52.6, 52.3, 36.0, 23.1. 19F NMR (376 MHz, Chloroform-d) δ -105.69 – -111.27 (m). HRMS (ESI) m/z [M+H]+ calculated for C12H15FNO3S: 272.0751, found 272.0750.

Methyl N-acetyl-S-(perfluorophenyl)-L-cysteinate (3i) was made according to GP2 on a 1.725 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 235.6 mg (0.67 mmol, 40%) of 3i as a yellow oil within 150 seconds residence time.

1H NMR (400 MHz, Chloroform-d) δ 6.35 (d, J = 6.21 Hz, 1H), 4.79 (dt, J = 7.11, 4.62 Hz, 1H), 3.67 (s, 3H), 3.51 – 3.24 (m, 2H), 2.00 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 170.2, 169.7, 148.7, 146.4, 138.9, 132.8, 129.2, 111.1, 52.8, 52.0, 36.5, 22.9. 19F NMR (376 MHz, Chloroform-d) δ -131.69 (dd, J = 22.97, 8.69 Hz), -151.41 (t, J = 20.90 Hz), -160.52 (td, J = 22.39, 6.40 Hz). HRMS (ESI) m/z [M + H]+ calculated for C12H11F5NO3S: 344.0380, found 344.0377.

Methyl N-acetyl-S-(perfluoro-[1,1’-biphenyl]-4-yl)-L-cysteinate (3j) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 206.4 mg (0.42 mmol, 42%) of 3j as a yellow oil. The reaction according to GP2 on a 1.875 mmol scale afforded 403.1 mg (0.84 mmol, 45%) of 3j within 30 seconds residence time. 1H NMR (399 MHz, Chloroform-d) δ 6.42 (d, J = 7.09 Hz, 1H), 4.87 (dt, J = 7.04, 4.36 Hz, 1H), 3.65 (s, 3H), 3.61 – 3.50 (m, 2H), 2.00 (s, 3H). 13C NMR (100 MHz, Chloroform-d) δ 170.2, 170.1, 148.4, 145.9, 143.3, 143.0, 142.8, 139.3, 136.9, 116.5, 52.9, 52.4, 36.1, 23.0. 19F NMR (376 MHz, Chloroform-d) δ -131.55 – -131.79 (m), -137.34 – -137.57 (m), -149.55 – -149.74 (m), -160.13 – -160.33 (m). HRMS (ESI) m/z [M + H]+ calculated for C18H11F9NO3S: 429.0310, found 429.0353.

Methyl N-acetyl-S-(4-trifluoromethylphenyl)-L-cysteinate (3k) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 192.6 mg (0.60 mmol, 60%) of 3k as a yellow oil. The reaction according to GP2 on a 1.700 mmol scale afforded 511.2 mg (1.59 mmol, 89%) of 3k within 30 seconds residence time. 1H NMR (400 MHz, Chloroform-d) δ 7.51 (d, J = 8.27 Hz, 2H), 7.44 (d, J = 8.21 Hz, 2H), 6.38 (s, 1H), 4.87 (dt, J = 7.31, 4.70 Hz, 1H), 3.60 (s, 3H), 3.55 (dd, J = 14.19, 4.78 Hz, 1H), 3.39 (dd, J = 14.18, 4.65 Hz, 1H), 1.88 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 170.5, 169.9, 152.8, 140.3, 129.2, 125.7 (q, J = 3.81 Hz), 125.2, 52.6, 52.2, 35.2, 22.8. 19F NMR (376 MHz, Chloroform-d) δ -62.67. HRMS (ESI) m/z [M+H]+ calculated for C13H15F3NO3S: 322.0718, found 322.0724.

Methyl N-acetyl-S-(3-trifluoromethylphenyl)-L-cysteinate (3l) was made according to GP2 on a 1.775 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 518.9 mg (1.62 mmol, 81%) of 3l as a yellow oil within 30 seconds residence time. 1H NMR (400 MHz, Chloroform-d) δ 7.63 (s, 1H), 7.56 (d, J = 7.58 Hz, 1H), 7.43 (dt, J = 15.53, 7.84 Hz, 2H), 6.32 (s, 1H), 4.88 (dt, J = 7.36, 4.51 Hz, 1H), 3.58 (s, 1H), 3.54 (dd, J = 14.32, 4.74 Hz, 1H), 3.41 (dd, J = 14.25, 4.39 Hz, 1H), 1.91 (s, 3H).13C NMR (101

4


MHz, Chloroform-d) δ 170.7, 169.9, 136.7, 133.6, 131.6 (q, J = 32.51 Hz), 129.6, 127.0 (q, J = 3.86 Hz), 123.8 (q, J = 272.59 Hz), 123.7 (q, J = 3.76 Hz), 52.8, 52.4, 36.3, 23.1.19F NMR (188 MHz, Chloroform-d) δ -62.86. HRMS (ESI) m/z [M+H]+ calculated for C13H15F3NO3S: 322.0718, found 322.0730.

Methyl N-acetyl-S-(4-trifluoromethoxyphenyl)-L-cysteinate (3m) was made according to GP2 on a 1.85 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 389.0 mg (1.15 mmol, 62%) of 3m as a yellow oil within 30 seconds residence time. 1H NMR (400 MHz, Chloroform-d) δ 7.49 – 7.39 (m, 2H), 7.20 – 7.08 (m, 2H), 6.22 (d, J = 6.2 Hz, 1H), 4.94 – 4.78 (m, 1H), 3.57 (s, 3H), 3.56 – 3.28 (m, 2H), 1.89 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 170.7, 169.9, 148.4, 133.7, 132.6, 121.8, 119.2, 52.6, 52.5, 36.9, 23.0; 19F NMR (188 MHz, Chloroform-d) δ -58.01. HRMS (ESI) m/z [M+H]+ calculated for C13H15F3NO4S: 338.0669, found 338.0673. HRMS (ESI) m/z [M+H]+ calculated for C13H15F3NO4S: 338.0674, found 338.0673.

Methyl N-acetyl-S-(2-bromophenyl)-L-cysteinate (3n) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 254.7 mg (0.79 mmol, 79%) of 3n as a yellow oil. 1H NMR (400 MHz, Chloroform-d) δ 7.53 – 7.46 (m, 1H), 7.37 (dd, J = 7.85, 1.18 Hz, 1H), 7.24 – 7.17 (m, 1H), 7.01 (td, J = 7.87, 1.30 Hz, 1H), 6.32 (d, J = 6.17 Hz, 1H), 4.81 (dt, J = 7.47, 4.74 Hz, 1H), 3.57 (s, 3H), 3.39 (qd, J = 14.01, 4.75 Hz, 2H), 1.86 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 170.7, 169.9, 135.9, 133.4, 131.5, 128.3, 128.0, 125.9, 52.8, 52.1, 35.9, 23.1. HRMS (ESI) m/z [M+H]+ calculated for C12H15BrNO3S: 331.9951, found 331.9951.

Methyl N-acetyl-S-(2-chlorophenyl)-L-cysteinate (3o) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 224.7 mg (0.78 mmol, 78%) of 3o as a yellow oil. 1H NMR (400 MHz, Chloroform-d) δ 7.41 (ddd, J = 24.53, 7.74, 1.61 Hz, 2H), 7.18 (dtd, J = 20.96, 7.47, 1.57 Hz, 2H), 6.36 (d, J = 6.68 Hz, 1H), 4.86 (dt, J = 7.46, 4.71 Hz, 1H), 3.61 (s, 3H), 3.51 – 3.37 (m, 2H), 1.92 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 170.7, 169.9, 135.6, 133.8, 131.9, 130.1, 128.3, 127.4, 52.7, 52.1, 35.5, 23.1. HRMS (ESI) m/z [M+H]+ calculated for C12H15ClNO3S: 288.0456, found 288.0463.

Methyl N-acetyl-S-(4-chlorophenyl)-L-cysteinate (3p) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 117.6 mg (0.62 mmol, 62%) of 3p as a brown oil.1H NMR (399 MHz, Chloroform-d) δ 7.31 (d, J = 8.69 Hz, 2H), 7.24 (d, J = 8.69 Hz, 2H), 6.34 (d, J = 6.91 Hz, 1H), 4.82 (dt, J = 7.48, 4.71 Hz, 1H), 3.57 (s, 3H), 3.37 (ddd, J = 55.08, 14.21, 4.72 Hz, 2H), 1.89 (s, 3H). 13C NMR (100 MHz, Chloroform-d) δ 170.7, 169.9, 133.4, 133.2, 132.3, 129.2,

52.6, 52.3, 36.7, 23.0. HRMS (ESI) m/z [M+H]+ calculated for C12H15ClNO3S: 288.0456, found 288.0461.

Methyl N-acetyl-S-(4-iodophenyl)-L-cysteinate (3q) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 133.2 mg (0.35 mmol, 35%) of 3q as a yellow oil. 1H NMR (399 MHz, Chloroform-d) δ 7.60 (d, J = 8.52 Hz, 2H), 7.12 (d, J = 8.52 Hz, 2H), 6.24 (d, J = 6.69 Hz, 1H), 4.84 (dt, J = 7.38, 4.59 Hz, 1H), 3.59 (s, 3H), 3.52 – 3.26 (m, 2H), 1.90 (s, 3H). 13C NMR (100 MHz, Chloroform-d) δ 170.7, 169.8, 138.1, 135.0, 132.5, 131.2, 129.2, 92.2, 52.7, 52.4, 36.4, 23.1. HRMS (ESI) m/z [M+H]+ calculated for C12H15INO3S: 379.9812, found 379.9817.

Methyl N-acetyl-S-(3-iodophenyl)-L-cysteinate (3r) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 155.6 mg (0.41 mmol, 41%) of 3r as a yellow oil. 1H NMR (399 MHz, Chloroform-d) δ 7.73 (t, J = 1.65 Hz, 1H), 7.53 (dt, J = 7.85, 1.17 Hz, 1H), 7.34 (dt, J = 7.83, 1.26 Hz, 1H), 7.00 (t, J = 7.87 Hz, 1H), 6.29 (d, J = 5.83 Hz, 1H), 4.87 (dt, J = 7.32, 4.43 Hz, 1H), 3.60 (s, 3H), 3.41 (ddd, J = 51.73, 14.25, 4.44 Hz, 2H), 1.92 (s, 3H). 13C NMR (100 MHz, Chloroform-d) δ 170.7, 169.9, 138.8, 137.4, 136.0, 130.6, 129.7, 94.6, 52.8, 52.4, 36.3, 23.1. HRMS (ESI) m/z [M+H]+ calculated for C12H15INO3S: 379.9812, found 379.9824.

Methyl N-acetyl-S-(4-acetylphenyl)-L-cysteinate (3s) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 230.6 mg (0.78 mmol, 78%) of 3s as a yellow oil. 1H NMR (400 MHz, Chloroform-d) δ 7.87 (d, J = 8.52 Hz, 2H), 7.40 (d, J = 8.52 Hz, 2H), 6.24 (d, J = 6.76 Hz, 1H), 4.91 (dt, J = 7.24, 4.67 Hz, 1H), 3.65 (s, 3H), 3.52 (ddd, J = 62.48, 14.15, 4.68 Hz, 2H), 2.57 (s, 3H), 1.91 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 197.2, 170.7, 169.9, 142.3, 135.0, 129.0, 128.4, 52.9, 52.3, 34.9, 26.7, 23.1. HRMS (ESI) m/z [M+H]+ calculated for C14H18NO4S: 296.0951, found 296.0946.

Methyl (R)-2-((2-acetamido-3-methoxy-3-oxopropyl)thio)benzoate (3t) was made according to GP1 on a 1.0 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 233.6 mg (0.75 mmol, 75%) of 3t as a yellow oil. 1H NMR (399 MHz, Chloroform-d) δ 7.85 (d, J = 7.55 Hz, 1H), 7.46 (s, 2H), 7.25 – 7.19 (m, 1H), 6.55 (s, 1H), 5.02 – 4.72 (m, 1H), 3.92 (s, 3H), 3.68 (s, 3H), 3.57 – 3.36 (m, 2H), 1.91 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 170.8, 170.1, 167.3, 138.3, 132.3, 130.9, 130.2, 128.6, 125.5, 52.8, 52.4, 51.9, 35.2, 23.0. HRMS (ESI) m/z [M+H]+ calculated for C14H18NO5S: 312.0900, found 312.0885.

Methyl N-acetyl-S-(6-chloropyridin-3-yl)-L-cysteinate (3u) was made according to GP2 on a 1.875 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 1:1) yielding 371.1 mg (1.29 mmol, 69%) of 3u as a yellow oil within 30

4


seconds residence time. 1H NMR (400 MHz, Chloroform-d) δ 8.39 (dd, J = 2.6, 0.7 Hz, 1H), 7.71 (dd, J = 8.3, 2.6 Hz, 1H), 7.29 – 7.26 (m, 1H), 6.24 (d, J = 7.0 Hz, 1H), 4.84 (dt, J = 7.2, 4.7 Hz, 1H), 3.64 (s, 3H), 3.53 – 3.30 (m, 2H), 1.96 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 170.5, 169.9, 151.4, 150.4, 141.3, 131.1, 124.6, 52.9, 52.3, 36.8, 23.1. HRMS (ESI) m/z [M+H]+ calculated for C11H13ClN2O3S: 289.0408, found 289.0422.

Methyl-N-((tert-butoxycarbonyl)-L-alanyl)-S-(4-(trifluoromethyl)phenyl)-L-cysteinate (8a) was made according to GP1 on a 0.26 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 2:1) yielding 70.2 mg (0.156 mmol, 60%) of 8a as a yellow solid. 1H NMR (200 MHz, Chloroform-d) δ 7.50 (q, J = 9.10 Hz, 4H), 6.94 (s, 1H), 4.90 – 4.75 (m, 1H), 4.71 (d, J = 8.42 Hz, 1H), 4.22 – 4.00 (m, 1H), 3.62 (s, 3H), 3.54 – 3.43 (m, 2H), 1.46 (s, 9H), 1.29 (d, J = 7.11 Hz, 3H). 19F NMR (376 MHz, Chloroform-d) δ -62.67. HRMS (ESI) m/z [M+H]+ calculated C19H26F3N2O5S: 451.1509, found 451.0895.

Methyl-N-((tert-butoxycarbonyl)-L-alanyl)-S-(4-fluorophenyl)-L-cysteinate (8b) was made according to GP1 on a 0.125 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 2:1) yielding 28.2 mg (0.07 mmol, 56%) of 8b as a yellow solid. 1H NMR (400 MHz, Chloroform-d) δ 7.42 (dd, J = 8.89, 5.23 Hz, 2H), 7.00 (t, J = 8.63 Hz, 2H), 6.88 (s, 1H), 4.78 (s, 1H), 4.13 (s, 1H), 3.65 (s, 1H), 3.57 (s, 3H), 3.42 – 3.25 (m, 2H), 1.47 (s, 9H), 1.31 (d, J = 7.01 Hz, 3H). 19F NMR (376 MHz, Chloroform-d) δ -113.89 (m). HRMS (ESI) m/z [M-BOC+2H]+ calculated for C13H18FN2O3S: 301.1022, found 301.1033.

Methyl-N-((tert-butoxycarbonyl)-L-alanyl)-S-(4-tert-butyl-phenyl)-L-cysteinate (8c) was made according to GP1 on a 0.25 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 2:1) yielding 46.6 mg (0.105 mmol, 42%) of 8c as a yellow solid. 1H NMR (400 MHz, Chloroform-d) δ 7.34 (q, J = 8.64 Hz, 4H), 6.87 (d, J = 7.00 Hz, 1H), 4.81 (dt, J = 7.61, 4.51 Hz, 1H), 4.59 – 4.44 (m, 1H), 4.12 (s, 1H), 3.56 (s, 3H), 3.38 (ddd, J = 47.26, 14.35, 4.45 Hz, 2H), 1.47 (s, 9H), 1.29 (s, 9H), 1.26 – 1.20 (m, 3H). 13C NMR (101 MHz, Chloroform-d) δ 172.2, 170.5, 150.7, 131.5, 131.4, 126.4, 126.3, 52.7, 52.6, 50.0, 35.8 (d, J = 223.40 Hz), 31.4, 28.5, 18.1. HRMS (ESI) m/z [M-BOC+2H]+ calculated for C17H27N2O3S: 339.1742, found 339.1760

Methyl-N-((tert-butoxycarbonyl)-L-leucyl)-S-(4-(trifluoromethylphenyl)-L-cysteinate (9a) was made according to GP1 on a 0.25 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 2:1) yielding 75.2 mg (0.15 mmol, 61%) of 9a as a yellow solid. The reaction according to GP2 on a 0.9 mmol scale afforded 366.2 mg (0.75 mmol, 86%) of 9a within 30 seconds residence time. 1H NMR (399 MHz, Chloroform-d) δ 7.58 – 7.43 (m, 1H), 6.92 (d, J = 6.26 Hz, 4H), 4.83 (dt, J = 7.18, 4.97 Hz, 1H), 4.62 (d, J = 4.71 Hz, 1H), 4.14 – 4.04 (m, 1H), 3.62 (s, 3H), 3.55 – 3.38 (m, 2H), 1.71 – 1.59 (m, 2H), 1.45 (s, 9H), 1.41 – 1.31 (m, 1H), 0.92 (t, J = 5.82 Hz, 6H). 13C NMR (100 MHz, Chloroform-d) δ 206.9,

172.8, 170.3, 155.6, 140.5, 129.1, 128.2 (q, J = 34.47 Hz), 125.7 (q, J = 3.68 Hz), 122.6 (q, J = 272.27 Hz), 114.0, 53.0, 52.5, 51.9, 40.9, 35.1, 30.8, 28.2, 24.6, 22.9, 21.7. 19F NMR (188 MHz, Chloroform-d) δ -62.60. HRMS (ESI) m/z [M-BOC+2H]+ calculated for C17H24F3N2O3S: 393.1460, found 393.1498.

Methyl-N-((tert-butoxycarbonyl)-L-leucyl)-S-(4-fluorophenyl)-L-cysteinate (9b) was made according to GP1 on a 0.25 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 4:1) yielding 51.1 mg (0.12 mmol, 46%) of 9b as a yellow solid. The reaction according to GP2 on a 0.75 mmol scale afforded 260.2 mg (0.59 mmol, 78%) of 9b within 30 seconds residence time. 1H NMR (399 MHz, Chloroform-d) δ 7.52 (d, J = 8.49 Hz, 2H), 7.45 (d, J = 8.24 Hz, 2H), 6.96 (d, J = 5.60 Hz, 1H), 4.82 (dt, J = 7.31, 4.98 Hz, 1H), 4.68 (d, J = 6.88 Hz, 1H), 4.10 (s, 1H), 3.61 (s, 3H), 3.55 – 3.33 (m, 2H), 1.62 (s, 3H), 1.44 (s, 9H), 0.91 (t, J = 5.92 Hz, 6H). 13C NMR (100 MHz, Chloroform-d) δ 172.7, 172.5, 170.4, 163.5, 161.0, 155.5 (d, J = 10.35 Hz), 134.0 (d, J = 8.09 Hz), 129.6 (d, J = 3.46 Hz), 116.2 (d, J = 21.85 Hz), 59.5, 53.5, 52.4, 51.9, 37.6, 29.8, 28.3, 24.7, 22.9, 21.8. 19F NMR (376 MHz, Chloroform-d) δ -126.91 (tt, J = 8.66, 4.46 Hz). HRMS (ESI) m/z [M-BOC+2H]+ calculated for C16H24FN2O3S: 343.1492, found 343.1503.

Methyl-N-((tert-butoxycarbonyl)-L-leucyl)-S-(perfluorophenyl)-L-cysteinate (9c) was made according to GP2 on a 0.9 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 7:1) yielding 185 mg (0.36 mmol, 40%) of 9c as a yellow solid within 150 seconds of residence time. 1H NMR (399 MHz, Chloroform-d) δ 7.01 (d, J = 7.23 Hz, 1H), 4.86 (d, J = 7.56 Hz, 1H), 4.73 (dt, J = 7.44, 4.93 Hz, 1H), 4.16 – 4.00 (m, 1H), 3.65 (s, 3H), 3.36 (d, J = 4.94 Hz, 2H), 1.76 – 1.58 (m, 3H), 1.44 (s, 9H), 0.93 (t, J = 6.59 Hz, 6H). 19F NMR (188 MHz, Chloroform-d) δ -131.77 (dd, J = 23.92, 7.71 Hz), -151.44 (t, J = 20.87 Hz), -160.50 (dt, J = 21.09, 11.73 Hz). 13C NMR (100 MHz, Chloroform-d) δ 172.6, 169.9, 155.6, 148.9, 139.0, 136.4, 107.9, 100.0, 80.4, 53.2, 52.7, 51.9, 41.0, 36.4, 28.2, 24.7, 22.9, 21.8. HRMS (ESI) m/z [M-BOC+2H]+ calculated for C16H20F5N2O3S: 415.1115, found 415.1113.

Methyl-N-((tert-butoxycarbonyl)-L-leucyl)-S-(4-tert-butylphenyl)-L-cysteinate (9d) was made according to GP1 on a 0.25 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 4:1) yielding 38.6 mg (0.08 mmol, 46%) of 9d as a yellow solid. The reaction according to GP2 on a 0.73 mmol scale afforded 215 mg (0.45 mmol, 61%) of 9d within 30 seconds residence time.

1H NMR (400 MHz, Chloroform-d) δ 7.32 (q, J = 8.60 Hz, 1H), 6.97 (d, J = 7.16 Hz, 4H), 4.78 (dt, J = 7.59, 4.77 Hz, 1H), 4.60 (d, J = 7.59 Hz, 1H), 4.11 (s, 1H), 3.52 (s, 3H), 3.33 (m, 2H), 1.72 – 1.51 (m, 2H), 1.45 (s, 9H), 1.27 (s, 9H), 1.22 – 1.16 (m, 1H), 0.95 – 0.86 (m, 6H). 13C NMR (100 MHz, Chloroform-d) δ 172.4, 170.5, 155.6, 150.5, 131.5, 131.2, 126.2, 80.2,

4


64.3, 52.4, 41.0, 36.9, 34.6, 31.3, 28.4, 25.3, 24.7, 23.1, 21.8. HRMS (ESI) m/z [M-BOC+2H]+ calculated for C20H33N2O3S: 381.2212, found 381.2244.

Methyl-N-((tert-butoxycarbonyl)-L-tryptophyl)-S-(4-tert-butylphenyl)-L-cysteinate (10a) was made according to GP1 on a 0.25 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 2:1) yielding 55.6 mg (0.098 mmol, 39%) of 10a as a yellow solid.

1H NMR (400 MHz, Chloroform-d) δ 8.34 (s, 1H), 7.66 (d, J = 9.42 Hz, 1H), 7.37 (d, J = 8.06 Hz, 1H), 7.31 – 7.18 (m, 5H), 7.14 (t, J = 7.45 Hz, 1H), 7.09 (s, 1H), 6.71 (d, J = 7.41 Hz, 1H), 5.11 (s, 1H), 4.80 – 4.67 (m, 1H), 4.46 (s, 1H), 3.44 (s, 3H), 3.35 – 3.13 (m, 4H), 1.47 (s, 9H), 1.29 (s, 9H). 13C NMR (101 MHz, Chloroform-d) δ 171.5, 170.3, 161.7, 150.6, 143.0, 132.6, 131.4, 131.0, 129.5, 127.8, 126.2, 123.3, 122.4, 119.9, 119.0, 111.3, 52.4, 52.2, 37.1, 34.7, 31.3, 28.5, 22.1. HRMS (ESI) m/z [M-BOC+2H]+ calculated for C25H32N3O3S: 454.2164, found 454.2161.

Methyl-N-((tert-butoxycarbonyl)-L-tryptophyl)-S-(4-tert-butylphenyl)-L-cysteinate (11a) was made according to GP1 on a 0.5 mmol scale. The crude product was purified by flash chromatography (petroleum ether: ethyl acetate 4:1) yielding 144.7 mg (0.28 mmol, 55%) of 11a as a yellow solid.

1H NMR (400 MHz, Chloroform-d) δ 7.53 (d, J = 8.14 Hz, 2H), 7.44 (d, J = 8.20 Hz, 2H), 7.31 (q, J = 8.24, 7.55 Hz, 3H), 7.22 (d, J = 6.23 Hz, 2H), 6.80 (d, J = 6.68 Hz, 1H), 4.91 – 4.78 (m, 2H), 4.46 – 4.27 (m, 1H), 3.60 (s, 3H), 3.44 (qd, J = 14.07, 4.99 Hz, 2H), 3.18 – 2.92 (m, 2H), 1.43 (d, J = 2334.78 Hz, 9H). 19F NMR (376 MHz, Chloroform-d) δ -62.54. 13C NMR (101 MHz, Chloroform-d) δ 171.3, 170.1, 140.3, 136.5, 129.4, 129.3, 128.8, 127.1, 125.9 (q, J = 3.73 Hz), 125.4, 122.7, 53.6, 52.7, 52.2, 38.0, 35.5, 29.8, 28.3. HRMS (ESI) m/z [M-BOC+2H]+ calculated for C20H22F3N2O3S: 427.1298, found 427.1382

ASSOCIATED CONTENT

The Supporting Information for this article is available free of charge on the Wiley Publications website at DOI: 10.1002/anie.201706700.

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CHAPTER 5

Visible light induced trifluoromethylation of highly functionalized arenes and heteroarenes in continuous flow


Abdiaj, I.; Bottecchia, C.; Alcazar, J.; Noël, T, Synthesis 2017, 49, 4978-4985

5

118 Chapter 5 Visible light induced trifluoromethylation in continuous flow 119

ABSTRACT

In the following chapter, a continuous-flow protocol for the trifluoromethylation of

arenes, heteroarenes and benzofused heterocycles is described. This photoredox

methodology relies on the use of solid CF3SO2Na as trifluoromethylating agent

and an Ir complex as photoredox catalyst. A diverse set of highly functionalized

heterocycles proved compatible with the methodology, and moderate to good

yields were obtained within 30 minutes residence time.

INTRODUCTION

Novel methodologies for the trifluoromethylation of arenes and heteroarenes

are highly demanded in the chemical and pharmaceutical industry.[1] In structure-

activity relationship (SAR) studies, the introduction of fluorine atoms can greatly

impact the electronic properties, acidity and lipophilicity of drug candidates.[2]

These effects are due to the high electronegativity of the fluorine atom, to its

relatively small radius and to the less polarizable nature of C–F bonds compared

to C–H bonds.[1b] The replacement of methyl groups with their trifluoromethyl

counterparts represents a conservative substitution in terms of steric hindrance,

while constituting a valuable strategy to block potential metabolically labile sites

in drug candidates, prolonging their half-life and metabolic stability.[1a]

The initially reported trifluoromethylation protocols relied on transition-

metal catalyzed cross-coupling methods, but suffered from the need for pre-

functionalized substrates and for stoichiometric amounts of metal salts.[3] The

vast majority of metal-promoted aromatic trifluoromethylation reactions require

the use of copper (with only few examples describing aromatic C-CF3 bond

formation in presence of Pd or Ni species).Moreover, viable reaction conditions

typically require increased temperatures.[3b] More recently, several strategies

reported in the literature demonstrated the utility of photocatalytic protocols

for the trifluoromethylation of alkenes, thiols, heterocycles and arenes.[4] The

most commonly used trifluoromethyl sources include expensive Togni and

Umemoto’s reagents, unstable triflyl chloride (CF3SO2Cl), gaseous CF3I and readily

available trifluoroacetic anhydride.[4e, 5] In addition, Langlois’ reagent (CF3SO2Na)

can be regarded as an easy-to-handle, inexpensive and solid trifluoromethylating

agent, capable of generating CF3 radicals in the presence of a strong oxidant (e.g.

tBuOOH).[6] [7]

As part of our interest to develop efficient continuous-flow protocols as enabling

tools for drug discovery, we envisioned a photocatalytic strategy for the

trifluoromethylation of a variety of highly functionalized heteroarenes, which

are of interest in medicinal chemistry.[4a, 4b, 8] Such substrates are often ignored in

many reports since these compounds are known to be highly challenging and thus

low yielding. In order to develop a practical and widely applicable methodology,

we opted to use the stable, inexpensive and solid Langlois reagent (CF3SO2Na) as

trifluoromethyl source.

5



We commenced our investigations by performing luminescence quenching studies,

which allowed us to rapidly select the optimal photocatalyst for our transformation

(see Supporting Information).[9] Among the photocatalysts tested, the

luminescence of both fac-Ir(ppy)3 and [Ir{dF(CF3)ppy}2](dtbpy)]PF6 was significantly

quenched by increasing equivalents of CF3SO2Na, as depicted in Error! Reference

source not found.. This suggests that the excited state of both photocatalysts

can be oxidatively quenched by the Langlois reagent thus generating a CF3 radical.

In particular, a high luminescence quenching percentage of 58% was obtained

for [Ir{dF(CF3)ppy}2](dtbpy)]PF6 in the presence of 300 equivalents of Langlois’

reagent, while 2500 equivalents of CF3SO2Na were needed to obtain a quenching

percentage of only 44% in the case of fac-Ir(ppy)3 (Figure 5.1). The higher

quenching efficiency observed with [Ir{dF(CF3)ppy}2](dtbpy)]PF6 is consistent with

the higher excited state reduction potential reported for this catalyst compared

to fac-Ir(ppy)3 (1.21 V vs 0.31 V respectively, both values are reported versus the

saturated calomel electrode (SCE)).[10] As reported by Glorius and co-workers, a

quenching percentage higher than 25% should be considered as significant and

relevant for photocatalytic reaction purposes.[9b] Therefore, we selected [Ir{dF(CF3)

ppy}2](dtbpy)]PF6 as the photocatalyst for our further investigations.

Figure 5.1: : Luminescence quenching percentage of [Ir{dF(CF3)ppy}2](dtbpy)]PF6 (squares) and fac-Ir(ppy)3 (dots) in presence of increasing amounts of Langlois reagent. Experimental conditions: the solutions of both photocatalysts were prepared in degassed acetonitrile with a 10μM concentration. Solutions with increasing concentrations of quencher

(CF3SO2Na) were prepared in degassed acetonitrile and tested. Compared to the amount of catalyst present, the concentrations of CF3SO2Na employed were the following: 25 mM, 50 mM, 100 mM and 300 mM. For [Ir{dF(CF3)ppy}2](dtbpy)]PF6 a concentration of 0.3 mM (corresponding to the optimized reaction conditions) was also tested. For more details on the procedure followed and for the calculation of the quenching percentages see the experimental section.

The trifluoromethylation of caffeine was selected as the benchmark reaction

for our optimization studies in flow as this was deemed a substrate of interest

for our industrial collaborators (Table 5.1). Moreover, a report published while

this research was carried out described a photochemical trifluoromethylation

with Langlois reagent, trifluoroacetic anhydride and acetone which included

caffeine as a viable substrate.[11] However, this method required UV irradiation,

inert atmosphere and prolonged reaction time (12 h). The photoflow reactor we

employed consists of a Vapourtec UV-150 photoreactor equipped with a 10 mL

capillary reactor (I.D. 1.3 mm), which was subjected to 450 nm irradiation (54 blue

LEDs; 24W). DMSO was chosen as a suitable solvent, which ensured high solubility

of the densely functionalized substrates and thus avoided the occurrence of

microreactor clogging. DMSO was chosen as a suitable solvent, which ensured

high solubility of the densely functionalized substrates and thus avoided the

occurrence of microreactor clogging. At 40°C, unsatisfactory yields were observed

in flow for the trifluoromethylated caffeine (Table 5.1, Entries 1 and 2). We

rationalized that the addition of an oxidant might assist the re-aromatisation of

the radical intermediate to the final product. Indeed, in presence of 1 equivalent

of (NH4)2S2O8 as an oxidant, an improved LC-MS yield of 48% was obtained (Table

5.1, Entry 3). Next, diacetoxyiodobenzene was tested as the oxidant, however, this

resulted in a lower 32% isolated yield (Table 5.1, Entry 4).

Increasing the amount of Langlois reagent to 3 equivalents further boosted

the LC-MS yield to 54% (Table 5.1, Entry 5) (45% isolated yield). Notably, in the

same reaction conditions the reaction with fac-Ir(ppy)3 gave 38% yield (Table 5.1,

Entry 6), thus confirming the choice of photocatalyst based on the luminescence

quenching studies. Increasing the reaction temperature to 60°C did not lead to a

further improvement of the reaction yield (Table 5.1, Entry 7). Control experiments

revealed the photocatalytic nature of our protocol as little to no product was

observed in the absence of either light or photocatalyst (Table 5.1, Entries 8 and

9). Finally, irradiation of the reaction mixture with a 365 nm UV lamp resulted in

46% of the target compound, which can be attributed to the UV-tailing absorption

of the iridium photocatalyst (Table 5.1, Entry 10). Nevertheless, it should be

noted that, especially for the synthesis of densely functionalized drug candidates,

5


irradiation with low-energy blue light is preferred over higher-energy ultraviolet in

order to minimize the occurrence of side reactions and compound degradation.[12]

Table 5.1: Optimization of reaction conditions for the trifluoromethylation of caffeine in continuous flowa

Entry Changes from optimized conditionsa LC-MS Yield (%)

1 20 min residence time, no oxidant 5

2 No oxidant, 1.5 eq CF3SO2Na 12

3 1 eq (NH4)2S2O8, 1.5 eq CF3SO2Na 48

4 1 eq Diacetoxyiodobenzene, 1.5eq CF3SO2Na 32b

5 none 54 (45b)

6 fac-Ir(ppy)3 as photocatalyst 38

7 60°C 50

8 No light 6

9 Light, no [IrdF[CF3(ppy)2](dtbpy)]PF6 n.r.

10 365 nm LEDs 46aReaction conditions: 0.2 mmol of caffeine 1a, 1 mol% [Ir{dF(CF3)ppy}2](dtbpy)]PF6, 3 equivalents

CF3SO2Na, 1 equivalent (NH4)2S2O8 in 2 ml DMSO (0.1M). Reactions performed in a commercially

available Vapourtec UV-150 photoreactor, irradiation with 450 nm blue LEDs and 30 min residence

time. bIsolated yield.

With the optimized reaction conditions in hand, our photocatalytic

trifluoromethylation strategy was evaluated on a wide range of heteroarenes

and arenes as well as benzofused heterocycles (Scheme 5.1). We focused our

attention specifically to halogen-bearing substrates, which are of high value

for drug discovery programs. In these programs, such functionalized substrates

are key building blocks for the very popular cross-coupling methods which

allow to construct carbon-carbon or carbon-heteroatom bonds.[13] 3-methyl and

2-methylindole derivatives 2b and 2c were obtained in satisfactory yields (62%

and 69% respectively). Notably, the presence of an iodo substituent on the

indole was well tolerated (2d; 50% isolated yield; C2:C3, 1:1). We then explored

our transformation on a series of benzimidazole derivatives. Unlike indoles,

benzimidazoles showed higher reactivity for the C4 and C6 position on the

aromatic ring.[14]

Scheme 5.1: Scope of the trifluoromethylation of heteroarenes, benzofused heterocycles and arenes. Reaction conditions: 0.5 mmol of substrate, 1 mol% [Ir{dF(CF3)ppy}2](dtbpy)]PF6, 3 equivalents CF3SO2Na, 1 equivalent (NH4)2S2O8 in 2 ml DMSO (0.1M). Reactions performed in a vapourtec UV-150 photoreactor, irradiation with 450 nm blue LEDs and 30 min residence time, isolated yields. bLC-MS yields cRegioselectivity was determined by 1H NMR analysis of the crude reaction mixture.

For example, trifluoromethyl benzimidazole 2e was obtained in 50% yield as a

separable mixture of C4:C6 (3:2) isomers as well as the 2-Br trifluoromethyl derivative

2f (52% yield; C4:C6, 3:2, separable mixture). Interestingly, 5-Cl benzimidazole

showed a different selectivity, and was trifluoromethylated at positions C4 and C7

30 min, 40°C, DMSO

1 mol % Ir photocat.3 equiv. CF3SO2Na1 equiv. (NH4)2S2O8

O NMe

NMe

N

NMe

O

O NMe

NMe

N

NMe

O

CF3

1a 2a

Vapourtec10 mL, 450 nm40°C, 30 min

X

Y

or

X

Y

1

X

Y

or

X

Y

2,3

CF3

CF3

NH

CF3NH

Me N

HNMe CF3

N

N

N

NCF3

Me

Me

MeO

O

NH

I

N

HN

Br

CF32

3

CF3

4

CF3

4

6

N

HN

CF3

2a, 45% 2c, 69%2b, 62% 2d, 50%(C2:C3 1:1)

2e, 50%(C4:C6 3:2)

2f, 52%(C4:C6 3:2)

4

7

N

NH

N

N

CF3

OMeMeO

OMe

N NH2

OMe

O

CF3

NH

O

CF3Br

NNH

F3C

NN

NH2

CF3

ON I N

N

Me

CF3OMeF3C Me

3a, 80% 3b, 60% 3c, 60%

3d, 40% 3h, 45%3g, 28%3e, 35% 3f, 53%b

(mono:di 3:1)

2g, 45%(C4:C7 2:1)

CF3

MeMe

MeN

CF3Br

CF3

MeMe

Me

I

Me

3j, 53%b3i, 38%b,c 3k, 80%b

6

Cl

CF3

Me

Me

5


(2g, 45%; C4:C7 2:1) probably due to the electronic and steric effect of the chlorine

atom. Notably, the possibility to easily separate the regioisomers obtained in

compounds 2d to 2g renders our strategy advantageous for the simultaneous

synthesis of fluorinated analogues relevant for medicinal chemistry SAR studies.

Next, we tested pyridone and pyrimidone, which are frequently used scaffolds in the

synthesis of novel active pharmaceutical ingredients (APIs).[15]Trifluoromethylated

pyrimidone 3a and Br-pyridone 3b were obtained in good to excellent yields (80%

and 60% respectively). We further investigated the reactivity of pyridine, obtaining

trifluoromethylated pyridine derivatives 3d, 3e and 3f in modest yields (40%, 35%

and 53%). Furthermore, 4-Ph pyrazole could be trifluoromethylated on the phenyl

substituent and isolated in 28% yield (3g). Conversely, the tetrahydropyran-

substituted 4-amino pyrazole derivative 3h was trifluoromethylated at position C5

on the pyrazole ring and isolated in a reasonable 45% yield. Br-dimethyl pyridine 3i

was successfully trifluoromethylated in 38% yield. Finally, we explored the scope of

our methodology with regard to unactivated arene substrates. Trifluoromethylated

mesitylene and iodo mesitylene 3j and 3k were obtained in good to excellent

yields, 53% and 80% respectively. Trifluoromethylation of unactivated arenes is a

particularly challenging transformation, often requiring long reaction times (18-24

hours), and so far rarely reported in photoredox-based protocols.[4h, 11, 16] Therefore,

we were pleased to observe significant product formation in our system within

only 30 minutes reaction time. The main reason for the remarkable acceleration of

the reaction rate observed in our protocol lies in the improved irradiation of the

reaction mixture obtained in the microflow reactor.[12, 17]

Moreover, to showcase the potential of microreactors in terms of productivity,

we performed a scale-up experiment on Br-pyridone.[17c, 18] The vapourtec UV-

150 photoreactor was continuously run for 3.5 hours without any intervention,

affording 545 mg of trifluoromethylated derivative 3b (56%).

A proposed mechanism for the trifluoromethylation reaction is depicted in

Scheme 5.2.

Despite the fact that no in depth mechanistic studies were conducted , we can

hypothesize that the first step involved in our photocatalytic trifluoromethylation

would be the reductive quenching of the excited state of [Ir{dF(CF3)ppy}2](dtbpy)]

PF6 by the Langlois reagent, thus generating the trifluoromethyl radical of interest.

This can be based on the evidence that the Langlois reagent quenches the emission

of [Ir{dF(CF3)ppy}2](dtbpy)]PF6. Subsequent trapping of the trifluoromethyl radical

by the substrate would generate the radical intermediate A that upon oxidation

with persulfate (intermediate B) and deprotonation would generate the final

product. We postulated that persulfate might be involved in the closing of the

photocatalytic cycle as well.

Scheme 5.2: Proposed catalytic cycle for the photocatalytic trifluoromethylation reaction.

CONCLUSION

In conclusion, we developed a continuous-flow trifluoromethylation strategy for

arenes, heteroarenes and benzofused heterocycles. Luminescence quenching

studies were employed to accelerate initial protocol optimization and to select

the best photocatalyst for the transformation. Easy to handle CF3SO2Na (Langlois’

reagent) was successfully used as trifluoromethylating agent. A variety of

substrates of high interest in drug discovery programs were trifluoromethylated

in good to excellent yields. Moreover, bromo, chloro and iodo containing

substrates were well tolerated, thus demonstrating the compatibility of our

methodology with cross-coupling methods. Process intensification in a microflow

reactor afforded reduced reaction times (30 minutes residence time) and a high

productivity. Therefore, we anticipate that our methodology will find application

in the late stage functionalization of pharmaceutical ingredients as well as in the

preparation of key intermediates in drug discovery programs.


General procedure for fluorescence quenching studies: All samples used in the luminescence quenching studies were prepared under oxygen free conditions. The quencher NaSO2CF3 and the photocatalysts to be screened were weighed into vials, dissolved separately in

IrIII*

Reductivequenching

cycleh

1/2 S2O8-

SO42-

CF3SO2Na

CF3+SO2

NH

NCF3H

NH

NCF3H

NH

N

NH

N

CF3-e-Deprotonation

Rearomatization

IrIII

IrII

Product

A

B

1/2 S2O8-

SO42-

+e-

-e-

5


acetonitrile and degassed by freeze-pump-thaw. For each measurement, the appropriate amount of the solution of photocatalyst and quencher were added to a cuvette and diluted to 1 mL with degassed acetonitrile. The experiments were conducted with a photocatalyst concentration of 10μM. The luminescence emission spectrum of each photocatalyst was measured multiple times (three different samples were prepared and measured each twice) and an average was taken as the standard reference spectrum. Solutions of increasing concentrations of quencher (CF3SO2Na) were prepared in acetonitrile and tested. The concentrations employed are the following: 25 mM, 50 mM, 100 mM and 300 mM. The samples containing the photocatalyst and different concentrations of quencher were each measured three times and an average was taken. The emission intensity (I) observed in presence of the quencher was compared with the one recorded for the photocatalyst alone (I0). The decrease in the intensity of the emission was then quantified as a “quenching percentage” (F) defined by the following formula:

All continuous flow experiments were carried out in a commercially available Vapourtec photoreactor UV-150 fixed on an E-series Vapourtec equipment.

General continuous-flow procedure: In an oven-dried vial equipped with a magnetic stirrer and a PTFE septum, 5.6 mg (1 mol %) of [Ir{dF(CF3)ppy}2](dtbpy)]PF6 was added to a mixture of the hetero-arene substrate (0.5 mmol, 1 equiv.), NaSO2CF3 (1.5 mmol, 3 equiv.) and (NH4)2S2O8 (0.5 mmol, 1 equiv.) in 5 ml of DMSO. The solution was pumped into the Vapourtec Photoreactor (fluoropolymer tube, 1.3 mm ID, 10 mL) and the liquid flowrate was set at 0.33 mL/min (30 minutes residence time). The reactor was irradiated with 54 blue LEDs (450 nm, total power 24W). The reaction mixture collected from the outlet was diluted with H2O and extracted with Et2O (3x). The combined organic layers were washed with brine, dried over MgSO4 and concentrated in vacuo. The crude was then pre-adsorbed onto silica, dried in vacuo and purified by flash chromatography to yield the trifluoromethylated product.


1H-Purine-2,6-dione,3,9-dihydro-1,3-dimethyl-8-(trifluoromethyl)(2a): Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethyl acetate 75/25 afforded the desired product as a white solid (59 mg, 45%. M.p. = 130.85 °C). 1H NMR (500MHz, CDCl3): δ = 4.14-4.19 (m, 3H), 3.60 (s, 3H), 3.42 ppm (s, 3H). 13C NMR (101MHz, CDCl3): δ = 155.5, 151.3, 146.5, 138.9, 119.5, 109.6, 33.2, 29.9, 28.2 ppm. 19F NMR (471MHz, CDCl3): δ = -62.37 ppm (s). HRMS (ESI) m/z calcd for C9H9F3N4O2 + [M+H]+ 263.0749, found 263.0742.

3-methyl-2-(trifluoromethyl)-1H-indole (2b); Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethyl acetate 80/20 afforded the desired product as a white solid (61.8 mg, 62%. M.p. = amorphous solid). 1H NMR (400MHz, CDCl3): δ = 8.18 (br s, 1H), 7.64 (d, J=8.1 Hz, 1H), 7.37-7.41 (m, 1H), 7.29-7.35 (m, 1H), 7.15-7.23 (m, 1H), 2.45 ppm (q, J=1.8 Hz, 3H). 13C NMR (101MHz, CDCl3): δ = 135.2, 128.1, 124.8, 124.6, 121.6, 122.2, 120.1, 114.1, 8.4 ppm. 19F NMR (471MHz, CDCl3): δ = -58.65 ppm(s). HRMS (ESI) m/z calcd for C10H8F3N + [M+H]+ 200.0680, found 200.0683

2-methyl-3-(trifluoromethyl)-1H-indole (2c): Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethyl acetate 80/20 afforded the desired product as a white solid (68,7 mg. 69%. M.p. = 147.45 °C).1H NMR (500MHz, CDCl3): δ = 8.18 (br s, 1H), 7.67 (br d, J=7.5 Hz, 1H), 7.24-7.32 (m, 1H), 7.09-7.22 (m, 2H), 2.49 ppm (s, 3H). 13C NMR (101MHz, CDCl3): δ = 133.9, 121.8, 121.3, 120.2, 119.1, 117.3, 113.7, 110.7, 99.3, 12.4 ppm. 19F NMR (471MHz, CDCl3): δ = -54.63 ppm; (s). HRMS (ESI) m/z calcd for C10H8F3N + [M+H]+ 200.0680, found 200.0686.

5-iodo-3-(trifluoromethyl)-1H-indole (2d); Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethyl acetate 90/10 afforded the desired product as a transparent oil (39 mg, 25%). 1H NMR (400MHz, CDCl3): δ = 8.47 (br s, 1H), 8.10 (s, 1H), 7.56 (dd, J=8.6, 1.6 Hz, 1H), 7.51 (dd, J=2.7, 1.3 Hz, 1H), 7.23 ppm (s, 1H). 13C NMR (101MHz, CDCl3): δ = 138.0, 134.9, 132.2, 128.4, 125.1, 125.0, 124.2, 113.5, 85.2 ppm. 19F NMR (471MHz, CDCl3): δ = -57.37 ppm(s). HRMS (ESI) m/z calcd for C9H5F3IN [M]- 309.9345, found 309.9368.

5-iodo-2-(trifluoromethyl)-1H-indole (2d): Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethylacetate 80/20 afforded the desired product as a transparent oil (39 mg, 25%). 1H NMR (500MHz, CDCl3): δ = 8.52 (br s, 1H), 8.03 (s, 1H), 7.58 (d, J=10.4 Hz, 1H), 7.22 (d, J=8.4 Hz, 1H), 6.85 ppm (s, 1H). 13C NMR (101MHz, CDCl3): δ = 135.2, 133.2, 130.9, 129.1, 126.7, 126.4, 121.9, 113.7, 103.4, 84.5 ppm. 19F NMR (471MHz, CDCl3): δ = -60.73 ppm(s). HRMS (ESI) m/z calcd for C9H5F3IN [M]- 309.9345, found 309.9368.

4-(trifluoromethyl)benzimidazole (2e, C4:C6 3:2): Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethylacetate 80/20 afforded the desired product as a white solid (27 mg, 29% M.p. = amorphous solid). 1H NMR (500MHz, CD3OD): δ = 8.20 (s, 1H), 7.63-7.88 (br s, 1H), 7.48 (br d, J=7.2 Hz, 1H), 7.31 ppm (t, J=7.8 Hz, 1H). 13C NMR (126MHz, CD3OD): δ = 164.1, 143.0, 133.2, 125.2, 123.1, 100.0, 99.8 ppm. 19F NMR (471MHz, CD3OD): δ = -64.21 ppm(s). HRMS (ESI) m/z calcd for C8H5F3N2 [M]- 185.0331, found 185.0337.

𝐹𝐹𝐹𝐹(%) = 100 �1 −II0�%

5


6-(trifluoromethyl)benzimidazole (2e C4:C6 3:2): Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethylacetate 80/20 afforded the desired product as a yellow oil (19 mg, 20%). 1H NMR (400MHz, CDCl3): δ = 8.25 (s, 1H), 7.99 (s, 1H), 7.74 (d, J=8.6 Hz, 1H), 7.57 ppm (d, J=8.6 Hz, 1H). 13C NMR (101MHz, CDCl3): δ = 142.6, 128.7, 126.0, 125.7, 125.4, 123.3, 120.1 ppm. 19F NMR (471MHz, CDCl3): δ = -62.20 ppm(s). HRMS (ESI) m/z calcd for C8H5F3N2 [M]- 185.0331, found 185.0337.

2-bromo-4-(trifluoromethyl)benzimidazole (2f C4:C6 3:2): Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethylacetate in gradient from 100/0 to 50/50 afforded the desired product as a white solid (40.2 mg, 30%. M.p. = 214.7 °C). 1H NMR (400MHz, CD3OD): δ = 7.85 (s, 1H), 7.68 (d, J=8.6 Hz, 1H), 7.55 ppm (d, J=8.6 Hz, 1H). 13C NMR (101MHz, CD3OD): δ = 140.8, 131.2, 130.1, 126.2, 122.2, 120.8, 115.7, 113.7 ppm. 19F NMR (471MHz, CD3OD): δ = -62.40 ppm (s). HRMS (ESI) m/z calcd for C8H4BrF3N2 [M]- 262.9436, found 262.9434.

2-bromo-6-(trifluoromethyl)benzimidazole (2f C4:C6 3:2): Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethylacetate in gradient from 100/0 to 50/50 afforded the desired product as a white solid (26.7 mg, 20%. M.p. = 214.7 °C). 1H NMR (400MHz, CDCl3): δ = 8.25 (s, 1H), 7.99 (s, 1H), 7.74 (d, J=8.6 Hz, 1H), 7.57 ppm (d, J=8.6 Hz, 1H). 13C NMR (101MHz, CDCl3): δ = 142.6, 128.7, 126.0, 125.7, 125.4, 123.3, 120.1 ppm. 19F NMR (471MHz, CDCl3): δ = -62.20 ppm(s). HRMS (ESI) m/z calcd for C8H4BrF3N2 [M]- 262.9436, found 262.9434.

5-chloro-2-methyl-7-(trifluoromethyl)-1H-benzimidazol (2g C4:C7 2:1): Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethylacetate 80/20 afforded the desired product as a white solid (23.8 mg, 20%.. M.p. = amorphous solid). 1H NMR (400MHz, CDCl3): δ = 9.48 (br s, 1H), 7.82 (br s, 1H), 7.47 (s, 1H), 2.68 ppm (s, 3H). 13C NMR (101MHz, CD3OD): δ = 157.3, 128.1, 126.1, 123.4, 120.7, 105.2, 14.3 ppm. 19F NMR (471MHz, CD3OD): δ = -62.87 ppm(s). HRMS (ESI) m/z calcd for C9H6ClF3N2 [M]- 233.0098, found 233.0108.

5-chloro-2-methyl-4-(trifluoromethyl)-1H-benzimidazol (2g C4:C7 2:1): Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethylacetate 80/20 afforded the desired product as a white solid (35.2 mg, 30%. M.p. = amorphous solid). 1H NMR (500MHz, CDCl3): δ = 9.50 (br s, 1H), 7.74 (d, J=8.4 Hz, 1H), 7.34 (d, J=8.4 Hz, 1H), 2.66 ppm (s, 3H). 13C NMR (126MHz, CDCl3): δ = 153.3, 143.5, 131.9, 127.0, 125.1, 122.9, 15.3 ppm. 19F NMR (471MHz, CDCl3): δ = -57.09 ppm(s). HRMS (ESI) m/z calcd for C9H6ClF3N2 [M]- 233.0098, found 233.0108.

5-(trifluoromethyl)pyrimidin-4(3H)-on (3a): Product prepared according to the general procedure, the solvent was evaporated in Genevac turboevaporator in overnight, the rests of the reaction mixture were washed with dichloromethane and the solvent was evaporated, purification in flash chromatography on silica gel MeOH/NH4OH( 9:1)/Dichloromethane 0-10% the desired product as a yellow oil (65.9 mg, 80%). 1H NMR (400MHz, DMSO-d6): δ = 8.44 (s, 1H), 8.40 ppm (s, 1H). 13C NMR (126MHz, CDCl3): δ = 154.7, 152.1, 129.7, 121.9 ppm 19F NMR (471MHz, CDCl3): δ = -65.35 ppm(s). HRMS (ESI) m/z calcd for C5H3F3N2O [M]- 163.0124, found 163.0129.

5-bromo-3-(trifluoromethyl)-1H-pyridin-2-one (3b): Product prepared according the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethylacetate 50/50 afforded the desired product as a yellow solid (72.6 mg 60%. M.p. = 213.39ºC). 1H NMR (500MHz, CDCl3): δ = 12.52-14.24 (m, 1H), 7.92 (d, J=2.0 Hz, 1H), 7.76 ppm (d, J=2.3 Hz, 1H). 13C NMR (126MHz, CDCl3): δ = 160.0, 143.8, 139.4, 121.5, 121.9, 97.7 ppm. 19F NMR (471MHz, CDCl3): δ = -65.98 ppm. HRMS (ESI) m/z calcd for C6H3BrF3NO [M]- 239.9277, found 239.9272.

2,4,6-trimethoxy-5-(trifluoromethyl)pyrimidine (3c): Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethylacetate 80/20 afforded the desired product as a pink solid (71.4 mg, 60%. M.p. = 123.6ºC). 1H NMR (500MHz, CDCl3): δ = 4.03 (s, 6H), 4.01 ppm (s, 3H). 13C NMR (101MHz, CDCl3): δ = 169.8, 165.0, 123.5, 89.3, 55.1, 55.0 ppm. 19F NMR (471MHz, CDCl3): δ = -55.97 ppm(s).

4-methoxy-3-(trifluoromethyl)pyridin-2-amine (3d): Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethylacetate 40/60 afforded the desired product as a yellow solid (38.5 mg, 40%. M.p. = 213.4ºC). 1H NMR (400MHz, CDCl3): δ = 8.07 (d, J=5.8 Hz, 1H), 6.31 (dd, J=5.9, 0.8 Hz, 1H), 5.00-5.20 (m, 2H), 3.88 ppm (s, 3H). 13C NMR (101MHz, CDCl3): δ = 166.3, 156.6, 152.8, 124.9, 98.3, 56.1 ppm. 19F NMR (471MHz, CDCl3): δ = -55.42 ppm(s). HRMS (ESI) m/z calcd for C7H7F3N2O [M]- 191.0437, found 191.0423.

2-iodo-3-methoxy-5-(trifluoromethyl)pyridine (3e):Product prepared according the general procedure, purification in flash chromatography on silica gel using as eluent pentane/diethyl ether 90/10 afforded the desired product as a transparent oil (52.9 mg, 35%). 1H NMR (400MHz, CDCl3): δ = 7.59 (d, J=8.6 Hz, 1H), 7.05 (d, J=8.6 Hz, 1H), 3.98 ppm (s, 3H). 13C NMR (101MHz, CDCl3): δ = 157.4, 140.4, 134.2, 121.0, 116.1, 111.7, 56.7 ppm. 19F NMR (471MHz, CDCl3): δ = -66.70 ppm. HRMS (ESI) m/z calcd for C7H5F3INO [M+H]+ 303.9488, found 303.9492.

5


3-[4-(trifluoromethyl)phenyl]-1H-pyrazole (3g): Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethylacetate 40/60 afforded the desired product as transparent oil (29.7 mg, 28%). 1H NMR (400MHz, CDCl3): δ = 7.90 (d, J=8.1 Hz, 2H), 7.65-7.70 (m, 1H), 7.62-7.74 (m, 2H), 6.70 ppm (d, J=2.3 Hz, 1H). 13C NMR (101MHz, CDCl3): δ = 136.0, 131.8, 129.5, 129.0, 128.8, 126.0, 125.7, 124.6, 122.6, 103.3 ppm. 19F NMR (471MHz, CDCl3): δ = -62.58 ppm(s). HRMS (ESI) m/z calcd for C10H7F3N2 [M]- 211.0488, found 211.0486.

1-tetrahydropyran-4-yl-5-(trifluoromethyl)pyrazol-4-amine (3h): Product prepared according to the general procedure, purification in flash chromatography on silica gel using as eluent heptane/ethylacetate 50/50 afforded the desired product as a dark yellow oil (52.9 mg, 45%). 1H NMR (400MHz, CDCl3): δ = 7.16 (s, 1H), 4.24 (tt, J=11.5, 4.0 Hz, 1H), 4.10 (dd, J=11.8, 4.6 Hz, 2H), 3.50 (td, J=12.2, 2.0 Hz, 2H), 3.28-3.42 (m, 2H), 2.26 (qd, J=12.4, 4.6 Hz, 2H), 1.78-1.92 ppm (m, 2H). 13C NMR (101MHz, CDCl3): δ = 130.8, 130.0, 121.8, 114.9, 67.1, 57.0, 32.9 ppm. 19F NMR (471MHz, CDCl3): δ = -56.70 ppm(s). HRMS (ESI) m/z calcd for C9H12F3N3O [M]- 234.2859, found 234.2855.

3-bromo-2,6-dimethyl-5-(trifluoromethyl)pyridine (3i); Product prepared according the general procedure. The organic layer was evaporated and the crude was analysed by LC-MS (38%). 1H NMR (400MHz, CDCl3): δ = 7.97 (s, 1H), 2.68 (s, 3H), 2.62-2.64 ppm (m, 3H). 19F NMR (471MHz, CDCl3): δ = -62.23 ppm.

1,3,5-trimethyl-2-(trifluoromethyl)benzene (3j); Product prepared according the general procedure. The organic layer was evaporated and the crude was analysed by LC-MS (53%).

2-iodo-1,3,5-trimethyl-4-(trifluoromethyl)benzene (3k); Product prepared according the general procedure. The organic layer was evaporated and the crude was analysed by LC-MS (80%).

ASSOCIATED CONTENT:

The Supporting Information for this article is available free of charge on the Thieme publication website at DOI: 10.1055/s-0036-1588527.

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132 Chapter 5

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CHAPTER 6

De novo design of organic photocatalysts: bithiophene derivatives for the visible-light induced C-H functionalization of heteroarenes


Bottecchia, C.; Martin, R.; Abdiaj, I.; Crovini, E.; Alcazar, J.; Orduna, J.; Blesa, M. J.; Muñoz, Carillo J.R.; Prieto, P.; Noël, T., Adv. Synth. Catal. 2018, DOI: 10.1002/adsc.201801571

6

136 Chapter 6 De novo design of organic photocatalysts 137

ABSTRACT

In this chapter, the de novo synthesis and characterization of a series of substituted

bis-thiophene derivatives as novel and inexpensive organic photocatalysts

is described. DFT calculations were used to predict a priori their absorption

spectra and redox potentials, which were then confirmed with empirical data.

The photocatalytic activity of this novel class of organic photoredox catalyst was

demonstrated in two visible-light mediated strategies for the C-H functionalization

of heteroarenes. The implementation of these strategies in a continuous-flow

photo-microreactor afforded moderate to excellent yields within few minutes

of reaction time. Due to their straightforward synthesis, low cost and good

photocatalytic properties, the proposed bis-thiophene derivatives could represent

a novel class of organic photoredox catalysts.

Visible-light photoredox catalysis received increasing attention in the last decade as

a powerful strategy enabling novel transformations under mild reaction conditions

(i.e. room temperature, atmospheric pressure and visible-light irradiation).[1] In

photoredox catalysis, the ability of a photocatalyst to absorb light in the visible

light range, reach an excited state and then undergo either energy or electron

transfers with the substrates of interest is paramount.[2] Generally speaking, Ir and

Ru polypyridyl complexes are among the most employed photocatalyst, offering

excellent redox potentials in their excited states and long-lived exciton lifetimes.[3] However, the main drawbacks associated with their use are their prohibitive

costs and scarce availability.[4] More recently, a number of studies demonstrated

that organic photocatalysts often provide valuable and inexpensive alternatives to

transition metals.[4-5] Nevertheless, the choice of a suitable organic photocatalyst

might be limited for reactions proceeding in acidic medium (e.g. Eosin Y is pH

sensitive) or in reducing environments promoting photobleaching.[6]

In this light, the development of novel classes of organic photoredox catalysts with

good redox properties, and largely available or easily prepared in high yields would

contribute to further advance research in photoredox catalysis.[7] Due to their

functional properties and versatility, deriving from the ease of functionalization of

the ring as well as the lengthening of the chain, thiophene based materials are of

broad interest across multiple disciplines. For example, in organic electronics their

semiconductor properties are exploited in organic field-effect transistors (OFETs)

and in photovoltaic devices.[8]

However, examples showing their use in the field of photoredox catalysis remain

scarce.[9] Thus, we became interested in probing the photocatalytic properties

of a series of 2,2’-bithiophene-based derivatives decorated with different

electron donating and electron withdrawing groups. In order to rapidly evaluate

the suitability of said structures as photoredox catalysts, we reasoned that

computational studies might represent a useful tool to predict their properties

a priori. Specifically, through TD-DFT[10] calculations (M06-2X[11], using CPCM[12],

CHCl3, see experimental section for further details), the absorption maximum and

emission maximum of this class of compounds were calculated, together with their

redox potentials. Based on this theoretical information, and taking into account

the commercial availability of the required starting materials, we selected a cluster

of six molecules to be synthesized (Scheme 6.1). Photocatalysts A to F were all

obtained in good to excellent yields through a one-step double Sonogashira cross-

coupling, starting from inexpensive and readily available alkynyl precursors (see

general procedure 1 in the experimental section).[13] The optimized procedure

6


was performed with minimal amount of solvent, under microwave irradiation and

employing recycled Pd catalyst.

Gratifyingly, the calculated theoretical values for the maximum absorption were

found to be in good agreement with the experimental measurements (Scheme 6.1

and experimental section). The UV-Visible spectra of all of the studied compounds

showed a unique absorption band in the 400 nm region with significant tailing

towards the blue light region, thus supporting our initial hypothesis that these

compounds might be useful in visible-light driven catalytic reactions.

Scheme 6.1: Overview of the six 2,2’-bisthiophene-based photocatalyst synthesized and comparison of their calculated vs experimental properties. All potential values are reported vs SCE (saturated calomel electrode). Calculated values for Absmax, Emmax and redox potentials for the proposed compounds were obtained through DFT calculations in chloroform solution using the conductor-like polarizable continuum model (CPCM)[1] at CPCM-M06-2X/6-311+G(2d,p)//CPCM-M06-2X/6-31G* in chloroform. These values were obtained by our collaborators in UCLM. For more details see experimental section. The excited state potentials were estimated

The redox properties of the reported bithiophenes were followed by Cyclic

Voltammetry (CV) and Differential Pulse Voltammetry (DPV) in CHCl3, and matched

closely the calculated theoretical values (Scheme 6.1). As expected, compounds

A, B and C, bearing electron-donating groups, showed lower oxidation potential

than compounds D and E, bearing electron-withdrawing groups. Moreover, a good

correlation between the experimental oxidation potentials and the Hammett

constant values for the substituents on the arene ring was found (see Figure 6.9

experimental section).

Notably, such correlation hints at the possibility to carefully design other members

of this photocatalytic series with increasing redox potential by further modulating

the nature and position of the substituents on the aryl ring. For all members of

the series, the theoretical reduction potentials were calculated to be higher than

-2V, and thus could not be confirmed experimentally due to the limited potential

window of CHCl3.[14] By combining the information derived from the absorption

spectra and the oxidation/reduction potentials, an approximation of the excited

state potentials for compounds A to F was made (Scheme 6.1), which appeared

in good agreement with the theoretical calculations. Notably, all compounds

of the series showed excited state potentials comparable to commonly used

photocatalysts (see also Table 6.3 in the experimental section).[3-4]

To test the activity of the bi-thiophene derivatives as photoredox catalysts, we

commenced our investigations by employing them in the C-H functionalization of

heteroarenes with malonate derivatives.[15] The same transformation was previously

reported to proceed with either Ru(bpy)32+ or with an organic photocatalyst, and

was therefore selected as benchmark reaction.[9, 15] As depicted in Table 6.1, we

initially tested the ability of our photocatalysts to catalyse the reaction between

3-Me indole (1a) and Br-malonate in presence of triphenylamine as sacrificial

electron donor. Subjected to blue LED irradiation, 87% of the desired alkylated

indole (2a) was obtained within 5 hours with 1 mol% of photocatalyst D (Table 6.1,

entry 1). Gratifyingly, our photocatalyst outperformed Ru(bpy)32+(Table 6.1 entry

3), and compared positively with the benzothiadiazole-based organic photocatalyst

Th-BT-Th (Table 6.1, entry 2, reference [9]). In order to better match the irradiation

wavelength with the photocatalyst maximum absorption, we then conducted the

reaction under irradiation with 400 nm LEDs, obtaining 75% of product within only

30 minutes. (Table 6.1, entry 4). Higher concentrations of photocatalyst did not

result in increased amounts of product (Table 6.1 entry 5 and 6).

S

S

R

R

A:

OMeMeO

MeO

2,2'-bithiophene-basedorganic photocatalysts

F:D: E:

NC

C:

MeO

B:

OMe

MeO

S

CF3

Calc. Absmax

Exp. Absmax

C

401 nm

393 nm

A B E FD

Calc. E1/2ox

Exp. E1/2ox 1.08 V

1.25 V

399 nm

388 nm

1.22 V

1.26 V

407 nm

390 nm

1.02 V

1.03 V

401 nm

390 nm

1.34 V

1.28 V

410 nm

398 nm

1.34 V

1.51 V

417 nm

400 nm

1.33 V

1.26 V

Calc. E1/2red -2.22 V -2.18 V -2.24 V -2.0 V -1.90 V -2.05 V

E*1/2 red=PC*/PC 0.57 V 0.68 V 0.74 V 0.78 V 0.58 V

-1.49 V -1.38 V -1.46 V -1.17 V -1.37 VE*1/2 ox=PC /PC* -1.49 V

0.53 V

6


Table 6.1: Optimization of reaction conditions for the C-H functionalization of 3-Me indole with Br-malonate

Entry Changes from initial reaction conditionsa GC-FID Yield (%)b

1 none 87

2 1 mol% Th-BT-Th as photocatalystc 79

3 1 mol% Ru(bpy)3Cl2•6H2O as photocatalyst 41

4 400 nm LEDs, 30 min reaction time 75

5 2 mol% D, 400 nm LEDs, 30 min reaction time 82

6 5 mol% D, 400 nm LEDs, 30 min reaction time 70

7 No light, no photocatalyst 0

8 Light (either 465 or 400 nm), no photocatalyst 0

9 Continuous flow, 400 nm LEDs, 7 min reaction timee 81 (70d)aReaction conditions: 0.4 mmol 3-Me indole (1a, 1 eq), Br-malonate (2 equiv), Ph3N (1.5 equiv), 1.0

mol% photocatalyst D in 2.5 ml DMF (0.16 M), irradiation with 465 nm LEDs, 5 hours reaction time. bYield determined by GC-FID with decafluorobiphenyl as internal standard. cbenzothiadiazole-based

photocatalyst, see ref.[9] for more information and for the structure. dIsolated yield. eFor detailed flow

conditions, see experimental section.

Control experiments revealed that both the presence of light and of the

photocatalyst were essential to achieve product formation (Table 1 entry 7, and

8). Finally, in order to overcome the limitations associated with photochemical

reactions performed in batch reactors (i.e. limited light penetration, sub-optimal

mixing), we implemented the use of a continuous flow capillary microreactor

(I.D. 760 μm, 2.5 mL reactor volume).[16] Owing to the efficient irradiation of the

reaction mixture achievable in microflow reactors, a good isolated yield of 2a

(70%) was obtained within only 7 minutes of residence time (Table 6.1, entry 9). All

other members of the series were tested under identical conditions and resulted

in similar or inferior yields, (Table 6.2).

With optimized conditions in hand, we then explored the scope of this

transformation (Scheme 6.2). A series of heteroaromatic compounds were tested:

both N-protected and unprotected indoles underwent the reaction in good to

excellent yields (2a-b). The functional group tolerance towards halides (2c) and

esters (2d) was demonstrated. The reaction proceeded with good yield also on

a protected tryptophan moiety (2f), thus suggesting a possible application of

this methodology in the context of amino acid modification and, provided that

selectivity towards tryptophan can be achieved, in bioconjugation methods.[17]

3-Me benzofuran was also successfully alkylated (2e).

Table 6.2: Alkylation of 3-Me Indole with Br-malonate performed with all different photocatalysts or with different catalyst loadings of D.

Entry Photocatalyst (1 mol% unless specified) Residence time GC-FID Yield (%)b

1 A 7 minutes 72

2 B 7 minutes 74

3 C 10 minutes 81

4 D 7 minutes 81 (70)c

5 E 7 minutes 40

6 F 7 minutes 78

7 D, 0.5 mol% 7 minutes 72c

8 D, 2 mol% 7 minutes 82 9 D, 5 mol% 7 minutes 70Reaction conditions: 0.4 mmol 3-Me indole (1 eq), Br-malonate (2 equiv), Ph3N (2.0 equiv), photocatalyst

(A-F) in 2.5 ml DMF (0.16 M), irradiation with 400 nm LEDs, 7 to 10 minutes residence time. bYield

determined by GC-FID with decafluorobiphenyl as internal standard. For this substrate, no other

side-products were observed in the GC-FID run, therefore the yield also represents the conversion of

starting material. cIsolated yield.

To further demonstrate the utility of this transformation, a series of 5-membered

heterocycles were tested: pyrroles (3a, 3b), thiophene (3c) and furans (3d, 3e)

were all found to be competent substrates, yielding the corresponding alkylated

products in moderate to excellent yields.

Encouraged by the positive results obtained for the C-H functionalization of

heteroarenes with malonate derivatives, we turned our attention to the possibility

of employing our organic photocatalysts for the C-H trifluoromethylation

and difluoroalkylation of heteroarenes. Due to the importance of fluorinated

moieties in structure-activity relationship (SAR) studies, novel methodologies for

the introduction of fluorine atoms in drug candidates are in high demand in the

pharmaceutical industry.[18]

1a 2a

photocatalyst (1 mol%),Ph3N, Br-malonate

DMFRT, 5 h

NH

Me

NH

MeCOOEt

COOEt

or

1a 2a

NH

Me

NH

MeCOOEt

COOEtDMF, rt

1mol% PCPh3N, Br-malonate

6


Scheme 6.2: Scope of the C-H functionalization of heteroarenes with malonate derivatives. Reaction conditions: 0.4 mmol of substrate, 1 mol% D, 1.5 equiv. of Ph3N, 2 equiv. Br-malonate in 2.5 ml DMF (0.16 M). Reactions performed in a capillary microflow reactor, irradiation with 400 nm LEDs, with different residence times depending on the substrate, isolated yields b Amount of di-substituted product obtained.

In this context, mild methodologies for the late-stage trifluoromethylation of

potential drug candidates occupy a prominent role. Several photoredox-based

trifluoromethylation methodologies have been reported in the last years,

relying mostly on the use of expensive transition metal-based photoredox

catalyst.[19] We commenced our attempts toward the trifluoromethylation of

heteroarenes in continuous flow by employing photocatalyst D, gaseous and

inexpensive trifluoromethyl iodide as trifluoromethylating agent and N,N,N′,N′-Tetramethylethane-1,2-diamine (TMEDA) as sacrificial electron donor.[20]

Notably, the formation of a gas-liquid slug flow regime in our continuous system

resulted in improved mixing, while granting an efficient and homogeneous

irradiation of the reaction mixture and affording a safe handling of the gas

reactant.[16] The results obtained for the trifluoromethylation of a diverse set of

heteroarenes are summarized in Scheme 6.3. Both trifluoromethylated indole (4a)

Scheme 6.3: Scope of the trifluoromethylation and difluoroalkylation of heteroarenes. Reaction conditions: 0.5 mmol of substrate, 1 mol% D, 3 equiv. TMEDA, 4 equiv. CF3I/ 2 equiv. BrCF2COOEt in 5 ml DMF (0.1M). Reactions performed in a Vapourtec UV-150 photoreactor, irradiation with 450 nm blue LEDs and 20 min residence time, isolated yields. bLC-MS yield. For a detailed procedure for compounds 5f, 5g and 5h, see experimental section.

and imidazo[1,2-a]pyridine (4b) derivatives were obtained in synthetically useful

yields, while the trifluoromethylated pyrrole derivative 4c was obtained with a

56% yield. Pyridine derivatives 4d and 4e were isolated in modest yields. Next, the

applicability of our methodology to other nitrogen containing heterocycles was

demonstrated with compounds 4g and 4h. Finally, the aniline derivative 4f was

obtained in 54% yields as a separable mixture of ortho- and para- regioisomers.

DMF, rtX

Y

or

2,3

1mol% DPh3N, Br-malonate

X X

Yor

X

NH

Me

2a, 70% 2b, 97% 2c, 39%2d, 79%

2e, 79% 3a, 56% (26%b) 3b, 90%

3d, 46%3c, 33%

COOEt

COOEt

N COOEt

COOEt

Me

N COOEt

COOEt

Me

Br

NMe

COOMe

EtOOCCOOEt

O

Me

COOEt

COOEt

2f, 55%

NH

COOEt

COOEt

COOMe

BocHN

NCOOEt

COOEtMeN

COOEt

COOEtMe

OHC

OCOOEt

COOEtS

COOEt

COOEtMe

3e, 75%

OCOOEt

COOEtMe

COOEt

COOEt

COOEt

COOEt

DMF, rtX

Y

or

4,5

NH

CF3

4a, 51% 4c, 56%b4b, 29%

4f, 54%o-/p- 1:5

4d, 26% 4e, 30%,C3:C5 1.5:1

1mol% D, TMEDA,CF3I or BrCF2COOEt

X

X

Me N

N

CF3

NH

CF3

COMe

NEt

Et

CF3po

N

CF3

NH2

N

CF3

OMe

NH2

35

4g, 26%b

NH

CF3

O

Br

4h, 27%

NN

NH2

CF3

O

Trifluoromethylation:

Difluoroalkylation:

NH

5a, 48% 5c, 49%

CF2COOEtN

N

CF2COOEt

5e, 22%

NH

C4F9

O

Br

Me

5d, 50%

NH

CF2COOEt

O

Br

NH

F F

OiPr

5f, 27%

NH

F F

OMeO

5g, 43%

NH

F F

OMe

5h, 38%

NH

5b, 43%

CF2COOEt

Me

X

Y

or X

X

CF3CF3

CF3

6


In order to further demonstrate the reactivity of the develop photocatalysts, we

then focused on the introduction of a difluoroester moiety (Scheme 6.3), which is

a precursor of high interest in the preparation of radio-labeled 18F compounds for

positron-emission tomography (PET) scan.[21],[22]

By employing Br-CF2COOEt as fluorinating agent, the corresponding indoles (5a, 5b)

and imidazo[1,2-a]pyridine (5c) derivatives were obtained in good yields. Similarly,

the Br-pyridone derivative 5d was obtained in 50% yield, while the corresponding

perfluoroalkylated derivative 5e, obtained via the use of IC4F9 as fluorinating

agent, was isolated in a 22% yield. Interestingly, when aniline derivatives were

employed for this transformation a one-pot tandem difluoroalkylation/amidation

was observed, resulting in the formation of difluorooxindoles derivatives 5f-h.[23]

Fluorescence quenching studies on the reactions studied demonstrated that both

reductive and oxidative single electron transfers (SET) are viable pathways for

the excited state of photocatalyst D. Such versatility in terms of redox properties

further highlights the potential of this novel photocatalyst series in a broad range

of photocatalytic reactions. To conduct these studies, the automated set-up

described in Chapter 7 was used.[24]

For the reaction with Br-malonate fluorescence quenching studies to assess

which reaction component can quench the excited state of photocatalyst D were

conducted. The results obtained are summarized in Figure 6.1.

Figure 6.1: Results obtained for the fluorescence quenching studies for the alkylation of 3-Me indole with Br-malonate

As Br-malonate was found to be the only quencher of the excited state of D, we

conducted a Stern-Volmer analysis to demonstrate the emission intensity of D

decreases linearly with an increase in concentration of Br-malonate (Figure 6.2).

Thus, in accordance with these results an oxidative quenching cycle in which the

first step is the generation of the malonate radical is proposed (). This hypothesis

would also be consistent with the calculated excited state potentials of catalyst D

(E*red = 0.74V, E*

ox = -1.46V vs SCE) and of the other reaction components (Ered Br-

malonate = -1V, Eox Ph3N = 0.89V, both vs SCE).[9] In other words, the excited state

reduction potential of D is not sufficient to abstract one electron from Ph3N, but

the excited state oxidation potential of D seems sufficient to donate an electron

to Br-malonate (-1.46 V vs -1V), thus generating the desired radical specie.

Figure 6.2: Stern-Volmer plot on the quenching of photocatalyst D with Br-malonate.

For the difluoroalkylation/trifluoromethylation reaction, mechanistic proposals

resulted more complex. Fluorescence quenching studies revealed that TMEDA,

CF3I and CF2COOEt can all quench the excited state of photocatalyst D (Figure 6.3,

and Figure 6.4 to 6.6 for the Stern-Volmer plots).

This means that both a reductive quenching cycle in which the excited state of

the photocatalyst is quenched by TMEDA (Eox ~ 0.8V vs SCE), and an oxidative

quenching cycle in which either CF3I (Ered= -1.22V vs SCE) or BrCF2COOEt (Ered=

-0.57V vs SCE) are reduced by the excited photocatalyst could be viable (Scheme

6.5). The results of the fluorescence quenching studies are in-line with the redox

A1 2 3 4

A BFluorescence quenching studies

Screening resultsList of tested quenchers

A1 : BlankA2: TriphenylamineA3: Br-malonateA4: 3-Me Indole

6


potentials of photocatalyst D and of the other reaction components. Moreover,

performing the reaction in presence of a radical trap (i.e. TEMPO) resulted in no

product formation, supporting the fact that the reaction proceeds via radical

intermediates.

Scheme 6.4: Proposed mechanism for the alkylation of heteroarenes with Br-malonate. After oxidative quenching of the excited state of catalyst D, the malonyl radical is trapped by 3-Me indole to generate intermediate A. closing of the catalytic cycle can then occur with A) intermediate A or B) with triphenyl amine.

Figure 6.3: Results obtained for the fluorescence quenching studies for the difluoroalkylation\trifluoromethylation of 3-Me indole with BrCF2COOEt or CF3I.

Thus, both a reductive and oxidative quenching cycle might be involved in the

reaction mechanism.

Figure 6.4: Stern-Volmer plot on the quenching of photocatalyst D with TMEDA.

D

D*


Supported byautomated quenching

experiments

- e-

Proposed mechanism for the Alkylation of heteroarenes with Br-malonate

Intermediate A

Intermediate A

COOEtBr

COOEt

COOEt

COOEt

NH

Me

NH

MeCOOEt

COOEt

NH

MeCOOEt

COOEt

Product

NH

MeCOOEt

COOEt

D



experiments

-1e-,- H+

Rearomatization

Intermediate A

COOEtBr

COOEt

COOEt

COOEt

NH

Me

NH

MeCOOEt

COOEtNH

MeCOOEt

COOEt

Product

Ph3N

Ph3N

A)

B)

D

D

+ e-

+ e-

- e-

D*

- H+Rearomatization

A1 2 3 4

A BFluorescence quenching studies

Screening resultsList of tested quenchers

A1 : BlankA2: 3-Me IndoleA3: TMEDAA4: CF3IA4: BrCF2COOEt

5

6


Figure 6.5: Stern-Volmer plot on the quenching of photocatalyst D with CF3I.

Figure 6.6: Stern-Volmer plot on the quenching of photocatalyst D with BrCF2COOEt.

Scheme 6.5: Possible mechanistic cycles involved in the difluoroalkylation/trifluoromethylation of heteroarenes with photocatalyst D.

In conclusion, we reported the rational de novo synthesis of a novel class of

2,2’-bisthiophene-based organic photocatalysts. DFT calculations were used

to predict a priori the spectroscopic properties and the redox potentials of an

array of molecules, of which six were then synthesized and characterized. The

experimental data were found to be in good agreement with the theoretical values.

The activity of this class of molecules as photocatalyst was then showcased in the

D*

REDUCTIVEQUENCHING CYCLE


experiments

+ e-

-1e-,- H+

Rearomatization NH

NH

CF3

or

CF3I or BrCF2COOEt

CF3 or CF2COOEt

Proposed mechanisms for difluoroalkylation/ trifluoromethylation of heteroarenes

TMEDA

TMEDA

D

D

or

MeMe

NH

CF2COOEt

Me

NH

CF2COOEt

Me

NH

CF3

Me



experiments

- e-

-1e-,- H+

Rearomatization NH

Me

NH

Me

NH

Me

CF3

TMEDA

TMEDA

B)

D

CF3I or BrCF2COOEt

CF3 or CF2COOEt

CF3

+ e-

- e-

A)

NH

Me

NH

Me

CF2COOEt CF2COOEt

or or

D*

D

6


C-H functionalization of heteroarenes with malonate derivatives. The activity of

the tested compounds was comparable with that of other commonly used organic

or transition-metal photocatalysts (i.e. Th-BT-Th and Ru(bpy)32+), and a series of

derivatives was obtained in good to excellent yields. Process intensification in a

microflow photoreactor afforded increased yields and reduced reaction times.

Moreover, the implementation of our photocatalyst for the trifluoromethylation

and difluoroalkylation of heteroarenes and benzofused heteroarenes was also

demonstrated. Mechanistic insight via fluorescence quenching studies and Stern-

Volmer analysis indicated that these photocatalyst can undergo both reductive and

oxidative quenching pathways. Due to their inexpensive nature, good absorption

in the 400 nm range and their favourable redox potentials, we believe that this

novel class of 2,2’-bithiophene-based organic photocatalyst could find interesting

applications in photoredox catalysis.


General procedure for the synthesis of photocatalysts A-F (GP1)

A mixture of 5,5’-dibromo-2,2’-bithiophene (0.100 g, 0.31 mmol), the corresponding acetylene derivative (0.77 mmol), DBU (0.094 g, 0.62 mmol), CuI (0.0017 g, 0.015 mmol) and Pd-EncatTM TPP30 (0.026 g, 0.035 mmol) was charged under argon to a dried microwave vessel. MeCN (1 mL) was added. The vessel was closed and irradiated at 140 °C for 25 min. The crude reaction product was purified by chromatography, eluting with hexane/ethyl acetate to give analytically pure photocatalysts A to F.

General procedure for the C-H functionalization of heteroarenes with malonate derivatives in continuous flow (GP2)

In an oven-dried vial equipped with a magnetic stirrer and a PTFE septum, 2.0 mg (1 mol %) of RA48 was added to a mixture of the hetero-arene substrate (0.4 mmol, 1 equiv.), Br-malonate (0.8 mmol, 2 equiv.) in 1.25 ml of DMF. In another vial, Ph3N (0.6 mmol, 1.5 equiv) was dissolved in 1.25 ml of DMF. The two solution were merged with a T-mixer prior entering the microcapillary flow reactor (PFA tubing, 0.76 mm ID, 2.5 mL reactor volume). The reactor was irradiated with 400 nm purple LEDs. For the residence time of every substrate we refer to the compound characterization in SI. The reaction mixture collected from the outlet was diluted with H2O and extracted with Et2O (2x) and EtOAc (1x). The combined organic layers were washed with brine, dried over MgSO4 and concentrated in vacuo. The crude was then pre-adsorbed onto silica, dried in vacuo and purified by flash chromatography to yield the desired alkylated product.

General procedure for the trifluoromethylation and difluoroalkylation of heteroarenes in continuous flow (GP3)

In an oven-dried vial equipped with a magnetic stirrer and a PTFE septum, 2.5 mg (1 mol %) of RA48 was added to a mixture of the hetero-arene substrate (0.5 mmol, 1 equiv.), BrCF2COOEt (1.0 mmol, 2 equiv.) in 2.5 ml of DMF. In another vial, TMEDA (1.5 mmol, 3 equiv) was dissolved in 2.5 ml of DMF. The two solution were merged with a T-mixer prior entering the Vapourtec Photoreactor (fluoropolymer tube, 1.3 mm ID, 5 mL) and the total liquid flowrate was set to 0.25 mL/min (20 minutes residence time).

For the difluoroalkylation/amidation reaction (compounds 5f, 5g, 5h) optimal conditions were found with inverted stoichiometry of the reagents: the starting aniline was used in excess (3 eq, 1.5 mmol), while BrCF2COOEt was used as limiting reagent (1eq, 0.5 mmol). Moreover, increased yield for the cyclization reaction were obtained with ICF2COOEt (1 eq, 0.5 mmol) instead of BrCF2COOEt. Isolated yields were calculated considering 0.5 mmol as maximal theoretical yield of product.

For trifluoromethylation reaction, gaseous CF3I was added via a mass flow controller (2.45 ml/min, 4eq) and the gaseous stream was merged with the liquid one with a T-mixer prior to entering the photoreactor. The reactor was irradiated with 54 blue LEDs (450 nm, total power 24W). The reaction mixture collected from the outlet was diluted with H2O and extracted with Et2O (2x) and EtOAc (1x). The combined organic layers were washed with brine, dried over MgSO4 and concentrated in vacuo. The crude was then pre-adsorbed onto silica, dried in vacuo and purified by flash chromatography to yield the desired alkylated product.

The continuous-flow set-ups employed in this chapter are almost identical to those reported in Chapter 2 (see Scheme 2.4 for a schematic overview).

PHOTOCATALYST CHARACTERIZATION

Theoretical calculations

The initial goal of the project was the development of a new series of 2,2’-bithiophene derivatives with the intention to test them as organic photoredox catalysts. Thanks to the collaboration with the University of Castilla La Mancha, computational studies were carried out as an efficient way to predict a priori molecule properties.

With the aim of carrying out a rational synthesis of 2,2’-bithiophene-based photocatalysts, our collaborators initially designed a series of 15 derivatives bearing different electron withdrawing and electron donating groups (for the initial series of compounds see SI of the article). Firstly, our collaborators calculated the theoretical absorption spectra of these 15 derivatives. The theoretical spectra were calculated in chloroform solution using the conductor-like polarizable continuum model (CPCM)[12, 25] and the time dependent density functional theory (TD-DFT) approach[10b, 26]. The M06-2X[11] meta-exchange functional was

6


employed. The UV-Visible spectra of all of the studied compounds show a unique absorption band close in the 400 nm region, with significant tailing towards the blue light range absorption which reveals the potential use of these compounds in visible-light photoredox catalysis. In all compounds of the series, both molecular orbitals are spread over the whole molecule, resulting in a unique absorption band typical of a one-electron HOMO-LUMO transition with large oscillator strength (f).

In order to predict the redox properties of the series, the redox potentials were also calculated (Table S 1). Ground state redox potentials (Eox and Ered) were calculated from the difference in Gibbs free energy between neutral molecules and oxidized (or reduced) radical ions in solution using the CPCM solvation model. The calculations of Gibbs free energies used M06-2x/6-311-G(2d,p) energies with thermal corrections calculated at the M06-2x/6-31G* level. The excited-state oxidation and reduction potentials were calculated from the ground state electron oxidation and reduction potentials according to the following equations:

E*ox = E1/2(PC+/PC*) = Eox - E0,0 (eq. 6.1)

E*red = E1/2(PC*/PC-)= Ered + E0,0 (eq. 6.2)

where E0,0 is the energy difference between the zeroth vibrational levels of the ground state and first excited electronic state.

Considering the theoretical information and taking into account the commercial availability of the alkynyl precursors needed, six members of the series were selected and synthesized. For these six catalysts, an overview of the absorption values and of the calculated redox potentials is depicted in Scheme 6.1. For all the other molecules of the initial series, all data can be found in the SI of the published article.

Experimental values

For photocatalysts A-F, a thorough experimental characterization was carried out as well. All experimental data matched closely with the calculated values.

Absorption and Emission spectra:

Absorption spectra were obtained on a Shimadzu UV-2501PC. All spectra were recorded in dichloroethane (DCE) with a 1 cm quartz cuvette (Figure 6.7). Fluorescence spectra were obtained on an Agilent Cary Eclipse Fluorescence Spectrophotometer in DCE with a 1 cm quartz cuvette (Figure 6.8).

Figure 6.7: UV-vis absorption spectra overlay of all photocatalysts (50 µM in DCE)

Figure 6.8: Emission spectra overlay of all photocatalysts (50 µM in DCE)

6


Redox potentials:

The redox properties of the reported 2,2’-bithiophene derivatives were followed by Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV). Cyclic Voltammetry measurements were performed with a µ−Autolab ECO-Chemie potentiostat, using a glassy carbon working electrode, Pt counter electrode, and Ag/AgCl reference electrode. The experiments were carried out under argon, in CHCl3, with Bu4NPF6 as supporting electrolyte (0.1 M). Scan rate was 50 mV·s-1. The runs of Pulse differential voltammetry measurements were carried out under argon in CHCl3 (0.5·mM), with Bu4NPF6 as supporting electrolyte (0.1 M). Scan rate was 0.01 V·s-1, modulation amplitude 0.025 V and modulation time 0.05 s-1. The oxidation potential was determined by the anodic peak of the voltammogram (DPV). An overview of the obtained experimental redox potentials can be found in Table 6.3, together with a comparison with the most commonly used photoredox catalysts.

Hammett constant and oxidation potentials

The experimental values obtained for the oxidation potentials of photocatalysts A to E were found to be in good correlation with the Hammett constants for the corresponding substituents. The correlation is illustrated in the graph below (R2 = 0.98).

Figure 6.9: Hammett plot showing the correlation between sigma values for benzene substituents and measured ground state oxidation potentials. For compound D (o-CF3) the tabulated value for p-CF3 was used, based on the fact that sigma values are mainly depending on electronic contributions.[27] Sigma values were taken from reference [28]

1,083,4,5-OMe

1,223,5-OMe

1,02p-OMe

1,34o-CF3

1,34p-CN

R² = 0,9787

-0,4 -0,2 0 0,2 0,4 0,6 0,8

Expe

rimet

nal E

ox (V

)

σ value

Hammett plot

Tab

le 6

.3: O

verv

iew

of

the

pro

per

ties

mea

sure

d f

or

pho

toca

taly

sts

A-F

and

co

mp

aris

on

wit

h so

me

of

the

mo

st c

om

mo

nly

used

pho

tore

dox

ca

taly

sts.

Ent

ryP

CE

1/2 ox

(PC

+ /P

C*)

a

(SC

E, V

)

E1/

2 re

d(P

C*/

PC

- )a

(SC

E, V

)

E1/

2 o

x(P

C+ /

PC

)(S

CE

, V)

Cal

cb /E

xpc

E1/

2 re

d (

PC

/P

C- )b

(SC

E, V

)

Exc

itat

ion

λ max

(nm

)C

alcb /

Exp

Em

issi

on

λ max

(nm

)C

alcc /

Exp

Ad

iab

atic

en

ergy

E0,

0d

(eV

)

1A

-1.4

90.

531.

25/1

.08

-2.2

240

1/39

350

6/45

12.

74

2B

-1.4

90.

571.

26/1

.22

-2.1

839

9/38

852

1/47

02.

75

3C

-1.3

80.

681.

03/1

.02

-2.2

440

7/39

051

0/47

42.

65

4D

-1.4

60.

741.

28/1

.34

-2.0

040

1/39

052

2/47

12.

74

5E

-1.1

70.

781.

51/1

.34

-1.9

041

0/39

853

4/45

92.

68

6F

-1.3

70.

581.

26/1

.33

-2.0

541

7/40

054

7/46

52.

63

7R

u(b

py)3

Cl2

-0.8

10.

771.

29-1

.33

452

615

2.10

8fa

c-Ir

(ppy

)3-1

.73

0.31

0.78

-2.2

037

551

82.

50

9M

es-A

cr-M

e+n.

a.2.

06n.

a.-0

.57

430

570

2.63

10e

Th-B

T-Th

-e-e

1.12

-1.1

5~4

65-e

-ea E

xcit

ed s

tate

red

ucti

on/

oxid

atio

n p

ote

ntia

ls w

ere

esti

mat

ed f

rom

gro

und

sta

te r

edox

po

tent

ials

and

E0,

0 fo

llow

ing

eq

. 1 a

nd 2

co

mp

uted

at

CP

CM

-M06

-2X

/6-

311+

G(2

d,p

)//C

PC

M-M

06-2

X/6

-31G

* in

chl

oro

form

; b C

om

put

ed a

t C

PC

M-M

06-2

X/6

-311

+G(2

d,p

)//C

PC

M-M

06-2

X/6

-31G

* in

chl

oro

form

; c Det

erm

ined

by

cycl

ic

volt

amm

etry

/DP

V i

n ch

loro

form

ver

sus

Ag

/Ag

Cl

(no

rmal

ized

to

SC

E);

dE 0

,0 i

s th

e en

erg

y d

iffer

ence

bet

wee

n th

e g

roun

d a

nd t

he e

xcit

ed s

tate

, b

oth

tak

en

at t

heir

zer

o v

ibra

tio

nal

leve

ls.

Co

mp

uted

at

CP

CM

-M06

-2X

/6-3

11+G

(2d

,p)/

/CP

CM

-M06

-2X

/6-3

1G*

in c

hlo

rofo

rm.

Val

ues

for

entr

y 7

to 9

wer

e ta

ken

fro

m

refe

renc

e [4

] and

[29]

, n.a

. = n

ot

app

licab

le f

or

Mes

-Acr

as

this

co

mp

oun

d d

oes

no

t un

der

go

oxi

dat

ive

que

nchi

ng c

ycle

s b

ut o

nly

red

ucti

ve q

uenc

hing

cyc

les.

e

Th-B

T-Th

is a

ben

zoth

iad

iazo

le-b

ased

org

anic

pho

toca

taly

st r

epo

rted

by

Zhan

g e

t al

.[9] V

alue

s m

issi

ng f

or

Th-B

T-Th

co

uld

no

t b

e re

trie

ved

fro

m t

he li

tera

ture

.

S

S

R

R

AOMe

MeO

MeO

Organicphotocatalystcore

F:D:

E:

NC

C:

MeO

B:OMe

MeO

S

CF 3

6



Photocatalysts

5,5’-bis((3,4,5-trimethoxyphenyl)ethynyl)-2,2’-bithiophene (A): Product prepared according to GP1 with 5-ethynyl-1,2,3-trimethoxybenzene (0.148 g, 0.77 mmol) as corresponding acetylene. Purification with flash chromatography on silica gel using as eluent hexane: ethyl acetate 3:1 afforded the desired product as a brown solid (0.126 g, 75%); 1H NMR (500 MHz, Chloroform-d) δ = 7.17 (d, J = 3.9 Hz, 2H, H3-thioph), 7.08 (d, J = 3.9 Hz, 2H, H4-thioph), 6.76 (s, 4H, o-Ph), 3.88 (s, 6H, p-OCH3), 3.87 (s, 12H, m-OCH3). 13C NMR (125 MHz, Chloroform-d) δ = 153,1; 139,1; 137,9; 132,7; 123,9; 122,5; 117,6; 108,6; 94,6, 81,6, 60,9; 56,1 MS calcd for (C26H18O2S2) M+· 546.117, found 546.540. M.p.: 160-162 °C.

5,5’-bis((3,5-dimethoxyphenyl)ethynyl)-2,2’-bithiophene (B): Product prepared according to GP1 with 1-ethynyl-3,5-dimethoxybenzene (0.125 g, 0.77 mmol) as corresponding acetylene. Purification with flash chromatography on silica gel using as eluent hexane: ethyl acetate 4:1 afforded the desired product as a dark brown solid (0.125 g, 83%); 1H NMR (500 MHz, Chloroform-d) δ = 7.18 (d, J = 3.9 Hz, 2H, H3-thioph), 7.09 (d, J = 3.9 Hz, 2H, H4-thioph), 6.67 (d, J = 2.9 Hz, 4H, o-Ph), 6.48 (t, J = 2.5 Hz, 2H, p-Ph), 3.81 (s, 12H, OCH3). 13C NMR (125 MHz, Chloroform-d) δ = 160.6, 138.1, 133.0, 124.0, 123.9, 122.5, 109.1, 102.2, 94.6, 82.1, 55.5 MS calcd for (C28H22O4S2) M+· 486.096, found 485.011. M.p.: 185-187°C

5,5’-bis((4-methoxyphenyl)ethynyl)-2,2’-bithiophene (C): Product prepared according to GP1 with 1-ethynyl-4-methoxybenzene (0.102 g, 0.77 mmol) as corresponding acetylene. Purification with flash chromatography on silica gel using as eluent hexane: ethyl acetate 9:1 afforded the desired product as a brown solid (0.116 g, 88%); 1H NMR (500 MHz, Chloroform-d) δ =. 7.45 (d, J = 8.8 Hz, 4H, m-Ph), 7.13 (d, J = 4.0 Hz, 2H, H3-thioph), 7.06 (d, J = 4.0 Hz, 2H, H4-thioph), 6.88 (d, J = 8.8 Hz, 4H, o-Ph), 3.84 (s, 6H, OCH3). 13C NMR (125 MHz, Chloroform-d) δ = 159.8, 137.7, 132.9, 132.3, 123.8, 122.9, 114.8, 114.1, 94.5, 81.2, 55.3. MS calcd for (C26H18O2S2) M+· 426.075, found 424.531. M.p.: 154-155 °C

5,5’-bis((2-(trifluoromethyl)phenyl)ethynyl)-2,2’-bithiophene (D): Product prepared according GP1 with 1-ethynyl-2-(trifluoromethyl)benzene (0.131 g, 0.77 mmol) as corresponding acetylene. Purification with flash chromatography on silica gel using as eluent hexane: ethyl acetate 7:1 afforded the desired product as a brown solid (0.108 g, 70%); 1H NMR (500 MHz, Chloroform-d) δ =. 7.69 (d, J = 7.8 Hz, 2H, H3), 7.65 (d, J = 7.8 Hz, 2H, H6), 7.53 (t, J = 7.5 Hz, 2H, H5), 7.43 (t, J = 7.5 Hz, 2H, H4), 7.24 (d, J = 4.0 Hz, 4H, H3-thioph), 7.13 (d, J = 4.0 Hz, 4H, H4-thioph) 13C NMR (125 MHz, Chloroform-d) δ = 138.8, 133.6, 133.4, 131.4, 128.1, 126.0, 125.9, 124.3, 122.0, 120.1, 121.0, 90.6, 87.9. MS calcd for (C26H12F6S2) M+· 502.028, found 502.534. M. p.: 227-229 °C

4,4’-([2,2’-bithiophene]-5,5’-diylbis(ethyne-2,1-diyl))dibenzonitrile (E): Product prepared according to GP1 with 4-ethynylbenzonitrile (0.098 g, 0.77 mmol) as corresponding acetylene. Purification with flash chromatography on silica gel using as eluent hexane: ethyl acetate 7:1 afforded the desired product as a brown solid (0.077 g, 60%); 1H NMR (500 MHz, DMSO-d6) δ: 7.91 (d, J = 8.3 Hz, 4H, o-Ph), 7.75 (d, J = 8.3 Hz, 4H, m-Ph), 7.52 (d, J = 4.0 Hz, 2H, H3-thioph), 7.49 (d, J = 4.0 Hz, 2H, H4-thioph). 13C NMR (125 MHz, DMSO-d6) δ = 139.0, 134.0, 132.1, 131.7, 128.6, 128.4, 127.6, 124.5, 111.7, 93.1, 86.8. MS calcd for (C26H12N2S2) M+· 416.044, found 419.367. M. p.: 175-177 °C

5,5’-bis(benzo[b]thiophen-2-ylethynyl)-2,2’-bithiophene (F): Product prepared according to GP1 with 2-ethynylbenzo[b]thiophene (0.122 g, 0.77 mmol) as corresponding acetylene. Purification with flash chromatography on silica gel using as eluent hexane: ethyl acetate 9:1 afforded the desired product as a brown solid (0.048 g, 40%); 1H NMR (500 MHz, Chloroform-d) δ: 7.78 (m, 4H, o-benzothiophene), 7.52 (s, 2H, H- benzothiophene-C≡C), 7.38 (m, 4H, m-benzothiophene), 7.24 (d, J = 3.9 Hz, 2H, H3-thioph), 7.13 (d, J = 3.9 Hz, 2H, H4-thioph). 13C NMR (100 MHz, Chloroform-d) δ = 139.9, 138.4, 139.9, 132.8, 124.4, 124.3, 126.1, 123.5, 123.3, 122.8, 122.0, 120.0, 91.6, 86.4. MS calcd for (C26H12N2S2) M+· 477.998, found 478.096. M.p.: 162-164°C

Diethyl 2-(3-methyl-1H-indol-2-yl)malonate (2a)[15]: Product prepared according to the GP2 with a 7 minutes residence time on a 0.4 mmol scale, purification with flash chromatography on silica gel using as eluent cyclohexane: DCM 2:1 afforded the desired product as a yellow solid (81 mg, 70%); 1H NMR (399 MHz, Chloroform-d) δ = 8.91 (s, 1H), 7.55 (d, J=7.8, 1H), 7.36 (d, J=8.1, 1H), 7.20 (d, J=7.0, 1H), 7.11 (t, J=7.5, 1H), 4.98 (s, 1H), 4.32 – 4.17 (m, 4H), 2.32 (s, 3H), 1.29 (t, J=7.1, 6H). 13C NMR (100 MHz, Chloroform-d) δ = 167.5, 136.0, 128.4, 124.6, 122.5, 119.3, 118.9, 111.2, 110.7, 62.4, 49.4, 14.2, 8.6.

Diethyl 2-(1-methyl-1H-indol-2-yl)malonate (2b)[15]: Product prepared according to GP2 with a 10 minutes residence time on a 0.4 mmol scale, purification with flash chromatography on silica gel using as eluent cyclohexane: DCM 1:1 afforded the desired product as a yellow solid (113 mg, 97%); 1H NMR (399 MHz, Chloroform-d) δ = 7.55 (d, J=7.9, 1H), 7.27 (d, J=8.2, 1H), 7.19 (t, J=8.1, 6.9, 1H), 7.06 (t, J=7.4, 1H), 6.55 (s, 1H), 4.89 (s, 1H), 4.30 – 4.17 (m, 4H), 3.68 (s, 3H), 1.26 (t, J=7.1, 6H). 13C NMR (100 MHz, Chloroform-d) δ = 167.1, 138.0, 131.0, 127.4, 122.1, 120.9, 119.8, 109.4, 103.1, 62.3, 51.4, 30.4, 14.2.

Diethyl 2-(5-bromo-1-methyl-1H-indol-2-yl)malonate (2c): Product prepared according to GP2 with a 20 minutes residence time on a 0.4 mmol scale, purification with flash chromatography on silica gel using as eluent cyclohexane: DCM 1:1 afforded the desired product as a yellow solid (57 mg, 39%); 1H NMR (399 MHz, Chloroform-d) δ = 7.70 (d, J=1.7, 1H), 7.30 (dd, J=8.7, 1.8, 1H), 7.17 (d, J=8.7, 1H), 6.53 (s, 1H), 4.90 (s, 1H), 4.28 (dd, J=7.1,

6


4.2, 3H), 3.70 (s, 6H), 1.30 (t, J=7.1, 5H). 13C NMR (100 MHz, Chloroform-d) δ = 166.8, 136.7, 132.3, 129.0, 124.9, 123.3, 113.1, 110.9, 102.7, 62.5, 51.4, 30.6, 14.2.

Dimethyl 2-(2-(methoxycarbonyl)-1-methyl-1H-indol-3-yl)malonate (2d)[15]: Product prepared according to GP2 with a 30 minutes residence time on a 0.4 mmol scale, purification with flash chromatography on silica gel using as eluent cyclohexane: ethyl acetate 5-20% afforded the desired product as a yellow solid (110 mg, 79%); 1H NMR (400 MHz, Chloroform-d) δ = 7.73 (d, J=7.4, 1H), 7.40 – 7.32 (m, 2H), 7.18 – 7.13 (m, 1H), 5.76 (s, 1H), 4.31 – 4.16 (m, 4H), 4.02 (s, 3H), 3.95 (s, 3H), 1.25 (t, J=7.1, 6H). 13C NMR (101 MHz, Chloroform-d) δ = 168.6, 162.6, 138.8, 126.3, 125.8, 125.5, 122.1, 120.9, 114.6, 110.4, 61.8, 51.9, 50.1, 32.4, 14.2.

Diethyl 2-(3-methylbenzofuran-2-yl)malonate (2e)[9]: Product prepared according to GP2 with a 30 minutes residence time on a 0.4 mmol scale, purification with flash chromatography on silica gel using as eluent cyclohexane: ethyl acetate 5-20% afforded the desired product as a yellow solid (92 mg, 79%); 1H NMR (400 MHz, Chloroform-d) δ = 7.73 (d, J=7.4, 1H), 7.40 – 7.32 (m, 2H), 7.18 – 7.13 (m, 1H), 5.76 (s, 1H), 4.31 – 4.16 (m, 4H), 4.02 (s, 3H), 3.95 (s, 3H), 1.25 (t, J=7.1, 6H). 13C NMR (101 MHz, Chloroform-d) δ = 168.6, 162.6, 138.8, 126.3, 125.8, 125.5, 122.1, 120.9, 114.6, 110.4, 61.8, 51.9, 50.1, 32.4, 14.2.

Diethyl (S)-2-(3-(2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-1H-indol-2-yl)malonate (2f)[15]: Product prepared according to GP2 with a 30 minutes residence time on a 0.4 mmol scale, purification with flash chromatography on silica gel using as eluent petrol ether: ethyl acetate 8:2 afforded the desired product as a yellow solid (105 mg, 55%); 1H NMR (399 MHz, Chloroform-d) δ = 8.93 (s, 1H), 7.54 (d, J=7.9, 1H), 7.35 (d, J=8.1, 1H), 7.19 (t, J=7.5, 1H), 7.10 (t, J=7.5, 1H), 5.25 (d, J=7.6, 1H), 4.98 (s, 1H), 4.66 (d, J=6.5, 1H), 4.34 – 4.17 (m, 4H), 3.61 (s, 3H), 3.28 (t, J=5.8, 2H), 1.42 (s, 9H), 1.30 (t, J=7.1, 6H). 13C NMR (101 MHz, Chloroform-d) δ = 172.8, 167.5, 155.4, 136.1, 127.8, 126.7, 122.9, 119.9, 119.2, 111.4, 109.5, 80.0, 62.7, 54.3, 52.4, 49.4, 28.4, 27.2, 14.1.

Diethyl 2-(1-methyl-1H-pyrrol-2-yl)malonate (3a): Product prepared according to GP2 with a 40 minutes residence time on a 0.4 mmol scale, purification with flash chromatography on silica gel using as eluent cyclohexane: ethyl acetate 5-10% gradient afforded the desired product as a yellow solid (40 mg, 42%); 1H NMR (400 MHz, Chloroform-d) δ = 6.62 (t, J=1.9, 1H), 6.20 (t, J=1.7, 1H), 6.10 (t, J=2.6, 1H), 4.71 (s, 1H), 4.32 – 4.17 (m, 4H), 3.59 (s, 3H), 1.29 (t, J=7.1, 3H). 13C NMR (101 MHz, Chloroform-d) δ = 167.6, 123.8, 123.4, 110.0, 107.4, 62.1, 50.8, 34.4, 14.2.

Diethyl 2-(5-formyl-1-methyl-1H-pyrrol-2-yl)malonate (3b): Product prepared according to GP2 with a 50 minutes residence time on a 0.4 mmol scale, purification with flash chromatography on silica gel using as eluent cyclohexane: ethyl acetate 5-10% gradient

afforded the desired product as an orange oil (96 mg, 90%); 1H NMR (400 MHz, Chloroform-d) δ = 9.52 (s, 1H), 6.89 (d, J=4.2, 1H), 6.34 (d, J=4.1, 1H), 4.75 (s, 1H), 4.33 – 4.19 (m, 4H), 3.91 (s, 3H), 1.29 (t, J=7.1, 7H). 13C NMR (101 MHz, Chloroform-d) δ = 179.9, 166.3, 134.3, 133.0, 124.2, 111.3, 62.6, 50.4, 32.9, 14.1.

Diethyl 2-(5-methylthiophen-2-yl)malonate (3c): Product prepared according GP2 with a 40 minutes residence time on a 0.4 mmol scale, purification with flash chromatography on silica gel using as eluent cyclohexane: cyclohexane: dichloromethane 30-50% gradient afforded the desired product as a green oil (34 mg, 33%); 1H NMR (400 MHz, Chloroform-d) δ = 6.86 (d, J=3.5, 1H), 6.65 – 6.59 (m, 1H), 4.81 (s, 1H), 4.33 – 4.16 (m, 4H), 2.46 (d, J=0.9, 3H), 1.28 (t, J=7.1, 6H).13C NMR (101 MHz, Chloroform-d) δ = 167.6, 141.1, 131.0, 127.9, 124.8, 62.2, 53.5, 15.4, 14.1.

Diethyl 2-(furan-2-yl)malonate (3d): Product prepared according to GP2 with a 30 minutes residence time on a 0.4 mmol scale, purification with flash chromatography on silica gel using as eluent cyclohexane: ethyl acetate 5% afforded the desired product as a yellow solid (96 mg, 46%); 1H NMR (400 MHz, Chloroform-d) δ = 7.40 (dd, J=1.8, 0.8, 1H), 6.43 (d, J=3.3, 1H), 6.38 (dd, J=3.2, 1.9, 1H), 4.77 (s, 1H), 4.33 – 4.18 (m, 4H), 1.28 (t, J=7.1, 6H). 13C NMR (101 MHz, Chloroform-d) δ = 166.4, 146.1, 142.9, 110.8, 109.4, 62.3, 52.3, 14.1.

Diethyl 2-(5-methylfuran-2-yl)malonate (3e): Product prepared according to GP2 with a 30 minutes residence time on a 0.4 mmol scale, purification with flash chromatography on silica gel using as eluent cyclohexane: ethyl acetate 5% afforded the desired product as a yellow oil (72 mg, 75%); 1H NMR (400 MHz, Chloroform-d) δ = 6.28 (d, J=3.1, 1H), 5.95 (dd, J=0.9, 1H), 4.70 (s, 1H), 4.31 – 4.18 (m, 4H), 2.27 (s, 3H), 1.28 (t, J=7.1, 6H). 13C NMR (101 MHz, Chloroform-d) δ = 166.6, 152.6, 144.1, 110.1, 106.8, 62.1, 52.3, 14.1, 13.7.

2-methyl-3-(trifluoromethyl)-1H-indole (4a)[20b]: Product prepared according to GP3 with a 20 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent heptane: ethyl acetate 0-10% gradient afforded the desired product as a yellow solid (50 mg, 51%); 1H NMR (400 MHz, Chloroform-d) δ 8.12 (s, 1H), 7.75 (d, J = 7.3 Hz, 1H), 7.36 (dd, J = 6.5, 2.6 Hz, 1H), 7.30 – 7.22 (m, 2H), 2.60 (q, J = 1.6 Hz, 3H). 13C NMR (101 MHz, Chloroform-d) δ 134.5, 122.6, 121.4, 120.2, 119.2, 117.3 (q, J = 4.9 Hz), 113.8, 110.7, 99.7, 12.8.19F NMR (471 MHz, Chloroform-d) δ -54.68. HRMS (ESI) m/z calcd for C10H7F3N [M]-: 198.0536; found: 198.0529.

3-(trifluoromethyl)imidazo[1,2-a]pyridine (4b)[19a]: Product prepared according to GP3 with a 30 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent heptane: ethyl acetate 0-20% eluent afforded the desired product as a yellow oil (27 mg, 27%); 1H NMR (500 MHz, Chloroform-d) δ 8.24 (d, J = 6.9 Hz, 1H), 7.96 (s, 1H), 7.73 (d, J = 9.2 Hz, 1H), 7.42 – 7.34 (m, 1H), 7.00 (t, J = 7.2 Hz, 1H). 13C NMR (126

6


MHz, Chloroform-d) δ 147.5, 134.9, 129.9, 126.7, 124.9, 124.7 – 118.6 (m), 118.5, 114.1. 19F NMR (471 MHz, Chloroform-d) δ -61.48. HRMS (ESI) m/z calcd for C8H6F3N2 [M]+: 187.0477; found: 187.0484.

1-(3-(trifluoromethyl)-1H-pyrrol-2-yl)ethan-1-one (4c): Product prepared according to GP3 with a 20 minutes residence time on a 0.5 mmol scale, LC-MS yield (see LC-MS of crude reaction mixture in SI).

3-(trifluoromethyl)pyridin-4-amine (4d)[19a]: Product prepared according to GP3 with a 20 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent heptane: ethyl acetate 20-70% eluent afforded the desired product as a yellow oil (21 mg, 26%); 1H NMR (500 MHz, Chloroform-d) δ 8.50 (s, 1H), 8.29 (s, 1H), 6.58 (s, 1H), 4.68 (s, 2H). 19F NMR (471 MHz, Chloroform-d) δ -62.47.

4-methoxy-3-(trifluoromethyl)pyridin-2-amine (4e-C3)[19a]: Product prepared according to GP3 with a 20 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent heptane: ethyl acetate 0-45% gradient afforded the desired product as a yellow oil (18 mg, 18%); 1H NMR (400 MHz, Chloroform-d) δ 8.05 (d, J = 5.9 Hz, 1H), 6.30 (d, J = 5.8 Hz, 1H), 5.14 (s, 2H), 3.87 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 166.3, 156.6, 152.8, 124.9 (q, J = 272.9 Hz), 98.2, 96.5, 56.1. 19F NMR (471 MHz, Chloroform-d) δ -55.43.HRMS (ESI) m/z calcd for C7H8F3N2O [M]+: 192.05105; found: 193.0592.

4-methoxy-5-(trifluoromethyl)pyridin-2-amine (4e-C5): Product prepared according to GP3 with a 20 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent heptane: ethyl acetate 0-60% gradient afforded the desired product as a yellow oil (12 mg, 12%); 1H NMR (500 MHz, Chloroform-d) δ 8.13 (s, 1H), 5.97 (s, 1H), 4.78 (s, 2H), 3.87 (s, 3H). 13C NMR (126 MHz, Chloroform-d) δ 165.0, 162.5, 147.5, 124.9 (q), 110.0, 89.8, 55.5. 19F NMR (471 MHz, Chloroform-d) δ -61.16. HRMS (ESI) m/z calcd for C7H8F3N2O [M]+: 192.05105; found: 193.0592.

N,N-diethyl-4-(trifluoromethyl)aniline (4f-para): Product prepared according to GP3 with a 20 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent 100% heptane afforded the desired product as a transparent oil (90 mg, 45%); 1H NMR (500 MHz, Chloroform-d) δ 7.42 (d, J = 8.7 Hz, 2H), 6.66 (d, J = 8.9 Hz, 2H), 3.39 (q, J = 7.1 Hz, 4H), 1.18 (t, J = 7.1 Hz, 6H). 13C NMR (126 MHz, Chloroform-d) δ 149.8, 126.5 (q, J = 3.8 Hz), 110.5, 110.0, 49.4, 44.4, 12.4. 19F NMR (471 MHz, Chloroform-d) δ -60.74. HRMS (ESI) m/z calcd for C11H15F3N [M]+: 218.1151; found: 218.1157.

N,N-diethyl-2-(trifluoromethyl)aniline (4f-ortho): Product prepared according to GP3 with a 20 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent 100% heptane afforded the desired product as a transparent oil

(18 mg, 9%); 1H NMR (500 MHz, Chloroform-d) δ 7.63 (d, J = 9.2 Hz, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 2.97 (q, J = 7.1 Hz, 4H), 0.99 (t, J = 7.1 Hz, 6H). 19F NMR (471 MHz, Chloroform-d) δ -59.75. HRMS (ESI) m/z calcd for C11H15F3N [M]+: 218.1151; found: 218.1157.

5-bromo-3-(trifluoromethyl)pyridin-2(1H)-one (4g)[19a]: Product prepared according to GP3 with a 20 minutes residence time on a 0.5 mmol scale, LC-MS yield (see LC-MS of crude reaction mixture in SI).

1-(tetrahydro-2H-pyran-4-yl)-5-(trifluoromethyl)-1H-pyrazol-4-amine (4h)[19a]: Product prepared according to GP3 with a 20 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent heptane: ethyl acetate 0-25% gradient afforded the desired product as a yellow oil (31 mg, 27%); 1H NMR (500 MHz, Chloroform-d) δ 7.15 (s, 1H), 4.22 (tt, J = 11.5, 4.1 Hz, 1H), 4.09 (dd, J = 11.7, 4.7 Hz, 2H), 3.49 (td, J = 12.2, 1.9 Hz, 2H), 3.36 (s, 2H), 2.25 (qd, J = 12.6, 4.6 Hz, 2H), 1.86 (ddd, J = 12.7, 3.9, 1.8 Hz, 2H). 13C NMR (126 MHz, Chloroform-d) δ 130.9, 129.6, 122.0 (q, J = 267.3 Hz), 115.0 (q, J = 37.4 Hz), 67.2, 57.1, 33.0. 19F NMR (471 MHz, Chloroform-d) δ -56.70. HRMS (ESI) m/z calcd for C9H13F3N3O [M]+: 236.1005; found: 236.1011.

Ethyl 2,2-difluoro-2-(3-methyl-1H-indol-2-yl)acetate (5a)[20b]: Product prepared according to GP3 with a 20 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent heptane: ethyl acetate 0-25% gradient afforded the desired product as a yellow oil (62 mg, 48%); 1H NMR (400 MHz, Chloroform-d) δ 8.35 (s, 1H), 7.63 (dd, J = 7.98, 1.05 Hz, 1H), 7.38 (d, J = 8.31 Hz, 1H), 7.30 (ddd, J = 8.15, 6.91, 1.11 Hz, 1H), 7.18 (ddd, J = 8.02, 6.93, 1.06 Hz, 1H), 4.35 (q, J = 7.13 Hz, 2H), 2.44 (t, J = 2.37 Hz, 3H), 1.34 (t, J = 7.14 Hz, 3H). 13C NMR (101 MHz, Chloroform-d) δ 163.5 (t, J = 35.91 Hz), 135.5, 128.6, 124.3, 123.4 (t, J = 29.69 Hz), 120.1, 119.8, 113.9 (t, J = 3.48 Hz), 111.5, 108.9, 63.5, 13.9, 8.5. 19F NMR (376 MHz, Chloroform-d) δ -101.50 (d, J = 2.61 Hz). HRMS (ESI) m/z calcd for C13H12F2NO2 [M]-: 252.0842; found: 252.0838.

Ethyl 2,2-difluoro-2-(2-methyl-1H-indol-3-yl)acetate (5b): Product prepared according to GP3 with a 20 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent heptane: ethyl acetate 0-25% gradient afforded the desired product as a yellow oil (54 mg, 43%); 1H NMR (400 MHz, Chloroform-d) δ 8.15 (s, 1H), 7.74 (d, J = 7.1 Hz, 1H), 7.29 (dd, J = 6.6, 2.0 Hz, 1H), 7.20 – 7.10 (m, 2H), 4.27 (q, J = 7.1 Hz, 2H), 2.56 (t, J = 2.1 Hz, 3H), 1.29 (t, J = 7.1 Hz, 3H. 13C NMR (101 MHz, Chloroform-d) δ 164.9, 135.8, 134.8, 129.3, 125.9, 122.3, 121.0, 119.8, 114.6, 110.6, 62.9, 14.1, 13.1. 19F NMR (471 MHz, Chloroform-d) δ -98.29. HRMS (ESI) m/z calcd for C13H12F2NO2 [M]-: 252.0842; found: 252.0836.

6


Ethyl 2,2-difluoro-2-(imidazo[1,2-a]pyridin-3-yl)acetate (5c): Product prepared according to GP3 with a 20 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent heptane: ethyl acetate 0-20% gradient afforded the desired product as a yellow solid (58 mg, 49%); 1H NMR (500 MHz, Chloroform-d) δ 8.47 (d, J = 6.93 Hz, 1H), 7.89 (s, 1H), 7.76 (d, J = 7.69 Hz, 1H), 7.39 – 7.33 (m, 1H), 6.97 (t, J = 6.77 Hz, 1H), 4.40 (q, J = 7.15 Hz, 2H), 1.37 (t, J = 7.15 Hz, 3H). 13C NMR (101 MHz, Chloroform-d) δ 135.6 (t, J = 4.44 Hz), 126.6, 126.2 (t, J = 3.64 Hz), 118.4, 113.8, 113.7, 63.9, 22.8, 14.1 19F NMR (471 MHz, Chloroform-d) δ -101.75. HRMS (ESI) m/z calcd for C11H11F2N2O2 [M]+: 241.0783; found: 241.0426

Ethyl 2-(5-bromo-2-oxo-1,2-dihydropyridin-3-yl)-2,2-difluoroacetate (5d): Product prepared according to GP3 with a 20 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent heptane: ethyl acetate 25-50% gradient afforded the desired product as a yellow solid (73 mg, 50%); 1H NMR (500 MHz, Chloroform-d) δ 12.73 (s, 1H), 7.92 (d, J = 2.6 Hz, 1H), 7.74 (d, J = 2.2 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, Chloroform-d) δ 162.7 (t, J = 33.0 Hz), 160.5 (t, J = 4.6 Hz), 142.6 (t, J = 7.2 Hz), 138.4, 125.4 (t, J = 24.7 Hz), 110.8 (t, J = 249.4 Hz), 98.7, 63.5, 14.0. 19F NMR (471 MHz, Chloroform-d) δ -106.57.HRMS (ESI) calculated for C9H7BrF2NO3 [M]-: 293.9578; found: 293.9519.

5-bromo-3-(1,1,2,2,3,3,4,4-octafluoropentyl)pyridin-2(1H)-one (5e): Product prepared according to GP3 with IC4F9 as fluorinating agent and a 20 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent cyclohexane: ethyl acetate 5-25% afforded the desired product as a yellow solid (43 mg, 22%); 1H NMR (500 MHz, Chloroform-d) δ 13.31 (s, 1H), 7.86 (d, J = 2.7 Hz, 1H), 7.75 (d, J = 2.7 Hz, 1H). 13C NMR (126 MHz, Chloroform-d) δ 160.1, 146.5 (t, J = 8.7 Hz), 140.2, 120.1 (t, J = 23.4 Hz), 118.6 (d, J = 32.5 Hz), 117.4 – 115.9 (m), 114.7 (t, J = 33.9 Hz), 113.7 – 112.4 (m), 97.9. 19F NMR (471 MHz, Chloroform-d) δ -80.87, -111.29, -121.74, -125.90. HRMS (ESI) calculated for C9H2BrF9NO [M]-: 389.9181; found: 389.9182.

3,3-difluoro-5-isopropylindolin-2-one (5f):[23] Product prepared according to GP3 with a 30 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent heptane: ethyl acetate 25% afforded the desired product as a transparent oil (29 mg, 27%); 1H NMR (500 MHz, Chloroform-d) δ 8.01 (s, 1H), 7.42 (d, J = 1.7 Hz, 1H), 7.30 (d, J = 9.2 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 2.92 (p, J = 6.9 Hz, 1H), 1.25 (d, J = 6.9 Hz, 6H). 13C NMR (126 MHz, Chloroform-d) δ 167.0, 145.4, 138.8, 131.8, 123.2, 121.2 – 120.0 (m), 113.1, 111.4, 33.9, 24.1.19F NMR (471 MHz, Chloroform-d) δ -111.34. HRMS (ESI) calculated for C11H10F2NO [M]-: 210.0735; found: 210.0733.

3,3-difluoro-5-methoxyindolin-2-one (5g): Product prepared according to GP3 with a 30 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent heptane: ethyl acetate 25% afforded the desired product as a transparent oil (42 mg, 43%); 1H NMR (400 MHz, Chloroform-d) δ 8.01 (s, 1H), 7.12 (d, J = 1.9 Hz, 1H), 6.98 (ddd, J = 8.6, 2.5, 1.3 Hz, 1H), 6.86 (dt, J = 8.6, 1.3 Hz, 1H), 3.82 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 156.8, 134.1, 121.3 (t, J = 22.8 Hz), 119.1, 112.5, 111.2, 108.7, 104.1, 56.1. 19F NMR (471 MHz, Chloroform-d) δ -111.55. HRMS (ESI) calculated for C9H6F2NO2 [M]-: 198.0372; found: 198.0369.

3,3-difluoro-5-methylindolin-2-one (5h)[23]: Product prepared according to GP3 with a 30 minutes residence time on a 0.5 mmol scale, purification with flash chromatography on silica gel using as eluent heptane: ethyl acetate 0-25% gradient afforded the desired product as a transparent oil (35.2 mg, 38%); 1H NMR (500 MHz, Chloroform-d) δ 7.77 (s, 1H), 7.36 (s, 1H), 7.24 (d, J = 8.1 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 2.36 (s, 3H). 13C NMR (126 MHz, Chloroform-d) δ 165.6, 134.1, 131.1, 129.9, 125.8, 111.3, 110.1, 109.0, 21.1. 19F NMR (471 MHz, Chloroform-d) δ -111.36. HRMS (ESI) calculated for C9H6F2NO [M]-: 182.0422; found: 182.0417

ASSOCIATED CONTENT:

The Supporting Information for this article is available free of charge on the Wiley publication website at DOI: 10.1002/adsc.201801571.

6


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CHAPTER 7

A fully automated continuous-flow platform for fluorescence quenching studies and Stern-Volmer analysis


Bottecchia, C.; Kuijpers, K. P. L.; Cambie, D.; Drummen, K.; König, N. J.; Noël, T., Angew. Chem, Int. Ed. 2018, 57, 11278-11282

7

168 Chapter 7 Automated flow platform for fluorescence quenching studies and Stern-Volmer analysis 169

ABSTRACT

In the following chapter, the first fully automated continuous-flow platform for

fluorescence quenching studies and Stern-Volmer analysis is reported. All the

components of the platform were automated and controlled by a self-written

Python script. A user-friendly software allows even inexperienced operators to

perform automated screening of novel quenchers or Stern-Volmer analysis, thus

accelerating and facilitating both reaction discovery and mechanistic studies. The

operational simplicity of our system affords a time and labor reduction over batch

methods, while increasing the accuracy and reproducibility of the data produced.

Finally, the applicability of our platform is elucidated through relevant case studies.

In the last decade, photoredox catalysis emerged as a powerful strategy for the

catalytic activation of organic molecules.[1] In photoredox reactions, the ability

of a photocatalyst to absorb visible light, reach an excited state and ultimately

engage in a single electron transfer (SET) with an organic substrate is exploited

as a powerful trigger to induce selective and unique transformations.[1a, 2] In

this context, a good deal of efforts have been devoted over the years to the

understanding of the photophysical and photochemical aspects governing SET

processes.[3] The translation of this knowledge to the field of organic synthesis has

represented a key advantage in identifying novel photoredox transformations.[4]

Among the techniques employed to determine the reactivity of the excited state

of a photocatalyst, fluorescence quenching studies occupy a prominent role.

Once in their excited states, photocatalysts decay to their ground energy levels

following either a radiative or non-radiative process (according to the kinetic

constant k0, which is a property of every chromophore).[3b] In other words, a

photocatalyst dissipates the energy acquired through light absorption by either

emitting light or heat. However, in presence of an organic molecule that can act as

either energy acceptor or electron donor/acceptor, energy dissipation is averted

and a productive transfer can occur, thus generating radical species of interest.[1a]

Therefore, observing a decrease in the emission of an excited photocatalyst can be

considered as a tangible proof of its interaction with an organic substrate and is

the principle on which fluorescence quenching studies are based.[5]

To determine the rate at which an organic substrate can quench the excited state

of a photocatalyst, a Stern-Volmer analysis can be conducted.[3b, 6] In this case, the

emission of a photocatalyst is measured at increasing concentrations of quencher.[7] However, despite their relevance in elucidating the interaction between organic

substrates and photocatalysts, Stern-Volmer experiments are time consuming

and often air-sensitive. This is due to the fact that molecular oxygen is a known

quencher of excited states, therefore a thorough Stern-Volmer analysis requires

strictly inert conditions.[8] Moreover, because of the inevitable errors associated

with human labour, the reproducibility and accuracy (R2) of consecutive Stern-

Volmer measurements is often problematic.[9] In addition, fluorescence quenching

studies can also serve as a powerful tool to screen novel reactivities.[10] In order to

facilitate, standardize and accelerate both quenching screening experiments and

Stern-Volmer analysis, we envisioned an automated continuous-flow platform able

to perform these analyses via in-line monitoring of the photocatalyst fluorescence

(Figure 7.1).

7


Figure 7.1: Conceptual overview of the automated continuous-flow platform for fluorescence quenching studies and Stern-Volmer analysis

Recent reports demonstrated the implementation of automated platforms

designed to assist chemists in the routinely aspects of synthesis, leaving room for

the intellectual pursuit of new chemical reactivities.[11] Bearing this prospect in

mind, the automated platform we conceived minimizes the errors and the labour

associated with fluorescence quenching experiments. Furthermore, our system

has the potential to facilitate the way we perform these insightful experiments,

thus offering a practical strategy for preliminary mechanistic investigations and

accelerated reaction discovery in the context of photoredox catalysis.

When designing our system, we reasoned that continuous-flow technology

would constitute a powerful mean to put into practice an automated platform

for fluorescence quenching studies. Specifically, the confined dimensions of

continuous-flow microreactors offer the advantage to minimize the volumes

necessary for analysis and provide a closed environment that sustains inert

conditions.[12] This aspect is especially relevant in light of the prohibitive costs of

many photoredox catalysts.

Figure 7.2: Overview of the automated set-up for fluorescence quenching studies and Stern-Volmer analysis

An overview of the designed set-up is depicted in Figure 7.2. Continuous UV-Vis

analysis of the catalyst fluorescence was achieved with a quartz flow cuvette

(100 μL volume) connected through optical fibres to both a light source and a

spectrophotometer with a 90° angle. Both the spectrophotometer and the light

source are controlled by a computer. Moreover, dedicated HPLC pumps regulate

the stream to the flow cuvette in an automated fashion. Desiring to access a fully

automated system suitable for both screening experiments and Stern-Volmer

analysis, we focused on flexibility as the key property of our set-up. The optimized

set-up can easily be operated in two modes: automated screening or Stern-Volmer

analysis. When performing screening experiments, an autosampler is incorporated

to inject all the samples to test in a solvent stream. This stream is then combined

with a catalyst solution before entering the flow cuvette. When performing

Stern-Volmer analysis, a third HPLC pump delivers the quencher solution, while

the autosampler remains inactive. Another aspect further contributing to the

flexibility of our system is the possibility to conveniently change LED light source

in order to match the absorbance of the photocatalyst in use.

In both operational modes, all components were automated and controlled by a

self-written Python script (the source code is freely available at GitHub).[13] The

software features a user-friendly interface (Figure 7.3), which allows the user to

easily fill in all experimental parameters without the need for any programming

knowledge. All data collected during experiments are stored into a SQLite

What does the platform provide?

NIr

N

N

Calculation ofquenching rate constant (Kq)

Mechanistic insight

Quenching studies


AutomatedQuenching?

< 25%25 - 50%> 50%

screening results

Screening for quenchers or photocatalyst

Accelerated reaction discovery

Labor intensiveLimited accuracy Reproducibility issuesSensitive to oxygen interference

Manual batch procedure: Automated continuous-flow platform (this work)

Completely automated High accuracy(R2>0.98)High reproducibilityClosed system(oxygen interference minimized)

0.0 0.1 0.2 0.3 0.4

1.00

1.25

1.50I/I

0 Pho

toca

taly

st

Concentration quencher (M)

Q1

Q2

Q3

Fluorescence quenching studies & Stern-Volmer Analysis

LED light source

Autosampler

or Flow cuvette

SpectrophotometerComputer

Waste

Catalyst

Solvent Quencher

UV-Vis

Control interface

7


database, therefore progressively creating an easily accessible quencher library

for known photocatalysts.

Figure 7.3: Screen capture of some of the graphical user interfaces of the software developed for our automated platform.

Automated screening

Our automated platform was first calibrated and tested with 9-mesityl-10-methyl

acridinium perchlorate (Mes-Acr) as model catalyst and with a series of amine

quenchers (i.e. DIPEA, TMEDA and TEA, Figure 7.4).[14] The raw data set acquired

via the in-line monitoring of the photocatalyst fluorescence was automatically

processed with a moving average function in order to minimize the fluctuations of

the system (see experimental section Figure 7.6).

Figure 7.4: Mes-Acr fluorescence quenching studies with a series of known amine quenchers. A) Raw-data from spectrophotometer. B) Graphical output provided by the automated platform. C) Automated colour-assignment of the tested quenchers and their meaning D) Tested quenchers.

Secondly, a time-based data extraction allowed us to cut the raw data set, thus

minimizing storage space, facilitating data management and reducing the

computing power necessary to generate a graphical output (see experimental

section Figure 7.6). Through data parsing, the software was then programmed to

colour-code the graphical peaks, each corresponding to the quenching degree of

every quencher tested, in order to clearly identify the outcome of the screening

test. Specifically, samples that afforded a quenching degree higher than 50% were

assigned a green colour, while yellow and red were chosen as colours for samples

with a quenching degree between 50-25% and below 25% respectively. Finally, the

conclusions drawn from the quenching assessment can be analysed by the user

and converted to the results of the screening experiment, as exemplified in Figure

7.4B and Figure 7.4C.


We further dedicated our efforts to the automation of Stern-Volmer analysis.

According to the Stern-Volmer relationship (Figure 7.5), plotting the fraction

of emission (i.e. photons emitted in absence of quencher over photons emitted

in presence of quencher) against the concentration of quencher yields a linear

relation, also known as Stern-Volmer plot (Figure 7.5B).[3b] If the lifetime of the

excited state (τ0) of the photocatalyst is known, the quenching rate constant Kq can

be derived from the slope of the Stern-Volmer plot.

Figure 7.5: Stern-Volmer equation and features of the automated measurement. A) Representation of the raw data for the quenching of Mes-Acr with increasing amounts of TMEDA. B) Automated plot generation.

A B

A1

A2

A3

A4

A5

A6

A7

A8

Quenching assessmentBA Raw-data

Automated data analysis and visualization

D

CQuenching?

< 25%25 - 50%> 50%

A1 A2 A3 A4 A5 A6 A7

Screening results

List of tested quenchers

A1 BlankA2 BlankA3 DIPEAA4 Blank

A5 TMEDAA6 BlankA7 TEA

N NN

N

DIPEA TMEDA TEA

A1 A2 A3 A4 A5 A6 A7

A Automated plot generationBRaw-data analysis

Automated Stern-Volmer analysis

Data processing

Automated (R2 = 0.99)

Measured for every [Q]

Measured in absence of Q

Automated quencher dilution(via �ow rate control)

Quenching constant = slope of the line

Lifetime of the excited state

0.00 0.01 0.02 0.030

1

2

3

4

I 0/I M

es-A

cr

TMEDA Concentration (M)0 5 10 15 20

0

50000

100000

150000

200000

Inte

nsity

(a.u

.)Time (min)

7


We first conducted a Stern-Volmer analysis to determine the rate at which

N,N,N′,N′-tetramethylethane-1,2-diamine (TMEDA) can quench the excited state

of Mes-Acr (for more details on the calibration of the system see experimental

section and Figure 7.7). A representation of the raw data collected is shown in

Figure 7.5A. The obtained ladder-like profile is consistent with the progressive

decrease of emission intensity, which is in turn correlated with the injection of

increasing concentrations of quencher. We programmed the software to include

the raw data into an Excel file and to automatically generate the corresponding

Stern-Volmer plot (explicitly reporting the equation of the line and the R2 value),

thus relieving the user from this elementary task (Figure 7.5B). To showcase the

reliability and the operational advantage that our automated platforms offers, we

performed multiple repetitions of the same Stern-Volmer analysis. As depicted in

Figure 7.5B, ten consecutive experiments gave consistent results both in terms of

accuracy (R2) and reproducibility. The precise slope and quenching rate constant

obtained are depicted in Table 7.1. In this light, we believe that our system can be

regarded as a convenient tool providing accurate, precise and reproducible results

within a small time frame.

Table 7.1: Slope values obtained for 10 consecutive Stern-Volmer analysis on the quenching rate of TMEDA on Mes-Acr. These analysis were used for the calibration of the automated platform. The average slope value and the coefficient of variation are also reported.

Moreover, it should be noted that despite the high quenching rate of molecular

oxygen on the excited state of many commonly employed photocatalysts, the

confined dimensions and the closed environment provided by our platform greatly

contribute in reducing the impact of atmospheric oxygen on the measurements.[7]

Case studies

With our automated platform in hand, we investigated its application in the

elucidation of the first step involved in the mechanism of a photocatalytic reaction.

We chose to investigate the Ir(ppy)3-catalyzed photocatalytic decarboxylation of

α,β–unsaturated carboxylic acids, a reaction developed in our laboratory (Scheme

7. 1A).[15] By comparing the excited state potentials of Ir(ppy)3 (E1/2 Ir4+/Ir*3+ = −1.73

V and E1/2 Ir*3+/ Ir2+ = 0.31 V vs SCE) with the reduction potentials of BrCF2COOEt

(Ered = −0.57 V vs SCE, see SI) and cinnamic acid (Ered = −1.09 V vs SCE, see SI), we

reasoned that the reaction would most likely proceed through the oxidative

quenching of the fluorinating agent, thus generating the CF2COOEt radical of

interest.[16] Nevertheless, due to the extremely high excited state oxidation

potential of Ir(ppy)3, we were curious to probe whether the reduction of cinnamic

acid, albeit unexpected, could take place (Scheme 7.1B). Thus, we performed a

series of automated Stern-Volmer experiments to determine the rate at which

ethyl bromodifluoroacetate or cinnamic acid can quench the excited state of

Ir(ppy)3. The results obtained are summarized in Scheme 7.1. To our surprise, our

initial results showed a higher quenching rate constant for cinnamic acid compared

to BrCF2COOEt (3.37 x 108 vs 1.84 x 108 [L·mol-1·s-1]). However, because of the fact

that an excess of base is required in this reaction to generate trans-cinnamate,

which is more prone to decarboxylation than its conjugated acid, we reasoned

that the quenching rate of trans-cinnamate should be considered instead. The

quenching rate constant of trans-cinnamate was found to be 1.24x108 [L·mol-1·s-1].

Thus, based on the quenching rate constants obtained for BrCF2COOEt and trans-

cinnamate and taking into account their different concentrations in the optimized

reaction conditions, the quenching fraction for every component was calculated

according to the following equation (Eq. 7.1):

(7.1)

where kq is the quenching constant, C is concentration and τ0 is the lifetime of

the excited state of the photocatalyst. The obtained quenching fraction for Br-

CF2COOEt was found to be 81% vs 18.2% for trans-cinnamic acid. Thus, we propose

that the first step involved in the photocatalytic cycle is most likely the oxidative

quenching of Ir(ppy)3 by BrCF2COOEt. This hypothesis is further supported by

the fact that a CF2COOEt adduct was found when performing a radical trapping

experiment with butylated hydroxytoluene (BHT). Notably, all the work required

to confirm our initial mechanistic conclusion was performed by our automated

system within less than an hour.

Entry Slope

1

2

3

151.9

152.2

155.9

4 154.7

5 152.2

Entry Slope

6 154.8

7 155.8

8 152.4

9 152.7

10 154.9

Average 153.8 CV% 0.009%

Results obtained for 10 consecutive Stern-Volmer analysis to determinethe quenching rate of TMEDA on Mes-Acr

𝑓𝑓𝑓𝑓 = 𝑘𝑘𝑘𝑘𝑞𝑞𝑞𝑞1𝐶𝐶𝐶𝐶1

𝜏𝜏𝜏𝜏0−1 + 𝑘𝑘𝑘𝑘𝑞𝑞𝑞𝑞1𝐶𝐶𝐶𝐶1 + 𝑘𝑘𝑘𝑘𝑞𝑞𝑞𝑞2𝐶𝐶𝐶𝐶2

7


I n a

Scheme 7.1 A) Overview of the reaction conditions for the photocatalytic decarboxylation of α,β–unsaturated carboxylic acids. B) Possible first steps involved in the photocatalytic cycle. C) Results of the automated Stern-Volmer analysis.

second case study, we became interested in the Ir-catalyzed decarboxylative

alkylation of N-containing heteroarenes with N-(acyloxy)phthalimides (Scheme

7.3). Initially reported by Fu and co-workers, this reaction was found to proceed

optimally when catalyzed by Ir[dF(CF3)ppy]2(dtbbpy)PF6 and in presence of an

excess of strong acid (i.e. 2 eq. TFA).[17] Interested in replacing a costly iridium-

based photocatalyst with an inexpensive alternative, we reasoned that a

rapid photocatalyst screening performed with our automated platform would

quickly present us with a viable substitute. A selection of catalysts to be tested

(Scheme 7.2) was based on their absorption range and on their reported excited

state potentials. Unaware of which catalytic cycle would be possible with the

photocatalyst of choice, phthalimide 4, isoquinoline 5 and isoquinoline 5 + TFA were

tested as quenchers. As depicted in Scheme 7.3B, we found that no photocatalyst

was quenched by phthalimide 4, with the exception of Ir(ppy)3. These results

seemed logical considering that Ir(ppy)3 is the only photocatalyst with an excited

state oxidation potential (E1/2 Ir4+/Ir*3+ = −1.73 V) sufficient for the direct reduction

of phthalimide 4 (Ered = −1.57 V vs SCE, see SI).[16]

Scheme 7.2: List of photocatalyst tested. The values reported for the absorption maximum and the excited state potential were obtained from the literature.[16, 18]

We also found that inexpensive organic photocatalysts 4CzIPN and 2CzPN both

showed quenching in presence of protonated isoquinoline, while no quenching

was observed with isoquinoline alone.[18a, 18b]. We rationalized these results by

considering that, as documented in the literature, the protonation of isoquinoline

Photocatalytic decarboxylation of , -unsaturated carboxylic acids

Possible quenching pathways of the photocatalyst

Ph

Ir3+

COO

Ir3+*

Ir4+

SET SET

Br CF2CO2Et

CF2CO2Et

PhCOO

CO2H CF2CO2Etfac-Ir(ppy)3 (1 mol%)2 equiv NaHCO3EtO2CF2C Br1,4-dioxane (0.1M)

rt, 24 h

+

1 2 3 (E/Z)

BHT

BHT CF2CO2Et

Results of the automated Stern-Volmer analysis

Entry Quencher Slope Rate constant (M-1s-1)

1

2

3

R2

BrCF2COOEt 350.05 0.992 1.84 x 108

640.47 0.995 3.37 x 108

trans-cinnamate 236.05 0.999 1.24 x 108

Cinnamic acid

A

C

B

List of photocatalysts tested:

Abs max: 375 nmE1/2 PC+/PC* = -1.73 VE1/2 PC*/PC- = 0.31 V

Abs max: 480 nmE1/2 PC*/PC- = 2.06 V

N

N

RuN

N

N

N

N

IrN

N

2+

NMe ClO4

Me

MeMe

Cbz

CbzCbz

Cbz

CNNC


Cbz

CN

Cbz = carbazolyl


Cbz

CN

All potentials are reported vs SCE


Ir(ppy)3 Ru(bpy)32+ Mes-Acr+

4CzIPN 2CzPN

7


5 might result in a decrease of its reduction potential, thus rendering quenching

with 4CzIPN and 2CzPN possible.[19]

Naturally, both organic photocatalysts were then employed in the reaction of

interest in a microcapillary flow reactor, obtaining excellent isolated yields of the

desired product 6 within 30 minutes residence time (Table 7.2).[12b] The results

obtained for the other photocatalysts were all confirmed experimentally (Table

7.2, entry 1-6). Finally, we conducted an automated Stern-Volmer analysis to

determine the rate at which the protonated isoquinoline can quench the excited

state of 4CzIPN and observed a quenching rate constant of 2.9 x 106 [L·mol-1·s-1]

(R2 = 0.96).

Scheme 7.3: A) Overview of the reaction conditions for the decarboxylative alkylation of N-containing heteroarenes with N-(Acyloxy)phthalimides. B) Results of the automated fluorescence quenching studies. C) Possible quenching pathways for the first mechanistic step.

Table 7.2: Photocatalyst screening in batch and results obtained with 4CzIPN and 2CzPN in flow.

Entry Photocatalyst light source (nm) Yield (%)b

1 [Ir(dF-CF3-ppy)2(dtbpy)]PF6 400 90c

2 fac-[Ir(ppy)3] 400 78

3 4CzIPN 465 84;65d

4 2CzPN 400 80

5 [Mes-Acr]ClO4 465 NR

6 Ru(bpy)3Cl2 465 37

7 none no light NR

8 none 400 10

9 none 465 NR

10 4CzIPN (flow, 30 min rt)e 450 83, 80f

aReaction conditions: 2.0 mol % photocatalyst, 0.2 mmol 4, 2 equiv 5, 2.0 equiv TFA, 2 mL DMA, performed

under Argon atmosphere, 5 hours. bYield determined via GC-FID analysis with decafluorobiphenyl as

internal standard; c result from the literature; dreverse stoichiometry (2 equiv 4 and 1 equiv 5), ; eReaction

performed in a capillary microreactor, for detailed conditions see experimental section, fIsolated yield.

Based on the results from the quenching studies, we suggest an oxidative

quenching cycle in which protonated isoquinoline can quench the excited state of

4CzIPN (Scheme 7.3C).

In summary, we developed a fully automated continuous-flow platform for

fluorescence quenching studies and Stern-Volmer analysis. The operational

simplicity of our system reduces the time and labor associated with these

analyses, while increasing the accuracy and reproducibility of the data produced.

We demonstrated the development, calibration and application of our system to

two case studies of interest. Ultimately, our automated platform has the potential

to facilitate reaction discovery in photoredox catalysis. In this light, we believe that

our automated continuous-flow platform will be of high interest both to a chemist

and engineering audience and will inspire the design of similar machine-assisted

screening systems.

A Decarboxylative alkylation of N-containing heteroarenes with N-(Acyloxy)phthalimides

B Fluorescence quenching study

R2

4CzIPN

4CzIPN*

SET SET

1 mol % PC2 equiv TFA

DMA (0.1M)rt, 5 h

N

O

O

O

O

N

N+

C Possible quenching pathways of 4CzIPN

Photocatalyst 4 5 5 + TFA

4 5 6

TFA

Ir(ppy)3

Ru(bpy)32+

Mes-Acr+

4CzIPN

2CzPN

> 50 % 25 - 50 % < 25 %Degree of quenching:

4CzIPN+

OPhth

O

NH

NHOPhth

O

Unfavored due

to mismatch

of potentials

Demonstrated with

automated quenching

experiments

1 mol % PC2 equiv TFA

DMA (0.1M)rt, 5 h

N

O

O

O

O

NN+

4 56

7



General flow procedure for the synthesis of compound 6 (GP1): In an oven-dried glass vial equipped with a magnetic stirrer, photoredox catalyst 4CzIPN (4 mg, 1 mol%) was added to Phthalimide ester 4 (136.5 mg, 0.5 mmol) in DMA (2.5 mL). The vial was fitted with a PTFE septum and subjected to three cycles of vacuum/Argon. In a second oven-dried glass vial isoquinoline was weighed (2 equiv, 1 mmol, 129 mg). The vial was then fitted with a septum and subjected to three cycles of vacuum/Argon and TFA was added through a syringe (77 μL, 1 mmol) and diluted with 2.5 mL of DMA. The two solutions were transferred into two 5 mL BD Discardit plastic syringes and introduced into the photo-microreactor through a syringe pump. The two liquid streams were merged with a T-Mixer (ID = 500 μm) before entering the reactor. The flow rate was set to 0.33 ml/min (0.17 mL/min per syringe), thus resulting in 30 minutes residence time (volume of reactor = 5 mL). The reaction sample was collected in a vial kept in the dark under argon atmosphere. The volume collected was measured and the sample was then diluted with water and extracted with Et2O (2x) and EtOAc (1x). The organic layer was washed with brine, dried with MgSO4 and evaporated under reduced pressure. The resulting crude compound was absorbed on silica gel and purified via column chromatography (0-15% EtOAc in Cyclohexane). The isolated compound was analysed by GC-MS and NMR.

General procedure for the synthesis of 4CzIPN (GP2): NaH (60% in oil, 1.53 g, 37.5 mmol, 7.5 eq) was added slowly to a stirred solution of carbazole (4.175 g, 25.0 mmol, 5 eq) in dry THF (50 mL) under a nitrogen atmosphere at room temperature. After 30 min, tetrafluoroisophthalonitrile (1 g, 5.00 mmol, 1 eq) was added. The reaction mixture was stirred at room temperature for 12 h and then quenched with few mL water (to quench the excess NaH). The resulting mixture was then concentrated under reduced pressure and washed by water and EtOH to yield the crude product, which was purified by recrystallization from acetone/n-hexane to give 3.39 g of 4CzIPN (86%).

General procedure for the synthesis of 2CzPN (GP3): NaH (60% in oil, 735 mg, 18 mmol, 3.6 eq) was added slowly to a stirred solution of carbazole (2.045 g, 12.2 mmol, 2.4 eq) in dry THF (50 mL) under a nitrogen atmosphere at room temperature. After 30 min, 4,5-difluorophthalonitrile (803 mg, 5.0 mmol, 1 eq) was added. The reaction mixture was refluxed for three hours and then quenched with few mL water (to quench the excess NaH). The resulting mixture was then concentrated under reduced pressure and washed with water. The crude product was dissolved in DCM and adsorbed on silica. Purification via column chromatography (50% to 60% DCM in cyclohexane) yielded the product, which was then further purified by recrystallization from acetone/n-hexane to give 1.0 g of 2CzPN (45%).

Set-up

The total set-up developed for automated fluorescence quenching studies and Stern-Volmer analysis was described in the main text of this chapter. As mentioned earlier, the set-up can be operated in two different configurations: one used to perform fluorescence quenching studies and one employed for the automated Stern-Volmer analysis. Notably, the two configurations can be easily switched by simply including or excluding the autosampler or the quencher pump. In most cases, fluorescence quenching experiments and Stern-Volmer analysis were conducted with a total flow rate of 1ml/min. When employing solvents with higher viscosity (e.g. DMA), a total flow rate of 4ml/min was found necessary to ensure appropriate washing/replenishing of the flow cuvette within measurements. A picture of the entire set-up and of different parts can be found in the SI.

Sample preparation

All samples prepared for the fluorescence quenching studies were prepared by adding a stock solution of solvent (degassed by argon purging) to 1.5 ml vials containing either a quencher or a photocatalyst of interest. For the automated Stern-Volmer analysis three stock solutions were prepared: one solution of photocatalyst, one solution of quencher and one stock solution of solvent. All three solutions were bubbled with Argon prior to their use. No freeze-pump-thaw method was employed on any of the samples, except when performing a batch Stern-Volmer analysis for comparison.

Figure 7.6: Overview of the data produced by the automated flow platform.

0

20

40

60

80

100A1 Blank A3 A4 B1

A1

2

3

4

B

A

0 10 20 300

20k

40k

Inte

nsity

(a.u

.)

Time (min)

Time-based data extractionBRaw-data smoothing

Automated data analysis and visualization

DC Quenching assessment Screening results

Spectrometer dataMoving average

Quenching?

< 25%25 - 50%> 50%

7


Step-by-step data analysis and processing for fluorescence quenching studies

The raw data acquired via the in-line spectrophotometer is automatically processed with a moving average function (step size 5, width=20) in order to minimize the fluctuations of the system (Figure 7.6A). The time-based data extraction allowed us to cut the raw data set, thus minimizing storage space by a factor of 5 (Figure 7.6B). The software assessed and color-codes the graphical peaks, each corresponding to the quenching degree of every quencher tested, in order to clearly identify the outcome of the screening test (Figure 7.6C). An overview of the screening performed is then provided as pdf file (Figure 7.6D).

Figure 7.7: Results of the blank Stern-Volmer analysis. A) raw data (no decrease in intensity observed) and B) automatically generated Stern-Volmer plot.

Calibration of the system for automated Stern-Volmer analysis

As described in the main text, the calibration of our system for Stern-Volmer analysis was tested with 9-mesityl-10-methyl acridinium perchlorate (Mes-Acr) as photocatalyst and with TMEDA as quencher (results are depicted in Figure 7.5and in Table 7.1). Notably, the increasing concentrations of quencher necessary for the Stern-Volmer analysis were achieved by increasing the flow rate of the quencher solution and lowering the flow rate of the solvent, while maintaining constant both the catalyst and total flow rate. This decision was based on the fact that quantification of the quenching constant kq can be easily done when assuming pseudo-first order conditions. In order to make such an assumption, the catalyst concentration needs to be kept constant throughout the experiment, while the quencher concentration needs to be at least one to two orders of magnitude higher than the one of the photocatalyst. Moreover, to demonstrate that the observed decrease in the intensity of the photocatalyst emission does not derive from a simple dilution of the catalyst solution, we performed two control experiments. Firstly, we conducted several

blank Stern-Volmer analysis, in which a substance not able to quench the excited state of the photocatalyst was used as “quencher”. A typical result obtained in a control analysis is depicted in Figure 7.7. Secondly, we constantly monitored for an entire Stern-Volmer analysis the total volume exiting the flow cell. The exiting volume was found to be constant throughout the experiment, thus proving that no errors in the flow rate of the three solutions occurred (and therefore no dilution of the photocatalyst).

As mentioned in the main text, to determine which components of the reaction mixture could quench the excited state of Ir(ppy)3 a screening experiment was conducted. The results showed that all components quenched the excited state of Ir(ppy)3 (Figure 7.8A). We reasoned that the quenching observed in presence of BrCF2COOEt results in its reduction (Ered = −0.57 V vs SCE), thus generating the radical of interest. We also speculated an oxidative quenching pathway in the case of cinnamic acid and trans-cinnamate based on their reduction potentials (Ered = −1.09 V vs SCE for cinnamic acid, Ered = −1.20 V vs SCE for trans-cinnamate).

Figure 7.8: Fluorescence quenching studies and Stern-Volmer analysis for the photocatalytic decarboxylation of α,β–unsaturated carboxylic acids.

A BRaw-data

Blank: Stern-Volmer analysisStern-Volmer plot

0.00 0.02 0.04 0.06 0.080.0

0.2

0.4

0.6

0.8

1.0I 0/

I Mes

-Acr

*

Blank Concentration (M)0 5 10 15 20

0

10k

20k

30k

40k

Inte

nsity

(a.u

.)

Time (min)

A BQuenching studies

First case study: results

Stern-Volmer plot

1 2 3 4

1: Blank2: BrCF2COOEt3: Cinnamic acid4: Trans cinnamate

R2= 0.999

0.000 0.002 0.004 0.0060

1

2

3

4

5

I 0/I I

r(ppy

) 3*

Cinnamic acid concentration (M)

R2= 0.995

0.000 0.003 0.006 0.009 0.0120

1

2

3

4

5

6

I 0/I I

r(ppy

) 3*

BrCF2COOEt concentration (M)

R2= 0.992

0.000 0.002 0.004 0.006 0.0080.0

0.5

1.0

1.5

2.0

2.5

3.0

I 0/I I

r(ppy

) 3*

trans-cinnamate concentration (M)

7


Moreover, by comparing the reduction potential of the excited state of the photocatalyst (E1/2 Ir*3+/ Ir2+ = 0.31 vs SCE) with the calculated oxidation potential of cinnamic acid and trans-cinnamate (Eox= 0.54 vs SCE for cinnamic acid, Eox= 0.81 vs SCE for trans-cinnamate), we also concluded that a reductive quenching pathway should not be possible in the reaction.[16] To determine which component exhibited the highest quenching rate, an automated Stern-Volmer analysis was performed (Figure 7.8B). An overview of the quenching rate and R2 values obtained can be found in Scheme 7. 1.

Because of the fact that an excess of base (NaHCO3) is required in the reaction, it is correct to assume that all cinnamic acid is present in its deprotonated form (trans- cinnamate). Thus, in order to compare the quenching rate of these two reagents, only the value of Br-CF2COOEt and trans-cinnamate should be considered.

Figure 7.9: Fluorescence quenching studies and Stern-Volmer analysis for the decarboxylative alkylation of N-containing heteroarenes with N-(Acyloxy)phthalimides.

Second case study

For the decarboxylative alkylation of N-containing heteroarenes with N-(Acyloxy)phthalimides, a rapid photocatalyst screening was conducted with the automated platform with the aim of replacing costly photocatalyst Ir[dF(CF3)ppy]2(dtbbpy)PF6 with an inexpensive alternative ( the photocatalyst tested and the results obtained are reported in Scheme 7.2

and Scheme 7.3 respectively). Phthalimide 4, isoquinoline 5 and isoquinoline 5 + TFA were tested as quenchers (TFA alone was also tested as control experiment).

As depicted in Figure 7.9B, in order to verify the results obtained via the quenching study multiple Stern-Volmer analyses were conducted. These experiments were done to verify that no other mixture of components in the reaction mixture could quench the excited state of 4CzIPN. The Stern-Volmer plot for Phthalimide + TFA and TFA are reported in Figure 7.9(bottom left and right corner respectively). As noticeable from the graphs, no quenching was observed even at increasing concentrations of quenchers, thus confirming that neither of these compounds can interact with the excited state of 4CzIPN. On the contrary, the analysis with isoquinoline + TFA gave a good Stern-Volmer plot (Figure 7.9, top right corner slope 14.5, rate constant 2.9x106).


1-cyclohexylisoquinoline (6) was made according to GP1 on a 1.0 mmol scale employing p-toluenesulfonic acid monohydrate (2 eq). The crude product was purified by flash chromatography (0-15% ethyl acetate in cyclohexane) yielding 168.7 mg (0.8 mmol, 80 %) of 6 as a transparent oil. 1H NMR (399 MHz, Chloroform-d) δ 8.47 (d, J = 5.69 Hz, 1H), 8.21 (d, J = 8.41 Hz, 1H), 7.77 (d, J = 8.00 Hz, 1H), 7.65 – 7.51 (m, 2H), 7.45 (d, J = 5.69 Hz, 1H), 3.55 (tt, J = 11.61, 3.24 Hz, 1H), 2.05 – 1.76 (m, 7H), 1.62 – 1.33 (m, 3H). 13C NMR (100 MHz, Chloroform-d) δ 165.7, 141.9, 136.4, 129.6, 127.6, 126.9, 126.3, 124.8, 118.9, 41.6, 32.7, 27.0, 26.3.

1,2,3,5-tetrakis(carbazol-9-yl)-4,6-dicyanobenzene (4CzIPN) was made according to GP2 and based on a procedure adapted from literature on a 5.0 mmol scale.[18b] The crude product was purified by recrystallization (acetone/n-hexane) yielding 3.39 g (4.3 mmol, 86 %) of 4CzIPN as a yellow solid. The NMR signals matched the data from literature.1H NMR (399 MHz, DMSO-d6) δ 8.35 (d, J = 7.69 Hz, 2H), 8.19 (d, J = 8.23 Hz, 2H), 7.86 (d, J = 7.13 Hz, 4H), 7.79 – 7.71 (m, 6H), 7.56 – 7.43 (m, 6H), 7.18 – 7.04 (m, 8H), 6.81 (t, J = 7.33 Hz, 2H), 6.70 (t, J = 7.77 Hz, 2H).

1,2-bis(carbazol-9-yl)-4,5-dicyanobenzene (2CzPN) was made according to GP3 and based on a procedure adapted from literature on a 5.0 mmol scale.[18b] The crude product was purified by column chromatography (50% to 60% DCM in cyclohexane) and recrystallized (acetone/n-hexane), yielding 1.02 g (2.25 mmol, 45 %) of 2CzPN as a pale green solid. The NMR signals matched the data from literature. 1H NMR (399 MHz, DMSO-d6) δ 8.83 (s, 2H), 7.94 – 7.89 (m, 4H), 7.33 – 7.28 (m, 4H), 7.12 – 7.05 (m, 8H).

ASSOCIATED CONTENT:

The Supporting Information for this article is available free of charge on the Wiley publication website at DOI: 10.1002/anie.201805632.

A BQuenching studies

Second case study: results

Stern-Volmer plot

Legend4: Cyclohexyl phthalimide ester5: IsoquinolineTFA: Trifluoroacetic acid

Photocatalyst 4 5 5 + TFA TFA

Ir(ppy)3

Ru(bpy)32+

Mes-Acr+

4CzIPN

2CzPN

> 50 % 25 - 50 % < 25 %Degree of quenching:

R2= 0.961

7


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[19] a) J. Zou, P. S. Mariano, Photochem. Photobiol. Sci. 2008, 7, 393; b) T. Damiano, D. Morton, A. Nelson, Org. Biomol. Chem. 2007, 5, 2735-2752; c) P. S. Mariano, Tetrahedron 1983, 39, 3845-3879.

Summary & Conclusions

List of abbreviations

List of publications

Acknowledgements

About the author

Summary & Conclusions 191

Summary & Conclusions

The research presented in this thesis focused on the implementation of visible-

light photoredox catalysis for bioactive molecule modification. Owing to the mild

reaction conditions (i.e. room temperature, use of visible light) and to the high

functional group tolerance, photoredox catalysis represents an ideal strategy

towards the chemoselective modification of this broad class of compounds. In

order to facilitate a uniform irradiation of the reaction medium and to accelerate

reaction kinetics, continuous-flow technology was chosen as the ideal platform to

enable these novel chemistries.

The work described in this dissertation can be divided in two main research lines.

Firstly, efforts towards the development of mild methodologies for cysteine

modification were discussed. Owing to the powerful combination of photoredox

catalysis with continuous flow technology, three strategies suitable for cysteine

modification at the single amino acid level or in peptide sequences were presented.

In Chapter 2 a fast and straightforward continuous-flow protocol for the visible

light-induced trifluoromethylation and perfluoroalkylation of cysteine was

described. By employing Ru(bpy)32+ as photocatalyst and either gaseous CF3I

or inexpensive perfluoroalkyl iodides as fluorinating agents, a broad array of

fluorinated cysteine derivatives and cysteine-containing dipeptides was obtained

in excellent yields within 5 minutes of reaction time. The implementation of a

continuous-flow microreactor afforded an enhanced gas-liquid mass transfer

between the two immiscible phases, while allowing a safe handling of gaseous

reactants. Mechanistic studies revealed that Ru(bpy)32+ undergoes a reductive

quenching cycle, but quantum yield measurements suggested that the presence

of concurrent radical chain mechanisms is likely. Further developments of this

work should focus on its application to more complex peptide substrates.

Specifically, more work is required to demonstrate site-selectivity towards

cysteine in sequences containing other amino acids that might interfere (e.g.

serine, lysine, histidine, and tryptophan). Moreover, a careful re-optimization of

reaction conditions (e.g. solvent, type of base, etc.) might facilitate the application

of the methodology to proteins as well. Finally, in the context of exploiting this

method for the preparation of large amounts of fluorinated cysteine derivatives, a

further improvement could be the creation of a two-step flow sequence allowing

the fluoroalkylation of cysteine followed by a deprotection step at the carboxylate

site. Such a telescoped two-step continuous-flow system would grant access to

192 Summary & Conclusions 193

gram scale of fluorinated cysteine derivatives ready to be employed in solid phase

peptide synthesis.

In Chapter 3, a mild and versatile protocol to accelerate the formation of

disulfide bonds was established. This visible light induced photocatalytic aerobic

oxidation of thiols was optimized with TiO2 as a heterogeneous photocatalyst.

The chemistry proved amenable to intramolecular S–S bond formation of cysteine

residues in complex peptides under biocompatible reaction conditions (buffer

solution, visible light, simple purification by filtration or centrifugation). Moreover,

a packed-bed reactor was prepared to allow the implementation of a continuous

protocol, resulting in an efficient and reliable scale-up of the reaction conditions.

A diverse set of symmetrical and unsymmetrical disulfides was obtained in good to

excellent yields within few minutes of reaction time. Future endeavours to improve

this methodology could be aimed at improving the reactor design. Specifically,

the disposition of the light source with the respect of the flow reactor could be

changed. Moreover, from a chemical point of view, more research on the use of

this methodology for the formation of unsymmetrical disulfides would be of high

interest. As example, the methodology could be further expanded to achieve

labeling of short peptides sequences with any linker/compound of interest.

Chapter 4 describes a mild and selective C-H arylation of cysteine. This

biocompatible, metal-free procedure relies on the use of aryldiazonium salts as

arylating agents and employs catalytic amounts of Eosin Y to afford the formation

of the desired aryl radicals. The batch method described was applied to the

chemoselective arylation of a model peptide in biocompatible reaction conditions

(room temperature, PBS buffer) within a short reaction time. Furthermore, the

implementation of a continuous-flow protocol afforded the in-situ formation of

diazonium salts, thus limiting the risks associated with their isolation. A broad

series of arylated cysteine derivatives and arylated cysteine-containing dipeptides

were prepared in flow within few seconds of residence time. Owing to the ease

of scalability, the flow protocol could be easily employed to obtain arylated

cysteine derivatives on gram scales, thus allowing the preparation of sufficient

quantities for automated solid phase peptide synthesis (SPPS). To improve the

relevance of this methodology, further work is required to establish whether

this arylation methodology might be suitable for cysteine arylation in longer

peptides and, possibly, in proteins. Towards this goal, a key point will be to

establish whether arylation of other amino acid residues (e.g. arylation at indole

moiety of Trp) will compete with the desired reaction. Moreover, similarly to what

discussed for Chapter 3, the methodology should be expanded to achieve labeling

of peptides with either linkers, drugs or biological probes. A potential hurdle to

be circumvented is the synthetic difficulty in preparing labels of interest with a

diazonium moiety. A possible solution would be to have the diazonium moiety as

part of a clickable linker, which could be attached to the label of interest after the

photocatalytic reaction at cysteine.

In the second part of the thesis, a parallel research line focused on the combination

of photoredox catalysis and microreactor technology to attempt to overcome some

of the drawbacks currently limiting the widespread use of photoredox catalysis

in the pharmaceutical industry. In particular, in Chapter 5, a continuous-flow

protocol for the trifluoromethylation of arenes, heteroarenes and benzofused

heterocycles was developed. Novel methodologies for the trifluoromethylation

of these compounds are highly demanded in the pharmaceutical industry, as the

introduction of fluorine atoms can greatly impact the electronic properties, acidity

and lipophilicity of drug candidates. The methodology relies on the use of solid,

shelf-stable CF3SO2Na as trifluoromethylating agent and of an iridium species as

photoredox catalyst. For the reaction scope, the focus was kept on substrates

of high value for drug discovery programs. Specifically, the applicability of the

methodology to nitrogen-containing heteroarenes/benzofused heterocycles

bearing halogen substituents was showcased. Moreover, process intensification in

a microflow reactor afforded reduced reaction times (30 minutes residence time)

and a high productivity. The main current limitation of this methodology resides

in the use of an expensive Ir-based photocatalyst. To improve the applicability

of this synthetic methods at the industrial scale, some measures could be taken.

Firstly, research into the use of an alternative, inexpensive organic catalysts (such

as those described in Chapter 6) could render the Ir species obsolete. Secondly, a

continuous-flow synthesis of the Ir photocatalyst could be designed starting from

relatively cheaper starting materials. Compared to a batch synthesis (typically

requiring high temperatures and long operation times for this class of transition-

metal complexes), the preparation of an Ir photocatalyst in flow could result in

increased yields and higher quality of the synthesized product thanks to the fine

control over the reaction temperature achievable in continuous systems.

As previously mentioned, one of the main obstacles currently preventing the

implementation of photoredox catalysis for the large scale production of fine

chemicals is the prohibitive cost and scarce availability of transition metal-based

photoredox catalysts (i.e. Ir and Ru polypyridyl complexes). Organic photocatalysts

often provide valuable and inexpensive alternatives to these complexes.

Therefore, the development of new classes of organic photoredox catalysts with

194 Summary & Conclusions 195

good redox properties, largely available or easily prepared in high yields would

contribute to further advance research in this field. In Chapter 6, the de novo

synthesis and characterization of a series of substituted bi-thiophene derivatives

as novel and inexpensive organic photocatalysts was described. DFT calculations

from our collaborators were used to predict a priori their absorption spectra

and redox potentials, which were then confirmed with experimental data. The

photocatalytic activity of this novel class of organic photoredox catalyst was then

demonstrated in two visible-light mediated strategies for the C–H functionalization

of heteroarenes. The activity of the tested compounds was comparable with that

of other commonly used organic or transition-metal photocatalysts. Starting

from the results obtained for this series of six derivatives, further research can

lead to the preparation of a second generation of improved bi-thiophene-based

photoredox catalyst. Specifically, the design of photocatalysts with different and

alternative substitution patterns, together with the use of DFT calculations to

predict their optical properties, can lead to photocatalyst with improved redox

potentials, different absorption spectra and increased chemical stability.

An important aspect in photoredox catalysis is the understanding of the

photophysical and photochemical aspects governing the interaction between the

excited state photocatalyst and the reaction substrates. Among the techniques

employed to determine the reactivity of the excited state photocatalyst,

fluorescence quenching studies and Stern-Volmer analysis are of high interest.

However, despite their relevance, these analyses are time consuming and often

air-sensitive. Desiring to facilitate, standardize and accelerate quenching screening

experiments and Stern-Volmer analysis, we developed a continuous-flow platform

able to perform these analyses in an automated fashion. As described in Chapter 7,

via in-line monitoring of the photocatalyst fluorescence, the interaction between

the excited state of a photocatalyst and an array of quenchers of interest can be

studied. Owing to the high level of automation, the system affords a time and

labor reduction, while granting access to accurate and reproducible data. The

potential of this platform to accelerate reaction discovery and to facilitate the

understanding of photocatalytic reaction mechanisms was showcased when it was

employed to rapidly select a suitable photocatalyst for a reaction of interest and

then to perform a Stern-Volmer analysis to elucidate the underlying mechanism

involved. Further improvement on this platform could result in an optimized set-

up capable of performing both fluorescence quenching studies and stern-Volmer

analysis at a higher pace. Such improvements could be achieved via minimization

of the dead volumes in the channels and by replacing the commercially available

flow cuvette with a specifically designed alternative.

In conclusion, the positive combination of photocatalysis and continuous-

flow technology resulted in the development of three strategies for cysteine

modification. The mild reaction conditions typical of photoredox catalysis,

together with the uniform irradiation of the reaction mixture enabled by microflow

reactors, proved to be especially suited to develop novel chemistries for amino

acid modification. At the beginning of this research, only few examples in the

literature showed the application of photocatalytic methodologies for biomolecule

modification, and examples on the implementation of continuous-flow reactors

for this purpose were yet to be reported. However, over the course of the last four

years an increased interest in the potential application of photoredox catalysis

in chemical biology lead to a plethora of publications elaborating on this matter.

As a result, photocatalytic approaches for the modification of cysteine, tyrosine,

methionine and tryptophan now exist. Despite these successful examples,

further improvements to ameliorate the applicability of these methodologies

beyond the single amino acid level and towards complex peptides and proteins

are paramount. In this context, an approach to obtain improved regioselectivity

in complex structures might be to target one or more residues with known redox

potential and match it with the redox potential of an appropriate photocatalyst.

Alternatively, taking example from the field of dual catalysis, approaches relying

on the use of organic photocatalysts and other catalytic systems could be designed.

The main advantage granted by a photoredox-transition metal dual catalytic

system lies in the possibility to generate radicals in mild reaction conditions and

in their subsequent addition to a transition-metal complex (i.e. single-electron

transmetalation). Through careful selection of both catalytic systems, improved

reaction conditions (e.g. faster kinetics, improved selectivity) could thus be

achieved.

Finally, the use of enabling technologies, such as microreactor technology and

automation protocols, might significantly facilitate the development of novel

photoredox methodologies for protein modification. For instance, they could

contribute in reducing the amount of materials needed for optimization while

allowing to easily control multiple reaction conditions.

196 List of Abbreviations 197

LIST OF ABBREVIATIONS

LIST OF ABBREVIATIONS USED IN THIS THESIS:[(Mes-Acr)ClO4] 9-Mesityl-10-methylacridinium perchlorate

2-MeTHF 2-methyl tetrahydrofuran

2CzPN 1,2-bis(carbazol-9-yl)-4,5-dicyanobenzene

4CzIPN 1,2,3,5-tetrakis(carbazol-9-yl)-4,6-dicyanobenzene

ACN Acetonitrile

AcOH Acetic acid

API Active pharmaceutical ingredient

BDE Bond dissociation energy

Boc tert-butyloxycarbonyl

BPR Back pressure regulator

CFL Compact fluorescent lamp

CF3SO2Cl Trifluoromethanesulfonyl chloride

CF3SO2Na Langlois reagent, Sodium trifluoromethanesulfinate

CHCl3 Chloroform

CTFR Copper tube flow reactor

CuAAC Copper-catalyzed alkyne-azide cycloaddition

CV Cyclic voltammetry

DCC N,N’-Dicyclohexylcarbodiimide

DCE Dichloroethane

DCM Dichloromethane

DFT Density functional theory

Dha Dehydroalanine

DIPEA N,N-Diisopropylethylamine

DLS Dynamic light scattering

DMAc Dimethylacetamide

DMF N,N-Dimethylformamide

DMSO Dimethyl sulfoxide

DPA 1,9-diphenylanthracene

DPV Differential pulse voltammetry

EtOAc Ethyl acetate

fac-[Ir(ppy)3] Tris[2-phenylpyridinato-C2,N]iridium(III)

FEP fluorinated ethylene propylene

GC-FID Gas chromatography flame ionization detector

GC-MS Gas chromatography-mass spectrometry

H2O Water

HOBt Hydroxybenzotriazole

HCl Hydrochloric acid

HPLC High pressure liquid chromatography

HRMS High resolution mass spectroscopy

ID Internal diameter

i-PrOH Isopropanol

IR Infrared

KOH Potassium hydroxide

LC-MS Liquid chromatography–mass spectrometry

LED Light emitting diode

MeCN Acetonitrile

MeOH Methanol

MgSO4 Magnesium sulfate

N-Ac-L-Cys-OMe N-(acetyl)-L-cysteine methyl ester

N-Boc-L-Cys -OMe N-(tert-Butoxycarbonyl)-L-cysteine methyl ester

Na2SO4 Sodium sulfate

NaHCO3 Sodium hydrogen carbonate

NaOH Sodium hydroxide

n-BuOH n-Butanol

NH3 Ammonia

(NH4)2S2O8 Ammonium persulfate

NMR Nuclear magnetic resonance

OFET Organic field-effect transistor

PBS Phosphate-buffered saline

PE Petrol Ether

PEEK Polyether ether ketone

PET Positron Emission Tomography

PFA Perfluoroalkoxy alkane

Ph3N Triphenyl amine

PTFE Polytetrafluoroethylene

p-TsOH p-toluenesulfonic acid

Ru(bpy)3Cl2 Tris(bipyridine)ruthenium(II) chloride

SAR Structure-activity relationship

SCE Saturated calomel electrode

SEM Scanning electron microscopy

SET Single electron transfer

SI Supporting information

List of Publications 199198

SPPS Solid phase peptide synthesis

tBuONO tert-butyl nitrite

tBuOOH Tert-butyl hydroperoxide

TD-DFT Time-dependent density functional theory

TEA Triethylamine

TEMPO 2,2,6,6-Tetramethylpiperidine 1-oxyl

TFA Trifluoroacetic acid

TMEDA Tetramethylethane-1,2-diamine

TiO2 Titanium oxide

TLC Thin layer chromatography

tr Residence time

TsOH para-toluenesulfonic acid

UV Ultra-violet

XRD X-ray diffraction

LIST OF PUBLICATIONS

PEER REVIEWED ARTICLES:Bottecchia, C.; Martin, R.; Abdiaj, I.; Crovini, E.; Alcazar, J.; Orduna, J.; Blesa, M. J.;

Muñoz, Carillo J.R.; Prieto, P.; Noël, T., De novo design of organic photocatalysts: bithiophene derivatives for the visible-light induced C-H functionalization of heteroarenes, Adv. Synth. Catal, 2019, doi:10.1002/adsc.201801571 (selected as VIP article).

Bottecchia, C.; Noël, T. Photocatalytic modification of amino acids, peptides and proteins. Chem. – Eur. J. 2019, 25, 26-42.

Bottecchia, C.; Kuijpers, K. P. L.; Cambie, D.; Drummen, K.; König, N. J.; Noël, T. A fully automated continuous-flow platform for fluorescence quenching studies and Stern-Volmer analysis. Angew. Chem, Int. Ed. 2018, 57, 11278-11282.

Abdiaj, I.; Bottecchia, C.; Alcazar, J.; Noël, T. Visible-light-induced trifluoromethylation of highly functionalized arenes and heteroarenes in continuous flow. Synthesis 2017, 49, 4978-4985. (Highlighted as news article in SynForm, 5/9/2017).

Bottecchia, C.; Rubens, M.; Gunnoo, S. B.; Madder, A.; Noël, T. Visible-Light-Mediated Selective Arylation of Cysteine in Batch and Flow. Angew. Chem, Int. Ed. 2017, 56, 12702-12707. (Highlighted in “Some Items of Interest to Process R&D Chemists and Engineers” in OPRD, 2017, 21, 1873-1883).

Bottecchia, C.; Erdmann, N.; Tijssen, P.; Milroy, L. G.; Brunsveld, L.; Hessel, V.; Noël, T. Batch and Flow Synthesis of Disulfides by Visible-Light-Induced TiO2 Photocatalysis. ChemSusChem 2016, 9, 1781-1785. (Highlighted in “Some Items of Interest to Process R&D Chemists and Engineers” in OPRD, 2016, 20, 1691-1701).

Bottecchia, C.; Wei, X.-J.; Kuijpers, K. P. L.; Hessel, V.; Noël, T. Visible light-induced trifluoromethylation and perfluoroalkylation of cysteine residues in batch and continuous flow. J. Org. Chem. 2016, 81, 7301-7307.

Bottecchia, C.; Cambié, D.; Straathof, N. J. W.; Hessel, V.; Noël, T. Applications of continuous-flow photochemistry in organic synthesis, material science, and water treatment. Chem. Rev. 2016, 116, 10276-10341.

BOOK CHAPTERS:Bottecchia, C.; Noël, T. Intermolecular transition-metal catalyzed C-C coupling

reactions in continuous flow for Science of Synthesis Reference Library: Flow Chemistry in Organic Synthesis, Thieme, Stuttgart, Germany, 2018 (in press).

CONFERENCE PROCEEDINGS:Oral presentations:

Bottecchia, C.; Noël, T. Photoredox catalysis in continuous-flow microreactors, Photo4Future conference, 12-13th November 2018, Eindhoven, The Netherlands.


Bottecchia, C.; Noël, T. An automated continuous-flow platform for fluorescence quenching studies and Stern-Volmer analysis, Photo4Future winter school, 11-14th December 2017, Toledo, Spain

Bottecchia, C.; Noël, T. Photoredox catalysis in flow for chemical biology applications, CHAINS, Chemistry As Innovating Science, 5-7th December 2017, Veldhoven, The Netherlands

Bottecchia, C.; Noël, T. Evaluation of novel organic photoredox catalysts, Photo4Future summer school, 12-15th Jun 2017, Bordeaux, France

Bottecchia, C.; Hessel, V.; Noël, T. Development of batch and flow visible light photocatalytic methodology for application in chemical biology CHAINS, Chemistry As Innovating Science, 6-8th December 2016, Veldhoven, The Netherlands

Bottecchia, C.; Noël, T. Biomolecule functionalization via photoredox catalysis: cysteine arylation, Photo4Future winter school, 8-11th November 2016, Cordoba, Spain

Bottecchia, C.; Noël, T., Strategies for the site selective modification of amino acids via photoredox catalysis, Photo4Future summer school, 6-9th Jun 2016, Leuven, Belgium

Bottecchia, C.; Noël, T. TiO2 catalyzed aerobic oxidation of thiols in a photo-microreactor Photo4Future winter school, 7-10th December 2015, Leipzig, Germany

Bottecchia, C.; Noël, T. Photocatalytic continuous-flow fluoroalkylation of bioactive molecules, Photo4Future Summer School, 8-11th September 2015, Eindhoven, Netherlands

Poster presentations:

Bottecchia, C.; Noël, T. Photochemistry in flow for chemical biology applications, Dutch peptide symposium 8th June 2017, Eindhoven, Netherlands

Bottecchia, C.; Noël, T. Photochemistry in flow for chemical biology applications, Flow Chemistry Europe 7-8th February 2017, Cambridge, UK.

ACKNOWLEDGMENTS

The research presented in this thesis would not have been possible without the

help and support of all the great people I had the pleasure to work with in the past

four years.

First of all, I would like to express my sincere gratitude to my promotor and PhD

supervisor, Dr Timothy Noël. Tim, working with you has been a pleasure since day

one, and I cannot find enough words to thank you for the continuous support

you gave me throughout the PhD. You always found a way to motivate me, and

convinced me in believing in myself. I really appreciated your support and advice

both on a professional and personal level. You always cared about all your students

and I am very happy I got the chance to work in your group.

I also want to take the opportunity to thank Prof Volker Hessel for being my PhD

promotor in the first part of my PhD and for the interesting conversations we had

during the years (scientific and non).

A special thank you goes to Prof. Fausto Gallucci for accepting to take over the role

of co-promotor in the last moths of my PhD.

My gratitude also goes to all the other members of my graduation committee, who

took the time to give valuable suggestions on this dissertation and supported my

work.

I also would like to thank all the group members of the SPE group. A special thanks

goes to Marlies and Peter for always helping with all the analytical equipment and

orders, to Carlo for keeping us all safe in the lab and to Erik for his precious help

in finalizing our flow set-ups. To José and Denise, thank you so much for always

helping me with all the paper work and for being such great organizers (every

group activity you planned for us was really fun!).

To my lab mates: thank you all! I enjoyed every single day we spent in the lab and I

am very grateful to all of you for being such supportive and fun colleagues.

A Dario, chi mai avrebbe pensato che ci saremmo ritrovati non solo compagni di

universitá ma anche di PhD! Grazie mille di tutto, il mio dottorato non sarebbe

stato lo stesso senza di te. Sei una persona speciale sotto molti punti di vista e

sono sicura avrai una fantastica carriera nel mondo accademico. Grazie sia a te che

a Nadia per la vostra compagnia ed amicizia in questi anni: vi auguro tutto il meglio

per il futuro.


A Gabriele, che piacere é stato conoscerti! Grazie mille per il tuo supporto morale

in questo ultimo anno per me un po’ difficile: i tuoi consigli e le chiacchiere tra

una sigaretta e l’altra hanno significato molto per me. Hai davanti a te un futuro

brillante, e sono certa il tuo sogno di diventare professore si realizzerá presto.

To Koen, thank you for all the fun times we spent together. You have always been

very welcoming with me and I will always cherish all the nice moments we shared

at work and in our free time.

My sincere gratitude also goes to all my other colleagues: XiaoJing, Natan, Minjing,

YuanHai, Fang, Nico, Carlo, Sacha, Alessandra, Paola and Yiran. It was always a

pleasure to work with all of you and I wish you all the best for your future. During

these four years of PhD I also had the pleasure to supervise some students who

helped me in my research projects. Thank you Maarten, Mark and Ettore!

To all the other PhDs in the Photo4Future network: thank you! We started as a

group of strangers working on different topics and became a real group of friends.

I enjoyed every moment of our winter and summer schools and I really hope to

keep in touch with all of you in the future. Un grazie speciale ad Irini, per tutto il

bel tempo passato insieme sia ad Eindhoven che a Toledo. Sono molto orgogliosa

di essere diventata tua amica e ti auguro tutto il meglio per il tuo postdoc negli

Stati Uniti. To Philipp, I was always impressed by your ability to understand science

on a deeper level. Thank you for all the nice trips we took (Sevilla was the best!).

To Lana, Mark and Miro: thank you guys for taking care of me this last year. You are

a wonderful family and you deserve all the happiness in this world.

A special thanks goes to the “nothing cooler than absolute zero” team: thank you

Jasper, Erik, Robin, Jolanda and Koen for all the nice Thursday evenings at the

Carrousel!

To my friends from Ghent, Smita, Matthias and Eva: thank you for inspiring me in

pursuing a PhD and thank you for all the lovely times we spent together throughout

the years. You are all great scientists and I am proud of all of you.

Ai miei cari amici Luca, Irene, Leonardo, Athena e Marco: grazie ragazzi perché ci

siete sempre e comunque.

Ai miei genitori Carmen e Alberto un grazie infinito per avermi sempre supportato,

sopportato ed ascoltato.

ABOUT THE AUTHOR:

Cecilia Bottecchia was born on December 29th 1989 in Milan, Italy. In 2014, she

received her M.Sc. degree in Chemistry and Pharmaceutical Technologies at the

University of Milan, Italy. During her studies, Cecilia joined the group of Prof.

Annemieke Madder at Ghent University in Belgium, where she conducted her

master thesis on the topic of novel peptide-based hydrogels for drug delivery

systems. In 2015, she started her doctorate studies at the Eindhoven University of

Technology (TU/e) in the Micro Flow Chemistry and Synthetic Methodology group,

under the supervision of Dr. Timothy Noël and Prof. Volker Hessel. Her PhD project

focused on the development of novel photocatalytic strategies for biomolecule

modification, with a focus on continuous-flow microreactors as enabling

technology to carry out these transformations. The main results of her research

are presented in this dissertation. Her research was funded by the European Union

as part of the Photo4Future Marie Skłodowska-Curie Innovative Training Network

(ITN). During her last year of PhD, Cecilia was selected as one of the participants

for the Syngenta chemistry workshop for talented PhD students, held every other

year at the Syngenta headquarters in Stein, Switzerland.

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