Mechanism and electrostatic acceleration of

2-(1-hydroxybenzy1)thiamin fragmentation

Stewart Chan

A thesis submitted in conforrnity with the requirements for the degree of Master of Science,

Graduate Department of Chemistry, University of Toronto

O Copyright by Stewart Chan 1997

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To grandma, grandpa,

and my Father who keeps them,

Mechanism and electrostatic acceleration of

2-(I-hydroxybenqy~ thium*~ fragmentation

Stewart Siu-Cheuk Chan,

Department of Chemistry, University of Toronto,

Masters of Science, 1997

The condensation of thiamin and benzaldehyde gives 2-(1-hydroxybenzyl)

thiamin (HBzT). Met hylation of HBzT gives 1 ' -methyl-2-(1 -hydroxybenzy 1) thiamin

(MBzT). MBzT fragments irreversibly in water to form a phenyl thiazoIy1 ketone (PTK)

and a dimethyl pyrimidine (DMP). The difference between HBzT and MBzT is a single

fked charge at the NI' position of MBzT. This charge may promote the fiagmentation

of MBzT in several ways. If the transition state is carbanionic in character, inductive or

resonance stabiliiation by electron-withdrawing substituents on the pyrimidine portion

should increase the rate of fkagmentation. However, this is not observed for the

electron-withdrawing nitrobenzyl analogues that were studied. Thus, since

fiagmentation occurs only if Ni' is charged, a significant electrostatic effect is necessary

t o lower the barrier to fiagmentation. In constrast, benzoylformate decarboxylase uses

HBzT pyrophosphate as an intermediate without fiagmentation. Thus, the active site

should suppress fiagmentation, perhaps by specifically stabilizing charged structures.

Finally, the detailed pathway of fragmentation appears to involve an

unprecedented 1,2 proton transfer fiom nitrogen to carbon. This deserves carefùl

exarnination. Ab initia studies evaluate the feasibility of various routes for this proton

transfer. In addition, analogues of MBzT focused attention on the portion of the

molecule that control the fragmentation.

1 would like to thank the many people who have helped me through these past

two years. First and foremost, 1 thank Ron for teaching me how to be greatly

independent. Without him, this project would not have been possible. 1 would also like

to thank the many students and post-docs who have been a part of the Kluger lab. You

have made these past couple of years enjoyable. In particular 1 thank Pete, Vittorio, and

Jodi, not only for giving many helpfûl insights into problems, but more importantly, for

their fkiendship.

1 am greatly in debt to Professor Csizmadia. On top of allowing me to use his

SGI for most of the computationat work mentioned in this paper, he has been incredibly

encouraging, patient and understanding. 1 also thank him for his helpful comments and

suggestions with regards to this thesis.

1 thank Demy, Bruce and Leah for their helpfiil discussions.

To al1 of my fiiends outside of school who have been supportive of my endeavor,

1 want to say a great big thank you.

I also want to thankMom, Dad, Stan and Mary for always looking out for me. 1

wish to say a special thank-you to Mae who always lifted my spirits when things were

down. Lastly, 1 would like to thank my Lord who gives me strength to endure al1 things.

1. INTRODUCTION

1.1 Background 1.1.1 Metabolic function of thiamin 1.1.2 The thiamin ylide 1.1.3 Mechanism of thiamin caüdysis

1.2 Reactions involving HBzT 1.2.1 The benzoin condensation 1.2.2 Proposed mechanism of HBzT fragmentation 1.2.3 Effect of charges on fragmentation

1.3 Benzoylformate decarboxylase

1.4 Putative intermediate of CoA acetylation 1.4.1 Mechanism of acetylation 1.4.2 Problems with studies involving acetyl-TPP 1.4.3 Benzoylthiazolium analogues

2. CONFORMATIONAL STUDY OF HBZT

2.1 Background 2.1.1 Ab initio calculations involving thiamin analogues 2.1.2 Search for an appropnate mode1 compound

2.2 Geaeral methods 2.2.1 First tnincation 2.2.1.1 Geometry and behaviour of a simple thiazolium species 2.2.1.2 Effect of electron-withdrawing substituents on leaving group 2.2.1.3 Mechanism of proton transfer - More O'Ferrall diagram 2.2.1.4 Transition state optimization

2.2.2 Second tnincation 2.2.2.1 Geometry of an acyclic system 2.2.2.2 Tnangular transition-state 2.2.2.3 Stabilization of transition state

2.2.3 Third truncation 2.2.3.1 Geometry of third îruncation 2.2.3.2 Conformational analysis of 3" truncation 2.2.3.3 E enolate preferred over Z enolate 2.2.3.4 One-dimensional conformational analysis of the en01 and enolate 2.2.3.5 2D conformational andysis from selected energy minima 2.2.3.6 Solvation of proposeci reaction intermediates with water 2.2.3.7 Enolate and ylide resonance forms and correspondhg tautomers 2.2.3.8 Hydroxyl proton abstraction lads to fragmentation

2.3 Acyl thiamin analogues

2.4 Conclusions from conformationai study

2.5 Caveats

3.1 Overview of chapter

3.2 Experimental procedures 3 -2.1 Synthesis of compounds

3.2.1.1 hydroqbenzylthiazolium analogues 3.2.1.2 Benzylthiazolium bromides 3.2.1.3 3-(3-pyridy1)thiazolium derivatives 3.2.1.4 Benzoylthiazoles

3.2.2 Methods 3.2.2.1 Method involving aromatic aldehydes 3.2.2.2 Method involving pyrimidine analogues 3.2.2.3 Method involving MHET 3.2.2.4 Method involving benzoylthiazolium analogues

3.3 Discussion 3.3.1 Studies on fragmentation

3.3.1.1 Fragmentation tendency from rnodifjing phenyl moiety of HBzT 3.3.1.2 Effect of m o w n g pyrimidine moiety of HBzT 3.3.1.3 No fragmentation of MHET

3.3.2 Synthesis of N-alkyl-2-substituted thiazoles 3.3.2.1 N-methyl pyridyl thiazole 3.3.2.2 Amino group required for substitution

3.3.3 Acylthiazolium ions 3.3.3.1 N-alkylation of 2-acylthiazole 3 -3.3.2 Hyddysis of 2-benzoylthiazoles

3.4 Conclusions

References

Appendices

................................................................................. Figure 1.1 Thiamin and thiamin pyrophosphate 1 0 ............................................................................................................... Figure 1.2 The thiamin ylide I I

....................................................................................................... Figure 1.3 Benzoylthiazolium ion 2 1 ...................................................................................................... Figure 2.1 Torsional designa fions -24

................................................................................................ Figure 2.2 Structure of 1" tmncation 25 .................................................................................. Figure 2.3 Fragmentation plot of 1" hwncation 2 6

..................................................... Figure 2.4 Model of 1 including electron-withdrawing substituents 27 ........ Figure 2.5 Energy profile of the fragmentation of 1 bearing an electron-withdrawing substituen 28

........................................................ Figure 2.6 Putative intermediates of a hydride shifl mechanism 2 9 ........................................................................... Figure 2.7 More O 'Ferra11 plot of a proton transfr -30

............................................................................................. Figure 2.8 Mode1 for second hrrncation -32 ......................................... Fjgure 2.9 Putatjve shucture of trjangulm transition stde of 2.d fnrcalion 32

........................................................... Figure 2.1 O Comparison of energies of several analogues of 2 3 4 ................................................................................... Figure 2.11 Atypical ylide fiom hrrncation of 2 35

rd ............................................................................................... Figure 2.12 Strucf ure of 3 truncation -36 ........................................................................ ........... Figure 2.13 Torsional designa tions for 3 3 7

.............................................................................................. Figure 2.14 S-CrCÎaO torsion de#ned -38 ............................................................................................ Figure 2.15 Enolate and en01 forms of 3 3 9

................................................ Figure 2.16 Poten tial energy surface scans of mode1 compound 6, J 43 Figure 2.17 The energy profile of 3, - 3d ............................................................................................. 45

.............................................................................................................. Figure 2.18 Solvation models 47 ........................................................................... Figure 2.19 Enolate and ylide resonance forms of3 4 8

........................................................................................................ Figure 2.20 tautomers 3, and 3; 49 .......................................................... Figure 2.21 Fragmentation of 3,01 and fragmentation of Saohle 50

.................................................................................. Figure 2.22 an ti and syn orientation of 4 and 4'. 51 ................................................................................................. Figure 3.1 Substituent effects of HBzT 55

......................................................................... Figure 3.2 Resonance stabilization of transition state 64 Figure 3.3 Thiamin analogues -pyrimidine is replaced with various nitro-substituted benzyl groups .. 65

..................................................................... Figure 3.4 3-pyridylthiazolium analogue of thiamin 6 7

vii

A acetyl-TPP ADMT BDMT CoA DDW DMS AE EW) E HBzT HET HETPP HF 'HNMR MBzT MHET NMR PDB PTK QCLDB STO 21 22 .r3 TLC TPP TST w

angstroms (10"' m) 2-acetyl-thiamin pyrophosphate 2-acetyl-3,4-dimethylthiazolium iodide 2-benzoyl-3,4-dimethylthiazolium chloride coenzyme A distilled deionized water dimethyl sulphate Relative energy with respect to an arbitranly chosen minimum in kcaVmo1 computed molecular Hartree-Fock energy in Hartree atomic units energy 241 - hy droxybenzyl) thiamin 2-(1 -hydroxyethyl) thiamin 2-(1-hydroxyethyl) thiamin pyrophosphate hartree-fock proton NMR 1 '-methyl-2-(1 -hydroxybenzyl) thiamin methyl hydroxyethyl thiamin nuclear magnetic resonance protein databank phenylthiazolyl ketone, 2-benzoyl-4-methyl-5-(2-hydroxyethyl)thie quantum chemistry literature database Slater-type orbitals torsion 1 (see Figure 2.13) torsion 2 (see Figure 2.13) torsion 3, defined by S-C2-C*=-O (see Figure 2.13) thin layer chromatography thiamin pyrophosphate (thiarnin diphosphate) (2-trimethylsi1yl)thiazole ultraviolet

. . . vlll

Chapter 1

Introduction

I a II I L I VUUbLIUI I

1.1 Background

1.1.1 Metabolic function of thiamin

Thiarnin, vitamins Bi, is an essential nutrient that is present in al1 organisms.'

Deficiency in the vitamin can lead to the conditions known as beriberi and Korsakoff s

syndrome? The coenzyme, thiamin pyrophosphate (TPP), is utilked in living

organisms for the catalytic decarboxylation of a-keto acids (Figure 1.1). The absence

of thiamin leads to the accumulation of pyruvate (an a-keto acid) in the blood leading to

lactic acidosis. In plants, TPP is also important since it is used in the biosynthetic

pathway leading to branched-chah amino acids.' It is clear that thiamin is of vital

importance to living organisms. However, up until the 1950's the exact role that thiarnin

played in these reactions was not fully understood.' Although the general mechanism of

thiamin catalysis is now known, many other reactions involving thiamin remain unsolved.

thiamin

thiarnin pyrophosphate (TPP)

Figure 1. I Thiamin and thiamin pyrophosphate.

TPP-dependent enzymes catalyze the cleavage and formation of electrophilic C-C

and C-H bonds. One exarnple is pyruvate decarboqdase which converts pymvate to

acetaldehyde and carbon dioxide. It was observed that thiarnin, independent of the

enzyme, can itself catalyze the sarne reaction as pyruvate decarbo~~lase .~ From this

observation, Breslow dernonstrated that thiamin is in equilibrium with the ylide and

correctly proposed that the thiamin ylide is directly involved in the mechanism of

catalysis (Figure 1.2). It was rationalized that the enhanced acidity of the Czproton of

thiamin over normal carbon acids was due to the presence of two strongly electron-

withdrawing atoms (S and ?Y+) adjacent to the carbon centre.lo7" The close relationship

between non-enzymatic and enzymatic systems was realized soon afterward when a

covalent intermediate derived fiom TPP and acetaldehyde was isolated fiom an

enzymatic reaction. 5,6,12

Figure 1.2 me thiamin ylide.

1.1.3 Mechanism of thiamin catalysis

Thiamin is effective in catalyzing the cleavage of electrophilic C-C bonds in a-

keto acids and related compounds. This can be seen in the reaction of decarboxylation

of pymvate. The mechanism involves the addition of the thiamin ylide to the a-carbonyl

of pyruvate to form lactyl thiamin (Scheme 1)' Decarboxylation of lactyl thiarnin results

in the formation of a carbanionic species, termed the second carbanion (under this

naming convention the thiamin ylide is considered the first carbanion).13 The success of

al1 reactions which are catalyzed by thiamin lies in thiamin's capacity to stabilize the

charge of this second carbanion. Resonance structures can be drawn to show that the

thiamin serves as an electron sink for reactions which proceed through this carbanionic

int ermediate.

The intermediate formed fiom the decarboxylation of lactyl thiamin can be

viewed simply as the combination of thiamin with acetaldehyde. Therefore one rnight

expect analogous intermediates to be formed fiom the reaction of thiamin with other

aldehydes in alkaline solution. This is s h o w to be tme for various aldehydes and

isolation of the covalent species is possible. 14~15 Of significance to the reactions

discussed in this paper is the reaction of thiarnin with benzaldehyde, which under the

appropriate conditions gives 2-(1-hydrowbenzyl) thiamin (HBzT). l6

pyrwic acid 1

Scheme I

1.2 Reactions in volving HBz T

1.2.1 The benzoin condensation

The benzoin condensation converts two equivalents of benzaldehyde into

benzoin, a synthetically usefùl reagent. l7 Thiamin catalyzes this reaction by first forming

a covalent intermediate, HBzT (Scheme 2), through the nucleophilic addition of thiamin

to benzaldehyde. In effect, HBzT activates the hydrogen of the aldehyde towards

proton-removal since the conjugate base of HBzT is stabilized through resonance. This

-- - - - - - - - - - - - - - - - - - - - -- ---- "' ---- ---------- ------ '- -- ---- ---' --------- J ------- -- lactyl thiamin.14 The conjugate base of HBzT can then add a second equivalent of

benzaldehyde, which is followed by elimination of the benzoin product.

Scheme 2

1.2.2 Proposed mechanism of HBzT fragmentation

Since HBzT can be isolated as the interrnediate fiom the benzoin condensation in

good quantity, its has been the subject of numerous studies. One study by Washabaugh

reports the conversion of HBzT to thiamin and benzaldehyde which proceeds by general

base catalysis.'* However, a more carefil examination under analogous conditions

showed that a PTK and DMP (see Scheme 3) are also formed fiom HE3zT. 1920*21

Recently, studies involving 1'-methyl-2-(1-hydroxybenzyl) thiamin (MBzT) show that

irreversible fragmentation of this species occurs in acidic, neutral and basic solutions

with no detectable quantity of the elimination products of benzaldehyde and 1'-methyl

thiarnin." This observation suggests that the rate at which HBzT fragments is greatly

enhanced by protonation of the pyrimidine moiety. A detailed study of the fragmentation

of MBzT gives dues to the mechanism by which HBzT fragments.

catalysis in which a proton is necessarily removed in the rate-determining step.22 One

might conclude that deprotonation of the Cza proton (see Scheme 2 for proton

designation) is the rate-determining-step in the reaction. However, NMR studies show

the rate of exchange of this proton in deuterium oxide is faster than the rate of

fiagmentation. This implies that the removal of this hydrogen cannot be the rate-

deterrnining-step of the overall reaction. The rate-determining-step must therefore

involve the removal of a proton fiom an intermediate formed in another step. The rate-

determining-step could be the deprotonation of the hydroxyl proton fiom the en01 (or

enamine) that is formed fiom removal of the C*, hydrogen. However, if this were the

complete mechanism the reaction would be second order in hydroxide ion concentration,

but kinetic measurements show that the reaction is clearly first order in hydroxide ion.

By protonating the thiazole nitrogen in a step prior to fiagmentation, the overall scheme

is one that is in agreement with the observed rate. The mechanism shown below is one

which is consistent with all of the above observations (Scheme 3).

Although protonation of the thiazole nitrogen satisfies the observed kinetics of

the reaction, its effect on the system is not fùlly understood. Addition of a proton to the

thiazole nitrogen may facilitate a proton transfer to pyrimidine during fiagrnentation.

Altematively, if the importance of protonation at this position is to generate a positive

charge at the nitrogen centre then fragmentation can be rationalized in tenns of a

contribution fiom electrostatic effects.

1.2.3 Effect of charges on fragmentation

DMP

PTK

The rate of fiagmentation of HBzT is enhanced by the presence ofa positive

charge on the pyrimidine portion of the ~ o r n ~ o u n d . ~ This was demonstrated fiom

studies showing that MBzT in water leads exclusively to fragmentation products.

However, the effect of a single charge on the behaviour of MBzT is unclear. From the

proposed mechanism of HBzT fragmentation, a second positive charge must be

introduced into the system in order to satisfy kinetic observations. If these two charges

are necessary for fragmentation to occur then one might rationalize that fragmentation

occurs as a result of the through-space electrostatic field-repulsion effect of the remote

charges. It is also possible to rationalize the observed fragmentation in ternis of a

significant inductive effect. The charge on pyrimidine provides a significant inductive

fi-agmentation. If inductive effects are sufficient to cause the species to fragment then

strongly electron-withdrawing substituents on the pyrimidine part of the system should

also lead to the corresponding fiagmentation products. Observations from this study

suggest that the pyrimidine moiety of HBzT and similar analogues require the charge in

order for the fragmentation to occur.

1.3 Benzoylformate decarboxylase

The TPP-dependent enzyme, benzoylformate decarboxylase, is present in bacteria

that make use of mandelic acid as a carbon source? This enzyme catalyzes the

decarboxylation of mandelic acid through the formation of the 2-mandelyl thiamin

pyrophosphate intermediate. The optimum pH range of this reaction is between 6 and

8? However, since the reaction must proceed through the formation of the conjugate

base of HBzT pyrophosphate (HBzTPP), fiagmentation of this intermediate is expected

to occur." The fact that fragmentation is not observed suggests that structural features

of the enzymatic site are present to promote catalysis. One way for the enzyme to

promote catalysis is to encourage elimination of benzaldehyde. This can be

accomplished by directing a proton towards the Cz, position of the intermediate.

Therefore the carbanion that is formed from the decarboqlation step is converted to

HBzTPP before fragmentation cm occur (Scheme 4). A base that is provided by the

enzyme may then deprotonate the hydroxyl hydrogen resulting in the elimination of

benzaldehyde and regeneration of thiamin.

Scheme 4

1.4 Putative intermediate of CoA acetylation

1.4.1 Mechanism of acetylation

Acetylation of coenzyme A (CoA) is catalyzed by the TPP-dependent enzyme,

pyruvate dehydrogenase. The mechanism by which this takes place has been proposed

to occur as follows: 1) addition of thiamin to pyruvate with subsequent decarboxylation

of the covalent intermediate, 2) oxidation of lactyl thiamin and acetylation of lipoate, 3)

acetylation of CoA (Scheme 5).25*26"

H&%O - O ___C)

O OPP

OPP

HS-CoA

HS K

H3C S C O A HS LA

LA

Scheme 5

The mechanism involving the transfer of an acetyl equivalent fkom hydroxyethyl

thiamin pyrophosphate (HETPP) to lipoic acid has been the subject of some debate.

There are two possible pathways for this reaction -- paths A and B (Scheme 6). In path

A, HETPP is oxidized to give Zacetyl thiamin pyrophosphate. Nucleophilic attack by

the reduced form of lipoic acid at the carbonyl of the intermediate and subsequent

cleavage of the bond gives acetylated lipoic In path B HETPP combines

with the oxidized form of lipoic acid, presumably through a simple nucleophilic addition,

to give S-acetylated lipoate without the formation of an acylthiazolium intermediate.

Some evidence for the formation of the acyl intermediate is provided by Frey and

coworkers who have synthesized acetylTPP and observed that it undergoes rapid

hydrolysis in aqueous solution.28

Pynivate +

TPP

Scheme 6

1.4.2 Problems with studies involving acetyl-TPP

Measurement of the rate of hydrolysis of acetyl-TPP is complicated by the

presence of two other foms that are identifiable in mildy acidic solution (Scheme 7).29

The carbinolamine form at low pH can be avoided by making an analogue lacking this

arnino group which would give a system where hydrolysis is not complicated by

intramolecular participation of the amine. Studies involving 2-acetyl-3,4-

dimethylthiazolium iodide (ADMT) show that it is only in equilibrium with a hydrate

form leading to hydrolysis rate measurements which are less cornpli~ated.~~ However,

detailed studies of analogues of acetyl-TPP have been difficult because these species

hydrolyze rapidly. In recent studies, these acetylthiazolium derivatives have also been

observed to undergo a reversible deuterium exchange in the acetyl portion of the

thiazolium species, indicating that enolization cornpetes with hydrolysis.3'~2 As a result,

alternative compound for the study of the mechanism of hydrolysis of acylthiazoles is

benzoylthiazolium chloride. Such aromatic-substituted analogues can also be used to

probe electronic effects.

Carbinolamine

Scheme 7

1.4.3 Benzoylthiazolium analogues

The synthesis of 2-benzoyl-3,4-dimethylthiazolium chlonde (BDMT) has only

been accomplished in extremely low yields.26 The yield-limiting synthetic step involves

methylation of the acylthiazole. Steric hindrance fiom the 4-methyl group is the likely

cause of this problem. The yield is expected to improved by using an analogue that lacks

this methyl group. This modification of the ring may also allow for the synthesis of

larger acylthiazolium derivatives because of the less stericaily hindered system. In

particular, the synthesis of 3-benzyl-2-acylthiazoIium species and similar derivatives

rate of hydrolysis of the acylthiazolium ion (Figure 1.3).

Figure 1.3 BenzoyIthicxzolium ion

Studies involving BDMT show that the compound cleaves in aqueous solution to

3,4-dimethylthiazoliurn iodide and bemic acid.16 These results are consistent with the

findings of Breslow and McNelis who found that ADMT hydrolyzed to give a compound

with a spectrum characteristic of 3,4-dimethylthiazolium i~dide. '~ BDMT reacts rapidly

in water and methanol. Gas chromatographie analysis of methyl benzoate from

methanolysis of BDMT shows that the half-life of the reaction is less than 15 seconds.

Calorimetnc measurements suggest a half-life of less than 5 seconds. Studies involving

the less hindered thiazole would presumably reduce the reactivity of the compound

because the acylthiazolium species is more stable.

Chapter 2

Conformational study of HBzT

2.1 Background

2.1.1 Ab initio calculations involving thiamin analogues

The quantum chemistry literature database (qcldb) shows no ab initio

calculations perfonned on thiamin or thiamin-related systems. The size and complexity

of thiamin and reactions pertaining to the compound have only allowed for calculations

at the serni-empirical level. 34,35,36 However, the present study lends itself to ab iniiio

methods since a truncated model of the thiamin system is representative of the actual

system that it rnimics. The truncated model is also cornputationally manageable. In

addition, it would be difficult to gain information on the intricate details of a proton

transfer reaction, which is the reaction under study, in any other way.

The present study examines the theoretical feasibility of the fragmentation of

HBzT via the mechanism proposed by ~ l u ~ e r . ' In order to fully understand the proton

transfer that occurs in the final step of this mechanism, it is necessary to locate the

transition state of the reaction. To accomplish this task, it was first necessary to locate

minimum energy conformations of the reaction intermediates. Transition state

optirnizations of structures based on these geometries is then possible. Although these

transition state have yet to be optimized, their structures have been closely

approximated. The geometry of the reaction intermediates that were constructed and

optimized can be used to locate the transition states to fragmentation in the future.

2.1.2 Search for an appropriate model compound

As for al1 intense ab-inifio computations, truncation of the molecule under study

is required in order to create a system that is computationally manageable. However,

key components of the system must remain in order to make a valid comparison between

the original and modified systems. For these reasons, several different models were

generated and examined. This section sumrnarizes the calculations performed for each of

charge and interatomic distances, are found in the appendices.

2.2 Generalmethods

Ail calculations were perfonned with the GAUSSIAN 9237 or 9438 programs on

either a Silicon Graphics Indy or Indigo 2 work station with IEUX operating system

versions 5.2, 5.3, or 6.0. Rasmol 2.5 or 2.6 was used as a graphicd interface to view

optimized structures. Gnuplot 3.5 was used to plot 3D energy surface diagrarns.

Cdculations were initially performed at the STO-3G level of theory on al1 mode1

systems. Further rigid one- and two-dimensional conformational scans were performed

at the 3 -2 1 ~ ~ ' level of theory on 3-21 G Mnimized geometries. Intemal rotational scans

were performed about three torsions (Figure 2.1). All subsequent local minima obtained

fiom these scans were fùlly re-optirnized at the 3-2 1 G level of theory to determine if

these apparent minima were non-degenerate. Wherever appropriate, transition state

optimizations were attempted.

Figure 2.1 Torsional designations.

2.2.1 .l Geometry and behaviour of a simple thiazolium species

The general susceptibility of 3-alkylthiazolium ions to fiagmentation can be

studied by employing a 2,3-disubstituted thiazole model (Figure 2.2). The structure

mimics the putative precursor to HBzT fragmentation and gives information about the

geometry of this species. The optimized geometry of 1 shows non-planarity at the C2

carbon which suggests that the ylide resonance contributor is favoured over the enolate

for this model. More is said about the relative stabilities of the ylide and enolate in a

later section (see section 2.2.3.7).

Figure 2.2 Structure of Ist hwncation.

Perturbation of 1 by lengthening the &C-N bond at fixed increments allows for

the determination of energies at each point along the reaction path. This process of bond

lengthening should give energy values that mirnic the fiagmentation pathway of HBzT.

The calculated C-N bond distance corresponding to the minimum energy geometry is

-1.5 A. Using this value as a guideline, the distance between carbon and nitrogen was

constrained and minirnized at increments between 1.6 - 2.8 A. AU energies obtained

were made relative to the optimized minimum energy structure. A plot of these energies

against the corresponding C-N bond distance shows that a smooth increase in energy

occurs at al1 points of the plot up to the point of fiagmentation. A steep drop in energy

occurs just d e r a C-N bond distance of 2.55 A (Figure 2.3).

C H dlstanœ (Angstroms)

Figure 2.3 Fragmentation plot of lst huncation

2.2.1.2 Effect of electron-withdrawing substituents on leaving group

The enhanced rate of fragmentation of HBzT is a result of the pyrimidine moiety

being charged. One hypothesis to explain this phenornenon is that the electron-

withdrawing inductive effect of the charged region stabilizes a negatively charged

transition state in the rate-determining-step of fiagmentation. If this is true, electron-

withdrawing substituents that can help stabilize a negatively charged centre should also

help promote fiagmentation. Modifications of 1 were made with halogens on the methyl

carbon (Figure 2.4).

Figure 2.4 Maiel of 1 including electron-withdkawing subslituents.

The system was optimized while maintaining the C-N distance constraint. The

energies obtained from these optimizations were plotted against the corresponding C-N

bond length (Figure 2.5, see also appendix A). A lowering of the energy barrier to

reaction is observed on changing to an electron-withdrawing leaving group. A

comparison of the energy differences of the reactant and the highest energy structure

(the estimate of the transition state) shows the magnitude by which the transition state is

stabilied. It is found that energy barrier to fragmentation products is halved on simply

replacing a hydrogen of the leaving group with a chlorine (85 kcal for l ~ , -45 kcal for

Ici). The transition state is also earlier for the electron-deficient systems occurring

approximately 0.3 A sooner for Ici than for lH. A comparison of la with la shows that

lcl produces a greater lowering of the activation energy even though chlonne is less

electronegative than fluorine and would therefore stabilize the carbanion to a lesser

extent in the transition state. This behaviour is likely the result of significant electronic

participation of the halide, but this phenornenon was not examined in detail. What is

important is that an electro-negative substituent is able to stabilize a negatively-charged

transition state significantly.

Figure 2.5 Energy profie of the fragmentation of 1 bearing an electron-withdlawng

substituent: + JH; Il F; A ICI

2.2.1.3 Mechanism of proton transfer - More O'Ferrall diagram

Two different mechanisms of hydrogen transfer in the rate-determining-step of

HBzT fiagmentation are considered here. One involves a hydride shift, and the other a

proton abstraction. If the fiagrnentation occurs as the result of a hydride shift (Figure

2.6), the pathway could proceed through an El, Elcb, or E2 mechanism. The El reaction

involves the formation of a positive charge at the carbon centre as the methyl group

cleaves before the hydnde transfer. This is in contrast to the observation that electron-

withdrawing substituents on the leaving group increase the rate of reaction. A hydride

shift following an Elcb mechanism generates a pentacoordinate carbon bearing a negative

charge. Although consistent with the hypothesis that a negative charge forms at the

carbon centre, a pentacoordinate carbon cannot be an intermediate in a reaction

mechanism.

Figure 2.6 Putative intermediates of a hyaide shift rnechanism

A second possible mechanism involves a proton transfer Rom nitrogen to carbon

(Figure 2.7). An El pathway to fragmentation would f o m a pentavalent carbon.

However, an Elfb mechanism creates a system with carbanionic character at the

methylene carbon. Electron-withdrawing substituents on carbon stabilize the charge at

this centre. This is consistent with the observation that electronegative halogens stabilize

the transition state of the rate-deterrnining-step in the fragmentation reaction. In the

proposed mechanism of HBzT fragmentation the negative charge resides at a benzylic

position that is stabilized through resonance by the pyrimidine ring. The positively

charged nitrogens of MBzT and N-protonated HBzT do not provide furlher resonance

stabilization of charge; they provide an inductive stabilization of the transition state.

increasing

- 1 distance I

- increasing N-H distance

Figure 2.7 More O 'Ferra22 plot of a proton tramfer

Requiring the thiazole nitrogen to be protonated in the mechanism of

fiagrnentation leads to another important consideration. That is, the success of the

reaction of fiagrnentation can be viewed as being dependent on two competing factors.

First, the proton is necessarily situated in the vicinity of the reaction centre therefore the

nitrogen must accommodate a positively charged proton. There must also be a great

enough electron-withdrawing ability of the leaving group to accommodate the negative

charge that is fomed upon C-N fragmentation. The rate of reaction increases only if the

electron-withdrawing strength of the substituent balances these effects.

2.2.1.4 Transition state optimization

Attempts were made to find the transition state of this reaction at low levels of

theory (STO-3G and 3-21G) starting with the geometry corresponding to a C-N bond

structure of 1 it most closely resembles the distance in the transition state (Scheme 8).

However, al1 attempts to optimize a transition state structure were unsuccessful. One

reason for the failure to find an optirnized transition state species may be that it does not

exist. However, it is likely that this mode1 system does not properly refIect the HBzT

system and therefore fiagmentation of 1 may not be expected to occur. In particular the

methyl substituent is not a good analogue of the aromatic pyrimidine system of HBzT.

Scheme 8

2.2.2 Second truncation

2.2.2.1 Geometry of an acyclic system

A change to the geometry of 1 was required to provide a system that more

accurately reflects the actual HBzT system. The result is 2, a larger systern, but one in

which the methylene carbon is in an allylic position that better represents the aromatic

pyrimidine than the first truncation (Figure 2.8). To reduce computational time, an

acyclic analogue of the thiazole moiety was used. The orientation of the en01 was

constructed as shown and rnay be different from the corresponding intermediate of the

HBzT fkagmentation in solution. However complications arose in the optirnization of

these compounds that required the en01 to be in a Z conformation (where the

conformation is defined by S-C2-C2,-O).

Figure 2.8 M d e l for second truncation

2.2.2.2 Triangular transition-state

In order for fiagmentation to occur for the en01 of 2 it is possible that structure

of the transition-state is one in which the proton on nitrogen is located between the

nitrogen and methylene carbon (Figure 2.9).

~ T S

Figure 2.9 Putative structure of triangular transition state of znd lnrfation

Protonation and geornetry optimization of 2 followed by the re-orientation of the

protons gives 2 ~ s . Minimization of this structure results in its complete fiagmentation to

the expected products (Scheme 9). Although this is oniy a very arbitrary designation of

the coordinate position of the proton, it shows that a triangular geometry (N-C-H)

causes enough strain in the system to result in fiagmentation. Attempts at optimizing the

transition state for this species were unsuccessfiil.

Scheme 9

2.2.2.3 Stabilization of transition state

Several analogues of 2 were optimized at STO-3G and 3-21G levels of theory

and their energies were compared to give relative energy differences of the calculated

species (Figure 2.10, see appendix B). The energy of the putative reaction intermediates

are shown. The transition state has been approximated by carbanionic species and is

valid i fa signincant negative charge exists in the actual transition state of fragmentation.

A drastic lowering in the energy of this transition state (-400 kcaWmo1) is observed by

placing a charge at the allylic Ntrogen. This large stabiliiation of the carbanionic species

is consistent with the observations of the first tmcation. However, it seems that a charge

at the allylic nitrogen of 2 stabilizes the transition state to a greater extent than simple

electron-withdrawing groups on 1. This is consistent with the results found fiom

experiments perfonned on HBzT analogues where electron-withdrawing substituents on

the pyrimidine portion do not give fiagmented material. This implies that a signincant

electrostatic effect assists in the fragmentation process.

- -

O. 5

Reaction coordinat@

Figure 2.10 Cornparison of energies of several analogues of 2. Energies for the reactants of above species are in Appendix B.

A unique intermediate that bears a negative charge on the carbon adjacent to the

ring nitrogen (an ylide) was optirnized, but would certainly not exist to any appreciable

extent in an aqueous environment (Figure 2.11). However, the fact that there is a

minimum energy structure of this geometry suggests that any negative charge formed on

the methylene carbon is partially stabilized by both the adjacent positive charge on

nitrogen as well as the allylic position of the anionic centre. It is likely that the bridging

methylene carbon in the transition state bears a significant negative charge. This is

consistent with the results obtained fiom the first tmncation.

Figure 2.11 A typical ylidem truncation of 2.

The acyclic structure creates complications during structure optirnizations. The

negatively charged oxygen in the vicinity of the positively-charged nitrogen abstracts a

proton fiom either the proton on nitrogen or the alkyl proton to form the enolic species.

The reason for this behaviour is due to the flexibility of the acyclic structure which

provides an extra dimension of rotation over a cyclic system allowing for the enolate to

corne into a favourable geometric orientation to abstract the dkyl proton. Another

concern that became apparent was that calculations in the gas-phase may not give

satisfactory conditions which properly reflect the actual system of HBzT fiagmentation

in aqueous solution. Solvation of the negatively charged oxygen may, in fact, lead to a

relatively stable zwitterionic intermediate (see section on solvat ion effects).

2.2.3 Third truncation

2.2.3.1 Geometry of third truncation

The model system which most closely approximate the HBzT molecule is one

which includes an allylic representation of the pyrimidine moiety with a terminal arnino

group in the position of the NI' ring nitrogen of HBzT. The cyclic structure is

maintained to reduce flexibility. Yet the model is sufficiently small for low-level

calculatiow. The cyclic HBzT analogue, 3, was optirnized at the 3-2 1G level of theory

and this geometry was used as a starting structure for later minirnizations.

Figure 2.12 Structure of srd truncution

The senes of reactions that lead to fiagrnented products are shown (Scheme 10).

Although the properties associated with the immediate precursor to fiagrnentation (the

zwitterionic enolate) are of the most interest, the structure and energy of the

intermediates that form the entire reaction sequence give a general overview of the

complexity of the whole reaction. The following scheme shows the proposed

intermediates and is the template on which the calculations were based.

HO- -

HO- - b&T

Scherne 10

2.2.3.2 Conformational analysis of 3rd truncation

Each of the intermediates in the scheme above has more than one rotational

degree of freedorn. Global energy minimum structures were deduced by locating al1

minimum energy conformations of each of these species and cornparhg them. There are

three important dihedral angles to consider in these structures, labeled r l , 22 and 23

(Figure 2.13). Keeping the cyclic thimle structure avoids having to consider an extra

rotational degree of fieedom (the torsion about the S-C2 bond).

Figure 2.13 Torsional designutions for 3. 'R ' represents the various C2 substitaents of

various intemediate qecies.

2.2.3.3 E enolate preferred over Z enolate

Al1 intermediates containing an en01 moiety were given a fixed 23 (S-C2-C2,-O)

torsional angle of either 0" (Z) or 180" (E) since only at these two torsional angles is the

system properly onented to allow for maximum x-MO overlap Figure 2.14, see also

'Enolate and ylide resonance forms and corresponding tautomers', pg. 48). The

unconstrained optimization of 3 resulted in 23 torsional values which do not difEer

significantly fiom the values of 0" and 180°, hence justifjing the use of constraints. A

rigid rotational scan about 23 employing the optirnized geometry of 3 showed that

energy minima occur near the 0" and 180" conformations. The E orientation is slightly

-.

for other intermediates of similar geometry.

3E 32

Figure 2.14 S-CrC2& torsion defied. JE: 23=18O 3 32.' ~ 3 = 0 O

The observed preference for the 3E conformation may be due to the electrostatic

attraction of the positively charged nitrogen centres and the negatively charged oxygen

of the enolate intermediates. In a system where a large bullcy group replaces the

aldehydic hydrogen of this mode1 compound (for example, a phenyl ring), one may

reason that a torsion of 23=180° is favoured because of the steric bulk of the substituent.

As a result of this observation, the subsequent calculations involving rotations about

torsional angles -cl and 22 were performed while keeping 23 fixed at 180".

2.2.3.4 One-dimensional conformational analysis of the enol and enolate

In order to obtain global energy minima of each of these species, rigid rotational

scans about z l and 22 were first performed employing either the STO-3G or 3-21G basis

sets. In each case, the global minimum energy structure was found by obtaining the

coordinate matrices of the structures that were in the vicinity of minimum energy wells,

as shown in the rigid conformational scans. These structures were fblly relaxed and re-

optimized at the 3-2 1 G level to give global energy minima.

Figure 2.15 Enolate and enol f o r s of 3.

Several torsional scans were performed on the en01 and enolate of 3 in order to

detennine local and global minima (Figure 2.15). Energy minima are easily observed in

the 2-dimensional plots (energy versus torsional angle) for each of these. Starting fkom

an arbitrary local energy minimum, ngid potential energy scans were performed about

the various torsional angles in order to determine the number and location of

conformational minima in the system. After each individuai torsional profile was

examined and minima located, 71 and 1.2 were held fixed at these values and a rigid scan

was performed again on 23 (Figure 2.16).

Energy (Hartrees)

Energy (kcaumol)

Energy (Hartrees)

k 2

k 2 k O> ii

w W P Vi

Energy (kcaumol)

- roo

Figure 2.16 Potential energy surface scans of model compound (3,,$. Rigid rotational scms perfomed about: a) r3; b) 71; c) r.2; d) rotational scan about 71, with 73 held at minimum foundfrom plot (a); e) rq 23 torsions flxed at minima found@om previous scans, then scanned about 71. Rotational scan perfomed on enolate of 3 about.$ 73; a) 22

Relaxation of torsional constraints and fil1 optimization of the minimum energy

conformers of the en01 and enolate results in global minimum structure for the enol.

However, the enolate was still able to abstract a proton fiom a neighboring site (the

proton on nitrogen) to give the corresponding enol. It became evident that the charged

enolate species would not optimize to give a stable geometry in which the oxygen is

unprotonated. As previously mentioned, energies fiom the calculations are of species in

the gas-phase. A solvation model where the negatively charged oxygen is suppressed by

a molecule of water also shows a lack of structural stability in the enolate (see section on

solvation, pg. 46). The apparent high-energy enolate intermediate could imply that it is

very near to the transition state of fragmentation. Alternatively, abstraction of the enolic

may not be possible.

2.2.3.5 2D conformational analysis from selected energy minima

The set of models which are analogues of the putative intermediates in the

fragmentation reaction of HBzT are shown (Scheme 11). En01 models are used since

unwanted proton abstraction occurs in models involving the enolate ion. Energy minima

were determined for each of these compounds through a preliminary optimization at the

STO-3G level of theory. The z l and z2 torsion values were fixed at values between 0°

to 360° at 30° increments (see Figure 2.13 for torsional definitions). A single point

energy calculation was performed at the STO-3G level of theory at each point.

Therefore 144 energy values were obtained from 144 different rigid geometries of each

model. This process was repeated for each of the compounds 3i - 3n.

Scheme I I

allows for the determination of global and local minima. To better visualize the areas of

minimum energy, a contour map is included to display areas where potential global

minima may lie (see Appendix G). These energies and the corresponding structures were

compared to determine the number of non-degenerate energy minima (Figure 2.17). Any

apparent local minima were fully re-optirnized at the 3-2 1 G level of theory to determine

the energy of the relaxed structure. The lowest energy optimized structure of al1

apparent minima corresponds to the global energy minimum structure (see Appendix C).

1 2 3 4 5 6

Reaction Pathway

Figure 2.1 7 The energy profile of 3i - 3,

The overall reaction is only slightly thermodynarnically favourable (1.5 kcal), an

amount comparable to a torsional rotation about a single bond. The energy barrier to

found. However, if the reaction pathway is to proceed through the proposed dicationic

intermediate, 3,, a substantial rise in energy of 260 kcaVmol must occur. This is the

energy difference between and 3,. The value is exaggerated since the charges present

in the system would be partially solvated in aqueous solution. However, the energy of 3,

is so large relative to starting reagents and products that it is reasonable to assume that

only a smdl perturbation in this system is required to drive the species to fiagrnent.

From Hammond's postdate, if it cm be assumed that the enolate is close in energy

relative to 3,, then the structure of the enolate should also be similar to 3,. A single

point energy calculation of the enolate, the geometry of which is based on the geometry

of 3 , would give a good indication of the energy of the transition state. Such a

calculation is attempted and is described in a later section (see section 2.2.3.8 'Hydroqd

proton abstraction leads to fiagrnentation').

2.2.3.6 Solvation of proposed reaction intermediates with water

The fiagmentation of HBzT occurs in aqueous solution, hence, the solvation of

this system most likely plays a major role in the stabilization of the proposed high-energy

intermediate. Optimization of 3,,,i,1, may be possible by introducing a molecule of

solvation. A single water molecule is used to solvate charge in al1 of these calculations.

It is proposed that deprotonation of the en01 occurs in the rate-determining step

of the reaction. Up until now attempts have been made at optimizing the enolate of 3 to

determine whether this high-energy intermediate exists. Al1 of these attempts have failed

which suggests that the rnechanism is one in which deprotonation of 3, is concerted with

fiagmentation. However there is still the possibility that deprotonation is occurring at a

site other than the hydroxyl proton of the enol. Since the enolate can also be represented

by its ylide resonance form (see Figure 2.19), it is conceivable that deprotonation in the

rate-determiiing-step is actually occumng fiom proton abstraction of the C2= proton and

not the en01 proton. Optimization of 3 with a molecule of solvation positioned at the

oxygen compared with the energy of solvation with the molecule of water at the Cz

environment (Figure 2.18). Attempts to optimize these systems at the 3-21G level of

theory were successful for only two of the set of species defined (see Appendix D).

Even the introduction of a single charge at the m s t remote site causes an unpredictable

proton transfer to occur and optimization of the structure fails to give a meaningful

result .

Figure 2.18 Solvation models

A cornparison of the energies of the two solvated forms shows a greater

stabilization for 30 over 3c by 14.8 kcaWmo1. This diflerence in energy is assumed to be

similar for SoH and 3rn in which the remote nitrogen is protonated. No estimate can be

made of 30HH and 3cm because the charged ring nitrogen is expected to stabilize an

adjacent negative charge greatly. In this case it might be that a solvated ylide structure is

thermodynarnically favoured over a solvated enolate.

The enolate form of 3 can atso be drawn to show that the negative charge can

reside on the C2 carbon of the thiazole moiety, which would give the ylide of 3 (Figure

2.19). By minimizing an arbitrary starting conformation of the enolate or ylide and

noting the geometry about the Ca carbon, one can infer the preference of the system to

adopt either one of the two resonance forms; if a planar trigonal geometry about the C2

carbon results, then the system favours the enolate form, however, a tetrahedral

geometry would suggest that the ylide is the preferred orientation. It is evident that the

Cz carbon of 1 is tetrahedral which suggests that the ylide is the greater resonance

contributor. Unfortunately, 3,,,1,(, does not optimize and so its major resonance

contributor cannot be determined fiom o b s e ~ n g its geometry.

enolate y1 i de

Figure 2.19 Enolute undylide resonance fonns of 3.

Examination of the energies of the en01 of the tautomers of 3 (a proton at the oxygen vs.

the proton at the C2 position) have been determined at the STO-3G and 3-21G levels of

theory Figure 2.20, see Appendix D). 3,* is thermodynamically more stable than 3, by

2.4 kcal/mol. If this energy difference is significant, then a pre-equilibrium

tautomerization of 3, to 3,. is possible. This implies that the rate-determining-step in the

mechanism of fragmentation of HBzT is actually the C2 proton abstraction from 3,. and

not the proton abstraction fiom 3,.

Figure 2.20 tautomers 3, and 3;

2.2.3.8 Hydroxyl proton abstraction leads to fragmentation

Elongation of the C-N bond of the third truncation should also produce an

energy curve that can describe the fiagmentation of HBzT in a similar manner to that of

the first truncation (refer back to Figure 2.1). By using the minimum energy structure

obtained fiom the conformational scan of an earlier calculation, the incremental

lengthening of the C-N3 bond at distances between 1.6 and 2.6 A with 0.2 A increments

gives a potential energy curve showing the energy required to break that bond (Scheme

12). As noted earlier, optimization of a species which contains an unsuppressed negative

charge on the oxygen results in a proton migration to give the corresponding enol. The

enolate is a highly unstable intermediate in the reaction pathway suggesting that and

deprotonation of the corresponding en01 may even be concerted with fiagmentation.

Scherne 12

Up to now, the enolate has been noted as a reactive species, but has not led to

the fragmentation of the truncated molecule as predicted. Starting with the global

energy minimum geometry of 3,, an approximate structure of the enolate with the same

C-N3 distance can be generated. At these distances removing the enolic proton fkom the

- - - V Y . -*

species wilI approximate an energy for the enolate at the vanous points along the

putative reaction pathway to fiagmentation. Full unconstrained optimization of this

species without any bond or torsion constraints shows the tendency of the stretched

molecule to either revert back to a moIecule with the C-N3 bond intact or fiagrnent into

two parts (see Appendix E).

1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2

C-N distance (A)

Figure 2.21 Fragmentation of Jm01 andpagmentation of 3,0he; end STO-3G: ienol STO-3G/3-2lG (geometry optimization at STO-3G followed by a single point

energy caledation at 3-21G); Aenolate STO-3G/3-21G.

The plot of bond distance versus the calculated energy of the system at various

levels of theory shows that the enolate requires considerably less perturbation in order

for fragmentation to occur (Figure 2.2 1). Conversely, the protonated en01 resists

fragmentation completely, up to a bond distance of 2.8 8. Without constraints a full

optimization of the geometry of the enolate shows that it fragments irreversibly at a bond

fiagrnentation is in exact agreement with the base-catalyzed mechanism observed for

~ z T .

2.3 Acyl thiamin analogues

Energies for acylthiazole and acylthiazolium systems have been determined for

structures optimized at the 3-21 G and 6-3 lG* levels of theory (see Appendix F). The

preferred conformation of the Cz substituent of acylthiazolium species can be rationalized

as the combination of electronic and steric effects of the system. The thermodynamicaily

preferred orientation of the carbonyl is anti to the ring suf i r for the species in which the

thiazole nitrogen is not alkylated. However, the preferred orientation changes to syn

upon introduction of a simple substituent at the thiazole nitrogen.

4 R = lone pair of electrons

4' R=CHs

Figure 2.22 anti and syn orientation of 4 and 4'

The hydrolysis of acylthiazolium species is rapid. This has been attributed to the

positively charged nitrogen which is able to stabilize the negative charge at the C2

position upon hydrolysis. However, an equally important consideration is the orientation

of the C2 substituent. Since the synthesis of these thiazolium species is normally

accomplished by alkylating acylthiazole, the orientation of the Cz carbonyl for synthetic

acylthiazolium ions is likely to be syn to the ring sulfur. For example the methylation of

- - - *

in direct interaction with the methyl group. The result is an observed rate of hydrolysis

which is greater than the rate of hydrolysis of acetylthiazolium species. The rate

acceleration caused by sterics is not present in the hydrolysis of acetylthiamin since the

formation of the acyl species is proposed to occur fiom the oxidation of the hydroxyallqd

precursor. Al1 efse aside, the acetylthiamin species would be oriented in an anti fashion

because of the presence of the charged nitrogen. Hence, studies relating the hydrolysis

or reactivity of acylthiazolium ions to the reactivity of native acetylthiamin should be

questioned with this in mind.

2.4 Conclusions from conformational study

Ab i i t io calculations on the thiamin system have been perforrned and

summarized in this paper. Al1 local and global minimum structures of 3 and derivatives

of 3 have been deterrnined. It has been found that the truncated structure of 3, is a

reasonable representation of a precursor of the fragmentation of HBzT. Fragmentation

of the N-protonated dicationic mode], 3 , is observed to occur only with deprotonation

of the hydroxyl proton of the enol. A simple representation of the pyrimidine moiety of

HBzT, 1, sufficiently details the effect of electron-withdrawing substituents on the

leaving group. Analogues of an acyclic structure, 2, were analyzed. It was observed

that a carbanionic species with a negative charge at the methylene carbon optirnizes to

give a local energy minima. This is evidence for the ability of the species to contain a

negative charge at that position, consistent with the theory that the fragmentation of

HBzT goes through a transition state with carbanionic character at the allylic carbon.

Atternpts at the solvation of tenolate with a single water molecule was

unsuccessful. A greater number of molecules of solvation seems necessary to solvate the

enolate. Solvation was possible with the singly-charged enolate and ylide species, 30 and

3c. The solvated en01 is thermodynamically more stable than the solvated ylide.

However, the conjugate acid of the ylide is lower in energy than the enol. This suggests

that tautomenzation of the en01 to form the conjugate acid of the ylide occurs in a pre-

- -- proposed intermediates on the pathway to fiagmentation have been determined. These

mode1 compounds can be use as starting structures for the purposes of locating any

transition states that may exist in the pathway to fragmentation.

The conformation of 2-acylthiazoles and 2-acylthiazolium species have been

compared. It is found that introduction of a charge at the ring nitrogen reverses the

orientation of the C2-substituent. This implies that the orientation of the Cz-substituent

is different with respect to the approach taken to synthesizing the acylthiazolium species.

The orientation of the substituent has a significant effect on the observed rate of

hydrolysis of the acylthiazolium compound.

2.5 Caveats

The synthesis of HBzT results in a racemic mixture with chirality at the CÎa

carbon. However, for calculational purposes formaldehyde was used in place of

benzaldehyde therefore there is no stereocentre formed at this carbon on condensation of

the thiamin fragment with forrnaldehyde.

The 23 torsion was constrained to give two distinct geometries. This torsion is

assumed not to change significantly upon conformational rotations about the other two

torsions. The torsion is also assumed to remain constant on perturbing (lengthening) the

C-N bond that leads to fiagmentation.

A molecule of water was introduced into the de-protonated structure of 3 in

order to stabilize the negative charge residing on the oxygen of the formaldehyde

residue. Solvation was necessary since rninor torsional perturbations about 23 resulted in

the abstraction of the methylene carbons adjacent to the thiazole nitrogen. This addition

is justified by the fact that the reaction occurs in aqueous environment. However, this is

onIy one molecule of water out of an entire solvation shell.

Chapter 3

Synthesis and analysis of HBzT and Acylthiamin

3.1 Ovewiew of chapter

The direction taken in studying the detailed mechanism of thiamin fragmentation

was to observe and rationalize the behaviour of analogues bearing eIectron-withdrawing

or electron-donating substituents near the site of fragmentation. This was accomplished

by making modifications, first to the phenyl group at the Cz, position, then to the

pyrimidine rnoiety of thiamin (Figure 3.1). The tendency for these analogues to m e n t

was observed and trends, with respect to the strength of the electron-withdrawing

substituent, were noted. It was found that the synthesis and isolation of some of the

hydroxybenzyl thiazolium species was difficult. A rationalization for these difficulties is

given in the present chapter.

Figure 3.1 Substituent effects of HBzT

3.2 Ekperimental procedures

3.2.1 Synthesis of compounds

Thiamin chloride hydrochloride was provided by Novopharm. Aromatic

aldehydes and organic reagents were purchased fiom Aldrich and used without fiirther

purification. NMR spectra were mn on a Gemini 200 spectrometer. TLCs were run on

-

Perkin Elmer Lambda 2 or Lambda 19 spectrophotometer employing quartz cuvettes

(1x1) in a thermostated ce11 holder. The spectrophotometer was interfaced with an BM-

PC compatible computer running Perkin-Elmer PECSS or W C S S data acquisition

software. The procedure for the synthesis of benzylthiazolium chloride, including the

synthesis of precursors, is a culmination of work by several authors. Modifications to

these existing synthetic procedures are included and described.

3.2.1.1 hydroxybenzylthiazolium analogues

2-(1-Hydroxybenzy1)thiamin chloride (HE~zT)' (5); To thiamin chloride hydrochloride

(10.0g) was added water (-20 mL) until the thiamin just dissolved. Methanol(40 mL)

was added to the solution and the pH was adjusted to 8 with 5M NaOH. An excess of

benzaldehyde (35 mL) was added, the reaction vesse1 seded, and the solution left to stir

for 6 hours. The solution was quenched with concentrated HCI to a pH of 2. Ethyl

acetate (10 rnL) was added to the solution to give two immiscible layers. The solution

was seeded with previously recrystallized HBzT and lefi to crystallize at 4OC. After 15

hours the product crystallized between the layers as an off-white solid and was filtered

by vacuum with 3 washes of cold 5% HCl solution, and the filter cake was dned under

vacuum giving 5 (2.2 g, 19%); 'HNMR 6 @O) 2.3 1 (s, 3H), 2.39 (s, 3H), 3-17 (t, 2H,

J=6.2), 3.90 (t, 2H, J=6.2), 5.25 (dd, 2H, J=16.1), 6.38 (s, lH), 6.60 (s, lH), 7.18 (m,

5 w -

1'-methyC2-(1-hydroxybenyl)thiamin perchlorate (MBzT)~ (6); 5 (4.5 g) was

dissolved in a minimal amount of water ( 4 5 mL) with warming. Sodium bicarbonate

was added to bring the pH of the solution to 6.5. Calcium carbonate was added to

scavenge any remaining acid. Dimethyl sulphate OMS) (1.5 rnL) was added drop-wise

and the solution was stirred for 45 minutes. An aqueous solution of sodium perchlorate

(4.5 g in 5 mL H20) was added forming a thick white precipitate. The solution was

filtered under vacuum and the filter cake was recrystallized fiom water. The crystals that

a *

give 6 (2,8 g, 60%); 'HNMR 6 2.38 (s, 3H), 2.54 (s, 2H), 3.22 (t, 2H, J=5.9), 3.52 (s,

3H), 3.96 (t, 2H, J=5.9), 5.31 (dd, 2H, J=18), 6.40 (s, IH), 6.49 (s, IH), 7.30-7.50 (m,

SHI.

2-benzoyl-4-methyl-5-(2hydroxyethyl)thide (phenyl thiazole ketone, PTK)~ (7); 6

(2.4 g) was dissolved in H20 (70 mL) with warming. The pH of the solution was

adjusted to 8 with 0.5 M KOH. The pH of the solution was monitored on and off and

readjusted to pH 8 when the pH had fallen below 7. M e r 24 hours a yellow oil formed

fiom solution and was extracted with ethyl acetate (3 x 30 mL). The extracts were

combined and dried with anhydrous magnesium sulphate. The dry solution was filtered

by gravity and the filtrate was concentrated under reduced pressure to give a yellow oil,

7 (650 mg, 54%); 'HNMR 6 2.50 (s, 3H), 3.10 (t, 2H, J=6.2), 3.91 (t, 2H, J=6.2), 7.45-

7.68 (m, 3H), 8.38-8.47 (m, 2H).

2-(1-Hydroxybenzy1)thiazole (8)'; To 7 (100 mg) was added an excess of sodium

borohydride (30 mg in 2 mL H20). The solution was stirred for 24 hours. The solution

was extracted with chloroform (5 x 2 m.). The chloroform layer was concentrated

under reduced pressure to give a hard off-white solid, 8 (70 mg, 70%); 'HNMR 6 2.16

(s, 3H), 2.89 (t, 2Y J=6.4), 3.69 (t, 2H, J=6.4), 5.92 (s, lH), 7.29-7.42 (m, 5H).

2-Benzoyl-3-benzyl-4-methyl-5-(Z-hydroxyethyl)thiazolium chloride (9) (attempted

synthesis); Benzyl bromide (60 mg) was added to 8 (70 mg). The mixture was heated

to 100°C for 6 hours in a constant temperature oil bath. A solid mass with a very deep

red colour resulted. The solid was dissolved in hot absolute ethanol and filtered by

vacuum. The NMR spectrum of the solid product showed that little or no reaction had

occurred.

I

The benzyl bromide was added to 4-methyl-5-(2-hydroxyethy1)thiazole (1.5

equivalents) and the mixture was heated to 100°C in a thennostated oil bath for 6 hours.

The mixture was allowed to cool to forrn a solid mass. The sample was triturated with

hot absolute ethanol and filtered by gravity. The filter cake was dried under vacuum to

yield the thiazolium chloride:

(a) 3-benzyl-4-methyl-5-(1-hydroxyethy1)thioi bromide (10);

@) 3-(4-nitrobenzyl)-4-methyl-5-(l-hydroayetolium bromide (11), 'HNMR

6 2.33 (s, 3H), 3.10 (t, 2H, J=6.0), 3.79 (t, 2H, J=6.0), 5.80 (s, 2H), 7.4 1 (d, 2H,

J=9.6), 8.24 (d, 2H, J=9.6), 9.81 (s, 1H);

(c) 3-(2,4-dinitrobenzyl)-4-methyl-5-(l-hydroxyethyl)thiolium bromide (12), 1 HNMR 6 2.41 (s, 3H), 3.19 (t, SH, J=5.6), 3.88 (t, 2H, J=5.6), 6.20 (s, 2H), 7.26

(d, lH, J=9.8), 8.57 (m, lH), 9.17 (d, lH, J=3.0), 9.76 (s, 1H).

1'-methyl-2-(1-hydroxyethy1)thiamin chloride (MHET) (13); 2-(1-hydroxyethyl)

thiamin chloride (HET) was prepared by the method of Washabaugh et al? HET (1.3

g) was dissolved in a minimal amount of water (5 mL). Sodium bicarbonate (0.2 g) was

added to bring the pH of the solution to 6.5. Calcium carbonate was added to scavenge

any remaining acid. Dimethyl sulphate (1.5 mL) was added drop-wise and the solution

was stirred for 45 minutes. Sodium perchlorate (100 mg) in water (1 mL) was added

and the solution was cooled at 4" for 15 hours. Methanol that was present in the

solution was removed by rotary evaporation. The solution was fieeze-dried to give 14

(850 mg, 65 %); 'HNMR6 1.72 (d, 3H, J=7.3), 2.61 (s, 3H), 2.67 (s, 3H), 3.19 (t, 2w

J=5.6), 3.78 (s, 3H), 3.91 (t, 2H, J=5.6), 5.44 (dd, 2H, J=6.9), 5.52 (s, lH), 7.21 (s,

lm.

3.2.1.3 3-(3-pyridyl)thiazolium derivatives

3-brornomethyl-1-methylpyridine (14); HBr (48%) was added to 3-pyridyl carbinol

(15g) while keeping the mixture cold in an ice-bath. The solution was allowed to warm

to room temperature over the course of 15 hours, then placed again in an ice bath and

NaI/acetone test precipitated NaBr inicating the presence of an alkyl bromide. The

sample was concentrated under reduced pressure to give a powder. The resulting solid

(5 g) was dissolved in water (10 mL) and the pH of the solution brought to 6 with

NaHC03. DMS (5 rnL) was added dropwise and the solution was allowed to stir for 2

hours. Sodium perchlorate (150 mg in 1 mL water) was added to the solution and it was

left to crystallize overnight. The crystals that formed were filtered to give a slightly

impure sample of 15, ' H N M R ~ 4.87 (s, 2H), 8.01 (t, lH, J=7.8), 8.46 (d, lH, J=8.2),

8.68 (d, lH, J= 8.1), 8.76 (s, 1H).

Tetrabutylammonium 2-aminonicotinate (15); To a solution of 2-aminonicotinic acid

(470 mg) in water (25 mL) was added tetrabutylammonium hydroxide (1M) until the pH

of the solution became 7. The solution was extracted into dichloromethane (5 x 20 rd).

The extracts were recombined and concentrated under reduced pressure to give an oil, 9

(870 mg, 74%).

Tetrabutylammonium Zaminonicotinoxide (16); To 15 (200 mg) was added THF

(20 mL) until it al1 dissolved. LiAlH in THF (0.8 mL, 1M) was added drop-wise and the

solution allowed to stir for 45 minutes, becorning deep yellow and then a light yellow

colour. The solution was slowly quenched with water (3 mL), filtered to remove solids,

and rinsed with fresh THF. The filtrate was concentrated under reduced pressure to give

a red oil, 16; 'HNMR 6 0.98 (t, 12H, J=7.5), 1.30-1.55 (m, 8H), 3.18-3.32 (m, 8H),

3.55-3.72 (t, 8H, J=5. l), 4.58 (s, 2H), 6.51-6.62 (m, lH), 7.24-7.35 (m, lw, 7.90-8.00

(m, 1H).

Zaminonicotinic chloride (17);' 16 (60 mg) was slowly dissolved in a solution of

thionyl chloride (3 mL) and stirred for 1 hr at room temperature. Excess thionyl chloride

was removed by rotary evaporation to give a red oil, 17. Attempts at separating 17 fiom

tetrabutylammonium chloride using dowex 50W cation-exhange resin (4% or 8%

Gl U;sJllIinGUJ 1 GDulLGU i l 1 wllar appcuzi L u u~ ilyu1 u l y m ~ u1 I I y l ~ l u u i è j au I I I C L ~ U L ~ U I ~

mixture of 17 and the corresponding alcohol by 'HNMR.

3.2.1.4 Benzoylthiazoles

2-Benzoylthiazole (18); Following the procedure of p on do ni^' et al., 2-

(trimethylsily1)thiazole (TST) (3 10 mg) was diluted with dichloromethane (40 mL).

Freshly distilled benzoylchloride (720 mg) in dichloromethane (30 mL) was added to the

TST solution and left to stir 15 hours. NaOH (40%, 50 mL) was added with a catalytic

amount of tetrabutylarnrnonium hydroxide (< 5 pL) to the solution to hydrolyze excess

benzoylchloride. The layen were separated with 3 washes with DDW and the organic

layer dried with MgSO4. Removal of organic solvent under reduced pressure resulted in

a clear oil, 18 (205 mg, 55%), 'HNMR 6 7.46-7.70 (m, 3H), 7.73 (d, 1% J=3.4), 8.1 1

(d, 1Y J=3.4), 8.43-8.52 (III, 2H).

2-Benzoyl-3-methylthide (18); To 17 (100 mg) was added iodomethane (.5 mL).

The solution was placed under argon, sealed with a rubber septum and the temperature

kept at 60°C for 2 hours. The solution become dark red in colour. The reaction mixture

was allowed to cool to room temperature and then left for 24 hours upon which clear

white crystals formed in solution. The mixture was washed with diethyl ether (3 x 3 rnL)

and the resulting crystals fieed of solvent by rotory evaporation. The 'HNMR spectrum

showed no presence of the desired product.

3.2.2 Methods

3.2.2.1 Method involving aromatic aldehydes

Thiamin chloride hydrochloride (1 .O g) was dissolved in water (5 mL). Methanol

(10 mL) was then added and the solution maintained at pH 8 by the drop-wise addition

of sodium hydroxide (lN). The aromatic aldehyde (3 equivalents) was added ta the

solution containing thiamin and the combined solution was Ieft to stir for 6 hours at 40°C

temperature were dissolved in a small amount of methanol before addition to the reaction

vessel. The solution pH was allowed to fa11 to 6 and then was stirred for an additional

hour. Solid precipitate was filtered off and the filtrate was extracted with ethyl acetate

(2 x 20 a). The ethyl acetate portions were combined and concentrated under reduced

pressure to give either a solid or oily residue. The 'HNMR spectrum of the resulting

product was taken to determine the presence or absence of a PTK. The reaction was

specificalIy carried out once in the absence of the aromatic aidehyde. No peak in the

'HNMR corresponding to fragmentation product was observed.

3.2.2.2 Method involving pyrimidine analogues

The method employed for determining the fragmentation tendency of 3-aryl-2-

hydroxybenzyithiazolium chlorides is similar to that used for the aromatic aldehydes.

The 3-aryl-2-hydroxybenzylthiazolium chlonde (1 .O g) was dissolved in water (5 mL).

Methanol(10 mL) was added and the solution was adjusted to pH 8 by the drop-wise

addition of sodium hydroxide (lN). Benzaldehyde (3 equivalents) was added to the

solution and left to stir for 6 hours at 40' C in a thermostated oil bath. M e r the reaction

had completed the solution pH was 6. Solid precipitate was filtered fkom the solution

and the filtrate was extracted twice with ethyl acetate. The ethyl acetate portions were

combined and concentrated under reduced pressure to give either a solid or oily residue.

The 'HNMR spectrum of the resulting product was obtained to determine the presence

of fragmented material.

3.2.2.3 Method involving MHET

An aqueous solution of MHET was adjusted to pH 8 and lefi to stir for 15 hrs.

The solution was extracted with ethyl acetate (3 x 10 ml;). The combined extracts were

drkd with anhydrous magnesium sulphate. The filtered solution was concentrated under

reduced pressure to give an oil which was analyzed by 'HNMR to determine the

presence or absence of the ketone.

3.2.2.4 Method involving benzoylthiazolium analogues

The benzoylthiazole was dissolved in f?eshly distilied acetonitrile to a

concentration of 1 x lu3 M. Bufer solutions of ionic strength 0.1 M were made to pH

3.8, 5.0 and 8.3 with acetate buffer. The W holder was maintained at 40°C with a

constant temperature bath. The buffer with an injection of acetonitnle was used as the

reference. The benzoylthiazole solution (100 pl,, final ce11 concentration is -1 x 10" M)

was injected wîth an eppendorf pipette into the UV ce11 containing the buffer (2 mL) and

the spectrum of the sarnple recorded. The spectrum was followed over the course of 24

hours. The procedure was repeated using an unbuffered solution of HCl at pH 2, but no

change in absorbante occurred over the 24 hour period.

3.3 Discussion

3.3.1 Studies on fragmentation

3.3.1 .l Fragmentation tendency frorn modifying phenyl moiety of HBzT

Numerous attempts to isolate hydroxyaryl thiamin intermediates by the method of

Okal were unsuccesshil (with the exception of HBzT). However, since a mixture

containing thiamin and the arornatic aldehyde is expected to be in equilibrium with the

hydroxyaryl thiamin intennediate in aqueous solution, the same intennediate should also

be expected to fiagrnent under the conditions observed for HBzT fiagrnentation (Scheme

13). Combining thiamin with aromatic aldehyde without isolation of the intermediate

and noting the presence of fragmentation products in the work up step shows whether

the hydroxyaryl intermediate dissociates to the expected fi-agmentation products. The

reaction can be monitored by noting distinct triplet peaks in the 'HNMR spectnim of the

sample that correspond to the PTK, a product of fragmentation. Since it is possible that

these peaks correspond to thiamin itself (thiamin gives a peak in the same region,

between 3.5 ppm and 4.0 ppm in DzO), a controlled test was performed in the absence of

thiamin was observed. A table of the products from thiamin and substrate shows the

fiagmentation tendency of thiamin with various aidehydes (Table 3-1). Although little

information can be learned about the transition state for fiagmentation involving these

aldehydes, the fact that fiagmentation is observed indicates that the reaction is not only

isolated to HBzT systems.

Table 3-1 fiagmentation of hydkoxyaryl thiamin

Aromatic aldehyde X Does it Fragment? ... f..................................... .................................................................................. ...*....*............ tolualdehyde 4-CH3 Y benzaldehyde 4-H Y anisaldehyde 4-OMe Y p-hydroxybenzaldehyde 4-OH N O-nitrobenzaldehyde 2-NO2 N p-nitrobenzaldehyde 4-NO2 N

3.3.1.2 Effect of modifying pyrimidine moiety of HBzT

Modification of the pyrimidine moiety of HBzT allows for the determination of

the electronic requirements that are necessary for fragmentation to occur. Replacement

of the pyrimidine fiom HBzT with a benzylic group bearing a strongly electron-

IV LLLLUL U V V L L I ~ ~ . ~ U O L L L U I ~ A L L . 111 b 1 1 w pu1 u u l v1 L l 1 U p U P l L l V l 1 J lllQJ UG G l l V U ~ l l L U D L Q U l l L L G L11G

transition state. In addition to inductive effects these substituents provide a resonance

stabilization of the putative carbanionic transition state (Figure 3.2). However, it was

found that fiagmentation occurs only for the cases where a charge is present on the

pyrimidine moiety.

Figure 3.2 Resonance siabilization of transition state

The three thiarnin derivatives examined bore an increasing number of electron-

withdrawing nitro substituent on the benzyl moiety. Zero, one, and two nitro

substituents are present on 10, 11, and 12 respectively (Figure 3.3). No fiagmentation

products were recovered from the reaction of any of these substituted species under the

conditions known to firipent HBzT. Assuming that the thiamin analogue and

benzaldehyde are in equilibrium with the hydroxybenzyl form, it appears that in addition

to electron-withdrawing inductive effects an electrostatic repulsive effect fkom a charged

pyrimidine system contribut es significantly to the destabilization of the precursor to

fiagmentation which (ie. the dicationic species). This is in agreement with the results

obtained fiom the optimization of the dicationic species in which the energy of this

species is found to be unusually high.

Figure 3.3 Thiarnin anuZogues -pyrimidine is replaced with various nitro-substituted be& groups

3.3.1.3 No fragmentation of MHET

Karimian and coworkers were able to synthesize MHET by the reaction of HET

with iodomethane." An alternative procedure that is described in this paper uses

dirnethyl sulphate in aqueous solution to generate the desired product. This species,

analogous to HBzT, was studied by placing it under the same conditions known to

fragment HBzT. MHET was found to resist fiagmentation in aqueous solutions of pH 6

to 8. The reason is that the C2a proton of MHET is not very acidic. Unlike HBzT, the

conjugate base of MHET is not stabilized through resonance. Moreover it does not form

the en01 (or enamine) precursor required for fiagmentation. Although MHET does not

fragment to any appreciable extent in water, it is possible that it eliminates acetaldehyde

to give the corresponding aldehyde and methylthiamin products. This reaction would be

analogous to the elimination of benzaldehyde fkom HBzT. However, it is unclear if this

elimination reaction occurred since the sample analysis performed in this study did not

include a close examination and identification of al1 of the products in the reaction

mixture. It is certain, however, that no fiagmentation products are fonned.

A desired analogue of HBzT is one in which the pyrimidine moiety of thiamin is

replaced by an aromatic ring bearing various substituents. Two approaches were taken

in synthesizing these compounds. The first approach involved coupling of the thiamin

analogue with aldehyde, a reaction that should be analogous the benzoin condensation

and give the desired product in reasonable yield. However, isolation of these species

proved to be difficult because a good technique couId not be found to separate product

from excess starting materiai. The second method involves the nucleophilic substitution

of hydroxybenqlthiazole ont0 an appropriate alkyl (or aryl) chioride. The synthesis by a

nucleophilic displacement of a good leaving group by the ring nitrogen of 2-

hydroxybenzyl-4-methyl-5-(2-hydroxyethyl)thiazole (4) yields no product. Other

attempts at alkylating the hydroxybenzylthiazole (with iodomethane for exemple) results

in poor yields (<5%). This section provides some reasons b e h d the difficulties in the

synthesis of the desired intermediates.

3.3.2.1 N-methyl pyridyl thiazole

Fragmentation of HBzT is greatly enhanced when a charge is present on the

pyrimidine moiety of the species. If this charge were the main cause for the species to

fragment, then an analogue where the N-methylpyrirnidine of HBzT is replaced with N-

methylpyridine should also fiagrnent under similar conditions. The N-rnethylpyridine

analogue is expected to have properties similar to MBzT and therefore is expected to

fiagrnent in base (Figure 3.4). The first step in the synthesis of this N-methylpyridine

analogue is the coupling of picolyl bromide to 4-methyl-5-(2-hydroxyethy1)thiazole to

make the 3-pyridyl thiazole. The synthesis was unsuccessfùl even after many attempts

under various reaction conditions. Methylation of the picolyl bromide before

nucleophilic substitution of the bromide with the thiazole also failed to give the desired

compound. The difficulty in forming the coupled adduct may lie in the necessity for the

electrophile to contain an amino substituent to activate the leaving group.

Figure 3.4 3-pyridyZthiazoZium analogue of thiamin

3.3.2.2 Amino group required for substitution

Coupling the appropriate aminopyrimidine broMde and thiazole gives thiamin

bromide in good yield. If the key to this synthesis lies in the presence of an amino-group

on the electrophile, then one would expect the coupling 2-amino nicotinic bromide with

thiazole to also give the desired product. It is possible that an arnino substituent or other

activating group is required to be present on the pyridyl group in order for coupling to

occur. If the success of the coupling of these compounds is due to the amino

substituent, then a mechanism for the synthesis of these compounds can be inferred. The

mechanism is one that involves amino-assisted displacement of the leaving group pnor or

during the addition of the nucleophile (Scheme 14). The leaving group is activated

towards displacement by a nucleophile. Evidence for this mechanism cornes from the

difficulty in isolating 2-arninonicotinic chloride. These attempts have led to the

hydrolysis of the chloride to give the corresponding alcohol which suggests that the

electrophile is quite reactive.

Scheme 14

67

It is possible to isolate a relatively pure sample of the aromatic acylthiazolium

compound, PTK, from MBzT fiagmentation. This adduct allows for the study of

reactions pertaining to acylthiazoles. In particular, acylthiazolium ions are known to

hydrolyze to the thiazolium and corresponding acid, so the hydrolysis of acylthiazoles,

under appropriate conditions, is also expected to generate sirnilar products (Scheme 15).

N-alkylation of the PTK generates a charge at the nitrogen of the thiazole moiety and

creates a benzoyl analogue of acetylthiamin. The effect of a phenyl group on reaction

rates of acylthiazolium species cm give some insight into the detailed mechanism of

acetylthiamin hydrolysis.

Scheme 15

N-alkylation of PTK (an acylthiazole) would give a desired benzoylthiazolium

that can be used in the study of the hydrolysis of acylthiazolium ions. However, attempts

at coupling the acylthiazole with benzyl chloride to fonn the acylthiazolium were

unsuccessfùl. Ingraham, Lienhard and others also found the synthesis of similar species

to be difficult. DifficuIty in N-allqlation of the thiazole is likely the result of the ring

nitrogen (i.e. the nucleophile) being in close proximity (P) to the carbonyl. The

- - - -

electron-withdrawing carbonyl group.

Although a charge could not be fixed at the thiazole nitrogen from N-alkylation,

it is possible that protonation of the nitrogen is sufficient to generate a charge that can

promote hydrolysis of the acylthiazole in water. It is expected acylthiazoles themselves

hydrolyze in rnildly acidic solution since protonation of the thiazole nitrogen should give

a system which is analogous to the putative acylthiazolium precursor to the acetylation

of lipoic acid.

3.3.3.2 Hydrolysis of 2-benzoylthiazoles

The hydrolysis of ayclthiazolium compounds has been measured for some

acylthiarnin analogues (Frey, Daigo). Comparison of the rates of hydrolysis of two

similar acylthiazolium species shows that the rate of hydrolysis of ADMT is faster than

the methanolysis of BDMT (Table 3-2, Table 3 - 3 ) . " ~ ~ Rate data for the hydrolysis of

BDMT is not known because the rate at which it occurs is extremely fast (hydrolysis

occurs upon solvolysis). The hatf-life of the reaction has been estimated to be Iess than

one second.

Ifthe charge on the thiazole nitrogen is the main factor in promoting the

hydrolysis of acylthiazoles, then benzoylthiazoles rnight also be expected to undergo

hydrolysis in acidic solution. It has been found with two different benoylthiazoles that

these compounds do not hydrolyze in acid or base, a finding consistent with Lienhard's

work with acetylthiazoles. This leads one to consider the reason for why hydrolysis does

not occur. The overall rate is the combination fiom hydrolysis of charged thiazole and

the hydrolysis of uncharged thiazole. Consider the acylthiazole that is protonated at low

pH. This species is analogous to acylthiazolium compounds. The charge on thiazole is

expected to increase the rate of hydrolysis because the leaving thiazole generates a

negative charge at the C2 position. This charge is stabilized by the positively-charged

nitrogen in the same way the negative charge on the thiamin ylide is stabilized by its

adjacent charge. However, at low pH the concentration of hydroxide ion is small and

small. The rate constant for the hydrolysis of the uncharged species is also expected to

be small since the negative charge of the leaving group is not stabilized. Therefore no

hydrolysis occurs for acylthiazoles at these higher pHs.

Table 3-p6 hyalrolysis of acetyl P P

Bulyer 1 Concn (mM) 1 pH 1 k,bs (min') 1 t~ (s) ......... ............. ..........*.......... ......... ....... . .......*....... ............. ............ ....... .....m..- .. ........

Table 3-323 methanok'ysis of BDMT

acetate MOPS

3.4 Conclusions

The fragmentation of HBzT is greatly enhanced by a charge that is present on the

pyrimidine. However, strongly electron-withdrawing substituents in the ortho or para

positions in a benzylthiazolium system are not enough to stabilize the putative

carbanionic transition state. This implies that a significant electrostatic effect exists to

promote the dissociation of MBzT into a PTK and a DMP. This effect should also be

present in other systems such as 3-(3-pyridyl)thiazolium, and fragmentation of this

species observed. However, synthesis of these cornpounds are difficult. The problem

lies in the coupling of the thiazole with the chloropyridine. The presence of an arnino

substituent strongly activates the leaving group of the electrophile. This is evidenced by

the ease by which the arninopyridine hydrolyzes in water.

Benzoylthiazoles do not hydrolyze in the pH range of 2 to 8. At low pH the

thiazole nitrogen is protonated, creating a species that is analogous to acylthiazolium

50.0 50.0

5.1 7.14

0.0868 0.673

479 62

hydrolysis because the leaving thiazole forms a negative charge at the CZ position which

is stabilized by the positively-charged nitrogen. This species is analogous to the thiamin

ylide. However, at low pH the concentration of hydroxide ion is small and no hydrolysis

is observed. At higher pHs (> -5) the concentration of charged thiazole is small.

Hydrolysis of the uncharged species is possible, but the rate for this reaction is also

expected to be slow since the negative charge of the leaving group is not stabilized.

Hydrolysis of the benzoylthiazole does not occur.

In conclusion, the reaction of irregular compounds, such as HBzT, are very

sensitive to destabilization by the presence of multiple positive charges. The detailed

mechanism of the fragmentation process remains a formidable question.

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Appendices

Appendk A : Model cornpound of HBzT resulting from the 1' truncation A - 1

Appendix B : Model cornpound of ~ i ï r e s u l t i n g / r o m the yd &uncationnR5

Appendix C : Model coinpound of HBzT resulting from the fd &uncation-cl 0

Appendk D : 30,3C and 3v' 0-29

Appendijc E : 3,d and 3,,r, E-32

Appendi* F : 4, und 4&- F-34

Appendix G : Energy su face diagram of various HBzT undogues 6-36

Appendk H : Graphical representations of optimized mode2 compounds - H-42

Table A-1 Table A-2 Table A-3 Table A-4 Table A-5 Table A-6 Table A-7 Table A-8 Table B-1 Table B-2 Table 23-3 Table B-4 Table B-5 Table C-1 Table C-2 Table C-3 Table C-4 Table C-5 Table C-6 Table C-7 Table C-8 Table C-9

Energies of 1" truncation A-1 MeH RHF/STO-3g atomic charges A-1 MeH RHF/6-31g* atomic charges A-2 MeF RHF/STO-3g atomic charges A-2 MeCi RHF/STO-3g atomic charges A-3 MeH RHF/6-31g* coordinate matrix A-3 MeH RHF/6-31 g * interatomic angles A-4 MeH RHF/6-31g* distance matrix (A) A-4 2"d huncation RHF/3-2lg energies B-5 2 RHF/3-21g coordinates and charges B-6 2 MF/33-21g distance maMx (4 . B-7 RHF/3-21 G single point energies for the analogs of 2 B-8 2 RHF.13-2lg interatomic angles B-9 3 RNF/3-21g global minimum energies C-10 Reaction path energies (2-10 RHF/STO-3g singlepoint energies for torsional scans of Ji C-Il 3i R?IF/3-21g coordinates and charges C-12 3i RHF/3-21g distance mat& (4 C-13 RHF/3-21g single-point energiesfor torsional scans of 3if C-14 3ii RnF/3-21g coordinates and charges C-15 3ii RHF/3-21g distance matrix (4 C-16 RNF/STO-3g single-point energies for torsional scans of 3iii C-17

Table C-1 O 3rir RHF/3-21g coordinates and charges C-18 Table C-II 3i3 RHF/3-21g distance matrix (4 C-19 Table C-12 RHF/STTO-3g single-point energies for torsional scans of 3, C-20 Table C-13 3iv RHF/3-21g coordinates and charges C-21 Table C-14 3iV RHF/3-21g distance matrix (4 C-22 Table C-15 RHF/STO-3g single-point energies for torsional scans of3, C-23 Table C-16 3, RHF/3-21g coordinates and charges C-24 Table C-17 3, RHF/3-21g distance matrix ( ' C-25 Table C-18 R H F m - 3 g single-point energies for torsional scans of 3d C-26 Table C-19 3d RHF/3-21g coordinates and charges C-27 Table C-20 3, RHF/3-21g distance matrix (4 C-28 Table D-1 30, 3= RHF/3-21g energies 0-29 Table 0-2 3 , 3 , p comparison of energies 0-29 Table 0-3 3,t MF/3-21g coordinates and charges 0-30 Table 0 -4 39 W / 3 - 2 1 g distance matrix 0-31 Table E-1 Con~parison of and 3,,1d, energies E-32 Table E-2 3,01 RHF/STO-g/3-21g atomic charges E-32 Table E-3 3,,1d, RHF/STO-3g/3-21g atomic charges. E-33 Table F-1 4 and 4' energies F-34 Table F-2 4 atomic charges F-35 Table F-3 4' utomic charges F-35

III

Figure A. 1 I numbering system for calculations A-1 Figure B. 1 2 numbering system for calculations B-5 Figure C. 1 3i numbering system for calculations C-11 Figure C. 2 3it num bering systern for calculations C-14 Figure C.3 3i13 numbering system for calculations C-17 Figure C.4 3, numbering system for calculations C-20 Figure CS 3, numbering system for calculations C-23 Figure C. 6 3a numbering systern for calculations. C-26 Figure D. 1 3,. numbering systern for calculutions 0-29 Figure F. 1 4anti numbering system for calculations F-34 Figure F.2 4'anti numbering system for calculations F-34 Figure G. 1 3i energy surface diagram G-36 Figure G.2 energy surface diagram G-37 Figure G.3 3133 energy surface diagram G-38 Figure G. 4 3i, energy surface diagram G-39 Figure G. 5 3, energy surface diagram G-40 Figure G. 6 3d energy surface diagram G-41 Figure N.1 1 H-42 Figure H.2 2 H-43 Figure H.3 3i H-44 Figure H. 4 33 H-45 Figure H.5 3& H-46 Figure H. 6 3,, H-47 Figure H. 7 3, H-48 Figure H. 8 3,. H-49 Figure H. 9 3d H-50

Appendix A : Model compouncl of H6zT resuitrng rrom m e 1'' truncation

Figure A. I 1 mmbering system for calculations

Table A-I Energies of bancation

C-N (A) min 1 -6 1.8 2.0 2.2 2.4 2.5 2.6 2.8

Table A-2 MeH RHF/STO-3g atomic charges

MeCl E (HF) AE(kcal1mol)

-1 165.6843 O

MeH E (HF) AE(kcal/mol)

-71 1.69463 O

MeF E (HF) AE(kcal/mol)

-809.1 4538 O

.*................... 1 S 2 C 3 C 4 N 5 C 6 C 7 H 8 H 9 H

10 O 11 C 12 H 13 H 14 H 15 H

C-N distance (A) 1.6 1.8 2.0 2.2 2.4 2.5 2.6 .....................................................................*...................................................*.*..................................

0.003 0.007 0.008 0.007 0.020 0.029 -0.008 -0.636 0.638 -0.376 -0.182 -0.060 -0.022 0.266 0.105 0.095 0.057 0.047 0.028 0.028 -0.017 0.218 0.222 0.282 0.080 -0.041 -0.063 -0.386

-0.187 -0.167 -0.225 -0.156 -0.105 -0.097 0.066 0.580 0.580 0.381 0.318 0.298 0.294 0.296 0.049 0.049 0.075 0.074 0.079 0.079 0.068 0.076 0.063 0.099 0.094 0.096 0.101 0.035

-0.142 -0.143 -0.084 -0.054 -0.039 -0.034 -0.010 -0.421 -0.435 -0.405 -0.368 -0.341 -0.330 -0.295 -0.241 -0.239 -0.192 -0.270 -0.370 -0.434 -0.283 0.245 0.228 0.151 0.203 0.239 0.249 0.055 0.110 0.119 0.063 0.063 0.062 0.064 0.055 0.097 0.108 0.078 0.065 0.061 0.062 0.071 0.145 0.149 0.089 0.078 0.072 0.072 0.087

Table A '4 MeF RHF/STO-3g atomic charges

C-N distance (A) 1 -6 1.8 2.0 2.2 2.4 2.5 2.6

C-N distance (A) 1.6 1.8 2.0 2.2 2.4 2.5 2.6 ....................................................*.........*....................*.......*..........*...........*......................................*....

0.008 0.021 0,010 0.041 0.000 -- 0.000 -0.626 -0.503 -0.210 -0.038 0.291 --- 0.31 4 0.109 0.071 0.055 0.055 -0.007 -- -0.036 0.197 0.343 0.040 0.019 -0.375 --- -0.41 0

-0.203 -0.234 -0.149 -0.142 0.024 -- 0.076 0.594 0.418 0.331 0.333 0.238 - 0.230 0.056 0.077 0.080 0.082 0.068 -- 0.076 0.078 0.099 0.097 0.122 0.056 - 0.032

-0.148 -0.095 -0.054 -0.026 0.034 -- 0.033 -0.416 -0.405 -0.363 -0.307 -0.282 -- -0.281 -0.047 0.171 0.120 :0.266 0.078 - 0.077 0.277 0.155 0.235 0.264 0.012 --- 0.01 9

-0.1 82 -0.244 -0.275 -0.31 6 -0.243 --- -0.247 0.165 0.064 0.039 0.086 0.062 -- 0.069 0.138 0.063 0.043 0.093 0.045 -- 0.050

Table A-6 MeH RHF/6-31g* coordinate maïrix

Center Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Atomic Nurnber

16 6 6 7 6 6 1 1 1 8 6 1 1 1 1

Coordinates (Angstroms) X Y z

0.260796 1.367387 -0.605903 0.485975 -0.368558 -0.309093

-1 -036931 1 -41 9325 0.567576 -0.797993 -0.8791 15 0.262049 -1 -471 208 0.236669 0.952606 1 -659547 -0.863966 0.3001 29

-1.461 775 2.346557 0.899349 -2.266397 -0.003398 1.628469 1.500196 -1.833998 0.8161 59 2.771 179 -0.402286 0.250650

-1.688340 -1.504847 -0.751 978 -0.557203 -1 .59l 593 0.932685 -2.608592 -1.826588 -0.280048 -1 -1 6341 9 -2.341 358 -1.1 86066 -1.893282 -0.767456 -1.51 1085

lnteratomic angles:

Table A-8 MeH RHF/6-31g* distance matrix (A)

Figure B. 1 2 mrnibering system for calculations

Table B-l znd hrncation RHF/3-221g energies

2i 2, Ziii Ziv

Energies EW) AE(kcaVmo1) -767.25047 472.5 -767.59405 256.9 -767.67658 205.1 -768.00340 0.0

Table B-2

Center Number ........**..*...*.........

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

2 RHF/3-21g coordinates and charges

Atomic Number ......*................ #

16 1 6 6 7 8 1 1 1 6 6 1 1 6 1 7 1 1 1 1 1

Coordinates (Angstroms) X Y Z ...........................f.........................*......,.......,.......................................~..............

1 -9581 26 1 S38665 -0.229775

Atomic Charges

-0.077

torsion 1 2v 2vi 2vii

lnteratornic angles: .......................................................... H2-S1-C3= 94.560

Table C-l 3 RHF/3-21g global minimum energies

Table C-2 Reaction path energies

3i 3ü 3& 3& 3, 3d

1 Energy

Energy E O AE(kcaVmo1)

-734.75370 --- -848.01390 148.5 -848.25052 0.0 -847.96238 180.8 -848.20860 26.3 -847.60002 408.2

Figure C. 1 3; mmbering system for calculalions

Table C-3 RHF/STO-3g single-point energies for torsional scans of 3i

Number Number Charge

0.794 -0.159 -0.777 0.1 89

-0.671 0.342 0.354

-0.21 1 -0.423 0.232

-0.903 0.286 0.294 0.252 0.271 0.379 0.374 0.378

Center

- Atomic Coordinates (Angstroms) 1 Atomic

-

I

Figure C.2 3u mbering system for calcula fions

Table C-6 RHF/3-21g single-point energies for torsional s c m of 3 ü

Center Number

Atomic Number

16 6 6 6 7 6 t 8 1 6 1 1 6 1 1 1 6 1 7 1 1 1

Coordinates (Angstroms) X Y Z

-2.319288 1.122671 0.069797

Atomic Charges

0.868

J

Figure C. 3 3, numbering ystern for calcularions

Td le C-9 RHFISTO-3g single-point energies for torsional scans of 3,

Center Number .........................

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2 1 22 23

Atomic Number ........................

16 6 6 6 7 6 1 8 1 6 1 1 6 1 1 1 6 3 7 1 1 1 1

Coordinates (Angstroms) Atomic X Y Z Charges ,...*.*....**................*.....................*..................................*......*.... ..*.........*..........*....

-2.441 81 0 0.907754 -0.326065 0.961

S.......,.........

0.000 2.1 41 4.516 4.1 37 2.523 1 .O65 4.1 53 3.097 3.396 2.764 5.444 3.933 3.359 5.072 4.252 5.1 49 5.937

Figure C. 4 3, num bering system for calculations

Table C- 12 RHF/STO-3g single-point energies for torsional scans of 3;,

Center Number

Atomic Number

Coordinates (Angstroms) 1 Atomic X Y- - Z

-2.243299 -0.042268 -0.773992 Charges

0.607

Figure C.5 3, numbering system for calmlations

Table C-15 RHF/STO03g single-point energies for torsional scans of 3,

Center Number

Atomic Number

16 6 6 6 7 6 1 8 1 1 6 1 6 1 1 1 6 1 7 1 1 1 1

Y - Coordinates (Angstroms) 1 Atomic

0.681 81 1 1 Sl732l 2.31 1028 O.? 37045

-0.127716 0.61 7088 3.525547 2.012448 0.086497

-0.870857 -0.932493 -2.302777 -0.600230 -0.694533 4.148779

-3.21 SI 14 -2.555985 -4.669234 -3.0471 87 -4.838893 -5.028298 -5.226956

X Y Z 1.9761 59 1.222723 -0.626286

Charges 0.772

Figure C. 6 3, nimbering system for calculatiom.

Table C-18 RHF/STO-3g single-point energies for torsional scans of 3~

Tl 1 T2

Center Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2 1

Atomic Number

16 6 6 6 7 6 1 8 1 6 1 6 1 1 1 6 1 7 1 1 1

Coordinates (Angstroms) Atomic X Y Z Charges

-2.4001 94 1.1 26724 0.01 7027 0.536

Table D-l JO, 3= RHF/3-21g energies

Table D-2 3, 3,~ cornparison of energies

Figure D. 1 3,~ numbering system for calculaiions

Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2 1 22 23

........................ Number

16 6 6 6 7 6 1 8 1 1 6 1 6 1 1 6 1 7 1 1 1 1 1

Charges

Table E-1 Cornparison of 3-1 and JaOue energies

C-N

Table E-2 3aiiol RHFISTO-3g/3-21g atomic charges

C-N bond distance (A) 1 -6 1.8 2.0 2.2 2.4 2.6 2.8

C-N bond distance (A) 1.6 1.8 2 2.2 2.4 2.6 2.8 ,...*..........*.................................................................*..........................................*......*........................

0.046 0.025 0.057 0.084 - 0.138 0.161 -0.079 -0.079 -0.1 17 -0.149 --- -0.163 -0.184 -0.361 -0.272 -0.237 -0.1 31 - 0.051 0.101 0.308 0.278 0.285 0.279 --- 0.278 0.291 0.269 0.157 0.147 0.071 -- -0.128 -0.232

-0.293 -0.286 -0.252 -0.214 -- -0.147 -0.080 0.232 0.228 0.234 0.241 - 0.242 0.243

-0.579 -0.568 -0.539 -0.490 - -0.422 -0.409 0.032 0.044 0.053 0.071 - 0.092 0.094 0.192 0.215 0.221 0.242 - 0.310 0.343

-0.027 -0.090 -0.1 52 -0.269 - -0.510 -0.646 0.139 0.184 0.165 0.174 - 0.196 0.190

-0.1 04 -0.065 -0.068 -0.039 - 0.039 0.085 0.1 19 0.132 0.133 0.139 - 0.166 0.196 0.1 18 0.136 0.145 0.151 - 0.172 0.201

-0.1 91 -0.1 89 -0.227 -0.294 - -0.401 -0.423 0.224 0.185 0.191 0.181 - 0.161 0.153

-0.366 -0.329 -0.295 -0.269 - -0.276 -0.284 0.243 0.239 0.237 0.235 - 0.234 0.233 0.347 0.344 0.335 0.327 - 0.324 0.322 0.362 0.356 0.343 0.331 -- 0.325 0.324 0.369 0.351 0.342 0.330 - 0.321 0.320

Figure F. I 4anti mmbering system for calculations. 4syn is the structure with torsion 1-2-6-10 equal to 0 4

Figure F.2 4'anti numbering system for calculations. 4'syn is the shwcture with torsion 1-2-6-10 equal to 0 4

Tabb F-1 4 and 4' energies EWF)

Basis set .*..........................., 3-21 g 6-31 g

4anti S...................................

-676.44956 -679.82645

4syn ..... *..............*............... -676.45897 -479.83556

4'anti -71 5.63325 -71 9.201 O0

4'syn I.............................*............................................. -71 5.62904 -71 9.1 9801

4anti m...........,............,

0.51 7 -0.21 6 -0.41 3 0.096

-0 S67 0.360 O ,244 0.255 0.1 81

-0.457

4syn W...............

0.575 -0.21 5 -0.457 0.095

-0.572 0.354 0.241 0.253 0.21 9

-0.494

Table F-3 4' atomic charges

4'anti 4' syn 0.885 0.924

nppwiu1A u . ciiei y y aui iabc uiayi aiira ui var ivua m u r i

analogues

Figure G. I Ji energy surface diagram

Figure G. 2 3ü energy surface diagram

Figure G. 3 3, energy surface diagram

Figure G. 4 3, energy surface diagram

Figure (3.5 3, energy surface diagram

Figure G. 6 3, energy surface diagram

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