PrologOverview publications

JOURNAL PUBLICATIONS/PATENTS

•Pieter Otten and Benjamin Littler, “Heat Flow Profiling as a Tool to Assess the Scale-up of Biphasic Reaction”, In Progress for Organic Process Research and Development•“Fluorescent Magnesium Indicators”, Robert E. London, Pieter A. Otten, and Louis A. Levy. US Patent 6,706,528•Yu, J.; Otten, P.; Ma, Z.; Cui, W.; Liu, L.; Mason, R. P., “Novel NMR Platform for Detecting Gene Transfection: Synthesis and Evaluation of Fluorinated Phenyl b-D-Galactosides with Potential Application for Assessing LacZ Gene Expression”. Bioconjugate Chemistry, 15(6); 1334: 2004•Weina Cui, Pieter Otten, Yingming Li, Kenneth Koeneman, Jianxin Yu, and Ralph P. Mason, “Novel NMR Approach to Assessing Gene Transfection: 4-Fluoro-2-Nitrophenyl-β-D-Galactopyranoside as a Prototype Reporter Molecule for β-Galactosidase”. Magnetic Resonance in Medicine, 51 (3); 616:2004•Pieter A. Otten, Robert E. London, and Louis A. Levy, “4-Oxo-4H-quinolizine-3-carboxylic Acids as Mg2+ Selective, Fluorescent Indicators”. Bioconjugate Chemistry, 12; 203: 2001 •Pieter A. Otten, Robert E. London, and Louis A. Levy, “A New Approach to the Synthesis of APTRA Indicators”. Bioconjugate Chemistry, 12; 76: 2001•Pieter A. Otten, Syb Gorter, and Arne van der Gen, “A Structural Study of Selenobenzamides. Crystal Structures and Dynamic 13C NMR”. Chem. Ber./Recl. Trav. Chim. Pays-Bas, 130; 49: 1997•Pieter A. Otten, Honorine M. Davis, Jan Hein van Steenis, Syb Gorter, and Arne van der Gen, “Stereoselective Synthesis of (Z)-1-Chlorovinyl Sulfoxides”. Tetrahedron, 53; 10527: 1997•Pieter A. Otten, Njord Oskam, and Arne van der Gen, “A Horner-Wittig Approach to S,N-Ketene Acetals. Acid-catalyzed Hydrolysis of S,N-Ketene Acetals to (S)-Thioesters”. Tetrahedron, 52; 11095: 1996•Pieter A. Otten, Honorine M. Davis, and Arne van der Gen, “A Horner-Wittig Synthesis of 1-Chlorovinyl Sulfoxides”. Tetrahedron Letters, 36; 781: 1995•Pieter A. Otten and Arne van der Gen, “The Reaction of α-Amino-substituted Diphenyl Phosphine Oxide Anions with Elemental Sulfur and Selenium. A New Route to Thio- and Selenoamides”. Recl. Trav. Chim. Pays-Bas, 113; 499: 1994

Horner-Wittig Reagents in Sulfur and Selenium Chemistry

Pieter Otten and Arne van der Gen

Leiden Institute of Chemistry

Leiden University, The Netherlands

P S Se

Advantages of Diphenylphosphine Oxides

•Crystalline materials•Reactive anions•Superb anion stability•Excellent stereoselectivity•Water soluble by-product

O

(C6H5)2P

O

O

OTBS

OTBS

O

OH

OH

(C6H5)2P

O

R

N

O

O

Fenuculin (fennel, star anise)(Z)-a-Bisabolene (oil from Opoponax)

Fecapentaene-12

(Z)-6-Heneicosen-11-one, sex attractant of the Douglas fir tussock moth

Thioamide SynthesisAminomethylphosphine oxides, excellent reagents for enamine formation, show a

unique reactivity towards chalcogens to form amides

(C6H5)2P

O

R

N

O

R N

S

O

F Cl

O

Br

S N

S

N

N

N

H

N

O 1) BuLi or LDA

2) 2 eq. S

R =

65-90%

50-70%

(C6H5)2POEt + RCHO +

R = aryl: reaction at r.tR = alkyl: reaction at 0oC to quell deprotonation of formed thioalkanamides by the HW reagent

Selenoamide Synthesis

(C6H5)2P

O

R

N

O

R N

Se

O

Cl Br S N

H

1) BuLi or LDA

2) 2 eq. Se

R =

56-80%

64% 56% (red selenium)

“Red selenium”, Se8, more reactive than metallic or gray Se; allows isolation of acidic selenoalkanamides at lower temperatures

Reaction is sluggish at ambient.

Mechanistic Considerations

(C6H5)2P

O

N(C6H5)2P

O

N

SS

SS

SS

SS

(C6H5)2P

O

N

SLi

N

S

(C6H5)2P

O

N

(C6H5)2P

O

N

SMe

[ ]Li+

+(C6H5)2POLi

(C6H5)2POSLi

"S"

Li+Li+

MeI

S8

68%

One eq. of S gives < 50% yield, recover phosphine oxide

All intermediates isolated and characterized. Mass balance accounted for.

Intermediate trappedat low temperatureswith MeI

Independently confirmed

Crystal Structure SelenobenzamidesAmino group reduces dihedral angle Θ

C=Se: 1.840 ÅC-N: 1.331 ÅΘ = 53.3º

C=Se: 1.824 Å

Θ = 81.1º

N

Seα

β

γ

α+β+γ = 360o

C(5) is sp2-hydrid.

Other Selenocarbonyls

C=Se of selenoamides close to otherN- or C=C-conjugated selenocarbonyls

VT 13C NMR Study: Rotational BarriersC=S and C=Se strongly e-withdrawing groups, σ+

p fits only

More polarizable Se more sensitive to σ+p than S

N

Se

OX

X (σ+p) =

H (0)

Me (-0.3) SMe(-0.6)

OMe(-0.78)

NMe2(-1.7)

o = Se* = S

Eyring eq.: ∆G*rot = 19.5Tc x [9.971 + log(Tc/∆ν)]

Synthesis of (Thiomethyl)- and (Selenomethyl)phosphine Oxides

(C6H5)2P

O

SR1Cl SR1

(C6H5)2P

O

SR1(C6H5)2P

O

OTs

(C6H5)2P

O

SR1(C6H5)2P

O

SR1

Cl

(C6H5)2P

O

SR1

Cl

O

(C6H5)2POEt +neat

∆

NaH + R1SH, THF, rtor

(R1S)2 + NaBH4, EtOH, rt

NCS

C6H5Cl, rt

m-CPBA

DCM, 0 oC

R1 = Me, n-Bu, t-Bu, c-Hex, Ph, p-Tol, p-(CF3)Ph

All steps >80%

(C6H5)2P

O

SeR3(C6H5)2P

O

OTs

NaH + R3SeH, THF, rtor

(R3Se)2 + NaBH4, EtOH, rt

R3 = Me, Ph, p-ClPh, p-(CF3)Ph, m-(CF3)Ph, p-Tol, 1-Naphthyl

59-97%

Synthesis of Vinyl Selenides

(C6H5)2P

O

SeR3R2

R1

SeR31) LDA

2) R1CHO or R1C(=O)R2

•>98% E-selectivity if R1, R3 = aryl•Reacts w/ acidic ketones, HW reagent weakly basic•E/Z ratio by NMR and GC

Mechanistic Considerations(Curtin-Hammett Principle)

Trapped HW-adducts (R3 = Ph -60 oC, H2O):R1 = n-Pr: pro(E)/pro(Z) = 1/1, quant.R1 = c-Hex: pro(E)/pro(Z) = 1/1+ 22% (E)-vinyl selenide!Sterics facilitate elimination, kE increases

R1 = phenyl, pro(E)/pro(Z) = 3/2. quant,pro-(E) and pro(Z) must rapidly equilibrate viareverse to aldehyde and/or epimerizationand (E)-isomer is thermodynamic sink

pro-(E)

Pro-(Z)

Do HW adducts equilibrate?

No vinyl selenide derived from propionaldehyde observed: no equilibrationvia reverse reaction with aliphatic aldehydes. Cannot rule out epimerization.

Fast equilibration with aromatic aldehydes to explain discrepancy betweenpro-(E) and pro-(Z) ratio for isolated HW adduct (3/2) and strong E-selectivityfor completed HW reaction.

SePhPH

HHO

Ph

OPh SePhP

H

HO

HPh

OPh

+

1) LDA, THF, -50oC

2) 5 eq propionaldehydeslow warm-up to ambient

SePh

SePh+

ratio: 1 to 1

ratio: 1 to 1

Alternate Intermediate

Warren proposes this late-stage intermediateto explain high pro-(Z) selectivity for simplealkyl phosphine oxides (R1 = alkyl)

Pro-(E)

Six-centered transition state. R1 equatorial to avoid 1,3-interaction with equatorial Ph. Lowenergy difference between SeR3 equatorialor axial, reflected by observed low stereoselectivityIn HW-adducts.

Pro-(Z)

Formation of 1-Chlorovinyl SulfoxidesA New Class of Compounds (1994)

(C6H5)2P

O

SR1

Cl

O

SR1

Cl

O

F FF

CH3H

OCl S

S

1) LDA, THF, -40 oC

2) R2CHO, rtR2

R1 = R2 =

20 examples

>96% Z-selectivity if R2 = aryl, alkenyl and/or R1 = aryl

Crystal Structure Confirms Z-geometry

P-1, a = 13.3146, b = 11.326, c = 9.395 Å; α = 125.15β = 97.88, γ = 96.76; V = 1097.45 Å3, Z = 4, ρ = 1.396 kg/dm3

S

Cl

O

O

Mechanistic Considerations

Phosphine oxide anion and carbonylare in equilibrium w/ adduct. Fast whenR1 = anion stabilizing aryl

Rotation sets up for the oxaphosphetane.

Sterics favor pro-(Z) intermediate

Thermodynamic sink

Elimination fast if R2 stabilizesdouble bond: aryl, vinyl

Mechanistic Considerations: Alternate ApproachSurprisingly poor stereochemistry with bulky c-hexanecarboxaldehyde

Cl

S

OO

(C6H5)2P

O

S

O

Cl

S

O

Cl

Z/E = 3.3/1+

+

Li+ ligated between P=O and S=O. R1 equatorial to avoid axial Ph.Aldehyde approaches with R2 pointing away from axial Ph. Must be anintermediate trough on the energy surface

Michael Addition

Cl

SO

O

O

O

O

H

S

O O

O

O

Op-TolNa+

THF

r.t.

(R)C(R)S, d.e >95%

72%

P21, a = 6.371, b = 7.646, c = 12.364 Å; α = 90.0

β = 98.45, γ = 90.0; V = 595.7 Å3, Z = 2, ρ = 1.50 kg/dm3

Mechanistic ConsiderationsA compact sodium complex is formed, dictating stereochemistry

Malonitrile gives 1:1 mixture of diastereoisomers

Synthesis of S,N-ketene Acetals and Thioesters

(C6H5)2P

O

SR1

Cl

X

N

(C6H5)2P

O

SR1

N

X

(C6H5)2P

O

SR1

N

O

N

SR1

OSR1

O

Br

O

S

X = CH2, O

neat, 80 oC

69-72%

1) LDA, -10 oC

2) R2CHO, rtR2

R2

THF, HClaq.

R1 = Me,

57-81%80-90%

R2=aryl: pure ketene acetale after extractive work-up only.R2=alkyl, contaminated w/ condensation products. Carry thru to thioester for good use

Goals Achieved

•User-friendly access to thio- and selenoamides•Proved role of substitution on aryl ring in conjugation in selenobenzamides•Stereoselective formation of 1-chlorovinyl sulfoxides•Demonstrated diastereoselective Michael addition on 1-chlorovinyl sulfoxides•General synthesis of vinyl selenides•Facile synthesis of S,N-ketene acetals•Homologation of aldehydes to (S)-thioesters

Fluorescent Magnesium Indicators

Pieter Otten, Louis Levy, and Robert London, National Institute of Environmental Health Sciences, RTP, NC

Physiological Importance of Mg2+

Mg2+, most abundant divalent cation:

•300 enzymatic reactions

•Energy production

•Hormone regulation

•DNA synthesis

•Muscle contraction

Mg-deficiency linked to:

•Atherosclerosis

•Hypertension

•Kidney stones

•Migraines

•Psychiatric problems

H

BeLi B

Na Al

KCa

Mg

C

Sc

Ideal, Fluorescent Mg2+ IndicatorHowever, fluoresc. behavior of difficult to predict

•Selective for Mg•Ratioable•Polycarboxylate•Excitation > 340 nm•Emission > 500 nm

•Photostable•Non-toxic

O

N COO

COO

O COO

NO

COO

4 K+

Mag-fura-2 [Mg2+] = Kd[(R - R0)

(Rsat -R)] F0,λ2

Fsat,λ2

R = F,λ1

F,λ2

Why APTRA (Aminophenol Triacetic Acid)?At physiol. Mg2+, BAPTA binds two ions. Cut BAPTA in half to get

to APTRA.

NNCOO

COO COOCOO

O O

NN

COO

OOC

OOC

COO

COO

O

N

COO

COO

EDTA

BAPTA, Tsien

APTRA, London, Levy

Pd coupling: fast, one step approach to quicklyinvest structural diversity

OH

F

NO2 Br

F

NO2

O

OH

NO2

O

NO2

O

OS

OO

F FF

K2CO3, DMF

NaOHaq., DMSO

Tf2O, pyr

DCM

95%

95%

73%

COO

O

N

COO

COO

Q

Y z

O

X

NO2

Ph

Q

Y z

M

+

M = B, SnX = Hal., OTf

1) Suzuki, Stille2) H2, Pd/C3) BrCH2COOMe

4) NaOHaq.

Suzuki Coupling

KD, Mg = 2.3 mM

KD, Ca = 70 µM

O

OTf

NO2

Ph

OB(OH)2

O

O

NO2

Ph

O

O

N

COOH

COOH

COOH

+Pd[P(Ph)3]4

DME, Na2CO3aq.

rfx

1) H2 (30 psi), 5% Pd on C, EtOH

2) BrCH2COOMe, NaI, Proton Sponge, ACN, rfx

3) NaOHaq., MeOH

4) HClaq.

81%

KD, Mg = 2.1 mMKD, Ca = 28 µM

KD, Mg = 1.8 mM

KD, Ca = 17 µM

O

N

COOH

COOH

COOH

O

N

COOH

COOH

COOH

Suzuki coupling: 92%Suzuki coupling 91%

Fluorescence Excitation Titration

O

N

COOH

COOH

COOH

Response to Mg and Ca is not identical, which was often assumed to correct for Ca-spikes

Synthesis of 4-Oxo-4H-quinolizine-3-carboxylatesKnown complexers of Mg2+ to shut down bacterial DNA-gyrase w/ KD = 1 mM

N

X

Y

z

1) LDA, THF

2) EMME N

X

Y

OEt

COOEt

EtOOC

z

Xylene

rfx

N

O

X

Y

Z

COOEt

X = Y = Z = H: 55%X = Cl; Y = Z = H: 63%

X = H, Y,Z = : 45%

N

O

X

Y

Z

COOEt

Electrophilic substitution

Nucleophilic substitutionPd-catalysis

Explore reactivity to diversify quickly

Electrophilic Aromatic Substitution

N

O O

OEtN

O O

OEt

NO2

N

O O

OEt

O H

N

O O

OEt

Br

H2NO3/(Ac)2O

50%

93%

POCl3/DMF

Br2/HOAc

71%

4-Oxo-4H-quinolizine-3-carboxylic Acids

N

O

O

O

KD,Mg = 1.1 mM

N

O

O

O

Cl

N

O

O

O

KD,Mg = 5.0 mM KD,Mg = 4.6 mM

Compare:

Representative Examples; Tri-acids

N

O

O

O

NOOC COO

N

O

O

O

OOC COO

N

O

O

O

O

N COO

COO

N

O

O

O

OOC

COO

Kd(Mg) = 0.5 mM

λem. = 548 nmKd(Mg) = 0.5 mM

λem. = 429 nm

Kd(Mg) = 1.0 mM

λem. = 402 nmKd(Mg) = 0.4 mM

λem. = 411 nm

Synthesis Bromo-substituted TriacidIntroduce 3rd ester, as decarboxylation could not be prevented

N

O

COOEt

Cl

Na[CH(COOMe)2]

DMSO, rt

N

O

COOEt

COOMe

MeOOC

63%

1) NaH

2) BrCH2COOEt

N

O

COOEt

COOMe

MeOOC

EtOOC74%

NaOHaq.

N

O

COOEt

COOMe

MeOOC

EtOOCBr

75%

N

O

COOH

HOOC

HOOC51%

KD(Mg) = 0.4 mM

Br2, HOAc

HClaq.

NaOHaq.

HClaq.

N

O

COOH

HOOC

HOOCBr

42%

KD(Mg) = 0.7 mMλem. = 437 nm

-CO2

Fluorescence Emission SpectrumThe first Mg-selective, ratioable fluorophore!

N

O

O

O

Br

O

O

O

OKD,Mg = 0.7 mM

Selected Fluorophores

Emission > 500 nm Ratioable

Suzuki Coupling

N

O

COOEt

Cl

O

B O

O

N

O

COOEt

O

N

O

O

COOH

N

O

COOH N

O

COOH

O

PdCl2[P(c-Hex)3]2

CsF, DMF, 110 oC50%

NaOHaq., MeOH, rfx

HClaq.

49%

51% (Pdcat. coupling) 48% (Pdcat. coupling)

KD(Mg) = 1.2 mM

λem. = 444 nm

KD(Mg) = 1.5 mM

λem. = 456nm

KD(Mg) = 1.3 mM

λem. = 459 nm

Poor aq solubilityFuture exploration:•Buchwald•Libraries of boronic acids anno 2009

Goals Achieved:

Developed a general synthesis of fluorescent APTRA indicators for intra-cellular Mg2+ and Ca2+

Showed that their response to Mg2+ and Ca2+ are not identical

Designed, synthesized, and evaluated new, ion-selective ratioable, fluorescent indicators for Mg2+ based on 4-oxo-4H-quinolizine-3-carboxylic acids

NMR-active, Fluorinated Reporter Molecules

Pieter Otten and Ralph P. Mason

Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX

6-FPOL

IδI-IδacidI

IδbaseI-IδIpH = pKa + log

pKa = 8.2

6-FPAM pKa = 7.05

CF3-POLTriple the fluorines, triple the signal

N

O

OO

N

O

OO

I

N

O

OO

I

Cl

N

O

I

O

O

N

O

O

O

F

F

F

N

O

OO

F

F

F

I2, K2CO3aq.

K2CO3, DMF, 60 oC

Acetone

p-TsOHcat.

Et3SiCF3

CuI, KF

DMF, NMP, 80 oC

1) 1M HCl, dioxane

2) H2, Pd on C, EtOH

65%

44% 91%

60%

pKa = 6.7∆δ = 1.6 ppm

Vitamin-B6

Does not penetrate rbc membrane

Fluorinated Gene Reporter

O

OHOH

OH

OH

O

F

NO2

β-D-galactopyranoside

Saline at 30 oC (♦)Plasma at 30 oC (□)Plasma at 37 oC (Δ)

Top: pH = 4.5; t = 30 oC; β-gal (Aspergillus Oryzae)Bottom: pH = 7.3 → 6.8; t = 37 oC; β-gal (E. Coli)

pKa = 6.85