"Chiral Acetate": The Preparation, Analysis, and ... - ACS Publications

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“Chiral Acetate”: The Preparation, Analysis, and Applications of Chiral Acetic Acid Addison Ault Department of Chemistry, Cornell College, Mount Vernon, IA 52314; [email protected]

In 1969, a research group based both in England and West Germany (1) and another group in Switzerland (2) reported the synthesis and analysis of chiral acetic acid, a species that contains a methyl group that is chiral by virtue of the presence of a proton, a deuteron, and a tritium atom.

methylmagnesium bromide, and the resulting mixture treated with deuterium oxide to produce the deuterium analog of phenylacetylene. This was reduced in a stereospecifically syn manner by diimide to give the Z isomer of 2-deutero-1phenylethylene, H

T D C H

φ

COOH

They also showed how this material could be applied to the determination of the stereochemistry at the methyl group of the enzymatic condensation of acetate with glyoxylate to form malate. −

COO

−

COO

C O

si at glyoxylate

D H C T

inversion at acetate

D

(R)-acetate + glyoxylate

D

H

C

C

T

which was shown to be diastereomerically pure by NMR. The deuterophenylethylene was then converted to the epoxide by perbenzoic acid to give a racemic mixture of a deuterated phenylethylene oxide by syn addition to the enantiotopic faces of the alkene. φ

H

H C

C

O D

O OH

COO−

−

COO

C

(Z)-2-deutero-1-phenylethylene

(R)-deuterotritioacetic acid “chiral acetate”

H

H C

(2S,3S)-malate

This research has remained largely unappreciated in the chemical literature for more than thirty years, and it is the purpose of this paper to present to a wider audience this tour de force that involves tracer levels of materials, the ultimate stereochemical phenomena, isotope effects, and the exquisite specificity of enzymatic reactions.

First, it is clear that the only sense in which “chiral acetate” is conceivable is as the result of isotopic substitution. That is, for a “chiral acetate” molecule to exist it must contain in its methyl group a proton, a deuteron, and a (radioactive) tritium atom. A practical consequence of the use of tritium at the tracer level is that the population of chiral molecules will be on the order of only one in every trillion, a concentration level about equivalent to that of a single person in the entire population of the world. While this low concentration required the development of a very clever way to link measurements of radioactivity to optical purity, the synthesis of acetic acid containing a low level of chiral and radioactive molecules could be done with procedures that were already available.

First Synthesis of Chiral Acetate The synthesis of chiral acetate described in ref 1 is summarized in Figure 1. Phenylacetylene was converted to its conjugate base by proton transfer to the methyl group of

C

C

φ

H D

enantiomers; racemic

This racemic mixture was then subjected to hydride reduction by lithium borohydride containing a tracer level of tritium. Hydride attack with inversion at the less substituted carbon gave the following pair of enantiomers as a racemic mixture. T

H

H

C

C

φ

T D

OH

Stereospecific Synthesis of Chiral Acetate

+

H

(+)-(1R,2R)-[2D,2T]1-phenylethanol

D

+

C

H

φ C H OH

(–)-(1S,2S)-[2D,2T]1-phenylethanol

At this point the racemic product was diluted with additional racemic 1-phenylethanol as a carrier. The resulting racemic mixture was resolved by fractional crystallization of the brucine phthalates into (+)-(1R )-1-phenylethanol and (᎑)-(1S )-1-phenylethanol, each of which contained, at the tracer level, methyl groups that contained both a deuterium atom and a tritium atom. These separated enantiomers were then oxidized with chromic acid to the corresponding acetophenones, and the acetophenones subjected to Baeyer– Villiger oxidation (oxidation by peroxy acids) to form the corresponding phenyl acetates. Alkaline hydrolysis of the separate acetate esters then produced the two desired samples of acetic acid, as indicated in Figure 1. This method of synthesis ensured that the acetic acid from the (+) enantiomer of 1-phenylethanol would contain, at the tracer level, molecules of (R )-deuterotritioacetic acid, and that the acetic acid from the (᎑) enantiomer of 1-phenylethanol would contain, at the tracer level, molecules of (S )-deuterotritioacetic acid.

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Br

φ

C

C

Mg

CH3

φ

H

C

C

Mg Br D 2O

φ

H

φ

H C

D

C O

perbenzoic acid

+ H

C

φ

H

H C

C D

D H H

LiBH3T + LiBH4

H

C

φ

D

D

+

C

C

φ

C

HO / H2O

D

H C C

φ

HO

H C C

O

T

φ C H

OH

−

φ O C

H2Cr2O7

(+)-(1R,2R)-[2D,2T]1-phenylethanol

resolution

H

T

C

H

D

OH

T

C

T

T


H

D

diastereomerically pure

H

T

C

H2NNH2 / H2O2 / Cu2+

φ

O C

C

O

trifluoroperacetic acid

D

D

(R)-deuterotritioacetic acid

O T

OH D


C

φ

T H

φ

H

C

H2Cr2O7

OH

H

−

HO / H2O

HO

O

T

(–)-(1S,2S)-[2D,2T]1-phenylethanol

T

D C O C

D C φ C

D C

H

O

trifluoroperacetic acid

H

C

(S)-deuterotritioacetic acid

O enantiomers; pure

Figure 1. First synthesis of chiral acetate.

C HO

OH

OH

O

NADH, lactate dehydrogenase

T

H C HO C

C

T

H C O C

methanol/acid

H3C

O

O

O 2-tritioglyoxylate

T

pure enantiomer

(2S)-[2T]-glycolic acid pure enantiomer

LiAlH4

H OH

H

H

C

C

H

BsCl ⬅ Br

S

OH

H

C

C

C

H

H

T H

Cl

O T

H

H

C

2-tritiobrosylate LiAlD4

O

OH

OBs

pure enantiomer H

O2 / Pt

+

+ H

C

C

H

H

H

C

C

H

T

OH each enantiomerically pure

Figure 2. Second synthesis of chiral acetate.

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p333-345.65

Journal of Chemical Education • Vol. 80 No. 3 March 2003 • JChemEd.chem.wisc.edu

334

+

D

1-tritiobrosylate each enantiomerically pure; constitutional isomers

C OH H

T

T

OH

C

D deuteroacetic acid not chiral

D

OBs

O

T

OH

2/11/03, 10:01 AM

HO

H C

C

D

O (R)-deuterotritioacetic acid enantiomerically pure


Second Synthesis of Chiral Acetate The synthesis of chiral acetate described in ref 2 is summarized in Figure 2. In this synthesis, 2-tritioglyoxylate, a substance that could be prepared from oxalic acid by partial reduction with magnesium metal and acid in tritiated water (3),

The mixture of ethanols was then oxidized by oxygen in the presence of platinum to the corresponding acetic acids. T

O C H

C

OH

+

H

O

D C −

O

T

O 2-tritioglyoxylate

was stereospecifically reduced by NADH in the presence of lactate dehydrogenase to give to give a product, (2S )-[2T]glycolic acid, whose configuration had been established. OH H C HO C

T

O (2S)-[2T]-glycolic acid enantiomerically pure

This acid was treated with methanol in the presence of acid to convert it to the corresponding methyl ester, which was then reduced by lithium aluminum hydride to (1S)-[1T]ethylene glycol. OH

H

H

C

C

H

T

OH

Analysis of Chiral Acetate The analysis of each isomer of chiral acetate was made via addition of the acetate to glyoxalate to form malate, and then exchange of the pro-R methylene proton of malate with solvent via the reversible dehydration of malate to fumarate in the presence of fumarase. These reactions are represented with achiral acetate by the following two equations. COOH

When this glycol was treated with p-bromobenzenesulfonyl chloride, “brosyl chloride”, a mixture containing a pair of constitutionally isomeric brosylates was formed, each of which was enantomerically pure.

H

OBs

H

C

H

C

C

H

C

H

T

+

H

COOH

C H

O

H H C H

malate synthase si

H

C

C

H

OH

COOH

COOH

T

H

(S)-malate

acetate

OH

OBs 2-tritiobrosylate

H H

The brosylates were then reduced with inversion by lithium aluminum deuteride, LiAlD4, to give a mixture of constitutionally isomeric deuterotritioethanols, each of which was enantiomerically pure. OH H

C

C

H

T T

+

D

COOH

1-tritiobrosylate

constitutional isomers; each enantiomerically pure

H

D

Since the original 2-tritioglyoxylate contained only tracer levels of tritiated material, almost all of the product of this synthesis was actually deuteroacetic acid, which was formed via unlabeled ethylene glycol. The configuration of those molecules that contained a chiral methyl group, about 1 in 109, was known because the sense of the stereospecificity of the reduction of glyoxalate in the presence of lactic dehydrogenase was known. The S enantiomer of chiral acetic acid was prepared analogously, using (2R )-[2T]-glycolic acid that was made by reducing 2-tritioglyoxylate with NADH in the presence of glyoxayate reductase from spinach leaves (4).

(1S)-[1T]-ethylene glycol enantiomerically pure

OH

H C

O (R)-deuterotritioacetic acid enantiomerically pure

deuteroacetic acid not chiral

C

HO

C

H

H

C

C

H

D

OH

constitutionally isomeric ethanols each enantiomerically pure

C

C

OH

H C

fumarase

H anti

COOH (S)-malate

HOOC

COOH

C H fumarate

It was known that acetate, as acetylCoA, adds stereospecifically to the si face of malate to form exclusively the S isomer of malate, as shown above. It was also known that the fumarase catalyzed elimination of water from (S )-malate to form fumarate was stereospecifically anti, and so involved exclusively the pro-R methylene proton of (S )-malate. The enzymatic elimination reaction can therefore be rewritten to indicate its stereospecificity.


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335


COOH pro-R

C

H

H

C

OH

H C

fumarase

H pro-S

anti

HOOC

COOH

C H

COOH

fumarate

(S)-malate

It still remains to be seen how these reactions, when carried out with chiral acetate, will allow the sense of the chirality of the acetate to be determined. The enzymatic aldol-like condensation of acetate with glyoxylate to form malate was known to be irreversible, and the reaction appeared to proceed by a rate-limiting enolization that involved no exchange of carbon-bound hydrogen with the medium. Since the postulated slow step involved the transfer of a hydrogen atom (in this case the transfer of a proton), this reaction was expected to show a kinetic hydrogen isotope effect. This expectation was confirmed by workers in both research groups when trideuteroacetylCoA was found to undergo the enzymatic reaction with glyoxylate somewhat more slowly than normal acetylCoA. When chiral acetate, as acetylCoA, reacts with glyoxalate in the presence of malate synthase to give malate, the reaction can involve either the loss of a proton, the loss of a deuteron, or the loss of the tritium atom from acetate. These possibilities are represented by the following three equations for the case of the (S )-acetate:

As stated above, the stereochemical course of the reaction involves addition to the si face of glyoxylate and is shown here with inversion at the methyl group of acetate. While at this point we should think of inversion at methyl as a guess, this turns out to represent the actual steric course of this reaction. The case for (R )-acetate can be represented analogously, and the equations for the R case would be just like the three for the S case but with the positions of the non-transferred isotopes reversed in the product, malate. COOH COOH

C H

O

D H C T

si, inversion H transfer

D

H

C

C

T

OH

COOH

COOH (R)-acetate

T is pro-S

COOH COOH

C H

O

T D C H

si, inversion D transfer

T

H

C

C

H

OH

COOH

COOH

T is pro-R

(R)-acetate

COOH COOH

COOH

H

C

C

C

D

C H

O

T H C D


T

OH

H H T C

COOH

COOH

(S)-acetate

T is pro-R

COOH O

D

H

si, inversion T transfer

H

COOH (R)-acetate

COOH COOH

C H

O

H D C T


H

H

C

C

T

OH

COOH

COOH

(S)-acetate

T is pro-S

COOH COOH

C H D T C

336

p333-345.65

O H

H

si, inversion T transfer

D

C

C

OH

H

COOH

COOH

(S)-acetate

no T present

OH

D

COOH no T present

If there were no kinetic hydrogen isotope effect in this reaction, the rates of these reactions would all be equal, and exactly the same amount of malate would be produced with tritium in the pro-R position as in the pro-S position in the case of both (R )-acetate and (S )-acetate. There is, however, a kinetic hydrogen isotope effect. In the case of the (S )-acetate there should be less malate produced with tritium in the non-exchangeable pro-S position than in the exchangeable pro-R position. The situation is just the reverse in the case of the (R )-acetate: more malate should be produced with tritium in the non-exchangeable pro-S position than in the exchangeable pro-R position. This contrast is summarized in Figure 3. When the experiments were done with (S )-acetate, only 30% of the tritium present in the malate was retained upon exchange via equilibration with fumarate, indicating that less malate was produced with tritium in the non-exchangeable pro-S position than in the exchangeable pro-R position. Correspondingly, when the experiments were done with (R )-acetate, about 70% of the tritium present in the malate was retained upon exchange via equilibration with fumarate,


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C

C

2/11/03, 10:01 AM


indicating that more malate was produced with tritium in the non-exchangeable pro-S position than in the exchangeable pro-R position. When tritiated acetate was subjected to the same process the (S )-malate obtained lost 50% of its tritium upon exchange via equilibration with fumarate. Two conclusions follow from this. First, (R )-acetate and (S )-acetate can be distinguished. Second, this reaction of acetate with glyoxylate involves inversion at the methyl carbon of acetate.

(S)-Acetate COOH

COOH

C H

O

T H C D


T

H

C

C

D

OH

more of this

Other Applications

COOH

COOH

T is pro-R exchangeable

(S)-acetate

COOH COOH

C H

O

H D C T


H

H

C

C

T

When a sample of possibly chiral acetate is analyzed, the sense of any chirality is indicated by whether the (S )-malate loses more (S enantiomer) or less (R enantiomer) than half of its tritium upon exchange via equilibration with fumarate. However, the magnitude of the deviation depends on both the chiral purity of the sample and upon the magnitude of the kinetic hydrogen isotope effect. Experiments with chiral acetate have given degrees of exchange as high as 90% [(S )acetate] and as low as 10% [(R )-acetate]. Many control experiments were done in order to ensure the validity of the conclusions, and a number of conditions had to be met in order that the method could succeed. One of the most important general conditions was that every molecule that contained T should contain D (the level of incorporation of D should be as close to 100% as possible). A second important condition was that the discrimination of fumarase with respect to the pro-R and pro-S positions be very high; fumarase is in fact famous for its extraordinarily high degree of diastereotopic discrimination (5).

OH

less of this

In the following year both the Cornforth group (6) and Rétey, Lüthy, and Arigoni (7) reported application of chiral acetate to the determination of stereochemical details in both the formation and cleavage of citrate. One set of experiments (6) showed that the cleavage of citrate in the presence of citrate lyase (pro-S) proceeds with inversion at the methylene group that becomes acetate. In the first experiment tritiated fumarate was hydrated in the presence of fumarase to (2S,3S)[2,3-T]-malate. T

COOH

COOH

(S)-acetate

T is pro-S non-exchangeable

C HOOC

C T

[2,3-T]-fumarate monotritiated, at the tracer level

(R)-Acetate COOH

COOH

COOH

C H

O

D H C T

si, inversion

H

C

C

T

D

H transfer

OH

more of this

T

C

C

T

H

OH

COOH

COOH

COOH

(2S,3S)-[2,3-T]-malate monotritiated

T is pro-S non-exchangeable

(R)-acetate

This was then oxidized by NAD+ and malate dehydrogenase to (3S )-[3T]-oxaloacetate in the presence of (R )-citrate and acetylCoA to irreversibly give citrate stereospecifically labeled with tritium in the pro-S branch.

COOH COOH

C H

COOH

O

T D C H


H

C

C

H

T

OH

COOH

COOH

T is pro-R exchangeable

(R)-acetate

Figure 3. Analysis of chiral acetate.

pro-R

COOH

less of this

H HOOC

C H H

C

C

OH

T pro-S

COOH citrate T stereospecifically pro-S in the pro-S branch


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2/11/03, 10:01 AM

337


T

COOH C

HOOC

citrate stereospecifically tritiated in the pro-S branch but in the opposite sense from the experiment summarized in Figure 4.

COOH

fumarase, H2 O

C

H

T [2,3-T]-fumarate

T

C

C

T

OH

pro-R

COOH H

HOOC

COOH

C T

(2S,3S)-[2,3-T]-malate

H

C

C

OH

H pro-S

COOH

malate dehydrogenase

citrate T stereospecifically pro-R in the pro-S branch pro-R

COOH H

HOOC

C H H

C

C

COOH

(R)-citrate synthase, acetylCoA

OH

C H

T pro-S

C

O

T

COOH

COOH citrate

(3S)-[3T]-oxaloacetate

citrate lysase (pro-S), D2O

glyoxylate; malate synthase

acetate

This citric acid was also isolated and then cleaved in D2O by the enzyme citrate lyase (pro-S) to give a sample of acetic acid that contained potentially chiral molecules of deuterotritioacetic acid. The acetic acid thus produced gave with glyoxylate and malate synthase a sample of malic acid that lost 39% of its tritium upon equilibration in the presence of fumarase. Thus the acetic acid contained an excess of chiral acetate of the R configuration, T D C

malate

COOH

Figure 4. Determination of the stereochemistry of citrate lyase (proS).

The citric acid was isolated and then cleaved in D2O by the enzyme citrate lyase (pro-S) to give a sample of acetic acid that contained potentially chiral molecules of deuterotritioacetic acid. This sequence is summarized in Figure 4. The acetic acid thus produced gave with glyoxylate and malate synthase a sample of malic acid that lost 56% of its tritium upon equilibration in the presence of fumarase. Thus the acetic acid contained an excess of chiral acetate of the S configuration, and therefore the cleavage of citrate in the presence of citrate lyase (pro-S) must proceed with inversion at the carbon that becomes the methyl group of acetic acid. H D C T COOH


indicating again that the cleavage of citrate in the presence of citrate lyase (pro-S) must proceed with inversion at the carbon that becomes the methyl group of acetic acid. Other Syntheses of Chiral Acetic Acid Several additional syntheses of chiral acetic acid have been reported in the thirty years after the appearance of the papers cited in ref 1 and 2. Two of these are presented here.

Synthesis of Chiral Acetic Acid by Rozzell and Benner The synthesis of chiral acetic acid reported by Rozzell and Benner (8) is outlined in Figure 5. The first step was the acid catalyzed hydrolysis of the methyl enol ether of cyclohexanone in tritiated water to give cyclohexanone randomly mono-tritiated in the alpha position.


This conclusion was supported by a complementary experiment (6) in which (2S,3R )-[3T]-malate was prepared by the fumarase catalyzed anti addition of water to fumarate,

H

CH3

O

O

T

tritated water, acid

COOH H T C

C

OH

H

COOH (2S,3S)-[3T]-malate

and then oxidized by NAD+ and malate dehydrogenase in the presence of acetylCoA and (R )-citrate synthase to give 338

p333-345.65

methyl enol ether of cyclohexanone

monotritiated cyclohexanone

The monotritiated cyclohexanone was then treated with deuterium oxide in the presence of the enzyme acetoacetate decarboxylase (AAD), which stereospecifically catalyzed the exchange of both of the two equivalent pro-R protons (and tritons) in the alpha positions, replacing them with deuterium.


338

2/11/03, 10:01 AM


O CH3

H D

O

O

D

H D

O

methyl enol ether of cyclohexanone

T

DOD, ADD

TOH, acid

D T

(2S,6R)-2-tritio-2,6-dideuterocyclohexanone just this enantiomer of the tritiated material

enantiomerically pure T

The product of this reaction, (2S,6R )-2-tritio-2,6dideuterocyclohexanone, was then treated with peroxytrifluoroacetic acid to give about equal amounts of the two possible products of the Baeyer–Villiger oxidation, which is known to involve retention of configuration at the migrating carbon.

racemic trifluoroperoxyacetic acid

O

O D O

D

O

+

H D

O

O

H D

T

H D

T

O

O

D T

+

H D

Baeyer–Villiger products

D T

Baeyer–Villiger products excess φ-Mg-Br in CH2Cl2, then acid

OH

φ

OH OH

φ

This mixture of lactones was added immediately to excess phenylmagnesium bromide to give, after hydrolysis, a mixture of isotopically substituted and chiral 1,1diphenylhexane-1,6-diols.

+

φ

DH

T D

OH

φ

HD

OH

DT

OTs

φ

φ

T D

+

φ

DH

T D

+

OH

φ

φ

DH

HD

DT

1,1-diphenylhexane-1,6-diols 85% from methyl enol ether of cyclohexanone

OH

OH

φ

OH

φ

p-toluenesulfonyl chloride in pyridine

OH

OTs

φ

HD

These were selectively converted to the corresponding primary p-toluenesulfonates (tosylates) by treatment with ptoluenesulfonyl chloride in pyridine.

DT

LiBEt3H

OH

OH OH

φ

φ

OH D H

T D

+

H

φ

φ

D H HD

OTs

φ

φ

T D

+

OTs

φ

φ

DH

HD

DT

H

1,1-diphenylhexane-1,6-diol-6-p-toluenesulfonates Kuhn–Roth oxidation

O

O D H

HO

+

D T

HO

The tosylates were then treated immediately with a threefold excess of lithium triethylborohydride (“Super-Hydride”) to displace the sulfonate by hydride with inversion of configuration at the chiral center to give the following pair of isotopically substituted 1,1-diphenylhexane-1-ols.

H

H deuteroacetic acid not chiral

(S)-deuterotritioacetic acid enantiomerically pure

Figure 5. The synthesis of chiral acetate by Rozzell and Benner.

OH

φ

φ

OH D H

T D

H

+

φ

φ

D T HD

1,1-diphenylhexane-1-ols


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H

2/11/03, 10:01 AM

339


Oxidation with a large excess of chromic oxide (Kuhn– Roth oxidation) served to degrade these substances to isotopically substituted acetic acids: deuteroacetic acid (non-chiral) from the substance shown on the left, and the S isomer of deuterotritioacetic acid from the substance shown on the right. O

Demonstration of the Chirality at C-6 of 1,1-Diphenyl-6-hexanol Rozzell and Benner confirmed the presence and degree of chiral purity at C-6 of the isotopically substituted 1,1diphenyl-6-hexanols prepared by this method through the series of reactions summarized in Figure 6.

O D H

HO

+

HO

H

φ

H (S)-deuterotritioacetic acid

deuteroacetic acid

O

D D

OH

φ

HD

DT

1,1-diphenylhexane-1,6-diol

They converted cyclohexanone to 2,2,6,6tetradeuterocyclohexanone by an acid-catalyzed exchange of all four of the ␣ protons with deuterium oxide, and then converted this to the S,S isomer of 2,6-dideuterocyclohexanone by an AAD-catalyzed exchange of the two pro-R ␣ deuterons with the protons of ordinary water.

O

exchange with DOD; acid catalysis

C-6

OH

D T

D D

O 2,2,6,6-tetradeuterocyclohexanone

cyclohexanone

O

D D

D D

D H

H D

exchange with HOH; AAD catalysis

O D H

2,2,6,6-tetradeuterocyclohexanone

H D

(S,S)-2,6-dideuterocyclohexanone

(S,S)-2,6-dideuterocyclohexanone

They next subjected this chiral C2 symmetric product to the Baeyer–Villiger oxidation and treated the product of that reaction with an excess of phenylmagnesium bromide to give a dideutero 1,1-diphenyl-6-hexanol.

trifluoroperoxyacetic acid

O O

OH excess φ-Mg-Br in CH2Cl2,

OH

φ

φ

DH

H

D H

O

O

dideutero-1,1-diphenyl6-hexanol

Baeyer–Villiger product

φ

H D

H D

acid

HD

OH

D

Baeyer–Villiger product

OH

φ

HD

D H

dideutero-1,1-diphenyl6-hexanol

The primary OH group of this diol was esterified by treatment with camphanyl chloride in pyridine

camphanyl chloride/pyridine

O OH

φ

O

φ

O

O

DH

O

O

Cl

methanesulfonyl chloride/triethylamine, -20 °C

HD 1

O

camphanyl chloride

O

to give an intermediate, 1,

φ

O O

φ D/H

O

O

HD

OH

O

2

φ Figure 6. Stereochemical analysis of the Rozzell and Benner isotopically substituted 1,1-diphenyl-6-hexanol.

340

p333-345.65

O

φ

DH

HD 1


340

2/11/03, 10:01 AM

O


that was dehydrated by treatment with methanesulfonyl chloride in the presence of triethylamine O Cl

S

CH3

O

methanesulfonyl chloride

to form a product, 2, that was analyzed by proton NMR spectrometry.

Synthesis of Chiral Acetic Acid by Floss et al. In the synthesis of chiral acetate by Floss et al. (9, 10), the enantiospecificity is achieved by the use of an enantiomerically pure chiral reducing agent, rather than by the use of an (enantiomerically pure) enzyme, as in the synthesis of Rozell and Benner. The synthesis, outlined in Figure 7, involved reduction of ethyl 3,5-dimethoxybenzoate by lithium aluminum deuteride to the corresponding dideuterobenzyl alcohol, which was then oxidized to the monodeutero aldehyde by manganese dioxide.

O

C-6

φ

O O

φ

OH

C H3

O

HD

D/H

O

C H3

O

O O

D D

Et LiAlD4

2 C-6 D is pro-S

O

When the proton NMR spectrum of unlabeled 2 was determined at 270 MHz in a CDCl3 solution in the presence of the “shift reagent” Eu(dpm)3, the diastereotopic C-6 protons were easily distinguished. When the same analysis was performed on the deuterium substituted 2 shown above, the downfield resonance for the diastereotopic C-6 protons was absent, indicating, according to previous studies, that the proton of the deuterium substituted 2 had to be in the proR position, and, therefore, that the deuterium atom was in the pro-S position, as the representation of 2 indicates. This result was consistent with the formation of the S isomer of deuterotritioacetic acid in the synthesis described above. The R isomer of deuterotritioacetic acid could be prepared by reversing the sources of any of the two labels, using, for example, water and LiBEt3D instead of deuterium oxide and LiBEt3H. In practice, however, it turned out to be more convenient to prepare (R )-deuterotritioacetic acid by inverting the configuration at C-6 of the 1,1-diphenyl-1,6hexanediols shown below by the Mitsunobu reaction and continuing with the inverted products. OH

φ

T D

CH3

ethyl 3,5-dimethoxybenzoate

dideuterobenzyl alcohol MnO2

OH

C H3 O

O

C H3 O

H D

D (+)-α-pinanyl-9-BBN

O

O CH3

CH3

S isomer only

monodeuterobenzaldehyde

1. NaH, 2. p-toluenesulfonyl chloride

C H3

OTs

C H3 O

H D

DH

O

T

LiBEt3T

OH OH

φ

O CH3

+

φ

DH

OH

φ

HD

O O

CH3

CH3

DT

1. O3 2. HgSO4 /H2SO4

p-toluenesulfonate

1,1-diphenyl-1,6-hexanediols DH

Mitsunobu

O T OH

φ

φ

OH D H

T D

OH

+

φ

φ

OH

D T HD

deuterotritioacetic acid S isomer, 95.5% ee

OH

inverted 1,1-diphenyl-1,6-hexanediols Figure 7. The synthesis of chiral acetate by Floss et al.


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341


O

C H3

OH

C H3 O

O Et

O

CH3

D D

O S O

C H3 O

O

O

H D

CH3

CH3 ester

O

alcohol

O CH3 p-toluenesulfonate

O

C H3 O

D

Reduction of the p-toluenesulfonate with lithium triethylborotritide took place by displacement of ptoluenesulfonate with inversion at the chiral center to give α-deuterotritio-3,5-dimethoxytoluene in which the isotopically substituted methyl group had the S configuration.

O CH3 aldehyde

The aldehyde was then enantiospecifially reduced at the si face by (+)-␣-pinanyl-9-borabicyclo[3.3.1]nonane (-BBN) to give the S enantiomer of ␣-deutero-3,5-dimethoxybenzyl alcohol.

C H3

DH

O

T

O CH3

B

(S)-␣-deuterotritio3,5-dimethyoxytoluene H (+)-␣-pinanyl-9-BBN

OH

C H3 O

H D

Oxidation of the chiral toluene derivative with ozone (followed by further oxidation with mercuric sulfate and sulfuric acid to remove contaminating formic acid) give the S enantiomer of deuterotritioacetic acid in a yield of 75% and with an enantiomeric excess of about 95%. DH O T OH

O CH3


(S)-[␣-D]-3,5-dimethyoxybenzyl alcohol

The (greater than 95%) enantiomerically pure (S )-[␣D]-3,5-dimethoxybenzyl alcohol was then converted to the corresponding p-toluenesulfonate by, first, deprotonation with sodium hydride, and, second, treatment of the conjugate base of the alcohol with p-toluenesulfonyl chloride.

O

H D

O CH3 conjugate base

342

p333-345.65

The optical purity of the (S)-[α-D]-3,5-dimethoxybenzyl alcohol was determined by conversion to the (–)-camphanic acid ester and analysis of the ester by proton NMR spectrometry in the presence of a shift reagent. Preparation of the R Enantiomer

O−

C H3

Determination of Optical Purity

The R enantiomer of deuterotritoacetic acid was prepared in three ways. One made use of the method just described but substituted the levorotatory enantiomer of α-pinanyl-9BBN, (–)-α-pinanyl-9-BBN, for the (+)-α-pinanyl-9-BBN. Another made use of unlabeled aldehyde, but deuterated (+)α-pinanyl-9-BBN, and a third involved inversion of the configuration of the S benzyl alcohol by alkaline hydrolysis of its p-toluenesulfonate.


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2/11/03, 10:01 AM


D

CH3

S O

C H3 O

O H D

DH

O

alkaline

H

H T

C

N

O

hydrolysis

O CH3 CH3

H (R)-methylamine

(R)-acetic acid

OH

O

OH

H T

O C H3

D

The chiral methylamine was first monotosylated by the action of p-toluenesulfonylchloride and base. This was converted to the ditosylate by deprotonation with sodium hydride, followed by a second addition of p-toluenesulfonyl chloride. CH3 D

Additional Applications of Chiral Acetate

O S

H T

During the thirty years since the publication of the first syntheses and analyses of chiral acetate (1, 2), the details of a number of subtle biosynthetic transformations have been deduced by determining the configurations of chiral centers that were derived from or could be converted to chiral acetate.

N O

O O

S

Biosynthesis of Indolmycin Floss et al. have reviewed the work of their group (9), and this example, concerning the biosynthesis of indolmycin, is drawn from their research (11).

CH3 ditosylate

D

H3C

O

OH

H T

O

N

N H

C D


N H

CH3

O

CH3

S

H T

N

NaN3 /H2SO4

monotosylate D

The two methyl groups of indolmycin, one carbonbound and the other on nitrogen, were shown by their earlier work (12, 13) to be introduced by methyl transfer from (S )-adenosylmethionine (AdoMet or SAM).

−

NH2

NH3 +

O

S O

N

CH3

p-toluenesulfonyl chloride/NaOH

H

H T

NaH/TsCl/ dimethylformamide

N H

(R)-deuterotritiomethylamine

CH3 D

N

H

N

O

+

N

−

O

H

N O

NH3

O S

H T

H

CH2

O

H

indolmycin

+

O

S

O O

S O

H

H

/sodium metal/HMPT

OH

OH +

(S)-adenosylmethionine; AdoMet; SAM

The stereochemical question of inversion or retention of the transferred methyl group was addressed by repeating the experiment with methionine that contained a chiral methyl group. This material was prepared as outlined in Figure 8. (R )-Deuterotritioacetic acid was converted to (R )-methylamine by a Schmidt reaction, which is known to proceed with retention of configuration.

O

CH3 NH3

D

S

−

S

ditosylate

H T

O “(L,S)-methionine”

Figure 8. The preparation of “(L,S)-methionine”.


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343


The two strongly electron-withdrawing sulfonyl groups sufficiently diminish the basicity of the nitrogen atom that the methyl group is available as an electrophile. Thus when (S )-benzyl-(L)-homocysteine is reductively debenzylated by sodium metal in liquid ammonia and the ditosylate of the chiral methylamine is added, the chiral electrophilic methyl group is transferred with inversion to the nucleophilic sulfur anion to give “(L,S )-methionine”. + −

transferred from (S)-adenosylmethionine to both carbon and nitrogen with inversion. R

DH T

O O

NH3

N

O

T

H

R

DH

O

4 indolmycin from “(L,S)-methionine”

(S)-benzyl-(L)-homocysteine +

O

N

H

S

N

NH3

D

S

Determination of the Chirality of the Methyl Groups of Indolmycin The indolmycin was hydrolyzed by base to give indolmycenic acid and methylamine.

−

H T

S O “(L,S)-methionine”

This version of methionine was used in a microbial synthesis of indolmycin in which the methionine was presumably converted in vivo to the corresponding (S )-adenosylmethionine, 3, bearing a chiral methyl group on sulfur, by displacement of triphosphate as a leaving group from adenosine triphosphate.

DH

DH

T

O

−

−

O −

O

P

O

P

O

N

−

O

O O

O

P

O

CH2

O

N

O

H

N

H H

H OH

NaOH

N

N

N H

−

D +

O

O

CH2 H

DH

N

N

H

T

O HO

OH

Kuhn–Roth oxidation

O

H N

O

N

H H OH

Anticipating the results of the analysis of the indolmycin produced in this experiment, the material formed can be represented as 4, indicating that the methyl groups had been

(R)-acetic acid

indolmycenic acid

The methylamine was converted to the ditosylate, which was then treated with potassium cyanide in HMPA to give, with inversion, the chiral acetonitrile. OTs N

T

KCN

DH

DH (R)-ditosylate

2/11/03, 10:01 AM

CN

T

HMPT

(S)-acetonitrile


344

OH

N

TsO

p333-345.65

DH

indolmycenic acid and methylamine

DH

3 (S)-adenosylmethionine

344

T

The indolmycenic acid was subjected to Kuhn–Roth oxidation with chromic anhydride and acid. The deuterotritioacetic acid thus formed was shown to have the R configuration, indicating that methyl transfer from methionine had taken place with inversion, as shown above.

NH2

H OH

N

OH

S H T

S

OH

H

H

DH

T

NH3

N

T

indolmycin

adenosine triphosphate; ATP

+

HO

N

H

O

H H

NH2

T

O


The nitrile was converted to acetic acid via the amide by hydrolysis with alkaline hydrogen peroxide followed by diazotization in aqueous acid. CN

T DH

O H 2 O2

T

O NaNO2

C NH2

NaOH

T

OH

DH

DH

(S)-acetamide

(S)-acetonitrile

C

H2SO4

(S)-acetic acid

Since the conversion of the N-bound methyl group of indolmycin to (S)-acetic acid involved one inversion, the configuration of the N-bound methyl group must have been R, also as shown above. Floss and Lee (9) summarize more than a dozen additional examples of enzymatic methyl transfer reactions to carbon, nitrogen, oxygen, and sulfur that occur with inversion of configuration at the methyl carbon. They also mention that methyl transfer from methanol to mercaptoethanesulfonic acid (coenzyme M) to give CH3SCoM is stereospecific and proceeds with net retention of configuration. T H D

T

OH

(S)-methanol

−

O

O S

HS

O

−

O

M. barkeri

+

physical organic course or in a biochemistry course. To make this material more easily used by others, I have summarized most of the chemistry in the figures. I mention also that (S )adenosylmethionine was profiled in the July 5, 1999, edition of Newsweek (14) in an article with an unusual emphasis on chemistry.

O S

H D

S

O

CH3SCoM

coenzyme M

The participation of a cobalt-containing corrinoid enzyme in this reaction suggests that net retention could be accounted for by a methyl transfer with inversion to cobalt, followed by a second methyl transfer with inversion from cobalt to coenzyme M. Suggestions for Use All of the concepts and most of the reactions cited in this paper are typically presented in the sophomore-year organic course, and I originally used much of the material presented here as a topic in an advanced organic chemistry course. It served as a review, but more importantly it showed how the most basic reactions and fundamental concepts of organic chemistry can be combined to create an extraordinary result. I believe this material could be presented in a

One Last Item A reasonable question at this point is: Where does the methyl of methionine come from? The short answer is: From our diet, as methionine is an essential amino acid for people, as well as for poultry and pigs. But then: Where does the methyl of methionine come from in those organisms that synthesize methionine? Well, that is another story… Literature Cited 1. Cornforth, J. W.; Redmond, J. W.; Eggerer, H.; Buckel, W.; Gutschow, Christine. Nature 1969, 221, 1212–1213 2. Lüthy, J.; Rétey, J.; Arigoni, D. Nature 1969, 221, 1213–1215. 3. Müllhofer, G.; Rose, I. A. J. Biol. Chem. 1965, 240, 1341– 1346, and references cited therein. 4. Krakow, G.; Vennesland, B. Biochem. Z. 1963, 338, 31. 5. Kasperek, G. J.; Pratt, R. F. J. Chem. Educ. 1977, 54, 515– 516. 6. Eggerer, H.; Buckel, W.; Lenz, H.; Wunderwald, P.; Gottschalk, G.; Cornforth, J. W.; Donninger, C.; Mallaby, R.; Redmond, J. W. Nature 1970, 226, 517–519. 7. Rétey, J.; Lüthy, J.; Arigoni, D. Nature 1970, 226, 519–521. 8. Rozzell, J. David, Jr.; Benner, Steven A. J. Org. Chem. 1983, 48, 1190–1193. 9. Floss, Heinz G.; Lee, Sungsook. Acc. Chem. Res. 1993, 26, 116–122. 10. Kobayashi, Koji; Jadhav, Prabhakar K.; Zydowsky, Thomas M.; Floss, Heinz G. J. Org. Chem. 1983, 48, 3510–3512. 11. Woodard, Ronald W.; Mascaro, Leonard, Jr.; Hörhammer, Rolf; Eisenstein, Stephen; Floss, Heinz G. J. Am. Chem. Soc. 1980, 102, 6314–6318. 12. Hornemann, U.; Hurley, L. H.; Speedie, M. K.; Floss, H. G. J. Am. Chem. Soc. 1971, 93, 3028–3035. 13. Speedie, Marilyn K.; Hornemann, Ulfert; Floss, Heinz G. J. Biol. Chem. 1975, 250, 7819–7825. 14. Cowley, Geoffrey; Underwood, Anne. Newsweek, July 5, 1999, pages 46–50.


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"Chiral Acetate": The Preparation, Analysis, and ... - ACS Publications

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