Development of a practical mass spectrometry based assay for determining enantiomeric excess. A fast and convenient method for the optimization of PLE-catalyzed hydrolysis of prochiral disubstituted malonates

A practical mass spectrometry-based enantioselectivity assay is presented which makes use of enantiomerically enriched, but not enantiomerically pure, probe molecules readily obtained from esterase hydrolysis of prochiral malonates. The technique presented here allows us to recycle materials obtained from esterase hydrolysis which give substantial, but synthetically insufficient, enantiomeric excess as probe molecules in an enantioselectivity assay. The enantiomerically enriched products are esterified using deuterium-labelled alcohol. The enantiomeric excess is measured using mass spectrometry (LC–MS and LDI) by measuring the D5/H5 ratio in the resulting products obtained from an enzymatic hydrolysis. The D5/H5 ratio is corrected to account for the enantiomeric purity of the probe. Herein we report the results obtained from Pig Liver Esterase hydrolyses of prochiral malonate esters and outline the strengths and limitations of this approach to enantioselectivity determinations. This assay strategy was able to identify reaction conditions that led to an improvement in ee from 70% ee to >97% ee in the PLE-catalyzed hydrolysis of a prochiral malonate used to prepare unnatural serine analogues.

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(R)-2-(4-(Benzyloxymethyl)-3-ethoxy-2-methyl-3-oxopropanoic acidC14H18O5[α]D21.8=+7.7 (c 0.208, MeOH)Source of chirality: asymmetric synthesisAbsolute configuration: (2R)

(R)-2-(4-(Benzyloxy)benzyl)-3-ethoxy-2-methyl-3-oxopropanoic acidC20H22O5[α]D22=-1.0 (c 0.066, CH2Cl2)Source of chirality: asymmetric synthesisAbsolute configuration: (2R)

(S)-Ethyl-3-(benzyloxy)-2-((4-methoxybenzyloxy)carbonylamino)-2-methylpropanoateC28H31NO6[α]D17.8=+24.2 (c 0.07, CH2Cl2).Source of chirality: asymmetric synthesisAbsolute configuration: (1S)

(S)-α-Methyl tyrosine ethyl esterC12H17NO3[α]obs21.7=-0.10 (c 1.2 M HCl)Source of chirality: asymmetric synthesisAbsolute configuration: (1S)