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

Article abstract of DOI:10.1016/j.tetasy.2009.05.037

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.

Full text of DOI:10.1016/j.tetasy.2009.05.037

Tetrahedron: Asymmetry 20 (2009) 1476–1486
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
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
*
Douglas S. Masterson , Dale A. Rosado Jr., Cassie Nabors
The University of Southern Mississippi, Department of Chemistry and Biochemistry, 118 College Drive #5043, Hattiesburg, MS 39406, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 April 2009
Accepted 29 May 2009
A practical mass spectrometry-based enantioselectivity assay is presented which makes use of enantio-
merically 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 hydro-
lysis. 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.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
is good but at unacceptable levels for synthetic purposes.5,16,21,22,15
There are numerous reports in the literature of using various co-
Synthetic chemists have made significant use of enzymatic
reactions in the recent years to provide high value synthetic inter-
mediates. These high value synthetic intermediates are usually
enantioenriched molecules where the enantioselectivity is pro-
vided by the enzymatic process being utilized. It is arguably the
case that the hydrolysis of esters is by far the most widely used
enzymatic reaction for the preparation of high value synthetic
intermediates.1–3 Enzymatic hydrolysis has been utilized in the
preparation of biologically active compounds containing stereo-
genic quaternary centers. The synthetic value of hydrolytic
enzymes is unquestioned and has been effectively demonstrated
in the preparation of various quaternary carbon containing unnat-
ural amino acids,4–6 anti-cancer agents,7 enzyme inhibitors,8,9 and
analgesics.10 It is anticipated that the use of enzymes in organic
synthesis will continue to increase now that the most versatile of
the hydrolytic enzymes, Pig Liver Esterase (PLE), has been cloned
and expressed in yeast.11,12
solvents and altered reaction conditions, which drastically improve
the enantioselectivity of the enzymatic hydrolysis.23,21,24–26 How-
ever, it appears that there is no general model which can be used
to predict which co-solvents or altered reaction conditions will
provide high levels of enantioselectivity for a particular sub-
strate/enzyme combination.21 At present, synthetic chemists must
use a combinatorial approach for the identification of optimal reac-
tion conditions for each substrate under consideration. This has led
to a significant research effort in the recent years toward the devel-
opment of methods which can rapidly determine enantioselectivi-
ty from such reactions.27
Traditionally accepted methods of determining enantiomeric
excesses include polarimetry,28 chiral chromatography (both GC
andHPLC),29–32 and NMR(derivativeswith chiralauxiliariesand chi-
ral shift reagents).33–35 Although these appear to be the most
straightforward methods for determining enantiomeric excess,
there are significant drawbacks to each of these which make their
use as a convenient and rapid technique questionable. For example,
polarimetry requires compounds of high purity as the measurement
It is well documented that hydrolytic enzymes (e.g., PLE) are
capable of hydrolyzing a wide variety of substrates with high levels
of enantiocontrol and chemical yield.13–20 However, there are
instances where the enantioselectivity of the enzymatic hydrolysis
of [a] is susceptible to the presence of both chiral and achiral
contaminates. Chiral chromatographic methods overcome many of
the issues associated with polarimetry, but in many cases the chro-
matographic methods are too time consuming to be practical in a
combinatorial approach. The use of chiral shift reagents in NMR also
* Corresponding author.
E-mail address: Douglas.Masterson@USM.edu (D.S. Masterson).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.05.037

D. S. Masterson et al. / Tetrahedron: Asymmetry 20 (2009) 1476–1486
1477
has considerable drawbacks such as insufficient resolution of the
diastereomeric NMR signals to obtain accurate signal integrations.
Chiral compounds can also be derivatized with enantiomerically
pure auxiliaries to generate diastereomeric pairs, which can be ana-
lyzed by NMR. However, this method requires additional manipula-
tion prior to analysis, resulting in reduced sample throughput.
Significant advances have been made with NMR allowing for high
throughput screening for enantioselectivity. Reetz has developed an
NMR technique based on the use of 13C-labelled pseudo enantiomers
andpseudomesocompounds.36,37 Thetechniquemakes useof apseu-
do racemate comprising one enantiomer that is 13C-enriched while
the other enantiomer is 12C-enriched. The reporting moiety in each
case is a methyl group. The products of the reaction are analyzed
using 1H NMR by integration of the singlet (12C-enriched methyl
group) and the doublet (13C-enriched methyl group) giving the
enantioselectivity of the reaction under investigation. The NMR
technique developed by Reetz et al. utilizes recent advances in
NMR flow cell technology and an auto-sampler resulting in a
throughput of approximately 1400 determinations per day.
Several groups have recently developed methods to determine
the enantioselectivity using various spectrophotometric reporter
systems. Anslyn et al. have developed UV–vis and fluorescence sys-
tems capable of determining the enantiomeric excess of various
compound classes.38–41 In one embodiment, Anslyn and co-work-
ers made use of amino acid coordination to a preformed chiral cop-
per complex containing an appropriate spectroscopic reporter.38
The reporter is displaced by coordination of the amino acid result-
ing in an absorbance change. The change in absorbance was found
to be proportional to the enantiomeric purity of the amino acid
under consideration. Anslyn has recently reported on an attractive
assay using CD spectroscopy to determine both enantiomeric
excess and concentration.42 Berkowitz has developed an ISES
(in situ enzymatic screening) assay43,44 that is capable of readily
determining enantiomeric excess and kinetic information by mak-
ing use of a dual cuvette and two reporter enzymes.45,46 The dual-
cuvette ISES assay was used successfully to screen catalysts for
their efficiency in the hydrolytic kinetic resolution of various race-
mic epoxides.45,46 The ISES assay was able to identify, in a combi-
natorial fashion, several catalysts capable of resolving epoxides
with acceptable levels of enantiomeric purity. Reetz has reported
on the use of FTIR to screen for enantioselectivity.47 The FTIR tech-
nique exploits the fact that a 13C-labelled carbonyl is more signif-
icantly shifted to a lower wave number (ꢀ40–50 cmꢁ1) in the
carbonyl region of the IR spectrum than is its 12C-enriched counter-
part. The FTIR screening method makes use of a pseudo racemic
mixture where one enantiomer has a 13C-labelled carbonyl while
the other enantiomer is a 12C-carbonyl. Applying the Beer–Lambert
law then allows for the calculation of enantiomeric excess. Unfor-
tunately, in order to apply the Beer–Lambert law, one must know
the molar extinction coefficient for each species under investiga-
tion. However, the FTIR method allows for up to 10,000 samples
per day to be processed using commercially available FTIR equip-
ment coupled to auto-sampler/plate reader devices.
pseudo racemate and pseudo meso compound strategy.54,55 Reetz
successfully used this strategy to assay enzymes for their ability to
perform kinetic resolutions. Reetz further refined the technique
using a specialized eight-channel multiplexed sprayer system cou-
pled to a mass spectrometer increasing the sample throughput to
approximately 10,000 samples per day.
Herein we report on our efforts to develop and implement a con-
venient assay for the determination of enantiomeric excess, which is
practical for the synthetic chemists with specific target compounds
under consideration. We are interested in exploiting hydrolase
enzymes, particularly PLE, to provide half-ester intermediates from
prochiral malonate esters for the preparation of various amino acid
classes.4 We have frequently encountered situations where the PLE
hydrolysis provides the half-ester intermediates in significant but
insufficient enantioselectivities (approximately 50–70%ee). Scheme
1 illustrates our assay strategy for enantioselectivity which allows
for the recycling of the half-ester products obtained from a signifi-
cant yet insufficient hydrolysis. The probes obtained from these
recycled products are then utilized, using the classical isotope dilu-
tion method,56 to screen for reaction conditions which provide
acceptable levels of enantioselectivity. The enantiomeric excess cal-
culated from the MS analysis can then be corrected to account for the
enantiomeric purity of the probe.35 To the best of our knowledge
thereareno reportsofrecyclingreactionproductsforassaypurposes
as illustrated in Scheme 1. We believe that the methodology
described below will find widespread use by synthetic chemists
given thatthe technique is straightforward and utilizesreadilyavail-
able instruments and synthetic methods.
2. Results and discussion
Weinitially attempteda PLEhydrolysisof malonate 1 as shown in
Scheme 2 using the standard PLE hydrolysis protocol to ultimately
prepare a series of unnatural serine analogues.4 Chiral HPLC analysis
of the half-ester product 2 obtained revealed that the hydrolysispro-
ceeded with only 70% ee. Although this is considerable enantioselec-
tivity, it is of little synthetic value for our purposes as we require
nearly enantiomerically pure material. Half-ester 2 was converted
into the pseudo prochiral probe 3 by preparing the acid chloride of
2 followed by treatment with ethanol-d6. We had attempted the
DCC coupling protocol to prepare probe 3, but found that the acid
chloride method required less extensive workup. The resulting com-
pound 3 was isolated by chromatography and completely character-
ized by NMR (1H and 13C) and high resolution MS analysis.
We first wanted to determine which MS technique would be the
most advantageous (MALDI or ESI). However, given the low molecu-
lar weight of our samples we could not use traditional MALDI due to
matrix interferences in the needed m/z range. Fortunately, our sam-
ples contain an aromatic ring and we were able to observe strong sig-
nal intensities of our half-ester products as potassium adducts using
laser desorption ionization (LDI). Unfortunately, obtaining useful LDI
data required extensive workup of the enzymatic reactions prior to
analysis.Theworkupinvolvedanextractionofthehalf-esterproducts
into methylene chloride to separate the products from the buffer in
the reaction medium. Attempts to directly analyze the reaction med-
ium proved difficult due to salt-promoted ion suppression. The
extractions required relatively large aliquots be taken from the enzy-
matic hydrolysis reactions and resulted in dramatically decreased
sample throughput. We also observed varying signal intensities from
sample to sample using LDI. We attributed the variability in signal
intensity to the varying thickness of the sample on the MALDI plate
from sample to sample. Using LC–MS proved to be more convenient;
the LC conditions were optimized such that the samples required lit-
tle preparation and analysis could be performed on small aliquots of
reaction medium. It was determined that useful data could be
obtained on samples in the low micromolar concentration range
Mass spectrometry has gained interest in the recent years as a
technique for the determination of enantiomeric excess. Several
techniques have been reported that make use of pseudo racemates
in which the pseudo enantiomers differ from one another by
mass.48–52 Pfaltz et al. have developed an MS-based enantioselectiv-
ity assay which was used to screen a library of ligands in palladium-
promoted allylic substitution reactions.53 In this embodiment one of
the pseudo enantiomers contains a methyl substituent while the
other pseudo enantiomer contains an ethyl substituent sufficiently
removed from the reaction center. This mass tagging strategy allows
for the rapid determination of enantiomeric excess by simply mea-
suring the ion intensities using electrospray ionization. Reetz has
exploited mass spectrometry in enantioselectivity assays using the

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D. S. Masterson et al. / Tetrahedron: Asymmetry 20 (2009) 1476–1486
O
O
O
O
Enzyme
Chiral HPLC
Analysis
EtO
OEt
EtO
OH
Standard Conditions
R
R
Substantial (yet insufficient)
Enantioselectivity
Known Enantiomeric Excess
Esterification
O
O
EtO
OEt(D5)
R
Probe
(known % ee)
1. Enzyme
(various conditions)
2. MS Analysis
I
m/z
Scheme 1. Mass spectrometry enantioselectivity assay using optically impure probes.
O
O
O
O
(R)
40000
35000
30000
25000
20000
15000
10000
5000
PLE
pH 7.4 buffer
EtO
OEt
EtO
OH
Chiral HPLC of 2 (PLE)
(S)
OBn
OBn
1
2
70% ee
NaOH
O
1. SOCl2
2. C2D6O
0
18
20
22
16
Time (min)
O
O
O
EtO
OH
EtO
OEt(D5)
OBn
OBn
3
+/- 2
35000
30000
25000
20000
15000
10000
5000
0
84% Yield
16
18
Time (min)
20
22
Scheme 2. Preparation and analysis of probe 3 (Chiralcel OJ-H column).
using select ion monitoring (SIM) of our half-ester products. Figures 1
and 2 show selected spectra obtained by LDI and LC–MS in the SIM
mode of the half-esters obtained by PLE hydrolysis of probe 3. The
LDI provided strong signals for potassium adducts as the buffer sys-
tem utilized in the PLE hydrolysis is potassium phosphate while the
LC–MS provided either intense proton or sodium adducts.
Our initial experiments were to test the hypothesis that enanti-
omerically impure probe molecules could be used in an MS-based
assay for enantioselectivity. The initial data were used to compare
the corrected ee values obtained by MS to the ee values obtained
by the traditional chiral HPLC method (without any correction of
the data). In order to accomplish this we needed half-ester
products whose ee values spanned a reasonable range of enantio-
meric purities. We obtained the necessary half-ester products in a
variety of ee values as shown in Scheme 3. Starting with probe 3,
we performed a typical PLE hydrolysis to obtain half-ester 4. We
performed both MS analysis and chiral HPLC analysis on 4 and
found the values to be in close agreement with one another. Ali-
quots of 4 were then spiked with varying amounts of racemic D5/
H5-labeled mixture 5 (50% H5/50% D5). The ee of the spiked sam-
ples was determined by chiral HPLC analysis and each sample
was analyzed by LC–MS in the SIM mode.
Figure 3 shows that the data obtained by chiral HPLC analysis
and the corrected MS values are in reasonable agreement with

D. S. Masterson et al. / Tetrahedron: Asymmetry 20 (2009) 1476–1486
1479
Figure 1. Laser desorption ionization (LDI) analysis of half-esters obtained by PLE hydrolysis of 3.
Figure 2. LC–MS analysis in SIM mode of half-ester products obtained by PLE hydrolysis of 3.
one another. Figure 3 illustrates a slope of near unity and an
acceptable fit to a straight line with a Y-intercept zero as expected.
This limited data set suggests that it is feasible to utilize enantio-
merically impure probes in a mass spectrometry-based enantiose-
lectivity assay by correcting for the enantiomeric purity of the
probe. Secondary kinetic isotope effects are expected to be insignif-
icant and within the limit of error for this method. The work
published by Reetz et al. shows an excellent correlation of MS ee
values with ee values obtained by chiral GC demonstrating that
the secondary isotope effects are insignificant.55
It has been demonstrated in several studies that hydrolytic
enzymescan be tuned to provide better enantioselectivities by alter-
ing the reaction medium, a process known as reaction medium engi-
neering.2 Although the natureof the effect is uncertain, it is clear that
the effect can be exploited in the preparation of advanced synthetic
intermediates.57,21 We sought to explore various conditions such as

1480
D. S. Masterson et al. / Tetrahedron: Asymmetry 20 (2009) 1476–1486
O
O
O
O
O
O
PLE
pH 7.4 buffer
EtO
OEt(D5)
OBn
+ Isotopologues of each
enantiomer
+ HO
OEt(D5)
OBn
EtO
OH
OBn
3
4
(70% ee (S))
70% ee by chiral HPLC
O
O
O
O
O
O
NaOH
+
EtO
OEt(D5)
EtO
OH
OBn
HO
OEt(D )
5
+ Isotopologues of each
enantiomer
OBn
OBn
5
+/-
3
+/-
5
+/-
Chiral HPLC
MS
4
4
( % ee < 70%)
(70% ee)
m/z
Scheme 3. Preparation of 4 with various % ee.
We turned our attention to the buffering species to determine if
the choice of buffer could have an effect on the outcome of the PLE-
catalyzed hydrolysis. We chose a variety of commonly used buffer
systems, such as phosphate (control), glycine–glycine, TRIS, imid-
azole, tricine, and bis–tris propane. This set of assays was
performed in a similar manner as described above for the assay
varying pH. We noticed that most of the buffer systems we chose
initially resulted in similar enantioselectivities to those obtained
using the simple phosphate buffer system. This suggests that the
choice of buffer is relatively unimportant. However, we did observe
significantly lower enantioselectivity compared to the control for
several of the buffer systems over time using probe 6 (Scheme
4). This observation suggests that extended reaction times in the
presence of nucleophilic buffering species can significantly de-
grade the enantioselectivity of the final product. We suspect these
result in lower enantiomeric excesses over time due to racemic
background hydrolysis.58,59
We next turned our attention to the addition of various co-sol-
vents to the PLE reaction medium. We decided to use the phos-
phate buffer system for the co-solvent study based on our results
from the buffer assay outlined above. We chose to study the
following miscible co-solvents: methanol, ethanol, isopropanol,
diglyme, triglyme, acetonitrile, DMF, dioxane, and DMSO. We
found that varying the co-solvent composition between 5% and
30% was tolerated by the phosphate buffer and all co-solvent
compositions were completely homogenous. Attempts to make
compositions greater than 30% co-solvent frequently resulted in
the crystallization of phosphate and/or two phase systems and
were not pursued further in this study. The assays were conducted
as described above substituting the co-solvent/phosphate buffer
mixtures for the phosphate buffer. The pH of the resulting co-sol-
vent/buffer solutions was not adjusted. The assay samples were
shaken vigorously at 25 °C and were analyzed by LC–MS.
100
80
60
40
20
0
m = 1.01
R2 = 0.95
e
0
20
40
60
80
100
% ee (HPLC)
Figure 3. Correlation of HPLC% ee with corrected% ee values from LC–MS analysis.
pH, buffer type, and co-solvent composition on the PLE-catalyzed
hydrolysis of 3 and 6 in the hope of obtaining conditions capable of
providing 4 and 8 with synthetically useful levels of enantioselectiv-
ity (Scheme 4). Compound 8 was chosen due to the fact that 8 can be
readily transformed into tyrosine analogues. Initially we decided to
determine if it would be possible to simply alter the pH of the phos-
phate buffer and observe significant changes in enantioselectivity.
We conducted our assays in small conical vials containing approxi-
mately 10 mg of either probe 3 or 6, 1.5 mL of phosphate buffer at
the desired pH, and 3 lL of PLE suspension (4 units/assay). The assay
samples were shaken vigorously at 25 °C for a specified time and
then small aliquots were removed for analysis by LC–MS, LDI, and
chiral HPLC. The data presented in Figure 4 clearly indicate that
changes in pH, over the narrow range studied here, have at best a
minimal influence on the enantioselectivity of the PLE-catalyzed
hydrolysis of probes 3 and 6. Figure 4 also illustrates that this general
trend is observed regardless of the analytical technique used (HPLC,
LDI, or LC–MS). This suggests that small changes in pH around the pH
optimum for PLE do not alter the dimensions of the chiral pocket to
any useful extent.16
We analyzed the primary alcohol co-solvent compositions first
as we were concerned about the possibility of transesterification
with the alcohol co-solvent. The chromatogram in Figure 5 shows

D. S. Masterson et al. / Tetrahedron: Asymmetry 20 (2009) 1476–1486
1481
O
O
O
O
O
O
PLE
1. SOCl2
2. C2D6O
EtO
OEt
EtO
OH
EtO
OEt(D5)
pH 7.4 Buffer
OBn
OBn
8
OBn
6
7
63% ee
PLE
(Various Buffers)
70
60
50
40
30
O
O
O
O
+
Tris
Imid
EtO
OH
HO
OEt(D5)
LC-MS
BTP
Gly-Gly
Tricine
1
2
3
4
5
6
7
8
Time (Days)
OBn
OBn
Scheme 4. Preparation of probe 6 and buffer assay results.
Probe 3
Probe 6
80
70
60
50
40
80
70
60
50
40
e
HPLC % ee
HPLC % ee
LDI-ToF Corr % ee
ESI-MS Corr % ee
LDI-ToF % ee
ESI-Ion Trap % ee
6.0
6.5
7.0
7.5
pH
8.0
8.5
6.0
6.5
7.0
7.5
8.0
8.5
pH
Figure 4. Effect of pH on the enantioselectivity of PLE hydrolysis of 3 and 6.
the total ion current chromatogram (TIC) and the reconstructed ion
chromatograms (RICs) for the assay performed in 10% methanol
co-solvent. It is apparent that several chromatographic peaks are
present indicating significant transesterification with the primary
alcohols. The identity of each of the chromatographic peaks was
determined using MS/MS analysis and comparison of the retention
times to those of authentic samples. MS analyses of the half-ester
products obtained using methanol and ethanol co-solvents pro-
duce flawed results due to the loss of the deuterium label by equil-
ibration with the co-solvent. This finding, although not entirely
unexpected, does place a limit on the assay in that highly nucleo-
philic co-solvents capable of forming transesterification products
with isobaric species must be avoided. A similar analysis with both
tert-butanol and isopropanol did not result in significant produc-
tion of transesterification products. Table 1 shows the results of
the co-solvent assays performed with probes 3 and 6. The non-
nucleophilic co-solvents studied demonstrate their ability to alter
the enantioselectivity of the PLE-catalyzed hydrolysis of 3. How-
ever, most of the co-solvents studied resulted in only marginal
improvements in enantioselectivity. The data in Table 1 show that
10% isopropanol co-solvent resulted in essentially enantiomeri-
cally pure material [ꢀ103% ee, (R)-isomer] for probe 3. The authors
realized that 103% ee is impossible.60 The value is obtained follow-
ing the correction for the probe purity. We chose to scale up the
PLE hydrolysis using 10% isopropanol co-solvent and diester 1.
The resulting half-ester was isolated in 60% yield and was analyzed
by chiral HPLC. Chiral HPLC indicated that the half-ester 2 was
produced in >97% ee and that the major isomer was of the (R) abso-
lute configuration (Scheme 5). This finding suggests that isopropa-
nol should be examined as a co-solvent in the PLE-catalyzed
hydrolysis of other prochiral malonates.24,61
Diester 7, a precursor to unnatural tyrosine analogues, is hydro-
lyzed by PLE in reasonable yield and moderate enantioselectivity
(63% ee). The half-ester 8 was recycled into probe 6 using the acid
chloride esterification method described above to produce 3
(Scheme 4). Probe 6 was subjected to PLE hydrolysis in various
co-solvents and was analyzed by LC–MS in the SIM mode. The data
in Table 1 identify 30% isopropanol co-solvent as the best reaction
medium for the hydrolysis of 8 with respect to enantioselectivity.
Diester 7 was hydrolyzed by PLE in 30% isopropanol co-solvent
in a scaled up reaction to produce 8 in <10% yield. The chiral HPLC
analysis of 8 obtained from the scaled up reaction confirmed the
production of the half-ester in 85% ee with the (R)-absolute stereo-
chemistry. The stereochemistry was confirmed by transforming
acid ester 8 into the
a-tyrosine analogue 10 via a Curtius rear-
rangement and comparison of the specific rotation with that of
authentic material (Scheme 6). Although the use of such high co-
solvent composition has a deleterious effect on the isolated yield

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D. S. Masterson et al. / Tetrahedron: Asymmetry 20 (2009) 1476–1486
Figure 5. LC–MS chromatogram of the PLE hydrolysis of 3 in methanol co-solvent.
of 8, the improvement in enantioselectivity is significant. We are
currently performing additional co-solvent assays on probe 6 to
identify reaction conditions that can provide the high levels of
enantioselectivity observed with the 30% isopropanol co-solvent
without detrimental effects on the yield.
obtained by LC–MS analysis with the values obtained by chiral
HPLC analysis. The linear regression of the data shows a line
of excellent fit with a slope of near unity and a Y-intercept near
zero. The excellent fit is noteworthy that the corrected MS-val-
ues contain the error of two independent measurements (the
observed % ee by MS and the % ee of the probe as determined
by chiral HPLC). This study demonstrates the viability of a ‘green’
methodology by recycling chiral compounds as probe molecules,
which can be used in assays designed to optimize reaction con-
ditions. The assay also allows us to determine the absolute
3. Conclusion
Much of the data obtained in this study are plotted in Figure
6 to illustrate the correlation of the corrected % ee values

D. S. Masterson et al. / Tetrahedron: Asymmetry 20 (2009) 1476–1486
1483
Table 1
100
Selected co-solvent assay data for probes 3 and 6
m = 0.95
Y-Int = 3.6
80
60
40
20
0
Co-solvent
Probe 3 corrected % ee
Probe 6 corrected % ee
R2 = 0.98
10%
20%
30%
10%
20%
30%
e
i-PrOH
t-BuOH
DMSO
MeCN
DMF
Dioxane
Diglyme
Triglyme
103
85
72
46
70
68
66
68
n.r.
n.r.
73
68
60
74
72
71
n.r.
n.r.
75
72
66
75
74
66
71
71
72
46
67
75
71
72
79
77
73
45
72
77
71
75
90
85
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
0
20
40
60
80
100
% ee (HPLC)
n.r. = not reported due to weak signal intensity.
n.a = not available.
Figure 6. Correlation of HPLC% ee with corrected% ee by LC–MS.
4. Experimental
4.1. General
stereochemistry of the hydrolysis reaction if the starting probe
configuration is known by simple inspection of the H5/D5 half-
ester ratio. We are currently using the MS assay described herein
to find optimum reaction conditions for enzymatic hydrolysis of
other prochiral malonates of interest in our laboratories and we
will report on those efforts in due course.
THFwasdistilledfromsodiumunderanitrogenatmosphereprior
to use. Methylene chloride and 1,2-dichloroethane were distilled
from CaH2 under a nitrogen atmosphere prior to use. Triethylamine
(R)
50000
40000
O
O
O
O
PLE
2 (PLE)
30000
EtO
OEt
EtO
OH
10% i Pr-OH/Buffer
20000
OBn
OBn
1
2
10000
0
(S)
60% Yield
> 97% ee
13
14
Time (min)
15
16
30000
25000
20000
15000
10000
5000
0
Racemic 2
13
14
15
16
Time (min)
Scheme 5. Scaled-up synthesis of 2 by PLE hydrolysis in 10% iPr-OH co-solvent (Chiralcel AD-H column).
O
O
O
H
N
O
OPMB
1. DPPA/Et3N
2. PMB-OH
1. H2, Pd/C
2. HCl
NH3Cl
EtO
EtO
OH
EtO
O
OBn
OBn
(R)-8
OH
(-)-10
9
O
O
NH2
EtOH/SOCl2
NH3Cl
HO
EtO
OH
(S)−α-Methyl-L-Tyrosine
OH
(-)-10
Scheme 6. Assignment of absolute stereochemistry as (S) for 11.

1484
D. S. Masterson et al. / Tetrahedron: Asymmetry 20 (2009) 1476–1486
was distilled from NaOH pellets under a nitrogen atmosphere prior
to use. Diphenylphosphorylazide (DPPA) was prepared using a liter-
ature procedure.62 All other chemicals and enzymes were obtained
from Aldrich Chemical and were used as received unless otherwise
noted. Diethyl 2-(benzyloxymethyl)-2-methylmalonate 1 was syn-
thesized according to a literature procedure.4
RP HPLC column (50 ꢂ 2.1 mm, ThermoFisher). The mobile phase
was 70:30 methanol/water at a flow rate of 100 L/min. The LC–
MS system was programmed to divert the initial 2 min of flow to
waste to desalt the sample. The data were collected for the chro-
matographic peak in SIM mode and the intensities were used to
calculate enantiomeric excess.
l
Mass spectrometry grade methanol was purchased from
Aldrich. HPLC grade water was purchased from Fisher. Acetic acid
was purchased from Aldrich. Auto sampler vials and caps were
purchased from VWR. Samples were analyzed using either a Ther-
moFisher LXQ ESI-Ion trap mass spectrometer coupled to a Ther-
moFisher Accela HPLC system or a Bruker Microflex MALDI mass
spectrometer in the LDI mode. All NMR spectra were acquired with
a Varian Mercury 300 MHz spectrometer and referenced to either
residual solvent protons or to TMS. IR spectra were acquired with
a Thermo-Nicolet Nexus 470-FT-IR using a diamond anvil ATR
accessory. UV–vis spectra were acquired with an HP 8452 spectro-
photometer. Optical rotation measurements were acquired with a
Rudolph Research Autopol III autopolarimeter using a 1 dm cell at
ambient temperature. TLC analysis was performed on EMD science
silica coated aluminum plates and was visualized using UV or
phosphomolybdic acid stain. Flash chromatography was per-
formed using Silicycle silica gel (Silia-P). Chiral HPLC was per-
formed using a LabAlliance Series III isocratic pump coupled to a
LabAlliance Model 500 UV–vis detector. HPLC chromatograms
were recorded using the PeaksimpleÒ data acquisition system
and software. Chiral HPLC was performed using a Chiralcel OJ-H
analytical column or a Chiralcel AD-H column from Chiral Technol-
ogies, Inc. HRMS analysis was performed at Old Dominion Univer-
sity on an Apex FT-MS using a 1:1 THF/MeOH solvent system with
added NaCl to observe sodium adducts of the compounds of inter-
est. Melting points were determined in an open capillary tube
using a Hoover melting point apparatus and are uncorrected.
4.4.1. (R)-2-(4-(Benzyloxymethyl)-3-ethoxy-2-methyl-3-
oxopropanoic acid 2
Synthesized using a literature procedure.4 The characterization
data match those of authentic material. The % ee was determined
by analytical chiral HPLC Chriacel OJ-H (257 nm, flow rate = 1 mL/
min, 4% iPr-OH/96% hexane) tR = 16.90 min, tR = 19.20 min or
ðRÞ
ðSÞ
Chiracel AD-H (45 °C, 257 nm, flow rate = 1 mL/min, 4% iPr-OH/
96% hexane) tR = 13.65 min, tR = 15.20 min.
ðRÞ
ðSÞ
4.4.2. D5-Diethyl 2-(benzyloxymethyl)-2-methylmalonate 3
Compound 2 (0.45 g, 1.7 mMol) was dissolved in 25 mL of
CH2Cl2 in a 50 mL round-bottomed flask with a magnetic stirbar.
Thionyl chloride (1.8 mL, 25.2 mMol) was added and the solution
was heated to reflux solvent overnight. The solution was concen-
trated in vacuo to remove excess thionyl chloride and solvent.
The acyl chloride was dissolved in 10 mL of dry CH2Cl2. A solution
of 220 lL of Et3N in 1 mL of ethanol-d6 was added dropwise to the
stirring acyl chloride under a dry nitrogen atmosphere. The reac-
tion mixture was stirred overnight at ambient temperature. The
reaction mixture was diluted with 20 mL of ether and was washed
three times each with 10% HCl and 1.0 M NaOH. The organic layer
was dried over MgSO4, filtered, and concentrated in vacuo to give 3
as a clear, viscous oil 0.43 g (1.41 mMol 84% yield) TLC (30% ether/
hexane) Rf = 0.28. IR (cmꢁ1) 1727, 1H NMR (300 MHz, CDCl3) 1.21
(3H, t, J = 7 Hz), 1.52 (3H, s), 3.82 (2H, s), 4.16 (2H, q, J = 7 Hz),
4.53 (3H, s), 7.28 (5H, m). 13C NMR (75 MHz, CDCl3) 14.2, 18.6,
55.0, 61.5, 72.8, 73.6, 81.8, 127.6, 127.8, 128.5, 138.2, 170.8. HRMS:
[C16H17D5O5Na+] calcd = 322.1679 obsd = 322.1670.
4.2. Enzyme assays (general procedure)
Approximately 10 mg of the probe under study 3 or 6 was
placed in a 2.0 mL microcentrifuge tube along with 1.5 mL of reac-
4.4.3. ( )-D5/H5-2-(4-(Benzyloxymethyl)-3-ethoxy-2-methyl-3-
oxopropanoic acid 5
tion media (buffer or buffer/co-solvent mixtures). A 3
l
L aliquot of
Compound 1 (4.00 g, (13.6 mMol) was dissolved in 11 mL of eth-
anol after which 0.790 g of KOH was added in a 50 mL round-bot-
tomed flask with a magnetic stirbar. After 48 h, the reaction
mixture was diluted with 20 mL of water and extracted three times
with20 mLof ether. TheaqueouslayerwasthenacidifiedtopH2and
extracted three times with 20 mL of CH2Cl2, the combined organic
layers were dried over MgSO4, filtered and concentrated in vacuo
to give the racemic half ester of 2 (2.21 g, 8.30 mMol, 61% yield).
The racemic half ester of 2 (0.446 g, 1.68 mMol) was then esterified
by the same procedure reported to prepare 3 to give 0.422 g
(1.41 mmol, 84% yield) of the racemic D5/H5 diester. The racemic
D5/H5 diester (0.357 g) was dissolved in a 4:1 iPr-OH/H2O solution
a 50 mg/mL PLE solution (in 0.1 M phosphate buffer) was added
(4.1 units total). The samples were loaded into an EppendorfTM
Thermomixer at 25 °C and 1400 RPM mixing rate. The samples
were incubated and mixed continuously for three to five days for
3 and 6, respectively. Aliquots were taken at the desired time inter-
vals and were analyzed by either LC–MS or LDI-ToF MS. The
observed ee values were corrected for the enantiomeric purity of
the probe by dividing the observed ee by the enantiomeric purity
of the probe as determined by chiral HPLC analysis.35
4.3. LDI-ToF MS
containing95.5 lLof a50%w/vaqueousNaOHsolution. Thereaction
A 200
acidified with 30
then extracted with 200
l
L aliquot of reaction media was placed in a glass vial and
L of 10% HCl solution. The acidified media were
L of CH2Cl2. A 2 L aliquot of the organic
was monitored by TLC and was complete after 48 h. The reaction
mixture was diluted with 20 mL of 1.0 M NaOH and was extracted
three times with ether. The aqueous layer was acidified to pH 2
and extracted three times with CH2Cl2, dried over MgSO4, filtered,
and concentrated in vacuo to give racemic D5/H5 half ester 5
[0.209 g, 0.775 mMol (based on average molecular weight), 65%
yield]. TLC (5% MeOH/CH2Cl2) Rf = 0.317. 1H NMR (300 MHz, CDCl3)
1.23 (1.5H, t, J = 7 Hz), 1.54 (3H, s), 3.81 (2H, s), 4.20 (1H, q, J = 7 Hz),
4.55 (2H, s), 7.28 (5H, m). The % ee was determined by analytical chi-
ral HPLC (Chiracel AD-H, 257 nm, flow rate = 1 mL/min, 4% iPr-OH/
96% hexane) tR = 14.97 min, tR = 17.62 min.
l
l
l
phase was spotted directly onto a polished steel MicroscoutÒ chip
(Bruker Daltonics). The sample was allowed to dry at ambient tem-
perature by gently passing a stream of air over the MicroscoutÒ
chip. An average of 800 laser pulses were collected for each sample
and the intensities of the peaks were used for the calculation of
enantiomeric excess.
4.4. Esi-ion trap ms
ðRÞ
ðSÞ
A 200
sampler vial and was diluted with 200
autosampler was programmed to inject 1
l
L aliquot of reaction media was placed in a glass auto-
L of methanol. The Accela
L onto a Hypersil Gold
4.4.4. Diethyl 2-(4-(benzyloxy)benzyl)-2-methylmalonate 7
Prepared in a similar fashion as reported in the literature.59A
250 mL three-necked round-bottomed flask was charged with
l
l

D. S. Masterson et al. / Tetrahedron: Asymmetry 20 (2009) 1476–1486
1485
100 mL of dry THF, a stirbar, and 1.70 g (41.9 mMol) of NaH (60% dis-
persion in mineral oil). The flask was fitted with a rubber septum, a
glass stopper, and a reflux condenser to which a nitrogen inlet was
attached. The flask was placed in an ice bath and allowed to stir for
15 min. A solution of diethyl methylmalonate (6.07 g, 34.9 mMol
in 5 mL dry THF) was added dropwise to the NaH suspension at
0 °C. The solution was allowed to stir at 0 °C until no further gas evo-
lution was observed. The reaction mixture was removed from the ice
bath and allowed to stir at ambient temperature for 90 min. A solu-
tion of 1-(benzyloxy)benzyl)-4-(chloromethyl)benzene (6.00 g,
38.3 mMol, in 30 mL dry THF) was added dropwise and the rubber
septumwasrapidlyreplacedwithaglassstopper. Theresultingsolu-
tion washeated at refluxovernight. Thesolutionwas thenallowedto
cool to ambient temperature and 1 mL of water was added to quench
any remaining NaH. The resulting suspension was diluted with
100 mL of ether and placed in a separatory funnel. The suspension
was then washed three times with brine and three times with 10%
HCl. The organic layer was dried over MgSO4, filtered, and concen-
trated in vacuo. The crude material was dissolved in warm pentane
and cooled to ꢁ25 °C for 5 h. The resulting white solid was isolated
by vacuum filtration and washed with three 20 mL portions of cold
pentane. 11.73 g (31.7 mMol, 91% yield) of 9 as a white amorphous
solid. TLC (20% EtOAc/hexane) Rf = 0.3. Mp = 49 °C, IR (cmꢁ1) 1727,
1H NMR (300 MHz, CDCl3) 1.25 (6H, t, J = 7 Hz), 1.33 (3H, s), 3.17
(2H, s), 4.19 (4H, q, J = 7 Hz), 5.02 (s, 2H), 6.92 (2H, d, J = 9 Hz), 7.04
(2H, d, J = 9 Hz) 7.38 (5H, m). 13C NMR (75 MHz, CDCl3) 14.3, 19.9,
40.5, 55.1, 61.5, 70.2, 114.7, 127.7, 128.1, 128.7, 128.8, 131.4,
137.2, 158.0, 172.2. HRMS: [C22H26O5Na+] calcd = 393.1678
obsd = 393.1672.
6.62 mmol, 76% yield) half ester ( )-8. Compound ( )-8 was then
esterified by the same procedure reported for 3 to give 0.391 g
(1.043 mMol, 71% yield) of the racemic D5/H5 diester. The racemic
D5/H5 diester (0.618 g, 1.646 mMol) was then dissolved in 13 mL of
a 4:1 iPr-OH/water solution containing 132 lL of 50% w/v aqueous
NaOH solution was added. The reaction was monitored by TLC (5%
MeOH/CH2Cl2) and was completed after 48 h. The reaction mixture
was diluted with 20 mL of 1.0 M NaOH and washed three times
with Et2O. The aqueous layer was then acidified to pH 2 and
extracted three times with CH2Cl2, dried over MgSO4, filtered,
and concentrated in vacuo to give 0.49 g (0.078 Mol, 87% yield)
of the racemic D5/H5 half ester 8b. TLC (5% MeOH /CH2Cl2)
Rf = 0.5. 1H NMR (300 MHz, CDCl3) 1.26 (1.5H, t, J = 7 Hz), 1.40
(3H, s), 3.13 (1H, d, J = 14 Hz), 3.25 (1H, d, J = 14 Hz), 4.20 (1H, q,
J = 7 Hz), 5.01 (2H, s), 6.88 (2H, d, J = 8 Hz), 7.08 (2H, d,
J = 8 Hz)7.38 (5H, m). Compound 8b was used to prepare samples
of 8 whose % ee was less than 63% to provide additional data points
for Figure 6 in a similar fashion to that shown in Scheme 3.
4.4.7. D5-Diethyl 2-(4-(benzyloxy)benzyl)-2-methylmalonate 6
Compound 10 (0.50 g, 1.46 mMol) (from PLE hydrolysis) was
dissolved in 15 mL of CH2Cl2 in a 25 mL round-bottomed flask with
a magnetic stirbar. Thionyl chloride (2 mL, 27.8 mMol) was added
and the solution was brought to reflux for 15 h. The solution was
concentrated in vacuo and the crude acyl chloride was dissolved
in 10 mL of dry CH2Cl2.Then, 220 lL of Et3N was dissolved in
1 mL of ethanol-d6 and added dropwise to the stirring acyl chlo-
ride. The reaction mixture was stirred for 15 h. at ambient temper-
ature under an N2 blanket. The reaction mixture was diluted with
20 mL of Et2O and washed three times each with 10% HCl and
1.0 M NaOH. The organic layer was dried over MgSO4, filtered,
and concentrated in vacuo to give 6 as a white amorphous so-
lid.10% EtOAc/hexane (Rf = 0.2). This gave 0.39 g (1.04 mMol, 71%
yield) of a white amorphous solid. Mp = 49 °C, IR (cmꢁ1) 1726, 1H
NMR (300 MHz, CDCl3) 1.24 (3H, t, J = 7 Hz), 1.33 (3H, s), 3.17
(2H, s), 4.18 (2H, q, J = 7 Hz), 6.86 (2H, d, J = 9 Hz), 7.04 (2H, d,
J = 9 Hz) 7.37 (5H, m). 13C NMR (75 MHz, CDCl3) 13.1 (sept,
J = 20 Hz), 14.2, 19.8, 40.4, 55.0, 60.6 (quint, J = 22 Hz), 61.5, 70.1,
114.6, 127.6, 128.0, 128.5, 128.7, 131.3, 137.1, 157.9, 172.1. HRMS:
[C22H21D5O5Na+] calcd = 398.1992 obsd = 398.1983.
4.4.5. (R)-2-(4-(Benzyloxy)benzyl)-3-ethoxy-2-methyl-3-
oxopropanoic acid 8
Compound 7 (4.00 g, 10.8 mMol) was dispersed in 600 mL of
rapidly stirring phosphate buffer (0.1 M, pH 7.4). Hundred milli-
grams of PLE (2100 units) was suspended in 1.0 mL of 3 M
(NH4)2SO4 and added to the buffer solution. The pH of the reaction
mixture was maintained using a 798 MPT Titrino in the pH stat
mode. The Titrino was set to titrate to a volume of 10.2 mL (1 equiv
of 1.06 M NaOH). The hydrolysis proceeded for 20 h, after which
time 40 mL of 1.06 M NaOH was added to make the solution suffi-
ciently basic. The aqueous solution was then extracted three times
with 300 mL portions of ether. The aqueous layer was acidified
using concentrated HCl to a pH of 2. The aqueous layer was then
extracted three times with CH2Cl2. The organic layer was dried
over MgSO4, filtered, and concentrated in vacuo to give 2.81 g
(8.21 mMol, 76% yield, 63% ee) of a white, amorphous solid. TLC
4.4.8. (S)-Ethyl-3–(benzyloxy)-2-((4-
methoxybenzyloxy)carbonylamino)-2-methylpropanoate 9
Compound 8 (0.310 g, 0.90 mMol) was dissolved in 10 mL of
dichloroethane in a 50 mL round-bottomed flask with a magnetic
stirbar. Then 215
(2.72 mMol, 3.0 equiv) of Et3N was added and the solution was
heated at reflux for 1.5 h, at which time 169 L (1.36 mMol,
lL (0.996 mMol, 1.1 equiv) of DPPA and 409 lL
(20% EtOAc/hexane) Rf = 0.25.
½
a 2D2
ꢃ
¼ ꢁ1:0 (c 0.066, CH2Cl2).
Mp = 99.0 °C 1H NMR (300 MHz, CDCl3) 1.22 (3.0 H, t, J = 7 Hz),
1.40 (3H, s), 3.15 (1H, d, J = 14 Hz), 3.25 (1H, d, J = 14 Hz), 4.21
(2H, q, J = 7 Hz), 5.02 (2H, s), 6.88 (2H, d, J = 8 Hz), 7.08 (2H, d,
J = 8 Hz)7.36 (5H, m), 11.50 (1H, br s). 13C NMR (75 MHz, CDCl3)
14.2, 20.1, 40.1, 55.2, 62.0, 70.1, 114.8, 127.7, 128.1, 128.2, 128.8,
131.4, 137.1, 158.1, 172.8, 178.0. HRMS: [C20H22O5Na+]
calcd = 365.1365 obsd = 365.1359. The % ee was determined by
analytical chiral HPLC (Chiracel AD-H, 282 nm, flow rate = 1 mL/
min, 4% iPr-OH/96% hexane) tR = 20.87 min, tR = 30.18 min.
l
1.5 equiv) of para-methoxybenzyl alcohol (PMB-OH) was added
and the solution was allowed to reflux for 15 h. The mixture was
then cooled and diluted with 20 mL of chloroform and washed
with 10% HCl. The organic layer was dried and concentrated in va-
cuo. The residue was purified by flash chromatography (SiO2, 50%
Et2O/50% hexane) to give 0.29 g (0.61 mMol, 67% yield, 63% ee) of
a
½ ꢃ
clear viscous oil. TLC (50% Et2O/hexane) Rf = 0.35.
a 1D7:8
¼ þ24:2 (c 0.07, CH2Cl2). IR 3426, 3354, 1716. 1H NMR
ðRÞ
ðSÞ
(300 MHz, CDCl3) 1.27 (3H, t, J = 7 Hz), 1.61 (3H, s), 3.09 (1H, d,
J = 13 Hz), 3.36 (1H, d, J = 13 Hz), 3.80 (3H, s), 4.18 (1H, m), 5.00
(2H, s), 5.00 (1H, d, J = 12 Hz), 5.10 (1H, d, J = 12 Hz), 5.44 (1H, br
s), 6.78 (2H, d, J = 9 Hz), 6.89 (4H, m), 7.37 (7H, m). 13C NMR
(75 MHz, CDCl3) 14.3, 23.8, 41.0, 55.5, 60.9, 61.9, 66.4, 70.1,
114.0, 114.7, 127.7, 128.1, 128.6, 128.8, 128.9, 130.3, 131.1,
137.2, 154.9, 157.9, 159.7, 173.8. HRMS: [C28H31NO6Na+]
calcd = 500.2049 obsd = 500.2044. The ee was determined by ana-
lytical chiral HPLC (Chiralcel AD-H, 282 nm, 4% iPr-OH/96% hex-
ane) tR = 34.38, tR = 39.35.
4.4.6. ( )-D5/H5-2-(4-(Benzyloxy)benzyl)-3-ethoxy-2-methyl-3-
oxopropanoic acid 8b
Compound 7 (3.24 g, 8.75 mMol) was dissolved in 20 mL of eth-
anol and 701 lL of a 50% w/v aqueous NaOH solution was added in
a 50 mL round-bottomed flask with a magnetic stirbar. After 24 h,
the reaction mixture was diluted with 40 mL of water and washed
three times with Et2O. The aqueous layer was then acidified to pH
2 and extracted three times with CH2Cl2, dried over MgSO4, fil-
tered, and concentrated in vacuo to give the racemic (2.27 g,
ðSÞ
ðRÞ

1486
D. S. Masterson et al. / Tetrahedron: Asymmetry 20 (2009) 1476–1486
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21:7
½
aꢃ
¼ ꢁ0:1 (c 1.2 M, HCl)) of 10 matched that of an authentic
obs
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(3H, s), 2.95 (1H, d, J = 14 Hz), 3.15 (1H, d, J = 14 Hz), 4.23 (2H,
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We would like to thank the National Science Foundation
(MCB0639817) for support of this work and (CHE 0639208 and
DBI 0619455) for funds to acquire the MS facilities used in this
study. We would also like to thank the Department of Chemistry
and Biochemistry at USM for start-up funds. DAR would like to
thank the Department of Education for a GAANN Fellowship
(#P200A060323). We would also like to thank Mrs. Tina Masterson
for reviewing the manuscript prior to submission.
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