Diastereoselective Hydrolysis of Branched Malonate Diesters by Porcine Liver Esterase: Synthesis of 5-Benzyl-Substituted Cα-Methyl-β-proline and Catalytic Evaluation

Article abstract of DOI:10.1002/ejoc.201700605

Malonate diesters with highly branched side chains containing a preexisting chiral center were prepared from optically pure amino alcohols and subjected to asymmetric enzymatic hydrolysis by Porcine Liver Esterase (PLE). Recombinant PLE isoenzymes have be

Full text of DOI:10.1002/ejoc.201700605

A Journal of
Accepted Article
Title: Diastereoselective hydrolysis of branched malonate diesters by
Porcine Liver Esterase: Synthesis of 5-benzyl substituted Cα-
methyl-β-proline and catalytic evaluation
Authors: Hari Kiran Kotapati, Jamarii Robinson, Daniel Lawrence,
Kimberly Fortner, Caleb Stanford, Douglas Powell, Rainer
Wardenga, Uwe Bornscheuer, and Douglas Scott Masterson
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201700605
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10.1002/ejoc.201700605
European Journal of Organic Chemistry
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Diastereoselective hydrolysis of branched malonate diesters by
Porcine Liver Esterase: Synthesis of 5-benzyl substituted Cα-
methyl-β-proline and catalytic evaluation
Hari Kiran Kotapati,[a] Jamarii D. Robinson,[a] Daniel R. Lawrence,[a] Kimberly R. Fortner,[a] Caleb W.
Stanford,[a] Douglas R. Powell,[b] Rainer Wardenga,[c] Uwe T. Bornscheuer,[d] Douglas S. Masterson*[a]
Abstract. Malonate diesters with highly branched side chains
containing a preexisting chiral center were prepared from optically
pure amino alcohols and subjected to asymmetric enzymatic
hydrolysis by Porcine Liver Esterase (PLE). Recombinant PLE
isoenzymes have been utilized in this work to synthesize
diastereomerically enriched malonate half-esters from enantiopure
malonate diesters. The diastereomeric excess of the product half-
esters was further improved in the later steps of synthesis either by
simple recrystallization or flash column chromatography. The
diastereomerically enriched half-ester was transformed into a novel
5-substituted Cα-methyl-β-proline analogue (3R, 5S)-1c, in high
optical purity employing a stereoselective cyclization methodology.
This β-proline analogue was tested for activity as a catalyst of the
Mannich reaction. The β-proline analogue derived from the
hydrolysis reaction by the crude PLE appeared to catalyze the
Mannich reaction between an α-imino ester and an aldehyde
providing decent to good diastereoselectivities. However, the
enantioselectivities in the reaction was low. The second
diastereomer of the 5-benzyl substituted Cα-methyl-β-proline, (3S,
5S)-1c was prepared by enzymatic hydrolysis using PLE isoenzyme
3 and tested for its catalytic activity in the Mannich reaction. Amino
acid, (3S, 5S)-1c catalyzed the Mannich reaction between
isovaleraldehyde and an α-imino ester yielding the “anti” selective
product with an optical purity of 99%ee.
thirds of current prescription drugs are chiral and the majority of
them being single stereoisomers.[1] These single stereoisomers
can be accessed by traditional synthetic procedures that involve
either asymmetric transformations or classic resolution
techniques. In asymmetric chemical transformations, the
stereoselectivity can be achieved by various methods that
include the use of chiral auxiliaries, chemical reactions that
involve chiral phase transfer catalysts or employ chiral
compounds that occur in nature.[2]
Porcine Liver Esterase (PLE) as a biocatalytic tool in asymmetric
synthesis
Porcine Liver Esterase (PLE) has proven to be a valuable
catalyst in the field of organic chemistry. PLE has been
successfully employed to carry out asymmetric transformations
on a broad variety of structurally different substrates providing
good to excellent stereoselective outcomes.[3] PLE is widely
used in organic synthesis to catalyze the asymmetric enzymatic
hydrolysis of malonate and other diesters to create optically
enriched chiral synthons in a highly selective fashion.[4] These
chiral synthons have been utilized in the synthesis of
compounds that are of tremendous biological significance.[5]
Over the past decade, our group has enjoyed success in using
PLE to synthesize various optically enriched chiral synthons and
transforming them into Cα-methyl substituted α-, β-, γ- and δ-
amino acids.[6] Our synthetic strategy involves desymmetrization
of the prochiral malonate diesters with branched side chains to
produce optically enriched half-ester intermediates. These
malonate half-ester intermediates serve as precursors to the Cα-
methyl substituted amino acids. In 2012, our group published the
first enantiodivergent synthesis of Cα-methyl-β-proline from an
optically enriched intermediate derived from PLE hydrolysis.[6c]
In this study, the enantiomerically enriched chiral half-ester from
PLE hydrolysis was transformed into a novel γ-lactam through a
stereoselective cyclization that is then converted to Cα-methyl-β-
proline. This amino acid was later reported to be an efficient
catalyst in the asymmetric anti-Mannich reactions performed
using organic solvents such as methylene chloride.[7] To
continue our efforts in investigating and expanding the substrate
scope for the PLE-catalyzed hydrolysis reactions with malonate
diester substrates, herein we present the results of PLE-
catalyzed hydrolysis of chiral malonate diesters and their
transformation to a novel Cα-methyl-β-proline analogue that has
proven to be an efficient anti-Mannich catalyst.
Introduction
The demand for chiral molecules, preferentially one
stereoisomer, has increased significantly due to the fact that two
[a]
Department of Chemistry & Biochemistry
The University of Southern Mississippi
118 College Drive, Box # 5043, Hattiesburg, Mississippi-39406, USA
E-mail: Douglas.Masterson@usm.edu
https://www.usm.edu/chemistry-biochemistry/faculty/douglas-
masterson
[b]
Department of Chemistry and Biochemistry
University of Oklahoma
101 Stephenson Parkway, Norman, Oklahoma 73019-5251.
Enzymicals AG
Walther-Rathenau-Str. 49a, 17489 Greifswald, Germany.
Department of Biotechnology & Enzyme Catalysis
University of Greifswald, Institute of Biochemistry
Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
[c]
[d]
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10.1002/ejoc.201700605
European Journal of Organic Chemistry
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Results and Discussion
dispense 1.0 N NaOH as the reaction progressed. The results of
the PLE-catalyzed reactions for substrate and co-solvent
screening are illustrated in Table 1.
Table 1. Asymmetric hydrolysis of malonate diesters, 4a-c, by crude PLE
enzyme
Substrate
EtOH
(Vol %)
CH2Cl2
(Vol %)
% Yield
Major
Product
(d.r.)[a]
Figure 1. Substrates utilized in the PLE-catalyzed hydrolysis studies
For our PLE-catalyzed hydrolysis studies, we chose to utilize
malonate diesters with a phthalimide group and a preexisting
chiral center in the side chain (Figure 1). These diesters were
readily prepared from optically active amino alcohols as shown
in Scheme 1.[8]
4a
4b
4c
2.5
2.5
-
-
-
75
12
10
(2R, 4S)-5a
(8:1)
(2R, 4S)-5b
(6:1)
0.8
(2R, 4S)-5c
(3.5:1)
4c
4c
4c
2.5
2.5
20
-
0.6
-
30
57
18
(2R, 4S)-5c
(4.7:1)
(2R, 4S)-5c
(4.1:1)
(2R, 4S)-5c
Scheme 1. Synthesis of malonate diesters 4a-c, from optically pure
(3.1:1)
amino alcohols
[a] d.r. determined from 1H NMR of crude product
Recently, Hoveyda et al reported
diastereoselective desymmetrization of
a
PLE-mediated
chiral malonate
a
Methylene chloride and ethanol were added as co-solvents
in the reaction. Ethanol is one of the common co-solvents used
to enhance the selectivity in the PLE hydrolysis reactions with
malonate diesters.[10] Methylene chloride was added to ensure
proper stirring of the reaction with malonate diester 4c as the
diester substrate’s viscous physical nature caused problems
with magnetic stirring of the reaction. Crude PLE, in the absence
of ethanol, produced the half-ester (2R, 4S)-5c with a d.r. of
3.5:1. Upon addition of 2.5% ethanol, the d.r. was improved to
4.7:1. This is in agreement with the previous results published
by our group on the improvement of selectivity in PLE-catalyzed
hydrolyses of malonate diesters upon addition of ethanol.[10] The
diastereomeric ratios of the PLE-hydrolysed product were
determined from the 1H NMR analysis of the crude product.
The absolute stereochemistry of the new quaternary chiral
center in the half-ester, (2R, 4S)-5c, was determined to be R
from the X-ray crystal analysis of its derivative (Scheme 3). The
stereochemistry of the major diastereomer, (2R, 4S)-5b, was
determined from the key correlations (Figure 2) observed in 2D-
NOESY NMR data of the lactam ester, (3R, 5S)-7b, that was
derived from the half-ester obtained from the hydrolysis
catalyzed by PLE (See experimental section for the synthesis of
(3R, 5S)-7b). The stereochemistry at the quaternary chiral
center of the half-ester 5a created during the PLE-catalyzed
hydrolysis is determined to be R from 1H NMR data. The shifts of
major diastereomeric hydrogens in the compound 5a were
compared to those in compounds (2R, 4S)-5b and (2R, 4S)-5c.
diester.[9] To the best of our knowledge, there are no published
reports of PLE-catalyzed hydrolysis of malonate diesters with a
preexisting chiral center and a phthalimide group. Our unique
approach, to utilize relatively inexpensive and optically active
starting materials, helped us avoid the possibility of producing a
mixture of enantiomers during the PLE-catalyzed hydrolysis
reaction. Here, the malonate diester was transformed into the
half-ester with the creation of a new chiral center at the
quaternary carbon. Since the stereochemistry of the preexisting
chiral center in the side chain of malonate diester substrate is
preset to be “S”, the PLE-catalyzed hydrolysis of this substrate
produces two possible diastereomeric half-esters (2R, 4S)-5a-c
and/or (2S, 4S)-5a-c as illustrated in Scheme 2.
Scheme 2. Asymmetric enzymatic hydrolysis of chiral malonate diesters
catalyzed by crude PLE
Malonate diesters, 4a-c, were subjected to asymmetric ester
hydrolysis by crude PLE enzyme (crude PLE refers to the
mixture of all isoenzymes). The PLE reaction was carried out at
pH 7.4 in phosphate buffer using an auto burette that was set to
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10.1002/ejoc.201700605
European Journal of Organic Chemistry
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4 were good, the yields were low. We believe that the benzyl
group, adjacent to the nitrogen, in malonate diester 4c is
probably too large to efficiently fit into the enzyme’s active site.
Also, these reactions proceeded rather slow and usually took 5-
7 days to go to completion as determined by the delivered
quantity of NaOH. The identity of the major product was
determined from 1H NMR chemical shifts and the reversal of
diastereopreference with isoenzymes 3 & 4 was confirmed from
1
the H NMR data of the product half-esters. The PLE-catalyzed
hydrolysis conditions for isoenzyme studies with substrate 4b
are similar to that for substrate 4c. These reactions were also
slow and normally proceeded for 4-5 days. During hydrolysis,
PLE 1, 2 & 5 did not show activity towards the substrate 4b. PLE
Figure 2. Key 2-D NOESY correlations of γ-lactam, (3R, 5S)-7b
PLE isoenzymes studies
3
&
6
showed an improvement in the yield and
Asymmetric hydrolysis of diesters 4a-c catalyzed by crude
PLE produced the half-esters in significant diastereomeric ratios.
However, the yields of the reaction with substrates 4b and 4c
were low compared to those using substrate 4a. In order to
optimize the enzymatic hydrolysis reaction for the yields and
diastereoselectivities, we had conducted PLE isoenzyme studies
with compounds 4b and 4c (Scheme 2). Crude PLE is a mixture
of at least six different isoenzymes that have different specific
activities, stereo preference, and turnover numbers compared to
that of the crude isoenzyme mixture.[11] The studies were
performed in phosphate buffer pH 7.4 using 2.5% ethanol and
0.8% methylene chloride as co-solvents. The yields and
selectivities of the half-esters obtained from the PLE isoenzyme-
catalyzed reactions are listed in Table 2.
diastereoselectivity of the resulting half-ester. The d.r. of the
product half-ester in the hydrolysis of diester 4b with PLE 6 was
improved significantly from 6:1 to 11:1, providing an improved
yield of 25%. However, the d.r. of the half-ester with PLE 4
diminished significantly (1.3:1). The stereoselective outcomes in
the hydrolysis of diester 4b with PLE isoenzymes remained the
same as that with crude PLE.
Synthesis of (3R, 5S)-5-benzyl-Cα-methyl-β-proline, (3R,
5S)-1c
With the product (2R, 4S)-5c (d.r.= 5:1) in hand we decided
to investigate the stereoselective cyclization reaction[6c] that
could provide access to a novel class of γ-lactams that can be
converted into 5-benzyl-Cα-methyl-β-proline. For this purpose
the malonate half-ester (2R, 4S)-5c was transformed into the p-
NO2-benzyl ester, (2S, 4S)-6c, as shown in Scheme 3.
Table 2. PLE isoenzyme studies of malonate diesters, 4b-c
Enzyme
Substrate
Major
d.r.
%Yield
Product
Crude PLE
Crude PLE
4b
4c
(2R, 4S)-5b
6:1
12
57
(2R, 4S)-5c
4.1:1
PLE 3
PLE 3
PLE 4
PLE 4
PLE 5
PLE 5
PLE 6
PLE 6
4b
4c
4b
4c
4b
4c
4b
4c
(2R, 4S)-5b
(2S, 4S)-5c*
(2R, 4S)-5b
(2S, 4S)-5c*
-
5.7:1
3.8:1
1.3:1
3.4:1
-
24
30
13
20
-
Scheme 3. Stereoselective cyclization to prepare γ-lactam (3R, 5S)-7c
Surprisingly the major diastereomer, (2S, 4S)-6c, readily
crystallized out of the crude reaction mixture during the work up
with diethyl ether in 67% yield. The improvement in
diastereopurity was confirmed by 1H NMR analysis. Crystals of
(2S, 4S)-6c, for X-ray analysis, were grown by the solvent
diffusion technique. Single-crystal X-ray analysis data of (2S,
4S)-6c revealed the stereochemistry at the quaternary chiral
center formed during the PLE hydrolysis to be S (Figure 3).
Recrystallized (2S, 4S)-6c was treated with hydrazine hydrate to
remove the phthalimide protecting group providing the primary
amine, which then went on to cyclize exclusively at the p-nitro
benzylic ester providing the lactam (3R, 5S)-7c in 70% yield.
Interestingly, no trace of cyclization on the ethyl ester side was
observed.
(2R, 4S)-5c
(2R, 4S)-5b
(2R, 4S)-5c
4.2:1
11:1
4.1:1
13
25
10
*Switch in diastereopreference
PLE isoenzymes 1 & 2 did not demonstrate activity with
malonate diester 4c. PLE 5 & 6 provided the diastereomer (2R,
4S)-5c as the major product (similar to crude PLE). Interestingly,
both PLE 3 & 4 showed a switch in the diastereopreference
providing the diastereomer (2S, 4S)-5c as the major product.
These results were of interest to us as the isoenzymes 3 & 4
would readily provide access to the (2S, 4S)-5c diastereomer
without additional chemical modifications. Even though the
diastereoselectivities of hydrolysis reactions using both PLE 3 &
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10.1002/ejoc.201700605
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Figure 3. Single X-ray crystal structure of (2S, 4S)-6c
Scheme 5. Synthesis of γ-lactam (3S, 5S)-7c from malonate diester 4c
The γ-lactam (3R, 5S)-7c was then transformed into 5-benzyl-
Cα-methyl-β-proline as illustrated in Scheme 4. The reduction of
the thiolactam previously reported by our group required N-
benzyl protection of the thiolactam.[6c] Our attempts to install a
benzyl group on the amide was not successful. So we have,
instead, chosen to avoid the N-benzyl protection and
deprotection steps. Upon reaction with Lawesson’s reagent,
lactam (3R, 5S)-7c was transformed into the thiolactam, (3S,
5S)-8c, in 93% yield.[12] Desulfurization of (3S, 5S)-8c was
The crude reaction mixture from the hydrazine hydrolysis
reaction was then purified via flash column chromatography to
isolate the desired diastereomer of the lactam ester, (3S, 5S)-7c,
in 29% yield (d.r. > 19:1) (Scheme 5). The stereochemistry at
the quaternary chiral carbon of the lactam ester (3S, 5S)-7c was
confirmed to be S from single X-ray crystallographic analysis
(Figure 4).
accomplished by treatment with Raney Ni under
a H2
atmosphere to provide (3R, 5S)-9c in 51% yield.[13] (3R, 5S)-9c
was then subjected to ester hydrolysis under refluxing acid
conditions using 6N HCl followed by cation exchange
chromatography to provide amino acid, (3R, 5S)-1c, in
quantitative yield (Scheme 4).
Figure 4. Single X-ray crystal structure of γ-lactam (3S, 5S)-7c
Compound (3S, 5S)-7c was also prepared from the
malonate diester 4c via the stereoselective cyclization
methodology using the half-ester derived from PLE isoenzyme 3
hydrolysis (Scheme 5). We have utilized PLE isoenzyme 3 to
cause the asymmetric hydrolysis of diester 4c to provide the
desired diastereomer (2S, 4S)-5c in 30% yield and a d.r. of 4:1.
The half-ester (2S, 4S)-5c was treated with p-nitro benzyl
bromide to provide the ester (2R, 4S)-6c in 91% yield and a d.r.
of 4:1. Our attempts to improve the diastereopurity at this point
were not successful. The 3.8:1 diastereomeric mixture of (2R,
4S)-6c was reacted with hydrazine hydrate under refluxing
conditions. The crude reaction mixture was analyzed by 1H NMR
and the data suggested that the cyclization had occurred
exclusively towards the p-nitro benzyl ester. The reaction
mixture was then purified via flash column chromatography to
resolve the diastereomers and provide the desired γ-lactam (3S,
5S)-7c in 31% yield. The γ-lactam, (3S, 5S)-7c, was then
transformed into 5-benzyl-Cα-methyl-β-proline, (3S, 5S)-1c, in
three steps (Scheme 6). Treatment of compound (3S, 5S)-7c
with Lawesson’s reagent produced the thiolactam (3R, 5S)-8c in
99% yield. In the next step, desulfurization of (3R, 5S)-8c was
carried out using Raney Ni under a H2 atmosphere to provide the
amino ester (3S, 5S)-9c in 59% yield. In the final step, hydrolysis
of the ethyl ester (3S, 5S)-9c in refluxing 6N HCl followed by
Scheme 4. Synthesis of 5-benzyl-Cα-Methyl-β-Proline, (3R, 5S)-1c
Synthesis of (3S, 5S)-5-benzyl-Cα-methyl-β-proline, (3S, 5S)-
1c
We have developed two different routes to the intermediate
γ-lactam (3S, 5S)-7c that was later transformed to 5-benzyl-Cα-
methyl-β-proline, (3S, 5S)-1c (Scheme 5). In the first route,
which involves a non-stereoselective cyclization and separation
of the diastereomers, 4c was reacted with hydrazine hydrate to
remove the phthalimide protecting group. Interestingly, upon
phthalimide deprotection, compounds (3R, 5S)-7c and (3S, 5S)-
7c were formed in a d.r. of 1.2:1.
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cation exchange chromatography furnished the target
diastereomer of 5-benzyl-Cα-methyl-β-proline, (3S, 5S)-1c, in
quantitative yield (Scheme 6).
the hydrogen bonding interaction between the carboxylic acid of
the amino acid (3R, 5S)-1c and the α-imino ester is diminished,
and thus results in lower enantioselectivities of the Mannich
reaction.
Scheme 6. Synthesis of Cα-Methyl-β-Proline analogue from γ-lactam (3S,
5S)-7c
Figure 5. Possible steric hindrance in the transition state for the Mannich
reaction by (3R, 5S)-1c
Mannich Reaction catalyzed by (3R, 5S)-1c & (3S, 5S)-1c
On the other hand, the second diastereomer of the designed
amino acid (3S, 5S)-1c proved to be a very efficient catalyst, for
the Mannich reaction between a preformed α-imino ester (11)
and isovaleraldehyde (12), yielding anti selective Mannich
products in a diastereomeric ratio of 23:1 (anti: syn) with
enantiomeric excess of 99% ee. Given the hydrophobic
quaternary methyl group and a benzyl group on the pyrrolidine
core of the amino acid, (3S, 5S)-1c, we chose to carry out the
Mannich reaction in methylene chloride at room temperature.
The catalyst, however, in its zwitterionic form was initially
insoluble in methylene chloride. The reaction mixture was
heterogeneous in the beginning and became more homogenous
as the reaction progressed. We were able to see improved
enantioselectivities in the Mannich reaction with the designed
amino acid (3S, 5S)-1c as catalyst. Based on the enamine
mechanism of proline catalyzed Mannich reaction we propose a
transition state (Figure 6) for the reaction, between preformed α-
imino ester and isovaleraldehyde, which accounts for the
observed enantioselectivities in the reaction. In the transition
state, the benzyl group and the carboxyl group on the pyrrolidine
core of amino acid, (3S, 5S)-1c, are on opposite sides as shown
in the Figure 6. This eliminates the possibility of steric hindrance
in the transition state and thus favors the strong hydrogen bond
interaction between the carboxylic acid and imine.[15]
With both diastereomers of 5-benzyl-Cα-methyl-β-proline in
hand, we proceeded to investigate the activity of these amino
acids in the Mannich reaction between a preformed N-PMP
protected α-imino ester (11) and isovaleraldehyde (12) (Scheme
7). We have performed this reaction with L-proline, which
provides “syn” selective products[14] and used it as a reference
with which to compare our results.
Scheme 7. Mannich reaction for catalytic evaluation of 5-benzyl-Cα-Methyl-
β-proline
The Mannich reaction, catalyzed by (3R, 5S)-1c in various
solvent systems, yielded anti products in moderate to excellent
diastereoselectivities (Table 3). However, the enantioselectivities
in the reaction were poor, suggesting a possible steric hindrance
in the transition state with amino acid (3R, 5S)-1c as catalyst.
We have performed this reaction in three different solvent
systems
enantioselectivity when the reaction was performed in methylene
chloride. Interestingly, we observed switch in the
and
observed
some
solvent
dependent
a
enantioselectivity of the anti diastereomer when the reaction was
performed in methylene chloride and in a 1:1 mixture of 2-
propanol/methylene chloride solvent systems (Table 3).
According to the enamine mechanism, the enamine formed from
the amino acid and the aldehyde component reacts with the
preformed imine (Figure 5). The carboxylic acid of the amino
acid should provide a hydrogen bond to the imine and control
the selective attack of the enamine on the imine component.
However, in the transition state the benzyl group and the
carboxylic acid on the pyrrolidine ring of amino acid (3R, 5S)-1c
are in the same side causing steric congestion between the
benzyl group of amino acid and imine as illustrated in Figure
5.[15] As a result of this steric congestion, we hypothesize that
Figure 6. Predicted transition state for the Mannich reaction catalyzed by
(3S, 5S)-1c
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Table 3. Table Caption Mannich reaction catalyzed by L-proline, and designed Cα-methyl-β-proline
Catalyst
Loading
Solvent
Time, Temp
% Yield
d.r.
%ee (Major)
L-Proline
(3R, 5S)-1c
(3R, 5S)-1c
20 mol%
5 mol%
5 mol%
DMSO
DMSO
4h, RT
6h, RT
2h, RT
96
79
82
24:1 (syn)
4:1 (anti)
19:1 (anti)
97% (2S, 3S)
9% (2R, 3S)
11% (2S, 3R)
i-PrOH:CH2Cl2
(1:1)
(3R, 5S)-1c
(3S, 5S)-1c
(3S, 5S)-1c
5 mol%
5 mol%
1 mol%
CH2Cl2
CH2Cl2
CH2Cl2
5h, RT
2.5h, RT
2.5h, RT
82
92
85
19:1 (anti)
23:1 (anti)
23:1 (anti)
32% (2S, 3R)
99% (2R, 3S)
98% (2R, 3S)
point analysis was performed using Thomas-Hoover “Uni-Melt” Melting
Point Apparatus and are uncorrected. Triflic anhydride was freshly
distilled on phosphorous pentoxide under Nitrogen atmosphere before
use according to the reported procedure.[16] High Resolution mass
spectra were obtained using positive electrospray ionization on a Bruker
12 Tesla APEX -Qe FTICR-MS with an Apollo II ion source.
Conclusions
Our unique strategy to employ optically pure starting
materials, enzyme Porcine Liver Esterase (PLE) and
stereoselective cyclization provides access to a novel class of
pyrrolidine-3-carboxylic acids that could have significant
biological applications in the field of chemistry. In this work, we
were able to show that PLE can be used as a valuable
biocatalyst in the asymmetric hydrolysis of chiral malonate
diesters providing decent diastereoselectivities. PLE isoenzyme
studies showed that PLE 3 & 4, when used in hydrolysis of
substrate 4c, switch the diastereoselectivity. We were also able
to demonstrate that the half-esters (2R, 4S)-5c and (2S, 4S)-5c,
derived from the PLE-catalyzed hydrolysis can be used as
valuable synthons that provide access to a novel class of Cα-
methyl-β-proline analogues. Asymmetric hydrolysis of substrate
4c by crude PLE provided the half-ester (2R, 4S)-5c in a d.r. of
4.6:1. This half-ester was then transformed to 5-benzyl-Cα-
methyl-β-proline analogue, (3R, 5S)-1c, via a diastereoselective
cyclization strategy. Substrate 4c, when subjected to hydrolysis
with PLE isoenzyme 3, provided half-ester (2S, 4S)-5c in a d.r.
of 3.8:1 that was then transformed into 5-benzyl-Cα-methyl-β-
Procedure for PLE Isoenzyme reactions
The reaction set up procedure for PLE isoenzyme reactions is similar to
that of the preparation of half-ester 5a. All the reactions were performed
in 60 mL of phosphate buffer pH 7.4 and PLE isoenzyme (250
units/mmol of the diester) was added as a suspension in 3.0 M
Ammonium Phosphate. For the PLE isoenzyme studies of diester 4b,
diester 4b (0.150g, 0.37 mmol) of was dissolved in appropriate co-
solvents as listed and added to the reaction buffer. For PLE isoenzyme
reactions of diester substrate 4c, diester 4c (0.150g, 0.34 mmol) was
dissolved in appropriate co-solvents and added to the reaction buffer. All
the reactions were carried out at room temperature and the pH of the
reaction was maintained at 7.4 with an auto burette set up that is set to
add 1.0N NaOH solution as the reaction proceeded. After the reaction
was completed, to the reaction was added 1.0N NaOH to bring the pH to
9.0. 40 mL of ethyl acetate was added to the above reaction buffer and a
gentle extraction was performed to remove any unreacted starting
material. The aqueous buffer solution was filtered through celite bed to
remove the enzyme. The filtered buffer solution was taken and the pH of
the solution was adjusted to 2.0 by adding 50% sulfuric acid slowly in a
drop wise manner while stirring. Then the aqueous buffer solution was
extracted carefully with dichloromethane (8 x 40 mL) to prevent the
formation of emulsion. All the organic portions were combined, dried over
MgSO4, filtered and the solvent was removed under vacuum to provide
crude half-esters. 1H NMR was obtained on the crude half-ester products
proline analogue, (3S, 5S)-1c, via
a
diastereoselective
cyclization. Both diastereomers of 5-benzyl-Cα-methyl-β-proline
analogue 1c were tested for catalytic activity in the Mannich
reaction between α-imino ester and isovaleraldehyde. Designed
amino acid (3S, 5S)-1c proved to be an efficient catalyst
providing “anti” selective products in a d.r. of 23:1 and %ee of up
to 99%.
1
and the diastereomeric ratios were determined. The H NMR data of the
product half-esters looked essentially pure in most cases. The yields of
the product half-esters was determined from the amount of half-esters
isolated after purification. Purification was performed using a flash
column chromatography, 40% ethyl acetate/60% hexanes to 100% ethyl
acetate (product eluted at 100% ethyl acetate). Our attempts to improve
diastereomeric ratio on flash column were not successful as the
diastereomers seemed to be inseparable.
Experimental Section
General Experimental Information
All anhydrous solvents were obtained by passage through a column of
activated silica. Flash column chromatography was performed using
SiliaFlash® P60 40-63µm (230-400 mesh) silica gel. NMR experiments
were performed on a Bruker 400 MHz NMR instrument and chemical
shifts were reported with reference to either TMS (for CDCl3) or residual
solvent proton peak (for CD3OD and D2O). Infra-Red analysis was
performed on a Thermo Nicolet nexus 470 FT-IR instrument. Melting
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10.1002/ejoc.201700605
European Journal of Organic Chemistry
FULL PAPER
Procedure for Mannich reactions
provide pure (2S, 4S)-6b (0.129 g, 0.253 mmol) in 59% Yield. Our
attempts to resolve diastereomers, to improve diastereomeric ratio, via
flash column purification were not successful at this point. (Rf = 0.38,
30% ethyl acetate / 70% hexanes). IR (cm-1): 2957, 1729, 1705. 1H NMR
(400 MHz, CDCl3) δ 8.21 – 8.11 (m, 2H), 7.87 – 7.66 (m, 4H), 7.39 (dd, J
= 9.0, 2.1 Hz, 2H), 4.83 (dd, J = 74.9, 13.5 Hz, 2H), 4.58 – 4.42 (m, 1H),
4.07 (q, J = 7.1 Hz, 2H), 3.10 – 2.97 (m, 1H), 2.28 – 2.09 (m, 2H), 1.53 (d,
J = 6.0 Hz, 3H), 1.50 – 1.34 (m, 2H), 1.12 (t, J = 7.1 Hz, 3H), 0.89 (dt, J =
12.8, 6.4 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 171.7, 171.3, 168.4,
167.3, 147.7, 142.5, 134.0, 128.2, 123.7, 65.4, 61.7, 52.9, 45.8, 42.5,
37.7, 25.0, 23.1, 21.8, 19.7, 13.9. HRMS (C27H30N2O8 Na+) calcd =
533.189437 m/z, obsd = 533.189437 m/z.
The preformed imine, 11, (0.207g, 1.0 mmol) was weighed and added to
a 5 mL sealed tube containing 2 mL anhydrous methylene chloride. Then
appropriate amount of catalyst was weighed and added to the reaction
vessel followed by 0.173 g (2.0 mmol) of isovaleraldehyde (12) as a
solution in 2.0 mL of anhydrous methylene chloride. Reaction tube was
tightly sealed and left to stir at room temperature. Reaction was
monitored by TLC for the disappearance of imine. Once all the starting
material had disappeared, reaction mixture was washed with saturated
NaCl (1 x 5 mL). The organic portion was dried over MgSO4, filtered and
the solvent was removed under vacuum to afford crude product. Crude
product was analyzed by 1H NMR to determine diastereomeric ratio of
the Mannich product. The crude product was then purified immediately
by flash column chromatography (20% ethyl acetate/80% hexanes) to
provide pure product. Flash column purification did not result in any
improvement of the diastereomeric ratio. The % yield of the reaction was
determined from the pure product isolated in the reaction. The pure
product was immediately analyzed by the HPLC using chiral stationary
phase to determine the percent enantiomeric excess. The data of the
products was compared to the reported to the literature and determined
to be consistent.[14-15, 17]
(3R, 5S)-ethyl 5-isobutyl-3-methyl-2-oxopyrrolidine-3-carboxylate,
(3R, 5S)-7b
(2S, 4S)-6b (0.120g, 0.242 mmol; d.r = 6:1) was dissolved in a
solvent mixture of 4 mL methanol and 2 mL methylene chloride in a 25
mL single neck round bottom flask. 0.1 mL of hydrazine hydrate (35% in
water) was added to the reaction flask and reaction mixture was heated
to reflux the solvent. Reaction was monitored by TLC. After stirring for 24
hrs an additional 0.060 mL of hydrazine hydrate was added to the
reaction to maintain the basic pH. Reaction was continued to stir for
another 17 hrs (total reaction time of 41 hrs) and then cooled to room
temperature. The white precipitate was washed with methylene chloride
(5 x 6 mL) and the contents were filtered via a micro column to remove
any white precipitate. The solvent was then removed under vacuum to
provide crude material. The crude product was taken up in methylene
chloride and the organic portion was washed with water (1 x 20 mL)
followed by brine (1 x 20 mL). Organic portion was then dried over
magnesium sulfate, filtered and the solvent was removed under vacuum.
The resulted crude product was carefully purified via gradient flash
column chromatography (45% ethyl acetate/55% hexanes to 80% ethyl
acetate/ 20% hexanes). Purification via flash column provided
diastereopure (3R, 5S)-7b (14 mg, 0.062 mmol) in 26% yield. The
improvement in diastereopurity was confirmed from the 1H NMR data
analysis. 2D-NOESY experiment was performed on this diastereopure
Synthesis of γ-lactam, (3R, 5S)-7b
Half-ester 5b (d.r. = 6:1) from PLE hydrolysis was reacted with 4-nitro
benzyl bromide to afford the ester 6b in 59% yield (Scheme 8).
Scheme 8. Synthesis of lactam (3R, 5S)-7b, via stereoselective cyclization
The 4-nitro benzyl ester (2S, 4S)-6b was then treated with hydrazine
hydrate to cause the phthalimide deprotection. This resulted in
stereoselective cyclization of the amine onto the carbonyl with 4-nitro
benzyl group to provide γ-lactam (3R, 5S)-7b (Scheme 8). There was no
trace of cyclization of the amine towards the ethyl ester observed from
the NMR data of the crude product. The crude material was then purified
via flash column chromatography to separate the diastereomers and
provide diastereopure γ-lactam (3R, 5S)-7b in 26% yield. The resulting
lactam (3R, 5S)-7b was analyzed by 2D-NOESY experiments to
determine the stereochemistry at the quaternary chiral center.
lactam and the stereochemistry at the quaternary chiral carbon was
24
determined to be R. Rf = 0.11(80% ethyl acetate / 20% hexanes). [α]D
=
+6.1 (c = 0.3, CH2Cl2).IR (cm-1): 3218, 2957, 2872, 1737, 1701.1H NMR
(400 MHz, CDCl3) δ 6.26 (s, 1H), 4.28 – 4.14 (m, 2H), 3.69 (p, J = 7.0 Hz,
1H), 2.33 – 2.24 (m, 1H), 2.13 (dd, J = 12.8, 7.0 Hz, 1H), 1.64 (tt, J =
13.1, 6.6 Hz, 1H), 1.58 – 1.48 (m, 1H), 1.47 – 1.44 (m, 3H), 1.39 (ddd, J
= 11.8, 7.3, 6.5 Hz, 1H), 1.32 – 1.24 (m, 3H), 0.93 (dt, J = 6.7, 3.3 Hz,
6H). 13C NMR (101 MHz, CDCl3) δ 176.4, 172.6, 61.6, 51.5, 49.6, 45.6,
40.3, 25.4, 22.8, 22.5, 20.3, 14.1. HRMS (C12H21NO3H+) calcd
=228.159420 m/z, obsd = 228.159218 m/z.
(S)-1-ethyl 3-(4-nitrobenzyl) 2-((S)-2-(1, 3-dioxoisoindolin-2-yl)-4-
methylpentyl)-2-methylmalonate, (2S, 4S)-6b
Supporting Information
(2R, 4S)-5b (0.160g, 0.426 mmol; d.r = 6:1) was dissolved in 4 mL
anhydrous DMF in a 100 mL, three neck flask at room temperature and
under N2 atmosphere. K2CO3 (0.059g, 0.426 mmol) was added to the
above flask and the contents were stirred for 10 minutes. Then, 4-
nitrobenzyl bromide (0.092g, 0.426 mmol) was added in a dropwise
manner as a solution in 2 mL anhydrous DMF to the above reaction flask.
Then the reaction mixture was allowed to stir overnight at room
temperature and under N2 atmosphere. After stirring overnight, 6 mL
water was added to the reaction flask and the organic contents were
extracted with diethyl ether (4 x 6 mL). All organic portions were
combined and washed with water (7 x 6 mL) followed by brine (3 x 6 mL).
The organic portion was dried over MgSO4 and the solvent was removed
under vacuum to provide crude product. Crude compound was purified
by flash column chromatography (10% ethyl acetate / 90% hexanes) to
NMR spectra and HPLC data are provided in the supporting
information. CCDC 1538677 and CCDC 1538678 contain the
supplementary crystallographic data for this paper. The data can
be obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/structures/
Acknowledgements
We acknowledge Dr. Faqing Huang (Ion Exchange
Chromatography) and Dr. Julie Pigza (2D-NMR data analysis)
for their valuable suggestions. We acknowledge Mr. Philipp
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10.1002/ejoc.201700605
European Journal of Organic Chemistry
FULL PAPER
[12] a) A. A. El-Barbary, S. Carlsson, S. O. Lawesson, Tetrahedron 1982,
38, 405-412; b) V. Beylin, D. C. Boyles, T. T. Curran, D. Macikenas, R.
V. I. V. Parlett, D. Vrieze, Org. Process Res. Dev. 2007, 11, 441-449.
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Süss for proof reading the manuscript. We thank the National
Science Foundation for a CAREER Award (MCB-0844478). We
thank the National Science Foundation for the Mass
Spectrometer and NMR Spectrometer instrumentation grants
(CHE-0639208, DBI-0619455, and CHE-0840390). We thank
the American Chemical Society, the Mississippi Section of the
American Chemical Society, and the Vice President for
Research (Dr. Gordon Cannon) for Project SEED funding (J.D.R.
& C.W.S). We thank the University of Southern Mississippi
McNair Scholars Program (K.R.F). We thank the Department of
Chemistry and Biochemistry at the University of Southern
Mississippi for continued support of our research programs.
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Keywords: Biocatalysis • anti-Mannich Catalyst •
Organocatalysis • Porcine Liver Esterase • Cα-Methyl-β-Proline
analogue
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10.1002/ejoc.201700605
European Journal of Organic Chemistry
FULL PAPER
FULL PAPER
Recombinant Porcine Liver Esterase
(PLE) has been used in this work to
Key Topic*
Enzyme catalysis, Organocatalysis
hydrolyse
malonate
optically
diesters
pure
and
chiral
provide
Authors:
diastereoenriched half-esters. Crude
PLE (mixture of six different
isoenzymes, PLE1-6) hydrolysed the
Hari Kiran Kotapati, Jamarii D.
Robinson, Daniel R. Lawrence, Kimberly
R. Fortner, Caleb W. Stanford, Douglas
R. Powell, Rainer Wardenga, Uwe T.
Bornscheuer, Douglas S. Masterson*
malonate diester 4c to provide the
half-ester in
a
d.r. of 5:1. PLE
isoenzyme 3 hydrolysed the diester 4c
to yield the half-ester in a d.r. of 4:1.
These half-esters were transformed to
novel 5-benzyl Cα-methyl-β-proline
analogues (3R, 5S)-1c and (3S, 5S)-
1c via a stereo selective cyclization
methodology. The synthesized amino
acid (3S, 5S)-1c is proved to be an
excellent catalyst in the Mannich
reaction between an aldehyde and an
α-imino ester providing anti selective
products in a d.r. of 23:1 and ee of
99%.
Page No. – Page No.
Title:
Diastereoselective hydrolysis of
branched malonate diesters by
Porcine Liver Esterase: Synthesis of
5-benzyl substituted Cα-methyl-β-
proline and catalytic evaluation
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