Model Studies on the Enzyme-Regulated Stereodivergent Cascade Passerini Reaction

Article abstract of DOI:10.1002/ejoc.202100760

The synthesis of chiral α-acyloxy carboxamides containing two stereogenic centers continues to be a challenging field of organic chemistry. Herein, we have proposed and proved the feasibility of an enzyme regulated-cascade reaction, which using the same substrates enables the formation of individual stereoisomers of α-acyloxy carboxamides with up to 99 % ee. The access to the individual stereoisomeric products has been achieved by a combination of the enzymatic kinetic resolution of racemic vinyl esters, subsequent Passerini reaction, and enzymatic kinetic resolution of formed α-acyloxy carboxamides. The presented studies are promising in exploratory proof-of-concept of enzyme-controlled stereodivergent cascade to form an important class of chiral compounds for medicinal chemistry.

Full text of DOI:10.1002/ejoc.202100760

Communications
doi.org/10.1002/ejoc.202100760
Model Studies on the Enzyme-Regulated Stereodivergent
Cascade Passerini Reaction
Monika Wilk,[a] Anna Brodzka,[a] Dominik Koszelewski,[a] Jan Samsonowicz-Górski,[a] and
Ryszard Ostaszewski*[a]
The synthesis of chiral α-acyloxy carboxamides containing two
stereogenic centers continues to be a challenging field of
organic chemistry. Herein, we have proposed and proved the
feasibility of an enzyme regulated-cascade reaction, which
using the same substrates enables the formation of individual
stereoisomers of α-acyloxy carboxamides with up to 99% ee.
The access to the individual stereoisomeric products has been
achieved by a combination of the enzymatic kinetic resolution
of racemic vinyl esters, subsequent Passerini reaction, and
enzymatic kinetic resolution of formed α-acyloxy carboxamides.
The presented studies are promising in exploratory proof-of-
concept of enzyme-controlled stereodivergent cascade to form
an important class of chiral compounds for medicinal chemistry.
Introduction
The development of the synthesis of chiral α-acyloxy carbox-
amides containing one or more stereocenters continues to be
an outstanding challenge in the way of synthesizing complex
Figure 1. The general structure of enantiopure lactate (1) and examples of
bioactive lactate derivatives.
molecules. The close relationship between biological activities
and stereoisomerism of chiral substances makes it crucial for
the pharmaceutical industry.[1] An important class of synthetic
precursors for medical chemistry is enantiopure lactate deriva-
tives (Figure 1). For example cyclohexyl lactic acid plays a key
role as an E-selectin inhibitor (2), which is essential for
mediating the interactions of leukocytes with the endothelium
during inflammation.[2] Furthermore, the phenyl lactic motif is
found in molecules exhibiting antiviral activities (3 and 4).[3] The
chiral scaffold of a lactate derivative with an amine substituent
(R=NHR’) can be found in the structure of various natural
products (5), peptidomimetics (6), antibiotics (7), and other
bioactive molecules, such as the anti-cancer drug[4] solimastat
(8).
A large number of natural products are chiral, and their
biological activity is highly enantiomer dependent. The applica-
tion of a racemate as a pharmaceutical is undesirable because it
usually requires a higher dose to elicit the desired response.
Additionally, the wrong enantiomer may have an adverse side
effect on an organism.[5] The demands for methods for the
synthesis of structurally different isomers and diastereoisomers
of chiral compounds have increased rapidly in response to the
requirements of the pharmaceutical industry. The application of
enzymes in organic synthesis is a promising solution to obtain
enantiomerically pure compounds. In particular, the biocata-
lyzed cascade reaction may be turned into an asymmetric
process leading to a non-racemic product.[6] Considering the
advantage of the broad family of substituted lactates, we have
studied the enzyme-regulated stereodivergent cascade leading
to diastereoisomeric α-acyloxy carboxamides. The classical
chemical approach leads to the inseparable mixture of stereo-
isomers. Therefore, we propose a one-pot system, which using
the same substrates enables the formation of a single stereo-
isomer with the enzymatic regulation of the stereoselectivity.
Moreover, the designed system is both more convenient
and more environmentally friendly since no separation of the
intermediate is required (Figure 2).
[a] M. Wilk, Dr. A. Brodzka, Dr. D. Koszelewski, J. Samsonowicz-Górski,
Prof. R. Ostaszewski
In stereodivergent processes, structurally identical com-
pounds are converted into products with diverse chemo-, regio-
or diastereoselectivity.[7] In particular, a diastereodivergent
transformation allows for the efficient transformation of inex-
pensive substrates into valuable products with different bio-
Institute of Organic Chemistry Polish Academy of Sciences
Kasprzaka 44/52, 01-224 Warsaw, Poland
E-mail: r.ostaszewski@icho.edu.pl
www.icho.edu.pl
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/ejoc.202100760
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Figure 2. The general concept of the proposed methodology.
logical activities. Moreover, an access to various stereoisomers
can be achieved by several strategies including changes of the
reaction conditions, distinct additives, or individual catalysts.[8]
Conceptually, a formation of products with a pair of stereogenic
centers can be divided into three general ways. The first
approach is based on using diverse catalysts working independ-
ently. At this concept the stereochemistry is controlled sepa-
rately at every step: an enantioselective formation of the first
stereocenter is followed by a second diastereoselective reaction
catalyzed by the distinct catalyst. The second catalyst requires
overcoming the stereochemical bias present in the chiral
compound formed at the first stage, which limits its utility. The
second pathway relies on the stepwise formation of stereo-
centers catalyzed by the same catalyst.[9] However, a significant
limitation of asymmetric synthesis is to obtain a chiral molecule
possessing multiple stereogenic centers in a single transforma-
tion driven by one catalyst. Such advantages are offered by the
cascade systems where a divergence can be entailed by the
combination of promiscuous enzymes. Interestingly, these
processes emulate biological systems, wherein complex mole-
cules are easily produced from simple substrates in continuous
processes driven by enzymes.[10]
Herein, we propose a new overall strategy of enantio- and
diastereodivergent dual biocatalytic process combined at a
cascade reaction. The designed cascade involves enzyme-
catalyzed kinetic resolution (EKR) of vinyl ester followed by the
Passerini reaction and subsequent enzymatic hydrolysis (EKR) of
formed α-acyloxy carboxamides. The sequential transformations
provide a powerful strategy for the fast production of complex
molecules with exquisite levels of enantio- and diastereocontrol
offered by multicomponent reactions and the enzyme promis-
cuity. The cascade reaction makes the processing time- and
cost-efficient. Recently, we have reported an elegant example
of a tandem reaction that utilizes the selectivity of enzymes and
synthetic potential offered by multicomponent reaction (MCR)
to the formation of enantioenriched α-hydroxy carboxamides.[11]
Taking advantage of the previous report herein we have tested
the utility of enzymes for the synthesis of compounds
containing two stereogenic centers in the cascade manner. We
have designed a chemoenzymatic diastereodivergent cascade
for the synthesis of chiral α-acyloxy carboxamides containing a
lactate scaffold (1). It is worth mentioning that the classical
syntheses are highly demanding and require the separated
control of stereochemistry at every step or overcoming the
stereochemical bias present in the chiral compound, wherein
the designed system proceeds smoothly in one pot. Moreover,
classical methods require three separated processes with 25%
of maximal yield. Herein, vinyl ester (9–12) is used as a
simultaneous precursor of irritant acetaldehyde and carboxylic
acid, which are the substrates of the Passerini reaction
(Scheme 1).
In the first step, the enzyme performs a kinetic resolution of
the racemic vinyl ester (9–12) leading to the formation of
enantioenriched carboxylic acid (14) and acetaldehyde (13).
Next, generated compounds follow the Passerini condensation
with an isocyanide, present in the reaction mixture. The utility
of acetaldehyde leads to the generation of new stereocenter in
the product of the Passerini reaction. The carboxylic acid
contains one stereogenic center in its structure, therefore the
second step results in the formation of a diastereomeric mixture
of α-acyloxy carboxamides (15–18). However, according to our
previous reports, hydrolytic enzymes can provide the kinetic
resolution of α-acyloxy carboxamides[11,12] formed in the Passer-
ini reaction to α-hydroxy carboxamides 19 and corresponding
carboxylic acid. Due to the high selectivity of an enzyme, we
expect that the last step should enable to obtain of diastereo-
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ylhydrocinnamate[13] (9) and p-methoxybenzylyisocyanide in a
phosphate buffer pH 7.4. We started our investigation with the
reaction in the absence of an enzyme, wherein neither the
formation of product 15 nor its subsequent hydrolysis product
were observed (Table 1, entry 1). Next, we tested the ability of
several commercially available lipases to drive the proposed
cascade (Supporting information, Table S1). The data in Table 1
presents the access to the full matrix of stereoisomeric products
of the Passerini reaction for several enzymes: Candida cylindra-
cea lipase (CCL), Pseudomonas fluorescens lipase (PFL), and
porcine pancreas lipase (PPL).
The condensation of 2-methylhydrocinnamic acid,
acetaldehyde, and p-methoxybenzyl isocyanide afforded the
desired compound 15 with 43% yield and as an inseparable
mixture of stereoisomers. (Table 1, entry 2) However, the
racemic vinyl ester 9 undergoes the enzymatic kinetic reso-
lution with different efficiency. The condensation step afforded
the desired product with a various contribution of α-acyloxy
carboxamide what enables the production of an individual
stereoisomer of the product. On basis of our hypothesis, the
simultaneous kinetic resolution of enantioenriched α-acyloxy
carboxamide 15 has a significant influence on the optical purity
of the remaining compounds. Depending on the enzyme used,
the proposed protocol gives access to all stereoisomers of the
diastereoisomeric Passerini product, which is highly difficult to
obtain using classical methods. Notably, PPL (Table 1, entry 6)
allows obtaining α-acyloxy carboxamide 15 containing more
than 50% of (SR)- stereoisomer in the products mixture with
63% of enantiomeric excess (ee). Unexpectedly, the increase of
the reaction time resulted in a decrease in the contribution of
(SS) and (SR)- stereoisomers and increase the content of
complementary (RR) and (RS)- products. Furthermore, based on
the chemical structure of the desired product, we intended to
change the molar ratio of the substrates. The application of 2
equivalents of vinyl ester 9 allows to obtain the stereoisomer
Scheme 1. Chemoenzymatic diastereo- and enantiodivergent preparation of
lactate derivatives via cascade I- EKR / Passerini/II- EKR reaction.
and enantiomerically enriched α-acyloxy carboxamides 15 in
one cascade system.
Besides, the stereochemistry of compounds 15 can be easily
inverted in the Mitsunobu reaction,[12] which significantly
increases the potential of the proposed methodology.
To overcome the principal limitation to design an elegant
cascade with compatible conditions for all steps, we have
applied various vinyl ester 9–12 and an isocyanide into a one-
pot reaction. As model reactants, we applied vinyl 2-meth-
(RS)- with the 99% of ee (Table 1, entry 9), wherein
3
equivalents gave the stereoisomer (RS) with 90% of ee (Table 1,
Table 1. The evaluation of reaction conditions for the racemic compound 9.[a]
Enzyme
Conv.[b]
Stereoisomer 15
ee 15a
[%]
ee 15b
[%]
Yield 19[d]
[%]
[%][c]
RR
SS
SR
RS
1
–
–
0
43
0
nd
25
nd
7
27
9
18
25
14
14
3
0
0
0
0
2[e]
3
25
25
25
0
BSA
CCL
PFL
PPL
PPL
PPL
PPL
PPL
PPL
nd
67
14
49
6
77
40
31
89
nd
21
84
63
48
8
99
90
92
nd
45
76
70
74
85
87
82
52
4
5
6
58
78
20
30
57
36
28
40
35
36
26
16
3
32
27
50
23
3
53
48
39
0
35
34
12
17
33
54
56
2
7[f]
8[g]
9[h]
10[i]
11[j]
3
46
[a] Reaction conditions: enzyme (100 mg) in phosphate buffer (10 mL, pH 7.4, 0.1 M), vinyl ester 9 (1 equiv., 0.5 mmol), p-methoxybenzylyisocyanide
(1 equiv., 0.5 mmol). Stirred for 2 days at rt. Product 15 was isolated by column chromatography and analyzed by HPLC. [b] Refers to the Passerini product
15 calculated as ((n15 +n19)/n9 ×100%). [d] The isolated yield of product 19 calculated as (n19/(n15 +n19)×100%). [e] Racemic 2-methylhydrocinnamic acid
(0.5 mmol) and acetaldehyde (0.5 mmol) were used instead of vinyl ester. [f] Reaction stirred for 3 days. [g] Reaction stirred for 6 days. [h] Reaction with
2 equiv. of vinyl ester 9. [i] Reaction with 3 equiv. of vinyl ester 9. [j] Reaction conducted in dichloromethane with 0.1% water content (v/v) according to
Żądło-Dobrolowska et al.[11]
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entry 10). Notable, the presented cascade consists of three
reactions proceeding with exquisite yields in each elementary
step.
The model reaction was carried in the phosphate buffer
(pH 7.4), but also other solvents were tested (Table S5, Support-
ing information). The reaction proceeding in dichloromethane
with 0.1% of water content, resulted in the formation of pure
diastereoisomeric Passerini products with 40% of yield for
unoptimized reaction conditions (Table 1, entry 11). It is worth
mentioning that two enantiomerically pure diastereoisomers
ity. Moreover, the obtained results are following the principles
of the biocatalytic process which requires suitable condition for
every transformation. The divergence of the proposed method-
ology relies on the enzyme promiscuity and can be optimized
for each stereoisomer if required, which is the privilege of the
designed enzymatic cascade reaction. Moreover, the presented
studies are promising in exploratory proof-of-concept of
enzyme-regulated diastereodivergent process.
can be easily separated by the column chromatography, giving Conclusion
the (SS)- and (SR)- acyloxy carboxamides with 89 and 92% of ee,
respectively and satisfactory yield. In comparison, the chemical
syntheses are hardy demanding and lead to the inseparable
mixture of stereoisomers with a maximal 25% of yield.
Owing to the high complexity of the proposed method-
ology, we tested the activity of various enzymes in the cascade
In summary, we have proposed an efficient diastereo- and
enantio-divergent methodology for the synthesis of enantio-
merically pure α-acyloxy carboxamides. The access to the
individual stereoisomeric products has been achieved by the
combination of the enzymatic kinetic resolution of racemic vinyl
esters, subsequent Passerini reaction, and enzymatic kinetic
resolution of formed α-acyloxy carboxamides. The course of the
cascade process is fully regulated by the enzyme and can be
optimized for each stereoisomeric product, what is the privilege
of the designed cascade reaction over the classical approach.
The presented studies are promising in exploratory proof-of-
concept of enzyme-regulated stereodivergent cascade to form
an important class of chiral compounds for medicinal chemistry.
reaction
with
vinyl
esters
10–12
and
p-meth-
oxybenzylisocyanide. The obtained results (Table S2–S4, Sup-
porting Information) proved that the reaction efficiency highly
depends on the enzyme type, which regulates the formation of
the corresponding diastereo- and enantioenriched α- acyloxy
carboxamides. To demonstrate the concept, in Figure 3, we
have collected the most promising results on way to obtain
diverse stereoisomeric products of Passerini reaction with esters
10–12. Interestingly the reaction of vinyl ester 11 in the
presence of Pseudomonas fluorescens lipase gives α-acyloxy
carboxamide 17a with excellent diastereo- and enantioselectiv- Acknowledgements
We gratefully acknowledge the financial support from the Na-
tional Science Centre (Poland), for the project: No. 2019/33/B/ST4/
01118.
Conflict of Interest
The authors declare no conflict of interest.
Keywords: Cascade reaction · Double kinetic resolution ·
Enzyme catalysis
methodology
·
Passerini reaction
·
Stereodivergent
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Manuscript received: June 28, 2021
Revised manuscript received: July 13, 2021
Accepted manuscript online: July 21, 2021
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