Enzyme-Promoted Asymmetric Tandem Passerini Reaction

Article abstract of DOI:10.1002/cctc.201700427

A versatile and highly efficient three-step, one-pot, enzyme-promoted Passerini tandem reaction has been developed. The chemoenzymatic sequence involved simultaneous enzyme catalyzed hydrolysis, subsequent Passerini reaction, and enzymatic kinetic resolution of the Passerini product. This methodology combines the diversity delivered by multicomponent reactions with the selectivity of biocatalysts, which results in efficient synthesis of the target compounds with excellent enantiomeric excesses of up to 99 %. With a small set of substrates, a large library of complex molecules was obtained within a short time by using the developed procedure.

Full text of DOI:10.1002/cctc.201700427

Accepted Article
Title: Enzyme-promoted Asymmetric Tandem Passerni Reaction
Authors: Ryszard Ostaszewski, Anna Zadlo, Dominik Koszelewski,
Daniel Paprocki, Arleta Madej, and Monika Wilka
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemCatChem 10.1002/cctc.201700427
Link to VoR: http://dx.doi.org/10.1002/cctc.201700427
A Journal of
www.chemcatchem.org

10.1002/cctc.201700427
ChemCatChem
FULL PAPER
Enzyme-promoted Asymmetric Tandem Passerini Reaction
Anna Żądło-Dobrowolska, Dominik Koszelewski, Daniel Paprocki, Arleta Madej, Monika Wilk, Ryszard
Ostaszewski[a]*
Abstract:
A
versatile and highly efficient three-step, one-pot,
time-consuming isolation and purification of intermediates.[10]
Over the past two decades the field of MCRs has incredibly
grown with the discovery and development of the new variations
of classical MCRs.[11] Isocyanide-based MCRs, such as Ugi and
Passerini reactions, have been of particular interest because of
the large number of applications in medicinal chemistry and drug
discovery,[12] combinatorial chemistry,[13] polymer chemistry,[14]
natural product synthesis,[15] agrochemistry.[16] The Passerini
three component reaction (P-3CR) involves the condensation of
a carbonyl compound, a carboxylic acid and an isocyanide
leading to an α-acyloxy carboxamide.[17] When carbonyl
enzyme-promoted Passerini tandem reaction has been developed.
The chemoenzymatic sequence involved simultaneous enzyme
catalyzed hydrolysis, subsequent Passerini reaction and enzymatic
kinetic resolution of the Passerini product was evaluated. This
methodology combining the diversity served by multicomponent
reactions with the selectivity of biocatalysts resulted in efficient
synthesis of the target compounds with excellent enantiomeric
excesses up to 99%. With a small set of substrates, large library of
complex molecules was obtained within
developed procedure.
a short time using
substrate different from formaldehyde is used,
a
new
stereogenic center is generated. However P-3CR has been
widely applied, the challenge of stereocontrol on a newly
generated stereogenic center is currently very limited. Number
of diastereoselective examples are known that use chiral
auxiliaries or chiral substrates,[18] but only a limited success has
been recorded in the field of enantioselective approach.[19]
Several problems must be addressed, e.g. the majority of
catalysts, especially metal complexes are toxic and expensive
and the products require complex purification procedures. They
cannot be used in the pharmaceutical and cosmetic industry,
due to pharmacopoeia limits of heavy metal contaminations
(below 5 ppm). Moreover, Lewis-acid-catalyzed P-3-CR could go
through both the imidate and the epoxide intermediate providing
two enantiomers of the α-acyloxy carboxamide, which led to the
erosion in enantiomeric excess of the products.[20]
Introduction
With the rise of biocatalysis, scientists inspired by nature, have
designed multi-enzymatic cascades to mimic living organisms in
building complex molecules from simple starting materials.[1]
A
lot of attempts have been made to reproduce such biomimetic-
type processes in vitro.[2] Consequently, numerous multi-
enzymatic cascades have been developed, ranging from the
simple combination of oxidoreductases with a suitable cofactor
regeneration system[3] to highly complex and specialized
synthetic routes.[4] Since cascades represent a very promising
approach, mainly due to the avoidance of purification steps and
reduction of both waste and production costs, they are more
readily employed by organic chemists.[5]
As an alternative to the commonly employed metalo-catalysis
or organo-catalysis, a biocatalytic approach can be expected to
be beneficial due to high selectivity of enzymes and mild
reaction conditions, thereby fulfilling the general principles of
green chemistry. The aim of the present work was to combine
the selectivity of biocatalysts with the diversity offered by a
Passerini reaction to obtain enantiomerically pure α-hydoxy
carboxamide scaffolds. To address this challenge we performed
studies toward coupling three reaction steps, involving enzyme
catalyzed hydrolysis, subsequent Passerini reaction and
enzymatic kinetic resolution (EKR), into a one-pot tandem
process. Synthetically valuable α-hydroxy carboxamides, which
could be easily transformed into the optically active natural and
non-natural amino acids, were chosen as the target compounds.
Compared with a cascade, where unstable intermediate is
generated, multi-step syntheses, which could be carried out
separately, is called tandem catalysis.[6] One of the most
common challenges for its practicability is the combination of
catalysts from different disciplines (biocatalysis, metal catalysis,
or organocatalysis) with multicomponent reactions (MCRs).[7] To
the best of our knowledge, literature recognized tandem
processes are limited only to two sequential reactions what
makes them inefficient in mimicking of the natural metabolic
pathways.[8]
MCRs involve condensation of more than two reactants in the
same pot to form a final product containing portions derived from
each of the reacting molecules, ideally all atoms.[9] In this way,
very high level of atom economy, efficiency and molecular
diversity could be achieved in a simple one-pot fashion, avoiding
Results and Discussion
[a]
Dr A. Żądło-Dobrowolska, Dr D. Koszelewski, M. Sc. D. Paprocki, M.
Sc. A. Madej, M. Sc. M. Wilk, Prof. R. Ostaszewski
Institute of Organic Chemistry
Biocatalytic retrosynthetic analysis suggested that the α-hydroxy
carboxamide skeleton
5
could be prepared from the
Polish Academy of Sciences
Kasprzaka 44/52, 01-224 Warsaw, Poland
E-mail: ryszard.ostaszewski@icho.edu.pl
corresponding vinyl esters 1 via a sequence involving enzyme-
catalyzed hydrolysis, Passerini reaction and EKR (Scheme 1).
Designing our synthetic route we took into consideration the
requirements assigned to multicatalyst asymmetric tandem
reactions, such as possibility of coupling the wide spectrum of
Supporting information available: Experimental procedures, 1H
NMR, 13C NMR spectra and chiral HPLC tracers.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201700427
ChemCatChem
FULL PAPER
substrates and reactions, catalysts compatibility and
stereochemical control.[21]
transformed with the inversion of configuration by Mitsunobu
reaction.[26a] Moreover, the generation of carboxylic acid 2, which
can repeatedly react as a substrate, provides a possibility of
self-regulation.
We started our investigation by performing simple
transformations. At first, we investigated Passerini reaction in a
phosphate buffer pH 7.4. As expected, the condensation of
acetic acid, acetaldehyde and p-methoxybenzyl isocyanide
afforded the desired compound 4a with good yield (45%, Table 1,
entry 1) after 24 hours. We also observed a small amount of a
subsequent spontaneous hydrolysis product 5a. Next, we have
examined enzymatic kinetic resolution of racemic compound 4a
in
a phosphate buffer pH 7.4. With the application of
Pseudomonas cepacia lipase (PcL), enantiomerically enriched
product (S)-5a was isolated with 43% yield and 90% ee (Table 1,
entry 2).
Table 1. Enzyme screening.
Entry
Enzyme
c
ee
yield
ee
E
[%]
4a [%]
5a [%]
5a [%]
1
-
45[a]
nd
50
50
44
47
47
33
28
32
48
nd
68
70
25
rac
44
rac
25
5
<5[a]
43
82
56
61
77
64
73
61
75
79
nd
90
20
20
rac
15
rac
26
3
nd
39
3
2
PcL [b]
PcL
3
4
Novozym 435
Lipozym
PfL
2
5
1
6
2
Scheme 1. Envisioned cascade process.
7
CrL
1
8
WGL
PLE
2
It is noteworthy that envisioned tandem process is a very
complicated system consisting of 3 reaction steps. In the first
step, enzyme catalyzes hydrolysis of vinyl ester 1, what should
lead to the corresponding carboxylic acid 2 and vinyl alcohol,
which spontaneously tautomerises to acetaldehyde 3,[22] Scheme
1. Vinyl esters are commonly used as acylating agents in
enzymatic transformations.[23] In this step, water acts as a
nucleophile and is compulsory for the initiation of enzymatic
hydrolysis. Second step involves the condensation of previously
generated carbonyl compound 3 and carboxylic acid 2 with an
isocyanide present in the reaction mixture, leading to an α-
acyloxy carboxamide 4.[24] Finally, the third reaction step was
designed based on previous studies, including pre- and post-
modifications of MCR products.[25] According to previously
reported protocol, in which hydrolytic enzymes were employed
for the kinetic resolution of α-acyloxy carboxamides,[26]
9
1
10
11
PPL
rac
38
rac
13
1
CaL
2
Conditions: A reaction mixture containing vinyl acetate (0.5 mmol), p-
methoxybenzyl isocyanide (0.5 mmol) and enzyme (20% by weight) in 20
mL of phosphate buffer (100 mM, pH 7.4) was stirred for 24 hours at rt. After
this time two products 4a and 5a were isolated by column chromatography;
Conversion refers to Passerini product 4a, calculated as ((n4a+n5a)/n1)·100%;
Yield 5a refers to isolated product after enzymatic kinetic resolution,
calculated as ((n5a)/n4a+n5a)·100%; nd – non defined; [a] acetic acid (0.5
mmol) and acetaldehyde (0.5 mmol) were used instead of vinyl acetate; [b]
Racemic 4a was used as a starting material.
compound
4
may be enzymatically hydrolyzed releasing
and
Then, to initially examine the feasibility of the envisaged
tandem process, vinyl acetate and p-methoxybenzyl isocyanide
were used as model reactants for the screening of a suitable
biocatalyst. In the classical procedure highly volatile (boiling
point 20 °C) and irritant acetaldehyde is required. Our elegant
protocol engages vinyl acetate (boiling point 72 °C) which is in
situ hydrolyzed by enzyme providing both substrates for P-3-CR;
enantiomerically enriched α-hydroxy carboxamide
5
carboxylic acid 2. Although enzymatic kinetic resolution is
always limited to a maximum yield of 50% of a single enantiomer,
a careful selection of the enzyme should result in both
enantimerically pure compounds 4 and 5. If only one enantiomer
of Passerini product is needed, compound 5 may be easily
This article is protected by copyright. All rights reserved.

10.1002/cctc.201700427
ChemCatChem
FULL PAPER
aldehyde and carboxylic acid. It is noteworthy that reaction
without enzyme did not run. Nine, various enzymes were
tertiary structure of proteins.[32] As shown in Table 2, Passerini
reaction does not proceed in ethers (Table 2, entries 5-6) and
proceeds poorly without solvent (Table 2, entry 7). Among the
screened solvents, dichloromethane (Table 2, entry 3) was
found to be the most suitable for the examined tandem reaction
leading to the product (S)-5a with 94% enantiomeric excess.
Reaction performed in toluene or hexane resulted in higher
yields but much lower enantioselectivities (Table 2, entries 2 and
4).
investigated in
a
three-step one-pot tandem reaction;
Pseudomonas cepacia lipase (PcL), Pseudomonas fluorescens
lipase (PfL), Novozym 435, Lipozym (immobilized lipase from
Mucor miehei) (MmL), Candida rugose lipase (CrL), wheat germ
lipase (WGL), porcine liver esterase (PLE), porcine pancreas
lipase (PPL) and Candida antarctica lipase (CaL). On the basis
of our hypothesis, acetic acid and acetaldehyde formed during
enzymatic hydrolysis of vinyl acetate, should smoothly react with
isocyanide present in the reaction mixture. Results shown in
Table 1, named as yield 5a, were calculated only for EKR step.
To our delight, all examined biocatalysts were active in the
first hydrolysis step (Table 1, entries 3-11) leading to the desired
acetic acid and acetaldehyde, which immediately formed product
4a with moderate to good yields (23-50%). Under the examined
conditions, α-acyloxy carboxamides 4a underwent subsequent
enzyme catalyzed hydrolysis providing enantiomerically
enriched product (S)-5a. Finally, Pseudomonas cepacia lipase
was found to be the best biocatalyst regarding the
enantioselectivity (E) and the yield (Table 1, entry 3) and was
consequently used in further experiments. Enantioselectivity
values were calculated according to Chen et al. equation.[27] It is
important to notice that the same enzyme maintained first
hydrolysis step and catalyzes enantioselectively the EKR, what
solves the compatibility problem.
Table 2. Solvent influence on the enzyme-driven Passerini reaction.
Entry
Solvent
Conversion
[%]
ee 4a [%]
Yield 5a
[%]
ee 5a [%]
E
1
2
3
4
5
6
7
PBS
50
41
44
38
0
70
36
rac
5
82
24
5
20
14
94
32
nd
nd
13
3
Toluene
DCM
1
34
2
Hexane
Et2O
13
0
nd
nd
rac
nd
nd
1
TBME
none
0
0
73
7
Comparison of the amount of product (S)-5a obtained for a
whole tandem reaction, starting from 1 (82% yield, 19% ee,
Table 1, entry 3) with a simple transformation, starting from
racemic 4a (43% yield, 90% ee, see Table 1, entry 2), revealed
that formation of acetic acid shifts the reaction equilibrium
toward products. However, such acceleration by acetic acid
influenced negatively on enantioselectivity. Moreover, acetic
acid formed during second hydrolysis step might react again as
a substrate in the P-3-CR, what resulted in 5% increase in
conversion (Table 1, entry 1 vs 3 and 4). This phenomena based
on acceleration of the Passerini reaction by the excess of
carboxylic acid is known.[28] The examined tandem process may
mimic metabolic pathways, which must be finely regulated to
maintain a constant homeostasis. In intrinsic regulation, the
metabolic pathway self-regulates to respond to changes in the
levels of substrates or products.[29] In our example, the overall
sequence is regulated by the amount of acetic acid. This type of
regulation remains out of reach in typical chemical tandems
performed in vitro.
Conditions:
A reaction mixture containing vinyl acetate (0.5 mmol), p-
methoxybenzyl isocyanide (0.5 mmol) in 20 mL of organic solvent (0.5% v/v),
and the enzyme (20% by weight) was stirred for 96 hours at rt. After this time
two products 4a and 5a were isolated by column chromatography; Conversion
refers to Passerini product 4a, calculated as ((n4a+n5a)/n1)·100%; Yield 5a
refers to isolated product after enzymatic kinetic resolution, calculated as
((n5a)/n4a+n5a)·100%; nd – non defined.
One of the major drawbacks in the implementation of one-
pot reactions is the selection of the optimal operating conditions.
When multiple reactions are run in the same pot, optimal
conditions for each individual reaction are not always identical.
To solve this problem, the optimization of the overall tandem
was performed. The results of a survey of reaction conditions
using Pseudomonas cepacia lipase as
a
catalyst and
dichloromethane (DCM) as a solvent are summarized in Table 3.
Further investigations revealed that increase of the amount
of isocyanide had a beneficial influence on the results. It was
proved that isocyanide undergoes slow spontaneous hydrolysis
in the presence of water.[26] Finally, increasing the molecular
ratio of isocyanide to vinyl acetate (3:1, n/n) resulted in the
product yield improvement from 5% to 21% with the
simultaneous increase of the enantiomeric excess (Table 3,
entry 3).
Next, we turned our attention to the optimization of the water
content. It is noteworthy that 2 equivalents of water act as a
nucleophile in the first and third reaction step and 1 equivalent is
produced in the second step. The best result with 21% yield and
>99% ee was achieved when 0.5% of water by volume was
added into the reaction (Table 3, entry 3). Decreasing the
Due to the high complexity of the studied tandem process
further optimization studies were undertake. Up to date, many
enzymes were shown to be catalytically active in a variety of
organic
solvents.[30]
Their
catalytic
efficiency
and
enantioselectivity in mixed aqueous-organic media are often
higher than in aqueous media alone.[31] Moreover, it is known
that Passerini reaction proceeds more productively in organic
solvents than in aqueous solutions.[25] For those reasons, we
decided to examine the influence of the organic solvent on the
reaction efficiency, Table 2.
A small amount of water (0.5% by volume) was crucial for
enzymatic hydrolysis, as well as for the stabilization of the
This article is protected by copyright. All rights reserved.

10.1002/cctc.201700427
ChemCatChem
FULL PAPER
amount of water resulted in lower efficiency (Table 3, entry 4),
while increasing the amount of water resulted in decreasing of
enantiomeric excesses to 80% (Table 3, entries 6-8).
examined as a catalyst in the studied tandem reaction. It is well
known that enzymes are highly selective and for each mixture of
substrates the other biocatalyst might be suitable.[33]
Elaborated protocol is applicable to a wide range of vinyl
esters and isocyanides, providing enantiopure α-hydroxy
carboxamides, which are valuable building blocks for the peptide
and peptidomimetics synthesis.[26] Application of our protocol
enables to obtain the final product with good yield (up to 50%)
and excellent enantiomeric excess (up to >99% ee) within 4
days at room temperature. Unsubstituted benzyl isocyanides
and isocyanides bearing electron donating groups in the para-
position were efficiently transformed by both enzymes with up to
50% conversion and high enantioselectivity (E>200) towards the
enantiomerically pure products (S)-5a and (S)-5b (Table 4,
entries 1,4,6 and 8). In order to assign the configuration of the
obtained products, compounds (S)-5a-c were synthesized from
commercially available, optically pure L-lactic acid, by
conversion into the S-configured α-hydroxy carboxamides 5a-c
(supplementary data). We would like to emphasize that benzyl
isocyanides can be replaced by cyclohexyl and hexyl isocyanide,
producing the corresponding products (S)-5c and 5d in
moderate yields (table 4, entries 9 and 10). The selectivity of the
reaction decreased when benzyl isocyanide was exchanged for
aliphatic isocyanide (Table 4, entries 8 vs 10), however aliphatic
vinyl esters were better enzyme substrates than aromatic esters
(Table 4, entries 1-4 vs 5). Application of vinyl acetate led to the
product (S)-5a in 21% yield (Table 4, entry 1), whereas the
elongation of the alkyl chain resulted in a lower yield, 5% for
vinyl propionate (Table 4, entry 3-4) and 9% for vinyl laurate
(Table 4, entry 6). Due to the high selectivity of the enzyme, in
some cases, the third step of the tandem reaction was catalyzed
by Pseudomonas cepacia lipase (Table 4, entries 5 and 10) or
by Novozym 435, exclusively (Table 4, entries 6 and 9). This
observation proves that careful selection of the enzyme is
necessary whenever the structure of substrate is changed.
Table 3. Reaction optimization.
Entry
Water
content
[%]
Vinyl
acetate
[equiv.]
Isocyanide
[equiv.]
c
[%]
Yield
(S)-5a
[%]
ee
(S)-5a
[%]
E
1
2
3
4
5
6
7
8
0.5
0.5
0.5
0.2
1
1
3
1
1
1
1
1
1
1
1
3
3
3
3
3
3
44
79
80
39
64
55
44
63
5
94
34
5
91
22
21
5
>99
>99
>99
98
>200
>200
>200
112
>200
9
14
11
14
6
2
5
99
10
80
Conditions: A reaction mixture containing (0.5 or 1.5 mmol) vinyl acetate, (0.5
or 1.5 mmol) 4-methoxybenzyl isocyanide, in 20 mL of dichloromethane, 0.2-
10% of water by volume, and the enzyme (20% by weight) was stirred for 96
hours at rt. After this time two products were isolated by column
chromatography; Conversion refers to Passerini product, calculated as
((n4a+n5a)/n1)·100%; Yield 5a refers to product after enzymatic kinetic
resolution, calculated as ((n5a)/n4a+n5a)·100%.
With the optimal conditions in hand, the generality of the
reaction was further investigated using a set of vinyl esters and
isocyanides. Since Pseudomonas cepacia lipase did not accept
all employed compounds as substrates, Novozym 435 was also
Table 4. Scope of limitations.
Entry
R1
R2
Product
5a
Enzyme
Conversion [%]
Yield (S)-5 [%]
ee (S)-5 [%]
E
1
2
3
4
5
6
7
8
9
10
Me
p-MeOBn
p-MeOBn
p-MeOBn
p-MeOBn
p-MeOBn
p-MeOBn
Bn
A
B
A
B
A
B
A
B
B
A
80
27
66
68
13
65
64
22
21
58
21
26
5
>99
90
>200
26
Me
5a
Et
5a
98
104
>200
28
Et
5a
20
15
9
>99
92
Ph
5a
CH3(CH2)10
5a
99
>200
>200
>200
1
Me
Me
Me
Me
5e
19
50
33
5
>99
97
Bn
5e
c-Hex
5f
7
Hex
5g
nd
nd
Conditions: A reaction mixture containing vinyl ester (0.5 mmol), p-methoxybenzyl isocyanide (1.5 mmol) in 20 mL of dichloromethane, water (0.5% v/v), and the
enzyme (20% by weight) was stirred for 96 hours in rt. After this time two products 4 and 5 were isolated by column chromatography; Conversion refers to
This article is protected by copyright. All rights reserved.

10.1002/cctc.201700427
ChemCatChem
FULL PAPER
Passerini product 4, calculated as ((n4a+n5a)/n1)·100%; Yield 5a refers to isolated product after enzymatic kinetic resolution, calculated as ((n5a)/n4a+n5a)·100%;
enzyme A refers to Pseudomonas cepacia lipase.; B refers to Novozym 435.
funnel filled with Celite and MgSO4. Then solvent was evaporated in
vacuum. Crude product 5 was purified by column chromatography (ethyl
acetate/hexane). The 1H NMR data were in accordance with those
recorded for racemates; (S)-(-)-5a: [α]D= -61.2 (c 1.0 CHCl3, >99% ee),
(S)-(-)-5b: [α]D= -29.8 (c 1.0 CHCl3, >99% ee), (S)-(-)-5c: [α]D= -11.4 (c
1.0 CHCl3, >99% ee).
Regardless of the substrates 4 structure, S-enatiomer of the
corresponding α-hydroxy carboxamides 5a-c was gained.
Obtained results open
a new field in enzyme-promoted
multicomponent reactions. Further studies on the optimization
and amplification of the chiral vinyl esters leading to products
with two stereogenic centers are still underway. In spite of the
described limitations the proposed approach diversifies already
known methods and leads to the enantiomerically pure products.
1[(4-Methoxybenzyl)amino]-1-oxopropan-2-yl acetate (4a)
White crystals; m.p. 122-123 °C; 1H-NMR (CDCl3, 400 MHz, ppm) δ 7.22-
7.12 (m, 2H, Ar-H), 6.98-6.84 (m, 2H, Ar-H), 6.40 (br. s., 1H, -CONH-),
5.22 (q, 1H, J=6.9 Hz, -OCH(CO)CH3-), 4.39 (d, 2H, J=5.6 Hz, -
NHCH2Ar-), 3.79 (s, 3H, -OCH3), 2.10 (s, 3H, -COCH3), 1.48 (d, 3H,
J=6.9 Hz, -CHCH3); 13C-NMR (CDCl3, 100 MHz, ppm) δ 170.2, 169.5,
159.1, 129.9, 129.0, 114.2, 70.7, 55.3, 42.7, 21.1, 17.9; HR-MS (ESI, [M
+ H+] calcd for: C13H17NO4Na, 274.1055, found, 274.1047; TLC: Rf = 0.45
(ethyl acetate/hexanes, 7:3 v/v).
Conclusions
In summary, we have developed a new, efficient, three-step
one-pot tandem reaction. A wide range of aromatic and aliphatic
vinyl esters were tolerated providing the corresponding α-
hydroxy carboxamides in high yields and excellent enantiomeric
excess (up to 99%). Access to enantiomerically pure products
has been achieved using a chemoenzymatic sequence involving
simultaneous enzyme catalyzed hydrolysis, subsequent
2-Hydroxy-N-(4-methoxybenzyl)propanamide (5a)
White crystals; m.p. 78-79 °C; 1H-NMR (CDCl3, 400 MHz, ppm) δ 7.23-
7.13 (m, 2H, Ar-H), 6.97-6.80 (m, 3H, Ar-H, -CONH-), 4.43-4.34 (m, 2H, -
NHCH2Ar-), 4.28-4.19 (m, 1H, -HOCH(CO)CH3-), 3.80-3.76 (s, 3H, -
OCH3), 2.76 (br.s., 1H, -OH), 1.43 (d, 3H, J=6.9 Hz, -CHCH3); 13C-NMR
(CDCl3, 100 MHz, ppm) δ 170.2, 169.5, 159.1, 129.9, 129.0, 114.2, 70.7,
55.3, 42.7, 21.1, 17.9; HR-MS (ESI, [M + H+] calcd for: C11H15NO3Na,
232.0950, found, 232.0940; TLC: Rf = 0.15 (ethyl acetate/hexane, 7:3
v/v). 2(S)-hydroxy-N-(4-methoxybenzyl)propanamide ((S)-5a) was
prepared from L-lactic acid (supplementary data); [α]D= -58.2 (c 1.0
CHCl3), HPLC: Chiralcel OD-H; hexane/isopropanol (80:20), λ = 225 nm;
1.0 mL/min, retention time of the racemic compound (in min): tR (R)= 9.6,
tR (S)= 11.9.
Passerini reaction and enzymatic kinetic resolution of
a
Passerini reaction product. Since developed protocol requires
only one enzyme, which catalyzes both hydrolysis steps, the
reaction was run as a one-pot three-step process. To the best of
our knowledge, this is the first chemoenzymatic tandem process,
consisting of more than two reactions, which may be performed
in organic solvents, as well as in water, without isolating the
intermediates.
1[(4-Methoxybenzyl)amino]-1-oxopropan-2-yl propanoate (4b)
1
Yellow oil; H-NMR (CDCl3, 400 MHz, ppm) δ 7.19 (d, 2H, J=8.4 Hz, Ar-
Experimental Section
H), 6.86 (d, 2H, J=8.8 Hz, Ar-H), 6.31 (br. s., 1H, -CONHCH2-), 5.24 (q,
1H, J=6.9 Hz, -OCH(CO)CH3-), 4.39 (d, 2H, J=5.6 Hz, -NHCH2Ar), 3.79
(s, 3H, -OCH3), 2.36 (q, 2H, J=7.6 Hz, CH3CH2CO-), 1.48 (d, 3H, J=6.9
Hz, CH3CH-), 1.13 (t, 3H, J=7.6 Hz); 13C-NMR (CDCl3, 100 MHz, ppm) δ
172.9, 170.3, 159.2, 129.9, 129.0, 114.2, 70.5, 55.3, 42.7, 27.6, 17.9,
9.0; HR-MS (ESI, [M + H+] calcd for: C14H19NO4Na, 288.1212, found,
288.1209; Rf = 0.58 (ethyl acetate/hexane, 7:3 v/v).
General Methods. 1H- and 13C-NMR spectra were recorded in CDCl3
solution. Chemical shifts are expressed in parts per million using TMS as
an internal standard. TLC was done on Kieselgel 60 F254 aluminum
sheets. All chemicals were commercial products of analytical grade.
Commercial enzymes were purchased from Sigma–Aldrich and
Novozymes.
1[(4-Methoxybenzyl)amino]-1-oxopropan-2-yl benzoate (4c)
General Screening Procedure in Phosphate Buffer. A vinyl ester (0.5
mmol) was added to the suspension of an enzyme (40 mg) in 20 mL of
phosphate buffer (pH 7.4), followed by addition of an isocyanide (0.5
mmol). The mixture was stirred for 24 hours at room temperature.
Reaction progress was controlled by TLC using hexanes:ethyl acetate
(3:7, v/v) as an eluent. The mixture was extracted with ethyl acetate,
organic layer was separated and dried with MgSO4. Then solvent was
evaporated in vacuum. The product was purified by column
chromatography (silica gel, ethyl acetate/hexane).
White crystals; m.p. 143 °C; 1H-NMR (CDCl3, 400 MHz, ppm) δ 8.12-7.99
(m, 2H, Ar-H), 7.69-7.52 (m, 1H, Ar-H), 7.52-7.40 (m, 2H, Ar-H), 7.29-
7.16 (m, 2H, Ar-H), 6.94-6.81 (m, 2H, Ar-H), 6.38 (br. s., 1H, -CONH-),
5.51 (q, 1H, J=6.9 Hz, -OCH(CO)CH3-), 4.42 (dd, 2H, J1=5.9 Hz, J2=3.2
Hz -NHCH2Ar-), 3.78 (s, 3H, -OCH3), 1.63 (d, 3H, J=6.9 Hz, -COCH3);
13C-NMR (CDCl3, 100 MHz, ppm) δ 170.2, 165.3, 133.6, 129.9, 129.7,
129.4, 128.9, 128.6, 114.2, 71.1, 55.3, 42.7, 17.9; HR-MS (ESI, [M + H+]
calcd for: C18H19NO4Na, 336.1212, found, 336.1205; Rf = 0.65 (ethyl
acetate/hexane, 7:3 v/v).
General Screening Procedure in Organic Solvents. A vinyl ester (0.5
mmol) was added to the suspension of an enzyme (40 mg) in 20 mL of
organic solvent, followed by addition of an isocyanide (1.5 mmol) and
water (1% v/v). The mixture was stirred for 4 days at room temperature.
Reaction progress was controlled by TLC using ethyl acetate/hexane (3:7,
v/v) as an eluent. The enzyme and water was then filtered off on the
1[(4-Methoxybenzyl)amino]-1-oxopropan-2-yl laurate (4d)
White crystals; m.p. 98-99 °C; 1H-NMR (CDCl3, 400 MHz, ppm) δ 7.25-
7.19 (m, 2H, Ar-H), 7.04-7.87 (m, 2H, Ar-H), 6.33 (br. s., 1H, -CONH-),
This article is protected by copyright. All rights reserved.

10.1002/cctc.201700427
ChemCatChem
FULL PAPER
5.27 (q, 1H, J=6.9 Hz, -OCH(CO)CH3-), 4.42 (d, 2H, J=5.6 Hz, -
NHCH2Ar-), 3.82 (s, 3H, -OCH3), 2.35 (t, 2H, J=7.6 Hz, -COCH2CH2-),
1.69-1.56 (m, 2H), 1.51 (d, 3H, J=6.9 Hz, -COCH3), 1.34-1.25 (m, 16H),
0.95-0.85 (m, 3H, -CH2CH3); 13C-NMR (CDCl3, 100 MHz, ppm) δ 172.3,
170.3, 159.2,129.9, 129.0, 114.2, 70.4, 55.3, 42.7, 34.3, 31.9, 29.6, 29.4,
29.3, 29.2, 29.1, 24.9, 22.7, 17.9, 14.1; HR-MS (ESI, [M + H+] calcd for:
70.7, 39.3, 31.4, 29.5, 26.5, 22.5, 21.1, 17.9, 13.9; HR-MS (ESI, [M + H+]
calcd for: C11H21NO3Na, 238.1419, found, 238.1416; TLC: Rf = 0.63
(ethyl acetate/hexane, 3:7 v/v).
N-Hexyl-2-hydroxypropanamide (5d)
C23H37NO4Na, 414.2620, found, 414.2621; TLC: Rf
acetate/hexane, 7:3 v/v).
= 0.70 (ethyl
Colourless oil; 1H-NMR (CDCl3, 400 MHz, ppm) δ 6.68 (br. s., 1H, -
CONHCH2-), 4.18 (q, 1H, J=6.9 Hz, -HOCH(CO)CH3-), 3.58 (br. s., 1H, -
OH), 3.37-3.16 (m, 2H, -NHCH2CH2-), 1.53-1.45 (m, 2H), 1.44-1.36 (d,
3H, J=6.8 Hz, -CHCH3), 1.35-1.19 (m, 6H), 0.97-0.78 (m, 3H, -CH2CH3);
13C-NMR (CDCl3, 100 MHz, ppm) δ 174.7,68.3, 39.2, 31.4, 29.5, 26.5,
22.5, 21.3, 14.0; HR-MS (ESI, [M + H+] calcd for: C9H19NO2Na, 196.1313,
found, 196.1310; TLC: Rf = 0.30 (hexane/ethyl acetate, 3:7 v/v).
1-(Benzylamino)-1-oxopropan-2-yl acetate (4e)
White crystals; m.p. 118-119 °C; 1H-NMR (CDCl3, 400 MHz, ppm) δ 7.34-
7.17 (m, 5H, Ar-H), 6.30 (br. s., 1H, -CONH-), 5.18 (q, 1H, J=6.7 Hz, -
OCH(CO)CH3-), 4.41 (dd, 2H, J1=5.9 Hz, J2=2.0 Hz, -NHCH2Ar-), 2.10-
1.98 (m, 3H, -COCH3), 1.44 (d, 3H, J=6.9 Hz, -CHCH3); 13C-NMR (CDCl3,
100 MHz, ppm) δ 170.3, 169.4, 137.9, 128.8, 127.7, 127.6, 70.7, 43.2,
21.1, 17.9; HR-MS (ESI, [M + H+] calcd for: C12H15NO3Na, 244.0950,
found, 244.0948; TLC: Rf = 0.52 (ethyl acetate/hexane, 7:3 v/v).
Acknowledgements
This work was supported by the Polish National Science Center
project Nos. 2014/14/M/ST5/00030.
N-Benzyl-2-hydroxypropanamide (5b)
Colorless oil; 1H-NMR (CDCl3, 400 MHz, ppm) δ 7.40-7.18 (m, 5H, Ar-H),
6.69 (br. s., 1H, -CONHCH2-), 4.46 (d, 2H, J=5.9 Hz, -NHCH2Ar-), 4.27 (q,
1H, J=6.8 Hz, -HOCH(CO)CH3-), 3.23 (br. s., 1H), 1.55-1.40 (m, 3H,
J=6.9 Hz, -CHCH3); 13C-NMR (CDCl3, 100 MHz, ppm) δ 174.5, 137.9,
Keywords: tandem • Passerini reaction • enantioselective •
multicomponent reaction • enzymatic kinetic resolution
[1] E. García-Junceda, I. Lavandera, D. Rother, J. H. Schrittwieser, J. Mol.
Catal. B Enzym. 2015, 114, 1-6.
128.7, 127.7, 127.6, 68.5, 43.1, 21.3; TLC: Rf
= 0.30 (ethyl
acetate/hexane, 7:3 v/v); 2(S)-hydroxy-N-(benzyl)propanamide ((S)-5b)
was prepared from L-lactic acid (supplementary data); [α]D= -28.5 (c 1.0
CHCl3), HPLC: Chiralcel IA; hexane/isopropanol (95:5), λ = 210 nm; 1.0
mL/min, retention time of the racemic compound (in min): tR (S)= 18.5, tR
(R)= 21.7.
[2] K. C. Nicolaou, T. Montagnon, S. A. Snyder, Chem. Commun. 2003, 551-
564.
[3] A Matsuyama, H Yamamoto, Y Kobayashi, Org. Process. Res. Dev. 2002,
6, 558–561.
[4] a) J. H. Sattler, M. Fuchs, F. G. Mutti, B. Grischek, P. Engel, J. Pfeffer, J. M.
Woodley, W. Kroutil, Angew. Chem. Int. Ed. 2014, 53, 14153-14157; b)
E. Busto, R. C. Simon, N. Richter, W. Kroutil, ACS Catal. 2016, 6,
2393-2397; c) S. P. France, S. Hussain, A. M. Hill, L. J. Hepworth, R. M.
Howard, K. R. Mulholland, S. L. Flitsch, N. J. Turner, ACS Catal. 2016,
6, 3753−3759.
1-(Cyclohexylamino)-1-oxopropan-2-yl acetate (4f)
Colorless oil; 1H-NMR (CDCl3, 400 MHz, ppm) δ 5.90 (br. s., 1H, -CONH-
), 5.15 (q, 1H, J=6.9 Hz, -OCH(CO)CH3-), 3.94-3.69 (m, 1H,-
NHCH(CH2)2-), 2.26-2.05 (s, 3H, -CHCH3), 1.91 (td, 2H, J1=4.1 Hz,
J2=2.1 Hz) 1.76-1.53 (m, 4H) 1.49-1.41 (d, 3H, J=6.8 Hz), 1.40-1.29 (m,
2H), 1.20-1.12 (m, 2H); 13C-NMR (CDCl3, 100 MHz, ppm) δ 169.4, 169.3,
70.7, 47.9, 33.0, 25.5, 24.8, 21.1, 17.9; HR-MS (ESI, [M + H+] calcd for:
[5] E. Ricca, B. Brucher, J. H. Schrittwieser, Adv. Synth. Catal. 2011, 353,
2239–2262.
[6] S. F. Mayer, W. Kroutil, K. Faber, Chem. Soc. Rev. 2001, 30, 332–339
[7] a) J.-L. Wang, X.-Y. Chen, Q. Wu, X.-F. Lina, Adv. Synth. Catal. 2014, 356,
999–1005; b) L. Banfi, A. Basso, L. Moni, R. Riva, Eur. J. Org. Chem.
2014, 10, 2005-2015.
C11H19NO3Na, 236.1263, found, 236.1257; TLC: Rf
acetate/hexane, 7:3 v/v).
= 0.54 (ethyl
[8] A. M. Deobald, A. G. Correa, D. G. Rivera, M. W. Paixao, Org. Biomol.
Chem. 2012, 10, 7681-7684.
N-Cyklohexyl-2-hydroxypropanamide (5c)
[9]
J. Zhu, H. Bienaymé, Multicomponent Reactions, Wiley-VCH,
Weinheim, 2005.
Colorless oil; 1H-NMR (CDCl3, 400 MHz, ppm) δ 6.52 (br. s., 1H, -
CONHCH2-), 4.16 (m, 1H, J=6.4 Hz, HOCH(CO)CH3-), 3.81-3.58 (m,
1H,-CH2CH(NH)CH2-), 3.47 (br. s., 1H, -OH), 1.89 (d, 2H), 1.77-1.65 (m,
2H), 1.65-1.54 (m, 1H), 1.43-1.29 (m, 5H), 1.23-1.07 (m, 3H); 13C-NMR
(CDCl3, 100 MHz, ppm) δ 173.7, 68.3, 47.8, 33.0, 25.5, 24.8, 21.3; HR-
MS (ESI, [M + H+] calcd for: C9H17NO2Na, 194.1157, found, 194.1152;
TLC: Rf = 0.25 (ethyl acetate/hexanes, 7:3 v/v). N-cyklohexyl-2(S)-
hydroxypropanamide ((S)-5c) was prepared from L-lactic acid
(supplementary data); [α]D= -152.5 (c 1.0 CHCl3), HPLC: Chiralcel IA;
hexane/isopropanol (90:10), λ = 210 nm; 1.0 mL/min, retention time of
the racemic compound (in min): tR (S)= 6.0, tR (R)= 8.0.
[10] C. de Graaff , E. Ruijter, R.V.A. Orru, Chem. Soc. Rev., 2012, 41, 3969-
4009.
[11] a) D. J. Ramón, M. Yus, Angew. Chem. Int. Ed. 2005, 44, 1602–1634; b)
A. Żądło-Dobrowolska, S. Kłossowski, D. Koszelewski, D. Paprocki, R.
Ostaszewski, Chem. Eur. J. 2016, 22, 16684–16689; c) A. L.
Chandgude, A. Dömling, Org. Lett. 2016, 18, 6396–6399
[12] a) S. Kłossowski, A. Muchowicz, M. Firczuk, M. Świech, A. Redzej, J.
Gołąb, R. Ostaszewski, J. Med. Chem. 2012, 55, 55–67; b) W.
Szymański, M. Zwolińska, S. Kłossowski, I. Młynarczuk-Biały, Ł. Biały,T.
Issat, J. Malejczyk, R. Ostaszewski, Bioorg. Med. Chem. 2014, 22,
1773-1781.
[13] L. Banfi, G. Guanti, R. Riva, Chem. Commun. 2000, 985-986.
[14] O. Kreye, T. Tóth, M. A. R. Meier, J. Am. Chem. Soc. 2011, 133, 1790–
1792.
1-(Hexylamino)-1-oxopropan-2-yl acetate (4g)
Colorless oil; 1H-NMR (CDCl3, 400 MHz, ppm) δ 6.10 (br. s., 1H, -CONH-
), 5.16 (q, 1H, J=6.8 Hz, -OCH(CO)CH3-), 3.29-3.20 (m, 2H), 2.12 (s, 3H,
-COCH3), 1.61-1.47 (m, 2H), 1.44 (d, 3H, J=6.8 Hz), 1.35-1.23 (m, 6H),
0.87 (t, 3H, J=6.8 Hz) ; 13C-NMR (CDCl3, 100 MHz, ppm) δ170.2, 169.4,
[15] B. B. Touré, D. G. Hall, Chem. Rev. 2009, 109, 4439–4486.
[16] C. Lamberth, A. Jeanguenat, F. Cederbaum, A. De Mesmaeker, M. Zeller,
H.-J. Kempf, R. Zeun, Bioorg. Med. Chem. 2008, 16, 1531–1545.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201700427
ChemCatChem
FULL PAPER
[17] a) D. Paprocki, D. Koszelewski, P. Walde, R. Ostaszewski, RSC Adv.
2015, 5, 102828-102835; b) R. Riva, L. Banfi, A. Basso in Science of
Synthesis: Multicomponent Reactions, Vol. 1 (Eds.: T. J. J. Müller),
Thieme, Stuttgart, 2013, pp. 327-414.
[25] a) S. Kłossowski, A. Redzej, S. Szymkuć, R.Ostaszewski, ARKIVOC 2013,
IV, 134-143; b) D. Paprocki, D. Koszelewski, P. Walde, R. Ostaszewski,
RSC Adv. 2015, 124, 102828-102835.; c) A. Znabet, S. Blanken, E.
Janssen, F. J. J. de Kanter, M. Helliwell, N. J. Turner, E. Ruijter, R. V. A.
Orru, Org., Biomol. Chem. 2012, 10, 941-944; d) A. Znabet, E. Ruijter,
F. J. J. de Kanter, V. Köhler, M. Helliwell, N. J. Turner, R. V. A. Orru,
Angew. Chem. 2010, 122, 5417–5420; e) V. Köhler, K. R. Bailey, A.
[18] a) L. Moni, L. Banfi, A. Basso, E. Martino, R. Riva, Org. Lett. 2016, 18,
1638-1641; b) B. Alcaide, P. Almendros, C. Aragoncillo, R. Callejo, M.
Pilar Ruiz, M. Rosario Torres, J. Org. Chem. 2012, 77, 6917−6928; c)
M. Bos, E Riguet, J. Org. Chem. 2014, 79, 10881−10889; c) S.
Marcaccini, D. Miguel, T. Torroba, M. Garcia Valverde, J. Org. Chem.
2003, 68, 3315−3318; d) S. Marcaccini, D. Miguel, T. Torroba, M.
Garcia Valverde, J. Org. Chem. 2003, 68, 3315−3318.
Znabet, J.
Raftery, M.
Helliwell, N.J.
Turner, Angew.
Chem. 2010, 122, 2228–2230.
[26] a) W. Szymanski, R. Ostaszewski, Tetrahedron Aymm. 2006, 17, 2667-
2671; b) W. Szymanski, R. Ostaszewski, Tetrahedron 2008, 64, 3197-
3203; c) W. Szymanski, M. Zwolinska, R. Ostaszewski, Tetrahedron
2007, 63, 7647-7653.
[19] a) U. Kusebauch, B. Beck, K. Messer, E. Herdtweck, A. Dömling, Org.
Lett. 2003, 5, 4021–4024; b) S. Denmark, Y. Fan, J. Am. Chem. Soc.
2003, 125, 7825–7827; c) S. Denmark, Y. Fan, J. Org. Chem. 2005, 70,
9667–9676; d) P. R. Andreana, C. C. Liu, S. L. Schreiber, Org. Lett.
2004, 6, 4231−4233; e) T. Yue, M.-X. Wang, D.-X. Wang, G. Masson, J.
Zhu, J. Org. Chem. 2009, 74, 8396-8399; f) S.-X. Wang, M.-X. Wang,
D.-X. Wang, J. Zhu, Angew. Chem. 2008, 120, 394–397; g) S.-X. Wang,
M.-X. Wang, D.-X. Wang, J. Zhu, Org. Lett. 2007, 9, 3615-3618; h) T.
Yue, M.-X. Wang, D.-X. Wang, J. Zhu, Angew. Chem., Int. Ed. 2008, 47,
9554–9457; i) J. Zhang, S.-X. Lin, D.-J. Cheng, X.-Y. Liu, B. Tan, J. Am.
Chem. Soc. 2015, 137, 14039−14042; j) U. Kusebauch, B. Beck, K.
Messer, E. Herdtweck, A. Dömling, Org. Lett. 2003, 5, 4021–4024.
[20] M. Christmann, S. Bräse, Asymmetric Synthesis: More Methods and
Applications Wiley-VCH, Weinheim, 2012.
[27] C.-S. Chen, Y. Fujimoto, G. Girdaukas, C.J. Sih, J. Am. Chem. Soc. 1982,
104, 7294-7299.
[28] S. Maeda, S. Komagawa, M. Uchiyama and K. Morokuma, Angew. Chem.,
Int. Ed. 2011, 50, 644–649.
[29] E. A. Newsholme, C. Start, Regulation in metabolism, Wiley, London,
1973.
[30] a) K. R. Natarajan, J. Chem. Educ. 1991, 68, 13; b) A. M. Klibanov,
Nature 2001, 409, 241-246; c) G. Carrea, S. Riva, Angew. Chem. Int.
Ed. 2000, 39, 2226-2254.
[31] V. V. Mozhaev, Y. L. Khmelnitsky, M. V. Sergeeva, A. B. Belova, N. L.
Klyachko, A. V. Levashov, K. Martinek, Eur. J. Biochem. 1989, 184,
597-602.
[21] J. Zhou, Chem. Asian J. 2010, 5, 422–434.
[32] A. Koskinen, A. Klibanov, Enzymatic Reactions in Organic Media;
Springer, London, 1995.
[22] A. K. Cederstav, B. M. Novak, J. Am. Chem. Soc. 1994, 116, 4073–4074.
[23] U. T. Bornscheuer, T. Yamane, J. Am. Oil Chem. Soc. 1995, 72, 193-197.
[24] D. Paprocki, D. Koszelewski, P. Walde, R. Ostaszewski, RSC Adv.
2015, 5, 102828-102835.
[33] R. G. Jensen, D. R. Galluzzo, V. J. Bush, Biocatalysis 1990, 3, 307-316.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201700427
ChemCatChem
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
Layout 1:
FULL PAPER
Access to enantiomerically pure α-
hydroxy carboxamides has been
achived using a chemoenzymatic
tandem reaction involving enzyme
catalyzed hydrolysis, subsequent
Passerini reaction and enzymatic
kinetic resolution. Reported protocol
may be applied for a broad substrate
scope, including various aromatic and
aliphatic carboxylic vinyl esters and
isocyanides.
Anna Żądło-Dobrowolska, Dominik
Koszelewski, Daniel Paprocki, Arleta
Madej, Monika Wilk, Ryszard
Ostaszewski*
Page No. – Page No.
Enzyme-promoted Asymmetric
Tandem Passerni Reaction
Layout 2:
FULL PAPER
Author(s), Corresponding Author(s)*
((Insert TOC Graphic here; max. width: 11.5 cm; max. height: 2.5 cm))
Page No. – Page No.
Title
Text for Table of Contents
This article is protected by copyright. All rights reserved.