Model studies on the first enzyme-catalyzed Ugi reaction

Article abstract of DOI:10.1021/ol3033829

Multicomponent reactions are powerful tools for organic chemistry, and among them, the Ugi reaction provides remarkable improvement in many fields of organic chemistry such us combinatorial chemistry, medicinal chemistry, and peptide chemistry. A new, enzyme-catalyzed example of the Ugi three-component reaction is presented. The studies include the selection of an enzyme as well as determination of the scope and limitations of the newly described reaction. The presented method combines the enzyme promiscuity and multicomponent reaction advantages in the first one-pot formation of dipeptide 1.

Full text of DOI:10.1021/ol3033829

ORGANIC
LETTERS
2013
Vol. 15, No. 3
566–569
Model Studies on the First
Enzyme-Catalyzed Ugi Reaction
_
Szymon Kzossowski, Barbara Wiraszka, Staniszaw Berzozecki, and
Ryszard Ostaszewski*
Institute of Organic Chemistry, PAS, Kasprzaka 44/52, 01-224 Warsaw, Poland
ryszard.ostaszewski@icho.edu.pl
Received December 11, 2012
ABSTRACT
Multicomponent reactions are powerful tools for organic chemistry, and among them, the Ugi reaction provides remarkable improvement in many fields of
organic chemistry such us combinatorial chemistry, medicinal chemistry, and peptide chemistry. A new, enzyme-catalyzed example of the Ugi three-
component reaction is presented. The studies include the selection of an enzyme as well as determination of the scope and limitations of the newly described
reaction. The presented method combines the enzyme promiscuity and multicomponent reaction advantages in the first one-pot formation of dipeptide 1.
Multicomponent reactions (MCRs) have great signifi-
cance in the efficient synthesis of diverse compound
libraries.1À3 Among the MCRs, the Ugi four-component
reaction (U-4-CR) has greatly contributed to modern syn-
thetic methods.4,5 The classic Ugi reaction is a condensation
of a primary amine, a carbonyl compound, carboxylic
acid, and isocyanide. This one-pot reaction results in R-ami-
doamide with water as the only byproduct.6 Its simplicity
and one-pot nature make the reaction a remarkable tool for
many fields of chemistry such as heterocyclic chemistry,7
medicinal chemistry,8 and the chemistry of peptides and
peptydomimetics.9 Thus, interest in the further develop-
ment of the Ugi reaction and other isocyanide-based
multicomponent reactions (IMCRs) grew rapidly in recent
years.10,11 Thisled tothe discoveryofnew, significanttypes
of IMCRs. Among them, interesting examples of catalytic
Ugi reactions leading to R-aminoamides were described. In
these Ugi three-component reactions(U-3-CRs), a catalyst
is required to activate an imine A in order to form nitrilium
intermediate B (Scheme 1).
Scheme 1. Catalytic Ugi Three-Component Reaction
(1) Zhu, J., Bienayme, H. Multicomponent Reactions; Wiley-VCH:
Weinheim, 2005.
€
(2) Domling, A.; Wang, W.; Wang, K. Chem. Rev. 2012, 112, 3083–
3135.
(3) Ramon, D. J.; Yus, M. Angew. Chem., Int. Ed. 2005, 103, 17–89.
€
(4) Domling, A.; Ugi, I. Angew. Chem., Int. Ed. 2000, 39, 3168–3210.
€
(5) Domling, A. Chem. Rev. 2006, 106, 17–89.
(6) Ugi, I. Angew. Chem., Int. Ed. 1962, 1, 8–21.
(7) Zhu, J. Eur. J. Org. Chem. 2003, 1133–1144.
(8) Akritopoulou-Zanze, I. Curr. Opin. Chem. Biol. 2008, 12, 324–
331.
(9) Wessjohann, L. A.; Rhoden, C. R. B.; Rivera, D. G.; Vercillo,
O. E. Cyclic Peptidomimetics and Pseudopeptides from Multicompo-
nent Reactions. Topics In Heterocyclic Chemistry; Springer-Verlag: Ber-
lin, 2010.
Pan and List have shown that such a reaction can be
catalyzed by Brønsted acids. Among a wide spectrum of
Brønsted acids examined, phenyl phosphinic acid has been
found to be the best catalyst.12 Further studies performed
€
(10) Berkel, v. S. S.; Bogels, B. G. M.; Wijdeven, M. A.; Westermann,
B.; Rutjes, F. P. J. T. Eur. J. Org. Chem. 2012, 19, 3543–3559.
(11) Kaim, L. E.; Grimaud, L. Tetrahedron 2009, 65, 2153–2171.
(12) Pan, S. C.; List, B. Angew. Chem., Int. Ed. 2008, 47, 3622–3625.
r
10.1021/ol3033829
Published on Web 01/23/2013
2013 American Chemical Society

by other groups revealed that similar U-3-CRs can also be
catalyzed by zinc chloride (R1 = 2-hydroxyphenyl) and
sulphonated cellulose in ethanol.13,14
Our model substrates were ethyl isocyanoacetate, iso-
valeric aldehyde, and benzylamine (1 equivof each). As itis
shown in Scheme 2, in the model reaction without enzyme,
no formation of expected aminoamide 2 occurred. Also
conversion of ethyl isocyanoacetate was rather poor, even
after 4 days of stirring the substrates. On the other hand, in
the presence of Novozym 435, we observed full conversion
of ethyl isocyanoacetate after 24 h. Unexpectedly, under
these reaction conditions, we did not observe formation of
aminoamide 2. It turned out that the compound with
structure 1 was isolated as a main product (43% isolated
yield). We also observed only the slight formation of
2-isocyano-N-(phenylmethyl)ethanamide (3c). Based on
the chemical structure of the main product, we decided
to change the molar ratio of the substrates. Applying 1:2:1
to the aldehyde/amine/isocyanide ratio, the reaction yield
significantly increased up to 75% (Table 1, entry 1).
The wide spectrum of commercially available enzymes
was screenedasa biocatalyst for this reaction.22 The results
are summarized in Table 1. In general, Candida antarctica
lipase (native and immobilized) as well as Amano PS lipase
catalyzed the Ugi reaction. Among them, the best yield was
obtained when using Novozym 435 (Table 1, entry 1). We
also performed additional experiments in order to confirm
thatthe enzyme activity, not the presenceof a protein itself,
is responsible for the formation of the product 1. For this
purpose, we used inactivated CALB, but no formation of
compound 1 was observed (Table 1, entry 6).
The goal of the present paper is the presentation of
enzymes as catalysts for U-3-CRs. We examined the ability
of the lipases to catalyze the three-component Ugi reaction
by activating the imine A. Lipases are widely utilized for
numerous transformations, and they are known to exhibit
unexpected promiscuities.15À19 With regard to Ugi reac-
tions, lipases were previously used for stereoselective hy-
drolysis of glutaric anhydrides that provided in situ an
acidic substrate for U-4-CRs.20 However, to the best of our
knowledge, no Ugi reaction catalyzed by an enzyme has
been reported so far.
We present, herein, the first example of an enzyme-
catalyzed Ugi condensation of an amine, aldehyde, and iso-
cyanide which leads to formation of dipeptide 1 (Scheme 2).
Scheme 2. Enzyme-Catalyzed Ugi Reaction
The model reaction was carried out in toluene, but also
other solvents were examined.
Table 2. Solvent Influence on Enzymatic Ugi Reaction
entry
solvent
yield [%]a
Table 1. Enzymatic Screening
1
2
3
4
5
toluene
75
64
45
0
entry
enzyme
yield [%]a
chloroform
water
1
2
3
4
5
6
Novozym 435
75
41
41
15
19
0
Candida antarctica lipase (native)
Candida antarctica lipase acrylic resin
Candida cylindracea lipase
ethanol 96%
tetrahydrofuran
0
a Isolated yields.
Amano lipase PS
Candida antarctica lipase (deacivated)
a Isolated yields. Reaction conditions: amine (2 equiv), aldehyde
The results presented in Table 2 show that initially
chosen toluene is the most efficient solvent; however a
high yield was also observed when chloroform was used as
a solvent (Table 2, entry 2). Compound 1 was not formed
in ethanol, despite the fact that it is considered to be a
proper solvent for Ugi reactions (as a protic solvent). Even
more significant is the observation that the reaction can
also be perfomed in water (Table 2, entry 3). Water is often
not compatible with the Ugi reaction due to isocyanide
decomposition which occurs in the presence of acids
in aqueous media. On the other hand, it is known that
(1 equiv), isocyanide (1 equiv, c = 0.02 M), enzyme (20%).21
(13) Shaabani, A.; Keshipour, S.; Shaabani, S.; Mahyari, M. Tetra-
hedron Lett. 2012, 53, 1641–1644.
(14) Mofakham, H.; Hezarkhani, Z.; Shaabani, A. J. Mol. Catal. A
2012, 360, 26–34.
(15) Bornscheuer, U. T.; Kazlauskas, R. J. Angew. Chem., Int. Ed.
2004, 43, 6032–6040.
(16) Wang, J.-L.; Liu, B.-K.; Yin, C.; Lin, X.-F. Tetrahedron 2011,
67, 2689–2692.
(17) Branneby, C.; Carlqvist, P.; Magnusson, A.; Hult, K.; Brinck,
T.; Berglund, P. J. Am. Chem. Soc. 2003, 125, 874–875.
(18) Svedendahl, M.; Hult, K.; Berglund, P. J. Am. Chem. Soc. 2005,
127, 17988–17989.
(19) Li, K.; He, T.; Li, C.; Feng, X. W.; Wang, N.; Yu, X. Q. Green
Chem. 2009, 11, 777–779.
(21) For detailed experimental procedure, see the Supporting
Information.
(22) For detailed list of enzymes, see the Supporing Information.
(23) Faber, K. Biotransformations in Organic Chemistry; Springer-
Verlag: Berlin, 2004.
(20) Fryszkowska, A.; Frelek, J.; Ostaszewski, R. Tetrahedron 2005,
61, 6064–607221.
Org. Lett., Vol. 15, No. 3, 2013
567

Table 3. Three-Component Ugi Reaction with Various Amines
and Aldehydes
Scheme 3. Possible Mechanism of the Presented Ugi Reaction
considering the amine and aldehyde components. The
reaction occurs with either ethyl or benzyl isocyanoacetate
with similar yields (Table 3, entries 1 and 2). However, the
ester moiety is required, as compound 1 was not obtained
when the ester group was replaced with an amide moiety
(isocyanide 3c) or alkyl group (cyclohexyl isocyanide).
This observation indicates that the ester group may be
involved in enzyme acylation. Based on this conclusion, we
presented a plausibleexplanation for the enzyme-catalyzed
Ugi reaction (Scheme 3). We proposed that, in the fisrt
step, the serine residue in the catalytic triad of the enzyme is
acylated with an ester group of isocyanide. This assump-
tion is based on the classic lipase-catalyzed ester hydrolysis
mechanism.23 Then, the imminium intermediate A is acti-
vated. It is possible that the acidic Asp residue in the active
site of the enzyme is responsible for the activation of imine
A as in the classic mechanism of the Ugi reaction presented
in Scheme 1. The subsequent steps are also consistent with
the U-3-CR mechanism described before.12 An activated
electrophilic imminium intermediate undergoes isocyanide
addition leading to nitrilium adduct B. Addition of water
results in intermediate C formation, followed by isomer-
ization in adduct D. The reaction with another amine
molecule finally leads to a cleaved dipeptide 1 and free
enzyme (Scheme 3). The proposed mechanism suggests
that water addition should improve, in general, the reac-
tion yield. On the other hand, the experimental results
indicate that this influence is not the same for all the
substrates. This observation can be explained by the
change of the tertiary structure of an enzyme. It is known
a Isolated yields. b Reaction conditions: amine (2 equiv), aldehyde
mac_opb;1 equiv), isocyanide (1 equiv, c = 0.02 M), Novozym 435 (20%).
c 0.5% water addition (v/v). d The reactions were stopped after 24 h.
lipase-catalyzed reactions are often more efficient in the
interface.23 Since no acids are used in the presented reac-
tion, the addition of water should not affect the isocyanide
stability. This conclusion prompted us to investigate the
influence of water on the presented reaction.
We performed a series of enzyme-catalyzed Ugi reac-
tions with and without the addition of water. The results
are presented in Table 3. In many cases, the addition of
water improved the reaction yield (Table 3, entries 3, 4, 7,
and 8) or actually enabled the formation of product
(Table 3, entry 5). On the other hand, in the other cases,
the addition of water decreased the yield of the reaction
(Table 3, entries 1, 2, and 6). Nevertheless, the results in
Table 3 clearly show that the reaction is relatively diverse
568
Org. Lett., Vol. 15, No. 3, 2013

that the tertiary structure of enzymes in organic solvent
strongly depends on the water content.24 Therefore, for
certain substrates, such a change in protein structure in the
active site may result in a decreased reaction rate.
We observed that, under the conditions studied, the
reactions are not stereoselective and the products are
obtained as racemic mixtures.25 The lack of stereoselectiv-
ity can be explained by the relatively high reactivity of the
isocyanide moiety. Therefore, the fact that the isocyanide
addition step (Scheme 3, AfB) proceeds rapidly but
reversibly may explain the lack of stereoselectivity in the
presented reactions.
reaction proceeds in either aqueous solution or organic
solvents. This makes the reaction attractive for applied
chemistry and inprints well within the green chemistry
concept. The presented U-3-CR is relatively diverse and
can be applied to the synthesis of dipeptides with unnatural
amino acids (4f) in a single step. The products are obtained
in moderate to excellent yields.
The presented studies revealed the unique and unpre-
dictible behavior of the enzymes which, combined with
multicomponent reactions, significanctly expanded the
synthetic methodology leading to peptide scaffolds.
In summary, we presented the first example of an en-
zymatically catalyzed multicomponent Ugi reaction. The
developed procedure utilizes the enzyme promiscuity and
advantages of the multicomponent reaction for the synthe-
sis of dipeptides 1, which have never been obtained in a
one-pot procedure before. In contrast to previously de-
scribed U-3-CR procedures (heating required), the enzy-
matic U-3-CR is carried out at room temperature and is
catalyzed by commonly avaible enzymes. Furthermore, the
Supporting Information Available. Detailed experimen-
tal procedures, spectral data, and additional information
are available.This material is available free of charge via
the Internet at http://pubs.acs.org.
Acknowledgment. This work was supported by project
“Biotransformations for pharmaceutical and cosmetics
industry” No. POIG.01.03.01-00-158/09-07 partly financed
by the European Union within the European Regional
Development Fund. S. K. thanks the National Science
Center for research grant no. DEC-2011/03/N/ST5/04353.
(24) Koskinen, A. M. P.; Klibanov, A. M. Enzymatic reactions in
organic media; Springer: London, 1996.
(25) For detailed description of the stereoselectivity studies, see the
The authors declare no competing financial interest.
Supporting Information.
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