Asymmetric michael reaction catalyzed by mimicked peptides

Article abstract of DOI:10.1007/s10562-013-1156-6

Peptides mimicked from active site of promiscuous aldo-ketoreductase were synthesized and tested as asymmetry catalysts in the Michael adduct reaction of aldehydes or ketones with nitroolefins to furnish the corresponding c-nitroaldehydes, γ-nitroketones

Full text of DOI:10.1007/s10562-013-1156-6

Catal Lett (2014) 144:222–228
DOI 10.1007/s10562-013-1156-6
Asymmetric Michael Reaction Catalyzed by Mimicked Peptides
•
Saadi Bayat Bimo Ario Tejo Abu Bakar Salleh
•
•
•
Emilia Abdulmalek Yahaya M. Normi
•
Mohd Basyaruddin Abdul Rahman
Received: 23 September 2013 / Accepted: 25 October 2013 / Published online: 27 November 2013
Ó Springer Science+Business Media New York 2013
Abstract Peptides mimicked from active site of promis-
cuous aldo-ketoreductase were synthesized and tested as
asymmetry catalysts in the Michael adduct reaction of
aldehydes or ketones with nitroolefins to furnish the cor-
responding c-nitroaldehydes, c-nitroketones with up to
93 % yield, 99:1 dr and 71 % ee at room temperature and
on eco-friendly solvents. Aspartic acid residue as second
amino acid produced greater enantioselectivity.
1 Introduction
The asymmetric Michael reaction is a well known funda-
mental method for the enantioselective construction of car-
bon–carbon bonds in organic synthesis [1]. With the recent
increase in demand for optically active pharmaceutical
compounds, much progress has been made in the develop-
ment of asymmetric organocatalysts, which provides for the
preparation of Michael adducts with high enantiomeric
purity [2]. In the past decade, a lot of research has been
conducted with asymmetric organocatalysts due to it being
environmentally benign (heavy metal free), low cost, and
stability in moisture and air [3, 4]. Since the pioneering work
of List et al. in the application of the amino acid proline as a
catalyst for the aldol reaction [5], a number of short peptides
have been synthesized and employed as asymmetric organ-
ocatalysts in various organic transformations [6–8]. Peptides
are structurally diverse and have multifunctional groups, and
are therefore able to create strong hydrogen bonds with ni-
troolefins, which provide the necessary conditions to
enhance stereoselectivity [9–11]. Design and synthesis of
the artificial enzyme that is able to catalyze various asym-
metric organic reactions have long been dream of chemists.
Therefore, peptides, which have an enzyme-like structure,
are ideal asymmetric artificial enzymes [12]. Type I aldol-
ase, which works through an enamine has inspired synthetic
chemists to delve into the possibility of miniaturizing these
enzymes into short peptides [13]. The active site of aldo-
ketoreductase (AKR) is comprised of tetrad amino acids
residues, Asp47, Tyr52, Lys77 and His110, which makes it
similar to aldolase. Several peptides were envisaged to able
to asymmetrically catalyze Michael reactions via covalent
and non-covalent interactions between catalysts and sub-
strates (Fig. 1). Peptides described in Fig. 1 were derived
from the AKR active sites.
Keywords Michael adduct Á Asymmetry Á Peptides Á
Aldo-ketoreductase Á Promiscuous
Electronic supplementary material The online version of this
article (doi:10.1007/s10562-013-1156-6) contains supplementary
material, which is available to authorized users.
S. Bayat Á B. A. Tejo Á A. B. Salleh Á E. Abdulmalek Á
Y. M. Normi Á M. B. Abdul Rahman (&)
Enzyme and Microbial Technology Research Centre, Universiti
Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia
e-mail: basya@upm.edu.my
S. Bayat Á B. A. Tejo Á E. Abdulmalek Á
M. B. Abdul Rahman
Department of Chemistry, Faculty of Science, Universiti Putra
Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia
A. B. Salleh
Department of Biochemistry, Universiti Putra Malaysia,
43400 Serdang, Selangor Darul Ehsan, Malaysia
Y. M. Normi
Department of Cell and Molecular Biology, Faculty of
Biotechnology and Biomolecular Sciences, Universiti Putra
Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia
M. B. Abdul Rahman
Structural and Synthetic Biology Research Centre, Malaysia
Genome Institute, Jalan Bangi, 43000 Kajang, Selangor Darul
Ehsan, Malaysia
123

Asymmetric Michael Reaction Catalyzed by Mimicked Peptides
223
temperature for 20 min, nitrostyrene (1 eq) was introduced.
The reaction mixture was stirred at the same temperature
for 15–72 h and concentrated with rotary evaporator under
reduced pressure, followed by precipitation by EtOAc. The
precipitate was filtrated for reuse, and the EtOAc layer was
concentrated. The residue was purified through column
chromatography on silica gel by using ethylacetate:n-hex-
ane (1:3) as an eluent to produce the corresponding
Michael adducts for further analysis.
2.3 Catalyst Preparation
All peptides were used for this work (Fig. 1). Synthesis and
purification of peptides were performed according to the
Fmoc solid-phase strategy. Briefly, the peptide was man-
ually synthesized from an Fmoc-Rink-Amide-Am-Resin,
using a 3-fold molar excess of amino acid derivatives with
HCTU/DIPEA as a coupling reagent and with double
coupling cycles. At the end of the synthesis, the peptides
were separated from the cocktail by using trifluoroacetic
acid (TFA, 92.5 %), triisopropylsilane (2.5 %), 1,4 dithi-
othritol (2.5 %) and water (2.5 %). 10 ml of the mixture
was used for 1 g resin with 2 h agitation. The peptide was
precipitated in dry diethyl ether and lyophilized. The purity
of the peptide (overall, more than 93 %) was measured by
Analytical Waters HPLC (Binary HPLC pump 1525 and
UV-Waters 2489), (RPC18, Xbridge 4.6 mm 9 250 mm),
flow rate 0.5 ml/min, 220 nm.
Fig. 1 Mimicked peptides based on AKR as asymmetry catalyst
2 Experimental
2.1 General Information
All chemicals were purchased and used without further
purification. Analytical thin layer chromatography (TLC)
was performed using Merck 60 F254 precoated silica gel
plate (0.2 mm thickness). Flash chromatography was per-
formed using Merck silica gel 60 (70–230 mesh). Fourier
transform infrared spectroscopy (FT-IR); Perkin Elmer
Spectrum 100 was used for the identification of functional
groups. NMR data was recorded on 500 MHz for 1H NMR
and 100 MHz for 13C NMR (JEOL JNM ECA) spectrom-
eter. The relative and absolute configurations (dr) of the
Michael adducts were determined through comparison with
1H NMR spectroscopic analysis and HPLC with chiral
columns. Mass spectra (MS) were measured with a spec-
trometer (DIMS QP5050A SHIMADZU). Liquid Chro-
matography–Mass Spectrometry (LC–MS, Agilent).
Optical rotations were measured on a JASCO P-2000
Polarimeter. Enantioselectivity was determined by HPLC
(Waters 1525 Binary Pump and UV-Water 2489) analysis
employing a Daicel ChiralPak OD-H and AD-H columns
(4.6 mm 9 250 mm).
3 Results and Discussion
Initially, the nitro-Michael reactions of propanal with
trans-b-nitrostyrene in the presence of peptides, 1, 2 and 3
(Fig. 1) were examined. The peptides were easily prepared
from readily available amino acids using standard proce-
dures of solid phase peptide synthesis [14, 15]. Catalytic
activities of peptide 1 under various conditions were
studied. A reaction with H2O without any additives, failed,
due to the insolubility of the substrate in water and the
acidic moiety of peptide (Table 1, entry 1). For good cat-
alytic activity, both catalyst and substrate should be soluble
in the reaction media. To create a facile enamine inter-
mediate, the pH needed to be adjusted. Therefore, iPrOH
and N-methylmorpholine (NMM) were used to increase the
solubility of the substrate and pH, respectively. The best
result was obtained when the ratios of solvent and pH were
(H2O:iPrOH, 1:1) and pH 5.5–6.0 (Table 1, entries 4, 5).
These results demonstrated that high solubility of cata-
lyst and substrates in solvents led to obtain high yield and
enantioselectivity. Slightly acidic pH helps to increase
enantioselectivity (Table 1, entries 4, 5). The influence of
catalyst loading was also evaluated and it was found that
2.2 General Procedure for Asymmetric Michael
Reaction
A catalyst (5 mol%) was added to a mixture of aldehydes
2 eq and iPrOH:H2O (0.5:0.5 ml). After stirring at ambient
123

224
S. Bayat et al.
Table 1 Optimization screening of Michael adduct reaction
Entry
Cat. (mol%)
NMM (pH)
Time (h)
Solv.
Yield (%)a
ee (%)b
1
2
3
4
5
6
7
8
9
10
5
No NMM (pH 2)
pH 4
72
24
24
24
24
24
24
24
20
15
H2O
NR
60
75
87
90
94
67
65
88
88
NR
35
43
44
44
34
40
42
44
44
5
H2O/iPrOH (8/2)
H2O/iPrOH (6/4)
H2O/iPrOH (5/5)
H2O/iPrOH (5/5)
H2O/iPrOH (5/5)
H2O/iPrOH (3/7)
H2O/iPrOH (5/5)
H2O/iPrOH (5/5)
H2O/iPrOH (5/5)
5
pH 5
5
pH 5.5
pH 6
5
5
pH 7
5
pH 5,5
pH 5.5
pH 5.5
pH 5.5
3
10
15
NR no reaction, NMM N-methylmorpholine
The Michael reaction between propanal (2 eq, 0.2 mmol, 11.6 mg) and trans-b-nitrostyrene (1 eq, 0.1 mml, 14.9 mg) in the presence of 5 mol%
of peptide 1 at ambient temperature
a
Yields of isolated products after column chromatography
b
Enantioselectivities were determined for syn-product by chiral-phase HPLC analysis (Daicel Chiralpak AD-H, OD-H)
5 mol% of catalyst furnished the best results (Table 1,
entry 4). A reduction in the amount of catalyst 1 and H2O
to 3 mol% and (H2O/iPrOH, 3/7) resulted in a relatively
slow reaction and still produced 65 % yield, 42 % ee and
67 % yield, 40 % ee within 24 h (Table 1, entries 7, 8).
Therefore, the reaction conditions were optimized with
5 mol% of peptides, NMM as pH adjustor (pH 5.5–6.0) and
mixture of H2O/iPrOH (1:1) as solvents for further studies.
The scope of the reaction was probed for different aromatic
nitroolefins and Michael donors with peptide 1 as catalyst
and NMM as an additive on H2O/iPrOH (Table 2). In all
cases, reactions afforded syn products. Surprisingly, when
a longer linear chain of aldehyde was applied, the enanti-
oselectivity decreased sharply from 44 to 27 % ee (Table 2,
entries 1 and 2). It can be explained that a longer chain caused
a larger steric hindrance between the nitroolefins and catalyst
to push it to both sides of the catalyst, resulting in a lower
yield of enantioisomers. Both electron-rich and electron-
deficient nitrostyrenes were shown to be good Michael
acceptors in reacting with propanal and the reactions all
occurred smoothly in aqueous media (Table 2, entries 3–7).
Michael adducts (Table 2, entries 1–7) were obtained in good
yields (81–93 %), moderate enantioselectivities (19–44 %
ee), and high diastereoselectivities (syn/anti ratio up to 99:1).
It was observed that reactions involving nitrostyrenes bearing
electron-withdrawing substituents in the ortho position pro-
duced higher enantioselectivity, but slightly lower yields and
diastereoselectivity when substituents in the para position
were used (Table 2, entries 3, 5, 6 vs 4). A cyclic ketone
donor—cyclohexanone—was tested under the previously
optimized conditions. The reaction of cyclohexanone with
trans-b-nitrostyrene resulted in a product with 85 % yield,
41 % ee and excellent diastereoselectivity syn/anti, 99/1
(Table 2, entry 7). The absolute configuration of g (depicted
in Table 2, entry 7) was determined to be 2S, 3R by com-
paring the optical rotation with those reported elsewhere [16–
18].
The influence of peptide length on stereoselectivity
outcome was investigated. Heptapeptides, 2 and 3, which
were derived from peptide 1 were synthesized and tested
on the model reaction under the optimum conditions
(Fig. 1). The results exhibited higher yield and stereose-
lectivity (Table 3, entry 1). Although both shorter hepta-
peptides had better yield and stereoselectivity compared to
peptide 1, slightly higher stereoselectivity was observed
when peptide 3 was applied as a catalyst. The difference
between peptides 2 and 3 is that the second amino acid
residue is glutamic acid in peptide 2 and is aspartic acid in
peptide 3. The difference between two amino acids is just
one methylene (–CH2–) group. It can be concluded that
with peptide 3, the carboxylic acid side chain is close
enough to nitroolefin to make better hydrogen bonds and
nucleophile attacks on the Michael acceptor at the opposite
side easier than glutamic acid (Fig. 2).
Proline as the N-terminus secondary amino acid creates
covalent interactions via enamine intermediates and
hydrogen bonding, which facilitates keeping the substrate
on one side of the catalyst, forcing the nucleophile to attack
123

Asymmetric Michael Reaction Catalyzed by Mimicked Peptides
225
Table 2 Examples of peptide 1
Entry
1
Product
Yield (%)a
87
dr (syn/anti)b
80/20
ee (%)c
44
catalyzed nitro-Michael
additions of different
aldehyedes and ketone to
nitroolefins
2
3
88
85
81/19
80/20
27
38
4
5
93
88
95/5
19
37
80/20
The Michael reaction between
aldehydes/ketone (2 eq,
0.2 mmol) and trans-b-
nitrostyrene (1 eq, 0.1 mmol) in
the presence of 5 mol% of
peptide 1 at ambient
temperature
a
Yields of isolated products
after column chromatography
6
7
81
85
66/34
99/1
32
41
b
Enantioselectivities were
determined for syn-product by
chiral-phase HPLC analysis
(Daicel Chiralpak AD-H, OD-H)
c
Determined by 1H NMR
analysis of the crude product of
chiral-phase HPLC analysis
(Daicel Chiralpak AD-H, OD-H)
the opposite side. The hydrogen bond between a water
molecule and the amide oxygen atom of the peptide could
increase the acidity of the amide bond and strengthen the
related hydrogen bond with the nitro group of the trans-b-
nitrostyrene. The additional positive effect of water could
be ascribed to the hydrogen bonds formed between the
nitro-group and the water molecule, which at the same time
forms the hydrogen bond with the COO– group of the
catalyst, thus, stabilizing the transition state.
catalysts, 2 and 3, with good yield (80–90 %) and moderate
to good enantioselectivities (39–71 %) (Table 3, entries
1–6). Catalyst 2 however, was found to exhibit moderate
enantioselectivity and good reactivity for the a-a-disub-
stituted aldehydes and niroolefin acceptors. Catalyst 3
exhibits greater reactivity and selectivity, when compared
to catalyst 2 for the Michael reaction of a-a-disubstituted
aldehydes and niroolefins (Table 3). The reactions of
substituted nitrostyrenes and linear aldehyde, n-pentanal,
were studied. Slightly lower yields and enantioselectivities
were obtained for both catalysts. The reactions between
aldehydes and nitrostyrenes, bearing both electron-donating
The scope of the reaction included a variety of alde-
hydes as donors and ketones reacting with nitroolefins of
variable electronic characteristics in the presence of
123

226
S. Bayat et al.
Table 3 Scope and limitation studies of Michael addition between aldehydes/ketone and nitroolefins catalyzed by peptides 2 and 3
Entry
1
Product
Cat.
Yield (%)a
%dr (syn/anti)b
ee (%)a
3
2
90
87
80/20
84/16
60
57
2
3
4
3
2
90
87
87/13
90/10
51
46
3
2
88
84
73/27
74/26
45
43
3
2
86
84
60/40
60/40
64
57
4
3
2
89
85
85/15
76/24
44
39
5
6
3
2
84
80
85/15
82/18
45
44
3
2
85
82
85/15
77/23
71
55
The Michael reaction between aldehydes/ketone (2 eq, 0.2 mmol) and trans-b-nitrostyrene (1 eq, 0.1 mmol) in the presence of 5 mol% of
peptides 2 and 3 at ambient temperature
a
Yields of isolated products after column chromatography
b
Enantioselectivities were determined for syn-product by chiral-phase HPLC analysis (Daicel Chiralpak AD-H, OD-H)
1
Determined by H NMR analysis of the crude product of chiral-phase HPLC analysis (Daicel Chiralpak AD-H, OD-H)
c
and electron-withdrawing substituents produced high yields
(80–89 %), moderate enantioselectivities (39–64 %), and
high stereoselectivities (syn/anti ratio up to 85/15) for both
catalysts (Table 3, entry 5). Obtained results demonstrated
that peptide 3 produced relatively higher yield and enanti-
oselectivity. The asymmetric addition of cyclohexanone to
123

Asymmetric Michael Reaction Catalyzed by Mimicked Peptides
227
Fig. 2 Proposed transition state
mechanism for Michael addition
catalyzed by peptides 2 and 3
trans-b-nitrostyrene using 2 and 3 as catalysts, respectively,
was also investigated. Good enantioselectivity was obtained
when 3 was applied (Table 3, entry 6, 71 % ee) and mod-
erate enantioselectivity was attained for 2 (Table 3, entry 6,
55 % ee).
moderate enantioselectivity was attained for 2 (Table 3,
entry 6, 55 % ee).
4 Conclusion
Peptide 2 exhibited moderate enantioselectivity and good
reactivity for the a-a-disubstituted aldehydes and niroolefin
acceptors. In comparison, peptide 3 exhibits greater reac-
tivity and selectivity. The reactions of substituted nitrosty-
renes and linear aldehyde n-pentanal were studied. Slightly,
lower yields and enantioselectivity was obtained for both
catalysts. The reactions between aldehydes and nitrosty-
renes, bearing both electron-donating and electron-with-
drawing substituents, produced high yields (80–89 %),
moderate enantioselectivities (39–64 %), and high stere-
oselectivities (syn/anti ratio up to 85/15) for both catalysts
(Table 3, entry 5). Results demonstrated that catalyst 3
produced relatively higher yield and enantioselectivity than
catalyst 2. The asymmetric addition of cyclohexanone to
trans-b-nitro-styrene using 2 and 3 as catalysts, respectively,
was also investigated. Good enantioselectivity was obtained
when catalyst 3 was applied (Table 3, entry 6, 71 % ee) and
In summary, three mimetic peptides were designed based
on promiscuous AKR active sites. Their catalytic activities
were evaluated in the Michael adduct reactions. Based on
experimental results, heptapeptides 2 and 3 demonstrated
higher reactivity. In order to investigate the role of very
similar structures of amino acid in the catalysis 3 was
synthesized. Aspartic acid as the second amino acid in
peptide 3 exhibited better asymmetric catalytic activity in
the Michael reaction than peptide 2. Water, as an eco-
friendly solvent also increased enantioselectivity.
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