Lipase/acetamide-catalyzed carbon-carbon bond formations: A mechanistic view

Article abstract of DOI:10.1002/adsc.201201080

A lipase B from Candida antarctica (CALB)/acetamide-catalyzed Michael addition of less-activated ketones and aromatic nitroolefins has been developed, which is particularly interesting because neither CALB nor acetamide can independently catalyze the reaction to any appreciable extent. This co-catalyst system was applicable to the Michael additions of cyclic and acyclic ketones to a series of aromatic and heteroaromatic nitroolefins. Hydrogen bonds between acetamide and cyclohexanone were confirmed for the observed activation by experimental facts, and new mechanistic insights into CALB/acetamide co-catalysis are presented. Copyright

Full text of DOI:10.1002/adsc.201201080

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DOI: 10.1002/adsc.201201080
Lipase/Acetamide-Catalyzed Carbon-Carbon Bond Formations:
A Mechanistic View
Xiao-Yang Chen,a Guo-Jun Chen,a Jun-Liang Wang,a Qi Wu,a,* and Xian-Fu Lina,*
a
Department of Chemistry, Zhejiang University, Hangzhou 310027, Peopleꢀs Republic of China
Fax : (+86)-571-8795-2618; e-mail: wuqi1000@yahoo.com.cn or llc123@zju.edu.cn
Received: December 9, 2012; Revised: January 28, 2013; Published online: March 15, 2013
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201201080.
Abstract: A lipase B from Candida antarctica
(CALB)/acetamide-catalyzed Michael addition of
less-activated ketones and aromatic nitroolefins has
been developed, which is particularly interesting be-
cause neither CALB nor acetamide can independ-
ently catalyze the reaction to any appreciable
extent. This co-catalyst system was applicable to the
Michael additions of cyclic and acyclic ketones to
a series of aromatic and heteroaromatic nitroole-
fins. Hydrogen bonds between acetamide and cyclo-
hexanone were confirmed for the observed activa-
tion by experimental facts, and new mechanistic in-
sights into CALB/acetamide co-catalysis are pre-
sented.
were mainly restricted to the reactions between a,b-
unsaturated carbonyl compounds and activated
carbon nucleophiles such as acetylacetone, acetoace-
tates, and methyl nitroacetate. As is well known, the
proton transfer is apparently dependent on the pKa
values of carbon nucleophiles. Thus some less-activat-
ed carbon nucleophiles with high pKa values like cy-
clohexanone were difficult to add to the Michael ac-
ceptor. How to improve this kind Michael addition is
an interesting scientific subject.
In nature, some enzymes can catalyze reactions in
the presence of small molecules, like oxidoreductase
and nicotinamide adenine dinucleotide (phosphate)
hydrogen [NAD(P)H],[6] transaminase and pyridoxal
phosphate (PLP).[7] Several artificial small molecules
have been used in enzymatic reactions to improve the
activity of enzymes or to broaden the substrate specif-
icity. For instance, in laccase-catalyzed oxidation reac-
tions where the substrate has a higher redox potential
than laccase, or the substrate is too large to penetrate
into the enzyme active site, the presence of a ꢁchemi-
Keywords: acetamide; co-catalyst system; enzyme
catalysis; hydrogen bonds; Michael addition
Lipase B from Candida antarctica (CALB) belongs to cal mediatorꢀ may be used to facilitate the reaction.[8]
the folding family of a/b hydrolases. Three amino Our group reported that N-methylimidazole signifi-
acid residues, the so-called catalytic triad 187Asp- cantly improved the lipase-catalyzed acylation of riba-
224His-105Ser, play a key role in the catalytic pro- virin.[9]
cess, including normal and promiscuous[1] reactions.
Inspired by the chemical ꢁcofactor or mediatorꢀ, we
Berglund and co-workers gave the first example of expect some small molecules to similarly improve the
carbon-carbon bond formation by a CALB-catalyzed enzymatic Michael addition of less-activated carbon
aldol addition of hexanal.[2] The authors found that nucleophiles. Fortunately, herein we report that the
the mutant Serl05Ala catalyzed this reaction faster CALB/acetamide co-catalyst system can efficiently
than the wild-type enzyme. Our group reported catalyze the Michael additions between less-activated
À
a CALB catalyzed C S bond addition and controlla- ketones and aromatic nitroolefins, which is particular-
ble selectivity in organic media.[3] CALB can also cat- ly interesting because neither CALB nor acetamide
[4]
[4c]
[5]
À
À
À
alyze the formation of C N, C S, C C bonds can independently catalyze the reaction to any appre-
via Michael-type additions. A common feature of all ciable extent. Furthermore, some proof-of-principle
these reaction mechanisms comprises the activation experiments were designed to provide us with mecha-
of a carbonyl function through an oxyanion hole, nistic insights into the role of acetamide as co-cata-
meanwhile facilitating proton transfer through the lyst.
basic nitrogen of the Asp-His pair during the catalytic
We began the investigations with trans-b-nitrostyr-
process. The few successful examples of CALB-cata- ene 1a as the Michael acceptor and cyclohexanone 2a
À
lyzed C C bond formations via Michael additions as the donor ketone, which could be catalyzed by or-
864
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Adv. Synth. Catal. 2013, 355, 864 – 868

Lipase/Acetamide-Catalyzed Carbon-Carbon Bond Formations: A Mechanistic View
Table 1. Control experiments of Michael addition.[a]
proved remarkably to 56% (Table 1, entry 1). Without
doubt, acetamide in the absence of CALB cannot cat-
alyze this reaction (Table 1, entry 2). When the reac-
tants were incubated with denatured CALB, empty
carrier or bovine serum albumin (BSA) no matter
with or without acetamide, the reactions were equal
to the background, implying that the active site of
CALB was responsible for the Michael addition reac-
tion (Table 1, entries 3–5).
Entry
Catalyst
Yield [%][b]
Yield [%][c]
A variety of structurally diverse nitroolefins and
less activated mono ketones also underwent the Mi-
chael addition smoothly under the catalysis of CALB/
acetamide to afford the corresponding adducts in ac-
ceptable yields. It must be noted that almost all of
these reactions cannot be efficiently catalyzed by
CALB alone and only trace products were detected.
The results are summarized in Table 2. Both electron-
donating and electron-withdrawing functionalities on
the benzene ring of the substrate were compatible,
but were less reactive than trans-b-nitrostyrene as the
Michael acceptor in the enzymatic reaction (Table 2,
entries 1, 2, and 8–11), probably due to a sterically
hindered effect. It was observed that butanone, 3-pen-
tanone, 1-hydroxypropan-2-one and 4-methylcyclo-
hexanone could also be applied as Michael donors
1
2
3
4
CALB
–
56
N.R.
N.R.
N.R.
N.R.
N.R.
–
N.R.
N.R.
N.R.
N.R.
72
denatured CALB[d]
empty carrier
BSA
5
6[e]
CALB
[a]
Reaction conditions: 0.5 mmol 1a, 1 mL 2a, 1 mmol acet-
amide, 50 mg enzyme, 48 h.
Determined by HPLC, and in the presence of acetamide.
N.R. means no reaction.
[b]
[c]
[d]
[e]
Without acetamide.
Pre-treated with urea at 1008C for 2 h.
Reaction conditions: 0.5 mmol 1a, 1 mL 2a, 0.5 mmol
acetamide, 20 mg enzyme, 72 h.
ganocatalysts[10] and enzymes.[11] After screening a lot albeit with a relatively lower yield (Table 2, entries 1–
of organic molecules (data not shown) and enzymes 7). In addition, furylnitroalkene also could be incor-
(see Supporting Information, Table S1), we found that porated into adducts of Michael addition in moderate
CALB/acetamide had the best effect on the above re- yields (Table 2, entries 12 and 13). The CALB/acet-
À
action. Actually, CALB alone cannot catalyze this re- amide catalyst system was also efficient for other C
action (Table 1, entry 1). However, surprisingly, under C bond forming reactions to different extents of suc-
the promotion of acetamide, the yield of 3a was im- cess, for example, the CALB-catalyzed Mannich reac-
Table 2. CALB/acetamide-catalyzed Michael addition of ketones to nitroolefins.[a]
Entry
R
Ketone
Product
Yield [%][b]
1
2
3
4
5
6
7
8
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
4-NO2-C6H4
4-NO2-C6H4
4-MeO-C6H4
4-MeO-C6H5
2-furan
2-furan
cyclohexanone
acetone
butanone
3-pentanone
cyclopentanone
4-methylcyclohexanone
1-hydroxypropan-2-one
cyclohexanone
acetone
cyclohexanone
acetone
cyclohexanone
acetone
3a
3b
3c
3d
3e
3f
3g
3h
3i
72
68
33
25
28
48
27
54
39
59
43
42
28
9
10
11
12
13
3j
3k
3l
3m
[a]
Reaction conditions: 0.5 mmol 1, 1 mL 2, 0.5 mmol acetamide, 20 mg CALB, 72 h.
Determined by HPLC.
[b]
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Xiao-Yang Chen et al.
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Table 3. Study of the Mannich reaction of (E)-N-(4-nitro-
amide and formamide had the best promotion effect,
and with the larger acyl group the lower was the acti-
vation effect (Table 4, entries 1–5), which may be as-
cribed to the more difficult access of the amide with
a larger volume into the enzyme active site and less
effective cooperative binding with substrates or enzy-
matic residues. Acrylamide with a higher activation
than propionamide possibly benefited by its stronger
acidity (Table 4, entries 3 and 4). But trifluoroaceta-
mide could not catalyze the reaction regardless of its
strong acidity, probably the highly polar trifluoro-
methyl group limited the combination of the amide
group with CALBꢀs hydrophobic residues (Table 4,
entry 8). It is worth noting that the NH2 of amide
group was considered to be significant for the activa-
tion, and any substitution of the amide will cause
a lost of the activation effect (Table 4, entry 2, 6, and
7). With all these results in hand, we suspected that
acetamide possibly activated the ketone through
benzylidene)aniline with cyclohexanone.[a]
Entry
CALB
Acetamide
Yield [%][b]
1
2
3
4
À
À
+
+
À
+
À
+
N.R.
N.R.
25
38
[a]
Reaction conditions: 0.5 mmol 6a, 1 mL 2a, 1 mmol acet-
amide, 50 mg CALB, 72 h.
Determined by HPLC. N.R. means no reaction.
[b]
tion, the yield of which was increased by 50% (from forming a hydrogen bond with it in the active site of
25% to 38%) after acetamide was added as co-cata- CALB.
lyst (Table 3).
Noticing that only acetamide and formamide had
Basing on this exciting observation, we were eager a good effect on the enzymatic reaction, we suspected
to know the role of acetamide in this CALB/acet- that amide bound in the stereospecificity pocket[12] of
amide co-catalyst system and designed some experi- CALB according to its 3D structure. A series of reac-
ments to confirm it step by step. We first performed tions was designed to confirm this (see Supporting In-
the experiment using acetylacetone as Michael donor formation, Figure S1). We found that acetamide de-
instead of cyclohexanone. The yield increased only creased the rate of the CALB-catalyzed acyl-transfer
from 64% to 74% after acetamide was added (see reaction of 1-phenylethyl alcohol, in which the stereo-
Supporting Information, Table S2). Comparing both specificity pocket was necessary. Acetamide had no
reactions, we suspected that acetamide played a role influence on the acyl-transfer reaction of benzyl alco-
to activate the less-active mono ketone such as cyclo- hol, because the stereospecificity pocket was not im-
hexanone, while acetylacetone was reactive enough as portant for this reaction.
Michael donor and the activation by acetamide was
not obvious. However, further results were needed to tion promoted by CALB/acetamide was proposed
determine what kind activation it was. (Scheme 1). Firstly, nitroolefin 1a bound to the oxyan-
Thus a mechanism for the Michael addition reac-
The screening results of various amides provided us ion hole, in the mean time cyclohexanone 2a was acti-
with some information about this activation effect of vated by acetamide. Then, His224 removed one of
acetamide. From the results listed in Table 4, we the a-protons of cyclohexanone to form an enolate A
found that both aliphatic and aryl amides could pro- stabilized by two hydrogen bonds with acetamide.
mote this Michael reaction to different extents. Acet- The two substrates are then close to each other in the
active site, which can result in a nucleophilic attack of
the carbon nucleophile on the b-carbon of nitroolefin
1a to form the intermediate B. After H+ transfer, fi-
nally the product 3a could then be released from the
enzyme active site.
Table 4. Influence of amide structure.[a]
Entry
Amide
Yield [%][b]
1
The evidence from H NMR and 13C NMR experi-
1
2
3
4
5
6
7
8
formamide (4b)
acetamide (4a)
propionamide (4c)
acrylamide (4d)
benzamide (4e)
N-methylacetamide (4f)
thioacetamide (4g)
trifluoroacetamide (4h)
72
72
11
29
ments recorded in CDCl3 or DMSO-d6 also confirmed
the ability of acetamide to form hydrogen bonds with
cyclohexanone and thus support the proposed mecha-
nism. The results are shown in Table 5. By comparing
the 13C NMR spectra (CDCl3) of cyclohexanone 2a
(neat) with those of a mixture of cyclohexanone and
acetamide 4a in different molar ratios, it could be ob-
served that there is a downfield shift of the carbonyl
carbon of cyclohexanone in the mixture, for example,
the downfield shift was up to 0.20 ppm in the 1 equiv.
8
N.R.
N.R.
N.R.
[a]
Reaction conditions: 0.5 mmol 1a, 1 mL 2a, 0.5 mmol
amide, 20 mg CALB, 72 h.
Determined by HPLC. N.R. means no reaction.
[b]
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Adv. Synth. Catal. 2013, 355, 864 – 868

Lipase/Acetamide-Catalyzed Carbon-Carbon Bond Formations: A Mechanistic View
Scheme 1. The proposed mechanism.
Table 5. The NMR experiments on the cyclohexanone-acet-
amide (2a/4a) complex.[a]
would cause a significant increase in the rate of NEt3-
catalyzed Henry reactions.[13] Also, the (thio)urea/pro-
line system combined via hydrogen bonds was widely
used as a catalyst in organic reactions.[14] Our NMR
data also demonstrated the existence of the hydrogen
bonds. Therefore, the proton of the a-carbon position
of cyclohexanone was easily deprived and the Michael
addition was promoted efficiently.
Solvent
CDCl3
Sample
dC (C=O) [ppm] dH (NH) [ppm]
2a
212.189
–
2a/4a=3/1 212.180
2a/4a=2/1 212.255
2a/4a=1/1 212.383
2a/4a=1/2 212.342
5.932
5.880
5.803
5.758
5.786
–
4a
–
To the best of our knowledge, a CALB-catalyzed
asymmetric addition has never been reported, be-
DMSO-d6 2a
210.757
2a/4a=1/1 210.785
4a
6.980
6.984
cause the active site of CALB is too voluminous[5b]
.
–
Although acetamide was binding with enzymatic resi-
dues in the stereospecificity pocket during the reac-
tion process, which led to a volumetric reduction of
the active site, no enantioselectivity was found in all
products (Table 2), and the products with two chiral
[a]
1
The H and 13C NMR spectra were recorded with TMS
as internal standard.
mixture of cyclohexanone and acetamide, indicating centers (Table 2, entry 1, 3–8, 10, and 12) had a low
the formation of a hydrogen bonding network with diastereoselectivity (diastereomeric ratio=1:1–1:3 ac-
the oxygen of the carbonyl group in cyclohexanone. cording to chiral HPLC). Nevertheless, we suspected
The hydrogen bonds were broken down in the strong- that a suitable molecule in size may cause an optimis-
ly polar solvent such as DMSO-d6, thus no similar tic progress. Further studies on enzyme/small mole-
shifts were observed in the 13C NMR recorded in cule-catalyzed asymmetric reactions are still in prog-
1
DMSO-d6. We also compared the H NMR spectra ress in our laboratories.
(CDCl3) of acetamide (neat) with thoser of a mixture
In conclusion, we have developed a CALB/acet-
of acetamide and cyclohexanone in different molar amide co-catalyst system for the Michael addition of
ratios, a downfield shift of the amino protons of acet- less-activated mono ketones and nitroolefins, which is
amide was similarly observed, verifying the existence particularly interesting because neither CALB nor
of hydrogen bonds of acetamide. In addition, no simi- acetamide can independently catalyze the reaction to
lar shifts were found in the 1H NMR spectra in any appreciable extent. A wide range of less-activated
DMSO-d6. In order to further confirm the activation ketones and substituted nitroolefins can be accepted.
of cyclohexanone by the hydrogen bonds forming The activation role played by acetamide for cyclohex-
with acetamide, (N,N-d2)-acetamide (58% deutera- anone was determined as the formation of hydrogen
tion, see Supporting Information, Figure S2) was also bonds between them in the active sites, which was
synthesized and used as co-catalyst for the model Mi- supported by experimental facts. The catalytic mecha-
chael addition. An obviously decreased reaction rate nism of CALB/acetamide was postulated based on
was observed compared with the reaction under the some proof-of-principle experiments. This cooperative
catalysis of CALB/acetamide (see Supporting Infor- co-catalyst system of lipase with small organic mole-
mation, Figure S3), which indicated the existence of cules will greatly expand the application potential of
a kinetic isotope effect. In the literature it has been enzymes in organic synthesis. More case studies of en-
reported that hydrogen bonds forming with acetamide hanced enzyme/small organic molecules catalyst sys-
Adv. Synth. Catal. 2013, 355, 864 – 868
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Xiao-Yang Chen et al.
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General Procedure for the Michael Additions
Nitroolefin (0.5 mmol), acetamide (0.5 mmol), CALB
(20 mg), and ketone (1 mL) were charged in a flask and the
reaction mixture was incubated at 508C for 72 h. The reac-
tion was terminated by filtering off the enzyme. The crude
residue was purified by silica gel column chromatography
with an eluent consisting of petroleum ether/acetone (4/1
v/v). Product-containing fractions were combined, concen-
trated, and dried to afford the respective product.
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The financial support from the National Natural Science
Foundation of China (No. 21072172, 21272208) and the Zhe-
jiang Provincial Natural Science Foundation (Project No.
2010-Z4090225) is gratefully acknowledged.
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