Promiscuous acylases-catalyzed Markovnikov addition of N-heterocycles to vinyl esters in organic media

Article abstract of DOI:10.1002/adsc.200505342

Three acylases, including D-aminoacylase from Escherichia coli, acylase "Amano" from Aspergillus oryzae and immobilized penicillin G acylase from Escherichia coli have been found to possess novel activity to catalyze the Markovnikov addition reaction of N

Full text of DOI:10.1002/adsc.200505342

FULL PAPERS
DOI: 10.1002/adsc.200505342
Promiscuous Acylases-Catalyzed Markovnikov Addition of
N-Heterocycles to Vinyl Esters in Organic Media
Wei-Bo Wu, Jian-Ming Xu, Qi Wu, De-Shui Lv, Xian-Fu Lin*
Department of Chemistry, Zhejiang University, Hangzhou 310027, Peopleꢀs Republic of China
Fax: (þ86)-571-8795-2618, e-mail: llc123@css.zju.edu.cn
Received: September 4, 2005; Accepted: December 28, 2005
Supporting Information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: Three acylases, including d-aminoacylase of these promiscuous acylases, a number of N-hetero-
from Escherichia coli, acylase “Amano” from Asper- cycles, including some pentacyclic N-heterocycles,
gillus oryzae and immobilized penicillin G acylase pyrimidines and purines, were successfully added to
from Escherichia coli have been found to possess nov- a series of vinyl esters in moderate to excellent yields
el activity to catalyze the Markovnikov addition reac- to prepare N-heterocycle derivatives. The acylase-cat-
tion of N-heterocycles to vinyl esters. The aza-Mar- alyzed Markovnikov addition reaction has provided a
kovnikov addition reactions of 4-nitroimidazle to vi- new strategy to perform the Markovnikov addition
nyl acetate catalyzed by d-aminoacylase, acylase and expanded the application of biocatalysts.
“Amano” and immobilized penicillin G acylase were
up to 1260-fold, 720-fold and 320-fold faster than
the respective non-enzymatic reaction. Some control Keywords: biotransformations; catalytic promiscuity;
experiments have been designed to demonstrate the enzyme catalysis; Markovnikov addition; N-heterocy-
catalytic specificity of acylases. Under the catalysis cles
Introduction
chael-type addition of trifluorinated a,b-unsaturated
carbonyl compounds in buffer solution.[5] Our group re-
With the discoveries of highly stereoselective enzymes ported that an alkaline protease from Bacillus subtilis
with broad substrate specificity, the study of enzyme cat- showed a remarkable activity to catalyze the Michael
alysts has expanded rapidly in the last decades.[1] During addition of N-nucleophiles to acrylates in organic sol-
the exploration of new activities of enzymes, a growing vents.[6] Several elegant works have described designed
number of them have been found to be capable of cata- combinations of experiments to investigate the possible
lyzing not only their “natural” reaction but also one or catalytic mechanism of “promiscuous” lipase.[7] Besides
more alternative reactions.[2] This “catalytic promiscui- Michael addition, aldol addition reactions were ach-
ty” of enzymes has potential impact on the evolution ieved under the catalysis of an engineered mutant of
of biocatalysts since duplication of the gene, followed CAL B.[8] In order to enrich enzymatic addition reac-
by a series of mutations to amplify the desired activity, tions, exploration of enzymes with new activities be-
may generate a new enzyme now having the progeni- comes particularly fascinating and remains a great chal-
torꢀs secondary activity as the primary one.[3] The cata- lenge.
lytic promiscuity also provides new tools for organic
The Markovnikov addition is one type of useful car-
and bioorganic transformations, and thus expands the bon-carbon, oxygen-carbon or nitrogen-carbon bond-
application of enzymes.[4] Research in this area has at- forming reaction. It is especially important to synthesize
tracted much attention of chemists and biochemists in bioactive N-heterocycle derivatives with a nitrogen-car-
recent years.
bon linkage which could be achieved by an addition re-
The addition reaction is among the most fundamental action. Such aza-Markovnikov additions can be tradi-
types of reactions in organic synthesis. However, there tionally performed under harsh chemical conditions in
were only scarce reports about enzymes which are able which bases, acids and strong heating were usually
to catalyze general addition reactions. Among them, used to promote the reaction.[9] In many cases, the yields
the Michael addition has been most extensively studied. and selectivities are far from satisfactory due to the oc-
Some hydrolytic enzymes were used to catalyze the Mi- currence of several side reactions. Enzyme catalysts
Adv. Synth. Catal. 2006, 348, 487 – 492
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487

Wei-Bo Wu et al.
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with high regioselectivity and stereoselectivity have
The reaction of 4-nitroimidazle (1a) with vinyl acetate
gained recognition as favorable, environmentally be- (2a) in the absence of enzyme led to the Markovnikov
nign alternatives. adduct in very low yield (<0.4%) even after 5 days. In
Previously, we reported the unprecedented Markov- contrast, the reactions in the presence of d-aminoacy-
nikov-type addition of allopurinol to vinyl ester by pen- lase, acylase “Amano” and immobilized penicillin G
icillin G acylase from Escherichia coli (PGA).[10] In the acylase were up to 1260-fold, 720-fold and 320-fold fast-
present work, we surprisingly found that another two er, respectively (entries 2, 6 and 7, Table 1). Besides, the
zinc-binding acylases, d-aminoacylase from Escherichia initial reaction rate is practically proportional to the en-
coli and acylase “Amano” from Aspergillus oryzae, zyme amount, also suggesting the catalytic effect of the
which naturally catalyze the hydrolysis of N-acyl-d-ami- enzyme (entries 2 and 3). When the reactants were incu-
no acids and N-acyl-l-amino acids, respectively, also bated with denatured d-aminoacylase (pre-treated with
possess the promiscuous activity to catalyze the Mar- urea at 1008C for 6 hours) or bovine serum albumin
kovnikov addition of a broad range of N-heterocycles (BSA), both the initial rates were almost equal to the
to vinyl esters and exhibit even higher activities. Under background reaction (entries 4 and 8), ruling out the
the catalysis of these promiscuous acylases, a number of possibility that the similar amino acid distribution on
pentacyclic N-heterocycles, pyrimidines and purines the protein surface has promoted the process. Catalysis
were successfully added toa series of vinyl esters in mod- of the reactions by two widely used hydrolases (lipase
erate to high yields. The N-heterocycle derivatives ob- from Candida cylindracea and lipozyme) could only ac-
tained are usually pharmacologically active and may celerate the process by 11- and 4-fold, respectively (en-
be applied as potential therapeutic alternatives.[11] The tries 9 and 10). To further establish that the reaction
catalytic specificity of acylases was demonstrated by takes place in the enzymeꢀs active site, control reactions
the combination of different control experiments. The were run with inhibited d-aminoacylase by adding
influence of the structure of the N-heterocycles or vinyl 50 mM non-competitive inhibitor ZnCl2. Ligation of
esters to the enzymatic reactions has been systematical- the inhibitory zinc ion by the highly conserved residues
ly evaluated. This novel Markovnikov addition activity Asp366, His67 and His69 in the active site would lower their
of acylases is of practical significance in expanding the pKa values and/or hold the nucleophile to perturb the
application of enzymes and in the evolution of new bio- proton shuttle and intermediate stabilization.[12] The in-
catalysts.
Results and Discussion
In the light of the above observations, we designed some
control experiments to demonstrate the catalytic specif-
icity of acylases. From the data in Table 1 and Figure 1,
reaction yields and initialrateswere calculated and com-
pared.
Scheme 1. Markovnikov addition of imidazoles to vinyl es-
ters.
Table 1. Markovnikov addition between 4-nitroimidazole (1a) and vinyl acetate (2a) in the presence of different catalysts.
Entry
Catalyst
Time [h]
Yield [%][a]
V0 (mM·minÀ1
)
Vr[b]
1
2
3
4
5
6
7
8
9
No catalyst
120
72
72
96
96
72
72
96
96
96
0.4
97.3
70.8
1.9
0.17
213.96
114.03
1.03
1.0
d-Aminoacylase
d-Aminoacylase[c]
Denatured[d]
Inhibited[e]
Acylase “Amano”
Immobilized PGA
BSA
1258.6
670.8
6.1
0.7
0.32
1.9
80.4
55.2
2.2
3.5
1.3
122.57
54.89
1.18
1.85
0.71
721.0
322.9
6.9
10.9
4.2
CCL
Lipozyme
10
Conditions: 4-nitroimidazole (0.6 mmol), vinyl acetate (4 equivs.), d-aminoacylase (100 mg), DMSO (2 mL), 508C.
[a]
Values in entries 2, 3, 6 and 7 are isolated yields and others were detected by HPLC.
Relative initial reaction rate to the reaction in absence of enzyme.
25 mg/mL d-aminoacylase was used.
d-Aminoacylase predenatured with urea at 1008C for 6 h.
[b]
[c]
[d]
[e]
d-Aminoacylase inhibited by 50 mM ZnCl2.
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Markovnikov Addition of N-Heterocycles to Vinyl Esters
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the hydrolytic reactions and favor the addition reaction
and to reach higher conversions, organic solvents and ra-
tios of vinyl esters have also been established. For imida-
zole or alkylimidazoles, an organic solvent with a higher
log P value was found to better facilitate the enzymatic
reactions (entries 10–12, Table 2). Consequently, the
hydrophobic solvent hexane was employed as the reac-
tion media. For nitroimidazoles, which could only dis-
solve in a strongly polar solvent, the reactions were car-
ried out in DMSO. When the formation of products was
followed by TLC and HPLC, it is noteworthy to mention
that the enzymatic addition reaction catalyzed by all
three acylases afforded no by-products resulting from
anti-Markovnikov addition, acylation reaction, hydro-
lytic reactions or other reactions.
Generally, the Markovnikov addition of imidazoles
1a–e to a variety of vinyl esters proceeded favorably,
furnishing the corresponding products in moderate to
excellent isolated yields. The influence of the structure
of the vinyl ester was evaluated using 4-nitroimidazole
(1a) as the substrate. The enzymatic addition reactivity
Figure 1. Progress curves of the Markovnikov addition of 4-
nitroimidazole (1a) to vinyl acetate (2a) catalyzed by differ-
ent catalysts.
hibited enzyme did not show any acylase activity to cat- was found to decrease as the chain length of the vinyl es-
alyze the hydrolysis of N-acetyl-d-methionine, and the ter increased (entries 1–6), and the Markovnikov addi-
specific activity for the Markovnikov addition was the tion of more sterically hindered vinyl esters provided
same as that of non-enzymatic reaction (entry 5). All lower yields (entries 2 and 3). When the chain lengths
these results suggest that the tertiary structure and the are comparable, divinyl dicarboxylates reacted faster
specific active site of acylases are responsible for the than monoacidic vinyl esters (entries 7 and 8). The Mar-
Markovnikov addition reaction.
kovnikov addition of 4-nitroimidazole to vinyl benzoate
We then examined the generality of the enzymatic (2i) is rather slow and provided only a 36% yield of prod-
conditions to imidazoles, and the results are listed in Ta- uct even with more equivalents of vinyl benzoate after a
ble 2. We focused on d-aminoacylase as biocatalyst since prolonged reaction time of192 h (entry 9). This may be
this enzyme has demonstrated high activity toward the due to the relatively weaker nucleophilicity of aromatic
Markovnikov addition reaction. In order to prevent acid vinyl esters in comparison to fatty acid vinyl esters.
Table 2. Markovnikov addition of imidazoles with vinyl esters catalyzed by d-aminoacylase.
Entry
Substrate
Vinyl ester
R3
Solvent
Product
Time [h]
Yield[a] [%]
No.
R1
R2
[equivs.]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1a
1a
1a
1a
1a
1a
1a
1a
1a
1b
1b
1b
1b
1b
1c
1d
1e
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
H
H
H
H
H
CH3
NO2
H
2a
2b
2c
2d
2e
2f
2g
2h
2i
2a
2a
2a
2b
2g
2a
2a
2a
CH3
4
8
8
8
8
8
8
8
16
8
8
8
8
8
8
8
8
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
toluene
hexane
hexane
hexane
hexane
DMSO
hexane
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3j
3j
3k
3l
72
84
84
95
96
95
84
96
192
84
84
84
96
96
96
96
96
97
94
88
95
93
74
92
95
36
48
55
85
82
88
80
55
63
(CH2)2CH3
CH(CH3)2
(CH2)3CH3
(CH2)4CH3
(CH2)6CH3
(CH2)2COOCH CH2
(CH2)4COOCH CH2
Ph
CH3
¼
¼
CH3
CH3
(CH2)2CH3
(CH2)2COOCH CH2
CH3
CH3
CH3
¼
3m
3n
3o
Conditions: substrate (0.6 mmol), vinyl ester (relevant equivs.), d-aminoacylase (100 mg), solvent (2 mL), 508C.
[a]
Isolated yields.
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Wei-Bo Wu et al.
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To confer versatility to this enzymatic Markovnikov
reaction, more complicated N-heterocycles, such as pyr-
imidines and purines, were introduced. The analogous
reaction of uracil (4d) with vinyl acetate (2a) provided
a very low yield of the Markovnikov adduct (5%) after
À
8 days, since the N H of the amide is much less nucleo-
philic than that of the amine (entry 4, Table 3). With an
electron-withdrawing group on the heterocycle, 5-fluo-
rouracil reacted faster (entry 5). Although the acylases
showed low activity toward pyrimidines, excellent regio-
selective was achieved and single N-1 adducts were pre-
pared (also monitored by TLC and HPLC). The enzy-
matic addition of allopurinol (4f) and 6-benzylamino-
purine (4g), two purine-type heterocycles, proceeded
smoothly to give Markovnikov adducts at the N-9 posi-
tion in moderately high yields (entry 6 and 7).
Figure 2. Other N-heterocyclic substrates used for the enzy-
matic Markovnikov addition.
The influence of different substituted groups on the imi-
We also have examined the acylase-catalyzed reac-
dazole ring was also examined. Addition of 4-nitroimi- tions between N-heterocycles and vinyl ethers, but no
dazole (1a) to vinyl acetate (2a) afforded a much higher products were observed. This strongly indicated that
yield (97%, entry 1) than imidazole (85%, entry 9) in a the carboxyl group in the vinyl ester plays a significant
shorter time with less equivalents of vinyl ester, indicat- role in the enzymatic process. With all these results in
ing that an electron-withdrawing group improves the ad- hand, we propose a tentative mechanism for the en-
dition reactivity of the N-heterocycle. Oppositely, the zyme-catalytic Markovnikov addition reaction (Scheme
additions of alkylimidazoles proceeded more slowly 2). The generally accepted acylase mechanism usually
(entries 15–17). A substituted group adjacent the N-1 involves the polarization of a carbonyl group by the
position sterically hindered the addition reaction dra-
matically, giving a much lower isolated yield (entries
16 and 17).
Having obtained favorable results with imidazoles, we
then examined the addition of other pentacyclic N-het-
erocycles to vinyl acetate (2a). The results are summar-
ized in Table 3. When similar processes were carried out
with pyrrole (4a), pyrazole (4b), and 1,2,4-triazole (4c),
comparable behaviors were observed. d-Aminoacylase
showed good Markovnikov activity toward these sub-
strates, providing exclusively compounds 5 g and 5b
and 5c with moderate to high yields (entries 1–3, Ta-
ble 3). The addition reactivity of the four pentacyclic
N-heterocycles examined in our research decreases in
the order of 1,2,4-triazole (4c), imidazole (1b), pyrazole
(4b), and pyrrole (4a). These results were in accordance
with their nucleophilicity.
Scheme 2. Proposed mechanism of d-aminoacylase-catalyzed
Markovnikov addition.
Table 3. Markovnikov addition of other N-heterocycles with vinyl acetate catalyzed by d-aminoacylase.[a]
Entry
Substrate
Product
Solvent
Time [h]
Yield [%][a]
Alkylated position
1
2
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
n-hexane
n-hexane
n-hexane
DMSO
DMSO
DMSO
96
96
84
192
120
96
64
76
89
5
24
71
77
N-1
N-1
N-1
N-1
N-1
N-9
N-9
3
4[b]
5
6
7
4g
5g
DMSO
96
Conditions: substrate (0.6 mmol), vinyl acetate (8 equivs.), d-aminoacylase (100 mg), solvent (2 mL), 508C.
[a]
Isolated yields.
[b]
This was carried out on a 5-mL scale, with same concentration of reactants and enzyme.
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Markovnikov Addition of N-Heterocycles to Vinyl Esters
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zinc ion bound in the active sites of d-aminoacylase and Experimental Section
acylase “Amano” (or by the oxyanion hole of PGA). A
highly conserved Asp (or a-amino group of N-terminal
Ser B1 of PGA) plays a key role in the proton transfer
Materials and General Methods
from the nucleophile water to the leaving group.[13]
The proposed mechanism would start with the accom-
modation of an addition acceptor (vinyl ester) in the ac-
tive site. For d-aminoacylase and acylase “Amano”, the
tightly bound zinc ion interacts with the carbonyl group
of the vinyl ester and draws electron density away. Ow-
ing to the electron-withdrawing effect of the carboxyl
group, the a-carbon of the vinyl group carries a partial
positive charge. When the substrate enters the active
site, the Asp functions as a general base and extracts
the N-proton and the nucleophile simultaneously adds
to the partially positive charged C-a position. The resul-
tant negative charge at the C-b carbon could be stabi-
lized by the zinc ion. Finally, the Asp, now functioning
as a general acid, would deliver the proton to complete
the reaction. For penicillin G acylase, the oxyanion
hole might play a similar role as the zinc ion, and the
a-amino group of N-terminal Ser B1 facilitates the pro-
ton transfer.
To our surprise, none or little optical activity was ob-
served for any tested Markovnikov adducts. A similar
result was also found in the exploration of the active-
site of CAL-B for catalysis of Michael-type additions.
No enantioselectivity could be achieved in that study.[7b]
These findings reveal that the active sites might perform
the promiscuous activity in some specific way. The rea-
sons for this will be the subject of further investigations.
1H and 13C NMR spectra were recorded on a Bruker AVANCE
DMX-500 spectrometer at 500 MHz and 125 MHz in DMSO-
d6, respectively. Chemical shifts are reported in ppm (d), rela-
tive to the internal standard of tetramethylsilane (TMS). HR-
MS were obtained on a Bruker 7-tesla FT-ICR MS equipped
with an electrospray source (Billelica, MA, USA). Melting
points were determined using an XT-4 apparatus and were
not corrected. All chemicals were obtained from commercial
suppliers and used without further purification. For all reac-
tions dry (molecular sieve), analytical grade solvents were
used. Solvents for column chromatography were distilled be-
fore use. d-Aminoacylase from Escherichia coli (EC 3.5.1.81,
lyophilized powder) and acylase “Amano”from Aspergillus or-
yzae (EC 3.5.1.14, lyophilized powder) were purchased from
Amano Enzyme Inc. (Japan). Immobilized penicillin G acylase
from Escherichia coli (EC 3.5.1.11, immobilized on acrylic
beads) was purchased from Hunan Flag Biotech Co. (P. R. Chi-
na).
General Procedure for the Enzymatic Markovnikov
Addition of N-Heterocycles to Vinyl Esters
A suspension of 1a (0.6 mmol) and 100 mg d-aminoacylase in
2 mL DMSO was incubated at 508C and 200 rpm. (orbitally
shaken) for 5 minutes. Then, the corresponding equivalents
of vinyl ester (2a–i) were added in order to initiate the reac-
tion. After the indicated time (Table 1), the enzyme was fil-
tered off to terminate the reaction and washed with MeOH
(3–5 mL). Solvent was evaporated under vacuum to dryness.
The crude residue was purified by flash chromatography on
silica gel using petroleum/ethyl acetate mixtures. Product-con-
taining fractions were combined, concentrated, and dried to
give 3a–i.
Conclusion
A facile biotransformation path to perform Markovni-
kov additions between N-heterocycles and vinyl esters
has been developed by utilizing three promiscuous acy-
lases as biocatalysts. The catalytic promiscuity of the
acylases was demonstrated by the combination of differ-
ent control experiments. These acylases could specify a
broad range of N-heterocycles as addition substrates, in-
cluding pentacyclic N-heterocycles, pyrimidines and pu-
rines. The influence of the structure of the N-heterocy-
cles and vinyl ester to the enzymatic addition has been
systematically examined. The ease and mildness of this
method provides an attractive route to the synthesis of
natural and unnatural N-heterocycle derivatives which
may used as pharmacological alternatives. The complete
study of the biological activity of these new derivatives
will be reported in due course.
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