Candida antarctica lipase B (CAL-B)-catalyzed carbon-sulfur bond addition and controllable selectivity in organic media

Article abstract of DOI:10.1002/adsc.200800207

A novel enzymatic, promiscuous protocol for Candida antarctica lipase B (CAL-B)-catalyzed carbon-sulfur bond addition is described. Some control experiments have been designed to demonstrate the catalytic specificity of CAL-B. Selectivity between anti-Markovnikov addition and Markovnikov addition was achieved in different organic media. A series of thioether-containing ester functional groups was synthesized under the catalysis of CAL-B at 50°C. All the products were characterized by spectroscopic methods (IR, NMR, ESIMS).

Full text of DOI:10.1002/adsc.200800207

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DOI: 10.1002/adsc.200800207
Candida antarctica Lipase B (CAL-B)-Catalyzed Carbon-Sulfur
Bond Addition and Controllable Selectivity in Organic Media
Feng-Wen Lou,a Bo-Kai Liu,a Qi Wu,a De-Shui Lv,a and Xian-Fu Lina,*
a
Department of Chemistry, Zhejiang University, Hangzhou 310027, Peopleꢀs Republic of China
Fax : (+86)-571-8795-2618; e-mail: llc123@zju.edu.cn
Received: April 3, 2008; Revised: May 26, 2008; Published online: August 13, 2008
Supporting information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: A novel enzymatic, promiscuous protocol
for Candida antarctica lipase B (CAL-B)-catalyzed
carbon-sulfur bond addition is described. Some con-
trol experiments have been designed to demon-
strate the catalytic specificity of CAL-B. Selectivity
between anti-Markovnikov addition and Markovni-
kov addition was achieved in different organic
media. A series of thioether-containing ester func-
tional groups was synthesized under the catalysis of
CAL-B at 508C. All the products were character-
ized by spectroscopic methods (IR, NMR, ESI-
MS).
availability and easy use and recycling. It is a very ef-
fective catalyst for the synthesis and resolution of a
wide range of compounds for pharmaceuticals and in-
dustry. In recent years, CAL-B showed promiscuity in
addition reactions. For example, Gotor used CAL-B
to catalyze the Michael addition of a variety of secon-
dary amines with acrylonitrile and obtained the corre-
sponding adducts.[6] Berglund reported that the
Ser105Ala mutant of CAL-B showed catalytic ability
for the aldol reaction and the Michael addition of nu-
cleophiles to a,b-unsaturated carbonyl compounds to
[7]
À
form C C bonds.
In the present work, CAL-B was first found to be
À
Keywords: anti-Markovnikov addition; catalytic
promiscuity; enzyme catalysis; regioselectivity;
thiols
able to catalyze C S bond addition. Controllable se-
lectivity in the Markovnikov and anti-Markovnikov
addition between thiols and vinyl esters was achieved
in different organic solvents. A series of thioethers
containing ester functional groups were prepared via
À
controllable C S bond addition under the catalysis of
CAL-B.
Enzymatic synthesis in organic media has attracted an
We first examined the addition reaction of benzyl
exponentially increasing interest in the field of organ- thiol (1a) and vinyl acetate (2a) (Scheme 1) in DMF.
ic synthesis.[1] The recent progress in catalytic promis- When 0.25 mmol 1a was added to a solution contain-
cuity of enzymes has greatly extended the application ing 10 mg Candida antarctica lipase B (CAL-B) and
of biocatalysis in organic chemistry. Subtilisin not 0.3 mmol 2a in DMF at 508C, a single product was
À
only catalyzed the hydrolysis of a sulfonamide S N
bond, but also showed a remarkable activity in the
Michael addition of N-nucleophiles to acrylates.[2]
À
Acylase proved to be able to promote C N bond for-
mation via the aza-Markovnikov reaction.[3] Subse-
quent studies revealed that it also displayed catalytic
ability for the Michael addition between aromatic N-
heterocycles and a,b-unsaturated compounds.[4] Some
lipases showed their great potential application as car-
boxylic acid esterases, thioesterases, peptidases, deha-
logenases, epoxide hydrolases and halo peroxidases,
etc.[5] Thus, the exploration of enzymes with new ac-
tivities becomes particularly fascinating and remains a
great challenge.
Among all the enzymes, Candida antarctica lipase
B (CAL-B) shows the advantages of commercial esters.
Scheme 1. CAL-B-catalyzed reaction of thiols with vinyl
Adv. Synth. Catal. 2008, 350, 1959 – 1962
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1959

Feng-Wen Lou et al.
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Table 1. anti-Markovnikov addition between benzyl thiol
(1a) and vinyl acetate (2a) in the presence of different cata-
lysts.[a]
Entry Catalyst Amount
[mg]
Yield
[%][b]
V0
Vr[c]
[mMminÀ1]
1
No cata-
lyst
-
0.6
3.63
1.0
2
3
4
5
6
7
8
9
CAL-B
CAL-B
10
5
91.6
61.8
2.9
28.0
13.6
33.3
1.5
550.13
246.51
17.57
111.7
82.3
132.8
9.1
20.6
151.6
67.9
4.8
30.8
22.7
36.6
2.5
CAL-B[d] 10
AYL[e]
AL[e]
10
10
10
10
10
10
PS-C[e]
BSA[e]
CCL[e]
CRL[e]
3.4
0.9
5.7
1.5
10
5.4
[a]
Reaction conditions: benzyl thiol (0.25 mmol), vinyl ace-
tate (0.30 mmol), enzyme, DMF (1 mL), 508C for 24 h.
Conversion was calculated from the GC results.
Relative initial reaction rate to the reaction in absence of
enzyme.
CAL-B predenatured with urea at 1008C for 10 h.
AYL: lipase AY 30; AL: Amano lipase M from Mucor
javanicus; CCL: lipase from Candida cylindracea; CRL:
lipase from Candida rugosa; PS-C: immobilized lipase
from Pseudomonas cepacia.
Figure 1. Progress curves of the anti-Markovnikov addition
of benzyl thiol (1a) to vinyl acetate (2a).
[b]
[c]
prepared in 86% isolated yield after 24 h. The struc-
[d]
[e]
ture of this compound assigned as 4a was confirmed
1
13
À
by IR, H NMR, C NMR. It showed that the C S
bond addition via an anti-Markovnikov addition reac-
tion in DMF was promoted by CAL-B. The progress
curves are shown in Figure 1 and the initial reaction
rates were calculated accordingly. The reaction pro-
ceeded efficiently under the catalysis of CAL-B and ceeded 508C, the activity decreased sharply, and the
the initial reaction rate was up to 550.13 mMminÀ1. yield at 658C was only 35% (Figure 2). This illustrates
91.6% yield was obtained in 24 h as detected by GC. that the existence of organic solvents increased the
As seen from Figure 1, the initial reaction rate is prac- thermal stability of enzymes.
tically proportional to the enzyme amount, suggesting
the catalytic effect of the enzyme.
In order to improve the activity of the enzyme,
some conventional organic solvents with different log
Taking into account these results, some control ex- P values were screened for the enzymatic reaction
periments were performed focusing on the specific and the results are shown in Table 2. Besides the anti-
catalytic effect of the anti-Markovnikov reaction Markovnikov addition product 4a, Markovnikov addi-
(Table 1). The reaction of benzyl thiol with vinyl ace-
tate in the absence of enzyme led to the anti-Markov-
nikov adduct in very low yield (Entry 1, Table 1) after
24 h. In contrast, the reactions in the presence of
CAL-B were up to 100-fold faster (entry 2, Table 1).
When the reactions were incubated with denatured
CAL-B or bovine serum albumin (BSA), both the ini-
tial rates were almost equal to the background reac-
tion (entries 4 and 8, Table 1), ruling out the possibili-
ty that the similar amino acid distribution on the pro-
tein surface has promoted the process. Another five
lipases, including AYL, AL, PS-C , CCL and CRL,
cannot catalyze the reaction efficiently in DMF (en-
tries 5–7, 9, 10, Table 1). All these results suggest that
the tertiary structure and the specific spatial confor-
mation of CAL-B are responsible for the anti-Mar-
kovnikov addition reaction of benzyl thiol to vinyl
acetate. Examining the influence of temperature
showed that the anti-Markovnikov addition activity of
CAL-B increased from 158C to 508C and reached the
highest activity at 508C. When the temperature ex- anti-Markovnikov addition.
Figure 2. Effect of temperature on the CAL-B catalyzed
1960
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Candida antarctica Lipase B (CAL-B)-Catalyzed Carbon-Sulfur Bond Addition
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Table 2. CAL-B catalyzed reaction of benzyl thiol (1a) with
vinyl acetate (2a) in different solvents (log P).[a]
yield was observed after 48 h in the absence of
enzyme. Thus, DMF and diisopropyl ether were
chosen as solvents in the following experiments. It has
been reported that organic solvents could cause a sig-
nificant change in enzyme conformation. Polar sol-
vents such as DMSO could affect the unfolding mech-
anism and increase the conformational flexibility of
enzyme.[8] In non-polar solvents, the hydrophilic sur-
face of CALB decreased by 10% in comparison to
that in water, while the hydrophobic surface is slightly
increased by 1%.[9] Based on these findings, we specu-
lated that the selectivites in DMF and diisopropyl
ether may be attributed to the conformational
changes of enzyme.
The scope of this method was investigated with dif-
ferent thiols and vinyl esters. The results are summar-
ized in Table 3. In diisopropyl ether, all the addition
reactions afforded exclusively the Markovnikov
adduct in high selectivity. Addition of benzyl thiol 1a
to 2a afforded Markovnikov adducts 3a in 81% isolat-
ed yield with 99.6% conversion, and the ratio of 3a to
4a was 99.6:0.4 (Table 3, entry 2). Benzyl thiol reacted
Entry Solvent
Log P CAL-B [mg] Product
[%][b]
3a
4a
1
2
3
4
5
6
7
8
9
DMSO
DMF
À1.3 10
À1.0 10
À0.33 10
2.1
2.3
3.4
4.8
15.3 25.8
83.9 0.3
56.0 2.6
58.7. 2.7
64.1 0.9
50.7
93.1
21.3
30.3
Acetonitrile
Tetrahydrofuran
tert-Butyl alcohol
Diisopropyl ether 1.9
Toluene 2.6
Methylcyclohexane 3.7
n-Hexane 3.9
Diisopropyl ether 1.9
0.49
0.80
10
10
10
10
10
10
-
10
1.3
0.1
[a]
Reactions were carried out with 0.25 mmol of benzyl
thiol, 0.3 mmol of vinyl acetate, and CAL-B in 1.0 mL of
solvent at 508C for 48 h.
Conversion was determined by gas chromatographic
analysis, using dodecane as an internal standard.
[b]
tion product 3a was detected by GC-MS in the reac- with 2b and 2c to give adducts in moderate yields
tion system. CAL-B showed higher anti-Markovnikov with high selectivity, and the ratio of Markovnikov
addition activity in DMF and led to a 93.1% GC yield adducts 3a to anti-Markovnikov adducts 4a exceeded
after 48 h (entry 2, Table 2). In other polar solvents 98:2 (entries 4 and 6). However, the reactivity of the
such as acetonitrile, tetrahydrofuran, tert-butyl alcohol acceptor decreased as the chain length increased.
and DMSO, the yields are far from satisfactory (en- Lower yields were obtained from the vinyl esters with
tries 3–5 and entry 1, Table 1). In solvents such as longer chain (entries 2, 4 and 6). Besides the accept-
methyl cyclohexane, diisopropyl ether, toluene, or n- ors, the influence of donors on the reaction was also
hexane, the anti-Markovnikov additions proceeded investigated. Butyl mercaptan and dodecyl mercaptan
very slowly and the yields were less than 3% (en- also facilitated the Markovnikov addition for the syn-
tries 6–9, Table 2). However, the Markovnikov addi- thesis of the corresponding adducts with good selec-
tion product was generated as a major product. The tivities (entries 8 and 12). However, n-dodecanethiol
Markovnikov addition in diisopropyl ether led to a showed rather lower activity because of the strong
83.9% GC yield after 48 h. The diisopropyl ether steric hindrance. As the activities of the mercaptan
could not promote the reaction, because only 1.3% became gradually weaker, the yields of the Markovni-
Table 3. Addition between thiols 1 and vinyl esters 2 in the presence of different catalysts.[a]
Entry
1
2
Solvent
1 Conversion [%][b]
3 Yield [%][c]
4 Yield [%][c]
Selectivity[b] 3/4
1
2
3
4
5
6
7
8
1a
2a
DMF
Diisopropyl ether
DMF
Diisopropyl ether
DMF
Diisopropyl ether
DMF
Diisopropyl ether
DMF
Diisopropyl ether
DMF
95.4
99.6
87.1
91.8
70.7
80.2
87.7
92.3
82.9
75.8
48.5
53.9
2.3
83.9
3.6
75.4
5.5
68.1
0.8
73.1
1.9
64.6
0.7
93.1
0.3
83.5
0.5
65.2
0.8
86.9
0.2
81.0
3.3
47.8
1.1
2.5/97.6
99.6/0.4
4.1/95.9
99.3/0.7
8.4/92.2
98.8/1.2
0.9/99.1
99.7/0.3
2.3/97.7
95.1/4.9
1.4/98.6
97.4/2.6
1a
1a
1b
1b
1c
2b
2c
2a
2b
2a
9
10
11
12
Diisopropyl ether
41.7
[a]
Reactions were carried out on 1.0 mmol scale of thiol with 4 equiv. of vinyl acetates in 4.0 mL of diisopropyl ether at
508C for 48 h.
[b]
[c]
Conversion was determined by gas chromatographic analysis, using dodecane as an internal standard.
1
Yields refer to those of pure isolated addition products characterized by IR, H and 13C NMR.
Adv. Synth. Catal. 2008, 350, 1959 – 1962
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Feng-Wen Lou et al.
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2006C11197) and Ph.D. Programs Foundation of Ministry of
Education of China (20060335031).
kov products decreased (entries 2, 8 and 12). A simi-
lar observation was found when the reactions were
carried out in DMF. The anti-Markovnikov products
were obtained, leaving a trace of the Markovnikov
products.
In conclusion, we herein report an unprecedented
CAL-B-catalyzed C S bond addition. The catalytic
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effect of the enzyme is demonstrated by the combina-
tion of different experiments. This new addition activ-
ity of CAL-B provides a clear example of catalytic
promiscuity. It is worthy of note that the selectivity of
CAL-B can be controlled by solvents. Anti-Markovni-
kov and Markovnikov addition were achieved in
DMF and diisopropyl ether, respectively. This novel
protocol provides a potential synthetic route for b-
(substituted thio)-ethanol acetate compounds.
Experimental Section
Materials and General Methods
1H and 13C NMR spectra were recorded on a Bruker AMX-
500 MHz spectrometer in CDCl3, respectively. Chemical
shifts are reported in ppm (d), relative to the internal stan-
dard of tetramethylsilane (TMS). HR-MS were obtained on
a Bruker 7-Tesla FT-ICR MS equipped with an electrospray
source (Billelica, MA, USA). All chemicals were obtained
from commercial suppliers and used without further purifi-
cation. For all reactions dry (molecular sieve) analytical
grade solvents were used. Solvents for column chromatogra-
phy were distilled before use. The progress curves of the en-
zymatic addition were analyzed by GC. Samples were ana-
lyzed using a GC (SE-54 capillary column, FID detection;
oven temperature: from 80 to 2408C, rate of heating
208CminÀ1) and using dodecane as an internal standard. All
the compounds were characterized (IR, 1H, 13C NMR and
MS) and analytically compared (GC) with authentic samples
prepared by conventional methods. The enzymes used in-
cluded CAL-B (lipase B acrylic resin from Candida antarcti-
ca, E.C. 3.1.1.3, 10,000 U/g), AYL (lipase AY 30, E.C.
3.1.1.3), AL (Amano lipase M from Mucor javanicus, E.C.
3.1.1.3, 10 U/mg), CCL (lipase from Candida cylindracea,
E.C. 3.1.1.3, 2.08 U/mg), CRL (lipase from Candida rugosa,
E.C. 3.1.1.3, 706 U/mg), PS-C (an immobilized lipase from
Pseudomonas cepacia, E.C. 3.1.1.3, 730 U/g).
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Acknowledgements
This investigation has enjoyed financial support from the
Natural Science Foundation of China (No. 20572099),the
Zhejiang Provincial Science and Technology Council (No.
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