Highly enantioselective acylation of chlorohydrins using Amano AK lipase from P. fluorescens immobilized on silk fibroin-alginate spheres

Article abstract of DOI:10.1016/j.tetlet.2014.07.032

Aromatic, allylic, and aliphatic compounds containing a chlorohydrin group were selected as substrates for the enzymatic kinetic resolution mediated by Amano AK lipase from Pseudomonas fluorescens immobilized in silk friboin-alginate spheres. Thus, the en

Full text of DOI:10.1016/j.tetlet.2014.07.032

Accepted Manuscript
Highly enantioselective acylation of chlorohydrins using a Amano AK lipase
from P. fluorescens immobilized in silk fibroin-alginate spheres
Irlon M. Ferreira, Rodolfo H.V. Nishimura, Ana B. dos A. Souza, Giuliano C.
Clososki, Sergio A. Yoshioka, André L.M. Porto
PII:
S0040-4039(14)01185-X
DOI:
http://dx.doi.org/10.1016/j.tetlet.2014.07.032
TETL 44875
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 June 2014
7 July 2014
8 July 2014
Please cite this article as: Ferreira, I.M., Nishimura, R.H.V., Souza, B.d.A., Clososki, G.C., Yoshioka, S.A., Porto,
A.L.M., Highly enantioselective acylation of chlorohydrins using a Amano AK lipase from P. fluorescens
immobilized in silk fibroin-alginate spheres, Tetrahedron Letters (2014), doi: http://dx.doi.org/10.1016/j.tetlet.
2014.07.032
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Highly enantioselective acylation of
chlorohydrins using a Amano AK lipase
from P. fluorescens immobilized in silk
fibroin-alginate spheres
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Irlon M. Ferreira,a Rodolfo H. V. Nishimura,b Ana B. dos A. Souza,a Giuliano C. Clososki,b Sergio A.
Yoshioka,a André L. M. Porto a,*

1
Tetrahedron Letters
jo u rn al h ome p ag e : w ww .e lse vie r .c om
Highly enantioselective acylation of chlorohydrins using a Amano AK lipase
from P. fluorescens immobilized in silk fibroin-alginate spheres
Irlon M. Ferreira,a Rodolfo H. V. Nishimura,b Ana B. dos A. Souza,a Giuliano C. Clososki,b Sergio A.
Yoshioka, c André L. M. Porto a,*
aLaboratory of Biocatalysis and Organic Chemistry, Institute of Chemistry of São Carlos, University of São Paulo, Av. João Dagnone, 1100, Ed. Environmental
Chemistry, Santa Angelina, 13563-120, São Carlos-SP, Brazil.
bResearch Center for Natural and Synthetic Products, University of São Paulo, Av. do Café, S/N, 14.040-903, Ribeirão Preto-SP, Brazil.
cLaboratory of Biochemistry and Biomaterials, Institute of Chemistry of São Carlos, University of São Paulo, Av. Trabalhador São-carlense, 400, 13560-970,
São Carlos, SP, Brazil.
ARTICLE INFO
ABSTRACT
Aromatic, allylic and aliphatic compounds containing a chlorohydrin group were selected as substrates
for the enzymatic kinetic resolution mediated by a Amano AK lipase from Pseudomonas fluorescens
immobilized in silk friboin-alginate spheres. Thus, the enantioselectivity of the process was sufficient
for the production of the desired alcohols and acetates in good yields and high enantiomeric purities.
This paper provides a simple, cheap and practical protocol for enantioselective and reinforces the
versatility of silk fibroin as supports.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
2009 Elsevier Ltd. All rights reserved.
biocatalysis
enzymatic kinetic
chiral chlorohydrins
silk fibroin
Pseudomonas fluorescens
In recent decades the concept of green chemistry has intensified
the search for sustainable synthetic methodologies.1 Among the
advantages of the application of enzymes in chemical synthesis,
features such as high catalytic activity, high chemo-, regio-, and
stereoselectivity.2 However, low stability and difficulties in
recovering and maintaining enzymatic turnover have frequently
precluded the application of biocatalysts in chemical synthesis.3
As a consequence of the growing importance of the biocatalysis
in the industry, the development of techniques for stabilizing
biocatalysts has received great attention.4 Thus, immobilization
of enzymes and whole cells is nowadays accepted as a key
strategy for developing efficient and economically viable
biocatalytical processes. However, in the case of lipases,
immobilization frequently promotes the increase of catalytic
activity in organic solvents as an extra advantage.5
Studies involving the use of enzymes in the enantioselective
preparation of optically active chlorohydrins are relevant since
these compounds find a great range of applications in organic
synthesis (Figure 1).10 The kinetic resolution of racemic 2-chloro-
1-phenylethan-1-ol has been successfully achieved by Amano
lipase from P. fluorescens leading to the corresponding (S)-ester
and (R)-alcohol with 92% and 97% ee respectively. In addition, a
combination of Pseudomonas cepacia (PS-C "Amano") and a
ruthenium catalyst were used in a dynamic kinetic resolution of
aromatic chlorohydrins. Moreover, the resolution of a (R)-α-
lipoic acid chlorohydrin precursor has been achieved via lipase
catalyzed enantioselective transacylation.11
Recently, Nishimura and coworkers synthesized several
chlorohydrins through the reaction of aldehydes with the mixed
lithium-magnesium carbenoid ClCH2MgCl.LiCl.12 In this work,
we have investigated the enzymatic kinetic resolution (EKR) of
these compounds using free and silk fibroin-alginate immobilized
Amano AK lipase from Pseudomonas fluorescens. Some
representative aromatic, allylic and aliphatic chlorohydrins were
selected as targets for this study.
Despite the great importance, some biomaterials used in the
immobilization of enzymes and microorganisms are still little
studied, such as, the silk fibroin (SF), chitosan nanofibres and
chitosan.6 The silk fibroin is a known class of biopolymers
derived from cocoons of the Bombyx mori silkworm that presents
a good biodegradability and biocompatibility.7,8
Thus, due to its unique tensile strength and elasticity, good
thermal stability, hygroscopicity, and microbial resistance,9
this biomaterial presents important features for enzymes
immobilization.
Figure 1. Some synthetic applications of chiral chlorohydrins.
* Corresponding author. Tel.: +55 16 3373 8103. Fax.: +55 16 3373
9952. E-mail address: almporto@iqsc.usp.br (A.L.M. Porto)

2
Tetrahedron Letters
3
Alg-Lip
Alg
Nr
Nr
Nr
Nr
Nr
Nr
Nr
Nr
4
Time (48 h)
5
6
7
8
Free lipase
Fib-Alg-Lip
Alg-Lip
95
99
Nr
Nr
68
53
Nr
Nr
41
35
Nr
Nr
80
339
Nr
Alg
Nr
Time (96 h)
9
Free lipase
Fib-Alg-Lip
Alg-Lip
91
98
Nr
Nr
95
94
Nr
Nr
49
48
Nr
Nr
124
327
Nr
10
11
In order to investigate the catalytic behavior of the free and
immobilized Amano AK lipase from P. fluorescens in the EKR
of chlorohydrins, we have chosen the (R,S)-2-chloro-1-
phenylethan-1-ol ((R,S)-1a) as a model substrate. In these
reactions vinyl acetate was used as an irreversible acyl donor
since the leaving group (enol) spontaneously tautomerize to
acetaldehyde.
12
Alg
Nr
aGeneral conditions: Immobilized Amano AK lipase from P. fluorescens in spheres
of silk fibroin-alginate (30 mg), substrate (40 mg), vinyl acetate 75 µL, n-hexane
(1 mL), 32 °C, 300 rpm.
bcCG-FID = eeS/eeP + eeS, as defined in Ref 13
cconversion: c = eeS/eeS + eeP.
.
dE = ln[eeP(1 - eeS) /(eeP + eeS)]/ ln[eeP(1 + eeS) /(eeP + eeS)].
Nr = No reaction.
The results are summarized in Table 1. Initial experiments on the
resolution of (R,S)-1a using the free lipase from P. fluorescens
have provided the desired acetate (S)-2a in 29% conversion and
97% ee (E = 98) within 24h (Table 1, entry 1). Interestingly,
when the spheres of Fib-Alg-Lip were used under the same
reaction time, the reaction conversion dropped to 22% but the
enantiomeric excess increased to >99% (E = 262, Table 1, entry
2). In contrast, no acetate formation was observed when the
reaction was performed with Amano AK lipase from P.
fluorescens immobilized in calcium alginate (Table 1, entries 3, 7
and 11). These unexpected results could be explained by changes
on the active site of the lipase under the immobilization process.
It is worth pointing out that the immobilized material was
subsequently lyophilized favoring the deactivation of the lipase.
Additionally, no acetylated products were observed in control
experiments carried out with spheres of calcium alginate in
absence of the lipase (Table 1, entries 4, 8 and 12).
Using Fib-Alg immobilized lipase spheres, the model reaction
was optimized by varying solvent and catalyst loadings. In
addition, to determine the optimum concentration of biocatalyst,
the EKR of (R,S)-1a were performed by using different
immobilized lipase amounts (0.015, 0.02, 0.03, 0.04, and 0.05 g
Table 2). After 48 hours reaction, the maximum ee (99%) was
obtained with 0.05 g of immobilized lipase (Table 2, entry 5).
Moreover, after 96 hours reaction, the increase of the catalyst
amount from 0.03 g (Table 2, entry 8) to 0.04 g and 0.05 g (Table
2, entries 9-10) had no effect on the yield and enantiomeric
excess of the desired product since all were obtained in 49%
conversion and 98% ee.
Table 2. Effect of the biocatalyst amount on the EKR of (R,S)-1awith
immobilized Amano AK lipase from P. fluorescens in spheres of silk fibroin-
alginate a.
When the EKR of (R,S)-1a using free and Fib-Alg immobilized
Amano AK lipase from P. fluorescens were analyzed after 48h,
the enantiomeric excesses were very similar to the observed with
24h reaction (95 and 99%) but the conversion increased to 41%
and 35%, respectively. Thus, it is evident that the immobilized
biocatalyst showed higher enantioselectivity than the free lipase
when used n-hexane as solvent (E = 339 and 80, respectively),
(Table 1, entries 5 and 6).
In 96 h of reaction, the immobilized lipase converted the (R,S)-
alcohol 1a into the desired (S)-2a in 48% (ee = 98%; E = 327)
(Table 1, entry 10). A similar conversion (49%) was observed
when the lipase was used in its free from, but the selectivity was
slightly lower (ee = 91, E = 124) when compared with 24h and
48h reaction (Table 1, entry 9).
immobilized
lipase (mg)
Time (48 h)
Entry
ee (%)
(S)-2ab
(R)-1ab
c (%)c
38
Ed
1
2
3
4
5
15
20
30
40
50
99
99
99
99
99
61
36
53
66
372
282
338
396
474
27
35
40
44
78
Time (96 h)
6
7
15
20
30
40
50
99
97
98
98
98
61
94
94
96
96
38
49
49
49
49
371
234
354
392
392
8
9
10
aGeneral conditions: Immobilized Amano AK lipase from P. fluorescens on
spheres of silk fibroin-alginate (30 mg), substrate (40 mg), vinyl acetate 75 µL, n-
hexane (1 mL), 32 °C, 300 rpm.
bcCG-FID = eeS/eeP + eeS, as defined in Ref 13
cconversion: c = eeS/eeS + eeP.
dE = ln[eeP(1 - eeS) /(eeP + eeS)]/ ln[eeP(1 + eeS) /(eeP + eeS)].
Results of Table 1 suggest that immobilization of Amano AK
lipase from P. fluorescens onto spheres of silk fibroin-alginate by
encapsulation, favored the reaction selectivity in comparison with
the free enzyme.
.
Table 1. Kinetic resolution of the chlorohydrin (R,S)-1a with free and
immobilized Amano AK lipase from Pseudomonas fluorescensa.
Biocatalysis in organic solvents offers numerous advantages with
a great impact on the enzyme activity which compelled us to
study their effect on the resolution of the chlorohydrin (R,S)-1a
by the free and immobilized Amano AK lipase from P.
fluorescens. The data are summarized in Table 3. A variety of
solvents with different polarities was studied. n-Hexane was
found to be the best solvent providing the desired product in 99%
and 98% yields, respectively (Table 3, entries 1 and 7). This
result is in agreement with the fact that hydrophobic solvents
with higher log P values do not have a tendency to strip the
tightly bound water molecules from the enzyme surface which
are essential for the catalytic activity of lipases. It is well know
that in some organic solvents enzymes may suffer from reduced
O
OH
OH
O
biocatalytic free or
immobilized
Cl
Cl
Cl
n-hexane, time (h),
300 rpm
1a
(R)-
1a
(RS)-
2a
(S)-
Entry
Biocatalyst
Time (24 h)
ee (%)
(S)-2ab (R)-1ab
c (%)c
29
Ed
1
2
Free lipase
97
41
28
98
Fib-Alg-Lip
>99
22
262

3
14
activity, selectivity or stability.
In our study, this effect has
result with the one obtained for the alcohol (R,S)-1d (Table 4,
been observed when ethyl ether, DMF and DMSO were used
(Table 3). Despite that the desired product had been observed
with high enantiomeric excesses in DMF and DMSO, the
conversions were much lower, even at 96 h of reaction. When
ether was used as solvent, not conversion was observed (Table 3,
entries 3 and 9). In toluene and chloroform the conversions were
moderate when compared with n-hexane as reaction solvent
(Table 3, entries 10-11). And it can be clearly seen that both the
conversion and the enantiomeric excesses are dramatically
dependent on the solvent, for lipase immobilized on Fib-Alg. In
our study the enzymatic resolution of (R,S)-1a with ethyl ether
and diisopropyl ether were not obtained with free and
immobilized lipase from P. fluorescens (experiments performed
in duplicate). This result was unexpected, since Oda et
al.11promoted the resolution of (RS)-1a successfully using the
same free enzyme.
entry 3) suggests that the position of substituent in the aromatic
ring has a strong influence on the interaction of the substrate with
the active site of the lipase.
Finally, compounds (S)-2e-h were synthesized with 99% ee and
with enantioselectivity E >200 (Table 4, entries 4-7). The
absolute configuration of the acetates (2a-h) was assigned as S
configuration by comparison with optical rotation values
described in the literature. Consequently, it was possible to
observe that the immobilized lipase has stereochemical
preference for S-acetates esterification. Thus, the observed
esterification preference is in agreement with the predictions of
the Kazlauskas rule.15
In order to evaluate the recycling potential of the lipase
immobilized on the Fib-Alg spheres, a series of repetitive EKR
experiments was carried out using (R,S)-2-chloro-1-
phenylethanol 1a as a substrate. After each process, the catalytic
spheres were collected by filtration and used again in a new
experiment. As shown in Figure 2, the immobilized enzyme
maintained its activity, exhibiting less than 55% decay after six
cycles. The conversion value and the enantiomeric excess of
product (S)-2a stabilized after the fourth cycle in 23% and 98%
respectively.
After finding the best reaction condition for the EKR of (R,S)-1a,
we have investigated the generality and scope of this protocol
through its application on the enzymatic resolution of different
chlorohydrins. Thus, compounds (R,S)-1b-h were reacted with
the Amano AK lipase from Pseudomonas fluorescens
immobilized on beads of silk fibroin-alginate (0,03 g) in hexane
at 32 ºC (300 rpm) using vinyl acetate as an acyl donor (Table 4).
The consumption of the starting materials was monitored through
the CG-FID or HPLC analysis of reaction aliquots, collected
every 48 and 96 hours (see Experimental Section).
100
100
With the exception of compound (R,S)-1d, that appeared as a
non-reactive substrate after 240 hour reaction (Table 4, entry 3),
all other substrates were resolved with E > 200 within 120 hours
reaction. Thus, the enantioselectivity of the process was
sufficient for the production of the most desired alcohols and
acetates in good yields and enantiomeric purities. For example,
resolution of (R,S)-1b afforded the corresponding acetate (S)-1b
gave ee = 99% and 45% of conversion (Table 4, entry 1 and
Figure 2).
80
95
60
90
e
e
c
40
85
20
80
0
1
2
3
4
5
6
number of cycle
Table 3. Effect of solvents in the EKR of (R,S)-1a with immobilized Amano
AK lipase from P. fluorescens in spheres of silk fibroin-alginatea.
Figure 2. Reuse of Amano AK lipase from Pseudomonas
fluorescens immobilized in Fib-Alg in the EKR of the (R,S)-2-
chloro-1-phenylethanol (1a). Conversion (▪); Enantiomeric
excess (*)
Entry
Solvents
48 h
ee (%)
(S)-2ab
(R)-1ab
c (%)c
35
Ed
1
2
3
Hexane
DMSO
99
>99
Nr
53
4
209
207
Nr
4.3
The enzymatic kinetic resolution of chlorohydrins mediated by a
Amano AK lipase from Pseudomonas fluorescens immobilized
in fibroin-alginate spheres has allowed the enantioselective
preparation of the desired compounds with high
enantioselectivity. In some cases, the enantioselective enzymatic
transesterification also provided the expected acetates in high
enantiomeric excess (> 99%). In summary, this paper provides a
simple, cheap and practical protocol for enantioselective
synthesis of chlorohydrins and reinforces the versatility of silk
fibroin as supports for heterogeneous catalysts since it is removed
easily from the reaction mixture by simple filtration and
maintains good yields and excellent enantioselectivity after
consecutive reactions.
Ethyl ether or
Disopropyl
ether
Nr
Nr
4
5
6
Toluene
>99
>99
99
4
3
4.3
3
207
205
200
Chloroform
DMF
0.5
0.6
96 h
7
8
9
Hexane
DMSO
98
96
Nr
94
3
49
3
354
50
Ethyl ether or
Disopropyl
ether
Nr
Nr
Nr
10
11
12
Toluene
98
98
83
17
13
1
15
12
1
116
112
11
Chloroform
Acknowledgments
DMF
aGeneral conditions: Immobilized Amano AK lipase from P. fluorescens in
spheres of silk fibroin-alginate (30 mg), substrate (40 mg), vinyl acetate 75
µL, n-hexane (1 mL), 32 °C, 300 rpm.
I. M. Ferreira thanks to CNPq for scholarship. The authors would
like to acknowledge FAPESP, CAPES and CNPq for the
financial support provided to this research.
bcCG-FID = eeS/eeP + eeS, as defined in Ref 13
cconversion: c = eeS/eeS + eeP.
.
dE = ln[eeP(1 - eeS) /(eeP + eeS)]/ ln[eeP(1 + eeS) /(eeP + eeS)].
Appendix A. Supplementary data
Supplementary material (1H NMR, 13C NMR spectra, GC and
HPLC chiral chromatography) associated with this article can be
found, in the online version, at http://dx.doi.org/xxxx
Interestingly, the alcohol (R,S)-1c was converted into (S)-2c in
30% with 99% ee in 120 h (Table 4, entry 2). Comparison of this

4
Tetrahedron Letters
Table 4. Kinetic resolution of chlorohydrins (R,S)-1b-h mediated by Amano AK lipase from Pseudomonas fluorescens immobilized on silk fibroin-alginate
spheresa.
Entry
chlorohydrins
ee (%)
Ed
c(%)e
Time (h)
(S)-1
(R)-2
1b
OH
99
82
510
45
120
Cl
F
1b
(RS)-
2b
3b
4c
99
Nr
99
43
Nr
43
304
30
Nr
30
120
240
120
Nr
OH
Cl
1d
CH3 (RS)-
304
5c
99
47
317
32
120
6c
39
99
99
15
292
230
71
13
120
120
OH
Cl
1g
(RS)-
7c
OH
O
Cl
O
1h
(RS)-
aGeneral conditions: immobilized Amano AK lipase from P. fluorescens on spheres of silk fibroin-alginate (30 mg), substrate (40 mg), vinyl acetate (75 µL), n-hexane (1
mL), 32 °C, 300 rpm.
bCCG-FID = eeS/(eeP + eeS), as defined in Ref 12
cCHPLC = eeS/(eeP + eeS), as defined in Ref 12
dE = ln[eeP(1 - eeS) /(eeP + eeS)]/ ln[eeP(1 + eeS) /(eeP + eeS)].
eConversion: c = eeS/(eeS + eeP).
(Nr) = No reaction.

5
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Berlin, 2000.
Experimental procedure for kinetic resolution of chlorohydrins (R,S)-1a-h by
immobilized Amano AK lipase from P. fluorescens: To a vial flask (3 mL)
were added solvent (1 mL), vinyl acetate (0.100 mL), immobilized lipase Fib-
Alg (30 mg) and the appropriate chlorohydrins (R,S)-1a-h (40 mg). The
reaction mixture was stirred in a mechanic agitation at 300 rpm, at 32 ºC. The
progress of the reactions was followed by removing samples (30 µL of
reaction mixture were added to 600 µL of EtOAc) for GC or HPLC analyses,
according to the time indicated in Table 4. After the reaction was complete,
the immobilized lipase was filtered off. The filtrate was evaporated under
reduced pressure and purified by column chromatography on silica gel using
hexanes/EtOAc (8:2) as eluent, yielding the enantiomerically enriched (R)-
alcohols 1a-h and (S)-acetates 2a-h. Reusability of the biocatalyst: At the end
of the reaction performed with 1a compound, following above-mentioned
procedure in Supplementary material. In addition, at the end of each recycle