Immobilization of Amano lipase from Pseudomonas fluorescens on silk fibroin spheres: an alternative protocol for the enantioselective synthesis of halohydrins

Article abstract of DOI:10.1039/c7ra00083a

The search for a new, efficient, cheaper and sustainable matrix for lipase immobilization is a growing area in biotechnology. Amano lipase from Pseudomonas fluorescens was immobilized on silk fibroin spheres and used in the enzymatic kinetic resolution of halohydrins, to obtain optically active epoxides (up to 99% ee), important precursors in the synthesis of derivative antifungal azoles. This paper reinforces the versatility of silk fibroin as a support for heterogeneous catalysts.

Full text of DOI:10.1039/c7ra00083a

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Immobilization of Amano lipase from
Pseudomonas fluorescens on silk fibroin spheres:
an alternative protocol for the enantioselective
synthesis of halohydrins†
Cite this: RSC Adv., 2017, 7, 12650
Irlon M. Ferreira, Sergio A. Yoshioka, Joao V. Comasseto and Andre L. M. Porto*a
ab
c
de
˜
´
The search for a new, efficient, cheaper and sustainable matrix for lipase immobilization is a growing area in
biotechnology. Amano lipase from Pseudomonas fluorescens was immobilized on silk fibroin spheres and
used in the enzymatic kinetic resolution of halohydrins, to obtain optically active epoxides (up to 99% ee),
important precursors in the synthesis of derivative antifungal azoles. This paper reinforces the versatility of
silk fibroin as a support for heterogeneous catalysts.
Received 3rd January 2017
Accepted 7th February 2017
DOI: 10.1039/c7ra00083a
rsc.li/rsc-advances
In between the biomaterials used in enzymes immobiliza-
Introduction
16
tion, the most popular is cotton ber. Some studies have also
17
18,19
Enzymes are versatile biocatalysts capable of turning a wide been carried out on proteins like lectins and silk broin.
range of reactions with high selectivity, including hydrolysis, However, it is necessary to advance in the studies of enzymatic
1–4
20,21
esterication and transesterication.
In many cases, the immobilization on silk microbroin.
Silk broin is a class of
native enzymes are not ideal for commercial applications owing biopolymers obtained from the Bombyx mori silk worm cocoon
to their tendency to denaturate under harsh industrial condi- that consists in a protein of molecular weight between 300 and
5
tions and separation, recovery and reutilization difficulties.
420 kDa. Silk is a protein of a block copolymer structure
To some extent, enzyme immobilization is an effective way to dominated by large hydrophobic domains and small hydro-
overcome such limitations, since multiple xing points of the philic spacers. This primary sequence, upon folding into
support may limit undesirable conformational changes in the assembled silk structures, leads to organized crystalline
enzyme protein chains in hostile environments and the insoluble domains (b-sheets) and less organized, more exible domains
2
2,23
supports can be more easily recovered than the soluble not (more hydrated).
immobilized. Currently, a variety of supports, organics and
6
Furthermore, silk broin biomaterials are stable to moisture
inorganics has been reported for the immobilization of enzymes, and changes in temperature and mechanically robust, due to
7
8
9
for example, chitosan, cellulose, silica xerogel, or hybrid the extensive network of physical cross-links (b-sheets) formed
10,11
12
organic–inorganic,
nanoower
such as, zeolitic imidazolate, crystal during the assembly process. Such biomaterial offers some
and copper hydroxysulfate nanocrystals.
13,14
15
important features that suggest its use as a support of enzymes
due to its unique tensile strength, elasticity, good thermal
24,25
stability, hygroscopicity and microbial resistance.
Azole-derivative agents have been used in various treatments
of anti-microbial infections. The azole portion of the molecule
a
Laborat ´o rio de Qu ´ı mica Org aˆ nica e Biocat a´ lise, Instituto de Qu ´ı mica de S a˜ o Carlos,
Universidade de S a˜ o Paulo, Av. Jo a˜ o Dagnone, Ed. Qu ´ı mica Ambiental, J. Santa
Angelina, 1100, 13563-120 S a˜ o Carlos, S a˜ o Paulo, Brazil. E-mail: almporto@iqsc.
usp.br
26,27
is essential for the high antifungal activity.
The therapeutic
properties of their enantiomers differ. Therefore pure enantio-
b
mers can be administered at doses half of those used for the
Universidade Federal do Amap a´ , Grupo de Biocat a´ lise e Biotransformaç a˜ o em
Qu ´ı mica Org aˆ nica, Rod. Juscelino Kubitscheck, KM 02, S/N – J. Marco Zero, 68903- racemic drug, which suppresses minor risks of side effects and
4
c
19, Macap a´ , AP, Brazil
unspecic toxicities derived from the administration of the non-
active S-enantiomer.
We recently, investigated the enzymatic kinetic resolution
(EKR) of chlorohydrins using Amano AK Pseudomonas uo-
rescens lipase immobilized in a blend of silk broin–alginate.
Laborat ´o rio de Bioqu ´ı mica e Biomateriais, Instituto de Qu ´ı mica de S a˜ o Carlos,
Universidade de S a˜ o Paulo, Av. Trabalhador S a˜ o-Carlense, 400, 13560-970 S a˜ o
Carlos, SP, Brazil
28
d
Instituto de Qu ´ı mica, Universidade de S a˜ o Paulo, Av. Professor Lineu Prestes 748,
0
5508-000 S
a˜ o Paulo, SP, Brazil
e
Instituto de Ci ˆe ncias Ambientais, Qu ´ı micas e Farmac ˆe uticas, Universidade Federal de The enantioselectivity of the process was sufficient for the
S a˜ o Paulo, Av. Prof. Artur Riedel, 275, 09972-270 Diadema, SP, Brazil
production of the acetates in good yields and high enantiomeric
excesses.
1
13
†
Electronic supplementary information (ESI) available: H NMR, C NMR
20,21
spectra, GC chiral chromatography. See DOI: 10.1039/c7ra00083a
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(S)-2a and (S)-2b, respectively, at 48 h reaction time (entries 6
and 8, Table 1). Rhizopus niveus lipase showed no activity for the
transesterication reaction of (R,S)-1a and (R,S)-1b. The best
ꢀ
result for a free lipase was obtained with P. uorescens at 32 C,
1
30 rpm for 48 h, yielding (S)-2a and (S)-2b with excellent optical
purities (98 and 92% ee ; respectively) and good conversions (c
35 and 48%; respectively) entries 4 and 2, Table 1.
Aer screening with different lipases was decided immobi-
p
¼
Fig.
1
Structures of antifungal agents (ꢁ)-miconazole and
(ꢁ)-econazole.
lize lipase from P. uorescens on silk broin spheres and to test
the behaviour of the immobilized enzyme in reaction with (R,S)-
1
a (Table 2) (see Experimental).
The enzymatic resolution for (R,S)-1a in reactions performed
Therefore, the present paper reports on the immobilization
of commercially available Amano AK Pseudomonas uorescens
lipase in a new material prepared from silk broin to obtain
halohydrins enantio-enriched, precursors derived from azoles
with different amounts of the immobilized lipase for 24 h led to
low conversions (6%, 10% and 12% respectively), however, with
excellent enantioselectivity (E > 200) in both cases (entries 1–3,
Table 2). When the reaction carried out with 150 mg of immo-
bilized lipase remained for 48 h in 130 rpm (entry 4, Table 2), the
conversion was 26% and 99% eep with excellent enantiose-
lectivity (E ¼ >200) for (S)-2a. When the reaction time was
increased to 72 h, the conversion increased to 39% (entry 5, Table
(Fig. 1). The main purpose of the use of biodegradable broin as
an immobilization support was the development of an envi-
ronmentally benign immobilization protocol that avoids the
use of harmful chemical reagents.
2
), the enantioselectivity (E ¼ 187) and enantiomeric excess (98%
Results and discussion
ee ) for (S)-2a. The comparison of the enantioselectivity of the
p
free lipase with the immobilized form under similar reaction
conditions demonstrates the lower enantioselectivity of the
lipase in the native form. These facts suggest that immobiliza-
tion of P. uorescens lipase on silk broin spheres promotes
changes on the enzyme conformation, leading to more effective
Initially a screening with different lipases was performed to
determine the most efficient for the kinetic resolution of hal-
ohydrin. Four free lipases were used as the catalysts. The results
are summarized in Table 1.
As shown in Table 1, Candida cylindracea lipase exhibited
a low conversions (c ¼ 17–36%) and selectivity of 73–48% ee
p
for
Table 1 Screening of lipases for the kinetic resolution of (R,S)-1a and (R,S)-1b
a
b
c
p
c
s
d
Entry
Lipase
X
Time (h)
c (%)
ee
(2)
ee
(1)
E
1
2
3
4
5
6
7
8
Pseudomonas uorescens
Br
Br
Cl
Cl
Br
Br
Cl
Cl
Br
Br
Cl
Cl
Br
Br
Cl
Cl
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
35
48
28
35
23
36
14
17
Nc
0.5
Nc
2.7
4
96
92
98
98
54
48
53
73
—
75
—
17
12
12
14
51
52
88
38
53
16
27
9
15
—
0.4
—
0.5
0.5
1
0.5
11
82
70
144
168
3.9
3.7
3.5
7.4
—
7
—
1.5
1.3
1.4
7
Candida cylindracea
Rhizopus niveus
13
14
15
16
17
18
19
20
Aspergillus niger
10
3
18
3.4
a
ꢀ
b
General conditions: substrate (0.10 mmol), lipase (20 mg), vinyl acetate (0.35 mol, 30 mg), n-hexane (1 mL), 32 C, 130 rpm. Conversion: c ¼ ee
s
/
c
d
(ee
s
+ ee
p
). ee: enantiomeric excess. E ¼ ln[ee
p
(1 ꢂ ee
s p s p s p s
)]/(ee + ee )/ln[ee (1 + ee )]/(ee + ee ). Nc: no conversion.
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Table 2 Kinetic resolution of (R,S)-2-chloro-1-(phenyl)ethanol (1a) by P. fluorescens lipase immobilized on silk fibroin spheres
a
b
c
p
c
s
d
Entry
Mass of the silk broin spheres/P. uorescens lipase (mg)
Time (h)
c (%)
ee
(%) (S)-2a
ee
(%) (R)-1a
E
1
2
3
4
5
50
24
24
24
48
72
6
99
99
99
99
98
10
11
14
34
62
>200
>200
>200
>200
187
100
150
150
150
10
12
26
39
a
General conditions: Pseudomonas uorescens lipase immobilized on silk broin spheres, substrate (0.12 mmol, 20 mg), vinyl acetate 25 mL (0.25
ꢀ
b
c
d
mmol), n-hexane (1 mL), 32 C, 130 rpm. Conversion: c ¼ ee
s
/(ee
s
+ ee
p
p s p s p s
). ee: enantiomeric excess. E ¼ ln[ee (1 ꢂ ee )]/(ee + ee )/ln[ee (1 + ee )]/
(ee + ee ).
p
s
binding of one of the enantiomers to the active site of the lipase aromatic ring. The enzymatic resolution of (R,S)-2-bromo-1-(4-
with favorable formation of the acyl-enzyme intermediate. bromophenyl)ethanol (1c) with P. uorescens lipase immobi-
In order to examine the scope and limitations of the process, lized on silk broin spheres for 96 h, led to (S)-acetate 2c in 35%
the protocol was realized on scale larger and extended to conversion and 99% ee (entry 3, Table 3). For compound (R,S)-1d
p
compounds (R,S)-1a–e, with different substituent groups in the under similar conditions, (S)-acetate-2d was achieved with 24%
a
Table 3 Kinetic resolution of (R,S)-1a–e by lipase from P. fluorescens immobilized on silk fibroin spheres
b
c
p
c
s
d
Entry
Substrate
Time (h)
c (%)
ee
(%) (S)-2
ee
(%) (R)-1
E
1
72
39
19
98
98
62
23
187
120
2
3
96
96
35
24
99
99
55
32
>200
>200
4
96
5
120
Nc
(—)
(—)
(—)
a
b
c
d
Determined by chiral GC-FID analysis. Conversion: c ¼ ee
+ ee ). Nc: no conversion.
s
/(ee
s
+ ee
p
). ee: enantiomeric excess. E ¼ ln[ee
p s p s p s
(1 ꢂ ee )]/(ee + ee )/ln[ee (1 + ee )]/
(ee
p
s
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conversion and 99% ee
p
(entry 4, Table 3), whereas compound fourth cycle, 12% conversion was observed and in the h cycle
ꢀ
(R,S)-1e was recovered unchanged aer 120 h at 32 C and a conversion of only 3% was achieved.
130 rpm, what suggests the protocol is sensitive to steric effects
This decrease in the conversion of (R,S)-1a to (S)-2a may be
since 1e presents a chlorine atom at the ortho position relative to due to the detachment of the enzyme from the support, since
the organic chain bearing the secondary alcohol.
both are linked only by mechanical and physical adsorption.
The chlorohydrin acetates (S)-2a–d (Table 3) were reacted
The thermograms of the samples, lipase on broin spheres
with LiOH in ethanol for 1 h so as to yield the corresponding and free lipase, showed similar trends (ESI†). The initial weight
ꢀ
enantiomerically pure epoxides (S)-3a–c (Table 4). This loss of the samples observed around 100–110 C is due to the
approach provided styrene oxides of high ee
p
.
loss of moisture, physically adsorbed water molecules seem to
The recycling of enzymes is important in biocatalytic be eliminated. In the thermogram (A), with increasing the
ꢀ
processes, therefore the reutilization of the immobilized P. temperature, the weight residue declined at around 330 C,

uorescens lipase on silk broin spheres was tested. The results probably due to the breakdown of side chain groups of amino
(
Fig. 2) show that the enantiomeric excesses values did not acid residues related to silk broin. Similar type of improved
ꢀ
29
signicantly decrease aer 5 consecutive operations at 32 C, stability was observed by Moraes et al. for the silk broin. The
1

30 rpm and 96 h reaction time, which indicates that the thermogram (B) is related to the broin supported lipase by
ꢀ
broin–lipase couple, can retain high enantioselectivity and adsorption, besides the peak at 330 C the thermogram showed
ꢀ
catalytic performance in the recycling process. However, the mass decrease also at 285 C, suggesting alteration in the
conversion of (R,S)-1a to (S)-2a declined over successive reuses. properties of the silk broin spheres. It is noteworthy that well
The second cycle yielded approximately 22% of the alcohol; in oriented bers typically have peak thermal decomposition acim
the third cycle the conversion suffered a slight decrease, con- to 300 C, in contrast, silk broin with sheet-b not oriented tend
verted 21% of the starting (R,S)-1a in the acetate, whereas in the to have next thermal decomposition at 290 C.
ꢀ
ꢀ
29
Table 4 Ring closure of the halo-acetates (S)-2a–d to the epoxides 3a–c
a
b
c
Entry
1
Substrate
Product
Yield (%)
ee (%)
95
98
98
98
2
3
98
95
99
4
99
a
The substrate (20 mg) was dissolved in 5 mL of EtOH and then LiOH (26 mg). The reaction was quenched by addition of NaHCO
Isolated yields. ee: enantiomeric excess regarding the corresponding chlorohydrin.
3
(6 equiv.).
b
c
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Scheme 1 Synthesis of a chiral analogue derivative of an antifungal
agent.
Relative activities for lipase immobilized from P. uorescens
on silk broin spheres were calculated as the ratio of the activity
of free lipase. The immobilized lipase showed relative activity of
82%. Losses of relative activity may be associated with the
drying process of the resulting of immobilized lipase (lyophili-
zation). More researches are needed to study the improvement
of immobilization efficiency in future.
Fig. 2 Reuse of FS-LPf in the EKR of (R,S)-1a. Conversion (-);
enantiomeric excess of the (S)-2a (*).
Conclusion
In conclusion, it was developed a methodology to immobilize
The broin microspheres showed irregular shapes. We guess Pseudomonas uorescens lipase on silk broin spheres, used to
that the unevenness on the surface of the microsphere can be promote the enzymatic kinetic resolution of halohydrins,
due to presences of proteins b-sheet structures. The broin ber precursor of chiral epoxides used in the synthesis of an
presents hydrophobic groups which interact with the hydro- analogue of antifungal agents in enantiomerically pure form.
phobic groups of the lipase of P. uorescens. In this way it was The methodology is eco-friendly and efficient in the acylation of
not able to morphological difference by microscopy the halohydrins. The lipase from P. ourescens displays good
immobilized of the not immobilized enzyme (Fig. 3).
activities and high selectivity when immobilized in silk broin
The absolute conguration of the acetates (2a–d) was spheres, and reinforces the versatility of silk broin as support
assigned as S conguration by comparison with optical rotation for heterogeneous catalysis since it is easily removed from the
values described in the literature. Consequently, it was possible reaction mixture by simple ltration. In all cases, the enantio-
to observe that the esterication preference is in agreement selective enzymatic transesterication provided the expected
30
with the predictions of the Kazlauskas rule.
acetates in high enantiomeric excess (99%).
Chiral b-imidazoles alcohols are important intermediates in
organic synthesis and are present in several bioactive mole-
cules. In this connection we decided to synthesize a chiral
analogue derivative of antifungal agents containing an imid-
Experimental section
Materials
azole rest (Scheme 1) using a chiral epoxide prepared as All the enzymes, lipases from Pseudomonas uorescens ($20 000
described earlier (see Table 4), to show the synthetic potential of units per mg), Candida cylindracea (4.9 units per mg), Rhizopus
the protocol described in this paper.
niveus (4.49 units per mg) and Aspergillus niger (184 units per mg)
Fig. 3 Scanning electron micrographs of silk fibroin sphere with lipase from P. fluorescens at (A) 400ꢃ and (B) 5000ꢃ.
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were purchased from Sigma-Aldrich Chemical Company and addition of 2 mg free lipase or 20 mg immobilized lipase
ꢀ
ꢀ
stored at 4 C. Reagents (vinyl acetate, a-halobenzophenones, preparation. The mixture was incubated at 32 C in a heating
sodium borohydride, acetic anhydride, pyridine, LiOH and block under 400 rpm. Aer 5 min, the reaction was terminated
imidazole) and solvents (ethyl acetate, n-hexane, methanol, DMF, by adding 0.5 mL of Na HCO (0.5 M) followed by centrifuging
2
3
isopropanol and chloroform) were purchased from Sigma-Aldrich for 10 min at 6000 rpm. The supernatant of 0.5 mL was diluted
USA) and Synth (Brazil). CaCO and CaCl were purchased from 10-fold with deionized water, the p-NP liberated was extracted
(
3
2
Synth-Brazil. The reactions were monitored by TLC with by the aqueous alkaline phase, and the extraction was detected
aluminum plates precoated with 60 F254 silica gel, eluting with n- at 410 nm against a blank without enzyme using a UV-vis
hexane and ethyl acetate, and visualized by spraying with phos- spectrophotometer against a blank without enzyme. Molar
ꢂ1
phomolybdic acid. The cocoon of the silkworm – Bombyx mori extinction coefficient of 12.444 mmol cm for p-nitrophenol (p-
was provided by sericultor Helio Hatshugai, SP-Brazil.
NP), determined from the absorbance of standard solutions of
p-NP in the reaction medium, was used.
Instrumentation
1
13
H NMR and C NMR spectra were recorded on an Agilent
General procedure for the synthesis of (R,S)-2-halo-1-(phenyl)
ethanols (1a–e)
Technologies 500/54 or Agilent Technologies 400/54 Premium
Shielded spectrometer using CDCl3 or CD OD as solvent and
3
Were added to a round-bottomed ask (100 mL), prior to small
portions of 2-chloro-1-phenylethanone (2.5 mmol, 0.498 g),
TMS as the internal standard. The chemical shis are given
in ppm and the coupling constants (J) in Hz. The data are re-
ported as follows: chemical shi (d), multiplicity (s ¼ singlet, d ¼
doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet) and integrated
4
methanol (25 mL), followed by the addition of NaBH (3 mmol,
ꢀ
0
1
.114 mg). The mixture was stirred for 30 min at 0 C and for
.5 h at room temperature. Methanol was evaporated under
1
3
intensity. The C NMR chemical shis are reported in ppm
reduced pressure and HCl (1 mL, 10%) was added to the
3
relative to the CDCl signal. FTIR spectra were recorded on
residue. The mixture was then extracted with ethyl acetate (3 ꢃ
a SHIMADZU IRAffinity-1 spectrometer samples were prepared as
thin lms on KBr disks (solid samples) or liquid lm (liquid
2 4
20 mL) and the organic phase was dried over Na SO and
evaporated under reduced pressure. The crude product was
puried by gel silica column chromatography with a mixture of
n-hexane and ethyl acetate (7 : 3) as eluent. The same procedure
was employed for other (R,S)-2-halo-1-(phenyl)ethanols (1b–e).
All spectral data of compounds 1a–e were in agreement with
ꢂ1
samples) in the 4000–400 cm region. Enzymatic kinetic reso-
lutions were carried out in a Tecnal TE-421 orbital shaker and
enzymatic reactions were analyzed in a Shimadzu GC 2010 gas
chromatographer equipped with an AOC 20i auto injector,
a ame ionization detector (FID), and a Varian chiral column CP-
Chiralsil-DEX (b-cyclodextrin) (25 m ꢃ 0.25 mm ꢃ 0.39 mm).
The chromatographic conditions employed were: carrier
those described in the literature (ESI†).
ꢂ1
2
-Chloro-1-phenylethanol (1a). C H ClO, 156.03 g mol
.
8
9
1
Yield 0.032 g (81%). Yellow oil; H NMR (400 MHz, CDCl
3
ppm)
nitrogen gas at 69.2 kPa, split ratio ¼ 1 : 10, injection volume ¼
d: 3.71 (dd, J ¼ 11.7 and 4.6 Hz, 1H), 3.78 (dd, J ¼ 11.7 and 8.0
ꢀ
1
2
.0 mL, injector temperature ¼ 250 C, detector temperature ¼
1
3
Hz, 1H), 5.95 (dd, J ¼ 8.0 and 4.6 Hz, 1H), 7.37–7.32 (m, 5H);
NMR (100 MHz, CDCl ) d (ppm): 50.9, 74, 126, 128.4, 128.6,
39.9; MS (EI, 70 eV) m/z (%): 156 (11), 107 (100), 79 (77), 51 (17);
C
ꢀ
ꢀ
50 C, oven temperature program initially at 120 C for 2 min
ꢀ
ꢂ1
ꢀ
3
and increased at a rate of 2 C min until 165 C for 8 min, total
time of analysis ¼ 32.5 min. The retention times for the (R,S)-
alcohols obtained were: 1a, (R) ¼ 20.5 min and (S) ¼ 19.8 min; 1b,
1
ꢂ1
IRvmax (cm ): 3404, 2956, 1494, 1064, 725.
-Bromo-1-phenylethanol (1b). C H BrO, 199.98 g mol
Yield 0.044 g (89%). Yellow oil; H NMR (400 MHz, CDCl ppm) d:
ꢂ1
2
.
8
9
(
2
R) ¼ 13.8 min and (S) ¼ 13.5 min; 1c (R) ¼ 28 min and (S) ¼
1
3
7.5 min; 1d, (R) ¼ 28.5 min and (S) ¼ 27.9 min; the retention
1
.57 (s, 1H), 3.48 (dd, J ¼ 10.5 and 8.8 Hz, 1H), 3.60 (dd, J ¼ 10.5
times for the (R,S)-acetates were: 2a, (R) ¼ 16.8 min and (S) ¼
and 3.4 Hz, 1H), 4.89 (dd, J ¼ 8.8 and 3.2 Hz, 1H), 7.25–7.46 (m,
1
6.1 min; 2b, (R) ¼ 10.7 min and (S) ¼ 10.1 min; 2c, (R) ¼ 18.3 min
1
3
3
5H); C NMR (100 MHz, CDCl ppm) d: 39.9, 73.1, 127.3, 128.8,
and (S) ¼ 18.6 min; 2d, (R) ¼ 23 min and (S) ¼ 22.5 min. SEM
1
34.2, 138.7; MS (EI, 70 eV) m/z (%): 200 (4), 107 (100), 79 (28), 51
images were obtained under a scanning electron microscope
ꢂ1
(12); IRvmax (cm ): 3398, 2954, 2926, 1429, 1454, 1377, 1068, 759.
(Seron Technology S-4800) and DTG analysis was carried out with
2
8 8 2
-Bromo-1-(4-bromophenyl)ethanol (1c). C H Br O, 279.96 g
sample mass of silk broin or immobilized P. uorescens lipase ca.
ꢂ1
ꢀ
32
mol . Yield 0.055 g (80%). White solid, mp: 59 C (lit. 69–71);
ꢂ1
5
.0 mg, in an alumina crucible, under a 50 mL min ow rate
1
H NMR (400 MHz, CDCl ppm) d: 3.49 (dd, J ¼ 10.8 Hz, 1H),
ꢀ
ꢂ1
3
dynamic nitrogen atmosphere and heating rates of 10 C min
from room temperature up to 600 C, in a TA Instrument Q-600.
3
(
1
(
.61 (dd, J ¼ 10.5, 4 Hz, 1H), 4.91 (m, 1H), 7.27 (d, J ¼ 8 Hz), 7.51
ꢀ
13
d, J ¼ 8 Hz); C NMR (100 MHz, CDCl
3
ppm) d: 39.9, 73, 122.3,
27.6, 131.7, 139.2; MS (EI, 70 eV) m/z (%): 280 (8), 185 (100), 157
Determination of activity relative
ꢂ1
23), 77 (75), 51 (20); IRvmax (cm ): 3398, 2954, 2926, 1492, 1454,
Ester hydrolysis activity was selected for the determination and 1068, 759, 700.
assessment of immobilized P. uorescens lipase of activity, 2-Bromo-1-(4-chlorophenyl)ethanol
performed with some modications according to the method of 236.51 g mol . Yield 0.050 g (86%). White solid, mp: 59 C (lit.
(1d).
8 8
C H BrClO,
ꢂ1
ꢀ
33
31
ꢀ
1
Ye et al. (2005) Were mixed in a 10 mL Falcon tube, 0.5 mL of 61–62 C); H NMR (400 MHz, CDCl ppm) d: 3.50 (dd, J ¼ 10.4
3
ethanol containing 14.4 mM p-NPP and 0.5 mL of phosphate and 8.8 Hz, 1H), 3.61 (dd, J ¼ 10.5 and 3.4 Hz, 1H), 4.91 (dt, J ¼
1
3
buffer solution (0.1 M, pH 7.0). The reaction was started by 8.6 and 3.1, 1H), 7.28–7.39 (m, 4H); C NMR (100 MHz, CDCl
3
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ppm) d: 138.7, 128.8, 127.3, 126.8, 73.0, 39.9; MS (EI, 70 eV) m/z n-hexane (500 mL) were added to Eppendorf® (2 mL) and stirred
ꢂ1
ꢀ
(
%): 236 (6), 141 (100), 113 (20), 77 (54), 51 (8); IRvmax (cm ): at 32 C under 130 rpm. The reaction was monitored by TLC and
3
390, 2960, 2865, 1597, 1429, 1091, 833, 727, 702.
the enantiomeric excesses (ee) of the alcohols [(S)-1a–b] and
acetates [(R)-2a–b] were determined by GC-FID analysis in chiral
column (Table 1). Aer the completion of the reaction, the
mixture was ltered and the solvent was evaporated under
reduced pressure and the residue was puried by silica gel
column ash chromatography eluted with n-hexane and ethyl
acetate (9 : 1).
General procedure for the preparation of (R,S)-2-halo-1-
phenyl)ethanol acetates (2a–e)
Acetic anhydride (0.6 mmol, 0.06 g, 56.3 mL) and pyridine
2 mmol, 0.16 g, 161.5 mL) were sequentially added to a round-
(
(
bottom ask containing the appropriate (R,S)-2-halo-1-(phenyl)
ethanols (0.5 mmol). The mixture was stirred overnight at room
temperature. The reactions were quenched by the addition of
Preparation of silk broin spheres
1
0% HCl (1 mL) and the acetate product was extracted with
ethyl acetate (3 ꢃ 20 mL). The combined organic phases were
dried over Na SO and the organic solvent was evaporated
The silkworm cocoon (3.0 g) was transferred to 500 mL of a 2%
ꢀ
Na
2
CO
3
solution, preheated at 100 C and stirred at this
2
4
temperature for 30 min. The solid material was separated by
under reduced pressure. The residue was then puried by silica
gel column chromatography using n-hexane and ethyl acetate

ltration and then washed with distilled water (3 ꢃ 1000 mL).
ꢀ
Finally, the ber was placed in an oven to dry (70 C) for 24 h.
The broin was shredded and pinked in a Wiley mill (SOLAB
SL31 – Embrapa – Brazil). Aer this step, 50 mL of a ternary
(9 : 1) as eluent.
2
-Chloro-1-phenylethyl acetate (2a). C10
H
11ClO
mol . Yield 0.055 g (80%); mp: 47 C; H NMR (500 MHz,
CDCl
ppm) d: 3.71 (dd, J ¼ 11.7 and 4.6 Hz, 1H), 3.78 (dd, J ¼
1.7 and 8 Hz, 1H), 5.95 (dd, J ¼ 8.4 and 4.9 Hz, 1H), 7.48–7.30
2
, 198.04 g
ꢂ1
ꢀ
1
2 2
solution of H O : EtOH : CaCl (8 : 2 : 1 molar proportion) were
3
used to solubilize the broin, which was transferred to a cellu-
lose dialysis tube with an exclusion limit of 16 kDa and dialyzed
in water for 3 d at room temperature. The dialysis water was
changed every 24 hours. The broin solution was stored at
1
(
1
3
m, 5H) ppm; C NMR (125 MHz, CDCl
3
ppm) d: 20.98, 46.50,
7
1
2
5.0, 126.6, 128.7, 128.8, 137.1, 169.8; MS (EI, 70 eV) m/z (%):
98 (3), 162 (15), 102 (12), 77 (23), 43 (100); IRvmax (cm ): 2960,
929, 1747, 1373, 1230, 1024, 761, 689.
ꢂ1
ꢀ
4
10 C. Aer the broin solution was centrifuged (6000 rpm for
0 min) to remove impurities and larger particles. The
1
2
-Bromo-1-phenylethyl acetate (2b). C10
H
11BrO
mol . Yield 0.022 g (95%). Colorless oil; H NMR (500 MHz,
CDCl
ppm) d: 7.40–7.28 (m, 1H), 5.05 (dd, J ¼ 7.5 and 6.2 Hz,
H), 4.46–4.39 (m, 2H), 2.05 (s, 3H); C NMR (125 MHz, CDCl
d (ppm): 169.7, 136.6, 131.9, 128.3, 122.9, 74.1, 33.7, 20.9; MS
2
, 241.99 g
concentration of the SF solution was adjusted to 5% (w/w) with
distilled water, the determination of the nal concentration was
performed by the gravimetric method. The broin solution was
transferred to a pressurized spray bottle glass. This solution was
sprayed in liquid nitrogen for formation of the SF spheres. Aer
evaporated the excess of liquid nitrogen and the spheres were
lyophilized for 24 h. SF spheres has passed in sieve (6 to 13
mesh) to increase the degree of standardization.
ꢂ1
1
3
1
3
1
3
)
(
(
EI, 70 eV) m/z (%): 242 (8), 198 (16), 141 (32), 103 (36), 77 (78), 40
100); IRvmax (cm ): 2964, 1745, 1489, 1236, 758.
2
ꢂ1
-bromo-1-(4-bromophenyl)ethyl acetate (2c). C H Br O ,
10 10 2 2
ꢂ1
1
3
22.00 g mol . Yield 0.028 g (86%). Colorless oil; H NMR (400
) d (ppm): 7.49 (d, J ¼ 8.5 Hz, 2H), 7.25–7.21 (m, 2H),
.90 (dd, J ¼ 7.6 and 5.1 Hz, 1H), 3.60 (dd, J ¼ 10.8 and 7.7 Hz,
MHz, CDCl
3
Lipase immobilization
5
1
1
3
The 1.0 g of spheres of broin, 10 mL of water and 100 mg of P.
H), 3.54 (dd, J ¼ 10.8 and 5.1 Hz, 1H), 2.12 (s, 3H); C NMR

uorescens lipase were added to ask (50 mL) and gently shook
(
100 MHz, CDCl
3
) d (ppm): 169.7, 136.6, 131.9, 128.3, 122.9,
for 60 min. The material containing the immobilized lipase was
7
(
4.1, 33.7, 20.9; MS (EI, 70 eV) m/z (%): 322 (0.3), 242 (15), 200
31), 183 (26), 157 (6), 102 (21), 77 (23), 43 (100); IRvmax (cm ):
ꢂ1
lyophilized (Edwards, Model: Freeze Dryer Modulyo) for 12 h.
ꢀ
The immobilized broin lipase spheres were stored at 4
C
2
962, 2926, 1745, 1489, 1371, 1236, 1070, 1012, 821, 723.
-Bromo-1-(4-chlorophenyl)ethyl acetate (2d). C10 10BrClO
77.94 g mol . Yield 0.021 g (75%). Colorless oil; H NMR (500
ppm) d: 7.34 (d, J ¼ 8.6 Hz, 2H), 7.28 (d, J ¼ 8.6 Hz,
H), 5.92 (dd, J ¼ 7.7 and 5.0 Hz, 1H), 3.60 (dd, J ¼ 10.8 and 7.7
before use.
2
H
2
,
ꢂ1
1
2
MHz, CDCl
3
Enzymatic kinetic resolution of (R,S)-1a with P. uorescens
lipase immobilized on broin spheres
2
Hz, 1H), 3.54 (dd, J ¼ 10.8 and 5.0 Hz, 1H), 2.12 (d, J ¼ 2.4 Hz,
The (R,S)-2-chloro-1-(phenyl)ethanol (0.12 mmol, 20 mg) (1a)
and vinyl acetate (0.35 mol, 0.030 mg) were added to a ask vial
1
3
3
1
(
(
H); C NMR (125 MHz, CDCl ppm) d: 169.66, 136.12, 134.70,
3
28.90, 128.00, 126.54, 74.07, 33.83, 20.89; MS (IE, 70 eV) m/z
%): 278 (0.15), 196 (20), 154 (45), 137 (40), 103 (14), 77 (17), 43
100); IRvmax (cm ): 3032, 2964, 1745, 1492, 1373, 1238, 1091,
27.
(2 mL). n-Hexane (1 mL) was added in different asks contain-
ing 50, 100 or 150 mg of immobilized lipase P. uorescens on silk
ꢂ1
ꢀ

broin spheres. Each reaction mixture was stirred at 32
C
8
under 130 rpm (Table 2) and monitored by GC-FID. The reaction
was ltered and extracted with EtOAc (3 ꢃ 15 mL), the organic
layers were combined, dried over Na SO , the solvents were
Enzymatic kinetic resolution of (R,S)-2-halo-1-(phenyl)ethanol
1a–b)
2
4
(
removed by distillation under reduced pressure and the residue
Appropriate alcohol (R,S)-1a or (R,S)-1b (0.12 mmol), vinyl was puried by silica gel ash column chromatography eluting
acetate (0.35 mol, 0.030 mg) and 5 mg of the appropriate lipase, with n-hexane and ethyl acetate (7 : 3).
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Scale-up of the enzymatic kinetic resolution of (R,S)-1a by P.
reduced pressure and yielded a crude reaction mixture that was
puried by silica gel ash chromatography eluted with
MeOH : EtOAc (5 : 9.5), yield 60% (52 mg) of (S)-4 as a yellowish

uorescens lipase immobilized on broin spheres
(R,S)-2-Chloro-1-phenylethanol (0.36 mmol, 60 mg) (1a) and
17
solid.
vinyl acetate (0.75 mol, 0.060 mg), 450 mg of P. uorescens lipase
immobilized on broin spheres and n-hexane (5 mL) were
(
S)-1-(4-Chlorophenyl)-2-(1H-1,2,3-triazol-1-yl)ethanol
(4).
ꢂ1
ꢀ
C
1
11
H
11ClN
2
O, 222.06 g mol . Yield 0.052 g (60%); mp: 185–
added to a vial (30 mL). The mixture was stirred at 32 C under
ꢀ
35
1
86 C (lit. 160–164); H NMR (400 MHz, CD
3
OD) d (ppm): 4.14
130 rpm and monitored by TLC. It was then ltered and
(
7
1
dd, J ¼ 14.1 and 6.9 Hz, 1H), 4.21 (dd, J ¼ 14.1 and 4.4 Hz, 1H),
extracted with EtOAc (3 ꢃ 15 mL). The organic layers were
.46 (s, 1H), 4.91 (dd, J ¼ 6.9 and 4.5 Hz, 1H), 7.03 (t, J ¼ 1.3 Hz,
combined and dried over Na SO . The solvent was removed by
2
4
13
H), 6.88 (s, 1H), 7.34–7.22 (m, 4H); C NMR (100 MHz, CD OD)
3
distillation under reduced pressure and the residue was puri-
ed by column ash chromatography with n-hexane and ethyl
d (ppm): 141.8, 139, 134.5, 129.4, 128.7, 128.4, 121.5, 73.4, 55;

MS (EI, 70 eV) m/z (%): 222 (5), 141 (19), 113 (13), 82 (100), 77
acetate (7 : 3) prior to the next synthetic step. The same proce-
dure was employed for other (R,S)-2-halo-1-(phenyl)ethanols
ꢂ1
(
7
40), 51 (7); IRnmax (cm ) ¼ 3116, 2929, 2856, 1514, 1408, 1072,
48, 661.
(1b–e). The yields of the acetates are showed in Table 3.
Absolute conguration
General procedure for the formation of epoxides (3a–c)
EtOH (1 mL), H O (500 mL) and LiOH (26 mg, 1.1 mmol) were
successively added to a solution of (S)-acetate 2a (22 mg, 0.10
mmol). The mixture was stirred at room temperature for 1 h.
EtOH was removed by distillation at reduced pressure, and
The optical rotations of puried alcohols 1a–d, acetates 2a–
d and epoxides 3a–c were measured in CHCl with a JASCO
3
P2000 polarimeter equipped with a Na-lamp 589 nm in 1 dm
cuvette. The absolute congurations of alcohols 1a–d, acetates
2
2
a–d and epoxides 3a–c were determined by comparing the
specic rotations measured with those reported in the literature
ESI†).
a solution of NaHCO (3 mL, 6 equiv.) and a saturated solution
3
of NaCl (5 mL) were added. The organic phase was extracted
(
with diethyl ether (3 ꢃ 30 mL) and the organic phases were
34
2 4
combined and dried over Na SO . The solvent was removed by
distillation under reduced pressure and puried by column Recycling of the biocatalyst

ash chromatography with a mixture of n-hexane and ethyl
Following the procedure described previously (section Experi-
acetate (9 : 1), affording (S)-3a as a colorless oil (quantitative).
The same procedure was employed for the other (S)-acetate (2b–
d). The yields of the epoxides (S)-3a–c are showed in Table 4.
mental), the catalyst was recovered, washed with n-hexane (3 ꢃ
1
mL) and reused. The process was repeated for four times (the
results are shown in Fig. 2).
ꢂ1
(
S)-2-Phenyloxirane (3a). C H O, 120.06 g mol . Yield
8
8
1
0
2
1
3
.015 g (95%); colorless oil; H NMR (400 MHz, CDCl ppm) d:
.82 (dd, J ¼ 5.5 and 2.6 Hz, 1H), 3.17 (dd, J ¼ 5.5 and 4.1 Hz, Acknowledgements
H), 3.88 (dd, J ¼ 4.0 and 2.6 Hz, 1H), 7.55–7.13 (m, 5H); NMR
1
3
I. M. F. acknowledge Conselho Nacional de Desenvolvimento
Cient ´ı co e Tecnol ´o gico o (CNPq Proc. 558062/2009-1) for
fellowship. A. L. M. Porto and J. V. Comasseto acknowledge to
CNPq (Proc. 558062/2009-1) and Fundaç ˜a o de Amparo `a Pes-
quisa do Estado de S ˜a o Paulo (FAPESP Proc. 2009/50688-3) for
the nancial support provided to this research.
3
C (100 MHz, CDCl ) d (ppm): 137.6 (s), 128.5, 128.1, 125.5,
52.3, 51.1; MS (EI, 70 eV) m/z (%): 120 (43), 119 (67), 91 (100), 77
ꢂ1
(10), 51 (20); IRnmax (cm ): 2913, 1495, 1453, 876, 760.
(
S)-2-(4-Bromophenyl)oxirane (3b). C H BrO, 197.07 g
8 7
ꢂ1
1
mol . Yield 0.010 g (98%). Colorless oil; H NMR (400 MHz,
CDCl
3
) d (ppm): 2.75 (dd, J ¼ 5.4 and 2.5 Hz, 1H); 3.14 (dd, J ¼
5
7
5
1
1
.3 and 4.2 Hz, 1H), 3.84–3.81 (m, 1H), 7.15 (d, J ¼ 8.4 Hz, 2H),
1
3
.47 (d, J ¼ 8.4 Hz, 2H); C NMR (100 MHz, CDCl
3
) d (ppm):
Notes and references
1.2, 58.8, 122, 127, 131, 137; MS (EI, 70 eV) m/z (%): 198 (13),
69 (20), 119 (55), 89 (100), 77 (7), 63 (32); IRnmax (cm ): 2958,
745, 1517, 1230, 1026.
ꢂ1
1 (a) H. Ferraz, G. Bianco, C. Teixeira, L. H. Andrade and
A. L. M. Porto, Tetrahedron: Asymmetry, 2007, 18, 1070; (b)
S. S. Ribeiro, I. M. Ferreira, J. P. F. Lima, B. A. Sousa,
R. C. Carmona, A. A. D. Santos and A. L. M. Porto, J. Braz.
Chem. Soc., 2015, 26, 1344.
2 (a) R. Ferrarini, J. V. Comasseto and A. Dos Santos,
Tetrahedron: Asymmetry, 2009, 20, 2043; (b) W. G. Birolli,
M. I. Ferreira, N. Alvarenga, D. A. Santos, I. L. De Matos,
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ꢂ1
(
S)-2-(4-Chlorophenyl)oxirane (3c). C H ClO, 154.07 g mol
.
8
7
1
Yield 0.012 g (95%); colorless oil. H NMR (400 MHz, CDCl )
3
d (ppm): 7.30 (d, J ¼ 8.5 Hz, 2H), 7.19 (d, J ¼ 8.5 Hz, 2H), 3.82
(
2
dd, J ¼ 3.7 and 2.8 Hz, 1H), 3.13 (dd, J ¼ 5.4 and 4.1 Hz, 1H),
1
3
.73 (dd, J ¼ 5.4 and 2.5 Hz, 1H) ppm; C NMR (100 MHz,
) d (ppm): 136.1, 133.9, 128.7, 126.8, 51.7, 51.2 ppm; MS
EI, 70 eV) m/z (%): 154 (23), 119 (69), 89 (100), 63 (20); IRnmax
CDCl
(
(
3
ꢂ1
cm ): 2927, 2854, 1215, 759.
Synthesis of (S)-azole (4). The 1,3-imidazole (100 mg, 1.5
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ꢀ
1
2
,4-dioxane (500 mL), and the mixture was stirred at 100 C for
4 h. The solvent was then removed by distillation under
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Paper
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12658 | RSC Adv., 2017, 7, 12650–12658
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