Chemoenzymatic synthesis of d-biotin intermediate lactone via lipase-catalyzed desymmetrization of meso diols

Article abstract of DOI:10.1016/j.molcatb.2013.09.014

A chemoenzymatic methodology for the asymmetric synthesis of d-biotin intermediate lactone ((3aS, 6aR)-tetrahydro-1,3-dibenzylhexahydro-1H-Furo[3,4-d] imidazole-2,4-dione) 1 has been demonstrated. The key step of the synthetic routes is Lipozyme RM IM catalyzed desymmetrization of meso-diols 3. The highest enantiomeric excess (e.e. > 98%) and yield (>90%) of the product was achieved with Lipozyme RM IM in Dioxane/Toluene (1:3, v/v) at 35 C. Furthermore, Lipozyme RM IM showed an excellent operational stability, retaining above 80% of the initial activity after 10 cycles of reaction. d-Biotin intermediate lactone 1 was obtained subsequently by Jones oxidation, basic hydrolysis and lactonization.

Full text of DOI:10.1016/j.molcatb.2013.09.014

Journal of Molecular Catalysis B: Enzymatic 98 (2013) 37–41
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Chemoenzymatic synthesis of d-biotin intermediate lactone via
lipase-catalyzed desymmetrization of meso diols
Jian-Yong Zheng, Sheng-Fan Wang, Yin-Jun Zhang, Xiang-Xian Ying,
Yu-guang Wang, Zhao Wang∗
College of Biological and Environmental Engineering, Zhejiang University of Technology, Hang Zhou 310032, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 June 2013
Received in revised form 2 September 2013
Accepted 9 September 2013
Available online 17 September 2013
A chemoenzymatic methodology for the asymmetric synthesis of d-biotin intermediate lactone ((3aS,
6aR)-tetrahydro-1,3-dibenzylhexahydro-1H-Furo[3,4-d] imidazole-2,4-dione) 1 has been demonstrated.
The key step of the synthetic routes is Lipozyme RM IM catalyzed desymmetrization of meso-diols 3. The
highest enantiomeric excess (e.e. > 98%) and yield (>90%) of the product was achieved with Lipozyme RM
IM in Dioxane/Toluene (1:3, v/v) at 35 ◦C. Furthermore, Lipozyme RM IM showed an excellent operational
stability, retaining above 80% of the initial activity after 10 cycles of reaction. d-Biotin intermediate lactone
1 was obtained subsequently by Jones oxidation, basic hydrolysis and lactonization.
Keywords:
Chemoenzymatic synthesis
Desymmetrization
© 2013 Elsevier B.V. All rights reserved.
d-Biotin intermediate lactone
Transesterification
1. Introduction
reaction, enzymatic reactions have an advantage status of green
chemistry over chemical synthesis [8]. Because of being carried out
d-Biotin (Vitamin H) is a water-soluble vitamin B which is
essential to the normal physiological activities of animals and
human life. It serves as the prosthetic group of some metabolic
enzymes in vivo known as biotin-dependent carboxylases [1–3].
Growth retardation and dysontogenesis will occur when poultry
lack biotin. d-Biotin has been extensively applied in medicine and
health protection, particularly as the feedstuff additive with large
commercial requirement. d-Biotin intermediate (3aS, 6aR)-lactone
1 is extremely vital to the synthesis of d-biotin [4,5]. Many scien-
tists devoted themselves to the synthesis of (3aS, 6aR)-lactone 1,
and the asymmetric synthetic routes of (3aS, 6aR)-lactone 1 have
been numerously reported in recent years. Fei Xiong et al. obtained
the (3aS, 6aR)-lactone 1 through a rapid cinchona alkaloid-based
sulfonamide-mediated enantioselective alcoholysis of meso-cyclic
anhydride [6]. A method through catalytic reactivity in the hydro-
genation of a cyclic anhydride to a biotin synthetic intermediate
has been investigated using Wilkinson Ru complex by Masahiro
Yoshimura et al. [7].
under mild conditions, enzymatic reactions have the advantage of
less side reaction (such as isomerization, racemization, epimeriza-
tion, and rearrangement of molecules). Enzymatic synthesis of (3aS,
6aR)-lactone 1 via asymmetric hydrolysis of meso-dicarboxylic
esters by pig liver esterase and subsequently following by Grig-
nard reaction was reported [9,10]. Zheng et al. reported that (3aS,
6aR)-lactone 1 could be efficiently prepared in excellent conversion
ratio (c ≥ 40%) and enantiomeric purities (e.e. ≥ 98%) via enantiose-
lective lactonization by the resting cell of Aspergillus oryzae WZ007
[11].
Hydrolases have been successfully used for the desymmetriza-
tion of different meso or prochiral alcohols, carboxylic acid esters,
anhydrides and nitriles [12]. Many authors have demonstrated that
enzymatic desymmetrization of the prochiral dimethyl carbon-
ate is a versatile and effective way to obtain the corresponding
monoester [13,14]. Two approaches have been employed for the
desymmetrization of different meso or prochiral alcohols: acylation
of the free alcohol by means of transesterification reaction [15,16],
and hydrolysis of an appropriate acyl derivative of alcohol [17,18].
Lipase-catalyzed transesterification may be an efficient method for
desymmetrization of meso-diols [19–22].
With the development of enzymatic reactions in organic chem-
istry, more and more chemists pay attention to the study of
enzymatic desymmetrization. Due to the ecological aspect of
In this paper, we described a novel synthetic route of (3aS, 6aR)-
lactone 1 by enantioselective transesterification of diols 3 with
Lipozyme RM IM and subsequently following Jones oxidation, basic
hydrolysis and lactonization (Scheme 1).
∗
Corresponding author. Tel. +86 0571 88320615; fax: +86 0571 88320781.
E-mail address: hzwangzhao@163.com (Z. Wang).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.09.014

38
J.-Y. Zheng et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 37–41
O
O
BnN NBn
O
O
BnN NBn
c
a
b
BnN NBn
BnN NBn
H
H
O
H
H
H
H
H
H
HOH2C CH2OH
MeOOC COOMe
AcOH2C CH2OH
O
3
2
4
1
Scheme 1. Synthetic scheme for the synthesis of d-biotin intermediate lactone 1. Reaction conditions: (a) EtOH, NaBH4, 0 ◦C to r.t. 50 ◦C, reflux 3 h 96%; (b) Lipozyme RM IM,
Dioxane/Toluene (1:3), 6 h, 35 ◦C, 90%; (c) CrO3, H2SO4, H2O, acetone, 0 ◦C, 2 h, then 2 M NaOH, 1 M HCl reflux, 3 h, 65%.
2. Experimental
that was purified by recrystallization from n-hexane affording
7.63 g of meso-diols 3 as a white solid (94%): 1H NMR(CDCl3): ı
7.36–7.27 (m, 10H), 4.92–4.89 (d, 2H, J = 15.40 Hz), 4.14–4.11 (d,
2H, J = 15.45 Hz), 3.84–3.82 (d, 2H, J = 13.00 Hz), 3.75–3.72 (d, 2H,
J = 12.90 Hz), 3.51 (s, 2H), 2.37 (s, 2H); 13C NMR(CDCl3): ı 161.5,
137.1, 128.8, 127.9, 127.8, 127.6, 57.7, 56.5, 46.6, 45.6; MS (m/z)
349 (M + Na)+; IR ꢀmax (cm−1): 3442, 3266, 2930, 1670, 1476, 1453,
1359, 1253, 1146, 1054, 1001, 741, 701, 657, 540.
2.1. General methods
The conversions were determined by Waters 1525 HPLC with
a UV detector. Enantiomeric separation of monoacetate 4 was
performed using the Chiralcel AD-H column (250 mm × 4.6 mm,
10 ␮m, Daicel, Japan) and a mixture of hexane and propan-2-ol
80:20 (v/v) as eluant. Flow rate was 0.8 mL/min and retention times
for (4R, 5S)-monoacetate-4 and (4S, 5R)-monoacetate-4 were 19.5
and 23.1 min, respectively. Enantiomeric separation of lactone 1
was performed using the Chiral CD-Ph column (250 mm × 4.6 mm,
10 ␮m, Shiseido, Japan) and a mixture of acetonitrile and water
60:40 (v/v) as eluant. Flow rate was 0.5 mL/min and retention times
for (3aR, 6aS)-lactone-1 and (3aS, 6aR)-lactone-1 were 12.8 and
14.2 min, respectively. 1H NMR spectra were recorded on a Bruker
500 Hz apparatus (TMS as an internal standard, 13C NMR 125 Hz).
Mass spectra were recorded on an Esquire 6000 spectrometer. IR
spectra were recorded on Bruker Tensor 27 spectrometer. Optical
rotations were determined using an Autopol IV Polarimeter at 25 ◦C
using a cell of 1 dm length.
2.4. Procedure for the synthesis of 4
2.4.1. General procedure for the chemical synthesis of racemic
monoacetates 4
To a solution of diols 3 (2.76 mmol) in dry CH2Cl2 (28 mL) were
added Et3N (120 ␮L, 8.40 mmol) and DMAP (111 mg, 0.90 mmol)
under nitrogen atmosphere. After the mixture was stirred for a
couple of minutes, Ac2O (265 ␮L, 2.8 mmol) was added in portions,
and the solution was stirred for an additional 4 h at room tempera-
ture. After the solvent was evaporated under reduced pressure, the
obtained reaction crude that was purified by flash chromatography
(EtOAc/petroleum benzene = 2:1) affording the monoacetate 4 as a
colorless oil (54% yield): Rf (EtOAc/petroleum benzene = 2:1) 0.46.
2.2. Materials
2.4.2. General procedure for the lipase-catalyzed synthesis of
monoacetates 4
Meso diester 2 and d-biotin intermediate lactone 1 was obtained
from Zhejiang Shengda Pharmaceutical Co., Ltd. (Zhejiang, China) as
a gift. Commercially available organic solvents were treated with
3 A molecular sieves. All other chemicals employed in this work
were obtained from various commercial suppliers.
A mixture of meso-diols 3 (326 mg, 1 mmol), Lipozyme RM IM
(100 mg), and vinyl acetate (555.4 ␮L, 6 mmol) was stirred in 10 mL
Dioxane and Toluene (1:3) at 35 ◦C for 6 h in water baths shaker.
After removal of polymer-supported enzyme by filtration, the
filtrate was evaporated under reduced pressure. Then the monoac-
etate 4 was obtained in a yield of 90% with the high enantiomeric
excess (e.e. > 98%). [␣]25D:−7.2(c 1.0 CHCl3); 1H NMR(CDCl3): ı
7.34–7.31 (m, 6H), 7.29–7.26 (m, 4H), 4.87 (d, 2H, J = 15.45 Hz), 4.77
(d, 2H, J = 15.40 Hz), 4.44 (d, 2H), 4.42 (d, 2H), 3.45 (s, 2H), 1.98
(s, 1H), 1.98(s, 3H); 13C NMR(CDCl3): ı 170.3, 161.2, 137.4, 137.1,
128.7, 128.6, 128.0, 127.9, 127.8, 127.5, 127.4, 61.6, 58.8, 56.4, 54.1,
54.0, 46.3, 46.2, 21.0, 20.7, 14.1. MS (m/z) 391 (M + Na)+; IR ꢀmax
(cm−1): 3354, 2901, 1744, 1658, 1477, 1451, 1356, 1231, 1044, 760,
739, 700, 457 (Scheme 2).
˚
Novozym 435 (component
B of the lipase from Candida
Antarctica immobilized on macroporous polyacrylate resin),
Lipozyme RM IM (Rhizomucor miehei immobilized on ionic resin)
were supplied by Novozymes A/S (Bagsvaerd, Denmark). Amano
lipase AK (Pseudomonas fluorescens lipase), Amano lipase
A
(Aspergillus niger lipase), Rhizopus niveus lipase, Amano PS IM
(lipase from Burkholderia cepacia immobilized on diatomaceous
earth), and Amano lipase AY (Candida rugosa lipase) were obtained
from Sigma–Aldrich (shanghai) Trading Co. Ltd. (shanghai, China).
Lipase from pig (porcine) pancreas (PPL) was obtained from Aladdin
Industrial Co. (shanghai, China).
Jones reagent was prepared as follows: chromium trioxide
(26.7 g) was dissolved in sulfuric acid (23 mL) and the resulting
mixture was diluted with water to 100 mL.
2.5. Procedure for the synthesis of 5
To a solution of the corresponding diols 3 (2.76 mmol) in
dry CH2Cl2 (28 mL) were successively added Et3N (2.34 mL,
16.62 mmol), DMAP (222 mg, 1.84 mmol), and Ac2O (1.04 mL,
11.08 mmol) under nitrogen atmosphere. The reaction was stirred
at room temperature during 4 h until complete consumption
of the starting material, and then the solvent was evaporated
under reduced pressure. The reaction crude was finally puri-
fied by flash chromatography on silica gel (EtOAc/petroleum
benzene = 2:1), yielding the corresponding diacetate 5 as an oil
(98%): Rf (EtOAc/petroleum benzene = 2:1) 0.69; 1H NMR(CDCl3):
ı 7.36–7.28 (m, 10H), 4.87 (d, 2H, J = 15.45 Hz), 4.33–4.32 (m, 2H,
J = 5.15 Hz), 4.30–4.29 (m, 2H, J = 5.05 Hz), 4.23 (s, 1H) 4.19 (s, 1H),
3.67 (s, 2H), 2.02 (s, 6H); 13C NMR(CDCl3): ı 170.2, 160.6, 137.1,
2.3. Procedure for the synthesis of 3 [23]
A
solution of diester 2 (9.53 g, 24.9 mmol) in anhydrous
CH3COOH (200 mL) was cooled to 0 ◦C and NaBH4 (11.3 g,
30.5 mmol) was carefully added during 10 min. The reaction
mixture was stirred for 5 h at room temperature, followed by
refluxing for 3 h at 80 ◦C. And then formic acid was dripped to
the above solution slowly until pH fell to about 4–5 at 0 ◦C. Salts
were filtered off through buchner funnel, and the residue was
washed with CH3COOH (3 mL × 20 mL), filtrate was combined and
evaporated under reduced pressure, the obtained reaction crude

J.-Y. Zheng et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 37–41
39
O
O
O
BnN NBn
BnN NBn
BnN NBn
Lipase
+
H
H
H
H
H
H
Vinyl acetate
AcOH2C
CH2OAc
HOH2C
CH2OH
AcOH2C
CH2OH
3
4
5
Scheme 2. Enzymatic desymmetrization of meso-diols 3.
Table 1
Lipase-catalyzed synthesis of monoacetate 4 using various lipases.
Table 2
The influence of the type of acylating reagent on the selectivity for the enzymatic
acylation of 3 by Lipozyme RM IM.
Entry
Enzyme
Conv. [%]a
Ratio 4/5b
e.e.(4)[%]c
4R, 5S
4S, 5R
Entry
Acylating reagent
Yield (5)[%]a
Yield (4)[%]a
e.e.(4)[%]a
1
2
3
4
5
6
7
Novozym435
Amano lipase A
Amano lipase AK
Amano PS IM
Lipozyme RM IM
PPL
26.22
2.29
60.61
92.96
90.90
5.74
93:7
66:33
96:4
98:2
94:6
50:50
98:2
33.06
24.95
90.83
92.62
–
–
–
–
–
1
2
3
4
5
6
Vinyl acetate
Ethyl acetate
Acetic acid
Acetic anhydride
Isopropenyl acetate
Butyl acetate
5.37
6.06
5.04
8.52
8.10
20.84
92.94
12.85
4.50
78.06
24.50
78.96
89.40
89.34
84.24
77.70
87.63
92.28
90.41
57.78
86.28
–
–
Amano lipase AY
65.66
Conditions: diols 3 (25 mmol/L), acylating reagent (150 mmol/L) and Lipozyme RM
Conditions: diols 3 (25 mmol/L), vinyl acetate (200 mmol/L) and lipases (0.05 g) in
IM (0.05 g) in CH2Cl2 (5 mL) were shaken at 30 ◦C for 24 h.
CH2Cl2 (5 mL) were shaken at 30 ◦C for 24 h.
a
e.e.% and yield of monoacetate 4 and the yield of diacetate 5 was determined by
a
Conv. [%] is the conversion of diols 3 was determined by HPLC.
Ratio 4/5 is the molar ratio of monoacetate 4 and diacetate 5 and was determined
HPLC.
b
by HPLC.
c
results, The enzymes could be classified in two groups (Table 1):
Entries 1–4: Amano lipase AK and Amano PS IM showed good
(60.61%) and high (92.96%) conversions, respectively, to the unde-
sired (4R, 5S)-monoacetate 4 in high (90.83%, 92.62%) e.e. Novozym
435 and Amano lipase A showed lower conversions (<26.22%) and
e.e. (24.95, 33.06%). Entries 5–7: Lipozyme RM IM and Amano lipase
AY showed high conversions (90.90%, 65.66%) to the desired (4S,
5R)-monoacetate 4 with high e.e. (90.41%, 86.28%). PPL showed low
(5.74%) conversion with moderate (57.78%) e.e. to the desired (4S,
5R)- monoacetate 4.
e.e.% of monoacetate 4 was determined by HPLC.
128.6, 128.0, 127.8, 127.5, 60.6, 53.7, 46.2, 20.6, 20.5; MS (m/z)
433 (M + Na)+; IR ꢀmax (cm−1): 3032, 2998, 2958, 1736, 1685, 1469,
1452, 1369, 1288, 1230, 1037, 922, 767, 700, 606, 565, 483.
2.6. Procedure for the synthesis of 1
To a solution of monoacetate 4 (0.2 g, 0.5 mmol) in acetone
(5.0 mL) was added Jones reagent (0.5 mL) at 0 ◦C. The mixture
was stirred for 2 h, i-propanol was added dropwise at the same
temperature until discoloration. Then the resulting mixture was
filtered through Celite, and the residue was washed with acetone
(3 mL × 5 mL). CH2Cl2 (5 mL) was added after the filtrate was com-
bined and evaporated under reduced pressure. The mixture was
washed with water (3 mL × 5 mL) until colorless, then the organic
phase was dried over anhydrous magnesium sulfate and evapo-
rated to afford the carboxylic acid. With the carboxylic acid in
hand, prepared THF (2 mL) and 2 M NaOH (5 mL) was added and
stirred at 80 ◦C for 5 h. The THF was evaporated, after which the
aqueous phase was extracted with CH2Cl2 (3 mL × 5 mL), then 1 M
HCl was added to the aqueous phase until pH fell to about 3–4.
After 1–3 h stirring at 60 ◦C, extraction of the aqueous phase with
CH2Cl2 (3 mL × 5 mL), washing the combined organic phases with
brine (10 mL), drying over MgSO4, and evaporation led to an pow-
der: mp 119–121 ◦C; [␣]25D: +59.1 (c 1.0 CHCl3) 1H NMR(CDCl3): ı
7.41–7.26 (m, 10H), 5.08 (d, 1H, J = 14.8 Hz), 4.67 (d, 1H, J = 15.15 Hz),
4.41–4.36 (m, 2H), 4.18 (d, 2H), 3.95 (d, 1H, J = 13.60 Hz), 3.75 (d,
1H); 13C NMR(CDCl3): ı 172.8, 158.2, 136.0, 135.9, 129.1, 128.9,
128.8, 128.3, 128.1, 127.9, 70.1, 54.4, 52.5, 47.0, 45.2; MS (m/z) 357
(M + Na)+; IR ꢀmax (cm−1): 3061, 2920, 1775, 1702, 1476, 1445,
1416, 1316, 1237, 1186, 998, 970, 789, 755, 670, 639, 528, 455. The
analytical data were consistent with those of the authentic sample
of 1.
To develop a scalable process for the biocatalytic synthesis of the
desired (4S, 5R)-monoacetate 4, further studies were conducted fol-
lowing up on the initial screening results. After analyzing several
reaction attributes, e.g. yield and e.e. of product, reaction rate and
cost of enzyme, Lipozyme RM IM was chosen for further develop-
ment of the enantioselective transesterification of diols 3 to the
desired (4S, 5R)-monoacetate 4.
3.2. Choice of acyl donor
The obtained results revealed that an acyl group donor has
an influence on both selectivity and reaction rate. Six acylating
agents were used in enzymatic transesterification, including vinyl
acetate, ethyl acetate, acetic acid, acetic anhydride, isopropenyl
acetate, and butyl acetate. The acylation reaction of 3 was carried
out in CH2Cl2 for 24 h with the set of different acylating reagents.
The results obtained were summarized in Table 2. As shown in
Table 2, the excellent enantioselectivity (e.e.92.28%) with a yield
(78.96%) was obtained when butyl acetate was used as the acy-
lating agent. But the yield of diacetate 5 reach 20.84% which was
undesirable. When other acylating agents were used, the outcome
indicated that vinyl acetate was the excellent acylating reagent for
the transesterification of diols 3 (Table 2, entry 1) leading to the
corresponding monoacetate in high yield (92.94%) and excellent
e.e. (89.40%) within 24 h.
3. Results and discussion
3.3. Choice of solvent
3.1. Screening of lipase
Since the hydrophobicity of organic solvents can influence the
enantioselectivity of lipase-catalyzed reaction, the choice of the
solvent used as the reaction medium is very significant in the enzy-
matic synthesis. A variety of organic solvents were examined. These
results are shown in Table 3.
Initially, we screened a series of lipases to identify potential cat-
alysts for the title reaction. Seven commercially available lipases
were screened, the results were listed in Table 1. Judging from these

40
J.-Y. Zheng et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 37–41
Table 3
45
90
88
86
84
82
80
Transesterification of diols 3 with vinyl acetate in different organic solvents.
40
35
30
25
20
15
10
5
Entry Solvent
Log Pa Yield (5)[%] Yield (4)[%] e.e.(4)[%]
1
2
3
4
5
6
7
8
Dioxane
Acetonitrile
Acetone
Tetrahydrofuran
Dichloromethane
TBME
Toluene
Cyclohenane
Hexane
−1.10
−0.33
−0.23
0.49
1.01
1.30
2.50
3.40
3.50
–
3.35
4.67
2.98
6.56
5.37
49.26
13.47
54.59
82.61
12.23
10.96
5.89
47.96
70.49
72.03
47.63
92.94
50.48
85.66
44.17
16.72
87.33
86.53
90.48
93.69
68.51
72.66
76.17
89.40
80.00
95.97
90.71
60.99
99.28
97.23
95.46
9
10
11
12
Dioxane/Toluene (1:3)
Dioxane/Toluene (1:1)
Dioxane/Toluene (3:1)
–
–
Conditions: diols 3 (25 mmol/L), vinyl acetate (150 mmol/L) and Lipozyme RM IM
(0.05 g) in different solvents (5 mL) were shaken at 30 ◦C for 24 h.
20
25
30
35
40
45
50
a
Logarithm of the octanol–water partition coefficient of the solvent [24].
Temperature (ºC)
Fig. 1. Effect of temperature on the reaction initial velocity and e.e. symbols: initial
velocity (ꢀ) and e.e. (ꢁ) of monoacetate 4. Conditions: diols 3 (25 mmol/L), vinyl
acetate (150 mmol/L) and Lipozyme RM IM (0.05 g) in Dioxane/Toluene (1:3) (5 mL)
were shaken at different temperatures. The initial velocity was calculated by the
conversion in 2 min.
As shown in Table 3, the remarkable influence of organic sol-
vent was observed. Solvents with log P < 1.30 showed low yields
of diacetate 5, while log P > 1.30 showed high yields of diacetate 5.
Dioxane (log P = −1.10) and dichloromethane (log P = 1.01) showed
highest e.e. of monoacetate 4 and lower yields of diacetate 5 (entry
1), toluene (log P = 2.50) showed a good yield (85.66%) and high
e.e. (95.97%) of monoacetate 4 (entry 7). When cyclohenane (log
P = −3.4) was used as the solvent, we obtained the monoacetate
4 in moderate yield (44.17%) and e.e.(90.71%). However the yield
of the undesired diacetate 5 reached 54.59% (entry 8). Consider-
ing the solubility of diols 3 in different solvent and the results in
Table 3, we tried to use mixed solvent to obtain a better result.
When a mixture of dioxane and toluene was used as the solvent,
we obtained an excellent result (entry 10–12). Excellent enantiose-
lectivity (e.e.99.28%) with a high yield (87.33%) was obtained when
diols 3 was treated with Lipozyme RM IM in dioxane/toluene (1:3)
compared with that in dioxane (yield 47.96%, e.e. 93.69%) and in
toluene (yield 85.66%, e.e. 95.97%) (entry 10). The mixed solvent
was chosen for further study.
reached maximum (95.0%) at 3 h. However, it began to decreased
after 3 h and dropt to 78% upon progression of the reaction. Further-
more, the production of diacetate 5 was almost not observed in the
first 1.5 h but it increased from 0.5% yield at 1.5 h to 5% yield at 3 h
and reached 20% at 24 h. Fortunately, the reuse of diacetate 5 was
developed by alkali treatment which was done when we obtained
d-biotin intermediate lactone 1 from monoacetate 4. On the other
hand, the enantiomeric excess of monoacetate 4 was increased
from 85% to 99% during the first 6 h, and then slowly decreased
to 97% at 24 h.
This is mainly because two steps is contained in the reaction and
monoacetates are in principle formed at unequal rates, and also
react further at unequal rates in a double step process. The first
step is desymmetrization, and the second step is kinetic resolution
[27]. Recently it has been demonstrated for several authors that a
particular immobilized form of lipases can be control this speci-
ficity towards the monoacetylated produced [28,29]. Lowering of
the reaction temperature led to an increased enantioselectivity, but
the rate ratio of the transformations of diol 3 to monoacetate 4
and of monoacetate 4 to diacetate 5 was not altered by lowering
the reaction temperature [30]. Hence monitoring of the reaction
3.4. Effect of temperature
Temperature has great effect on the activity, selectivity and sta-
bility of a enzyme and the thermodynamic equilibrium of a reaction
as well [25]. The thermal stability of biocatalysts is always taken
as one of the most important criteria for industrial applications
[26]. Therefore, the effect of temperature on the enantioselective
transesterification of diols 3 was studied. The initial velocity and
e.e. at different temperatures were shown in Fig. 1. The initial
velocity was calculated by the conversion in 2 min, and the con-
version was calculated by the concentration decrement of diols 3
by HPLC. Judging from Fig. 1, the initial velocity increased when the
reaction temperature was raised since higher temperature acceler-
ates molecular diffusion. However e.e. decreased while the reaction
temperature raised over 35 ◦C. This is due to the partial inactiva-
tion of the enzyme in organic solvent at high temperature for a long
time. Consequently, the optimal temperature was 35 ◦C because of
its highest e.e. and acceptable initial velocity.
100
80
60
40
20
0
100
80
60
40
20
0
3.5. Time-courses of Lipozyme RM IM catalyzed
desymmetrization of diols 3
0
5
10
15
20
25
Composition of the reaction mixture during the 24 h of acety-
lation of diols 3 is shown in Fig. 2. The amount of the diols 3 was
rapidly reduced to 9.2% from 100% at the first 1.5 h and was dropt
to 0.3% after the reaction for 3 h. That means the substrate diols 3
was complete reaction at the first 3 h. The yield of monoacetate 4
was notably increased to 82.9% during the fist 1.5 h of reaction and
Time (h)
Fig. 2. Time course of enzyme-catalyzed transesterification with the amount of diols
3 (ꢀ), monoacetate 4 (᭹) and diacetate 5 (ꢁ) (n [%]) and the e.e. of monoacetate 4
(ꢂ). Conditions: diols 3 (25 mmol/L), vinyl acetate (150 mmol/L) and Lipozyme RM
IM (0.05 g) in Dioxane/Toluene (1:3) (5 mL) were shaken at 35 ◦C for 24 h.

J.-Y. Zheng et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 37–41
41
100
80
60
40
20
100
4 by Jones oxidation, basic hydrolysis and lactonization. Currently,
we are investigating its implementation in the practical large-scale
preparation of d-biotin.
80
60
40
20
Acknowledgements
The authors are thankful to Zhejiang Shengda Pharmaceutical
Co., Ltd for supplying Meso diester 2 and d-biotin intermediate
lactone 1. This research was financially supported by Key Sci-
ence and Technology Innovation Team Project of Zhejiang province
(No. 2011R09043-03) and Natural Science Foundation of Zhejiang
Province (No. LY12B06011).
2
4
6
8
10
References
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In summary, we have developed an efficient methodology for
the chemoenzymatic synthesis of d-biotin intermediate lactone 1.
The key chiral monoacetate 4 was obtained by Lipozyme RM IM
catalyzed enantioselective transesterification of diols 3. The prefer-
able solvent (dioxane/toluene (1:3)) and reaction condition (35 ◦C,
200 rpm) were determined. Lipozyme RM IM was found to be effec-
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