Convenient enzymatic resolution of cis-6-benzyltetrahydro-1H-pyrrolo[3,4-b]pyridine-5,7(6H,7aH)-dione using lipase to prepare the intermediate of moxifloxacin

Article abstract of DOI:10.1016/j.molcatb.2014.10.009

A convenient and efficient route has been successfully developed for preparing (4aR,7aS)-6-benzyltetrahydro-1H-pyrrolo[3,4-b]pyridine-5,7(6H,7aH)-dione through enzyme-mediated kinetic resolution processes under mild and environmentally acceptable conditio

Full text of DOI:10.1016/j.molcatb.2014.10.009

Accepted Manuscript
Title: Convenient enzymatic resolution of
cis-6-benzyltetrahydro-
1
H-pyrrolo[3,4-b]pyridine-5,7(6H,7aH)-dione using lipase to
prepare the intermediate of moxifloxacin
Author: Yuanyuan Li Anming Wang Yingqiang Shen Pengfei
Zhang
PII:
S1381-1177(14)00284-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2014.10.009
MOLCAB 3047
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
10-9-2014
15-10-2014
16-10-2014
Please cite this article as: Y. Li, A. Wang, Y. Shen, P. Zhang, Convenient enzymatic
resolution of cis-6-benzyltetrahydro- 1H-pyrrolo[3,4-b]pyridine-5,7(6H,7aH)-dione
using lipase to prepare the intermediate of moxifloxacin, Journal of Molecular Catalysis
B: Enzymatic (2014), http://dx.doi.org/10.1016/j.molcatb.2014.10.009
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Research highlights:
(
1) The optically pure (4aR,7aS)-1 was successfully prepared efficiently using the enzymatic
resolution.
(
2) The target amine was obtained in conversion (49%) and an excellent degree of enantiomeric
purity (>99%) when Candida antarctica lipase B (CAL-B) was combinated with phenyl allyl
carbonates donors.
(
3) Based on the enzymatic acylation, the chemo-enzymtic dynamic kinetic resolution for
rac-cis-1 to obtain the amine (4aR,7aS)-1 would be approached in the coming future when
racemizing (4aS,7aR)-1 by interchange between carboxide and enol.
1
Page 1 of 29

Convenient enzymatic resolution of cis-6-benzyltetrahydro-
H-pyrrolo[3,4-b]pyridine-5,7(6H,7aH)-dione using lipase to
prepare the intermediate of moxifloxacin
1
1
1, 2
1
1
Yuanyuan Li , Anming Wang *, Yingqiang Shen , Pengfei Zhang
1
College of Materials, Chemistry and Chemical Engineering,
Hangzhou Normal University, Hangzhou 310036, P. R. China
2
Research Center of Bioconversion and Biopurification, Ministry of Education,
Zhengjiang University of Technology, Hangzhou 310014, P. R. China
*Corresponding author at:
College of Material, Chemistry and Chemical Engineering,
Hangzhou Normal University, Hangzhou 310036, P. R. China
TEL: +86-571-28865978
FAX: +86-571-28865630
E-mail: waming@hznu.edu.cn; anmwang@hotmail.com
2
Page 2 of 29

Abstact:
A convenient and efficient route has been successfully developed for preparing
4aR,7aS)-6-benzyltetrahydro-1H-pyrrolo[3,4-b]pyridine-5,7(6H,7aH)-dione through
(
enzyme-mediated kinetic resolution processes under mild and environmentally
acceptable conditions. Different reaction condition factors, including acyl agent,
solvent, reaction temperature, and the concentration of Et
to establish the optimal reaction conditions for the enzymatic kinetic resolution of
secondary amines. It was demonstrated that acyl agent, the concentration of Et N or
3
N, were optimized in order
3
the type of solvent have a greater influence on the selectivity and rate of the
asymmetric acylation reaction in the process of the enzymatic resolution, respectively.
The Target product amine was obtained in good conversion (49%) and an excellent
degree of enantiomeric purity (>99%) when Candida antarctica lipase B (CAL-B)
and phenyl allyl carbonates were combinated to use in the tert-butyl methyl ether
(TBME) medium. This method will be an alternative to the traditional route for
producing the optically pure intermediate of moxifloxacin.
Key Words:
moxifloxacin; Candida antarctica lipase B; enantioselective; asymmetric
acylation; dynamic kinetic resolution
3
Page 3 of 29

1
. Introduction
The optically pure (4aR,7aS)-6-benzyltetrahydro-1H-pyrrolo[3,4-b]
pyridine-5,7(6H,7aH)-dione has attracted considerable attention because it is an
important building blocks in synthesis of (4aS,7aS)-6-benzyloctahydro-1H-
pyrrolo[3,4-b]pyridine, which is commonly used as a crucial intermediate in
the preparation for moxifloxacin or its pharmacentically acceptable salts with
antibacterial effectiveness [1-3]. Particularly, moxifloxacin is one of the most
widely used fourth generation fluoroquinolones antibacterial agents with well
tolerated drug developed by Bayer AG, which can exert their powerful
antibacterial activity by interacting with two essential bacterial enzymes, DNA
gyrase and DNA topoisomerase IV [4-6]. In the preparation of moxifloxacin,
(
4aR,7aS)-6-benzyltetrahydro-1H-pyrrolo[3,4-b]pyridine-5,7(6H,7aH)-dione
(4aR,7aS)-1) is the key chiral intermediate and is often recieved in the ways to
(
split the chemically synthesized amine enantiomers such as recrystallization of
diastereomeric salts [7]. (-)-2,3:4,6-di-O–Isopropyl-idene-2-keto-L-gulonic
acid which is similar to tartaric acid has been reported to be well used in the
separation of the above-mentioned enantiomers [8]. Unfortunately, there are
some drawbacks in the chemical resolution of the enantiomer, such as harsh
reaction conditions (high reaction temperature), poor crystallinity, low yield,
moderate enantiomeric excesses and poor optical purity of products, etc.
Additionally, a large number of organic solvents are required which may cause
4
Page 4 of 29

a great waste and environmental pollution.
As the most common biocatalysis for enantioselective transformation,
enzymatic catalysis possesses many remarkable advantages such as high
enantioselectivity, mild reaction condition, small amount of organic solvent,
and the ability to be recycled by the simple separation of the immobilized
enzyme [9, 10]. Now, the methods for the resolution of chiral secondary amines
have been developed by using the specific enzymes such as aminoacylases [11],
proteases [12, 13] and amino oxidases [14, 15] which could be obtained by
direct evolution. As known to all, lipases among the enzymes of synthetic
interest usually were the most effective tool for the enzymatic resolution
synthesis of enantiomerically pure compounds over the last two decades
because of their outstanding merits of operability, low cost, and excellent
tolerance of a broad range of racemic substrates [16-18]. Generally, most
lipase-mediated kinetic resolution reactions involved acylation of chiral
alcohols [19-23] or hydrolysis of esters [24-26], while in few reports the
enzyme was used to prepare chiral primary and secondary amines despite the
fact that gentle reaction conditions are required for the fomation of these
nitrogenous compounds [27-31].
However, lipase was seldom used to catalyze the kinetic acylation or
alkoxycarbonylation of chiral cyclic secondary amines, even though they are
useful building blocks for preparing many valuable products [32-41]. This may
be due to the structure of the cyclic secondary amines which could cause the
5
Page 5 of 29

steric hindrance of substrate access to the enzyme active site [13].
In this work, several commercially available lipases were screened to
identify the ideal enzyme with the desired reactivity and enantioselectivity in
the acylation of the cyclic secondary amine enantiomers (Scheme 1). The
corresponding immobilized enzyme of screened lipase was further applied in
the enzymatic process to isolate the intermediate (cyclic secondary amines) of
moxifloxacin in an enantiomerically pure form. A comprehensive optimization
of the reaction parameters in terms of the temperature, solvent and acyl donor
was proceeded in order to obtain the high conversion rate and enantiomeric
excesses in the chiral separation process of the racemic intermediate (rac-cis-1)
of moxifloxacin.
2
. Experimental
2
.1.General
Racemic cis-6-benzyltetrahydro-1H-pyrrolo[3,4-b]pyridine-5,7(6H,7aH)-
dione as a gift was given by Zhejiang Apeloa Kangyu Pharmaceutical Co., Ltd..
Candida antarctica lipase type B (CAL-B, Novozyme 435, ≥5,000 U/g)，
Candida antarctica lipase type A (CAL-A, ≥5,00 U/g), Candida antarctica
lipase (CAL, ≥1.5 U/mg), lipase from Porcine pancreas type II, (PPL, 30-90
units/mg protein using triacetin) and lipase from Candida rugosa type VII,
(CRL, ≥700 unit/mg) were purchased from Sigma–Aldrich. rac-cis-1 was
stored over P O at 0 °C. Chemical reagents were used as received from
2
5
6
Page 6 of 29

commercial sources and used without further purification. Solvents were
distilled over an adequate desiccant under nitrogen. Anhydrous solvents were
stored over freshly reactivated 4 Å molecular sieves.
1
13
H NMR and C NMR spectra were recorded on a Bruker 500 MHz or
Bruker 400 MHz spectrometer at ambient temperature with CDCl or
3
DMSO-d as solvent unless otherwise noted and tetramethylsilane (TMS) was
6
1
used as the internal standard. H NMR data were reported as follows: chemical
shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd =
double– doublet, m = multiplet and br = broad), coupling constant ( J values,
13
Hz). C NMR data were reported in terms of chemical shift ( δ ppm). Mass
spectra were determined using an Agilent 1100 series GC/MSD Trap
instrument. High resolution mass spectra (HRMS) were determined using an
Agilent 6210 TOF LC/MSD Trap instrument. Analytical thin layer
chromatography (TLC) was performed on Merck precoated TLC (silica gel 60
F254) plates. Flash chromatography was performed using silica gel 60
(230–240 mesh). Measurement of the optical rotation was done in a
Perkin–Elmer 241 polarimeter. High performance liquid
chromatography(HPLC) analysis was carried out in a Waters 715 Ultra Wisp
UV detector using a Daicel Chiralcel IC column (25 cm × 4.6 mm I.D.)( 90%
n-hexane/2-propanol; 1.0 mL/min; 254 nm; 35 °C for rac-cis-1 and 80%
n-hexane/ethanol; 1.0 mL/min; 254 nm; 35 °C for rac-cis-3). Retention times
were as follows: for 1 [(4aR,7aS)-1 20.0 min, (4aS,7aR)-1 23.0 min], 3
7
Page 7 of 29

[(4aR,7aS)-3 12.8 min, (4aS,7aR)-3 15.0 min].
2
.2.Chemical synthesis of cis-allyl 6-benzyl-5,7-dioxooctahydro-1H-pyrrol-
o[3,4-b]pyridine-1-carboxylate rac-cis-3
Pyridine (14.5 uL, 0.18 mmol) was added to a solution of rac-cis-1 (244
mg, 1mmol) in dry CH Cl (2.5 mL) in a nitrogen atmosphere and the mixture
2
2
was cooled in an ice bath. Allyl chloroformate (130 uL, 1.1 mmol) was added
dropwise and the system was allowed to warm up to room temperature. After
stirring for 4 h, no starting material was detected by TLC analysis. The solvent
was evaporated under reduced pressure and the residue obtained was purified
by flash chromatography (10% EtOAc/hexane) to make 300 mg of carbamate
1
rac-cis-3 as a pale yellow oil (yield: 92%). Rf (10% EtOAc/hexane): 0.4; H
NMR (CDCl , 400 MHz): δ 7.29-7.36 (m, 5H), 5.93-5.94 (m, 1H), 5.22-5.38
3
(m, 3H), 4.66 (s, 4H), 3.92-4.03 (m, 1H), 2.77-3.02 (m, 2 H), 2.05 (s, 1H), 1.49
13
(
s, 3H)； C NMR (CDCl , 100 MHz): δ 176.85, 174.89, 156.17, 135.51,
3
1
2
32.51, 128.81, 128.73, 128.09, 117.88, 66.78, 52.95, 42.44, 41.50, 38.87,
+
4.28, 20.52; GC-MS: (m/e, 328.1, M+); HRMS(ESI , m/z) calcd for
+
+
(
C H N NaO ) (M + Na) 351.1315, found 351.1330. HPLC elution times:
18 20 2 4
(4aR,7aS)-3 12.8 min, (4aS,7aR)-3 15.0 min; conditions: Daicel Chiralcel IC
column (25 cm × 4.6 mm I.D.), eluent n-hexane/ethanol (80:20) at 1.0 mL/min
flow rate.These data are in accordance with the values reported in the literature.
2
.3.Typical procedure for the enzymatic kinetic resolution of racemic
cis-6-benzyltetrahydro-1H-pyrrolo[3,4-b]pyridine-5,7(6H,7aH)-dione rac-
8
Page 8 of 29

cis-1
Amine rac-cis-1 (49mg, 0.2mmol), and phenyl allyl carbonate (89mg,
.5mmol) were dissolved in dry TBME (2mL); 4 Ǻ molecular sieves (100mg)
0
and Et N (150uL, 1mmol) were added. The reaction was started by the addition
3
of Candida antarctica lipase type B (CAL-B, Novozyme 435) (100 mg) and
was shaken at 250 rpm on a rotary shaker at 45 °C for the necessary time to
achieve a good kinetic resolution. The progress and ee (%) of the reaction were
monitored by TLC and HPLC analysis. The reaction was stopped at close to
5
0% conversion by removal of the enzyme and molecular sieves and washed
with TBME (2mL). The TBME organic layer was washed with 2 M HCl (2 ×
mL), and the combined acid was then washed with dichloromethane (2 mL).
5
The pH of the aqueous layer was then adjusted to 9 with ammonia water (25%)
and then extracted with dichloromethane (2 × 3mL). The organic phase was
dried over Na SO and evaporated to obtain (4aS,7aR)-1 as a light yellow oil
2
4
(20mg, yield: 41%, ee: 97%). The TBME layer was mixed with 2 M NaOH (5
mL), dried over MgSO , and evaporated to obtain carbamate (4aR,7aS)-3 as a
4
pale yellow oil (19mg, yield: 39%, ee: >99%).
2
.4.Hydrolysis of carbamate (4aR,7aS)-3
A mixture of (4aR,7aS)-3 (0.1mmol) and concentrated hydrochloric acid
(1mL) was stirred refluxing at 110 ℃ for 5 h. After cooling to room
temperature, 2mL H O was added into the mixture and washed with
2
dichloromethane (2 × 2mL). The aqueous layer was then adjusted PH to 9 with
9
Page 9 of 29

ammonia water (25%) and then extracted with dichloromethane (2 × 3mL). The
organic phase was dried over Na SO and evaporated to obtain the crude
2
4
product (4aR,7aS)-1 as a light yellow oil (22mg, yield: 92%, ee: >99%). [α] =
+
1
-
25.10 (C = 5, EtOH); GC-MS: (m/e, 244.1, M ); H NMR (CDCl , 500 MHz):
3
δ7.25-7.36 (m, 5H), 4.64 (s, 2H), 3.82-3.84 (d, 1H, J=6.9Hz), 2.83-2.87 (dd,
1
1
1
2
H), 2.76-2.80 (m, 1H), 2.63-2.68(m, 1H), 2.20(br s, 1H), 1.92-1.99 (m, 1H),
1
3
.61-1.68 (m, 1H), 1.48-1.52 (m, 2H)； C NMR (CDCl , 100 MHz): δ177.94,
3
77.78, 135.84, 128.65, 128.40, 127.85, 55.39, 42.29, 42.12, 39.23, 23.02,
2.34. These data are in accordance with the values reported in the literature
[1].
3
. Result and discussion
All reactions were performed with the strict exclusion of water and
molecular sieves were added to absorb any traces of moisture as well as the
liberated alcohol. Primarily, the higher moisture of the reaction system was, the
fewer available acylating agent remained and as a result the reaction rate was
slow down. The adventitious hydrolysis of carbonate releases an acid, which
could inhibit the enzyme activity or cause the substrate protonation that was
unfavorable for reaction. It is worth mentioning that some activated esters are
partly hydrolyzed for the presence of water in the seemingly dry enzyme
preparation, always giving some acid in the reaction mixture. Thus, in all
experiments triethylamine was used as the non-nucleophilic base to neutralize
1
0
Page 10 of 29

the released acid to enhance the reaction rate and the enantioselectivity of the
process.
3
.1.Screen of enzyme
The enzymatic kinetic resolution of rac-cis-1 was then carried out using
lipase from different sources such as Candida antarctica lipase (CAL),
Candida antarctica lipase A (CAL-A), Candida antarctica lipase B (CAL-B),
Porcine pancreas lipase (PPL) and Candida rugosa lipase (CRL). Initially, we
focused on the enzymatic acylation of rac-cis-1 for enzyme screening to select
the most suitable lipase. The enol esters were chosen as the acyl donor because
of their fast rate of enzymatic acylation. The tautomerism of the enol to ketone
means that the process is irreversible and the separation of product after the
reaction is simple [36, 42]. Thus, vinyl acetate was used as the acyl donor in
toluene. Some of these lipases were suited for catalyzing the synthesis of the
amide, while the amine that remained was racemic and the reaction mixture
became brown because high background reactions would occur when these
enol esters were used as acylating agents. Moreover, many others activated
esters, including 2,2,2-Trifluoroethyl butyrate and ethyl acetate, which are
well-known in the enzyme-catalyzed acylation of alcohols and aliphatic amines
[
9], were also selected as acyl donors. Unfortunately, these acyl donors did not
allow the formation of the corresponding amides for the chiral secondary amine
4aR,7aS)-1 in our work. Based on the previous process that showed excellent
results to the enzymatic resolution of chiral secondary amines, in which allyl
(
1
1
Page 11 of 29

carbonates was used as alkoxycarbonylating reagent [12, 36, 38-41], we turned
our attention to the study of the alkoxycarbonylation reaction of rac-cis-1.
When phenyl allyl carbonate was selected as the alkoxycarbonylating
reagent for an initial enzyme activity screening in order to increase the rate and
selectivity, as shown in Table 1. PPL, CAL-A and CRL did not show any
acylating activity, while CAL-B showed a high enantioselectivity (>99%) and a
conversion of 4.56% to form the carbamate (4aR,7aS)-3 after 120 h (entry 1).
Candida antarctica lipase (CAL) was also used to catalyze the
biotransformation but even lower conversions than CAL-B and moderate
enantioselectivity were observed (entry 2). For CAL-B, the high
enantioselectivity for the chiral secondary amine (4aR,7aS)-1 was the key for
the subsequent resolution of the racemic intermediate (rac-cis-1).
3
.2.Effect of solvent and reaction temperature on the enzyme kinetic
resolution
The effect of some solvents with different polarities, including dimethyl
formamide (DMF), acetonitrile, ethanol, acetone, tetrahydrofuran,
dichloromethane, tert-butyl methyl ether (TBME), diisopropyl ether and
toluene, on the enzymatic kinetic resolution was investigated. As presented in
Table 2, TBME was the most efficient medium in carrying out the enzymatic
reaction. In this medium, both a higher conversion and a high enantioselectivity
were obtained. The DMF with high solvent polarity may lead to enzyme
denaturation through depriving the water from the surface of enzyme (entry 1).
1
2
Page 12 of 29

Other solvents, such as acetonitrile, acetone, tetrahydrofuran, dichloromethane,
and toluene led to low conversion (entries 2, 4-6, 9) and ethanol did not
promote the product formation (entry 3). The catalytic capacity of the
immobilized enzyme was essentially the same in moderate polar solvents,
including TBME and isopropyl ether. The conversions in the two mediums
were close to 50% along with excellent enantioselectivity. Nevertheless, the
shorter reaction time was obtained in TBME, which may result from TBME’s
higher dissolving capacity to the substrate allowing the possibility of increasing
the mass transfer. Thus, TBME is a good candidate for optimizing the reaction
conditions of the enzymatic resolution of rac-cis-1. In order to improve the
activity of enzyme, the effect of the temperature on the acylation was also
examined (Table 2). The results showed that the highest conversion, reaction
rate and ee values were observed when the temperature of the reaction mixture
p
was at 45 ℃ (entry 11).
3
.3.Effect of alkoxycarbonylating reagents and their concentrations on the
conversion
Various alkoxycarbonylating reagents were introduced into the CAL-B
catalytic reaction for the acylation the rac-cis-1 in TBME(Scheme 2). The
results in Table 3, showed that no corresponding amide product was formed
when the diallyl carbonate 2b was used as the alkoxycarbonylating reagent
(entry 2). However, nearly 50% of the conversion rate was yielded using the
1
3
Page 13 of 29

other three asymmetrical carbonates (entries 1, 3, 4). In addition, excellent
enantioselectivities (>99%) were all observed when using these three efficient
alkoxycarbonylating reagents. The appearance of the phenol by-product did not
cause enzyme inhibition and it could be removed easily with a base wash at the
end of the resolution. Noteworthily, the cost of the phenyl allyl carbonate 2a is
even lower than that of carbonates 2c and 2d.
As we well know, the concentration of the acyl donor is a key factor for the
reaction rate. Thus, the effect of the amount of acyl donor on the enzyme
catalytic reaction was investigated closely. The results were showed in Table 3.
Originally, a small amount of carbonate was used in acylation of substrate in
order to avoid any possible formation of by-products and reduce inhibitiory
effects because of high concentrations of reactants. When the amount of 2a was
added up to 2.5 equivalents, around 50% of the conversion was obtained with
excellent enantioselectivities (entry 1). However, the inhibitory effects at high
concentration were observed and the conversion rate decreased distinctly
(entries 6 and 7) when 5 and 10 equivalent amount of acyl donors were used.
3
.4.Effect of the concentration of Et N on the acylation reaction rate
3
To furthuer optimise the reaction coditions, the effect of the concentration
of Et N on the acylation reaction rate was developed (Figure 1). Interestingly,
3
with the increase of the amount of extra Et N, the reaction became
3
progressively faster, and both the enantiomeric excess (99%) and the
1
4
Page 14 of 29

enantioselectivity (>200) remained high. In some papers, it was reported that
the by-product acid could inhibit the enzyme or substrate protonation that was
unfavorable for acylation reaction [43-45]. Thus, we speculated that the base
has the ability to neutralize the by-product acid which was released from the
adventitious hydrolysis of carbonate by water in the seemingly dry enzyme
preparation.
3
.5.Carbamate hydrolysis
The catalytic hydrolysis of acid and base are two classic approaches to
hydrolyze carbamate. Here, common acid and base were used as the catalysts
to hydrolyze the carbamate group. We found that no any (4aR,7aS)-1 was
obtained when the sodium hydroxide (2 M) base-catalyzed hydrolysis of the
amide was under a high temperature. It was possible that excessive or improper
hydrolysis through sodium hydroxide occurred and led to the hydrolysis of
other amide chemical bond in the (4aR,7aS)-3 instead of only the carbamate
group. However, the proper and desired hydrolysis of the amide could be
achieved by refluxing with concentrated hydrochloric acid (12 M), resulting in
up to 92% high yields. Therefore, concentrated hydrochloric acid can
effectively solve the problem that hydrolyzing the amide (4aR,7aS)-3 to release
the amine (4aR,7aS)-1.
Conclution
1
5
Page 15 of 29

In this work, a practical synthetic route was presented for the production of
optically active secondary amine (4aR,7aS)-1 through enzymatic kinetic
resolution using the immobilized CAL-B. The optimal conditions for the
enzymatic resolution is the combination of the immobilized CAL-B with
phenyl allyl carbonates at 45℃ in TBME and a reaction mixture containing the
substrate and Et N (molar ratio, 1:5). After the hydrolysis of (4aR,7aS)-3, the
3
target product (4aR,7aS)-1 was successfully obtained with a good conversion
(49%) and an excellent degree of enantiomerical purity (99%). In conclusion,
this proposed way proved to be simple, effective and more practical, which is
expected to be an alternative to those previously described traditional methods
in producing optically active secondary amine (4aR,7aS)-1 after further
scale-up experiment. In addition, (4aS,7aR)-1, which couldn’t be acylated using
CAL-B, could be isolated and further catalyzed to (4aR,7aS)-1 to form the
enantiomer mixtures again. Based on this transformation, our work will focus
on the chemo-enzymtic dynamic kinetic resolution of rac-cis-1 to obtain the
amine (4aR,7aS)-1 in the coming future.
Acknowledgements
This study was supported by the National Natural Science Foundation of China
(20906016)，Natural Science Foundation of Zhejiang Province (LY13B060008),
the China Postdoctoral Science Foundation (2012M521198, 2013T60606), the
Technology Research and Development Program of Hangzhou (20120232B13),
1
6
Page 16 of 29

the special funds for Key-Sci-Tech Innovation Team of Zhejiang Province (No.
010R50017) and the Program for Changjiang Scholars and Innovative
2
Research Team in Chinese Universities (IRT1231). Thanks are due to the Prof.
Yuguo Zheng for the valuable discussions and to Shayne X. Wang, Ph.D. from
Canada and Stephen G. Wu from Washington University in St. Louis for
assistance in revising the manuscript.
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0
Page 20 of 29

Figure legends
Scheme 1 Enzymatic kinetic resolution of rac-cis-1 using phenyl allyl carbonate.
Scheme 2 Enzymatic kinetic resolution of rac-cis-1 using different allyl carbonates.
Solvent, TBME; molar ratio of amine versus carbonate, 1:2.5; reaction
temperature, 45 ℃.
3
Figure Effect of the substrate-to-Et N molar ratio on the enzymatic resolution.
Reaction conditions: rac-cis-1 (0.2mmol); CAL-B (50mg/ml); phenyl allyl
carbonate (0.5mmol); TBME (2 mL); 45 ℃; 250 rpm; 4 Ǻ molecular sieve
(
100mg). (A) substrate-to-Et
molar ratio, 1:1; (C) substrate-to-Et
substrate-to-Et N molar ratio, 1:10.
3
N molar ratio, 1: 0 ; (B) substrate-to-Et
3
N
3
N
molar ratio, 1:5; (D)
3
2
1
Page 21 of 29

Table captions
Table 1 Alkoxycarbonylation of rac-cis-1 with phenyl allyl carbonate catalyzed by
various lipases
Table 2 CAL-B enzymatic kinetic resolution of rac-cis-1 with phenyl allyl carbonate
using different solvents
Table 3 Enzymatic kinetic resolution of rac-cis-1 with different carbonates
2
2
Page 22 of 29

a
Table 1 Alkoxycarbonylation of rac-cis-1 with phenyl allyl carbonate catalyzed by various lipases
b
b
c
d
Entry
Enzyme
CAL-B
CAL
t (h)
120
144
ee
s
(%)
5
ee
p
(%)
c (%)
E
1
2
>99
56
5
2
>200
3
1
a
Reaction conditions: rac-cis-1 (0.2mmol); enzyme (50mg/ml); phenyl allyl carbonate (0.5mmol); Et
mmol); toluene (2 mL); 37 ℃; 150 rpm; 4 Ǻ molecular sieve (100mg).
Calculated by HPLC.
3
N (150uL,
1
b
c
S S P
c=ee /(ee +ee ).
d
S S
E= ln[(1- c)*(1-ee )]/ln[(1-c)*(1 + ee )].
2
3
Page 23 of 29

a
Table 2 CAL-B kinetic resolution of rac-cis-1 with phenyl allyl carbonate using different solvents
0
b
b
c
d
Entry Solvent
Log P
-1.0
-0.33
-0.24
0.29
0.49
1.2
T ( C) t (h)
ee
s
(%)
ee
p
(%)
c (%)
—
7
E
1
2
3
4
5
6
7
8
9
1
Dimethyl formamide
37
37
37
37
37
37
37
37
37
30
45
22
22
22
22
22
22
22
58
120
22
22
—
7
—
—
Acetonitrile
>99
—
>200
—
Ethanol
—
2
—
2
Acetone
>99
>99
>99
>99
>99
>99
>99
>99
>200
>200
>200
>200
>200
>200
>200
>200
Tetrahydrofuran
Dichloromethane
Tert-butyl methyl ether
Diisopropyl ether
Toluene
10
1
9
1
1.35
1.9
90
87
5
47
47
5
2.5
0
Tert-butyl methyl ether
Tert-butyl methyl ether
1.35
1.35
69
97
41
49
11
a
Reaction conditions: rac-cis-1 (0.2mmol); CAL-B (50mg/ml); phenyl allyl carbonate (0.5mmol); Et
150uL, 1mmol); solvents (2 mL); 250 rpm; 4 Ǻ molecular sieve (100mg).
Calculated by HPLC.
3
N
(
b
c
S S P
c=ee /(ee +ee ).
d
S S
E= ln[(1- c)*(1-ee )]/ln[(1-c)*(1 + ee )].
2
4
Page 24 of 29

a
Table 3 Enzymatic kinetic resolution of rac-cis-1 with different carbonates
e
b
b
c
d
Entry Acyl donor
Ratio
t (h)
22
22
22
22
22
22
22
ee
s
(%)
ee
p
(%)
c (%)
49
E
1
2
3
4
5
6
7
2a
2b
2c
2d
2a
2a
2a
1:2.5
97
—
93
92
53
43
26
>99
—
>200
—
1:2.5
1:2.5
1:2.5
1:1
—
>99
>99
>99
>99
>99
48
>200
>200
>200
>200
>200
48
35
1:5
30
1:10
21
a
Reaction conditions: rac-cis-1 (0.2mmol); CAL-B (50mg/ml); Et
50 rpm; 4 Ǻ molecular sieve (100mg).
Calculated by HPLC.
3
N (150uL, 1mmol); TBME (2 mL);
2
b
c
S S P
c=ee /(ee +ee ).
d
e
S S
E= ln[(1- c)*(1-ee )]/ln[(1-c)*(1 + ee )].
The ratio of amine/diallyl carbonate in molar.
2
5
Page 25 of 29

*Graphical Abstract (for review)
Page 26 of 29

Scheme 1
Page 27 of 29

Scheme 2
Page 28 of 29

Figure 1
Page 29 of 29