Tandem dynamic kinetic resolution and enzymatic polycondensation to synthesize mPEG-functionalized poly(amine-co-ester)-type chiral prodrugs

Article abstract of DOI:10.1002/pola.26594

Amphiphilic poly(amine-co-ester)s, which contain a single effective enantiomer of an asymmetric drug and thus can avoid potentially serious side effects, are difficult to prepare through nonselective chemical routes not only in the process of introducing chiral drugs to the polymer, but also in the synthesis of the polymer's backbone by metal catalysts. A model of racemic mexiletine, an important antiarrhythmic agent, was used to demonstrate the tandem combination of Candida antarctica lipase B (CAL-B)- and Pd/C-catalyzed dynamic kinetic resolution (DKR) and subsequent CAL-B-catalyzed polycondensation, as an efficient protocol to prepare poly(ethylene glycol)-functionalized poly(amine-co-ester)s containing (R)-mexiletine with 99% ee value. Chemoenzymatic DKR and enzymatic polymerization conditions were optimized, and the optical purity of incorporated (R)-mexiletine was confirmed through its hydrolysis from polyester. The copolymers can readily self-assemble into nanometer-scale-sized micelles with well-dispersed spheres, which have a size distribution that can be efficiently adjusted by changing the polymer concentration. 2013 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013 An efficient protocol to prepare amphiphilic poly(amine-co-ester) containing (R)-mexiletine with 99% ee by combining Candida antarctica lipase B/(Pd/C)-catalyzed dynamic kinetic resolution and subsequent polycondensation was developed. The copolymers can readily self-assemble into nanometer-scale-sized micelles with well-dispersed spheres, and their size distribution can be efficiently adjusted by changing the polymer concentration. Copyright

Full text of DOI:10.1002/pola.26594

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Tandem Dynamic Kinetic Resolution and Enzymatic Polycondensation to
Synthesize mPEG-Functionalized Poly(amine-co-ester)-Type Chiral Prodrugs
Xueqi Qian, Zhengyang Jiang, Xianfu Lin, Qi Wu
Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China
Correspondence to: Q. Wu (E-mail: llc123@zju.edu.cn)
Received 29 July 2012; accepted 6 January 2013; published online 14 February 2013
DOI: 10.1002/pola.26594
ABSTRACT: Amphiphilic poly(amine-co-ester)s, which contain a
single effective enantiomer of an asymmetric drug and thus
can avoid potentially serious side effects, are difficult to pre-
pare through nonselective chemical routes not only in the pro-
cess of introducing chiral drugs to the polymer, but also in the
synthesis of the polymer’s backbone by metal catalysts. A
model of racemic mexiletine, an important antiarrhythmic
agent, was used to demonstrate the tandem combination of
taining (R)-mexiletine with 99% ee value. Chemoenzymatic
DKR and enzymatic polymerization conditions were optimized,
and the optical purity of incorporated (R)-mexiletine was con-
firmed through its hydrolysis from polyester. The copolymers
can readily self-assemble into nanometer-scale-sized micelles
with well-dispersed spheres, which have a size distribution that
can be efficiently adjusted by changing the polymer concentra-
C
V
tion.
2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A:
Candida antarctica lipase
B
(CAL-B)- and Pd/C-catalyzed
Polym. Chem. 2013, 51, 2049–2057
dynamic kinetic resolution (DKR) and subsequent CAL-B-cata-
lyzed polycondensation, as an efficient protocol to prepare
poly(ethylene glycol)-functionalized poly(amine-co-ester)s con-
KEYWORDS: CAL-B; dynamic kinetic resolution; enzyme; micelles;
stereospecific polymers
INTRODUCTION Macromolecular prodrugs, which generally
show advantages such as high stability, prolonged half-life,
enhanced permeability and retention (EPR) effect, antigenic-
ity, and low immunogenicity, have attracted increasing atten-
tion from both academia and industry in recent years.1–3
More research has focused on designing new macromolecu-
lar prodrugs for medical applications.4–7 The typical
approaches to incorporating small molecular drugs into mac-
romolecular carriers include various chemically catalyzed
reactions. Most are very effective. However, when the target-
ing molecule contains chiral centers, the chemical route usu-
ally makes it difficult to prepare macromolecular prodrugs
that bear its single effective enantiomer. This is crucial for
many drugs, because two enantiomers of a chiral drug candi-
date often have differences in physiology, pharmacokinetics,
metabolic activities, and toxicology. Furthermore, the use of
toxic metal catalysts, required critical conditions such as
high temperature, and complicated protection/deprotection
steps for the incorporation of drugs through chemical routes
all cause problems of environmental or synthetic effi-
ciency.8,9 Poly(amine-co-ester), which is safe to use and easy
to produce as a nonviral gene vector because of its ability to
condense plasmid DNA via electrostatic interactions to form
polyplexes, is attracting more scientific attention.10 However,
few efficient synthetic methods are available for the prepara-
tion of amino-containing polyesters, primarily because metal
catalysts required for conventional polyester synthesis are
often sensitive to and deactivated by amino groups.11 The
study of more efficient methods to synthesize poly(amine-co-
ester) and selectively introduce one effective enantiomer of a
chiral drug into a macromolecular system has thus become
an important issue for pharmaceutical academia and
industry.
Lipase, because of its excellent selectivity, nontoxicity, ‘‘green’’
energy savings, and other advantages,12 has been widely
used as a powerful tool for the preparation of chiral inter-
mediates and the resolution of racemic drugs.13 Gotor et al.
have demonstrated Candida antarctica lipase B (CAL-B)-cata-
lyzed kinetic resolution (KR) of some pharmacologically im-
portant b-substituted isopropylamines to obtain the amides
with a 98% ee value.14 Koul developed a facile route for the
conversion of (R,S)-naproxen ester to (S)-naproxen (ee >
99%, E ꢀ500) using lipase from Trichosporon sp.15 The com-
bination of the enzymatic resolution and the macromolecular
synthesis may overcome the difficulties described above by
the incorporation of one effective enantiomer of racemic
drugs into a macromolecular system. Our group previously
reported a combinational strategy of lipase-catalyzed KR and
chemically free radical polymerization to prepare nonbiode-
gradable poly(vinyl alcohol) prodrugs of optically active
Additional Supporting Information may be found in the online version of this article.
C
V
2013 Wiley Periodicals, Inc.
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nonsteroidal anti-inflammatory drugs from vinyl ester mono-
mers.16,17 Although KR is useful for preparing optically active
monomers, its yield of the desired isomer is greatly limited
and cannot exceed 50%.
purchased from Sigma. Amano lipase PS-IM was purchased
from Sigma-Aldrich. The lipase catalyst was dried at 25 ꢁC
under 2.0 mmHg for 24 h before use. Pd/C (10% Pd) was
purchased from China National Medicine. Pd/BaSO4 (5% Pd)
was purchased from Aladdin.
The dynamic kinetic resolution (DKR) of racemates by
metal-catalyzed racemization combined with enzymatic KR is
attracting interest, because it is a promising route to over-
come the limitation of KR for the synthesis of optically pure
compounds from racemic mixtures without separating the
undesired enantiomer after the reaction.18–22 Reetz and
Schimossek reported the first example of chemoenzymatic
DKR for the preparation of enantiopure amines,23 obtaining
(R)-N-(1-phenylethyl) acetamide (99% ee) in a 64% yield
after 8 days at 50–55 ꢁC using palladium on charcoal and
CAL-B as catalysts. Kroutil and coworkers24 reported the en-
zymatic DKR for the preparation of enantiopure mexiletine
in 2009. Considering the high efficiency and wide application
of DKR, the powerful combination of enzymatic DKR and
enzymatic polymerization for the preparation of chiral
macromolecules or chiral polymeric prodrugs containing the
effective enantiomer is appealing. Recent pioneer works for
the synthesis of chiral oligomers or polyesters by combining
DKR and enzymatic polymerization have been reported by
Palmans, Heise, and Meijer25,26 and Howdle.27 However,
these works were mainly restricted to the preparation of
general polyester with a chiral center at the backbone or at
the ring-opening initiator. Synthesis of chiral polymeric pro-
drugs has seldom involved combining DKR and enzymatic
polymerization.
Methods
NMR spectra were measured with a Bruker DRX 400 NMR
spectrometer at 300 Hz using CDCl3 as the solvent. IR spec-
tra were recorded on a Nicolet Nexus FTIR 470 spectropho-
tometer. Samples were film-cast in chloroform onto sodium
chloride plates. High-resolution mass spectrometry (HRMS)
was obtained on a Bruker 7-tesla FT-ICR MS equipped with
an electrospray source (Billelica, MA). The number and
weight average molecular weights (Mn and Mw, respectively)
of copolymers were measured by gel permeation chromatog-
raphy (GPC) with a system equipped with refractive-index
detector (Waters 2414) and Waters Styragel GPC columns.
The GPC columns were standardized with narrow dispersity
polystyrene in molecular weights ranging from 6 ꢃ 105 to
500. The mobile phase was THF at a flow rate of 1.0 mL/
min. The enantiomer of (R,S)-MAPP was analyzed using AD-
H column and was detected at 220 nm. The enantiomer of
(R,S)-mexiletine acetamide was analyzed using OD-H column
and was detected at 220 nm.
General Procedure for DKR of Mexiletine
Pd/C-catalyzed DKR
A suspension containing racemic mexiletine 10 mg, Pd/C 10
mg, CAL-B 20 mg, MAP 70 mg, and Et3N 50 lL, in 1 mL
solvent was stirred at 50 ꢁC under 1 atm H2. After 5 days,
the reaction mixture was cooled to room temperature for
high-performance liquid chromatography (HPLC) analysis.
Herein, the resolution of mexiletine was selected as a model.
Mexiletine (1-(2,6-dimethylphenoxy)-2-amino-propane) is
classified as an antiarrhythmic agent, (R)-enantiomer, which
is more potent than its (S)-counterpart in experimental
arrhythmias and in binding studies on cardiac sodium chan-
nels.28,29 However, mexiletine in its racemic form is a risky
treatment for ventricular tachyarrhythmias. The DKR of the
racemic mexiletine with methyl 3-(bis (2-hydroxyethyl)
amino) propanoate (MAP) was achieved under the combina-
tional catalysts system of CAL-B and Pd/C. The obtained
((R)-3-(bis (2-hydroxyethyl) amino)-N-(1-(2, 6-dimethylphe-
noxy) propan-2-yl) propanamide ((R)-MAPP) can be copoly-
merized with divinyl dicarboxylate and poly(ethylene glycol)
(PEG) to prepare amphiphilic polymeric chiral prodrugs, the
micellization ability of which was further investigated
(Scheme 1).
Pd/BaSO4-catalyzed DKR
A suspension containing racemic mexiletine 10 mg, Pd/
BaSO4 10 mg, CAL-B 20 mg, and MAP 70 mg, in 1 mL tolu-
ene was stirred at 70 ꢁC under 1 atm H2. After 3 days, the
reaction mixture was cooled to room temperature for HPLC
analysis.
General Procedure for Synthesis of (R)-MAPP
A suspension containing racemic mexiletine 1 g, Pd/C 1 g,
CAL-B 2 g, MAP 7 g, and Et3N 5 mL, in 30 mL THF was
stirred at 50 ꢁC under 1 atm H2. After 5 days, the reaction
mixture was cooled to room temperature. The solution was
filtered and concentrated. The crude products were purified
by silica gel column chromatography with the mobile phase
of ethyl acetate/methanol (6:1, v/v). The separated yield of
(R)-MAPP was 63%.
EXPERIMENTAL
Materials
Diphenyl ether (99%), tetrahydrofuran (THF) (99%), and
dimethyl sulfoxide (DMSO) were purchased from Sinopharm
Chemical Reagent (China). Polyethylene glycol 1000 mono-
methyl ether was purchased from Fluka. (R,S)-mexiletine
hydrochloride was purchased from Jiangsu Jintan Yabang
Pharmaceutical. Lipase immobilized on acrylic resin from
CAL-B (EC 3.1.1.3, ꢂ10,000 U/g) was purchased from Sigma.
Lipase from porcine pancreas (PPL) was purchased from
Fluka. Lipase Type VII from Candida rugosa (CRL) was
1H NMR (500 MHz, D2O) d 7.05 (d, J ¼ 7.6 Hz, 2H), 7.01–
6.93 (m, 1H), 4.23 (dd, J ¼ 11.1, 6.5 Hz, 1H), 3.77 (ddd, J ¼
16.9, 10.0, 5.7 Hz, 2H), 3.63 (t, J ¼ 6.1 Hz, 4H), 2.86 (dq, J ¼
13.7, 6.9 Hz, 2H), 2.67 (t, J ¼ 6.0 Hz, 4H), 2.43 (t, J ¼ 7.1 Hz,
2H), 2.20 (s, 6H), 1.21 (d, J ¼ 6.6 Hz, 3H). 13C NMR (100
MHz, D2O) d 174.41, 154.21, 131.18, 128.99, 124.68, 74.22,
58.61, 54.71, 50.04, 45.77, 32.41, 16.07, 15.31. IR (/cm):
3292, 2948, 1643, 1552, 1202, 770. HRMS (EI) calcd for
338.2206, found: 338.2203. [a]2D5 ¼ þ21.1ꢁ in CHCl3.
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SCHEME 1 Combinational DKR and enzymatic polymerization for efficient synthesis of PEG-functionalized polyester enantiopure
prodrugs.
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SCHEME 2 Hydrolysis of mPEG-b-(R)-MAE4 and synthesis of (R)-mexiletine acetamide.
Preparation of mPEG-b-(R)-MAE Copolymers
Transmission Electron Microscopy Measurement
The mixture of divinyl diester (5 mmol), (R)-MAPP (5
mmol), and diphenyl ether (2 mL) were placed in a dried
test tube equipped with a magnetic stir bar. After the mix-
ture was stirred for 15 min, CAL-B was added. The tube was
Transmission electron microscopy (TEM) was carried out
with a JEM-1230 TEM at an accelerating voltage of 80 keV. A
drop of the micelle solution was placed onto a 230-mesh
copper grid coated with carbon and then dried in air.
ꢁ
immersed in an oil bath thermostated at 35 C for 18 h in a
Hydrolysis of mPEG-b-(R)-MAE4 and Analysis of Optical
Purity of Mexiletine in Copolymers
vacuum. Then mPEG (1.6 mmol) was added and kept at 75
ꢁC for another 6 h. The polymerization was quenched by
introducing a small amount of chloroform to the mixture.
The insoluble CAL-B was filtered away, and the organic solu-
tion was separated by filtration. The filtrate was dropped
into a large amount of ether to isolate the polymer product.
The resulting oily precipitates were obtained by centrifuga-
tion, then dried in a vacuum. The products were character-
ized by FTIR, GPC, and NMR spectroscopy.
A measured amount of mPEG-b-(R)-MAE4 (200 mg) was
placed in a dry test tube equipped with a magnetic stir bar.
Then 5 mL of THF and 10 mL of hydrochloric acid (12 mol/
L) was added. The tube was immersed in an oil bath ther-
mostated at 100 ꢁC for 6 h. The solution was dried on the
rotary evaporator to obtain the crude mexiletine hydrochlor-
ide. The product was dissolved in 30 mL of chloroform that
contained 5 mL Et3N. Because mexiletine must be derived
using acetyl chloride in order to determine the optical purity
of released mexiletine using HPLC, the acylation reaction of
mexiletine was carried out in room temperature for 30 min
after dropping 3 mL of acetyl chloride into the solution. The
mixture was then washed three times by 30 mL of saturated
NaCl solution. The product was prepared for chiral HPLC
analysis, and the ee value was 93% (Scheme 2).
mPEG-b-(R)-MAE2
1
Yield: 73%. H NMR (400 MHz, DMSO) d 6.97 (d), 6.94–6.83
(m), 4.31 (s), 4.23–4.06 (m), 3.68 (d), 3.63 (s), 3.36 (s), 2.38
(d), 2.38 (d), 2.27 (s), 2.23 (s), 1.58 (d), 1.37 (d). 13C NMR
(101 MHz, CDCl3) d 173.09, 171.65, 154.83, 130.61, 128.86,
123.96, 77.29, 76.97, 76.66, 73.75, 70.43, 61.88, 52.32,
51.11, 45.20, 33.60, 24.08, 17.56, 16.08. Mn (GPC): 2660 g/
mol.
RESULTS AND DISCUSSION
mPEG-b-(R)-MAE4
Yield: 86%. H NMR (400 MHz, CDCl3) d 6.99 (d), 6.95–6.83
1
Chemoenzymatic DKR of Mexiletine
(m), 4.10–3.60 (m), 3.39 (s), 3.15–2.66 (m), 2.46 (s), 2.46–
2.17 (m), 1.56 (s), 1.39 (d), 1.25 (s). 13C NMR (100 MHz,
CDCl3) d 173.55, 154.93, 130.62, 128.85, 123.93, 77.27,
76.95, 76.63, 73.78, 70.45, 61.78, 52.36, 51.18, 45.14, 34.04,
28.97, 28.76, 24.71, 17.61, 16.10. Mn (GPC): 3360 g/mol.
MAP was selected as the acyl donor of mexiletine, because
two hydroxyl groups of MAP can be further used as diols for
the subsequent polycondensation. Palladium complexes such
as Pd/C and Pd/BaSO4 were used as racemization catalysts
of chiral mexiletine. Scheme 1 shows the DKR of racemic
mexiletine with MAP under the racemization by Pd/C and
the transesterification by CAL-B. The influence of organic sol-
vents on the synthesis of (R)-MAPP was investigated.
Unfortunately, MAP was immiscible with many solvents, such
as ether and hexane, because of the high polarity of two
hydroxyl groups. Thus, some solvents commonly used in the
resolution reactions did not show good results (data not
shown). When using Pd/C as the racemization catalyst in the
screened polar solvents (Table 1, entries 1–5), all obtained
ee values were about 99%. Among these solvents, the reac-
tion catalyzed by Pd/C in THF gave the highest yield of 71%
after 120 h. Reactions carried out in 1,4-dioxane, and chloro-
form could give better results than that in 2-methyl-2-buta-
nol or acetone (entries 2, 3, vs. 1, 5 in Table 1). Compared to
Pd/C catalyst, Pd/BaSO4 could more efficiently racemize the
chiral mexiletine to provide a higher yield of 84% in a
Preparation of Micelles
The micelles of mPEG-b-(R)-MAE2 were prepared using a
simple dialysis method. A certain amount of amphiphilic co-
polymer was dissolved in DMSO. Ultrapure water was then
dropped into the organic solvents under vigorous stirring.
The solution was subjected to dialysis against 2000 mL of
distilled water for 48 h using a dialysis tube (molecular
weight cutoff of 3500). The distilled water was replaced ev-
ery 6 h to remove organic solvent. The resulting solution
was finally lyophilized.
Dynamic Light Scattering Measurement
The hydrodynamic size of formed micelles in the aqueous
phase was determined using dynamic light scattering (DLS)
techniques with a Nanoseries (Malvern, UK) zetasizer with
ꢁ
90ꢁ instrument. Measurements were carried out at 25 C.
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TABLE 1 Dynamic Kinetic Resolution of Racemic Mexiletinea
to prepare the final amphiphilic polymer. Generally, the first
step was not necessarily separated or purified. After suitable
reaction time, mPEG was added, and the second transesterifi-
cation was directly carried out under the specific conditions.
For example, when using CAL-B as the catalyst, the first step
was carried out at 35 ꢁC for 18 h, and after the addition of
mPEG, the temperature was increased to 75 ꢁC for 6 h for
further copolymerization. To confirm the success of the first
polycondensation step, the intermediate polymer (R-MAE)
was separated for structure analysis. The average molecule
weight of R-MAE determined by GPC was about 2950 g/mol
(polydispersity ¼ 1.32) (Supporting Information Fig. S5).
Both the separated process and cascade process can provide
the final copolymer.
eep
(%)b
Yield
(%)b
Entriesa
Solvent
1
2
3
4
5
6c
a
Acetone
99
99
99
99
99
92
34
40
46
71
21
84
1,4-Dione
Chloroform
THF
2-Methyl-2-butanol
Toluene
Reaction conditions: racemic mexiletine 10 mg, CAL-B 20 mg, Et3N 50
lL, Pd/C 10 mg, methyl 3-(bis (2-hydroxyethyl) amino) propanoate
(MAP) 70 mg, solvent 1 mL, 50 ꢁC, 120 h, 1 atm H2.
b
Determined by HPLC analysis using AD-H column.
c
In the whole cascade process without intermediate separa-
tion, both solvents must be brought into contact with each
other for the first or second step. Considering the boiling
point and polymerization effect, diphenyl ether was selected.
The influence of the enzyme source on the copolymerization
was also investigated. Of all the lipase tested, CAL-B showed
the highest activity, and the molecule weight of the obtained
polymer was about 3360 g/mol (entry 4, Table 2) (GPC was
shown in Supporting Information Fig. S11). Compared with
CAL-B, CRL, PPL, and PS-IM may be unsuitable for the poly-
merization, as the Mn of the obtained copolymers were not
more than 1800 g/mol (entries 1, 2, 3, Table 2). The polydis-
persities of the polymers were all among 1.1–1.3, which
were moderately narrow. Furthermore, divinyl adipate also
can be used as acyl donor, and the molecular weight of the
forming copolymer (mPEG-b-(R)-MAE2) was a bit lower than
that of divinyl sebacate (entries 4, 5, Table 2) (GPC was
shown in Supporting Information Fig. S7). Under the cataly-
sis of CAL-B, mPEG-b-(R)-MAE2 and mPEG-b-(R)-MAE4 were
obtained in yields of 73 and 86%, respectively.
Conditions: racemic mexiletine 10 mg, Pd/BaSO4 10 mg, CAL-B 20 mg,
MAP 70 mg, toluene 1 mL,70 ꢁC, 72 h, 1 atm H2.
shorter reaction time of 72 h. However, the stereoselectivity
of the Pd/BaSO4-CAL-B system (ee: 92%) was lower than
that of the Pd/C-CAL-B system. The high temperature of rac-
ꢁ
emization (70 C) required for Pd/BaSO4 led to the decrease
of enzymatic stereoselectivity. Finally, the DKR process pro-
vided (R)-MAPP with 99% ee value in a 71% yield.
Enzymatic Polymerization of Poly(amine-co-ester) and
Structure Characterization
To combine the advantages of polymer-drug conjugates and
polymer micelles carriers of drugs,30–32 we focused on the
macromolecular prodrugs micelles, which can provide a
much more effective way to control the level of drug-loading
and the rate of drug release than the general polymer
micelles. An amphiphilic macromolecular prodrug containing
R-mexiletine was designed, and its preparation starting from
(R)-MAPP, divinyl diesters, and mPEG (Mw ¼ 1000) is shown
in Scheme 1.
The chemical structures of the intermediate and final prod-
ucts were characterized by 1H and 13C NMR spectroscopy.
Figure 1 compares 1H NMR spectra of (R)-MAPP, the first
CAL-B-catalyzed polymerization product ((R)-MAE), and final
product (mPEG-b-(R)-MAE4). In the spectrum of (R)-MAPP
[Fig. 1(A)] recorded in D2O, peaks a and g belong to the pro-
tons of benzene ring and methyl group, and peaks p and u
belong to the protons of AN(CH2CH2OH)2. All of the above
specific signals of mexiletine appeared in the spectra of
the obtained (R)-MAE [Fig. 1(B)] and mPEG-b-(R)-MAE4
Enzymatic polymerization was performed in two steps: the
lipase-catalyzed polycondensation, and the sequential trans-
esterification. First, polycondensation of (R)-MAPP and
divinyl diesters provided the polyester (R)-MAE, which con-
tained a vinyl ester group at one end of the chain; mean-
while the byproduct vinyl alcohol was in situ transformed
into aldehyde, pushing forward the equilibrium of transester-
ification. The vinyl ester end of the polyester (R)-MAE was
easily reacted with mPEG through further transesterification
TABLE 2 Enzymatic Synthesis of Amphiphilic Copolymer
a
Diesters
(m)
Mn
Yield
(%)
Entries
Block Copolymer
Lipase
(g/mol)
PDIa
1
2
3
4
5
mPEG-b-(R)-MAE4a
mPEG-b-(R)-MAE4b
mPEG-b-(R)-MAE4c
mPEG-b-(R)-MAE4
mPEG-b-(R)-MAE2
CRL
4
4
4
4
2
1,770
1,470
1,710
3,360
2,660
24
17
21
73
86
1.2
1.3
1.1
1.3
1.2
PPL
PS-IM
CAL-B
CAL-B
a
Determined by GPC.
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FIGURE 1 1H NMR spectra of (A) (R)-MAPP in D2O, (B) (R)-MAE, and (C) mPEG-b-(R)-MAE4 in CDCl3.
[Fig. 1(C)]. In Figure 1(B), the appearance of the peaks f and
h was attributed to protons of ACH2AOA and terminal
methoxy group of mPEG, respectively. The signals of protons
in sebacate or adipate units (peaks b, c, and d) were also
found in the spectra. All these results confirmed the struc-
ture of mPEG-functionalized poly(amine-co-ester). To com-
pletely remove the unreacted mPEG1000 and the first poly-
condensation product (R)-MAE4, the final products were
rigorously separated and purified. 1H NMR spectroscopy
[Fig. 1(C)] of the obtained copolymer gave information to
determine whether the mPEG was removed. The peak of pro-
tons of ACH2OH end group in residual mPEG should appear
at 3.44 ppm. The peak of CH3OA end group (peak h at 3.37
ppm) was clear, but there was almost no peak at 3.44 ppm,
which confirmed the absence of mixed PEG in the copolymer.
Similarly, the vinyl protons at the end group of the first poly-
condensation product (R)-MAE4 [Fig. 1(B)] were not
observed in 1H NMR spectra of copolymers, ruling out the
existence of mixed (R)-MAE4 in the copolymer.
Hydrolysis of Copolymers
After the preparation of amphiphilic polymeric prodrugs con-
taining R-mexiletine, how well the configuration of R-mexile-
tine kept during the polymerization was investigated. The
hydrolysis of mPEG-b-(R)-MAE4 in hydrochloride acid was
performed to identify the configuration of mexiletine in the
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FIGURE 2 Chiral HPLC of acetamide derivatives of racemic mexiletine (A) and released (R)-mexiletine from polymeric prodrug
mPEG-b-(R)-MAE4 (B).
polymeric prodrug. Considering the separation difficulty of
mexiletine using chiral HPLC,33 the derivation of the released
mexiletine was carried out using acetyl chloride to form cor-
responding acetamide, which was analyzed by chiral HPLC
(Fig. 2). The analysis data showed that the ee value of
obtained mexiletine acetamide was greater than 93%, dem-
onstrating that the optical purity of R-mexiletine was kept
stable in the enzymatic polymerization, and the amphiphilic
polymeric chiral prodrugs were successfully developed.
Micellization of Copolymers in Aqueous Solution
The obtained amphiphilic block copolymers, consisting of
hydrophilic mPEG and hydrophobic MAE blocks, can self-
assemble to form micelles in water. Nanoparticles were pre-
pared from the amphiphilic copolymer using a dialysis
method. Specific solvents that could both dissolve the copol-
ymer and were miscible with water, such as DMSO,
were selected. The morphology and size distribution of
copolymer micelles were investigated by TEM. As TEM
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Mexiletine was transformed to the corresponding amide
monomer with high yields and above 99% optical purities.
The optically active drug monomer was copolymerized with
mPEG and divinyl dicarboxylates under the catalysis of CAL-
B. The obtained amphiphilic polymers can self-assemble into
nanometer-scale-sized particles in water. The diameters of
the micelles determined by DLS depend on the polymer con-
centration in DMSO. The chiral HPLC analysis of released
mexiletine demonstrates the stable optical purity of R-mexi-
letine during the polymerization, and successful preparation
of amphiphilic polymeric chiral prodrugs.
FIGURE 3 The TEM image of mPEG-b-(R)-MAE4 self-assem-
blies in water.
shows in Figure 3, the self-assembled micelles of mPEG-b-
(R)-MAE4 in the H2O system were regularly spherical and
well dispersed as individual nanoparticles. In order to know
whether the concentration of copolymer affects the size of
the formed micelles, a series of polymer concentrations from
0.01 to 1 mg/mL in DMSO were studied. The size distribu-
tion was measured by DLS, and its number-size plots are
shown in Figure 4. When the concentration of the copolymer
decreased from 1 to 0.2 mg/mL [Fig. 4(A–C)], the diameter
of micelles was nearly the same, most of which were among
40–60 nm. When the concentration decreased from 0.2 to
0.1 mg/mL [Fig. 4(C,D)], the diameter increased slightly. Fur-
ther decrease of the concentration to 0.01 mg/mL caused
the diameter of nanoparticles to increase to 90–110 nm. In
the DLS intensity size distribution plots (Supporting Informa-
tion Fig. S12), the micellar size similarly increased when
reducing the copolymer concentrations. This result indicates
that the size distribution of the micelles can be adjusted by
changing the concentration of copolymers. Concerning the
effect of concentration on the micellar size, the copolymer
mPEG-b-(R)-MAE2 at the lower concentration possibly had a
slower micellization rate, and the large aggregates formed
were possibly loose compound micelles or micellar clusters,
while the smaller aggregates formed at high polymer concen-
tration were well-defined micelles.34,35 More data regarding
the effect of concentration are needed to confirm this.
CONCLUSIONS
The powerful combination of chemoenzymatic DKR and
lipase-catalyzed polymerization has been demonstrated for
the preparation of macromolecular chiral prodrugs. The DKR
of mexiletine was efficiently achieved using Pd/C or Pd/
BaSO4, which are inexpensive and easy to obtain for
racemization together with CAL-B as the resolution catalyst.
FIGURE 4 The number-size distribution of mPEG-b-(R)-MAE2
self-assemblies in water measured by DLS. The concentrations
of copolymer in DMSO are (A) 1 mg/mL, (B) 0.5 mg/mL, (C) 0.2
mg/mL, (D) 0.1 mg/mL, and (E) 0.01 mg/mL.
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17 X. Q. Cai, N. Wang, X. F. Lin, Polymer 2006, 47, 6491–6495.
ACKNOWLEDGMENTS
18 B. Martin-Matute, J. E. Backvall, Curr. Opin. Chem. Biol.
2007, 11, 226–232.
The financial support of this work is from the National Natural
Science Foundation of China (No.20874086, No.20704037).
19 Y. Kim, J. Park, M. J. Kim, Chemcatchem. 2011, 3, 271–277.
20 Y. Sato, Y. Kayaki, T. Ikariya, Chem. Commun. 2012, 48,
3635–3637.
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