Regioselective synthesis of amphiphilic metoprolol-saccharide conjugates by enzymatic strategy in organic media

Article abstract of DOI:10.1016/j.procbio.2010.07.028

An efficient protocol to prepare metoprolol-saccharide conjugates by a selective enzymatic synthesis method was developed. Firstly, the transesterification of metoprolol with three divinyl dicarboxylates (divinyl succinate, divinyl adipate and divinyl sebacate) was performed. The influences of organic solvents, sources of enzymes and acylating reagents on the synthesis of N-(vinyloxycarbonyl)metoprolol were investigated. A series of lipophilic metoprolol derivatives with vinyl group were obtained by using a lipase from porcine pancreas (PPL) in anhydrous tetrachloromethane at 50 °C. Subsequently, alkaline protease from Bacillus subtilis catalyzed highly regioselective acylation of three monosaccharides (glucose, mannose and galactose) and two disaccharides (maltose and sucrose) with N-(5- vinyloxycarbonylpentanoyl)metoprolol in anhydrous pyridine at 50 °C to give metoprolol-saccharide conjugates in good yields. The partition coefficients of the products were investigated. The results indicated that the aqueous solubility of metoprolol-monosaccharide and metoprolol-disaccharide conjugates was improved markedly compared with the parent drug of metoprolol, and the aqueous solubility of metoprolol-disaccharide conjugates was much better than that of metoprolol-monosaccharide conjugates.

Full text of DOI:10.1016/j.procbio.2010.07.028

Process Biochemistry 46 (2011) 123–127
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Regioselective synthesis of amphiphilic metoprolol–saccharide conjugates by
enzymatic strategy in organic media
Cheng-Zhen Zheng, Jun-Liang Wang, Xia Li, Bo-Kai Liu, Qi Wu, Xian-Fu Lin∗
Department of Chemistry, Zhejiang University, Hangzhou 310027, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 April 2010
Received in revised form 26 June 2010
Accepted 23 July 2010
An efficient protocol to prepare metoprolol–saccharide conjugates by a selective enzymatic synthesis
method was developed. Firstly, the transesterification of metoprolol with three divinyl dicarboxylates
(divinyl succinate, divinyl adipate and divinyl sebacate) was performed. The influences of organic sol-
vents, sources of enzymes and acylating reagents on the synthesis of N-(vinyloxycarbonyl)metoprolol
were investigated. A series of lipophilic metoprolol derivatives with vinyl group were obtained
by using a lipase from porcine pancreas (PPL) in anhydrous tetrachloromethane at 50 ◦C. Subse-
quently, alkaline protease from Bacillus subtilis catalyzed highly regioselective acylation of three
monosaccharides (glucose, mannose and galactose) and two disaccharides (maltose and sucrose) with N-
(5-vinyloxycarbonylpentanoyl)metoprolol in anhydrous pyridine at 50 ◦C to give metoprolol–saccharide
conjugates in good yields. The partition coefficients of the products were investigated. The results
indicated that the aqueous solubility of metoprolol–monosaccharide and metoprolol–disaccharide con-
jugates was improved markedly compared with the parent drug of metoprolol, and the aqueous solubility
of metoprolol–disaccharide conjugates was much better than that of metoprolol–monosaccharide con-
jugates.
Keywords:
Metoprolol
Enzymatic synthesis
Metoprolol–saccharide conjugates
Regioselective
Solubility
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
However, chemical routes for the synthesis of those derivatives,
especially their selective modifications, are complicated because
of specific protection/de-protection steps. So exploring of new
Metoprolol,
1-(isopropylamino)-3-[4-(2-methoxyethyl)phe-
noxy]propan-2-ol, is a typical ␤-blocker, which has remarkable
efficacy in angina pectoris, hypertension, cardiac arrhythmias,
migraine headaches and other disorders related to sympathetic
nervous system [1]. However, drug candidates in developmen-
tal stage currently exhibit some limitations, such as very low
and highly variable bioavailability [2,3], and rapid elimination
with half-life between 3 and 4 h [4], which strongly restrict
their applications in clinic. To overcome those drawbacks, much
attention has been paid to searching for new derivatives and
extensive modifications of metoprolol [5,6]. Among the deriva-
tives, metoprolol–saccharide conjugates have attracted particular
interest [7]. The combinations of drugs and carbohydrates for parts
of their therapeutic actions have extended a wide range of drugs
[8–11]. Some antitumor drugs and vaccines have been modified by
galactose or lactose to improve their bioavailability [12–15]. The
properties of porphyrin–saccharide conjugates have been proved
to possess the abilities of targeting and incapacitating on cancer
cells [16].
approaches is highly demanded. The enzymatic method is a bet-
ter protocol due to its high regioselectivity under mild conditions
[17]. Since Klibanov and co-workers first demonstrated selec-
tive acylation of monosaccharides catalyzed by lipases in organic
media [18], various studies concerning similar biotransformations
have been reported [19,20]. Carbohydrates bearing vinyl esters
can offer a new family of functional water-soluble monomers
for preparing drug–saccharide conjugates by enzyme-catalyzed
selective transformations. Especially divinyl dicarboxylates, which
have been proved to be useful acylating reagents, have higher
transesterification reactivity for the synthesis of sugar/drugs vinyl
ester derivatives and drug–saccharide conjugates [21–24]. In addi-
tion, the aqueous solubility of drug–saccharide conjugates is still
a valuable and interesting topic, which would provide available
information for further investigation of the therapeutic benefit of
drugs and their derivatives.
In this paper, based on our previous work on enzymatic mod-
ification of pharmaceutics [25–28], three N-(vinyloxycarbonyl)
metoprolol monomers were prepared by PPL in CCl4. The obtained
derivatives were subjected to highly regioselective enzymatic
transformations with three monosaccharides (glucose, mannose
and galactose) and two disaccharides (maltose and sucrose) for the
∗
Corresponding author. Tel.: +86 571 87951588; fax: +86 571 87952618.
E-mail address: llc123@zju.edu.cn (X.-F. Lin).
1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.07.028

124
C.-Z. Zheng et al. / Process Biochemistry 46 (2011) 123–127
Scheme 1. Enzymatic synthesis of N-(vinyloxycarbonyl)metoprolol.
synthesis of metoprolol–saccharide conjugates (Scheme 1). And the
aqueous solubility of five kinds of metoprolol–saccharide conju-
gates was also studied.
7.26 (dd, 1H, J = 6.6 Hz, J = 13.8 Hz, –CH ), 7.13 (d, 2H, J = 8.4 Hz, Ar–H), 6.83 (d,
2H, J = 8.8 Hz, Ar–H), 5.51 (1H, OH), 4.87 (dd, 1H, J = 13.8 Hz, CH2), 4.57 (dd, 1H,
J = 6.0 Hz, CH2), 4.11–3.99 (m, 3H, NCH2 and NCH(CH3)2), 3.80 (d, 1H, J = 8.4 Hz,
CHOH), 3.62–3.54 (m, 3H, OCH2CH2 and OCH2CH), 3.40 (d, 1H, J = 14.8 Hz, OCH2CH),
3.35 (s, 3H, CH3O), 2.81 (t, 2H, J = 7.0, Ar–CH2), 2.42 (q, 4H, J = 9.2 Hz, CH2), 1.73 (s, 4H,
CH2), 1.26 (d, 3H, J = 6.4 Hz, CH3), 1.19 (d, 3H, J = 6.4, CH3). 13C NMR (CDCl3, ı, ppm):
175.3, 170.4 (C O), 156.9, 131.3, 129.8, 114.2 (Ar–C, metoprolol), 141.1 (OCH ),
97.7 ( CH2), 73.8, 72.3, 46.1, 35.2, 33.6, 33.2, 24.6, 24.2 (CH2), 69.5, 49.1 (CH), 58.6,
21.2, 20.7 (CH3). ESI-MS (m/z): 444.0 [M+Na]+.
2. Experimental
2.1. Materials
Lipozyme® (E.C. 3.1.1.1, an immobilized preparation of lipase from Mucor
miehei, 42 U/g, MML), lipase from porcine pancreas (E.C. 3.1.1.3, type II, pow-
der, 30–90 U/mg, PPL) and lipase from Candida cylindracea (E.C. 3.1.1.3, powder,
2.8 U/mg, CCL) were purchased from Fluka. Candida antarctica lipase acrylic resin
(E.C. 3.1.1.3, 10,000 U/g, CAL-B) and lipase type VII from Candida rugosa (E.C. 3.1.1.3,
powder, 706 U/mg, CRL) were purchased from Sigma. Lipase AY30 (E.C. 3.1.1.3, pow-
der, AY30) was purchased from Acrös. Alkaline protease from Bacillus subtilis (E.C.
3.4.21.14, a crude preparation of alkaline serine protease, 100 U/mg) was purchased
from Wuxi Enzyme Co., Ltd. (Wuxi, PR China). Metoprolol succinate was presented
by the research center of Aisen (Jinhua, PR China), and the free metoprolol was
prepared by neutralization of the aqueous solutions of salts with NaOH. All other
chemicals used in this work were of analytical grade and all solvents were first dried
2.3.3. Synthesis of N-(9-vinyloxycarbonylnonanoyl)metoprolol (3c)
The reaction time was 96 h and the yield of 3c was 52%. IR (KBr, cm−1): 3342 (OH),
1755 (OC O), 1645 (C C), 1512, 794, 776 (Ar). 1H NMR (CDCl3, ı, ppm): 7.26 (dd,
1H, J = 6.6 Hz, J = 13.4 Hz, –CH ), 7.13 (d, 2H, J = 8.4 Hz, Ar–H), 6.83 (d, 2H, J = 8.4 Hz,
Ar–H), 5.63 (s, 1H, OH), 4.86 (d, 1H, J = 14.0 Hz, CH2), 4.55 (d, 1H, J = 6.4 Hz, CH2),
4.13–3.97 (m, 3H, NCH2 and NCH(CH3)2), 3.78 (t, 1H, J = 8.4 Hz, CHOH), 3.63–3.54 (m,
3H, OCH2CH2 and OCH2CH), 3.39 (d, 1H, J = 14.8 Hz, OCH2CH), 3.35 (s, 3H, CH3O), 2.81
(t, 2H, J = 7.2 Hz, Ar–CH2), 2.45–2.34 (m, 4H, CH2), 1.64 (t, 4H, J = 6.8 Hz, CH2), 1.33
(s, 8H, CH2), 1.26 (d, 3H, J = 6.4 Hz, CH3), 1.18 (d, 3H, J = 6.8, CH3). 13C NMR (CDCl3,
ı, ppm): 176.1, 170.8 (C O), 156.9, 131.3, 129.8, 114.2 (Ar–C, metoprolol), 141.1
(OCH ), 97.4 ( CH2), 73.8, 72.4, 46.1, 35.2, 33.9, 33.6, 29.3, 29.1, 29.0, 28.9, 25.3,
24.5 (CH2), 69.5, 49.1 (CH), 58.6, 21.2, 20.7 (CH3). ESI-MS (m/z): 500.1 [M+Na]+.
˚
over 4 A molecular sieves.
2.2. Analytical methods
2.4. General procedure for the synthesis of metoprolol–saccharide conjugates
All reactions were monitored by TLC on silica gel plates eluted with petroleum
ether/ethyl acetate (1.5/1, v/v). The 1H and 13C NMR spectra were recorded
with TMS as internal standard using a Bruker AMX-400 MHz spectrometer at
400 and 100 MHz, respectively. Infrared spectra were measured with a Nico-
let Nexus FTIR 670 spectrophotometer. Analytical HPLC was performed using
Agilent 1100 system (Agilent, USA) with a reversed-phase Shim-Pack VP-ODS
column (150 mm × 4.6 mm) and a DAD detector (220 nm). Mobile phase for N-
(vinyloxycarbonyl)metoprolol was methanol/PBS (10 mmol/L, pH = 3.0) (60/40, v/v).
Mobile phase for metoprolol–saccharide conjugates was methanol/water (80/20,
v/v). Flow rate was adjusted to 1.0 mL/min.
A mixture of N-(5-vinyloxycarbonylpentanoyl)metoprolol (3b) (1 mmol), sac-
charides (4 mmol), alkaline protease from B. subtilis (250 mg) and 10 mL anhydrous
pyridine in 50 mL conical flask was kept at 50 ◦C and stirred at 200 rpm for 3 days.
The reaction was terminated by filtering off the enzyme and pyridine was evapo-
rated. The formation of metoprolol–saccharide conjugates was monitored by TLC.
The products were isolated by silica gel chromatography with an eluent consist-
ing of ethyl acetate/methanol/water (17/2/1, v/v/v). The regioselective enzymatic
synthesis of metoprolol–saccharide conjugates was shown in Scheme 2.
2.4.1. Synthesis of N-(5-(6-deoxy-d-glucopyranose-
6-yloxy)carbonylpentanoyl)metoprolol (3bGc)
2.3. General procedure for enzymatic synthesis of
N-(vinyloxycarbonyl)metoprolol (3a–3c)
The isolated yield of 3bGc was 69%. IR (liquid film, cm−1): 3373 (OH), 1732
(C O). 1H NMR (CDCl3, ı, ppm): 7.12 (t, 2H, J = 7.8 Hz, Ar–H), 6.81 (dd, 2H, J = 8.4 Hz,
J = 16.8 Hz, Ar–H), 6.43 (s, 1H, H of d-glucose), 5.26 (d, 1H, J = 8.8 Hz, H of d-glucose),
4.88 (s, 1H, H of d-glucose), 4.67 (s, 8H, H of d-glucose), 4.25 (q, 2H, J = 14.4 Hz,
H of d-glucose), 4.12–4.09 (t, J = 6.6 Hz, 1H, NCH2), 4.00 (q, 2H, J = 9.2, NCH2 and
NCH(CH3)2), 3.92–3.78 (m, 3H, CHOH and H of d-glucose), 3.71 (t, 2H, J = 8.0 Hz,
OCH2CH2), 3.52–3.41 (m, OCH2CH and H of d-glucose), 3.23 (s, 3H, CH3O), 2.71 (q,
2H, J = 6.6 Hz, Ar–CH2), 2.52 (s, 4H, CH2), 1.15 (d, 3H, J = 6.4 Hz, CH3), 1.12 (d, 3H,
J = 7.6, CH3). 13C NMR (CDCl3, ı, ppm): 175.9, 173.9 (C O), 156.9, 131.3, 129.8, 114.3
(Ar–C, metoprolol), 92.3, 73.7, 73.2, 71.6, 70.3, 63.7 (C of d-glucose), 75.3, 74.4, 45.8,
35.1, 33.7, 33.2, 24.5, 24.5 (CH2), 69.4, 49.2 (CH), 58.5, 21.1, 20.7 (CH3). ESI-MS (m/z):
580.1 [M+Na]+.
The reaction was initiated by adding 300 mg PPL to 20 mL anhydrous
tetrachloromethane containing metoprolol (2 mmol) and divinyl dicarboxy-
lates (8 mmol) in 50 mL conical flask. The suspension was kept at 50 ◦C and
stirred at 200 rpm for 48–96 h. The reaction was terminated by filtering off
the enzyme and tetrachloromethane was evaporated. The formation of N-
(vinyloxycarbonyl)metoprolol was monitored by TLC. The products were purified
by silica gel chromatography with an eluent consisting of petroleum ether/ethyl
acetate (2/1, v/v).
2.3.1. Synthesis of N-(3-vinyloxycarbonylpropanoyl)metoprolol (3a)
The reaction time was 48 h and the yield of 3a was 57% (except special expla-
nation, the yields were all determined by HPLC). IR (KBr, cm−1): 3335 (OH), 1757
(OC O), 1646 (C C), 1514, 794, 776 (Ar). 1H NMR (CDCl3, ı, ppm): 7.25 (dd, 1H,
J = 6.6 Hz, J = 13.4 Hz, –CH ), 7.12 (d, 2H, J = 8.0 Hz, Ar–H), 6.83 (d, 2H, J = 8.4 Hz, Ar–H),
5.20 (s, 1H, OH), 4.89 (d, 1H, J = 14.0 Hz, CH2), 4.58 (d, 1H, J = 6.0 Hz, CH2), 4.13 (t,
1H, J = 6.6 Hz, NCH2), 3.99 (q, 2H, J = 9.6 Hz, NCH2 and NCH(CH3)2), 3.78 (q, J = 8.6 Hz,
1H, CHOH), 3.61–3.54 (m, 3H, OCH2CH2 and OCH2CH), 3.43 (d, 1H, J = 13.4, OCH2CH),
3.35 (s, 3H, CH3O), 2.84–2.73 (m, 6H, CH2 and Ar–CH2), 1.28 (d, 3H, J = 6.8 Hz, CH3),
1.22 (d, 3H, J = 6.8, CH3). 13C NMR (CDCl3, ı, ppm): 173.6, 170.2 (C O), 156.9, 131.4,
129.8, 114.3 (Ar–C, metoprolol), 141.1 (OCH ), 97.9 ( CH2), 73.8, 72.1, 46.1, 35.2,
29.0, 28.0 (CH2), 69.5, 48.9 (CH), 58.6, 21.1, 20.6 (CH3). ESI-MS (m/z): 416.0 [M+Na]+.
2.4.2. Synthesis of N-(5-(6-deoxy-d-mannopyranose-
6-yloxy)carbonylpentanoyl)metoprolol (3bMn)
The isolated yield of 3bMn was 57%. IR (liquid film, cm−1): 3374 (OH), 1732
(C O). 1H NMR (DMSO-d6, ı, ppm): 7.11 (t, 2H, J = 7.8 Hz, Ar–H), 6.80 (dd, 2H,
J = 8.4, J = 18.0, Ar–H), 6.38 (s, 0.7H, H of d-mannose), 5.33 (s, 0.4H, H of d-mannose),
5.21 (s, 0.5 H, H of d-mannose), 4.86 (s, 2H, H of d-mannose), 4.58 (d, 8H, H of d-
mannose), 4.29 (d, 2H, J = 10.0, H of d-mannose), 4.03–3.80 (m, 4H, NCH2, NCH(CH3)2
and CHOH), 3.70 (d, 1H, J = 8.0, H of d-mannose), 3.55 (s, 1H, OCH2CH2), 3.49–3.30
(m, OCH2CH2, OCH2CH and H of d-mannose), 3.22 (s, 3H, CH3O), 2.74–2.71 (q, 2H,
J = 7.0 Hz, Ar–CH2), 2.40–2.26 (m, 4H, CH2), 1.49 (d, 4H, J = 9.2 Hz, CH2), 1.15 (d, 3H,
J = 6.4 Hz, CH3), 1.11 (d, 3H, J = 7.2, CH3). 13C NMR (DMSO-d6, ı, ppm): 173.6, 172.9
(C O), 157.0, 131.1, 130.0, 114.4 (Ar–C, metoprolol), 94.2, 71.5, 70.8, 70.6, 70.2, 68.9,
67.4, 64.4 (C of d-mannose), 73.3, 72.4, 44.6, 34.7, 33.6, 33.2, 24.6, 24.4 (CH2), 68.9,
48.1 (CH), 58.0, 21.0, 20.5 (CH3). ESI-MS (m/z): 580.2 [M+Na]+.
2.3.2. Synthesis of N-(5-vinyloxycarbonylpentanoyl)metoprolol (3b)
The reaction time was 72 h and the yield of 3b was 74%. IR (KBr, cm−1): 3335
(OH), 1752 (OC O), 1646 (C C), 1513, 794, 776 (Ar). 1H NMR (CDCl3, ı, ppm):

C.-Z. Zheng et al. / Process Biochemistry 46 (2011) 123–127
125
Scheme 2. Synthesis of metoprolol–saccharide conjugates.
2.4.3. Synthesis of N-(5-(6-deoxy-d-galactopyranose-
6-yloxy)carbonylpentanoyl)metoprolol (3bGt)
of sucrose), 3.22 (s, 3H, CH3O), 2.70 (t, 2H, J = 6.8 Hz, Ar–CH2), 2.40–2.30 (m, 4H,
CH2), 1.49 (t, 4H, J = 9.2 Hz, CH2), 1.14 (d, 3H, J = 6.0 Hz, CH3), 1.11 (d, 3H, J = 7.6, CH3).
13C NMR (DMSO-d6, ı, ppm): 173.5, 172.8 (C O), 157.0, 131.1, 130.0, 114.4 (Ar–C,
metoprolol), 103.3, 92.3, 82.9, 76.7, 73.5, 71.6, 70.2, 62.2 (C of sucrose), 73.3, 72.4,
47.0, 34.7, 33.6, 33.1, 24.6, 24.4 (CH2), 69.9, 48.1 (CH), 58.0, 21.1, 20.5 (CH3). ESI-MS
(m/z): 742.1 [M+Na]+.
The isolated yield of 3bGt was 41%. IR (liquid film, cm−1): 3373 (OH), 1733 (C O).
1H NMR (DMSO-d6, ı, ppm): 7.11 (t, 2H, J = 7.6, Ar–H), 6.80 (dd, 2H, J = 8.4, J = 17.6,
Ar–H), 6.27 (s, 0.5H, H of d-galactose), 5.22 (s, 1H, H of d-galactose), 4.99–4.91
(m, 1H, H of d-galactose), 4.61 (s, 7H, H of d-galactose), 4.23 (t, 2H, J = 8.4, H of
d-galactose), 4.09–3.98 (m, 3H, NCH2 and NCH(CH3)2), 3.91–3.80 (m, 3H, CHOH and
H of d-galactose), 3.67–3.55 (m, 3H, OCH2CH2 and OCH2CH), 3.50–3.41 (m, OCH2CH
and H of d-galactose), 3.23 (s, 3H, CH3O), 3.17 (s, 1H, CH2), 2.74–2.71 (t, 2H, J = 6.8 Hz,
Ar–CH2), 2.39–2.28 (m, 4H, CH2), 1.49 (d, 3H, J = 18.4 Hz, CH2), 1.15 (d, 3H, J = 6.4 Hz,
CH3), 1.11 (d, 3H, J = 7.2, CH3). 13C NMR (DMSO-d6, ı, ppm): 173.7, 173.2 (C O),
157.1, 131.2, 130.0, 114.4 (Ar–C, metoprolol), 92.8, 68.8, 68.7, 67.8, 67.5, 64.2 (C of
d-galactose), 73.2, 72.4, 47.1, 34.6, 33.5, 33.1, 24.6, 24.3 (CH2), 68.5, 48.2 (CH), 58.0,
21.0, 20.5 (CH3). ESI-MS (m/z): 580.1 [M+Na]+.
2.5. Determination of distribution coefficient
The distribution coefficients (D7.4) of drugs and drug–saccharide conjugates
were determined by dissolving 5 mg of different metoprolol–saccharide conjugates
in 2.5 mL of n-octanol and an equal volume of PBS (pH = 7.4) in screw-capped test
tube. The solutions were then mixed for 15 min, and centrifuged at 1 × 104 rpm for
5 min. The layers were separated and aliquots of 50 ␮L were diluted to 500 ␮L with
methanol. Samples were taken with 20 ␮L and determined by HPLC. The value of
D7.4 was the peak area ratio of up-layer to down-layer [29,30]. All experiments were
conducted in triplicate and the mean values were taken. The ꢀdetection of HPLC was
220 nm. The values of D7.4 for different metoprolol–saccharide conjugates were then
calculated and the results were listed in Table 3.
2.4.4. Synthesis of N-(5-(6ꢀ-deoxy-(4-yloxy-
(d-glucopyranosyl)-d-glucopyranose))carbonylpentanoyl)metoprolol (3bMt)
The isolated yield of 3bMt was 62%. IR (liquid film, cm−1): 3355 (OH), 1734
(C O). 1H NMR (DMSO-d6, ı, ppm): 7.11 (t, 2H, J = 7.0, Ar–H), 6.80 (d, 2H, J = 8.4,
J = 17.6, Ar–H), 5.70 (s, 1H, H of maltose), 5.37 (d, 0.5H, H of maltose), 4.98–4.90 (t,
1.5H, J = 15.2, H of maltose), 4.53 (s, 2H, H of maltose), 4.31–4.25 (dd, 1.5H, J = 8.6,
J = 15.0, H of maltose), 4.09–3.99 (t, 3H, J = 20.6, NCH2 and NCH(CH3)2), 3.89–3.80
(m, 2.5H, CHOH and H of maltose), 3.70–3.64 (m, 3H, H of maltose), 3.53–3.37 (m,
OCH2CH2, OCH2CH and H of maltose), 3.22 (s, 7H, CH3O and H of maltose), 2.70 (t,
2H, J = 6.8 Hz, Ar–CH2), 2.37–2.28 (m, 4H, CH2), 1.48 (t, 4H, J = 9.4 Hz, CH2), 1.14 (d,
3H, J = 6.0 Hz, CH3), 1.11 (d, 3H, J = 6.8, CH3). 13C NMR (DMSO-d6, ı, ppm): 173.7,
173.0 (C O), 157.1, 131.3, 130.1, 114.6 (Ar–C, metoprolol), 101.4, 97.1, 92.4, 81.0,
76.8, 75.4, 74.5, 73.3, 72.8, 72.6, 70.7, 70.3, 64.0, 60.6 (C of maltose), 73.4, 72.1, 47.2,
34.8, 33.6, 33.3, 24.8, 24.5 (CH2), 68.1, 48.3 (CH), 58.1, 21.2, 20.7 (CH3). ESI-MS (m/z):
742.1 [M+Na]+.
3. Results and discussion
3.1. Effects of enzymes and solvents on the synthesis of
N-(vinyloxycarbonyl)metoprolol
To identify suitable enzymes with high transesterification
activity in the synthesis of N-(vinyloxycarbonyl)metoprolol, five
commercially available enzymes were tested for the transesteri-
fication of metoprolol with divinyl adipate at 50 ◦C as shown in
Scheme 1. The reaction between metoprolol and divinyl adipate
could take place in most of organic solvents. In the absence of
enzymes, the reaction yields were all less than 2% in THF, DMF and
CCl4, so the three solvents were selected as the reaction media.
As seen from Table 1, the reaction media played an important
role in the enzymatic transesterification. PPL showed the high-
est catalytic ability in CCl4 (entry 6, Table 1). The yield of 3b was
2.4.5. Synthesis of N-(5-(1ꢀ-deoxy-(4-yloxy-(d-fructofuranosyl)-
d-glucopyranoside))carbonylpentanoyl)metoprolol (3bSr)
The isolated yield of 3bSr was 63%. IR (liquid film, cm−1): 3363 (OH), 1736 (C O).
1H NMR (DMSO-d6, ı, ppm): 7.11 (d, 2H, J = 7.6 Hz, Ar–H), 6.80 (d, 2H, J = 8.6 Hz,
J = 17.4 Hz, Ar–H), 5.35 (s, 1H, H of sucrose), 5.17 (d, 1H, H of sucrose), 4.93 (s, 2H,
H of sucrose), 4,46 (d, 4H, J = 13.6 Hz, H of sucrose), 4.27–4.24 (t, 1H, J = 6.6 Hz, H
of sucrose), 4.17–4.08 (m, 2H, NCH2 and NCH(CH3)2), 3.95 (d, 2H, J = 12.0 Hz, NCH2
and H of sucrose), 3.90–3.77 (m, 4H, CHOH and H of sucrose), 3.65–3.62 (m, 2H,
OCH2CH2), 3.56 (s, 4H, OCH2CH and H of sucrose), 3.52–3.39 (m, OCH2CH and H

126
C.-Z. Zheng et al. / Process Biochemistry 46 (2011) 123–127
Table 1
Table 3
The influences of enzymes and solvents on the reaction of N-(vinyloxycarbonyl)
The lipophilic parameters of metoprolol–saccharide conjugates and the parent drug.
metoprolola.
Entry
Compound
Lipophilicitya
Relative solubility
(mg/mL)b
Log Pc
Entry
Enzyme
AY30
Solvent
Yield (%)b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
THF
DMF
CCl4
THF
DMF
CCl4
THF
DMF
CCl4
THF
DMF
CCl4
THF
DMF
CCl4
25
10
26
< 1
6
74
13
8
3
2
8
16
19
1
1
2
3
4
5
6
Metoprolol
3bGc
3bMn
3bGt
3bMt
3bSr
2.1d
–
2.9
2.8
2.9
66.7
48.8
0.32
−0.14
−0.12
−0.14
−1.51
−1.37
0.73
0.76
0.73
0.031
0.043
PPL
CRL
MML
CCL
a
It was characterized by D7.4, distribution coefficient in n-octanol/phosphate
buffer (pH = 7.4); Eluent: methanol/water (80/20, v/v).
b
The solubility of drug–saccharide conjugates/the solubility of the parent drug.
Log P is the logarithm of partition coefficient in n-octanol/phosphate buffer
c
(pH = 7.4).
d
The data was from Ref. [31].
21
as the chain length of the alkyl of N-(vinyloxycarbonyl)metoprolol
increased. Thus, N-(5-vinyloxycarbonylpentanoyl)metoprolol was
selected as the substrate to investigate the influences of the struc-
tures of saccharides on the preparation of metoprolol–saccharide
conjugates.
a
Reaction conditions: enzyme (15 mg/mL), metoprolol (0.1 mmol/mL), divinyl
adipate (0.4 mmol/mL), solvent (1.0 mL), 50 ◦C, 200 rpm, 3 days.
b
Yields were determined by HPLC. Eluent: methanol/PBS (10 mmol/L, pH = 3.0)
(60/40, v/v).
Thereactions involvingN-(5-vinyloxycarbonylpentanoyl)meto-
prolol and saccharides, such as monosaccharides (glucose,
mannose and galactose) and disaccharides (maltose and sucrose)
were carried out. The selectivity and yields of enzymatic synthe-
sis of metoprolol–saccharide conjugates were listed in Table 2.
d-glucose exhibited higher reactivity than other monosaccharides,
resulting in a yield of 69% after 72 h (entry 1, Table 2). The structure
of monosaccharides showed no influence on the selectivity of
the transesterification. All monosaccharides were acylated at C-6
position (entries 1–3, Table 2). For disaccharides, the yields were
the same under the identical conditions. Maltose was acylated at
C-6ꢀ position (entry 4, Table 2), while sucrose was acylated at C-1ꢀ
position (entry 5, Table 2).
74% after 72 h. However, no positive result was observed in other
two solvents (entries 4 and 5, Table 1). The enzyme sources also
affected the transesterification. The other four lipases could not cat-
alyze the reaction effectively. Therefore, PPL was employed as the
catalyst and three N-(vinyloxycarbonyl)metoprolol (3a–3c) were
synthesized in CCl4 by the reaction of metoprolol with divinyl dicar-
boxylates.
3.2. Effects of the chain length of acylating reagents on the
synthesis of N-(vinyloxycarbonyl) metoprolol
The influences of the chain length of acylating reagents on reac-
tion yield were evaluated. Under the same reaction conditions, the
yield of N-(3-vinyloxycarbonylpropanoyl)metoprolol was 34% after
24 h, while that of N-(5-vinyloxycarbonylpentanoyl)metoprolol
and N-(9-vinyloxycarbonylnonanoyl)metoprolol were 31% and
17%, respectively. The yields decreased as the chain length of divinyl
dicarboxylates increased. It may be due to the more steric influ-
ences as the length increased.
3.4. Partition coefficients of metoprolol–saccharide conjugates
Finally, the apparent partition coefficients (log P) of meto-
prolol and its glycolipids were investigated, and the results are
shown in Table 3. The metoprolol–saccharide conjugates exhib-
ited better aqueous solubility compared with the parent drug.
The aqueous solubility of metoprolol–monosaccharide conju-
gates was ∼2.80-fold (entries 2–4, Table 3) of the parent drug.
For metoprolol–maltose and metoprolol–sucrose conjugates, the
aqueous solubility were 66.7-fold and 48.8-fold of metoprolol
(entries 5 and 6, Table 3), respectively. It was notable that the
aqueous solubility of metoprolol–disaccharide conjugates was
at least 16-fold more than that of metoprolol–monosaccharide
conjugates. It demonstrated that the aqueous solubility of
metoprolol–disaccharide conjugates was much better than that of
metoprolol–monosaccharide conjugates. Thus, the aqueous solu-
bility of metoprolol derivatives could be regulated by conjugating
with different saccharides.
3.3. Synthesis and characterization of metoprolol–saccharide
conjugates
The influences of the structures of N-(vinyloxycarbonyl)
metoprolol on the synthesis of metoprolol–glucose conjugates
were investigated by using N-(5-vinyloxycarbonylpentanoyl)
metoprolol
and
N-(9-vinyloxycarbonylnonanoyl)metoprolol
as acylating reagents. The yield of N-(5-vinyloxycarbonyl-
pentanoyl)metoprolol with glucose was higher (69%) than that of
N-(9-vinyloxycarbonylnonanoyl)metoprolol (30%) after 72 h under
the same reaction conditions. It indicated that the yields decreased
4. Conclusion
Table 2
The selectivity and yields of metoprolol–saccharide conjugatesa.
In conclusion,
a selective and facile strategy to prepare
Entry
Product
Yield (%)b
Acylation position
metoprolol–saccharide conjugates by enzymatic reaction was
developed. The influences of enzyme sources, organic solvents
and the chain length of acylating reagents on the reaction for the
synthesis of N-(vinyloxycarbonyl) metoprolol were systematically
investigated. PPL showed the best activity in tetrachloromethane
and was used to synthesize N-(vinyloxycarbonyl)metoprolol. Then,
a series of metoprolol–saccharide conjugates were obtained by
regioselective acylation with three monosaccharides and two
disaccharides. The aqueous solubility of metoprolol–saccharide
1
2
3
4
5
3bGc
3bMn
3bGt
3bMt
3bSr
69
57
41
62
63
6
6
6
6ꢀ
1ꢀ
a
Reaction
conditions:
N-(5-vinyloxycarbonylpentanoyl)metoprolol
(0.1 mmol/mL), saccharides (0.4 mmol/mL), Alkaline protease from Bacillus
subtilis (25 mg/mL), pyridine (10.0 mL), 50 ◦C, 3 days.
b
Yields were isolated yields.

C.-Z. Zheng et al. / Process Biochemistry 46 (2011) 123–127
127
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