Enantioselective hydrolysis of 3-hydroxy-1,4-benzodiazepin-2-one esters by pig liver microsomes

Article abstract of DOI:10.1016/j.molcatb.2014.01.019

The method of enantioselective hydrolysis of 3-hydroxy-7-bromo-5-phenyl-1, 2-dihydro-3H-1,4-benzodiazepin-2-one esters using pig liver microsomal fraction was developed. The S-enantiomers of three substrates were obtained with ee _s >97% and yie

Full text of DOI:10.1016/j.molcatb.2014.01.019

Accepted Manuscript
Title: Enantioselective hydrolysis of
3-hydroxy-1,4-benzodiazepin-2-one esters by pig liver
microsomes
Author: E.A. Shesterenko I.I. Romanovska O.V. Sevastyanov
S.A. Andronati V.I. Pavlovsky T.A. Yurpalova B. Wicher
V.Ch. Kravtsov A.A. Krysko
PII:
S1381-1177(14)00030-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2014.01.019
MOLCAB 2876
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
3-10-2013
16-1-2014
23-1-2014
Please cite this article as: E.A. Shesterenko, I.I. Romanovska, O.V. Sevastyanov,
S.A. Andronati, T.A. Yurpalova, B. Wicher, V.Ch. Kravtsov, A.A. Krysko,
Enantioselective hydrolysis of 3-hydroxy-1,4-benzodiazepin-2-one esters by
pig liver microsomes, Journal of Molecular Catalysis B: Enzymatic (2014),
http://dx.doi.org/10.1016/j.molcatb.2014.01.019
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ENANTIOSELECTIVE HYDROLYSIS OF 3-HYDROXY-1,4-
BENZODIAZEPIN-2-ONE ESTERS BY PIG LIVER MICROSOMES
E.A. Shesterenkoa, I.I. Romanovskaa, O.V. Sevastyanova, S.A. Andronatia,
V.I. Pavlovskya, T.A. Yurpalovaa, B. Wicherb, V.Ch. Kravtsovc, A.A. Kryskoa
aDepartment of medical chemistry, A.V. Bogatsky Physico-chemical Institute,
National Academy of Sciences of Ukraine, Lustdorfska dor., 86, Odessa, Ukraine
bFaculty of Chemistry, Adam Mickiewicz University, 60-780 Poznan, Poland
cInstitute of Applied Physics, Academy of Sciences of Moldova, Chisinau, Republic
of Moldova
ABSTRACT
The method of enantioselective hydrolysis of 3-hydroxy-7-bromo-5-phenyl-1,2-
dihydro-3H-1,4-benzodiazepin-2-one esters using pig liver microsomal fraction
was developed. The S-enantiomers of three substrates were obtained with ees
>97 % and yields 44-49 %, their absolute configurations were determined by X-ray
crystallography. It was shown, that the S-enantiomers of 3-hydroxy-7-bromo-5-
phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-one esters were 1.4-2.1 times more
potent ligands of CBR than the corresponding racemates. Pig liver microsomal
fraction was immobilized in calcium alginate beads. It was shown, that
immobilized preparation has 3 times greater thermal stability at 50 ºC compared to
the free microsomal fraction. Enantioselective hydrolysis of 1-methyl-3-acetoxy-7-
bromo-5-phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-one using immobilized
microsomal fraction was conducted for 12 cycles of use without loss of the esterase
activity.
Key words: enantioselective hydrolysis, microsomal fraction, 3-hydroxy-
1,4-benzodiazepin-2-one esters, immobilization.
1. Introduction
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The configuration of chiral biologically active compounds plays an
important role in processes of their transport, biotransformation and binding with
biomembranes [1, 2]. Furthermore, pharmacokinetics profile of one enantiomer
may be dependent on the presence of the other enantiomer [1].
Methods of asymmetric synthesis and resolution of enantiomers, such as
spontaneous crystallization, usage of optically active solvents, via diastereomers,
chromatography on chiral stationary phases are fraught with difficulties [3]. Thus
development of economical preparative biotechnological methods of enantiomers
resolution is prospective.
Carboxylesterases (EC 3.1.1.1) are the most studied enzymes, catalyzing the
enantioselective hydrolysis of an exceptional range of acyclic [4, 5], carbocyclic
[6-11] and heterocyclic [12, 13] compounds, including 3-hydroxy-1,4-
benzodiazepin-2-one esters [14-16].
Benzodiazepine drugs are the rather wide class of medicines, which clinical
effects include anxiolytic, anticonvulsant and hypnotic effects, muscle relaxation
[17]. But the number of publications, devoted to the enantioselective hydrolysis of
benzodiazepine derivatives is quite limited [14-16, 18-19].
Pig liver carboxylesterase is a member of an α,β-hydrolase- fold family and
is localized in the endoplasmic reticulum and cytosol of hepatocytes [20]. The
enzyme has many advantages: it is cheap, stable, does not require a coenzyme.
Commercially available preparations are mixtures of isozymes, which exhibit
essentially the same stereospecificity and can be used in asymmetric synthesis as a
single enzyme [21]. Nevertheless, the limitations of the carboxylesterase usage are
the high cost of commercial enzyme and its single usage.
Therefore, the application of more economical carboxylesterase as a
component of pig liver microsomal fraction, as well as development of it
immobilization on polymeric carriers [22, 23], such as calcium alginate [24, 25]
are actual tasks.
Alginate is biocompatible, non-toxic, non-immunogenic and biodegradable
biopolymer [26, 27]. It is an anionic copolymer, composed of D-mannuronic acid
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and L-guluronic acid units, arranged in an irregular blockwise pattern [25].
Alginate forms a hydrogel in the presence of divalent cations, such as Ca 2+, which
act as cross-linkers between the functional groups of alginate chains [26].
Present study describes the enantioselective hydrolysis of ester bonds in
substrates 1-3, catalyzed by pig liver microsomal fraction. Absolute configurations
of S-enantiomers of the substrates 1a-3a were determined by the X-ray
crystallography method. The method of the microsomal fraction immobilization in
calcium alginate beads was developed. Stability, esterase activity of the
immobilized microsomal fraction and its application in the enantioselective
hydrolysis of ester bonds in substrates 1-3 was investigated.
2. Materials and methods
2.1.Materials
Pig liver was obtained from local supermarket. Sodium alginate,
hydroxylamine hydrochloride, silica gel (60-100 mesh) were purchased from
Sigma Chem. Co, (Schnelldorf, Germany). Bovine serum albumin (BSA),
potassium dihydrogen phosphate, sodium dodecyl sulfate, 1-naphthyl acetate and
dimethyl sulfoxide, were purchased from Merck (Darmstadt, Germany). The other
chemicals used in this work were of analytical grade from local sources.
2.2. Isolation of microsomal fraction, protein and activity assay
Isolation of microsomal fraction was performed by calcium sedimentation
and low speed centrifugation [28] from pig liver. Protein concentration was
determined according to modified Lowry method [29] using BSA as the standard
protein. Esterase activity was determined by hydrolysis of 1-naphthyl acetate in
potassium phosphate buffer (16 mM, pH 7.0) and 40 % (v/v) DMSO at 37ºC.
Released 1-naphthol was quantified spectrophotometrically by reaction with p-
nitrophenyl diazonium tetrafluoroborate at 490 nm [30]. One unit of activity (1 U)
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was defined as the amount of enzyme (mg) releasing 1 μmol of 1-naphthol per
minute under the assay conditions.
2.3 Synthesis of the substrates (2,3)
2.3.1. (R,S)-1-Methyl-3-acetoxy-7-bromo-5-phenyl-1,2-dihydro-3H-1,4-
benzodiazepin-2-one (2)
(R,S)-3-acetoxy-7-bromo-5-phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-
one (1) was synthesized according to the literature procedure [31]. In a 50 ml flask
(R,S)-3-acetoxy-7-bromo-5-phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-one (1)
(3.73 g, 10 mmol), iodomethane (1.24 ml, 20 mmol), tetrabutylammonium
bromide (0.24 g, 1 mmol) and chloroform (20 ml) were mixed and saturated
solution of К2CO3 was added. The reaction mixture was stirred for 3 h. Next,
chloroform was separated, washed with water (10 ml x 3) and dried over CaCl2.
The solvent was removed under reduced pressure. The resulting oil was
crystallized from acetonitrile.
Mass-spectra of synthesized compounds were recorded on mass-
spectrometer VG 7070 at electron energy 70 eV and ion source temperature 200 ºC
using direct injection system of sample. The exact mass of molecular ions were
measured by the peak matching method using perfluorokerosene as internal
1
standard at resolution about 10000. H-NMR spectra were recorded on a Bruker
spectrometer at 500 MHz frequency, in CDCl3 and DMSO-d6, internal standard
о
TMS, at 25 ºC. Yield: 3.1 g (80 %); m.p. 267 – 268 С; EI-MS m/z 386 [M+];
1
HRMS calcd. for C18H15BrN2O3 [M]+ m/z, 386.0266; found, 386.0276; H-NMR:
(500 MHz, CDCl3) δ 7.64–7.74 (3H, m), 7.41–7. 52 (4H, m), 7.29–7.32 (1H, d),
5.94 (1H, s), 3.45 (3H, s), 2.33 (3H, s).
2.3.2. (R,S)- 1-Ethyl-3-acetoxy-7-bromo-5-phenyl-1,2-dihydro-3H-1,4-
benzodiazepin-2-one (3)
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In a 50 ml flask (R,S)-3-acetoxy-7-bromo-5-phenyl-1,2-dihydro-3H-1,4-
benzodiazepin-2-one (1) (3.73 g, 10 mmol), bromoethane (1.5 ml, 20 mmol),
tetrabutylammonium bromide (0.24 g, 1 mmol) and chloroform (20 ml) were
mixed and saturated solution of К2CO3 was added. The reaction mixture was
stirred for 3 h. Next, chloroform was separated, washed with water (10 ml x 3) and
dried over CaCl2. The solvent was removed under reduced pressure. The resulting
о
oil was crystallized from acetonitrile. Yield: 2.93 g (73 %); m.p. 202 – 205 С;
EI-MS m/z 400 [M+]; HRMS calcd. for C19H17BrN2O3 [M]+ m/z, 400.0423; found,
1
400.0434; H-NMR (500 MHz, CDCl3) δ 7.61–7.70 (3H, m), 7.41–7.51 (4H, m),
7.33–7.35 (1H, d, J = 8.8), 5.94 (1H, s), 4.24-4.31 (1H, m), 3.71-3.78 (1H, m),
2.31 (3H, s), 1.11-1.13 (3H, t, J = 7.2);
2.4. General procedure for the enantioselective hydrolysis of 3-hydroxy-7-
bromo-5-phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-one esters with the free or
immobilized microsomal fraction
To the substrates 1-3 (0.37-0.40 g, 1 mmol) dissolved in potassium
phosphate buffer (16 mM, pH 7.0) and 40 % (v/v) DMSO 13600-19040 U (20-28
mL) of free or immobilized microsomal fraction were added. The reaction mixture
was incubated at 37 ºC for 2.5 h and extracted with chloroform (150 ml x 3). The
combined organic layers were washed with water (300 ml x 2) and the solvent was
removed under reduced pressure. Enantiomers of substrates 1a-3a were purified by
silica gel column chromatography (chloroform/ethyl acetate = 3/1 or 1.5/2.5, v/v).
Quantitative analysis of enantiomers of substrates 1a-3a was performed
spectrophotometrically by ferric hydroxamate method [32].
Enantiomeric excesses of substrates (ees) were determined by HPLC using
Shimadzu LC-8A pump with a chiral column ChiraDex 5 μm eluted with
methanol/ water (70/30, v/v) at room temperature and a flow rate of 1 mL/min.
The retention times (tR) of peaks of the racemate 1 are 5.3 min - 49.93 % R-
enantiomer and 7.8 min - 50.07 % - S-enantiomer.
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tR of peaks of the obtained enantiomer 1a are 5.3 min - 1.2 % R-enantiomer
and 7.8 min 98.8 % - S-enantiomer. tR of peaks of 2 are 5.6 min - 48.9 % - R-
enantiomer and 8.1 min - 51.1 % S-enantiomer. tR of peaks 2a are 5.6 min - 1.3 %
- R-enantiomer and 8.1 min 98.7 % S-enantiomer. tR of peaks of 3 are 7.6 min -
48.6 % - R-enantiomer and 9.6 min – 51.4 % S-enantiomer. tR of peaks 3a are 7.6
min - 1.4 % - R-enantiomer and 9.6 min 98.6 % S-enantiomer.
The ees was calculated from the ratio (%) of [(S-form) − (R-form)]/[(S-form)
+ (R-form)].
2.4.1. Compound 1a: (S)-3-acetoxy-7-bromo-5-phenyl-1,2-dihydro-3H-1,4-
benzodiazepin-2-one
20 +116.9º (c = 1.0, CHCl3).
D


Yield; 49 %, ees > 97 %,
2.4.2. Compound 2a: (S)-1-methyl-3-acetoxy-7-bromo-5-phenyl-1,2-
dihydro-3H-1,4- benzodiazepin-2-one
20 +195.3º (c = 1.0, CHCl3).
D


Yield; 47 %, ees > 97 %,
2.4.3. Compound 3a: (S)-1-ethyl-3-acetoxy-7-bromo-5-phenyl-1,2-dihydro-
3H-1,4- benzodiazepin-2-one
20 +193.8º (c = 1.0, CHCl3).
D

Yield; 44 %, ees > 97 %,
2.5. X-ray crystal structure analysis of 1a-3a
The diffraction data for 1a and 3a were collected with a Xcalibur E single-
crystal diffractometer using MoKα radiation λ=0.71073 Å) and for 2a data were
collected with a SuperNova single-crystal diffractometer using micro-source
radiation CuKα λ=1.54184 Å). All experiments were carried out at room
temperature 293K. Data collection and reduction were performed with CrysAlisPro
software (versions 1.171.33.66 for 1a and 3a and 1.171.33 for 2a). The data were
Page 6 of 20

7
corrected for the Lorentz and polarization factors and for absorption. The structure
was solved by direct methods refined by full-matrix least-squares method on F2
with SHELXL-97 [33]. All non-hydrogen atoms were refined with anisotropic
displacement parameters. The hydrogen atom positions were found in difference
Fourier maps however, for further refinement H-atoms bonded with carbon atoms,
were determined on the basis of molecular geometry (C–H 0.93-0.98 Å) and their
displacement parameters were set equal to 1.5Ueq(C) for methyl groups and
1.2Ueq(C) for others. The absolute structure was determined from anomalous
dispersion effects [34].
Molecules of 1a-3a have S-configuration, the absolute structure Flack
parameters are: 1a -0.03(2), 2a-0.00(1), 3a-0.004(8).
Crystallographic data for the structure 1a, 2a, and 3a reported in this paper
have been deposited with the Cambridge Crystallographic Data Centre. Deposit
Nos. CCDC 916962, 789193, and 916961, respectively. Copies of the data can be
obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge
CB12 1EZ, UK (fax +44 1223 336033 or e-mail:deposit@ccdc.cam.ac.uk).
2.6. [3H] Flumazenil binding.
Affinity of compounds 1-3 and 1a-3a for CBR of rat brain was determined
and values of IC50 were evaluated as described previously [35, 36].
2.7. Immobilization of microsomal fraction in calcium alginate beads
The isolated microsomal fraction (1544 U or 15 ml) was mixed with 45 ml
of a solution of sodium alginate and then dropped into 200 ml of calcium chloride
solution (10 %) to form biocatalyst beads. The beads were washed with distilled
water and stored at 4 ºC for its further use.
2.8. pH- and thermal dependence of microsomal fraction activity
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The effect of pH on free and immobilized microsomes was studied by
assaying the preparations at different pH values (3.0-10.0) using the following
buffers: acetate (3.0-6.0), potassium phosphate (pH 6.0-8.0), Tris (8.0-9.0).
The effect of temperature on free and immobilized microsomal fraction was
determined by changing the reaction medium temperature in the range from 10 to
50 ºC in potassium phosphate buffer (16 mM, pH 7.0).
2.9. Thermostability of microsomal fraction
Free and immobilized microsomal fraction were incubated in potassium
phosphate buffer (16 mM, pH 7.0) at 50 ºC. Samples were taken after 0.25, 0.5, 1,
2, 3, 4 and 5 h. The residual activity was determined by hydrolysis of 1-naphthyl
acetate. The thermal inactivation constants were calculated from the slope of
residual activity value common logarithm vs. time dependence plot by the linear
regression method.
2.10. Reusability of immobilized microsomal fraction
The reuse activity of immobilized microsomal fraction was studied in
repeated batch experiments with hydrolysis of compound 2 (0.5 mM) dissolved in
potassium phosphate buffer (16 mM, pH 7.0) and 40 % (v/v) DMSO at 37 ºC for 1
h. At the end of each cycle, the immobilized microsomes were filtered and washed
thoroughly with distilled water.
3. Results and discussion
3.1. Synthesis of (R,S)-esters 2-3
(R,S)-1-Methyl-(2) and (R,S)-1-ethyl-3-acetoxy-7-bromo-5-phenyl-1,2-
dihydro-3H-1,4-benzodiazepin-2-one (3) were synthesized by alkylation of (R,S)-
3-acetoxy-7-bromo-5-phenyl-1,2-dihydro-3H-1,4-benzodiazepine-2-one (1) with
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iodomethane or bromoethane under phase-transfer catalysis conditions,
respectively.
R
N
H
O
O
N
Hal-R
Br
O
O
O
O
N
N
Br
1
2,3
R: H (1), CH3 (2), C2H5 (3)
Scheme 1. Synthesis of (R,S)-esters 2,3.
The structure of (R,S)-esters 2,3 were confirmed by mass spectrometry and
1Н-NMR spectroscopy.
3.2. Enantioselective hydrolysis of ester bonds in substrates 1-3 with free
microsomal fraction
Microsomal fraction was obtained from pig liver. Protein yield was
38.0 mg/g of liver tissue and esterase activity was 17.25 U/mg protein.
Enantioselective hydrolysis of ester bonds in compounds 1-3 was conducted using
the microsomal fraction (Scheme 2).
R
N
R
N
R
N
O
O
microsomal
fraction
O
+
O
O
OH
O
O
N
N
Br
Br
N
Br
1-3
R: H (1,4), CH3 (2,5), C2H5 (3,6)
4-6
1a-3a
Scheme 2. Enantioselective hydrolysis of (R,S)-3-hydroxy-7-bromo-5-
phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-one esters 1-3.
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The hydrolysis of 1 with the microsomal fraction was studied at different
temperatures and pH to find the optimal reaction conditions. Table 1 shows, that
the maximal conversion of 1 was observed at 37 ºC and pH 7.0.
Table 1
Effect of temperature and pH on enzymatic conversion of substrate 1
Conversion (%)
Conversion (%)
Temperature
рН
Free Immobilized
Free Immobilized
(ºС)
catalyst
13.0
19.0
32.8
50.5
47.8
20.5
13.0
catalyst
13.5
21.5
31.2
50.7
49.8
27.6
19.2
catalyst
catalyst
10
20
30
37
40
50
60
3.0
4.0
5.0
6.0
7.0
8.0
9.0
6.7
7.2
11.6
26.7
37.3
50.9
46.5
42.6
10.8
28.5
37.7
49.8
49.6
42.7
Next, we had investigated the effect of substituents in the first position of the
benzodiazepine moiety on the microsomal carboxylesterase specificity (fig. 1). It
was found, that to convert the compound 1 by 50 %, it was needed the least
amount of the microsomal fraction – 100 U/ml. 50 % conversion of 2 and 3 was
achieved with increasing the required amount of the catalyst [37].
140
120
100
80
60
40
20
0
1
2
3
Substrate
Fig.1. Effect of the substrate structure on the required amount of the
microsomal fraction.
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It was shown, that the products 4-6 underwent racemization during
hydrolysis and subsequent isolation, which is consistent with the literature [38].
But the absolute configurations of the substrate enantiomers 1a-3a were
unambiguously determined by single crystal X-ray diffraction analysis as (S)-
forms (ees > 97 %). Thus, the pig liver microsomal carboxylesterase is showing
higher specificity to R-enantiomers of esters 1-3.
a
b
d
c
Fig.2. ORTEP view of molecular structure of 1a (a), 2a (b), and two conformers in
the structure 3a (c and d) illustrate their absolute configuration. Displacement
ellipsoids are drawn at the 50% probability level.
The X-ray crystal structure analysis had shown the similar molecular
conformation of 1a-3a, Fig.2. Symmetrically independent molecules a and b in 3a
differ mainly by opposite orientation of ethyl substituent, torsion angle
C(2)N(1)C(1)C(12) equals - 74.0° and 80.7° for molecule a and b. The seven-
membered benzodiazepine moiety in all these compounds has a boat-like
conformation. The deviation of atoms N(1), C(2), N(4) and C(5), which constitute
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bottom of the boat, from the their mean plane is less than 0.004 Å in 1a and 2a and
0.022 Å in 3a, while atoms C(3), C(10) and C(11) are displaced from this plane in
the same direction by 0.75, 0.65 and 0.67Å in 1a, 0.77, 0.73 and 0.73Å in 2a, and
0.79/0.79, 0.75/0.80, and 0.70/0.76 Å in 3a. A distortion of ring from an ideal
cycloheptatriene “boat” with mirror symmetry Cs may be evaluated by Duix
asymmetry parameter ΔCs [39-40] which equal 3.32°, 6.85° for 1a, 2a, and 2.33°
and 7.14° for molecules a and b in 3a, respectively and are well within the range
found in 1,4-benzodiazepin-2-ones [40]. Bond lengths and angles in of 1a-3a are in
a good agreement. Distances N(4)-C(5) range within the values 1.276-1.294 Å and
indicate the double character of this bond. Distances C(3)-N(4), N(1)- C(2), and
N(1)-C(10) vary in the narrow intervals 1.435-1.442, 1.362-1.368, and 1.402-
1.428Å, respectively.
Using radioligand binding methods, affinity of S-enantiomers 1a-3a and
racemates 1-3 for CBR of rat brain was determined and values of IC50 were
evaluated. It was shown, that the S-enantiomers 1a-3a are 1.4-2.1 times more
potent ligands of CBR than the corresponding racemates 1-3.
250
200
150
100
50
0
1
2
3
S-enantiomers racemates
Fig. 3. In vitro binding affinity (Ki, nМ) for CBR of compounds 1-3
and 1a-3a
3.3. Immobilization studies
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In this study, we have determined the optimal concentration of alginate and
calcium chloride solutions (4 % and 5 %, respectively) to prepare the biocatalyst
beads. Characteristics of the obtaining biocatalyst are shown in Table 2.
Table 2.
Characteristics of immobilized microsomal fraction
Biocatalyst characteristics
Amount of protein, mg/g beads
Esterase activity, U/ g beads
Retention of initial activity, %
Beads diameter, mm
19.2 ± 0.7
230.9 ± 10.3
70.0
2.5 ± 0.5
15 ± 0.45
Beads weight, mg
As shown, there were no significant differences in pH and temperature
profile of free and immobilized microsomal fraction (Table 1). But investigation of
thermal inactivation of both biocatalysts showed an improvement of thermal
stability of the immobilized microsomal fraction. The thermal inactivation
constants were 0,8·10-3 min-1 and 1,5·10-3 min-1, for the free and the immobilized
biocatalysts, respectively (fig. 4).
Such enhancement of thermal stability seems to represent stabilization of
microsomal vesicles by the carrier, which prevents the membrane-bound proteins
denaturation, and as a consequence disaggregation of the vesicles [41].
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alog A - common logarithm of residual esterase activity
Fig. 4. Thermal stability of free and immobilized microsomal fraction at
50ºC
3.4. Enantioselective hydrolysis of ester 2 with immobilized microsomal
fraction
Enantioselective hydrolysis of ester 2 using immobilized microsomal
fraction was conducted in optimized reaction conditions in a batch process (Fig. 5).
After 8 cycles, the beads of immobilized microsomal fraction have lost
insolubility, because of the buffer solution influence, which contributes to the
gradual release of calcium ions from the beads [27, 42].
So, the ability of distilled water application instead of the buffer solution
was investigated. Application of distilled water made it necessary to increase the
required amount of the biocatalyst at 18 %. But it was shown, that after 14 cycles
of use the beads were not destroyed, and the 50 % conversion of 2 was found after
12 cycles of usage.
Fig. 5. Reusability of immobilized microsomal fraction in enantioselective
hydrolysis of the ester 2.
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4. Conclusions
The optimized reaction conditions of enantioselective hydrolysis of the 3-
hydroxy-7-bromo-5-phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-one esters using
pig liver microsomal fraction were developed. Three S-enantiomers of substrates
were obtained, so the pig liver microsomal carboxylesterase is showing higher
specificity to R-enantiomers of esters 1-3. It was found, that the S-enantiomers 1a-
3a are the most potent ligands of CBR as compared to the racemates 1-3. The
biocatalyst with improved thermal stability was developed using the
immobilization of microsomal fraction in calcium alginate beads. Obtained
biocatalyst was used for the enantioselective hydrolysis of the ester 2 for 12 cycles
of use in a batch process.
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Highlights:
The S-enantiomers of the 3-hydroxy-7-bromo-5-phenyl-1,2-dihydro-3H-1,4-
benzodiazepin-2-one esters were obtained (ees >97 %).
S-enantiomers were 1.4-2.1 times more potent ligands of CBR than the
corresponding racemates.
Pig liver microsomal fraction was immobilized in calcium alginate beads.
Obtained biocatalyst has been applied for the enantioselective hydrolysis of the
esters for 12 cycles of use.
[43]
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*Graphical Abstract (for review)
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