Bioorganic & Medicinal Chemistry Letters
Efficient synthesis of the intermediate of abacavir and carbovir using
a novel (+)-c-lactamase as a catalyst
Shuaihua Gao a, Shaozhou Zhu b, Rong Huang a, Yingxiu Lu a, Guojun Zheng a,
⇑
a State Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing 100029, People’s Republic of China
b Department of Chemistry, Biochemistry, Philipps-University Marburg, Hans-Meerwein-Strasse 4 and LOEWE-Centre for Synthetic Microbiology, D-35032 Marburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The enantiomers of 2-azabicyclo[2.2.1]hept-5-en-3-one (
c
-lactam) are key chiral synthons in the synthe-
-Lactamase can be used as a catalyst in the enzy-
-lactam. Here, (+)- -lactamase discovered from
Bradyrhizobium japonicum USDA 6 by sequence-structure guided genome mining was cloned, purified
and characterized. The enzyme possesses a significant catalytic activity towards -lactam. The active site
of the (+)- -lactamase was studied by homologous modeling and molecular docking, and the accuracy of
the prediction was confirmed by site-specific mutagenesis. The (+)- -lactamase reveals the great practi-
Received 21 April 2015
Revised 11 July 2015
Accepted 18 July 2015
Available online 26 July 2015
sis of antiviral drugs such as carbovir and abacavir. (+)-
c
matic preparation of optically pure (ꢀ)-
c
a
c
c
c
Keywords:
2-Azabicyclo[2.2.1]hept-5-en-3-one
Enzymatic resolution
c
cal potential as an enzymatic method for the efficient production of carbocyclic nucleosides of pharma-
ceutical interest.
(+)-c-Lactamase
Ó 2015 Elsevier Ltd. All rights reserved.
Site-specific mutagenesis
The enantiomers of 2-azabicyclo[2.2.1]hept-5-en-3-one (
c
-lac-
genome mining.15 (+)-
performance liquid chromatography (HPLC). In this letter, the
overexpression, purification and characterization of the (+)- -lac-
tamase was reported. The active site of the (+)- -lactamase was
studied by homologous modeling and molecular docking method.
-lactam with high yield (>49.9%)
and ee value (>99.8%) was achieved.
According to the sequence (GenBank accession no. BJ6T_02120),
primer P1 and P2 (Table 1) were designed for the amplification
c-lactamase activity was detected by high-
tam) are extremely versatile synthons in the preparation of carbo-
cyclic nucleoside analogues which possess therapeutic properties
such as antibiotic activity and antiviral activities.1–3 The optically
c
c
pure enantiomer (ꢀ)-
c-lactam has attracted much more attentions
during last two decades due to its great potential as a precursor in
The preparation of pure (ꢀ)-
c
the synthesis of carbovir and abacavir which are active against
HIV.4,5 The hydrolyzed (+)-
c
-lactam amino acid product is also
used in the synthesis of pharmaceutical targets such as mel-
ogliptin6, MK-08127 and piperidinium compound8 (Scheme 1).
process of the (+)-c-lactamase gene. The gene was cloned into
Many methods have been exploited for the preparation of (ꢀ)-
c
-
the expression vector pET-28a with a 6ꢁHis tag at the N terminus.
lactam. The enzymatic synthesis method proved to be an efficient
alternative to classical chemical method because of its high stere-
oselectivity and low cost.
The recombinant protein was overexpressed in E. coli Rosetta
under the induction of 0.08 M isopropyl-b-D-thiogalactopyranoside
(IPTG). The harvested cells were sonicated and followed by Ni-
affinity column chromatography and gel filtration by using a stan-
dard purification procedure. The gene encoded 504 amino acid
residues with a theoretical molecular weight of 49 kDa. The rela-
tive molecular mass was about 50 kDa on native SDS–PAGE after
gel filtration (Fig. 1, lane 3), which meant that the native protein
appeared as a monomer.
The physical properties of the enzyme were investigated. The
standard assay mixture (0.5 mL) contained 50 mM Tris–HCl buffer
(pH 7.5), 4.6 mM c-lactam and 4.8 lM purified enzyme. The activ-
(+)-
ity on (rac)-
of (+)-
-lactam, and then the optical (ꢀ)-
The kinetic resolution of -lactam by (+)-
c
-Lactamase was defined on the basis of its enantioselectiv-
-lactam. It enantioselectively catalyzed the hydrolysis
-lactam was obtained.
-lactamases has been a
-lac-
-lactamase was
c
c
c
c
c
popular method for the preparation of the optically pure (ꢀ)-
c
tam since the first wild type strain containing (+)-
c
screened.9
Up to now, only five (+)-
characterized in Escherichia coli expression system.10–14 In our pre-
vious report, we discovered (+)- -lactamase gene from
c-lactamases have been purified and
a
c
ity was determined by a Daicel Chiralpak AD-H column which was
eluted with a mobile phase consisting of 93% n-hexane and 7% iso-
propanol (volume ratio) at a flow rate of 0.75 mL/min. The UV
Bradyrhizobium japonicum USDA 6 by sequence-structure guided
absorbance of the eluted
c-lactam was measured at 230 nm. One
⇑
Corresponding author. Tel./fax: +86 10 64437507.
unit of (+)- -lactamase was defined as the amount of enzyme
c
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.