Bioorganic & Medicinal Chemistry Letters
Enzymatic preparation of optically pure
(+)-2-azabicyclo[2.2.1]hept-5-en-3-one by (ꢀ)-
c-lactamase
from Bradyrhizobium japonicum USDA 6
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Shaozhou Zhu , Lu Ren , Songzhu Yu, Cuiyu Gong, Dawei Song, Guojun Zheng
State Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing 100029, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 April 2014
Revised 27 July 2014
Accepted 5 August 2014
Available online 2 September 2014
Whole cells of Bradyrhizobium japonicum USDA 6 showed both (+)-
mase activity. Insight into the genome of B. japonicum USDA 6 revealed two potential
c
-lactamase activity and (ꢀ)-
-lactamases: a
-lactamase, making it the first strain to contain two totally different
enantioselective lactamases. Both recombinant enzymes could easily be used to prepare either optically
pure (+)-
c-lacta-
c
type I (+)-
c
-lactamase and a (ꢀ)-
c
c
-lactam ((+)-2-azabicyclo[2.2.1]hept-5-en-3-one) or optically pure (ꢀ)- -lactam ((ꢀ)-2-azabi-
c
cyclo[2.2.1]hept-5-en-3-one), which are versatile synthetic building blocks for the synthesis of various
Keywords:
carbocyclic nucleosides and carbocyclic sugar analogues. Bioinformatic analysis showed that the type I
c
-Lactam
(+)- -Lactamase
-Lactamase
(+)-
which consists of 274 amino acids, belongs to the hydrolase family. Here, we report that B. japonicum
-lactamase in addition to a (+)- -lactamase from this
-lactam with
c
-lactamase belongs to the amidase signature family, with 504 amino acids; the (ꢀ)-
c-lactamase,
c
(ꢀ)-
c
USDA contains a (ꢀ)-
c
c
-lactamase, and it is the (ꢀ)-
c
Biocatalysis
strain that is examined in detail in this Letter. Enzymatic synthesis of optically pure (+)-
c
Enzymatic resolution
nearly 50% isolated yield and >99% ee was achieved.
Ó 2014 Elsevier Ltd. All rights reserved.
Since the thalidomide tragedy, stricter regulations have been
introduced by the FDA on enantiomerically pure compounds. As
a consequence, chiral drugs are now almost exclusively developed
as single enantiomers.1 Recently, the enzymatic resolution of race-
mic drug intermediates has been gaining more and more attention
over the past few years due to high stereoselectivity and low cost.2
One successful example is the preparation of optically pure
microorganisms containing
far.3,4,7–14
c
-lactamase have been studied thus
c
-Lactamase is a type of enzyme that can cleave the enantiomer
-lactam (Fig. 1). This name is given according to its industrial
activity since its in vivo activity is unknown.15 Based on enantiose-
lectivity, -lactamase and
-lactamases can be divided into (ꢀ)-
(+)- -lactamase.
of
c
c
c
c
c-lactam by
c
-lactamase.3
In past research involving a rational genome mining method,
c
-Lactams such as ( )-2-azabicyclo[2.2.1]hept-5-en-3-one are
our group successfully predicted that Bradyrhizobium japonicum
important precursors to several pharmaceutically active
compounds, which were initially used for the synthesis of carbocy-
clic nucleosides.4 Later, it was found that both enantiomers are
extremely versatile synthons, and they are being used in a growing
USDA 6 may have the (+)-c
-lactamase gene.6 When we tested
the whole cell’s lactamase activity, we were surprised to discover
that the whole cell of B. japonicum USDA 6 also showed some
(ꢀ)-
c
-lactamase activity, making it the first example of a cell that
number of drug candidates.5 Different types of
c-lactamases that
contains both (+)-
c
-lactamase and (ꢀ)-
c
-lactamase.
could improve the efficiency of the preparation of these optically
pure synthons have been discovered by activity-based screening
methods and genome mining-based methods.3,6 Even so, few
A legume-root nodulating species, B. japonicum USDA 6 is a
symbiotic nitrogen-fixing bacterium. Under laboratory conditions,
we found that it is a weak biocatalyst for industrial application. The
strain grows very slowly, and because the whole cell contains both
(+)-
c
-lactamase and (ꢀ)-
c-lactamase, the stereoselectivity of the
⇑
Corresponding author. Tel./fax: +86 10 64437507.
whole cell is poor. Therefore, heterologous expression of these
enzymes in another host is needed.
We have already heterologously expressed (+)-
Escherichia coli and examined it in detail in a previous study; the
Current address: Department of Chemistry, Philipps-University Marburg,
Hans-Meerwein-Strasse 4 and LOEWE-Centre for Synthetic Microbiology, D-35032
Marburg, Germany.
c-lactamase in
à
These authors contributed equally.
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.