Angewandte
Communications
Chemie
Biocatalysis
Directed Evolution of a Fluorinase for Improved Fluorination
Efficiency with a Non-native Substrate
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Abstract: Fluorinases offer an environmentally friendly alter-
native for selective fluorination under mild conditions. How-
ever, their diversity is limited in nature and they have yet to be
engineered through directed evolution. Herein, we report the
directed evolution of the fluorinase FlA1 for improved
conversion of the non-native substrate 5’-chloro-5’-deoxyade-
nosine (5’-ClDA) into 5’-fluoro-5’-deoxyadenosine (5’-FDA).
The evolved variants, fah2081 (A279Y) and fah2114 (F213Y,
A279L), were successfully applied in the radiosynthesis of 5’-
deoxyadenosine (5’-FDA) and l-methionine (l-Met) through
[10]
a nucleophilic substitution (S 2) mechanism.
Pioneering
N
work by OꢀHaganꢀs group has demonstrated the application
1
8
[11]
of the fluorinase FlA in the radiosynthesis of 5’-[ F]FDA,
as well as the coupling of FlA with additional enzymes to
1
8
[12]
18
produce a range of F-labeled nucleosides
labeled sugar
and a F-
[
11b,13]
[14]
for PET applications.
To explore the substrate promiscuity for broader applica-
tions, FlA has been reported to accept 5’-chloro-5’-deoxy-
1
8
[15]
[16]
[
F]FDA, with overall radiochemical conversion (RCC) more
adenosine (5’-ClDA),
Met analogues,
2’-deoxyadenosine substrates,
l-
5’-
[
17]
[18]
than 3-fold higher than wild-type FlA1. Kinetic studies of the
two-step reaction revealed that the variants show a significantly
improved kcat value in the conversion of 5’-ClDA into S-
adenosyl-l-methionine (SAM) but a reduced kcat value in the
conversion of SAM into 5’-FDA.
a methylaza analogue of SAM,
chlorodeoxy-2-ethynyladenosine (ClDEA),
highly complex di- and tetra-cyclic peptide conjugates of
ClDEA. It is noteworthy that the catalytic efficiency of wild-
type fluorinases with non-native substrates is often re-
[19]
and even
[20]
[17–19]
duced.
as directed evolution
Powerful enzyme engineering approaches such
[21]
[22]
O
rganofluorine compounds play important roles in phar-
and rational protein design
can
[
1]
[2]
[3]
maceuticals,
agrochemicals,
materials,
and positron
potentially restore the impaired activity. Thomsen et al.
identified a FlA mutant with increased activity for the ethyl
analogue of l-Met through a series of rationally designed
[
4]
emission tomography (PET) imaging. Many advances
[
5]
have been made in CÀF bond formation, while selective
fluorination under mild conditions remains challenging. The
discovery and characterization of four naturally occurring
fluorinase enzymes (FlA, FlA1, FlA3 and NobA
offers an attractive prospect for using biocatalysts to produce
[
17]
point mutations. However, directed evolution has yet to be
employed to engineer fluorinases. Herein, we report directed
evolution of the fluorinase FlA1 to improve the fluorination
efficiency for the non-native substrate 5’-ClDA.
[
6]
[7]
[7]
[7–8]
)
[
9]
fluorinated compounds. These fluorinases convert S-adeno-
syl-l-methionine (SAM) and fluoride ions into 5’-fluoro-5’-
5’-ClDA is an attractive substrate that could be fluori-
[
15]
nated in the presence of l-Met (Scheme 1).
It can be
[
+]
[+]
[
*] Dr. H. Sun, W. L. Yeo, Prof. Dr. H. Zhao, Dr. E. L. Ang
Metabolic Engineering Research Laboratory (MERL)
Science and Engineering Institutes
Prof. R. C. Robinson
Department of Biochemistry, Yong Loo Lin School of Medicine
National University of Singapore, Singapore 117597 (Singapore)
and
Agency for Science, Technology, and Research (A*STAR)
31 Biopolis Way, Nanos #01-01, Singapore 138669 (Singapore)
NTU Institute of Structural Biology
E-mail: angel@merl.a-star.edu.sg
Nanyang Technological University (NTU)
5
and
9 Nanyang Drive, Singapore 636921 (Singapore)
Prof. Dr. H. Zhao
2
15 Roger Adams Laboratory, Box C3
University of Illinois at Urbana-Champaign
00 South Mathews Avenue, Urbana, IL 61801 (USA)
E-mail: zhao5@illinois.edu
School of Biological Sciences, NTU
60 Nanyang Drive, Singapore 637551 (Singapore)
6
and
[
+]
Lee Kong Chian School of Medicine
Dr. Y. H. Lim, X. Chew, Dr. K. P. Chan
50 Nanyang Avenue, Singapore 639798 (Singapore)
Institute of Chemical and Engineering Sciences (ICES), A*STAR
Dr. E. G. Robins
Singapore Bioimaging Consortium (SBIC), A*STAR
8
Biomedical Grove, Neuros #07-01/02/03, Singapore 138665
(
Singapore)
11 Biopolis way, #02-02, Singapore 138667 (Singapore)
Dr. D. J. Smith
Bioinformatics Institute, A*STAR
+
[
] These authors contributed equally to this work.
3
and
0 Biopolis Street, Matrix #07-01, Singapore 138671 (Singapore)
Biotransformation Innovation Platform
61 Biopolis Drive, Proteos #04–14, Singapore 138673 (Singapore)
Dr. B. Xue, Prof. R. C. Robinson
Institute of Molecular and Cell Biology (IMCB), A*STAR
61 Biopolis Drive, Proteos #03–15, Singapore 138673 (Singapore)
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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