An enzymatic Finkelstein reaction: Fluorinase catalyses direct halogen exchange

Article abstract of DOI:10.1039/c9ob01625b

The fluorinase enzyme from Streptomyces cattleya is shown to catalyse a direct displacement of bromide and iodide by fluoride ion from 5′-bromodeoxyadenosine (5′-BrDA) and 5′-iododeoxyadenosine (5′-IDA) respectively to form 5′-fluorodeoxyadenosine (5′-FDA) in the absence of l-methionine (l-Met) or S-adenosyl-l-methionine (SAM). 5′-BrDA is the most efficient substrate for this enzyme catalysed Finkelstein reaction.

Full text of DOI:10.1039/c9ob01625b

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: D. O'Hagan, P. T.
Lowe and S. L. Cobb, Org. Biomol. Chem., 2019, DOI: 10.1039/C9OB01625B.
Volume 15
Number 47
21 December 2017
Pages 9945-10124
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
Accepted Manuscripts are published online shortly after acceptance,
rsc.li/obc
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
ISSN 1477-0520
PAPER
I . J. Dmochowski et al.
Oligonucleotide modifi cations enhance probe stability for
shall the Royal Society of Chemistry be held responsible for any errors
single cell transcriptome in vivo analysis (TIVA)
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
rsc.li/obc

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 1 of 4
View Article Online
DOI: 10.1039/C9OB01625B
Organic & Biomolecular Chemistry
Paper
An enzymatic Finkelstein reaction: Fluorinase
catalyses direct halogen exchange
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
Phillip T. Lowe, Steven L. Cobb and David O'Hagan*a
DOI: 10.1039/x0xx00000x
The fluorinase enzyme from Streptomyces cattleya is shown to catalyse a direct displacement of bromide and
iodide by fluoride ion from 5’-bromodeoxyadenosine (5’-BrDA) and 5’-iododeoxyadenosine (5’-IDA)
respectively, to form 5’-fluorodeoxyadenosine (5’-FDA) in the absence of L-methionine (L-Met) or S-adenosyl-
L-methionine (SAM). 5’-BrDA is the most efficient substrate for this enzyme catalysed Finkelstein reaction.
www.rsc.org/
Introduction
5’-chloro-5’-deoxy adenosine (5’-ClDA) 1 by L-Met to generate SAM
Fluorine containing compounds have a remarkable record in tuning
the properties of medicinal,1,2 agricultural3,4 and materials5
chemistry products. As such, in recent years the development of
methodology to incorporate fluorine into biologically and
commercially relevant compounds under mild conditions has
intensified. The naturally occurring fluorinase enzyme (5’-fluoro-5’-
deoxy-adenosine synthase), originally isolated from Streptomyces
cattleya where it is involved in the C-F bond forming step in
fluoroacetate and 4-fluorothreonine biosynthesis, offers a rare
opportunity to introduce fluorine enzymatically. Since its discovery,
subsequent investigation has yielded 5 enzyme homologues from
actinomycete organisms (FLA,6,7 FLA1,8 FLA3,8 FLA49,10 and NobA8,11),
along with significant insight into its structure and mechanism of
action.12,13 In nature the fluorinase catalyses a C-F bond forming
reaction between S-adenosyl-L-methionine (SAM) 2 and fluoride ion
2.15 When this reaction is conducted in the presence of fluoride ion,
the resultant SAM 2 is then converted to 5’-FDA 3, to achieve a two-
step transhalogenation from 5’-ClDA 1 to 5’-FDA 3. The substrate
flexibility of this ‘two-step’ fluorination process extends to 2’-deoxy
analogues16 of 5’-ClDA 1 as well as C-2 decoration of the adenosine
base of 5’-ClDA 1 with an acetylene, or terminally functionalised
acetylene moieties.17-23 In this manner, fluorinase-mediated trans-
halogenation reactions have become established as a strategy for the
synthesis of fluorinated bioactive compounds under experimentally
benign conditions (ambient temperatures in buffer at pH 7.8). In
particular, the method has been utilised for the late-stage 18F-
radiolabelling of cancer relevant targeting peptides17-21 and A2A
adenosine receptor agonists23 for use in positron emission
tomography (PET). Recently, in an effort to diversify the substrate
scope and optimise the ‘two-step’ fluorination reaction, the
fluorinase was subject to directed evolution, and modest rate
improvements were achieved for the first step only, involving
displacement of chloride from 5’-ClDA 3 by L-Met.24-25 In this paper
we report that the fluorinase is able to mediate a direct fluorination
reaction of 5’-halogenated-5’-deoxy-adenosines, in a reaction which
does not require its natural substrates SAM or L-Met.
to generate 5’-fluoro-5’-deoxy-adenosine (5’-FDA)
3 and L-
methionine (L-Met).12-14 The enzyme also catalyses the reverse
reaction, for example the nucleophilic displacement of chloride from
NH2
N
NH2
N
NH2
N
N
N
N
N
N
N
Fluorinase
L-Met
Fluorinase
KF
O
Cl
S
F
N
N
N
O
O
O
O
pH 7.8, 37 °C
pH 7.8, 37 °C
NH3
NH2
N
NH2
N
OH OH
OH OH
OH OH
N
N
N
N
Fluorinase
KF
5'-ClDA 1
SAM
2
5'-FDA
3
X
F
X
Scheme 1 Fluorinase-catalysed transhalogenation of 5’-ClDA 1 to 5’-FDA 3 via
SAM 2.
N
N
O
O
pH 7.8, 37 °C
F
OH OH
OH OH
3
5'-FDA
1
4
5
X = Cl
X = Br
X = I
a. School of Chemistry, University of St. Andrews, North Haugh, St. Andrews, Fife,
KY16 9ST, UK. E-mail: do1@st-andrews.ac.uk
Scheme 2 Fluorinase-catalysed direct fluorination of 1, 4 and 5 halogenated-
5’-deoxy-adenosine substrates.
b. Now at Department of Chemistry, Durham University, South Road, Durham,
DH1 3LE, UK. E-mail: s.l.cobb@durham.ac.uk
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 2 of 4
ARTICLE
Journal Name
fluorinase
(125
M).
Yield
=
(concentration ViewoAfrticle5O’-nFlDinAe
Results and Discussion
generated/concentration of substrate) x 100%, data are representative of
three different experiments.
DOI: 10.1039/C9OB01625B
The halogenated nucleosides: 5’-ClDA 1, 5’-BrDA 4 and 5’-IDA 5
(Scheme 2) were assayed in initial time course experiments to
determine their ability to undergo a direct fluorination. Each
substrate, 5’-ClDA 1, 5’-BrDA 4 and 5’-IDA 5, was converted to
5’-FDA 3 in the presence of the fluorinase and inorganic fluoride
only (Fig S2), although the rates differed significantly depending
on the 5’-halogen (Br >>> I > Cl). The bromo substrate 5’-BrDA 4
was the most efficient, reaching conversions above 35% under
the conditions, whereas 5’-IDA 5 and 5’-ClDA 1 displayed
significantly lower conversions of 8% and 5% respectively.
To investigate the comparative rates of fluorination of 1, 4
and 5, time course experiments were conducted with the
fluorinase over a period of 24 h (Fig. 1). Assays were performed
with KF both either with or without added L-Met. As expected,
the fluorinase showed significantly greater initial rates for direct
fluorination (without L-Met) with 5’-BrDA 4 than for 5’-IDA 5
and 5’-ClDA 1, yielding 15%, 1.2% and 0.2% conversions
respectively after 4 h (Fig. 1). Direct fluorination kinetic
parameters were evaluated for 5’-BrDA 4 (Km = 9.7 ± 2.6 μM, kcat
= 0.017 ± 0.004 min-1). The Km was similar in magnitude to SAM
indicating similar affinities for the enzyme. When
transhalogenation assays to generate 5’-FDA were performed in
the presence of L-Met, 5’-ClDA 1 was the most efficient
substrate. 5’-BrDA 4 presented a slightly improved rate over
direct fluorination, indicating that it can also participate in the
Table 1 Comparative kinetic data of fluorinase substrates 5’-BrDA 4 and SAM.
KM
(μM)
kcat
[kcat/KM] (mM-
1min-1)
Substrate
5’-BrDA 4a
SAMb
(min-1)
9.7 ± 2.6
0.017 ± 0.004
2.1
6.5 ± 0.3
0.07 ± 0.001
10.8
a
b
Assays contain: 4 (10 – 500 μM), KF (200 mM), and fluorinase (8 μM).
Kinetic data from Zhu et al.13
substrate in the presence of L-Met but in the absence of KF
(Table 2). The observed conversions to SAM after a 4 h
incubation (1 (44%), 4 (27%) and 5 (<0.1%)) were consistent
with the rates for the overall two-step fluorination assays
favouring 5’-ClDA 1 and then 5’-BrDA 4. 5’-IDA 5 was not a
substrate for this reaction.
5’-BrDA
4
undergoes both direct and ‘two-step’
transhalogenation reactions with similar efficiencies in the
presence of fluoride ion to generate 5’-FDA, whereas 5’-ClDA 1
requires L-Met for efficient conversion and progresses most
efficiently via the two-step transhalogenation process. When
5’-BrDA 4 was assayed with other halides, e.g. by adding KCl or
KI as halide sources, there were no productive reactions to
generate 5’-IDA 5 or 5’-ClDA 1 (Scheme S4). Also the reverse
reaction to produce 5’-BrDA 4 could not be observed when 5’-
FDA 3 was incubated with the fluorinase and KBr (Scheme S5).
Previous X-ray crystallographic analysis of 5’-ClDA 1 bound
into the active site of the fluorinase revealed that it adopts two
poses (Fig 2).15 One orientates the 5’-chlorine atom into the
halide binding pocket which would otherwise bind fluoride ion
(Fig 2a). This orientation is essential for step one of the two-step
reaction to proceed as it accommodates the correct trajectory
for nucleophilic attack by the sulphur atom of L-Met to the 5’-
carbon of 5’-ClDA, for the synthesis of SAM. The second pose
orientates the 5’-chlorine towards the empty binding pocket for
the L-Met sulfonium sulfur (Fig 2b). This orientation is required
two-step process. 5’-IDA
5 did not demonstrate any
improvement in the presence L-Met and both regimes gave low
conversions with 5’-IDA 5.
In order to compare the relative ability of the fluorinase to
perform the initial step of the two step process, to generate
SAM from 1, 4 and 5, the enzyme was incubated with each
45
40
35
%
yield
30
25
20
15
10
5
Table 2 Comparative % SAM product yields of the fluorinase using 5’-ClDA 1,
5’-BrDA 4 and 5’-IDA 5.
NH2
NH2
ClDA + LMet + KF
BrDA + LMet + KF
BrDA + KF
N
N
N
N
Fluorinase
L-Met
N
N
O
R
S
N
N
O
O
O
pH 7.8, 37 °C
NH3
ClDA + KF
OH OH
OH OH
SAM (2)
R = Cl (1), Br (4), I (5)
IDA + KF
% Conversion to
SAM after 4ha
Substrate
ClDA (1)
BrDA (4)
IDA (5)
44 % ± 1.5
27 % ± 3.2
<0.1 % ± 0
0
0
10
20
Time (h)
a
Assays contain: substrate (0.2 mM), L-Met (0.2 mM), and fluorinase (125
mM).
Fig. 1 Reactions rates for the fluorinase with substrates 5’-ClDA (1), 5’-BrDA
(2) and 5’-IDA (5) at 37 ◦C over 24 h. Both conversions to 5’-FDA via
transhalogenation (with L-Met + KF) and direct fluorination (with KF, without
L-Met) are shown. Reaction conditions: substrates 1, 4, & 5 (0.2 mM), L-Met
(0.1 mM, excluded for direct fluorination experiments), KF (80 mM), and
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 4
Organic & Biomolecular Chemistry
Please do not adjust margins
Journal Name
ARTICLE
View Article Online
DOI: 10.1039/C9OB01625B
Experimental
A
See Supporting Information for experimental detail on:
Compound synthesis and characterisation, assay conditions and
fluorinase overexpression and purification.
Conflicts of interest
There are no conflicts to declare
B
Acknowledgements
We thank the Engineering and Physical Sciences Research
Council, UK, for a research grant.
References
(1) Zhou, Y.; Wang, J.; Gu, Z.; Wang, S.; Zhu, W.; Aceña, J. L.;
Soloshonok, V. A.; Izawa, K.; Liu, H. Chem. Rev., 2016, 116,
422-518.
Fig. 2 Structure of the fluorinase enzyme bound to 5’-ClDA 1 accommodating (2) Meanwell, N. A. J. Med. Chem., 2018, 61, 5822-5880.
two distinct poses within the active site: A - with its 5’-chlorine atom
(3) Jeschke, P. Pest Manage. Sci., 2010, 66, 10-27.
positioned within the halide binding pocket (orientation for two-step
(4) Jeschke, P. Pest Manage. Sci., 2017, 73, 1053-1066.
fluorination) and B - with its 5’-chlorine atom positioned within the L-
(5) Berger, R.; Resnati, G.; Metrangolo, P.; Weber, E.; Hulliger,
J. Chem. Soc. Rev., 2011, 40, 3496-3508.
Methionine binding pocket (orientation for direct fluorination).15
(6) O'Hagan, D.; Schaffrath, C.; Cobb, S. L.; Hamilton, J. T. G.;
Murphy, C. D. Nature., 2002, 416, 279-279.
(7) Schaffrath, C.; Deng, H.; O'Hagan, D. FEBS Lett. 2003, 547,
111-114.
(8) Deng, H.; Ma, L.; Bandaranayaka, N.; Qin, Z.; Mann, G.;
Kyeremeh, K.; Yu, Y.; Shepherd, T.; Naismith, J. H.; O'Hagan,
D. ChemBioChem., 2014, 15, 364-368.
(9) Huang, S.; Ma, L.; Tong, M. H.; Yu, Y.; O'Hagan, D.; Deng, H.
Org. Biomol. Chem., 2014, 12, 4828-4831.
to allow direct fluorination of 5’-ClDA 1 by attack of fluoride
from the fluoride ion binding site, however the process is not
efficient presumably due to the relative strength of the C-Cl
bond and a poor catalytic prowess of the fluorinase to achieve
chloride displacement. It follows that the bromine in 5’-BrDA 4
must reasonably locate in the methylsulfonium binding site, and
the weaker C-Br bond becomes displaced by fluoride ion (Fig
2b). The inability 5’-IDA 5 to undergo either the two-step
transhalogenation reaction or direct fluorination in an efficient
manner, suggests the iodine is too large to be accommodated
with an appropriate geometry for efficient nucleophilic
displacement in either site.
(10) Ma, L.; Li, Y.; Meng, L.; Deng, H.; Li, Y.; Zhang, Q.; Diao, A.
RSC Adv., 2016, 6, 27047-27051.
(11) Wang, Y.; Deng, Z.; Qu, X. F1000Research., 2014, 3:61. 1-14.
(12) Dong, C.; Huang, F.; Deng, H.; Schaffrath, C.; Spencer, J. B.;
O'Hagan, D.; Naismith, J. H. Nature., 2004, 427, 561-565.
(13) Zhu, X.; Robinson, D. A.; McEwan, A. R.; O'Hagan, D.;
Naismith, J. H. J. Am. Chem. Soc. 2007, 129, 14597-13655.
(14) Cadicamo, C. D.; Courtieu, J.; Deng, H.; Meddour, A.;
O'Hagan, D. ChemBioChem., 2004, 5, 685-690.
(15) Deng, H.; Cobb, S. L.; McEwan, A. R.; McGlinchey, R. P.;
Naismith, J. H.; O'Hagan, D.; Robinson, D. A.; Spencer, J. B.
Angew. Chem., 2006, 45, 759-762.
Conclusions
We have extended the substrate scope of the fluorinase
enzyme, revealing its capacity to mediate a direct fluorination
of 5’-BrDA 4 by fluoride ion and to a lesser extent using 5’-IDA 5
as a substrate. 5’-ClDA 1, though capable of this transformation,
is a very poor substrate. This is an enzymatic Finkelstein
reaction,
a transformation not previously reported in
enzymology. The one step process offers an advantage over the
previous two-step approach for accelerated evolution.[24,25]
Furthermore there is potential for a simplified one step protocol
for the preparation of [18F]-fluoride labelled radioligands for PET
imaging. To date [18F]-Fluoride incorporation has largely
focussed on two-step enzymatic reactions using 5’-ClDA
analogues and L-Met as a cofactor.[17-23]
(16) Cobb, S. L.; Deng, H.; McEwan, A. R.; Naismith, J. H.;
O’Hagan, D.; Robinson, D. A. Org. Biomol. Chem., 2006, 4,
1458-1460.
(17) Thompson, S.; Zhang, Q.; Onega, M.; McMahon, S.; Fleming,
I.; Ashworth, S.; Naismith, J. H.; Passchier, J.; O'Hagan, D.
Angew. Chem., 2014, 53, 8913-8918.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 4 of 4
ARTICLE
Journal Name
(18) Thompson, S.; Onega, M.; Ashworth, S.; Fleming, I. N.;
Passchier, J.; O'Hagan, D. Chem. Commun., 2015, 51, 13542-
13545.
View Article Online
DOI: 10.1039/C9OB01625B
(19) Thompson, S.; Fleming, I. N.; O'Hagan, D. Org. Biomol.
Chem., 2016, 14, 3120-3129.
(20) Zhang, Q.; Dall'Angelo, S.; Fleming, I. N.; Schweiger, L. F.;
Zanda, M.; O'Hagan, D. Chem. - Eur. J., 2016, 22, 10998-
11004.
(21) Lowe, P. T.; Dall'Angelo, S.; Fleming, I. N.; Piras, M; Zanda,
M.; O'Hagan, D. Org. Biomol. Chem., 2019, 17, 1480-1486.
(22) Lowe, P. T.; Dall'Angelo, S.; Devine, A.; Zanda, M.; O'Hagan,
D. ChemBioChem, 2018, 19, 1969-1978.
(23) Lowe, P. T.; Dall'Angelo, S.; Mulder-Krieger, T.; Ijzerman, A.
P.; Zanda, M.; O'Hagan, D. ChemBioChem, 2017, 18, 2156-
2164.
(24) Sun, H.; Yeo, W. L.; Lim, Y. H.; Chew, X.; Smith, D. J.; Xue, B.;
Chan, K. P.; Robinson, R. C.; Robins, E. G.; Zhao, H.; Ang, E.
L. Angew. Chem., 2016, 55, 14277-14280.
(25) Yeo, W. L.; Chew, X.; Smith, D. J.; Chan, K. P.; Sun, H.; Zhao,
H.; Lim, Y. H.; Ang, E. L. Chem. Commun., 2017, 53, 2559-
2562.
(26) Finkelstein, H. Ber. Dtsch. Chem. Ges., 1910, 43, 1528-1532.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins