Exploration of a potential difluoromethyl-nucleoside substrate with the fluorinase enzyme

Article abstract of DOI:10.1016/j.bioorg.2015.11.003

The investigation of a difluoromethyl-bearing nucleoside with the fluorinase enzyme is described. 5′,5′-Difluoro-5′-deoxyadenosine 7 (F₂DA) was synthesised from adenosine, and found to bind to the fluorinase enzyme by isothermal titration calorimetry with similar affinity compared to 5′-fluoro-5′-deoxyadenosine 2 (FDA), the natural product of the enzymatic reaction. F₂DA 7 was found, however, not to undergo the enzyme catalysed reaction with l-selenomethionine, unlike FDA 2, which undergoes reaction with l-selenomethionine to generate Se-adenosylselenomethionine. A co-crystal structure of the fluorinase and F₂DA 7 and tartrate was solved to 1.8 ?, and revealed that the difluoromethyl group bridges interactions known to be essential for activation of the single fluorine in FDA 2. An unusual hydrogen bonding interaction between the hydrogen of the difluoromethyl group and one of the hydroxyl oxygens of the tartrate ligand was also observed. The bridging interactions, coupled with the inherently stronger C-F bond in the difluoromethyl group, offers an explanation for why no reaction is observed.

Full text of DOI:10.1016/j.bioorg.2015.11.003

Bioorganic Chemistry 64 (2016) 37–41
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Exploration of a potential difluoromethyl-nucleoside substrate
with the fluorinase enzyme
⇑
Stephen Thompson, Stephen A. McMahon, James H. Naismith, David O’Hagan
School of Chemistry and Biomedical Sciences Research Centre, University of St Andrews, North Haugh, St Andrews KY16 9ST, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
The investigation of a difluoromethyl-bearing nucleoside with the fluorinase enzyme is described.
50,50-Difluoro-50-deoxyadenosine 7 (F2DA) was synthesised from adenosine, and found to bind to the
fluorinase enzyme by isothermal titration calorimetry with similar affinity compared to 50-fluoro-50-
deoxyadenosine 2 (FDA), the natural product of the enzymatic reaction. F2DA 7 was found, however,
Received 7 October 2015
Revised 15 November 2015
Accepted 17 November 2015
Available online 18 November 2015
not to undergo the enzyme catalysed reaction with
L-selenomethionine, unlike FDA 2, which undergoes
reaction with -selenomethionine to generate Se-adenosylselenomethionine. A co-crystal structure of the
L
Keywords:
Fluorinase
Difluoromethyl
Isothermal titration calorimetry
Protein crystallography
fluorinase and F2DA 7 and tartrate was solved to 1.8 Å, and revealed that the difluoromethyl group
bridges interactions known to be essential for activation of the single fluorine in FDA 2. An unusual
hydrogen bonding interaction between the hydrogen of the difluoromethyl group and one of the hydroxyl
oxygens of the tartrate ligand was also observed. The bridging interactions, coupled with the inherently
stronger C–F bond in the difluoromethyl group, offers an explanation for why no reaction is observed.
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
The difluoromethyl group is of interest for introduction into
bioactive molecules as it bears a hydrogen atom which is available
The fluorinase enzyme, isolated originally from the soil
bacterium Streptomyces cattleya [1] and recently identified in addi-
tional bacterial species [2], catalyses formation of a C–F bond from
fluoride ion and S-adenosylmethionine 1 (SAM), generating 50-flu
oro-50-deoxyadenosine 2 (FDA) (Scheme 1). The reaction has been
shown to proceed by an inversion of configuration [3,4], and
QM/MM calculations provide strong support for an SN2 reaction
mechanism [5]. The fluorination reaction is reversible, and the
enzyme has the capacity to produce SAM 1 and fluoride ion when
as a hydrogen bond donor, although a more lipophilic hydrogen
bond donor than the more common hydrophilic O–H and N–H
groups [13]. The lipophilic hydrogen bonding capacity of the
difluoromethyl group has led to its development as a bio-isostere
of alcohols [14], thiols [15] and hydroxamic acids [16]. Routes
to access the difluoromethyl motif involve reaction of an
aldehyde with sulfur tetrafluoride [17], reaction of nucleophilic
‘‘RCFꢀ2 ” species with electrophiles [18–21], reaction of a nucle-
ophilic C, S, N, or O nucleophiles with difluorocarbene [22–26],
or transfer of a difluoromethyl radical to an appropriate substrate
[27–29].
incubated with FDA 2 and L-methionine [6]. The fluorinase is of
interest to biotechnology through its use as a biocatalyst for the
introduction of fluorine into organic molecules, and this ability
has been exploited using [18F]fluoride for the synthesis of PET
radiotracers [7–10]. The difluoromethyl and fluoromethyl groups
remain comparatively unexplored in terms of their application to
bioactive molecules [11]. Enzymatic synthesis of the fluoromethyl
group (R–CH2F) has been extensively explored [12], and we were
interested in exploiting the ability of the fluorinase to activate
fluoride ion in water, to extend fluorinase biocatalysis to the syn-
thesis of a difluoromethyl group (R–CF2H) from an appropriate
substrate.
To explore the enzymatic synthesis of a difluoromethyl group, an
appropriate substrate was required. To investigate the putative
transformation in the forward direction, an S-adenosylmethionine
analogue bearing
be required, however such 50-modified SAM derivatives have not
been reported. Simple -fluorosulfonium species, including (fluoro
a
fluorine atom at the 50-position would
a
methyl)dimethylsulfonium tetrafluoroborate [30], and diaryl
fluoromethylsulfonium salts [31] have been investigated as
electrophiles, suggesting that the motif may be susceptible to attack
by a suitably activated fluoride nucleophile. While the literature
reports of the
a-fluorosulfonium motif suggest that such a fluori-
nated SAM derivative may be thermodynamically stable, synthesis
of this highly functionalised substrate would not be trivial.
⇑
Corresponding author.
E-mail address: do1@st-andrews.ac.uk (D. O’Hagan).
http://dx.doi.org/10.1016/j.bioorg.2015.11.003
0045-2068/Ó 2015 Elsevier Inc. All rights reserved.

38
S. Thompson et al. / Bioorganic Chemistry 64 (2016) 37–41
NH2
N
by-products. After optimisation of the reaction conditions, fluori-
nation of aldehyde 5 with DeoxofluorÒ in THF furnished 6 with
fewer fluorinated by-products, ultimately offering 6 in 10% yield
after purification. With protected F2DA 7 in hand, the benzoyl
groups were cleaved by reaction of 6 with a freshly saturated solu-
tion of ammonia in methanol, in a sealed tube at 60 °C for 16 h.
After isolation, the resultant acetonide was hydrolysed in a mix-
ture of TFA and water, to provide F2DA 7 as a colourless powder,
in good yield. With a sample of F2DA 7 in hand, we set out to
explore its interaction with the fluorinase enzyme.
NH2
N
COO
N
N
N
N
-Met
L
F
H3N
N
N
O
O
F
S
Me
fluorinase
OH
OH
HO
HO
SAM
1
FDA
2
Scheme 1. The reversible synthesis of a C–F bond from fluoride and S-adenosyl-
methionine 1 (SAM) by the fluorinase enzyme.
2.2. Isothermal titration calorimetry
We envisioned utilising the fluorinase-catalysed reaction in the
reverse direction, where incubation of a difluoromethylated sub-
strate (F2DA 7) was proposed to generate a fluoro–SeSAM interme-
The binding of F2DA 7 to the fluorinase was investigated using
isothermal titration calorimetry (ITC). Accordingly, a solution of
F2DA 7 in a phosphate buffer was titrated into a solution of the
fluorinase in the same buffer. The binding curve obtained is
illustrated below in Fig. 1A, and the data was fitted to a 1:1
isotherm assuming a single binding site. A similar experiment
was conducted by titrating FDA 2, the natural substrate, into the
fluorinase for comparison, and the resultant titration curve is illus-
trated in Fig. 1B. The titration curve revealed that F2DA 7 exhibited
strong binding to the fluorinase. The association constant (Ka) for
this interaction was calculated to be 27.1 2.01 ꢁ 105 Mꢀ1. Com-
parison of the association constant with that of FDA 2 revealed that
F2DA 7 bound with slightly higher affinity for the fluorinase, by a
factor of nearly two. Both compounds show favourable enthalpic
contributions, but F2DA 7 shows a greater exotherm upon binding,
likely due to increased lipophilicity of the difluoromethyl group
compared to the fluoromethyl group. The greater enthalpic contri-
bution for F2DA 7 is compensated for by a larger entropic penalty,
which results in the similar nett free energy change to that
observed for FDA 2. The presence of the second fluorine substituent
at C-50 appears to increase the affinity of the nucleoside for the
enzyme.
diate upon reaction with L-selenomethionine [6] as the incoming
nucleophile. In this paper, we describe a modified synthesis of
F2DA 7, and an exploration of this putative substrate for selective
substitution of one of the fluorine atoms of a difluoromethyl group,
attempting to capitalise on the fluoride-activating properties of the
fluorinase enzyme.
2. Results and discussion
2.1. Synthesis of F2DA 7
The synthesis of 50,50-difluoro-50-deoxyadenosine 7 has been
previously reported by Jarvi et al. [32,33], where fluorination of
N,N-dibenzoyl-20,30-O-isopropylidneadenosine-50-aldehyde 5 with
diethylaminosulfur trifluoride (DAST) was the key fluorination
step. In a modified version of their synthesis, adenosine 3 was
protected as its 20,30-acetonide with 2,2-dimethoxypropane in
acetone, catalysed by perchloric acid, as shown in Scheme 2. The
resultant acetonide was dibenzoylated in excellent yield with
TMSCl, and an excess of benzoyl chloride to furnish 4. Oxidation
of 4 under Moffatt conditions, but substituting DCC for EDCI.HCl,
gave excellent conversion of the alcohol to a mixture of aldehyde 5
and its hydrate, without the requirement for trapping as the ami-
nal as reported by Jarvi et al. [32,33]. Azeotropic removal of water
by repeated co-evaporation with toluene furnished aldehyde 5 in
quantitative yield, which was used without further purification.
Jarvi et al. [32,33] report a low yield for the fluorination of 5
(18%) using DAST in DCM, however, we found that use of the
reported conditions led to the formation of multiple fluorinated
2.3. Incubation of F2DA 7 with the fluorinase
Confident that the candidate substrate, F2DA 7, binds to the
enzyme, attention turned to investigating whether the fluorinase
was able to catalyse substitution of one of the fluorine atoms of
with
L-selenomethionine (L-SeMet) rather than L-methionine.
Selenium (Se) is a better nucleophile [34] than sulfur due to the
presence of a higher energy HOMO on the selenium atom, and
NH2
N
NBz2
N
NBz2
N
N
N
N
N
N
1. 2,2-dimethoxypropane,
HClO4
80%
EDCI, DMSO,
O
,
acetone,
dichloroacetic acid
N
N
N
N
O
O
O
HO
HO
H
pyridine,
2. TMSCl,
BzCl,
quant.
99%
O
O
O
O
HO OH
Me Me
Me Me
3
4
5
Deoxofluor®
THF
10%
NH2
NBz2
N
N
N
N
F
F
1. NH3
2.
,
35%
86%
MeOH,
N
O
N
N
N
O
F
F
H
TFA, 2O,
HO OH
O
O
Me Me
7
6
Scheme 2. Synthesis of F2DA 7.

S. Thompson et al. / Bioorganic Chemistry 64 (2016) 37–41
39
Fig. 1. Isothermal titration calorimetric determination of the binding affinity of A. F2DA 7 (0.72 mM) into the fluorinase (60.3
lM) and B. FDA 2 (1.03 mM) into the fluorinase
(53.6 lM).
the increased polarizability of Se over S [35]. In addition, it has
previously been demonstrated that, in the reverse direction, the
fluorinase catalysed reaction between FDA 2 and
6-fold faster [6] than with the -Met, suggesting that assays with
-SeMet are better suited for investigating putative substrates.
L-SeMet occurs
L
L
The anticipated transformation of F2DA 7 to FSeAM 8 is illustrated
above in Scheme 3.
F2DA 7 was incubated with L-SeMet (0.6 mM) and the fluorinase
enzyme in phosphate buffer. Reaction progress was monitored by
HPLC at t = 0, 1, 2, and 19 h, and the results are illustrated in
Fig. 2. The HPLC trace did not reveal any change in the concentra-
tion of F2DA 7 over 19 h, and there were no significant new peaks
evident in the chromatogram. A similar experiment, substituting
Fig. 2. HPLC time course of incubation of F2DA 7 with L-SeMet (0.6 mM) and the
L-Met for L-SeMet did not result in any conversion to new products.
fluorinase, with samples taken at t = 0, 1, 2 and 19 h. The chromatograms show no
change in concentration of F2DA 7, and no new products were evident, suggesting
that F2DA 7 is not a substrate for the fluorinase enzyme.
This data suggested that the fluorinase is not capable of catalysing
the proposed substitution.
The lack of any obvious reaction with either L-Met or L-SeMet
or its selenium analogue, distinguishing between equilibrium
effects or inherent lack of reactivity is not straightforward.
suggested that the inherent properties of F2DA 7, possibly steric
or electronic, or a combination thereof, prevent substitution of
one of the fluorine atoms at C-50. As the fluorinase reaction is
known to be reversible, the possibility also exists that the equilib-
rium for this transformation lies far in favour of the difluorinated
product. Without access to synthetic samples of a 50-fluoro-SAM
2.4. Crystallography of F2DA 7 with the fluorinase
To further probe the binding of F2DA 7 to the enzyme and to try
to understand the lack of reactivity, conditions were explored for
the preparation of a fluorinase-F2DA 7 co-crystal. A co-crystal suit-
able for diffraction was obtained and a structure solved to a reso-
lution of 1.8 Å (PDB code: 5FIU) by molecular replacement using
the original fluorinase crystal structure (PDB code: 1RQR) [36].
For direct comparison, the co-crystal structure of F2DA 7 was over-
NH2
N
NH2
N
COO
N
N
N
N
F
L-SeMet
H3N
F
F
N
N
O
O
Se
Me
F
fluorinase
OH
OH
HO
HO
laid with that of FDA 2 and L-Met bound in the active site of the flu-
FSeAM
8
F2DA
7
orinase [36]. The structures are shown above in Fig. 3, with the
bright coloured structure and ligand with grey C-atoms belonging
to the F2DA 7 structure, while pastel coloured structure and ligands
Scheme 3. Exploration of the enzymatic transformation of F2DA 7 to its corre-
sponding FSeAM analogue 8.
with yellow C-atoms belonging to the FDA-L-Met structure.

40
S. Thompson et al. / Bioorganic Chemistry 64 (2016) 37–41
Fig. 3. A. Overlay of the structure of fluorinase bound to FDA 2 (pastel colours) and F2DA 7 (bright colours) showing no gross conformational change when bound to F2DA 7. B.
F2DA 7 in the active site of the fluorinase, showing a hydrogen bonding contact between the hydrogen of the difluoromethyl group and one of the tartrate hydroxyl oxygens.
The two monomers of the fluorinase shown in blue and red. The structure was obtained with a molecule of tartrate bound in the active site. C. Close up of the active site
showing orientation of the fluorine atoms of F2DA 7 (grey carbon skeleton) compared to the orientation of the C–F bond of FDA 2 (yellow carbon skeleton). Distances (Å) from
the fluorine atoms to key residues are shown for FDA 2 (yellow dashes) and F2DA 7 (grey dashes).
Binding of F2DA 7 does not result in any gross conformational
change of the structure of the fluorinase, as shown in Fig. 3A.
F2DA 7 was located in the active site, along with a molecule of
tartrate (from the crystallisation buffer), as shown in Fig. 3B. Exam-
ination of the structure revealed that F2DA 7 engaged in numerous
contacts to active site residues, as had been observed with other
nucleosides co-crystallised with the enzyme. The adenine base
bonding interaction with one of the hydroxyl oxygen atoms of
the co-bound tartrate (CF2H–O distance = 2.1 Å), and does not form
close contacts to any amino acid residues in the active site.
The adenine bases overlay well, as does the ribose ring. It is
clear in Fig. 3C that the fluorine atom of FDA 2, the natural product,
lies between the two fluorine atoms of the difluoromethyl group of
F2DA 7. The location of the two fluorine atoms and their suggested
interactions with Thr-80 and Ser-158, as shown above, support the
suggestion that the fluoromethyl group bridges the interactions
usually engaged by the single fluorine atom of FDA 2.
The C–F bond is the strongest single bond observed in organic
molecules, having a bond dissociation energy, on average, of
105.4 kJ molꢀ1 [37]. In aliphatic systems, fluoride is well charac-
terised as a poor leaving group [38] a consequence of the strength
of the C–F bond. The fluorinase enzyme has, however, uniquely
evolved the ability to catalyse the cleavage of the C–F, bond with
was found to participate in
p–p stacking interactions with Phe-
254 and Trp-50, while the endo- and exo-cyclic nitrogen atoms
formed hydrogen bonding interactions to Ala-279 and Asn-215.
The 20,30-diol system of the ribose ring was found to participate
in a hydrogen bonding interaction with Asp-16. Tartrate occupies
the
where one of the carboxylates engages in polar interactions similar
to those observed for carboxylate group of -Met.
L-Met binding site, adopting a similar conformation to L-Met,
L
Closer examination of the difluoromethyl group revealed that
the fluorine atoms were located in the hydrophobic fluoride bind-
ing pocket. The interactions previously identified [36] as important
for reaction appeared conserved. One of the fluorine atoms lies
3.0 Å from the OH group of Thr-80, while the second fluorine
makes contacts with the amide NH and side chain OH of Ser-150
(3.2 Å and 2.9 Å respectively). The two fluorine atoms appear to
‘‘share” the contacts usually observed between the protein
and the fluorine atom of FDA 2. The hydrogen atom of the
difluoromethyl group appears to engage in a weak hydrogen
L-Met as an incoming nucleophile. The analogous transformation
has also been reported, using a range of Lewis acid or hydrogen
bond catalysts to activate fluoride as a leaving group [39]. Substi-
tution of both fluorine atoms in difluoromethylene and difluo-
romethyl groups is an even more challenging transformation. The
addition of a second electronegative fluorine atom is expected to
increase the strength of each of the C–F bonds, due to increased
positive character at carbon [40]. The non-bonding electrons of
the second fluorine atom are also expected to hinder approach of

S. Thompson et al. / Bioorganic Chemistry 64 (2016) 37–41
41
an incoming nucleophile [41]. Despite this, substitution of such
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Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2015.11.
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