Fluorinase-coupled base swaps: Synthesis of [18F]-5′- deoxy-5′-fluorouridines

Article abstract of DOI:10.1002/anie.200804040

(Chemical Equation Presented) Making F-ases: One-pot fluorination/base-swap biotransformations were developed by coupling the fluorinase enzyme to nucleoside phosphorylases to generate 5′-deoxy-5′-fluoronucleosides (FDAs). These biotransformations are amenable to radiolabeling syntheses starting from [¹⁸F]fluoride ion, as exemplified by the synthesis of [¹⁸F]-5′-deoxy-5′-fluorouridines (see scheme), and demonstrate a new application of fluorinase as a catalyst for ¹⁸F-C bond formation.

Full text of DOI:10.1002/anie.200804040

Angewandte
Chemie
DOI: 10.1002/anie.200804040
Radiolabeling
Fluorinase-Coupled Base Swaps: Synthesis of [18F]-5’-Deoxy-5’-
fluorouridines**
Margit Winkler,* Juozas Domarkas, Lutz F. Schweiger, and David OꢀHagan*
The application of positron emission tomography (PET) for
medical imaging and diagnostics[1] is a rapidly growing area,
and cyclotrons are being increasingly commissioned in major
hospitals. Developing methods for the introduction of the
appropriate isotopes (11C, 13N, 18F, and 15O) into organic
structural motifs is a major research activity at present.[2] The
relatively long half-life of 18F (t1/2 = 110 min) renders it an
attractive radioisotope for PET, and synthetic protocols
employing [18F]fluoride ion are particularly attractive as this
form of the isotope is generated in a very high specific activity
in which a cold carrier ([19F]fluoride) is not added.[3] Chemical
strategies for the incorporation of 18F into organic compounds
are being widely explored,[4] but enzymatic approaches offer a
unique, mild, and selective approach for fluorination. We
manner.[7] More generally, radiolabeled nucleosides are being
extensively studied as possible tracers for the assessment of
tumor biochemistry.[8] The presence of adenosine receptors[9]
and specific uridine receptors[10] in the brain increases the
significance of these compounds as tracers for neurological
studies. Combining fluorinase with nucleoside-converting
enzymes[11] offered an attractive strategy for the preparation
of radiolabeled nucleosides. Herein we report that 5’-deoxy-
5’-fluoronucleosides can be prepared using fluorinase com-
bined with appropriate nucleoside phosphorylases. The
reaction of 5’-FDA with a purine nucleoside phosphorylase
(PNP, E.C. 2.4.2.1) generates 5-deoxy-5-fluoro-a,d-ribose-1-
phosphate (5-FDRP) in situ. The reversibility of this reaction
offers the potential for swapping adenine for another purine
base. PNPs from various sources have been identified[12] but
only a few catalyze the depurination of adenosine. In this
study the PNP from S. cattleya was used because 5’-FDA is its
natural substrate. Accordingly, incubation of the fluorinase
and fluoride ion with PNP and 2,6-diaminopurine resulted in
the accumulation of the fluorinated nucleoside 1 (Scheme 1).
The substrate specificity of this PNP is restricted to adenine
analogues having an amine at C6 of the purine.[13]
have been interested in using the fluorinase enzyme (E.C.
18
À
2.5.1.63, Streptomyces cattleya) as a catalyst for F C bond
formation. The enzyme catalyzes the nucleophilic attack of
fluoride ion to the C5’ center of (S)-adenosyl-l-methionine
(SAM) to generate 5’-deoxy-5’-fluoroadenosine (5’-FDA) and
l-methionine.[5] Although the fluorinase is a relatively slow
enzyme, it is readily available by overexpression from
E. coli.[6] It is easily obtained in milligram per milliliter
quantities, is stable for long periods, and can be used at
micromolar concentrations. [18F]Fluoride is generated by the
cyclotron at nanomolar concentrations; therefore the enzyme
is usually in excess which can overcome sluggish reaction
rates. We recently demonstrated the synthesis of 5’-[18F]FDA
in high radiochemical yield (RCY) using fluorinase in this
To extend the versatility of the base-swap protocol,
reactions with
a pyrimidine nucleoside phosphorylase
(PyNP, EC 2.4.2.2 Bacillus stearothermophilus) were
explored. This enzyme catalyses the reversible phosphorolysis
of both uridine and thymidine by displacing the pyrimidine
[*] Dr. M. Winkler, Prof. Dr. D. O’Hagan
Centre for Biomolecular Sciences and School of Chemistry
University of St. Andrews
North Haugh, St. Andrews KY16 9ST (UK)
Fax: (+44)1334-463-808
http://chemistry.st-and.ac.uk/staff/doh/group
E-mail: mw60@st-andrews.ac.uk
do1@st-andrews.ac.uk
Dr. J. Domarkas, Dr. L. F. Schweiger
John Mallard Scottish PET Centre, School of Medical Sciences
University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (UK)
[**] We thank the Austrian Science Fund FWF for fellowship support (to
M.W., Project J2694) and J.D. acknowledges the Scottish Funding
Council and SINAPSE collaboration for financial support. We thank
Leslie Kinsland and Steven E. Ealick (Cornell University (USA)) for
providing recombinant pyrimidine nucleoside phosphorylase, and
we are grateful to Louise L. Major for the expression vector for
purine nucleoside phosphorylase. Caroline E. R. Horsburgh and
Catherine H. Botting (MS) as well as Melanja H. Smith and Tomas
Lebl (NMR) are acknowledged for analytical support and Stuart
Craib for 18F production.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200804040.
Scheme 1. Biotransformation to give 2-amino-5’-deoxy-5’-fluoroadeno-
sine.
Angew. Chem. Int. Ed. 2008, 47, 10141 –10143
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
10141

Communications
base in each case.[14] Previous reports of enzymatic trans-
glycosylation reactions have almost exclusively monitored the
overall displacement of a pyrimidine by a purine base.[11b,15] In
this case, the reaction equilibria have been optimized to
promote the reverse reaction and to generate pyrimidine
nucleosides 2–6 (Scheme 2).
Scheme 3. Preparation of the dehydroxyfluoro analogue of antiviral
ribavirin.
however, it proved to be a useful enzyme for transglycosy-
lations to generate 2–6.
Scheme 2. One-pot fluorinase/nucleoside phosphorylase reaction to
give 5’-fluorinated uridine derivatives (R=H: 2, R=Me: 3, R=F: 4,
R=Cl: 5, R=Br: 6).
Ribavirin is a nucleoside with broad spectrum antiviral
activity.[17,18] To additionally demonstrate the versatility of the
fluorination/base-swap protocol the fluorodehydroxy ana-
logue of ribavirin was prepared in a one-pot biotransforma-
tion (Scheme 3).
Finally, one-pot biotransformations to give radiolabeled
[18F]-5’-deoxy-5’-fluorouridines 2, 4, and 6 were executed
(Scheme 4). To improve the conversion, an additional
Thymidine phosphorylases (TP, EC 2.4.2.4) are reported
to display specificity for 2’-deoxyribonucleosides,[11,16] and to
accept a variety of pyrimidine bases, therefore emerging as an
attractive prospect to add structural diversity to the fluorina-
tion/base-swap reactions. The commercial preparation of this
enzyme contains 500 mm phosphate and uracil as a stabilizer,
therefore the enzyme was preferably subcloned from E. coli
(pET-28a). Unexpectedly, TP could be used in place of PyNP
in the reactions shown in Scheme 2 (also see Table 1). With
this observation the substrate specificity of TP was reassessed
by a comparison of the rate of phosphorolysis of thymidine
(2’-deoxyriboside) versus 5-methyluridine (riboside). Thymi-
dine is converted into 2-deoxy-a,d-ribose-1-phosphate and
the equilibrium is established within a few minutes; in
contrast it took 2 hours to establish the equilibrium with 5-
methyluridine. The Km values reflect the selectivity of TP
towards 2’-deoxy substrates (Km 535 mm versus 2.2 mm);
Scheme 4. Enzymatic radiolabeling of [18F]-5’-deoxy-5’-fluorouridines
from SAM.
Table 1: Conversions of 5’-FDA into the nucleoside products 2–6 in PNP/
PyNP or PNP/TP mediated base-swap reactions.[a]
enzyme, l-amino acid oxidase (l-AAO, from Crotalus ada-
manteus), was added. The l-AAO oxidizes l-methionine, the
byproduct of the fluorination reaction, thereby pushing the
equilibrium of the reaction towards 5’-[18F]FDA production.[7]
This protocol is particularly advantageous in radiolabeling
syntheses because of the reversibility of the fluorination and
the low concentration of [18F]fluoride (ca. 10À9 m) used,
relative to unlabeled reactions.
Entry
Base
NP[b]
Product
Conversion [%]
For the synthesis of [18F]2, SAM was incubated at 378C
with four enzymes (fluorinase, PNP, TP, and l-AAO) in the
presence of uracil, potassium phosphate, and [18F]fluoride
(Figure 1). The radiolabeling base-swap reaction was studied
with respect to the relative enzyme and substrate concen-
trations and the conversion of [18F]fluoride into [18F]2
increased from less than 1% to 33% after a typical reaction
time of 4 hours. With this optimized protocol in hand [18F]4
and [18F]6 were also prepared; for example, [18F]4 was
generated in 23% (RCY) after 2 hours (see the Supporting
Information). Balancing production of 18F-labeled nucleo-
sides against radiochemical decay, the optimal reaction time
1
2
3
4
5
6
7
8
9
10
uracil
uracil
PyNP
TP
PyNP
TP
PyNP
TP
PyNP
TP
2
2
3
3
4
4
5
5
6
6
29
26
24
61
32
30
19
13
15
11
thymine
thymine
5-F-uracil
5-F-uracil
5-Cl-uracil
5-Cl-uracil
5-Br-uracil
5-Br-uracil
PyNP
TP
[a] Conditions: Tris-HCl (100 mm, pH 7.5), synthetic 5’-FDA (1 mm),
PNP (0.3 mgmLÀ1), potassium phosphate (25 mm), PyNP or TP
(0.1 mgmLÀ1), base (10 mm), 378C, 3 h. [b] Nucleoside phosphorylase.
10142 www.angewandte.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 10141 –10143

Angewandte
Chemie
buffer (100 mm, pH 7.5 at 378C), and the reaction was incubated at
378C for 4 h. Every hour an aliquot (15 mL) was withdrawn from the
reaction mixture and the proteins were precipitated by the addition of
acetonitrile (30 mL) and then removed by centrifugation (14500 rpm,
2 min). Conversions were determined by HPLC, monitoring UV and
radioactivity simultaneously.
Received: August 15, 2008
Published online: November 25, 2008
Keywords: enzyme catalysis · fluorides · glycosylation ·
.
nucleosides
Figure 1. Time course of [18F]fluorideÀconsumption and product forma-
18
18
18
~
^
tion: ꢀ [ F]2; 5’-[ F]FDA; [ F]F . The data was obtained by area
normalization of HPLC traces (radiochemical detector), are decay
corrected and represent the average of two experiments.
[1] J. Czernin, H. R. Schelbert, D. H. S. Silverman, W. P. Melega,
PET: Molecular Imaging and its Biological Applications (Ed.:
M. E. Phelps), Springer, Berlin, 2004, pp. 321 – 584.
[2] a) Fluorine and Health—Molecular Imaging, Biomedical Mate-
rials and Pharmaceuticals (Eds.: A. Tressaud, G. Haufe),
Elsevier, Amsterdam, 2008, pp. 3 – 278; b) L. Mu, A. Hꢀhne,
P. A. Schubiger, S. M. Ametamey, K. Graham, J. E. Cyr, L.
Dinkelborg, T. Stellfeld, A. Srinivasan, U. Voigtmann, U. Klar,
Angew. Chem. 2008, 120, 5000 – 5003; Angew. Chem. Int. Ed.
2008, 47, 4922 – 4925.
[3] L. Cai, S. Lu, V. W. Pike, Eur. J. Org. Chem. 2008, 2853 – 2873.
[4] Recent examples for fluorination of nucleosides: a) H. P. Le,
C. E. Muller, Bioorg. Med. Chem. Lett. 2006, 16, 6139 – 6142;
b) R. C. Spitale, M. G. Heller, A. J. Pelly, J. E. Wedekind, J. Org.
Chem. 2007, 72, 8551 – 8554.
[5] a) D. OꢁHagan, C. Schaffrath, S. L. Cobb, J. T. G. Hamilton,
C. D. Murphy, Nature 2002, 416, 279; b) C. D. Cadicamo, J.
Courtieu, H. Deng, A. Meddour, D. OꢁHagan, ChemBioChem
2004, 5, 685 – 690.
[6] C. Dong, F. Huang, H. Deng, C. Schaffrath, J. B. Spencer, D.
OꢁHagan, J. H. Naismith, Nature 2004, 427, 561 – 565.
[7] H. Deng, S. L. Cobb, A. D. Gee, A. Lockhart, L. Martarello,
R. P. McGlinchey, D. OꢁHagan, M. Onega, Chem. Commun.
2006, 652 – 654.
for these reactions was found to be between 1.5 and 3 hours,
depending on the respective base (up to 13% conversion,
uncorrected for decay). This reaction time is well within two
half-lives of 18F and enables the isolation of respectable
amounts of these novel PET tracers for cancer cell uptake
studies.
In summary, we have developed one-pot fluorination/
base-swap biotransformations of fluoride ions into 5’-deoxy-
5’-fluoronucleosides by using combinations of fluorinase and
nucleoside phosphorylase enzymes. These biotransformations
are amenable to radiolabeling syntheses starting from
[18F]fluoride ion, an ideal source of isotope for PET synthesis.
Studies are ongoing to explore the uptake of prepared
radiolabeled compounds in various cancer cell lines.
Experimental Section
[8] a) J. Toyohara, Y Fujibayashi, Nucl. Med. Biol. 2003, 30, 681 –
685; b) L. I. Wiebe, Braz. Arch. Biol. Technol. 2007, 50, 445 –
459; c) M. M. Alauddin, A. Shahinian, R. Park, M. Tohme, J. D.
Fissekis, P. S. Conti, Nucl. Med. Biol. 2007, 34, 267 – 272.
[9] K. Ishiwata, Y. Kimura, E. F. de Vries, P. H. Elsinga, Cent. Nerv.
Syst. Agents Med. Chem. 2007, 7, 57 – 77.
[10] T. Kimura, I. K. Ho, I. Yamamoto, Sleep 2001, 24, 251 – 260.
[11] a) M. J. Pugmire, S. E. Ealick, Biochem. J. 2002, 361, 1 – 25;
b) E. S. Lewkowicz, A. M. Iribarren, Curr. Org. Chem. 2006, 10,
1197 – 1215.
[12] A. Bzowska, E. Kulikowska, D. Shugar, Pharmacol. Ther. 2000,
88, 349 – 425.
[13] Experiments using purine or its derivatives such as 6-methyl-
purine, 2-amino-6-chloropurine, 2-fluoro-6-chloropurine, hypo-
xanthine, or guanine failed to give fluorinated nucleoside
analogues.
[14] M. J. Pugmire, S. E. Ealick, Structure 1998, 6, 1467 – 1479.
[15] a) A. K. Prasad, S. Trikha, V. S. Parmar, Bioorg. Chem. 1999, 27,
135 – 154; b) I. A. Mikhailopulo, Curr. Org. Chem. 2007, 11, 317 –
335.
[16] a) W. E. Razzell, P. Casshyap, J. Biol. Chem. 1964, 239, 1789 –
1793; b) N. G. Panova et al., Biochemistry 2007, 72, 21 – 28.
[17] a) R. W. Sidwell, J. H. Huffman, G. P. Khare, L. B. Allen, J. T.
Witkowski, R. K. Robins, Science 1972, 177, 705 – 706; b) W. B.
Parker, Virus Res. 2005, 107, 165 – 171.
HPLC sample for compound 1: SAM (1 mm) and KF (10 mm) were
incubated overnight at 378C with fluorinase (40 mL, 2–6 mgmLÀ1),
PNP (40 mL, 1–13 mgmLÀ1), potassium phosphate (5 mm), and 2,6-
diaminopurine (10 mm) in Tris-HCl buffer (100 mm, pH 7.5) at 378C.
The reaction mixture was heated at 988C for 3 min and then subjected
to centrifugation to remove the protein. The supernatant was
analyzed by HPLC-UV (260 nm) and HPLC-MS methods. HPLC
samples for 2–7 were obtained using a similar protocol as that
described for 1, but the amount of PNP was decreased to 20 mL and
PyNP (20 mL, 0.4–2 mgmLÀ1) or TP (20 mL, 3.6–75 mgmLÀ1) were
added in addition to the respective nitrogenous bases (10 mm).
A semipreparative sample for 1: Synthetic 5’-FDA (1 mm) was
incubated for 18 h at 378C with PNP (600 mL, 5–10 mgmLÀ1),
potassium phosphate (10 mm), and 2,6-diaminopurine (10 mm) in
Tris-HCl buffer (100 mm, pH 7.5) at 378C in a final volume of 680 mL.
The reaction mixture was heated at 988C for 3 min and then subjected
to centrifugation to remove the protein. The sample was analyzed by
19F NMR methods (376 MHz) after addition of 100 mL D2O, and then
the products were purified by preparative-HPLC. HRMS electro-
spray mass spectrometry data was obtained from the freeze-dried
residues. Semipreparative samples of compounds 2–7 were obtained
as described for 1 but the PNP aliquot was decreased (300 mL) and
PyNP (300 mL, 1–2 mgmLÀ1) or TP (300 mL, 3.6–75 mgmLÀ1) added
in addition to the respective nitrogenous base (10 mm).
Typical radiolabeling experiment: [18F]fluoride (25 mL, 180 Æ
25 MBq) was added to a reaction mixture composed of SAM
(15 mL, 20 mm), fluorinase (50 mL, 49 mgmLÀ1), l-AAO (1 mg),
PNP (50 mL, 20 mgmLÀ1), TP (40 mL, 27 mgmLÀ1), potassium phos-
phate (10 mL, 500 mm, pH 7.5), and uracil (9 mL, 200 mm) in Tris-HCl
[18] V. N. Barai, A. I. Zinchenko, L. A. Eroshevskaya, E. N. Kalini-
chenko, T. I. Kulak, I. A. Mikhailopulo, Helv. Chim. Acta 2002,
85, 1901 – 1908.
Angew. Chem. Int. Ed. 2008, 47, 10141 –10143
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 10143