Highly efficient and facile synthesis of 5-azido-2'-deoxyuridine

Article abstract of DOI:10.1080/15257770.2010.487507

Previously reported syntheses of the photoaffinity label 5-azido-2'-deoxyuridine are rather inefficient and involve the tedious preparation of a 5-nitro intermediate. To overcome these inconveniences, we have developed a new approach from the commercially available 5-bromo-2'- deoxyuridine nucleoside. Our synthetic route makes use of a benzylamination reduction sequence. Using this strategy, the 5-azido-2'-deoxyuridine photolabel is prepared in three steps and quantitative yields. Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2010.487507

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Highly Efficient and Facile Synthesis of
5-Azido-2′-Deoxyuridine
Stéphanie Gourdain a , Christian Petermann a , Dominique Harakat b
& Pascale Clivio a
a Université de Reims Champagne Ardenne, Institut de Chimie
Moléculaire de Reims, CNRS UMR 6229, UFR de Pharmacie, Reims
cedex, France
b Université de Reims Champagne Ardenne, Institut de Chimie
Moléculaire de Reims, CNRS UMR 6229, UFR des Sciences Exactes et
Naturelles, Reims cedex, France
Published online: 28 Jun 2010.
To cite this article: Stéphanie Gourdain , Christian Petermann , Dominique Harakat & Pascale Clivio
(2010): Highly Efficient and Facile Synthesis of 5-Azido-2′-Deoxyuridine, Nucleosides, Nucleotides and
Nucleic Acids, 29:7, 542-546
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Nucleosides, Nucleotides and Nucleic Acids, 29:542–546, 2010
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2010.487507
HIGHLY EFFICIENT AND FACILE SYNTHESIS
OF 5-AZIDO-2ꢀ-DEOXYURIDINE
Ste´phanie Gourdain,1 Christian Petermann,1 Dominique Harakat,2
and Pascale Clivio1
1Universite´ de Reims Champagne Ardenne, Institut de Chimie Mole´culaire de Reims, CNRS
UMR 6229, UFR de Pharmacie, Reims cedex, France
2Universite´ de Reims Champagne Ardenne, Institut de Chimie Mole´culaire de Reims, CNRS
UMR 6229, UFR des Sciences Exactes et Naturelles, Reims cedex, France
Previously reported syntheses of the photoaffinity label 5-azido-2ꢁ-deoxyuridine are rather in-
2
efficient and involve the tedious preparation of a 5-nitro intermediate. To overcome these inconve-
niences, we have developed a new approach from the commercially available 5-bromo-2ꢁ-deoxyuridine
nucleoside. Our synthetic route makes use of a benzylamination reduction sequence. Using this strat-
egy, the 5-azido-2ꢁ-deoxyuridine photolabel is prepared in three steps and quantitative yields.
Keywords 5-Azidouracil nucleosides; benzylamination; azidation
Photoactivable molecules incorporating the 5-azidouracil moiety are
widely used to study a large variety of proteins including nucleoside drug
metabolizing enzymes,[1a] polymerases,[1b-d] transcription factors,[1e] ribo-
somal proteins,[1f] triphosphatases,[1g] and sugar transferases.[2] Recently,
we studied the photochemical properties of 5-azido-2ꢁ-deoxyuridine[3] (1)
and faced the problem of its convenient and efficient synthesis. Herein, we
disclose a simple and highly efficient method to prepare 1 from the com-
mercially available 5-bromo-2ꢁ-deoxyuridine 2.[4]
Compound 1 has already been synthesized[5,6] and the synthetic ap-
proach follows a main protocol developed in the nucleoside monophosphate
series.[7,8] The first step always consists in the nitration of 2ꢁ-deoxyuridine 3
into the 5-nitro derivative 4 using nitrosonium tetrafluoroborate (Scheme
1). Compound 4 is then reduced by the action of Zn/HCl[5,7] or 5% Pd-
[6,8]
C/H2
to afford the corresponding amino derivative 5. Diazotization of
Received 1 April 2010; accepted 16 April 2010.
ICSN/CNRS is acknowledged for a doctoral fellowship to S. G.
Address correspondence to Pascale Clivio, Universite´ de Reims Champagne Ardenne, Insti-
tut de Chimie Mole´culaire de Reims, CNRS UMR 6229, UFR de Pharmacie, 51 rue Cognacq Jay,
51 096 Reims cedex, France. E-mail: pascale.clivio@univ-reims.fr
542

Synthesis of 5-Azido-2ꢁ-Deoxyuridine
543
O
O
O
O
N
NaNO
HCl
2
5
O N
2
H N
N
2
3
NH
NOBF
DMF
NH
NH
4
NH
Zn/HCl
N
O
N
dR
4
O
or
Pd-C/H
N
dR
5
O
NaN
O
3
dR
2
dR
1
3
SCHEME 15,6 Known syntheses of 1 using a 5-nitro intermediate.
5 in the presence of sodium nitrite and 1N HCl followed by treatment with
NaN3 gives rise to 1. However, detailed experimental descriptions are lacking
and the overall yield, from 3 to 1, when reported, is rather low since it os-
cillate between 10% and 20%.[5] In addition, such synthesis requires a large
excess (10 to 25 equiv.) of moisture sensitive and expensive NOBF4 reagent
as well as tedious purification protocols. More embarrassingly, in our hands,
this first step that is described to be quantitative was not always reproducible
depending greatly on the quality of NOBF4 and the DMF moisture content.
Therefore, alternative and improved routes for the synthesis of 1 are highly
desirable.
5-Aminouracil nucleosides necessary to access 5-azido derivatives accord-
ing to this synthetic route can be directly prepared by nucleophilic sub-
stitution of 5-bromouracil precursors.[9] Applied to 2, this procedure that
requires treatment with liquid ammonia in a steel bomb and heating for
several days yielded 5 in 63% to 75% yield.[9b,c,f] However, this amination
reaction presents moderate and variable yields and also hazards associated
with handling anhydrous liquid NH3. It also requires appropriate material
and training. Owing to these drawbacks, we searched for a safer, more conve-
nient and efficient approach to introduce the amine function at position C5
of 2ꢁ-deoxyuridine starting from the bromo precursor 2. Benzylamination of
uracil aglycones from their 5-bromo counterpart is an efficient reaction.[10]
Therefore, we selected the benzylamination/reduction sequence to prepare
5 (Scheme 2) assuming that the presence of the 2-deoxyribose would not
interfere with this reactions sequence.
O
O
HCOONH
O
NaNO
AcOH
O
4
2
H
N
BnNH
2
5
10% Pd-C
Br
5
H N
2
N
3
NH
Bn
NH
NH
NH
90°C
NaN
3
N
O
N
O
N
dR
5
O
N
dR
1
O
dR
dR
2
6
SCHEME 2 Synthesis of 1 using a benzylamination/reduction sequence.
Treatment of 2 in the presence of neat benzylamine at 90◦C yielded
quantitatively the 5-benzylamino derivative 6 after an aqueous extraction
procedure. Then, catalytic hydrogenolysis of 6 using ammonium formate

544
S. Gourdain et al.
and 10% palladium-carbon catalyst in refluxing methanol afforded quanti-
tatively 5 after filtration.
In our hands, the azidation conditions described in the literature
(NaNO2/1N HCl at 0◦C and NaN3)[7,8] led to the hydrolysis of the N -
glycosidic bond of 5. Therefore, we used a milder protocol for the generation
of nitrous acid which makes used of 80% acetic acid and NaNO2.11 Under
these milder conditions, the azido compound 1 was formed quantitatively
after 1 hour at 0◦C in the presence of 1.1 equiv. NaN3.
In conclusion, we have herein described a quantitative, safe, convenient
and easily reproducible synthesis of 1 starting from commercially available
nucleoside 2. This synthetic route provides as well a new and reliable access
to 5-amino-2ꢁ-deoxyuridine 5 that is also an important biological tool.[9c,f]
Finally, our synthetic route to 1, in addition to its usefulness with regard
to photoaffinity labeling, should be also profitable for applications in the
copper-catalyzed Huisgen cycloaddition chemistry. Indeed, a recent exam-
ple illustrates the first use of 1 to attach an alkyne fluorescent dye for the
detection of DNA synthesis in cells.[6]
EXPERIMENTAL
5-Benzylamino-2ꢀ-deoxyuridine 6
5-Bromo-2ꢁ-deoxyuridine 2 (1g, 3.26 mmol.) was dissolved in benzy-
lamine (7.2 mL) and the solution was heated at 90◦C for 3 hours. After
cooling to room temperature, the reaction mixture was co-evaporated with
toluene. The residue was dissolved in CH2Cl2 (100 mL) and extracted with
water (3 × 100 mL). The aqueous extract was concentrated to give quanti-
1
tatively 6 as a white powder. H NMR (300 MHz, CD3OD) δ 7.40–7.21 (m,
5H), 6.76 (s, 1H), 6.34 (dd, J = 6.3; 7.3 Hz, 1H), 4.24 (d, J = 13.7 Hz, 1H,
ddd, J = 3.3; 3.6; 6.4 Hz, 1H), 4.14 (d, J = 13.7 Hz, 1H), 3.82 (ddd, J =
3.3; 3.6; 4.0 Hz, 1H), 3.63 (dd, J = 3.6; 11.8 Hz, 1H), 3.56 (dd, J = 4.0;
11.8 Hz, 1H), 2.11 (ddd, J = 3.6; 6.3; 13.5 Hz, 1H), 1.98 (ddd, J = 6.4;
7.3; 13.5 Hz, 1H); 13C NMR (75 MHz, CD3OD) δ 162.5, 150.4, 139.0, 129.2,
128.1, 128.0, 125.8, 113.5, 87.8, 85.6, 71.7, 62.6, 48.6, 40.2; HRMS (ESI): m/z:
[M+Na]+ calcd for C16H19N3O5Na: 356.1222, found 356.1221; Anal. Calcd
for C16H19N3O5N.1/4 H2O: C, 56.88; H, 5.82; N, 12.44. Found: C, 56.65; H,
5.66; N, 12.47.
5-Amino-2ꢀ-deoxyuridine 5
To a solution of 6 (288 mg, 0.86 mmol.) in methanol (12 mL) under
nitrogen was added 10% Pd-C (83 mg, 0.78 mmol., 0.9 equiv.) then ammo-
nium formate (97 mg, 1.54 mmol., 1.78 equiv.). The reaction mixture was

Synthesis of 5-Azido-2ꢁ-Deoxyuridine
545
vigorously stirred and refluxed for 1 hour. After cooling to room tempera-
ture, the reaction mixture was sonicated for 1h then filtered through Celite.
The Celite was washed with methanol and the filtrate and methanolic wash
were combined and evaporated in vacuo to furnish 5 as a white powder in
quantitative yield. 1H NMR (300 MHz, CD3OD) δ 7.29 (s, 1H), 6.29 (t, J = 6.8
Hz), 4.42 (dt, J = 3.6; 5.4 Hz, 1H), 3.99 (ddd, J = 3.5; 3.6; 5.0 Hz, 1H), 3.80
(dd, J = 3.5; 12.5 Hz, 1H), 3.72 (dd, J = 5.0; 12.5 Hz, 1H), 2.32 (dd, J = 5.4;
6.8 Hz, 2H); 13C NMR (75 MHz, CD3OD) δ 162.7, 150.8, 123.8, 119.5, 88.2,
85.7, 72.2, 63.1, 40.1; HRMS (ESI) m/z: [M+Na]+ calcd for C9H13N3O5Na:
266.0755, found 266.0753.
5-Azido-2ꢀ-deoxyuridine 1
To a stirred solution of 5 (263 mg; 1.08 mmol) in 80% acetic acid
(2.8 mL) at 0◦C was added NaNO2 (82 mg; 1.19 mmol., 1.1 equiv.). After 5
minutes, NaN3 (77 mg, 1.19 mmol., 1.1 equiv.) was added and the reaction
mixture was allowed to stir at 0◦C for 1 hour in the dark. Solvents were re-
moved by evaporation under reduced pressure without heating then coevap-
orated with toluene. Compound 1 was quantitatively obtained with 16 equiv.
of acetic acid. When necessary, full removal of acetic acid was achieved by a
silicagel purification of the residue using a gradient of methanol in CH2Cl2
(0 to 10%). Fractions eluting at 10% were collected and concentrated to
dryness to yield 1 as a white powder (116 mg, 40% yield): IR (thin film):
2119, 1690, 1468, 1403, 1359, 1310, 1266 cm−1; 1H NMR (300 MHz, D2O) δ
7.71 (s, 1H), 6.25 (t, J = 6.5 Hz, 1H), 4.43 (ddd, J = 3.9; 4.8; 6.3 Hz, 1H),
4.00 (ddd, J = 3.4; 3.9; 4.6 Hz, 1H), 3.82 (dd, J = 3.4; 12.6 Hz, 1H), 3.73
(dd, J = 4.6; 12.6 Hz, 1H), 2.34 (m, 2H); 13C NMR (75 MHz, D2O) δ 160.9,
149.9, 128.7, 115.7, 86.4, 85.2, 69.8, 60.9, 37.2; HRMS (ESI) m/z: [M+Na]+
calcd for C9H11N5O5Na: 292.0658, found 292.0664.
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