New synthesis of 5-carboxy-2′-deoxyuridine and its incorporation into synthetic oligonucleotides

Article abstract of DOI:10.1081/NCN-120022739

5-Carboxy-2′-deoxyuridine is a methyl oxidation product of thymidine. It can be formed by the menadione-mediated photosensitization of thymidine in aerated aqueous solution. Here in we present a new four-step synthesis of the 5-carboxy-2′-deoxyuridine pho

Full text of DOI:10.1081/NCN-120022739

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New Synthesis of 5-Carboxy-2′-deoxyuridine and Its
Incorporation into Synthetic Oligonucleotides
V. Guerniou a , D. Gasparutto a b , S. Sauvaigo a , A. Favier a & J. Cadet a
a Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique,
UMR 5046, CEA/DSM/Departement de Recherche Fondamentale sur la Matière Condensée,
Grenoble, Cedex, France
b CEA Grenoble, LAN/SCIB/DREMC, 17, Avenue des Martyrs, F-38054, Grenoble, France
Published online: 31 Aug 2006.
To cite this article: V. Guerniou , D. Gasparutto , S. Sauvaigo , A. Favier & J. Cadet (2003) New Synthesis of 5-Carboxy-2′-
deoxyuridine and Its Incorporation into Synthetic Oligonucleotides, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8,
1073-1075, DOI: 10.1081/NCN-120022739
To link to this article: http://dx.doi.org/10.1081/NCN-120022739
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1073–1075, 2003
New Synthesis of 5-Carboxy-20-deoxyuridine and Its
Incorporation into Synthetic Oligonucleotides
*
V. Guerniou, D. Gasparutto, S. Sauvaigo, A. Favier, and J. Cadet
´
´
Laboratoire Lesions des Acides Nucleiques, Service de Chimie Inorganique
et Biologique, UMR 5046, CEA=DSM=Departement de Recherche
`
´
Fondamentale sur la Matiere Condensee,
Grenoble, Cedex, France
ABSTRACT
5-Carboxy-20-deoxyuridine is a methyl oxidation product of thymidine. It can be
formed by the menadione-mediated photosensitization of thymidine in aerated
aqueous solution. Here in we present a new four-step synthesis of the 5-carb-
oxy-20-deoxyuridine phosphoramidite building block based on the alkaline hydro-
lysis of 5-trifluoromethyl-20-deoxyuridine. The phosphoramidite derivative has
been incorporated at defined sites into oligonucleotides using the solid phase
synthesis approach.
5-Carboxy-20-deoxyuridine (CdU) is an oxidized lesion of thymidine that can be
formed by menadione-mediated photosensitization of thymidine.[1] Therefore, it
is important to evaluate the possible biological effects of CdU. For assessing the
structural and biological features of this lesion, the use of site-specific modified
oligonucleotides appears to be a good approach. Recently, a synthesis has been
*Correspondence: D. Gasparutto, CEA Grenoble, LAN/SCIB/DREMC, 17, Avenue des
Martyrs, F-38054 Grenoble, France; Fax: þ33 4 3878 5090; E-mail: dgasparutto@drfmc.
ceng.cea.fr.
1073
DOI: 10.1081/NCN-120022739
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

1074
Guerniou et al.
Figure 1. a: CF3dT, H2O, NaOH pH10, 37 ꢀC, 36 h, 90% ; b: HOBT, DCCI, Bu3N, EtOH
anhydre, 15 h, 75% ; c: DMTrCl, DMAP, pyridine, 23 h, 55% ; d: 2-Cyanoethyl-diisopropyl-
chlorophosphoramidite, DIEA, CH2Cl2, 3 h under argon atmosphere, quantitative yield.
described.[2] Up to now no biological data have been reported in the litterature. Here
in we report a new method to synthesize 5-Carboxy-20-deoxyuridine-containing oli-
gonucleotides.
The phosphoramidite derivative of the protected ethyl ester of 5-carboxy-
20-deoxyuridine 5 was prepared from 5-trifluoromethyl-20-deoxyuridine 1 in 4 steps
(Fig. 1). Thus, alkaline hydrolysis of commercially available 5-trifluoromethyl-
20-deoxyuridine 1[3] followed by neutralization with 1M HCl and desalting on a
cation exchange Dowex resin gave 5-carboxy-20-deoxyuridine 2.a The carboxyle
function was then esterificated by an ethyl group as described.[2] The last two steps
are standard dimethoxytritylation and phosphitylation.
Using compound 5, 5-carboxy-20-deoxyuridine-containing oligonucleotides
were synthesized using an automated DNA synthesizer according to the standard
b-cyanoethyl phosphoramidite chemistry. After deprotection with 0.1M NaOH at
room temperature for 24 h, the oligonucleotides were purified using electrophoresis
on denaturing polyacrylamide gel containing 8M urea and then desalted.
The synthetic oligonucleotides were characterized by ESI-MS and MALDI-TOF
MS analyses. The enzymatic digestion of the oligonucleotides followed by HPLC-
MS=MS analysis has been performed, confirming the presence and the integrity of
5-Carboxy-20-deoxyuridine into the oligonucleotides.b
a2: ESI-MS (negative mode): [M ꢁ H]ꢁ ¼ 270.9 0.1 Da. 1H NMR (200 MHz, D2O) d 9.01 (s,
1H, H-6); 6.33 (t, 1H, H-10); 4.55 (m, 1H, H-30); 4.17 (m, 1H, H-40); 3.98 and 3.85 (m, 2H, H-50
and H-500); 2.62 and 2.48 (m, 2H, H-20 and H-200). 3: ESI-MS (positive mode): [M þ H]þ ¼
301.1 0.1 Da, [M þ Na]þ ¼ 323.2 0.1 Da. 4: ESI-MS (positive mode): [M þ H]þ ¼ 624.9
0.1 Da, [M þ Na]þ ¼ 646.9 0.1 Da. 5: ESI-MS (positive mode): [M þ H]þ ¼ 803.8 0.1 Da,
[M þ Na]þ ¼ 825.8 0.1 Da.
bThe following 22-mer-(5-carboxy-20-deoxyuridine-containing)-oligonucleotide was synthe-
0
50
sized: CAC TTC GGA XCG TGA CTG ATC T 3 , with X ¼ CdU. Purity was checked
by ESI-MS analysis, MALDI-TOFF MS analysis (calculated MW ¼ 6731.4 Da, found MW
(negative mode) ¼ 6731.1 Da) and enzymatic digestion followed by HPLC-MS=MS analysis.
All the analyses confirm the purity of the oligonucleotide together as well as the presence
and the integrity of CdU 2.

5-Carboxy-20-deoxyuridine
1075
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dation product of thymidine. J. Photochem. Photobiol. 1997, 104, 97–104.
´
2. Berthod, T.; Petillot, Y.; Guy, A.; Cadet, J.; Molko, D. Synthesis of oligonucleo-
tides containing 5-carboxy-20-deoxyuridine at defined sites. J. Org. Chem. 1996,
61, 6075–6078.
3. Jones, M.F. The stability of trifluorothymidine: Hydrolysis in buffered aqueous
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