Synthesis, incorporation into triplex-forming oligonucleotide, and binding properties of a novel 2'-deoxy-C-nucleoside featuring a 6-(thiazolyl-5)benzimidazole nucleobase.

[reaction: see text] 6-(Thiazolyl-5)benzimidazole (B(t)()) was designed as a novel nucleobase for the specific recognition of an inverted A.T base pair in a triple helix motif. It was successfully incorporated into an 18-mer triplex-forming oligonucleotide (TFO) using the 2'-deoxy-C-nucleoside phosphoramidite 16. The triple helix binding properties of the modified TFO were examined by means of thermal denaturation experiments targeting an oligopyrimidine.oligopurine 26-mer DNA duplex containing an A.T base pair inversion.

Thiation of 2'-deoxy-5,6-dihydropyrimidine nucleosides with Lawesson's reagent: characterisation of oxathiaphosphepane intermediates.

Treatment of 2'-deoxy-3',5'-dithexyldimethylsilyl-5,6-dihydrouridine with Lawesson's reagent led to the expected C4-thiolated derivative together with a number of oxathiaphosphepane isomers which resulted from the heat reversible incorporation of an AnPS2 unit within the 2'-deoxyribose moiety explaining the subsequent anomerisation of the 5,6-dihydropyrimidine nucleosides.

Photochemistry of 4-Thiothymine Derivatives in the Presence of N-9-Substituted-Adenine Derivatives: Formation of N-6-Formamidopyrimidines.

UV irradiation of aqueous solutions containing either 4-thiothymin-1-ylacetic acid (1b) and adenosine (2a), 4-thiothymidine (1a) and adenin-9-ylacetic acid (2b), or 1b and 2b led to 4,5-diamino-6-formamidopyrimidine (N-6-Fapy-Ade) derivatives as observed after irradiation of a mixture of 1a and 2a (J. Am. Chem. Soc. 1996, 118, 8142-8143). These new observations demonstrate that the replacement of one or both nucleoside sugar residues by a carboxymethyl group does not affect the regioselective course of the photochemical reaction. The thermal decomposition of 3a that resulted from irradiation of 1a in the presence of 2a, was examined along with its behavior under mild alkaline conditions. Finally, irradiation of N-3-methyl-4-thiothymidine (6a) in the presence of adenosine gave the N-3-methylcytidine derivative 7.

Synthesis and triplex binding properties of oligonucleotides containing a novel nucleobase.

The thiazolo-indole compound 1 bearing the complementary donor-acceptor-donor sites (dad) was designed for specific recognition of an AT inverted base pair in pyrimidine triple helix motif. It was successfully incorporated into 14-mer oligonucleotide using a serinol unit as sugar derivative. The triple helix hybridization studies were examined by means of thermal denaturation experiments with a 26-mer DNA duplex containing the AT inverted base pair.

Crystal structure and photochemical behavior in solution of the 3'-N-sulfamate analogue of thymidylyl(3'-5')thymidine.

The 3'-N-sulfamate analogue of thymidylyl(3'-5')thymidine (TnsoT, 1) exhibits a preference for a C3'-endo conformation in the solution and solid states. Its photochemical behavior in solution is compared to that of its natural counterpart, thymidylyl(3'-5')thymidine (TpT, 2), to get further insight into the significance of the C3'-endo conformation on the photoproduct formation at the single-stranded dinucleotide level. Irradiation at 254 nm of 1 led to the same type of photoproducts as observed with 2. However, 1 was significantly more photoreactive than 2, and accordingly, the initial rate of photoproduct formation was enhanced in accordance with its propensity to base stack compared to 2. The corresponding quantum yields were determined and showed that the enhancement factor (1 compared to 2) is moderate for the cyclobutane pyrimidine dimer (CPD) (1.26) and much higher for the (6-4) photoproduct (1.8). These data strongly suggest that the CPD and (6-4) photoproduct arise from distinct minor stacked conformations.

Introduction of a substituent at the 5"-position of N-Boc neomycin B under Mitsunobu reaction conditions.

Because of the peculiar reactivity of the idose part of N-Boc neomycin B , special care must be exercised to introduce a substituent at the 5"-position of the antibiotic when using Mitsunobu reaction conditions.

Synthesis of the TT pyrimidine (6-4) pyrimidone photoproduct-thio analogue phosphoramidite building block.

The phosphoramidite building block synthesis of the thio analogue at the 5,6-dihydropyrimidine C5 position of the thymidylyl(3'-5')thymidine (6-4) photoproduct 1 is presented. This compound was readily obtained from the appropriately protected dinucleotide P-methyl-5'-O-dimethoxytritylthymidilyl(3' --> 5')-4-thiothymidine 2 after irradiation at 366 nm, then S-sulfenylmethylation of the thiol function of the resulting (6-4) adduct, and phosphitylation of the 3'-hydroxyl group.

Further insight in the photochemistry of DNA: structure of a 2-imidazolone (5-4) pyrimidone adduct derived from the mutagenic pyrimidine (6-4) pyrimidone photolesion by UV irradiation.

Pyrimidine (6-4) pyrimidone photoproducts represent one of the major mutagenic and carcinogenic class of DNA damage produced by UV exposure. At present, besides their conversion to their Dewar valence isomer, (6-4) photoproducts are generally believed to be photostable, and the observed biological properties of Paterno-Büchi-derived photoproducts are, thus far, exclusively attributed to these two types of compounds. Using a model system (2) relevant to DNA photochemistry, we have observed that the 5'-base moiety of the (6-4) thymine dimer 3, under far-UV radiation, is able to undergo a ring contraction leading to a 2-oxoimidazoline, 1. This unprecedented secondary photochemical reaction constitutes the first report of a major photomodification affecting (6-4) photoproducts and strongly questions the biological stability of the (6-4) adducts under UV light with 2-imidazolone (5-4) pyrimidone adducts being possibly another source of endogenous DNA damage.

Design of artificial nucleobases for the recognition of the AT inversion by triple-helix forming oligonucleotides: a structure-stability relationship study and neighbour bases effect.

We report herein on the synthesis, the incorporation into triplex forming oligonucleotides (TFO) and the recognition properties of a series of synthetic nucleosides designed for the specific recognition of an inverted A x T base pair in a pyrimidine triple helix motif. These analogues were designed on the basis of the results obtained with our previously reported compounds S and B(t), in order to define a structure-stability relationship. We report also on the chemical nature effect of the bases flanking S in the case of S-containing TFOs, in order to get further informations about the recognition process within the A x TxS triplet. This study establishes guidelines for the conception of more potent analogues for the recognition of both A x T and G x C inverted base pairs.

Gamma-radiolysis DNA products: synthesis of 6-(alpha-thyminyl)-5,6-dihydro-4-thiothymine derivatives.

Far-UV photolysis of 4-thiothymidylyl(3'-5')thymidine led to the formation of three stable derivatives: one resulting from a combination between a 3'-end methylene radical and a 5'-end C(4) radical [4-(alpha-thyminyl) derivative] and two formed after a combination between a 3'-end methylene radical and a 5'-end C(6) radical [6-(alpha-thyminyl) derivative]. In the latter series, two stereochemical pathways took place during the reaction between the methylene and C(6) radicals. The major pathway occurred when the 5'-base glycosidic bond had an anti conformation leading to an S configuration of the C(6) Tp-end. The minor pathway, which had never been reported before in this series, involved a 5'-base in a syn conformation leading consequently to the R configuration at the C(6) Tp-end. The 5,6-dihydrothymine moiety of these two adducts presented a 5,6-trans diaxial substitution that resulted from the epimerization, at the 5,6-dihydropyrimidine 5-position, of a less stable cis-disubstituted intermediate.

Sugar conformational effects on the photochemistry of thymidylyl(3'-5')thymidine.

The synthesis and conformational analysis of 2'-O,5-dimethyluridylyl(3'-5')-2'-O,5-dimethyluridine (1a), the analogue of thymidylyl(3'-5')thymidine (TpT; 1b) in which a methoxy group replaces each 2'-alpha-hydrogen atom, are described. In comparison with TpT, such modification increases the population of the C3'-endo conformer of the sugar ring puckering at the 5'- and 3'-ends from 30 to 75% and from 37 to 66%, respectively. Photolyses of 1a and TpT at 254 nm are qualitatively comparable (the cis-syn cyclobutane pyrimidine dimer and the (6-4) photoproduct are formed), although it is significantly faster in the case of 1a. These results are explained by the increased propensity of the modified dinucleotide to adopt a base-stacked conformation geometry reminiscent of that for TpT.

Binding properties of oligonucleotides containing a modified 2'-deoxyuridine with a thymine ended linker to pair with 2'-deoxyadenosine.

Oligonucleotides containing in the place of thymidine the nucleoside 2, a 2'-deoxyuridine harbouring at C-5 a thymine ended linker, were found to undergo base pairing with the opposite 2'-deoxyadenosine. However, the corresponding duplexes are significantly destabilised as compared to the fully natural ones.

Stereocontrolled synthesis of heterocyclic C-nucleosides. Protecting group effect and molecular modeling studies.

We report herein a short stereocontrolled synthesis of heterocyclic C-nucleosides (indole, imidazole, benzimidazole, and 6-iodobenzimidazole). First, condensation of 2-lithiated heterocycles 2-5 with 5-(tert-butyldiphenylsilyl)-2,3-O-isopropylidene-D-gamma-ribonolactone (1) afforded the hemiacetals 6-9 in good yields. Then, borohydride reduction (NaBH(4)) of the protected hemiacetals proceeded stereoselectively to give predominantly the S diols 10-13, which upon Mitsunobu cyclization afforded the alpha-C-nucleosides 14-17. In contrast, the same PPh(3)/DEAD treatment of the 1:1 diastereomeric mixture of the free heterocyclic diols 10d and 11d gave exclusively the beta-anomers 14dbeta and 15dbeta, respectively, by a stereocontrolled process. The mechanisms of these stereocontrolled steps are discussed with the support of molecular modeling studies.