Abstract

New oligonucleotides with a long-chain linker (6,9-dioxa-3,12-diazatetradecane-1,14-diyl) in their backbone were synthesized, and their hybridization properties were studied by measurement of their T_m curves and fluorescence spectra. The T_m analyses revealed that these oligonucleotides could bind to their complementary strands despite the presence of the long-chain linker. We also demonstrated interesting fluorescence properties of oligodeoxynucleotides with an anthracen-9-ylmethyl group on one of the two N-atoms in the long-chain linker. The fluorescence intensity of these oligonucleotides increased upon their hybridization to the complementary strands and was sensitive to the presence of the mismatch base pairs at a specific position.

Deoxycytidine derivatives modified with various N-substituted carbamoyl groups at the 4-amino group were synthesized. The detailed ¹H NMR studies suggest that the 3′,5′-O-disilylated N-carbamoyldeoxycytidine derivative 11 exists as a species having an intramolecular hydrogen bond between the N³ atom and the carbonyl oxygen atom in MeOD but upon addition of CDCl₃, the amount of a homodimer species, having intermolecular hydrogen bonds, gradually increases. Oligodeoxyribonucleotides incorporating various 4-N-carbamoyldeoxycytidine derivatives were also synthesized. These modified oligodeoxynucleotides can hybridize with the complementary strands without disturbing the structure of DNA duplexes, hence changing the orientation of the carbamoyl group in such a manner that a stable Watson–Crick base pair can be formed with the guanine base at the opposite site. It should be noted that the base recognition ability (G against T, C and A) of these modified cytosine bases can be preserved satisfactorily. These results suggest that the 4-N-carbamoyl group is useful as a backbone structure of the linker between various functional residues and oligonucleotides. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

A TpT dimer analogue (U_2′sq_5′T), in which the 3′-5′ phosphodiester linkage was replaced by a 2′-5′ squaryldiamide linkage and the 5′-upstream T was replaced by a 3′-deoxyuridine, was synthesized in almost quantitative yield from diethyl squarate. This new dimer structural motif was designed to eliminate the squaryldiamide skeleton-induced overall strain in T_3′sq_5′T, previously incorporated into DNA fragments as a new TpT mimic, through the change in the connection mode from the 3′-5′ linkage to a 2′-5′ linkage. Spectral analyses of U_2′sq_5′T suggest that the overall structure of this dimer mimic is basically similar to that of TpT. A DNA 10mer 5′-d(CGCAU_2′sq_5′TAGCC)-3′ incorporating this dimer was synthesized. From the CD analysis, it turned out that the overall structure of a DNA duplex of 5′-d(CGCAU_2′sq_5′TAGCC)-3′/3′-d(GCGTAATCGG)-5′ is closer to that of the unmodified duplex than the DNA duplex of 5′-d(CGCAT_3′sq_5′TAGCC)-3′/3′-d(GCGTAATCGG)-5′. Interestingly, extensive Tm experiments suggest that d(CGCAU_2′sq_5′TAGCC)-3′ exhibits intriguing inherent hybridization affinity not only for the completely complementary oligodeoxynucleotide 3′-d(GCGAATCGG)-5′, but also for 3′-d(GCGTAGTCGG)-5′, with a mismatched dG. The unique property of the 3′-downstream dT moiety of U_2′sq_5′T − the ability to recognize both dA and dG − was also supported by more detailed computational analysis of U_2′sq_5′T and TpT. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

The new protecting groups 1a,b and 2a,b were developed for the 5′-OH group of deoxynucleosides by utilizing the unique characters of the sulfenate and sulfenamide linkage. These new protecting groups have a 2-(hydroxymethyl)benzoyl or 2-[(methylamino)methyl]benzoyl skeleton whose hydroxy O-atom or amino N-atom was blocked with a tritylthio-type substituent. They are removable by intramolecular cyclization following the oxidative hydrolysis of the tritylthio-type substituents under mildly oxidative conditions (Schemes 3 and 6). Among them, 2-{{[(4-methoxytrityl)sulfenyl]oxy}methyl}benzoyl (MOB; 2b) was found to be the most preferable for protection of the 5′-OH function of deoxynucleosides. MOB can be introduced at the 5′-OH groups of various deoxynucleosides without the protection of the 3′-OH functions (Scheme 5). The applicability of the MOB group to a new oligodeoxynucleotide synthesis protocol without acid treatment was demonstrated by the solid-phase synthesis of a tetrathymidylate (Scheme 8).

This paper deals with the synthesis and properties of oligodeoxynucleotides of a Dickerson−Drew sequence, d(CGCGAATTCGCG), in which one of the four cytosine bases has been replaced with an N-phosphorylated or N-diethylphosphorylated cytosine base in order to study the effect of the N-phosphoryl or N-diethoxyphosphoryl group on their structural property and hybridization affinity. The CD spectra of these self-complementary duplexes showed the typical B DNA-type shape with very minor changes. In contrast, it was found from T_m experiments that N-phosphoryl groups destabilized the DNA duplexes significantly through steric hindrance and electronic repulsion. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

This paper deals with kinetic studies of TMSOTf-mediated C1′ epimerization of β-thymidine to α-thymidine. The effect of neighboring group participation by various 5′-hydroxy protecting groups, such as toluoyl, Et₂CHC(O), Et₂NC(O), and Et₂NC(S), on the βα conversion is described in detail. The time dependence of the ratio of the α and β anomers in the C1′ epimerization was estimated by ¹H NMR. The α/β equilibrium constants K and the rate constants k_α and k_β were calculated on the basis of the experimental data. As the result, it was concluded that, in acetonitrile, the α/β equilibrium constants K are thermodynamically affected by steric hindrance from the 5′-hydroxy protecting group. On the other hand, the rate constants k_α and k_β are mainly influenced by the stability of the oxonium ion intermediate. In particular, formation of an intramolecularly cyclized iminium ion intermediate from the oxonium ion intermediate, due to the neighboring group participation by the diethylthiocarbamoyl group, tended to decrease the overall reaction rate. Finally, the α/β C1′ epimerization could be carried out with a high α anomer selectivity of 89% through the use of the Et₂CHC(O) group. Thus, 5′-O-pixyl-α-thymidine could be synthesized from β-thymidine as a key intermediate for the synthesis of α-DNA in a considerably improved overall yield of 40%.

This paper describes the synthesis of conformationally constrained dinucleoside cyclic phosphotriester derivatives and related compounds, such as Tpc3Um (2), Tpc3dU (3), Tpc2dU (4), pc3dU (16), and pc2dU (25), where c2 and c3 refer to ethylene and propylene bridges between the 5′-phosphate of the 3′-downstream nucleoside and the 5-position of the uracil residue. These studies found that the c3 linker is essential for fixing the 3′-downstream nucleoside residue in the C3′-endo puckering. The presence of the 2′-hydroxyl group at the 3′-downstream nucleoside is also crucial for stabilization of the C3′-endo conformation. Two sets of diastereomeric oligonucleotides, T₄(Tpc3Um)T₄ (30) and T₄ (Tpc3dU)T₄ (33), were synthesized, and each stereoisomer was isolated. The hybridization capability of these oligonucleotides was analyzed by melting point experiments.

Oligodeoxynucleotides [11a−i: d(T₆XT₆)] incorporating various N-acyldeoxycytidine derivatives (X) have been synthesized through use of a new DBU-labile 4,5-dichlorophthaloyl linker on polymer supports. The hybridization capabilities of these oligonucleotides with the complementary d(A₆GA₆) were examined with the aid of Tm experiments. It turned out that the Tm values of DNA duplexes decreased significantly with an increase in the number of the methylene groups in the aliphatic acyl group introduced into one strand of the DNA duplex. Ab initio MO calculations and ¹H NMR analysis suggested that the acyl groups in the tested derivatives were oriented in a manner that made the formation of conventional Watson−Crick-type (W−C-type) base pairs with the guanine residue possible, with the help of an intramolecular hydrogen bond between the amide carbonyl oxygen atom and the 5-vinyl proton of the N-acylcytosine residue. Moreover, all the aromatic N-acyl groups were found markedly to decrease the thermal stability of the DNA duplexes. Ab initio calculations suggested that the base pairs formed between 4-N-acyl-1-methylcytosine derivatives and 9-methylguanine have wholly planar structures as their most stable geometries. Detailed studies of the hydrogen-bond energy of the modified base pairs also suggested that the electronic repulsion between the heteroatom of the acyl group of X and the guanine residue resulted in significant destabilization of the X-G base pair and that the hydrogen-bond network structure of water molecules around the major groove of the DNA duplex is a key factor in the stabilization of DNA duplexes.