Kenzo Fujimoto

Template directed photochemical synthesis of branched oligodeoxynucleotides via 5-carboxyvinyldeoxyuridine

Abstract An efficient template directed DNA photoligation using 5-carboxyvinyldeoxyuridine ( CV U) is described. By the use of this photoligation, a new efficient method for the synthesis of branched oligonucleotides is reported.

Evidence for intrastrand C2′ hydrogen abstraction in photoirradiation of 5-halouracil-containing oligonucleotides by using stereospecifically C2′-deuterated deoxyadenosine

In order to investigate the nature of deoxyribose C1′ and C2′ H abstraction by uracilyl-5-yl radical in DNA, we have examined the photoreaction of hexanucleotides d(GCABrUGC)2 in which one of the C2′ hydrogens of the deoxyribose moiety was stereospecifically deuterated. Irradiation of (2′R)-[2′-2H]-2′-deoxyadenosine-containing hexamer (1) gave only a smaller amount of 8 compared to that of the unlabeled hexamer, whereas (2′S)-[2′-2H]-2′-deoxyadenosine-containing hexamer (2) gave essentially the same amount of 8 as that obtained for the unlabeled hexamer. The results indicated that the formation of C2′ oxidation product 8 occurs via a rate-limiting abstraction of (2′α)-H of the deoxyribose moiety by uracilyl-5-yl radical. The kinetic isotope effects (kHkD) obtained for the formation of 8 by photoirradiation of BrU- and IU-containing hexamers were 7.5 and 7.2, respectively.

PREFERENTIAL C1' HYDROGEN ABSTRACTION BY A URACILYL RADICAL IN A DNA-RNA HYBRID

Abstract Photoreaction of 5-halouracil contained in a DNA-RNA hybrid which forms an A form type structure was examined and the photoreactivity of this hybrid was compared with that for a DNA duplex. The results indicated that in a DNA-RNA hybrid, the uracilyl-5-yl radical preferentially abstracts the C1′ H of the deoxyribose that is on the 5′ side of the radical. NOE experiments of DNA hexamer and DNA-RNA hybrid demonstrated that there appears to be no correlation between the distances and the selectivity of H abstraction. In order to examine the origin of the selectivity of H abstraction, a putative transition state for H abstraction in a DNA duplex and a DNA-RNA hybrid was constructed and the conformational energy required to achieve their transition states was evaluated. The results provide a qualitative explanation of conformation-dependent H abstraction by the uracilyl-5-yl radical in a DNA duplex and DNA-RNA hybrid.

Template-directed reversible photocircularization of DNA via 5-vinyldeoxycytidine

Abstract An efficient and reversible template-directed photoligation of oligodeoxynucleotides (ODNs) using 5-vinyldeoxycytidine (VC) was described. When ODN containing VC at the 5′ end was photoirradiated with ODN containing thymine at the 3′ end in the presence of a template ODN, an efficient photoligation was observed. Reversible photopadlocking of a circular DNA by the photocircularization using VC was demonstrated.

Reversible DNA photocircularization on triple helix: effect of vinyl substituent on base stacking

Abstract An efficient method for the synthesis of circular oligodeoxyribonucleotides (ODNs) using reversible template-directed photoligation via 5-vinyldeoxycytidine (VC) is described. It is shown that the 5′-end VC of ODN reversibly reacts by photoirraditation with thymine at its own 3′-end in the presence of a single-stranded template via triple helix formation to produce circular DNA. Thermodynamic analysis indicated that the stability of triple helix is remarkably enhanced by the vinyl substituent of VC.

Deoxyribonolactone formation in photoirradiation of 5-bromouracil-containing oligonucleotides by direct C1′ hydrogen abstraction

Mechanistic studies on the formation of deoxyribonolactone by UV irradiation of hexamer d(GC1BrUGC)2 (ODN 2a) containing 1′-deuterio-2′-deoxyadenosine 1 was examined. The kinetic isotope effect (kH/kD) for the formation of C1′ oxidation product 3 was 1.7±0.1. ESI-MS of d(pUGC) (7) obtained from the photoreaction of ODN 2a indicated that 1′ deuterium was abstracted by uracilyl-5-yl radical and incorporated into 7 in 96% yield.

Direct strand cleavage via furanyladenine formation in anaerobic photoirradiation of 5-bromouracil-containing oligonucleotides

Abstract The mechanism of direct strand cleavage induced by anaerobic irradiation of 5-bromouracil ( Br U)-containing DNA was investigated. The formation of an oligonucleotide fragment containing 3′-furanyladenine end like 5 was observed as a major photoproduct for the direct strand breaks.

Sequence dependent photoreduction of 5-bromouracil-contaning oligonucleotides via electron transfer

in order to investigate photoactivation mechanism of 5-bromouracil (BrU)-containing oligonucleotides, deuterium incorporation experiments and fluorescence emission were examined. Photoreduction of BrU contained in a duplex oligomer in aqueous solution containing (CD3)2CDOH gave 5-deuterated uracil in high yield. It was found that highly photoreactive oligomers possessing 5′-ABrU-3′ sequence show a strong fluorescence emission at 370 nm by excitation at 308 nm. These experimental results support a photoactivation mechanism that involves one electron transfer from adjacent adenine at 5′-side to n,π∗ excited state of BrU in DNA duplex.