Claus-Dietmar Pein

Oligonucleotide cleavages by restriction endonucleases MvaI and EcoRII: a comprehensive study on the influence of structural

Abstract To elucidate the mechanism of action of the restriction endonucleases - isoschizomers Eco RII and Mva I - a study was made of their interaction with a set of synthetic oligonucleotide duplexes containing a single 5′-d(CC 4 / T GG)-3′ Eco RII ( Mvaz I) recognition site. The substrates had varying length and structure of the nucleotide sequences flanking the recognition site. The structure of the flanking sequence is important for the cleavage by Eco RII and Mva I enzymes; there is a structure which was found to speed up the Eco RII and Mva I action. The cleavage of oligonucleotide duplexes by Eco RII enzymes does not go to completion. Eco RII endonuclease cleaved extended substrates less efficiently thhan short ones. Extension of the flanking sequences, with the same nucleotide surrounding of the recognition site, substantially altered the whole kinetic pattern of Mva I hydrolysis. This was not observed with Eco RII enzyme. The restriction endonuclease Mva I distinguished between dA and dT residues in the recognition site, which was reflected in the higher rate of hydrolysis of the dA-containing strand of the quasipalindromic DNA duplex.

The influence of modifications on the cleavage of oligo-nucleotide duplexes by Eco R II and Mva I endonulceases

Abstract To study the interaction of the restriction endonucleases Mval and Eco RII with DNA we have synthesized some modified oligonucleotides. The results of hydrolysis demonstrate that both enzymes cleave their substrate by different mechanism.

An unusual reaction of difluoromethylene with pyrimidine nucleosides

Abstract Under inert conditions 4-0-silylated pyrimidines react with difluoromethylene to the corresponding 4-0-difluoromethylethers I. The reaction pathway proceeds via an insertion of difluoromethylene into the SiO-bond where I is formed by hydrolysis afterwards. The reactivity depends on the stability of the SiO-bond due to the fact, that using persilylated nucleosides an analogues insertion into the SiO-bond in the carbohydrate moiety could not be obtained. The selective difluoromethylation of the pyrimidine base permits the synthesis of 4-0-difluoromethylnucleosides with different carbohydrates (ribose, 2′-deoxyribose, arabinose) and additional the synthesis of the corresponding 5-substituted pyrimidine nucleosides. Hg(CF 3 ) 2 , CF 2 Br 2 , and CF 2 HCl are used for the preparation of difluoromethylene, only using Hg(CF 3 ) 2 the yields of transformation of the nucleosides are sufficient. Contrary 2, 4-0-bisdifluoromethyl- or 2-0-difluoromethyl- derivatives were formed in the case of the trasformation of pyrimidines depending on the sources of difluoromethylene. Some of the 4-0-difluoromethylnucleosides are inhibitors of the viral activity.