Dieter Cech

Automated incorporation of polyethylene glycol into synthetic oligonucleotides

(4, 4′-Dimethoxytrityl)-polyethylene glycol-(2-cyanoethyl-N, N-diisopropyl)-phosphoramidites and (4, 4′-dimethoxytrityl)-polyethylene glycol derivatized glass supports were synthesized and used to introduce PEG with polymerization degrees from about 6 to more than 100 at 3′- and 5′-ends of oligonucleotides by means of automated synthesis. The electrophoretic mobility of the conjugates is altered with respect to the unmodified oligonucleotides. The hydrophobicity of the conjugates increases with increasing length of the PEG chain.

The behaviour of 2'-deoxy-2'-fluorouridine incorporated into oligonucleotides by the phosphoramidite approach

Abstract 2′-Deoxy-2′-fluorouridine has been chemically incorporated into an oligodeoxynucleotide of the structure 5′ACGGAX 3′ (X=U(2′-F)) using the phosphoramidite method and the behaviour of the product has been studied. 5′-O-Monomethoxytrityl-2′-deoxy-2′-fluorouridine was fixed on silica gel at the 3′-end and the chain elongated on a DNA-synthesizer using nucleoside methoxyphosphoramidites. After alkaline work-up two products were observed. One was found to be the desired fluoro containing hexamer, whereas the other corresponds to an araU-hexamer (X=arabino-furanosyluridine). The latter compound is supposed to be a product of alkaline hydrolysis of the C-2′-F-bond. The oligomers containing 2′-fluoro- and ara-U at their 3′-end were chemically sequenced by a solid phase method on CCS-paper which confirmed the right primary structure.

Synthesis and Analytical Characterization of RNA-Polyethylene Glycol Conjugates

Abstract Polyethylene glycols with degrees of polymerization from 5 to more than 100 were incorporated into synthetic oligoribonucleotides by automated solid phase synthesis at 3′-terminal, 5′-terminal and internal positions. The conjugates were characterized by chromatographic, electrophoretic and mass-spectrometric methods. The influence of coupling site, polymer size and number of coupled polymers per oligonucleotide on the molecular properties of the conjugates is investigated.

A facile solid phase synthesis of 2′- and 3′-aminonucleoside triphosphates

A convenient and time-saving solid phase synthesis of different 2′- and 3′-amino-functionalized nucleoside-5′-triphosphates using polymer bounded triphenylphosphine is described.

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.

Chemische synthese von nonadesoxyribonucleotiden mit den abgewandelten basen uracil, 5-bromuracil und 5-methylcytosin nach dem triester-verfahren

Abstract In order to investigate the interaction of Eco RII restriction and modification enzymes with synthetic DNA fragments three nonadeoxyribonucleotides containing the modified bases uracil, 5-bromouracil and 5-methylcytosine were synthesized according to the phosphate tri-ester approach using TPS/l-methylimidazole as the condensation agent. The patterns of these modified DNA fragments obtained by Maxam/Gilbert sequence technique are presented.

Triester-festphasensynthese von oligodesoxyribonucleotiden an polystyren-teflon trägern

Zusammenfassung A simple solid phase method for the synthesis of oligodeoxyribonucleotides on a grafted polystyrene-teflon support has been employed using the phosphotriester approach in 5′-3′ direction. As a control of the reactions a semiquantitative pyrolysis mass spectrometry approach for analyzing of the polymerbound oligonucleotides was developed.

A New Approach to the Synthesis of 2-Aminopurine-2′-deoxyriboside via Tri-n-butyltin Hydride Reduction

Abstract A simple conversion of guanosine to 2-aminopurine-2′-deoxyriboside is described. Following the synthesis of the 2′-phenoxythiocarbonyl derivative of 6-chloroguanosine, 2-aminopurine-2′-deoxyriboside can be prepared by simultaneous reduction of both the 6- and 2′-position using tri-n-butyltin hydride as reducing agent. Several oligo-nucleotides containing 2-aminopurine-2′-deoxyriboside have been synthesized.

Zur synthese von difluormethylethern verschieden substituierter pyrimidinnucleoside durch reaktion mit difluorcarben

Abstract The reaction of CF 2 with different substituted persilylated pyrimidine nucleosides either in the ribo-, 2′-deoxyrigo- or in the arabino-series gave the corresponding 4-0-difluoromethylethers. Optimum yields were obtained using Hg(CF 3 ) 2 as source of CF 2 .

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.