A simplified and efficient route to 2'-O, 4'-C-methylene-linked bicyclic ribonucleosides (locked nucleic acid).

A novel efficient method for the synthesis of locked nucleic acid (LNA) monomers is described. The LNA 5',3'-diols containing thymine, 4-N-acetyl- and 4-N-benzoylcytosine, 6-N-benzoyladenine, and 2-N-isobutyrylguanine as nucleobases were prepared via convergent syntheses. The method is based on the use of the common sugar intermediate 1,2-di-O-acetyl-3-O-benzyl-4-C-methanesulfonoxymethyl-5-O-methanesulfonyl-D-erythro-pentofuranose (8) that easily can be prepared from D-glucose in multigram scale. Four different nucleobases were stereoselectively coupled to 8 using a modified Vorbrüggen procedure to give the corresponding 4'-C-branched nucleoside derivatives. Subsequent ring closing furnished the protected LNA nucleosides. The 5'-O-mesyl groups were efficiently displaced by nucleophilic substitution using sodium benzoate. Saponification of the 5'-benzoates followed by catalytic removal of the 3'-O-benzyl groups afforded the free LNA diols. The exocyclic amino groups of adenosine and cytidine were selectively acylated to give 4-N-acetyl- or 4-N-benzoyl-LNA-C and 6-N-benzoyl-LNA-A. The isobutyryl group of guanine was retained during the preparation of 2-N-isobutyryl-LNA-G. The LNA-T diol and base-protected LNA diols can be directly converted into LNA-phosphoramidites for automated chemical synthesis of LNA containing oligonucleotides.

Photochemical immobilization of anthraquinone conjugated oligonucleotides and PCR amplicons on solid surfaces.

Ligand immobilization on solid surfaces is an essential step in fields such as diagnostics, bio sensor manufacturing, and new material sciences in general. In this paper a photochemical approach based on anthraquinone as the chromophore is presented. Photochemical procedures offer special advantages as they are able to generate highly reactive species in an orientation specific manner. As presented here, anthraquinone (AQ) mediated covalent DNA immobilization appears to be superior to currently known procedures. A synthetic procedure providing AQ-phosphoramidites is presented. These reagents facilitate AQ conjugation during routine DNA synthesis, thus enabling the AQ-oligonucleotides to be immobilized in a very convenient and efficient manner. AQ-conjugated PCR primers can be used directly in PCR. When the PCR is performed in solution, the amplicons can be immobilized after the PCR. Moreover, when the primers are immobilized prior to the PCR, a solid-phase PCR can be performed and the amplicons are thus produced directly on the solid support.

Synthesis of diastereomeric mixtures of 5'-C-hydroxymethylthymidine and introduction of a novel class of C-hydroxymethyl functionalised oligodeoxynucleotides.

Novel 5'-C-hydroxymethyl substituted derivatives of thymidine have been prepared by dihydroxylation of the 5'C-methylene nucleoside 1. Osmium tetraoxide catalysed dihydroxylation of 1 afforded a 3:2 epimeric mixture of diols 2, whereas asymmetric dihydroxylation using AD-mix-alpha and AD-mix-beta resulted in mixtures 3 and 4 of the two epimeric diols, both enriched with the same diastereomer. Nucleosides 2 were transformed into phosphoramidites 8 which were used for solid phase synthesis of oligodeoxynucleotides (ODNs) containing 5'-C-(hydroxymethyl) functionalised thymidine monomers. This novel class of C-hydroxymethyl modified ODN-analogues exhibited promising affinity towards both complementary DNA and RNA as well as resistance towards 3'-exonucleolytic degradation.