Vasulinga T. Ravikumar

On the formation of longmers in phosphorothioate oligodeoxyribonucleotide synthesis

Abstract The extent of longmer formation in phosphorothioate oligodeoxyribonucleotide synthesis through amidete chemistry on solid support depends on base composition, contact time and acidity of the promotor used for activation of the phosphoramidite. A longmer formation mechanism that involves dedimethoxytritylation of the phosphite triester intermediate is proposed.

Efficient and selective enzymatic acylation reaction: separation of furanosyl and pyranosyl nucleosides.

Candida antarctica lipase-B (CAL-B) immobilized on lewatite selectively acylated the primary hydroxyl group of the furanosyl nucleoside in a mixture of 1-(alpha-D-arabinofuranosyl)thymine and 1-(alpha-D-arabinopyranosyl)thymine. This selective biocatalytic acylation of furanosyl nucleoside has enabled us an easy separation of arabinofuranosyl thymine from an inseparable mixture with arabinopyranosyl thymine. The primary hydroxyl selective acylation methodology of arabinonucleoside has also been successfully used for the separation of 1-(beta-D-xylofuranosyl)thymine and 1-(beta-D-xylopyranosyl)thymine from a mixture of the two, which demonstrate the generality of the enzymatic methodology for separation of furanosyl and pyranosyl nucleosides.

Human RNase H1 Uses One Tryptophan and Two Lysines to Position the Enzyme at the 3′-DNA/5′-RNA Terminus of the Heteroduplex Substrate

In a previous study, we showed that the RNA-binding domain of human RNase H1 is responsible for the positional preference for cleavage exhibited by the enzyme (Wu, H., Lima, W. F., and Crooke, S. T. (2001) J. Biol. Chem. 276, 23547–23553). Here, we identify the substituents on the heteroduplex substrate and the amino acid residues within the RNA-binding domain of human RNase H1 involved in positioning of the enzyme. The human RNase H1 cleavage patterns observed for heteroduplexes with various 3′-DNA/5′-RNA and 5′-DNA/3′-RNA termini indicate that the 5′-most cleavage site on the oligoribonucleotide is positioned 7 bp from the first 3′-DNA/5′-RNA base pair. The presence or absence of phosphate or hydroxyl groups at either the 3′-DNA or 5′-RNA terminus had no effect on the human RNase H1 cleavage pattern. Substitution of the 3′-deoxynucleotide with a ribonucleotide, 2′-methoxyethyl nucleotide, or mismatched deoxyribonucleotide resulted in the ablation of the 5′-most cleavage site on the oligoribonucleotide. Mutants in which Trp43 and Lys59-Lys60 of the RNA-binding domain were substituted with alanine showed a loss of the positional preference for cleavage. Comparison of the kcat, Km, and Kd for the alanine-substituted mutants with those for human RNase H1 suggests that Lys59 and Lys60 are involved in binding to the heteroduplex and that Trp43 is responsible for properly positioning the enzyme on the substrate for catalysis. These data suggest that Trp43, Lys59, and Lys60 constitute an extended nucleic binding surface for the RNA-binding domain of human RNase H1, with the entire interaction taking place at the 3′-DNA/5′-RNA pole of the heteroduplex. These results offer further insights into the interaction between human RNase H1 and the heteroduplex substrate as well as approaches to enhance the design of effective antisense oligonucleotides.

Phosphorothioate oligonucleotides: Largely reduced (N-1)-mer and phosphodiester content through the use of dimeric phosphoramidite synthons

Abstract Phosphorothioate oligonucleotides synthesized through an assembly of dimeric phosphoramidite synthons on controlled pore glass solid support show a significantly improved impurity profile compared to oligomers synthesized through a coupling of standard monomer phosphoramidites. A greater than 70% reduction of the (n-1)-mer population and a ca 50% reduction of phosphodiester linkages has been achieved.

Efficiency of sulfurization in the synthesis of oligodeoxyribonucleotide phosphorothioates utilizing various sulfurizing reagents

Abstract The synthesis of fully modified oligodeoxyribonucleotide phosphorothioates utilizing various sulfurizing reagents is described.

Efficient synthesis of deoxyribonucleotide phosphorothioates by the use of DMT cation scavenger1

Abstract Triethylsilane in the presence of dichloroacetic acid in dichloromethane is an efficient DMT cation scavenger during the synthesis of deoxyribonucleotide phosphorothioates and leads to increased overall yields.

Synthesis of oligonucleotides via phosphoramidite approach utilizing 2-diphenylmethylsilylethyl (DPSE) as a phosphorus protecting group

Abstract 2-Diphenylmethylsilylethyl (DPSE) is a new protecting group for internucleotidic bonds in the synthesis of oligodeoxyribo nucleotides by the phosphoramidite approach. This group is stable to acidic conditions and can be removed under mild β-fragmentation conditions using aqueous ammonium hydroxide.

Efficient synthesis of antisense oligodeoxyribonucleotide phosphorothioates

Abstract Efficient and economical synthesis of antisense oligodeoxyribonucleotide phosphorothioates utilizing 2 equivalents of phosphoramidite synthon is reported.

Solid Phase Synthesis of Phosphorothioate Oligonucleotides Utilizing Diethyldithiocarbonate Disulfide (DDD) as an Efficient Sulfur Transfer Reagent

Abstract Diethyldithiodicarbonate (DDD), a cheap and easily prepared compound, is found to be a rapid and efficient sulfurizing reagent in solid phase synthesis of phosphorothioate oligodeoxyribonucleotides via the phosphoramidite approach. Product yield and quality based on IP-LC-MS compares well with high quality oligonucleotides synthesized using phenylacetyl disulfide (PADS) which is being used for manufacture of our antisense drugs.

Applications of green chemistry in the manufacture of oligonucleotide drugs

We have modified the current phosphoramidite-based, solid-phase synthesis of antisense oligonucleotides to accommodate principles of green chemistry. In this article, we summarize key accomplishments that reduce or eliminate the use or generation of toxic materials, solvents, and reagents. Also discussed are methodologies that allow reuse of valuable materials such as amidites, solid-support, and protecting groups, thus improving the atom economy and cost-efficiency of oligonucleotide manufacture. Approaches to accident prevention and the use of safer reagents during oligonucleotide synthesis are also covered.