Chemgenes Corporation

Satya P Bajpai

Suresh C Srivastava

Naveen P Srivastava

Sant K Srivastav

Divya Pandey

The present invention is directed to n-alkylated synthetic nucleosides of high regiospecific purity and oligonucleotides that can be utilized for studies on reversal of cytotoxic and mutagenic DNA damage, and as diagnostic tools.

Nucleosides and oligonucleotides for studies on reversal of cytotoxic and mutagenic damage of DNA and as diagnostics tools

The present invention relates to novel phosphoramidites, A-n-bz, C-n-bz, C-n-ac, G-n-ac and U are produced with an HPLC purity of greater than 98% andP NMR purity greater than 99%. A novel process of reverse 5'&#x2192;3&#x2032; directed synthesis of RNA oligomers has been developed and disclosed. Using that method demonstrated high quality RNA synthesis with coupling efficiency approaching 99%.

RNA synthesis&#x2014;phosphoramidites for synthetic RNA in the reverse direction, and application in convenient introduction of ligands, chromophores and modifications of synthetic RNA at the 3'-end

This invention relates to n-alkylated synthetic nucleosides and phosphoramidites of high regio-specific purity and stability and for selective deprotection of the protecting group in oligonucleotides for the purpose of synthesis of high purity selectively n-alkylated sequence specific DNA and RNA. Such oligonucleotides are useful for study of mechanism of cytotoxic and mutagenic DNA damage, detection and reversal of cellular cytotoxic and mutagenic damages that occurs from the incorporation of methylated nucleosides, the corresponding phosphates and triphosphates and their precursors, via de novo DNA synthesis. The reagents could be extremely valuable tools as diagnostics and mutagenic reversal reagents. The present invention describes N-1-nitrogen alkylated purine and N-3 nitrogen alkylated pyrimidines with appropriate nucleobase amino protection, which are selectively deprotected with minimal migration of the alkyl substituent under the condition which are required for synthesizing high purity regiospecific N-1 and N-3 nitrogen alkylated oligonucleotides. A novel process has been developed to synthesize N-3 nitrogen alkylated cytidine.

Nucleosides and oligonucleotides for studies on reversal of cytotoxic and mutagenic damage of DNA potential diagnostics tools

Novel technology for RNA synthesis in the reverse direction, involving a new class of products, 3'-DMT-5'-CE ribonucleoside phosphoramidites and 3&#x2032;-DMT-5'-succinyl ribonucleoside solid supports, with per step coupling efficiency surpassing 99% in the RNA synthesis. This leads to high purity RNA. Examples of a large number of 20-21 mers and a few examples of long chain oligonucleotides are demonstrated. The data indicates dramatic improvement in coupling efficiency per step during oligonucleotide synthesis using the reverse RNA monomers (5&#x2032;&#x2192;&#x2032; direction) as compared to 3&#x2032;-CE ribonucleoside phosphoramidites used in the conventional method of RNA synthesis (3&#x2032;&#x2192;5&#x2032; direction). The new process requires shorter coupling cycle time, approx. 4 minutes as compared to approx. 10 minutes using conventional RNA synthesis method (3&#x2032;&#x2192;5&#x2032; direction). Furthermore, almost complete absence of M+1 impurities in the reverse RNA synthesis methodology were observed, even when the last phosphoramidite was a macromolecule. The process resulted in very high purity 3&#x2032;-modified oligonucleotides after HPLC purification. As a result of high purity of synthesized RNA and clean introduction of various 3&#x2032;-end modified RNA requiring long chain ligands, chromophores, fluorophores and quenchers, this method of RNA synthesis is expected to be a very useful method of choice for therapeutic grade RNA. The novel phosphoramidites of this invention, Rev-A-n-bz, Rev-C-n-bz, Rev-C-n-ac, Rev-G-n-ac and Rev-rU show HPLC purity greater than 98% andP NMR purity greater than 99.5%.

RNA synthesis-phosphoramidites for synthetic RNA in the reverse direction, and application in convenient introduction of ligands, chromophores and modifications of synthetic RNA at the 3'-end

Growing community of inventors

Lucknow, India

Satya P Bajpai