Synthesis of backbone modified morpholino oligonucleotides and chimeras using phosphoramidite chemistry
AssigneeThe Regents of the University of Colorado
InventorsMarvin Caruthers, Sibasish Paul
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Abstract
Amine substituted morpholino oligonucleotides, other than the classical N,N-dimethylamino PMO analogue, and methods of efficiently synthesizing these oligonucleotides with high yield are provided. Morpholino oligonucleotides having thiophosphoramidate, phosphoramidate, and alkyl phosphoramidate linkers. Chimeras containing unmodified DNA/RNA and other analogs of DNA/RNA can be prepared. These oligonucleotides form duplexes with complementary DNA or RNA that are more stable than natural DNA or DNA/RNA complexes, are active with RNAse H1, and may be transfected into cells using standard lipid reagents. These analogues are therefore useful for numerous applications.
Claims15
A method of preparing a phosphorodiamidite monomer having a phosphorodiamidite moiety attached to the 3′-nitrogen of a morpholino ring comprising:
- a. contacting a 5′-O-Dimethoxytrityl ribonucleoside with sodium periodate and ammonium biborate to produce a dihydroxyribose morpholino monomer, wherein the base is one of uracil, thymine, cytosine, adenine, hypoxanthine, and guanine, optionally protected at the nucleoside amino groups by a protecting group,
- b. reducing hydroxyl groups on the dihydroxyribose morpholino monomer with sodium cyanoborohydride to produce a morpholino monomer, and
- c. phosphitylating the morpholino monomer with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite and 4,5-dicyanoimidazole (DCI) in dichloromethane to produce a phosphorodiamidite monomer.
Dependencies are inferred from the claim text.