Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages.
Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.
Isis Pharmaceuticals, Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA bwan@isisph.com.
W Brad
WB
Wan
Isis Pharmaceuticals, Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
Michael T
MT
Migawa
Isis Pharmaceuticals, Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
Guillermo
G
Vasquez
Isis Pharmaceuticals, Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
Heather M
HM
Murray
Isis Pharmaceuticals, Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
Josh G
JG
Nichols
Isis Pharmaceuticals, Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
Hans
H
Gaus
Isis Pharmaceuticals, Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
Andres
A
Berdeja
Isis Pharmaceuticals, Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
Sam
S
Lee
Isis Pharmaceuticals, Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
Christopher E
CE
Hart
Isis Pharmaceuticals, Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
Walt F
WF
Lima
Isis Pharmaceuticals, Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
Eric E
EE
Swayze
Isis Pharmaceuticals, Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
Punit P
PP
Seth
D000818:Animals; D002478:Cells, Cultured; D005656:Fungal Proteins / Q000378:metabolism; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry*; D016914:Ribonuclease H / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013237:Stereoisomerism; D013696:Temperature
D005656:Fungal Proteins; D016376:Oligonucleotides, Antisense; D054735:Phosphorothioate Oligonucleotides; D016914:Ribonuclease H; C526098:Endonuclease S1, Aspergillus; D015719:Single-Strand Specific DNA and RNA Endonucleases