title: 2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides.
abstract: Oligonucleotides containing 2'-O-aminopropyl-substituted RNA have been synthesized. The 2'-O-(aminopropyl)adenosine (APA), 2'-O-(aminopropyl)cytidine (APC), 2'-O-(aminopropyl)-guanosine (APG), and 2'-O-(aminopropyl)uridine (APU) have been prepared in high yield from the ribonucleoside, protected, and incorporated into an oligonucleotide using conventional phosphoramidite chemistry. Molecular dynamics studies of a dinucleotide in water demonstrates that a short alkylamine located off the 2'-oxygen of ribonucleotides alters the sugar pucker of the nucleoside but does not form a tight ion pair with the proximate phosphate. A 5-mer with the sequence ACTUC has been characterized using NMR. As predicted from the modeling results, the sugar pucker of the APU moiety is shifted toward a C3'-endo geometry. In addition, the primary amine rotates freely and is not bound electrostatically to any phosphate group, as evidenced by the different sign of the NOE between sugar proton resonances and the signals from the propylamine chain. Incorporation of aminopropyl nucleoside residues into point-substituted and fully modified oligomers does not decrease the affinity for complementary RNA compared to 2'-O-alkyl substituents of the same length. However, two APU residues placed at the 3'-terminus of an oligomer gives a 100-fold increase in resistance to exonuclease degradation, which is greater than observed for phosphorothioate oligomers. These structural and biophysical characteristics make the 2'-O-aminopropyl group a leading choice for incorporation into antisense therapeutics. A 20-mer phosphorothioate oligonucleotide capped with two phosphodiester aminopropyl nucleotides targeted against C-raf mRNA has been transfected into cells via electroporation. This oligonucleotide has 5-10-fold greater activity than the control phosphorothioate for reducing the abundance of C-raf mRNA and protein.
authors.affiliation: Isis Pharmaceuticals, Carlsbad, California 92008, USA.
authors.forename: R H
authors.initials: RH
authors.lastname: Griffey
authors.forename: B P
authors.initials: BP
authors.lastname: Monia
authors.forename: L L
authors.initials: LL
authors.lastname: Cummins
authors.forename: S
authors.initials: S
authors.lastname: Freier
authors.forename: M J
authors.initials: MJ
authors.lastname: Greig
authors.forename: C J
authors.initials: CJ
authors.lastname: Guinosso
authors.forename: E
authors.initials: E
authors.lastname: Lesnik
authors.forename: S M
authors.initials: SM
authors.lastname: Manalili
authors.forename: V
authors.initials: V
authors.lastname: Mohan
authors.forename: S
authors.initials: S
authors.lastname: Owens
authors.forename: B R
authors.initials: BR
authors.lastname: Ross
authors.forename: H
authors.initials: H
authors.lastname: Sasmor
authors.forename: E
authors.initials: E
authors.lastname: Wancewicz
authors.forename: K
authors.initials: K
authors.lastname: Weiler
authors.forename: P D
authors.initials: PD
authors.lastname: Wheeler
authors.forename: P D
authors.initials: PD
authors.lastname: Cook
mesh_terms: D005092:Exonucleases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009693:Nucleic Acid Hybridization; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf; D012265:Ribonucleotides / Q000737:chemistry*
chemical_list: D016376:Oligonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D012265:Ribonucleotides; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D005092:Exonucleases