Synthesis and properties of second-generation 2-5A-antisense chimeras with enhanced resistance to exonucleases.
In order to stabilize 2-5A-antisense chimeras to exonucleases, we have synthesized chimeric oligonucleotides in which the last phosphodiester bond at the 3'-terminus of the antisense domain was inverted from the usual 3',5'-linkage to a 3',3'-linkage. The preparation of such analogues was accomplished through standard phosphoramidite chemistry with the use of a controlled pore glass solid support with a nucleoside attached through its 5'-hydroxyl, thereby permitting elongation at the 3'-hydroxyl. The structures of such terminally inverted linkage chimeras of the general formula pA4-[pBu]2-(pdNn3'-3'dN) were corroborated by a combination of snake venom phosphodiesterase digestion in the presence or absence of bacterial alkaline phosphatase. Most characteristically, the presence of the 3'-terminal-inverted phosphodiester linkage produced an unnatural dinucleotide of general composition dN3'p3'dM. These structures could be confirmed by independent synthesis and fast atom bombardment mass spectroscopy (FAB). 2-5A-Antisense chimeras of this structural class, pA4-[pBu]2-(pdNn'3-3'dN), were 5-6-fold more stable than their unmodified congeners, pA4-[pBu]2-(pdN)n, to degradation by a representative phosphodiesterase from snake venom. In 10% human serum, the new 2-5A-antisense chimeras, pA4-[pBu]2-(pdNn3'-3'dN), possessed a half-life that was 28-fold longer than that of the unmodified chimeras. These results provide entry to a second generation of 2-5A-antisense chimeras.
Li
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Section on Biomedical Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0805, USA.
Xiao
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Torrence
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D000227:Adenine Nucleotides / Q000737:chemistry*; D000469:Alkaline Phosphatase / Q000378:metabolism; D002678:Chimera; D004355:Drug Stability; D005092:Exonucleases / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics; D009843:Oligoribonucleotides / Q000737:chemistry*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D012910:Snake Venoms; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013329:Structure-Activity Relationship
D000227:Adenine Nucleotides; D007202:Indicators and Reagents; D016376:Oligonucleotides, Antisense; D009843:Oligoribonucleotides; D012910:Snake Venoms; C023505:2',5'-oligoadenylate; D005092:Exonucleases; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases