Synthesis of antisense oligonucleotides as potential antiviral and antitumor agents

Abstract

Antisense oligonucleotide란 바이러스 및 암세포의 mRNA에 존재하는 염기 서열에 상보적으로 결합하여 유전자 발현을 차단함으로써 단백질 합성을 억제하여 각종 바이러스 및 암세포의 증식을 선택적으로 억제하는 치료제이다. 이 duplex DNA를 안정화시키고 지용성을 증대시킨 Antisense oligonucleotide를 만들기 위하여 그 구성 단위가 되는 새로운 뉴크레오사이드를 합성하였다. 1,2-O-Isopropylidene-α-D-xylofuranose로부터 중간체인 1,2,5-tri-O-acetyl-3-[(terr-butyldiphenyl) silyloxymetyl]-D-ribofuranose를 합성하고 이것을 silylation된 thymine과 축합하였다. 여기서 3'-OH 기는 silylation하여 protection 한 후, 2'-OH기에 methoxyethoxy기를 도입하고 5'-OH기를 4,4'-dimethoxytrityl 기로 치환하고 그 뒤에 3'-OH기를 deprotection하는 방법으로 원하는 뉴크레오사이드를 합성하였다. 같은 방법으로 uracil을 축합한 뉴크레오사이드도 합성하였다. 이 중 thymidine 유도체를 이용하여 DNA synthesizer를 통해 oligonucleotide를 만들고 이것의 Tm을 측정하여 wild type standard의 Tm과 비교하여 본 결과 oligonucleotided의 Tm이 더 낮게 나타났다. 따라서 지용성을 증가시키면서도 steric hinderance가 나타나지 않는 oligonucleotide의 합성이 필요할 것으로 생각된다.;Antisense oligonucleotides were designed to bind viral and neoplastic mRNAs and inhibit gene expression. Any mechanism of antisense inhibition in cells requires hybridization of DNA oligonucleotides to a target mRNA which possesses its own secondary and tertiary structure. To increase the lipophlicity and the stability of the hybrid, new nucleosides, the unit of the anitsense oligonucleotides, were synthesized from 1,2-O-isopropylidene- α -D- xylofanose. Silylation of 1,2-O-isopropylidene- α -D-xylofanose with tert- butyldimetylsilyl chloride produced the 5-O-(tert-butyldimetylsilyl) derivative 2. Oxidation of 2 with chromium trioxide/pyridine/acetic anhydride complex (1 :2: 1, molar ratio) yielded the 3-keto derivative 3, which was then converted to the 3-methylene analogue 4 by Wittig reaction. Hydroboration-oxydation of 4 afforded the 3-deoxy-3-hydroxymethyl derivative 5. Treatment of 5 with benzyl bromide gave the benzylated derivative 6. Deblocking of 6 with 50% acetic acid was followed by acetylation to afford 7 which was debenzylated by catalytic hydrogenation with palladium hydroxide on carbon and cyclohexene in ethanol to give the 3'-deblocked derivative 8. And 3-OH group of 8 was silylated to yield 1,2,5-O-acetyl-3-[(tert-butyldiphenyl)silyloxymethyl]-3- deoxy-D-ribofuranose. Coupling of 9 with thymine in the presence of hexamethyldisilazane (HMDS), ammonium sulfate and TMSOTf yielded compound 10 in 62% yield. Treatment of compound 10 with benzoyl chloride in pyridine produced 3-N-benzoylated derivative 11, which was then deacetylated with K_(2)CO_(3) in MeOH to afford 12. And 12 was benzoylated to afford 13. The reaction of 13 with 2- methoxyethoxymethyl chloride (MEMCl) gave 14, which was then debenzoylated with methanolic ammonia to afford 5'- deprotected nucleoside 15. And 15 was treated with 4,4'-dimethoxytrityl chloride to give compound 16, which was then reacted with tetra-n- butylammonium fluoride (n-Bu4NF) in THF to yield desired nucleoside 17. And uracil homologue was also synthesized from intermediate 9 by the same methodology. Antisence oligonucleotides were synthesized from these nucleosides using DNA synthesizer and the Tm's of the oligonucleotides were measured and compared with those of natural oligonucleotides.