Cieślak Jacek, Grajkowski Andrzej, Livengood Victor, Beaucage Serge L
Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, Maryland 20892, USA.
J Org Chem. 2004 Apr 2;69(7):2509-15. doi: 10.1021/jo035861f.
The thermolabile 4-methylthio-1-butyl phosphate/thiophosphate protecting group for DNA oligonucleotides has been investigated for its potential application to a "heat-driven" process for either oligonucleotide synthesis on diagnostic microarrays or, oppositely, to the large-scale preparation of therapeutic oligonucleotides. The preparation of phosphoramidites 10a-d is straightforward, and the incorporation of these amidites into oligonucleotides via solid-phase techniques proceeds as efficiently as that achieved with 2-cyanoethyl deoxyribonucleoside phosphoramidites. The versatility of the 4-methylthio-1-butyl phosphate/thiophosphate protecting group is exemplified by its facile removal from oligonucleotides upon heating for 30 min at 55 degrees C in an aqueous buffer under neutral conditions or within 2 h at 55 degrees C in concentrated NH(4)OH. The deprotection reaction occurs through an intramolecular cyclodeesterification mechanism leading to the formation of sulfonium salt 18. When mixed with deoxyribonucleosides and N-protected 2'-deoxyribonucleosides or with a model phosphorothioate diester under conditions approximating those of large-scale (>50 mmol) oligonucleotide deprotection reactions, the salt 18 did not significantly alter DNA nucleobases or desulfurize the phosphorothioate diester model to an appreciable extent.
已对用于DNA寡核苷酸的热不稳定的4-甲硫基-1-丁基磷酸酯/硫代磷酸酯保护基团在“热驱动”过程中的潜在应用进行了研究，该过程可用于诊断微阵列上的寡核苷酸合成，或者相反，用于大规模制备治疗性寡核苷酸。亚磷酰胺10a-d的制备很简单，并且通过固相技术将这些亚磷酰胺掺入寡核苷酸的效率与用2-氰基乙基脱氧核糖核苷亚磷酰胺所达到的效率相同。4-甲硫基-1-丁基磷酸酯/硫代磷酸酯保护基团的多功能性体现在，在中性条件下于水性缓冲液中在55℃加热30分钟或在浓NH(4)OH中在55℃加热2小时后，它能从寡核苷酸上轻松除去。脱保护反应通过分子内环化脱酯机理发生，导致形成锍盐18。当在接近大规模（>50 mmol）寡核苷酸脱保护反应的条件下与脱氧核糖核苷和N-保护的2'-脱氧核糖核苷混合或与模型硫代磷酸二酯混合时，盐18不会显著改变DNA碱基，也不会使硫代磷酸二酯模型发生明显的脱硫。
