1 超高純度DNAの化学合成(口頭発表の部)

抄録

A new pathway to DNA oligomers of unprecedently high purity has been opened using allyl and allyloxycarbonyl (AOC) groups as protectors of internucleotide linkage and nucleobases, respectively, in conjunction with palladium chemistry. The efficiency of this method has been demonstrated by the synthesis of a 60mer d(^5'TA TGGGCCTTTTGATAGGATGCTCACCGAGCAAAACCAAGAACAACCAGGAGATTTTATT^3') known as a part of a DNA sequence of yolk sac tumor proteoglycan cDNA pPG1. The synthesis has been performed on controlled pore glass supports with a long-chain alkylamine spacer via the phosphoramidite method using monomer units in which all NH_2 moieties of 2'-deoxyadenosine, 2'-deoxycytidine, and 2'-deoxyguanosine are blocked by AOC groups and the phosphoramidite functions are protected by allyl groups. The coupling reaction proceeds in 99.3% average yield, attaining 66% overall yield. The fully protected product is deblocked by treatment with a mixture of tris(dibenzylideneacetone)dipalladium(0) chloroform complex (2.5 equiv/allyl), triphenyl-phosphine (25 equiv/allyl), and a large excess of butylamine and formic acid in THF at 50℃ for 1h, and then with sodium N,N-diethyldithiocarbamate solution for 0.5h. The target DNA is detached from the polymer supports by exposure to conc ammonia at room temperature for 2h. The autoradiogram and bio-image chromatogram of the ^<32>P-labeled 5'-phosphrylated product reveal that the 60mer possesses extremely high purity (70% content in the crude product) which is unaccessible by the conventional method using as protecting groups acyl for amino function of nucleobases and 2-cyanoethyl for internucleotid linkage (20% content).