CVII. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast. Synthesis of a dodecadeoxynucleotide and a hexadeoxynucleotide corresponding to the nucleotide sequences 1 to 12

Abstract

A dodecadeoxynucleotide having the sequence, d-T-G-G-T-G-G-A-C-G-A-G-T, and a hexanucleotide having the sequence, d-C-C-A-C-C-A, have been chemically synthesized. These compounds represent, respectively, the nucleotide sequence 1 to 12 of one strand and 1 to 6 of the complementary strand of the gene corresponding to yeast alanine transfer RNA. The synthesis of the dodecanucleotide started with the condensation of 5′- O-monomethoxytrityl thymidine (d-MMTr-T) with N-benzoyl-3′- O-acetyl deoxyguanosine 5′-phosphate (d-pg BZ- OAc) to give the dinucleotide, d-MMTr-TpG BZ. Successive condensations of suitability protected mononuoleotides with the 3′-hydroxyl end of the growing chain gave the protected heptanucleotide, d-MMTr-TpG BZpG BZpTpG BZpG BZpA BZ. The protected heptanucleotide was then condensed with the dinucleotide, d-pC ANpG BZ- OAc, to give the nonanucleotide, d-MMTr-TpG BZpG BZpTpG BZpG BZpA BZpC ANpG BZ. Condensation of the nonanucleotide with the protected trinucleotide, d-pA BZpG BZpT- OAc, gave the protected dodecanucleotide, d-MMTr-TpG BZpG BZpTpG BZpG BZ-pA BZpC ANpGr BZpA BZpG BZpT. The condensing agents used were dicyclohexylcarbodiimide, tri-isopropylbenzenesulfonyl chloride and mesitylenesulfonyl chloride. After removal of the protecting groups, the completely deprotected dodecanucleotide was further purified by anion-exchange chromatography in the presence of 7 M-urea. The steps involved in the synthesis of the hexanucleotide were: the condensation, of 5′- O-cyanoethyl phosphate of N (4)-anisoyl deoxycytidylyl-(3′ → 5′)MN (4)aniaoyl deoxycytidine, d-CEpC AnpC An, with d-pA BZ- OAc to give the protected trinucleotide, d-pC AnpC AnpA BZ, and the condensation of cyanoethyl derivative of the trinucleotide (d-CEpC AnpC AnpA BZ) with the trinucleotide, d-pC AnpC AnpA BZ- OAc, to give the protected hexanucleotide, d-pC AnpC AnpA BZpC AnpC AnpA BZ. After removal of the N-protecting groups the 5′-phosphate group was removed by treatment with bacterial alkaline phosphatase and the hexanucleotide, d-C-C-A-C-C-A, was isolated by paper chromatography. The yields varied between 20 and 80% at different steps.