Xu Y Z, Swann P F
Department of Biochemistry, University College and Middlesex School of Medicine, London, UK.
Nucleic Acids Res. 1990 Jul 25;18(14):4061-5. doi: 10.1093/nar/18.14.4061.
A route to prepare the cyanoethyl-phosphoramidite monomer of O4-alkylthymine and a method for the routine solid-phase synthesis of oligodeoxynucleotides containing O4-alkylthymine are described. This method has been used to make DNA sequences up to 48 bases in length. The amino function of the adenine and guanine in the sequence were protected with the phenoxyacetyl group, and that of cytosine with the isobutyryl group. The phosphodiesters were protected with the cyanoethyl group. This allowed complete deprotection of the oligomer with alkoxide ions (methanol/1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) for the oligomers containing O4-methylthymine, or ethanol/DBU for those containing O4-ethylthymine) thus avoiding the use of ammonia which is known to attack the O4-alkylthymine to form 5-methylcytosine. There was no detectable loss of the alkyl group to form thymine.
描述了一种制备O4-烷基胸腺嘧啶的氰乙基亚磷酰胺单体的方法以及一种常规固相合成含O4-烷基胸腺嘧啶的寡脱氧核苷酸的方法。该方法已用于制备长度达48个碱基的DNA序列。序列中腺嘌呤和鸟嘌呤的氨基用苯氧乙酰基保护，胞嘧啶的氨基用异丁酰基保护。磷酸二酯用氰乙基保护。这使得寡聚物能用醇盐离子（对于含O4-甲基胸腺嘧啶的寡聚物用甲醇/1,8-二氮杂双环[5.4.0]十一碳-7-烯（DBU），对于含O4-乙基胸腺嘧啶的寡聚物用乙醇/DBU）完全脱保护，从而避免使用已知会攻击O4-烷基胸腺嘧啶形成5-甲基胞嘧啶的氨。未检测到烷基形成胸腺嘧啶的损失。
