Bennett G N, Gough G R, Gilham P T
Biochemistry. 1976 Oct 19;15(21):4623-8. doi: 10.1021/bi00666a012.
A new procedure for the synthesis of the pyrophosphate bond has been employed in the preparation of nucleoside dipyrophosphates from nucleoside 3',5'-diphosphates. The method makes use of a powerful phosphorylating agent generated in a mixture of cyanoethyl phosphate, dicyclohexylcarbodiimide, and mesitylenesulfonyl chloride in order to avoid possible intramolecular reactions between the two phosphate groups on the sugar ring. That such reactions can readily occur was shown by the facile cyclization of deoxyguanosine 3',5'-diphosphate to P1,P2-deoxyguanosine 3',5'-cyclic pyrophosphate in the presence of dicyclohexylcarbodiimide alone. The phosphorylation reagent was initially tested in the conversion of deoxyguanosine 3',5'-diphosphate to the corresponding 3',5'-dipyrophosphate and was then used to phosphorylate 2'-O-(alpha-methoxyethyl)guanosine 3',5'-diphosphate, which had been prepared from 2'-O-(alpha-methoxyethyl)guanosine. In the latter case, the addition of the two beta phosphate groups was accomplished in 40% yield. Removal of the methoxyethyl group from the phosphorylated product gave guanosine 3',5'-dipyrophosphate, which was shown to be identical with guanosine tetraphosphate prepared enzymatically from a mixture of GDP and ATP. A modification of published procedures was also necessary to effect the synthesis of guanosine bis(methylenediphosphonate). Guanosine was treated with methylenediphosphonic acid and dicyclohexylcarbodiimide in the absence of added base. The product consisted of a mixture of guanosine 2',5' - and 3',5'-bis(methylenediphosphonate), which was resolved by anion-exchange chromatography. The 2',5' and 3',5' isomers are interconvertible at low pH, with the ultimate formation of an equilibrium mixture having a composition ratio of 2:3. The predominant constituent of this mixture has been unequivocally identified as the 3',5' isomer by synthesis from 2'-O-tetrahydropyranylguanosine.
一种合成焦磷酸键的新方法已用于从核苷3',5'-二磷酸制备核苷二磷酸。该方法利用在磷酸氰乙酯、二环己基碳二亚胺和均三甲苯磺酰氯的混合物中生成的一种强磷酸化剂，以避免糖环上两个磷酸基团之间可能发生的分子内反应。单独在二环己基碳二亚胺存在下，脱氧鸟苷3',5'-二磷酸容易环化形成P1,P2-脱氧鸟苷3',5'-环焦磷酸，这表明此类反应很容易发生。磷酸化试剂最初在将脱氧鸟苷3',5'-二磷酸转化为相应的3',5'-二焦磷酸的过程中进行了测试，然后用于磷酸化由2'-O-(α-甲氧基乙基)鸟苷制备的2'-O-(α-甲氧基乙基)鸟苷3',5'-二磷酸。在后一种情况下，添加两个β磷酸基团的产率为40%。从磷酸化产物中除去甲氧基乙基基团得到鸟苷3',5'-二焦磷酸，经证明它与由GDP和ATP混合物酶促制备的鸟苷四磷酸相同。为了实现鸟苷双(亚甲基二磷酸)的合成，还需要对已发表的方法进行改进。鸟苷在不添加碱的情况下用亚甲基二膦酸和二环己基碳二亚胺处理。产物由鸟苷2',5'-和3',5'-双(亚甲基二磷酸)的混合物组成，通过阴离子交换色谱法进行分离。2',5'和3',5'异构体在低pH下可相互转化，最终形成组成比为2:3的平衡混合物。通过从2'-O-四氢吡喃基鸟苷合成，已明确鉴定出该混合物的主要成分是3',5'异构体。
