Callahan L, Schwartz N, Ng K
Division of Cytokine Biology, Food and Drug Administration, Bethesda, Maryland 20892.
Anal Biochem. 1991 Aug 1;196(2):229-33. doi: 10.1016/0003-2697(91)90458-6.
Aliphatic diamines when used as single ion pairing reagents were capable of resolving 3'-,5'- and 2'-,5'- nucleotidyl diphosphates from one another while conventional ion pairing reagents did not separate these positional isomers. The use of 1,2-diamines resulted in the greatest resolution while increasing spacing between the amino groups progressively reduced the resolution while increasing the retention volume. A competitive ion pairing system was also developed using triethylamine as an additional ion pairing reagent. Using this system ethylenediamine, 1,2- and 1,3-diaminopropane were nearly equivalent in their ability to resolve adenosine 3'-phosphate 5'-phosphate, from adenosine 2'-phosphate 5'-phosphate, and adenosine 3'-phosphate 5'-beta-methylenephosphosulfate (3'-mePAPS) from adenosine 2'-phosphate 5'-beta-methylenephosphosulfate (2'-mePAPS), respectively. The ability to easily resolve these positional isomers allows the use of a more simplified synthetic procedure that does not involve the use selective protecting groups to specifically phosphorylate the 2' or 3' hydroxyl group. We have used this procedure on a semipreparative scale to obtain small quantities of both mePAPS and 2'-mePAPS for use in enzymatic studies.
脂肪族二胺用作单一离子对试剂时，能够分离3' - 、5' - 和2' - 、5' - 二磷酸核苷酸，而传统离子对试剂无法分离这些位置异构体。使用1,2 - 二胺时分辨率最高，而增加氨基之间的间距会逐渐降低分辨率，同时增加保留体积。还开发了一种竞争性离子对系统，使用三乙胺作为额外的离子对试剂。使用该系统时，乙二胺、1,2 - 二氨基丙烷和1,3 - 二氨基丙烷在分离3'-磷酸 - 5'-磷酸腺苷与2'-磷酸 - 5'-磷酸腺苷，以及3'-磷酸 - 5'-β - 亚甲基磷酰硫酸腺苷（3'-mePAPS）与2'-磷酸 - 5'-β - 亚甲基磷酰硫酸腺苷（2'-mePAPS）的能力上几乎相当。能够轻松分离这些位置异构体使得可以使用更简化的合成程序，该程序不涉及使用选择性保护基团来特异性磷酸化2'或3'羟基。我们已在半制备规模上使用该程序获得少量的mePAPS和2'-mePAPS用于酶学研究。
