Huang M C, Montgomery J A, Thorpe M C, Stewart E L, Secrist J A, Blakley R L
Arch Biochem Biophys. 1983 Apr 1;222(1):133-44. doi: 10.1016/0003-9861(83)90510-6.
Earlier results suggested that although the N-deoxyribosyltransferase from lactobacilli is a convenient tool for the preparation of analogs of 2'-deoxyadenosine, 8-substituted purines do not act as substrates. However, eight of nine 8-substituted purines that were examined proved to be substrates for the transferase from Lactobacillus leichmannii, and deoxyribonucleosides of four of these bases have been prepared. The substituents at C-8 of the purine greatly affect the rate of deoxyribosyl transfer to the base, and in all cases the rate is slower than transfer to purines lacking an 8-substituent. The 8-substituent also affects the nature of the nucleoside formed. With the electron-donating methyl group at position 8 of adenine, the transferase forms the expected 8-methyl-9-(2'-deoxyribofuranosyl)adenine. However, when purines bearing an electron-withdrawing substituent at the 8-position are used as substrates, the deoxyribosyl moiety is preferentially transferred to N-3 of the base. In the case of 8-trifluoromethyladenine the 3-deoxyribonucleoside is the only product detectable. With 8-bromo or 8-chloroadenine as substrate the 3- and 9-deoxyribonucleosides can both be isolated from the enzymatic reaction mixture. Time course studies indicated that with thymidine and 8-bromoadenine as substrates the 3-deoxyribonucleoside is initially the major product, but that the 9-deoxyribonucleoside becomes the major product after long incubation periods. Negligible interconversion of these nucleosides occurs in the absence of transferase, but conversion in either direction occurs readily in the presence of the enzyme. Significant hydrolysis of pyrimidine and purine deoxyribonucleosides occurs in the presence of the transferase. This was more obvious during the course of reactions involving 8-substituted purines because the slowness of deoxyribosyl transfer required longer incubation periods and larger amounts of enzyme. The hydrolysis is proportional to enzyme concentration, little affected by the nature of the base and is attributed to hydrolysis of a deoxyribosyl derivative of the transferase which is an obligatory intermediate of deoxyribosyl transfer. 8-Trifluoromethyl-3-(2'-deoxyribofuranosyl)adenine, 8-methyl-9-(2'-deoxyribofuranosyl)adenine, and 8-bromo-9-(2'-deoxyribofuranosyl)adenine were tested for their ability to inhibit the growth of CCRF-CEM cells in culture. Unlike the potent 2-halogeno-2'-deoxyadenosine derivatives, these three nucleosides cause less than 50% inhibition at concentrations up to 100 microM.
早期结果表明，尽管来自乳酸杆菌的N-脱氧核糖基转移酶是制备2'-脱氧腺苷类似物的便利工具，但8-取代嘌呤并非该酶的底物。然而，所检测的9种8-取代嘌呤中有8种被证明是莱氏乳杆菌转移酶的底物，并且已经制备了其中4种碱基的脱氧核糖核苷。嘌呤C-8位的取代基极大地影响脱氧核糖基向碱基的转移速率，在所有情况下，该速率都比向无8-取代基的嘌呤转移要慢。8-取代基还会影响所形成核苷的性质。当腺嘌呤的8位带有供电子的甲基时，转移酶会形成预期的8-甲基-9-(2'-脱氧呋喃核糖基)腺嘌呤。然而，当使用在8位带有吸电子取代基的嘌呤作为底物时，脱氧核糖基部分会优先转移到碱基的N-3位。以8-三氟甲基腺嘌呤为底物时，3-脱氧核糖核苷是唯一可检测到的产物。以8-溴或8-氯腺嘌呤为底物时，3-和9-脱氧核糖核苷都可以从酶促反应混合物中分离出来。时间进程研究表明，以胸苷和8-溴腺嘌呤为底物时，3-脱氧核糖核苷最初是主要产物，但长时间孵育后9-脱氧核糖核苷会成为主要产物。在没有转移酶的情况下，这些核苷之间的相互转化可以忽略不计，但在有酶存在时，两个方向的转化都很容易发生。在转移酶存在的情况下，嘧啶和嘌呤脱氧核糖核苷会发生显著水解。在涉及8-取代嘌呤的反应过程中，这种情况更为明显，因为脱氧核糖基转移较慢需要更长的孵育时间和更多的酶量。水解与酶浓度成正比，受碱基性质影响较小，这归因于转移酶的一种脱氧核糖基衍生物的水解，该衍生物是脱氧核糖基转移的必需中间体。对8-三氟甲基-3-(2'-脱氧呋喃核糖基)腺嘌呤、8-甲基-9-(2'-脱氧呋喃核糖基)腺嘌呤和8-溴-9-(2'-脱氧呋喃核糖基)腺嘌呤抑制培养的CCRF-CEM细胞生长的能力进行了测试。与强效的2-卤代-2'-脱氧腺苷衍生物不同，这三种核苷在浓度高达100 microM时引起的抑制作用小于50%。
