Abstract：
Enzyme-catalyzed late-stage functionalization (LSF), such as methylation of drug molecules and lead structures, enables direct access to more potent active pharmaceutical ingredients (API). S-adenosyl-l-methionine-dependent methyltransferases (MTs) can play a key role in the development of new APIs, as they catalyze the chemo- and regioselective methylation of O-, N-, S- and C-atoms, being superior to traditional chemical routes. To identify suitable MTs, we developed a continuous fluorescence-based, high-throughput assay for SAM-dependent methyltransferases, which facilitates screening using E. coli cell lysates. This assay involves two enzymatic steps for the conversion of S-adenosyl-l-homocysteine into H2 S to result in a selective fluorescence readout via reduction of an azidocoumarin sulfide probe. Investigation of two O-MTs and an N-MT confirmed that this assay is suitable for the determination of methyltransferase activity in E. coli cell lysates.
摘要：
酶催化的后期官能化(LSF)，如药物分子和铅结构的甲基化，能够直接获得更有效的活性药物成分（API）。S-腺苷-1-蛋氨酸依赖性甲基转移酶（MTs）可以在开发新的API中发挥关键作用，当它们催化O-的化学和区域选择性甲基化时，N-,S-和C-原子，优于传统的化学路线。为了确定合适的MT,我们开发了一种基于连续荧光的，SAM依赖性甲基转移酶的高通量检测，这有利于使用大肠杆菌细胞裂解物进行筛选。该测定涉及两个酶促步骤，用于将S-腺苷-1-高半胱氨酸转化为H2S，以通过还原叠氮香豆素硫化物探针来产生选择性荧光读出。对两种O-MT和一种N-MT的研究证实该测定适用于测定大肠杆菌细胞裂解物中的甲基转移酶活性。
