Synthesis of radiolabeled nicotinamide cofactors from labeled pyridines: versatile probes for enzyme kinetics.
Synthesis of radiolabeled nicotinamide cofactors from labeled pyridines: versatile probes for enzyme kinetics.
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DOI：
10.1016/j.ab.2012.08.012
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发表时间：
2012-11-15
影响因子：
2.9
通讯作者：
Kohen A
中科院分区：
文献类型：
--
作者：
Sen A;Stojković V;Kohen A
14 C标记的烟酰胺辅因子广泛用于生物医学研究，例如描绘代谢途径，阐明酶促机制，或作为动力学同位素效应（KIE）实验中的底物。14 C标记通常位于远离反应位置，经常在腺嘌呤环上。商业前体成本的上升，以及已建立的合成所需的酶的可用性的中断，最近使得标记的烟酰胺如[Ad-14 C]-NADPH的制备变得不可行。在这里，我们报告了几种替代品的合成和表征：[羰基-14C]-NADPH，4 R-[羰基-14C，4- 2 H]-NADPH和[羰基-14C，4- 2 H2]-NADPH。新方法利用[羰基-14 C]-烟酰胺作为起始材料，因为它比NADPH的其他商业14 C-前体便宜得多，并且仅需要一种商业上可获得的酶来制备NAD（P）+和NAD（P）H。羰基-14 C与反应中心的接近增加了不恰当的14 C同位素效应的风险。这种担忧已经通过与大肠杆菌二氢叶酸还原酶（EcDHFR）的竞争性KIE测量得到缓解，该测量使用这种特定的羰基-14 C NADPH。结合同位素效应和KIE测量的组合在酰胺羰基处没有产生显著的12 C/14 C同位素效应（KIE = 1.003 ± 0.004）。报道的程序提供了一个高产率，高纯度和成本效益的替代标记的烟酰胺辅因子通过先前公布的路线合成。
14C-labeled nicotinamide cofactors are widely employed in biomedical investigations, e.g. to delineate metabolic pathways, to elucidate enzymatic mechanisms, or as substrates in kinetic isotope effect (KIE) experiments. The 14C-label has generally been located remote from the reactive position, frequently at the adenine ring. Rising costs of commercial precursors, and disruptions in the availability of enzymes required for established syntheses, have recently made the preparation of labeled nicotinamides such as [Ad-14C]-NADPH inviable. Here, we report the syntheses and characterization of several alternatives: [carbonyl-14C]-NADPH, 4R-[carbonyl-14C, 4-2H]-NADPH, and [carbonyl-14C, 4-2H2]-NADPH. The new procedures utilize [carbonyl-14C]-nicotinamide as starting material, as it is significantly cheaper than other commercial 14C-precursors of NADPH, and require only one commercially available enzyme to prepare NAD(P)+ and NAD(P)H. The proximity of carbonyl-14C to the reactive center raises the risk of an inopportune 14C-isotope effect. This concern has been alleviated via competitive KIE measurements with Escherichia coli dihydrofolate reductase (EcDHFR), that use this specific carbonyl-14C NADPH. A combination of binding isotope effect and KIE measurements yielded no significant 12C/14C isotope effect at the amide carbonyl (KIE = 1.003 ± 0.004). The reported procedure provides a high-yield, high-purity and cost-effective alternative to labeled nicotinamide cofactors synthesized by previously published routes.