STEREOSELECTIVE PREPARATION OF DEUTERATED REDUCED NICOTINAMIDE ADENINE-NUCLEOTIDES AND SUBSTRATES BY ENZYMATIC-SYNTHESIS
STEREOSELECTIVE PREPARATION OF DEUTERATED REDUCED NICOTINAMIDE ADENINE-NUCLEOTIDES AND SUBSTRATES BY ENZYMATIC-SYNTHESIS
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DOI：
10.1016/0003-2697(79)90590-6
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发表时间：
1979-01-01
影响因子：
2.9
通讯作者：
CLELAND, WW
中科院分区：
文献类型：
--
作者：
VIOLA, RE;COOK, PF;CLELAND, WW
在NAD、醇脱氢酶和醛脱氢酶的存在下，通过乙醇-d 6逐步氧化为乙酸来制备A侧（4-R）-（4- 2 H）-还原的烟酰胺腺嘌呤二核苷酸（NADD）。利用G-6-P脱氢酶的葡萄糖脱氢酶活性，在40%二甲基亚砜中氧化葡萄糖-1-d，制备NADD的B侧（4-S）异构体。使用强碱性聚苯乙烯大孔树脂（AG MP-1）柱，用0.2M LiCl，pH 10洗脱，并将合并的NADD峰施加到聚丙烯酰胺凝胶（Bio-Gel P-2）柱上，实现还原的核苷酸的后续纯化。这些制剂获得的最终A260/A340 [吸光度260 nm/吸光度340 nm]比低于2.3。以这种方式制备氘代还原的核苷酸允许通过偶联的酶促合成产生特异性氘代底物。L-苹果酸-2-d是通过偶联合成A侧NADD和A侧酶苹果酸脱氢酶还原草酰乙酸来制备的。
A-side (4-R)-(4-2H)-reduced nicotinamide adenine dinucleotide (NADD) was prepared by a stepwise oxidation of ethanol-d6 to acetate in the presence of NAD, alcohol dehydrogenase, and aldehyde dehydrogenase. The B-side (4-S) isomer of NADD was prepared using the glucose dehydrogenase activity of G-6-P dehydrogenase to oxidize glucose-1-d in 40% dimethyl sulfoxide. Subsequent purification of the reduced nucleotides was achieved using a column of strongly basic polystyrene macroporous resin (AG MP-1) eluted with 0.2 M LiCl, pH 10, and applying the pooled NADD peak to a polyacrylamide gel (Bio-Gel P-2) column. The final A260/A340 [absorbance260nm/absorbance340nm] ratio obtained for these preparations was below 2.3. Preparation of the deuterated reduced nucleotides in this manner allows production of specifically deuterated substrates by coupled enzymatic synthesis. L-Malate-2-d was prepared by coupled synthesis of A-side NADD to the reduction of oxaloacetate by the A-side enzyme malate dehydrogenase.