Chemoenzymatic Preparation of 4′‐Thioribose NAD⁺

Abstract

This chemoenzymatic procedure describes a strategy for the preparation of 4′‐thioribose nicotinamide adenine dinucleotide (S‐NAD+), including chemical synthesis of nicotinamide 4′‐riboside (S‐NR), recombinant expression and purification of two NAD+ biosynthesis enzymes nicotinamide riboside kinase (NRK) and nicotinamide mononucleotide adenylyltransferase (NMNAT), and enzymatic synthesis of S‐NAD+. The first basic protocol describes the procedures for introduction of nicotinamide onto 4′‐thioribose and subsequent deprotection to generate S‐NR as the key intermediate for enzymatically synthesizing S‐NAD+. In the second basic protocol, experimental methods are detailed for the production of recombinant human NRK1 and NMNAT1 to catalyze conversion of S‐NR to S‐NAD+. The third basic protocol presents the enzymatic approach for the generation of S‐NAD+ from S‐NR precursor. © 2019 by John Wiley & Sons, Inc.

Improved Yield for the Enzymatic Synthesis of Radiolabeled Nicotinamide Adenine Dinucleotide

“…This was consistent with previously reported yields of ~70% in a combined reaction. 15 In contrast, the addition of PPase appeared to prevent the main reaction from reaching equilibrium and resulted in a 98% ± 1% incorporation of the 32 P signal into NAD + , corresponding to a yield of approximately 49 µM 32 P-traced NAD + (2.9 µM 32 P-NAD + and 46 µM unlabeled NAD + ) (Figure 1c,d). Expected to be also remaining in the mixture would be ~4 µM 32 P-traced ATP (240 nM 32 P-ATP and 3.8 µM unlabeled ATP), ~1 µM NMN, as well as NMNAT1 and PPase enzymes.…”

“…[13][14] This strategy had been adopted into a two-step synthesis to produce NAD + derivatives such as nicotinamide 4'-thioribose NAD + (S-NAD + ) at 70% yield, starting from modified 4-thio nicotinamide riboside. 15 We reasoned that the synthesis of labeled NAD analogs could be simplified into a single-step process by including radiolabeled analogs directly at the adenylyl transferase step (Figure 1a). We also wanted to test the consequence of including inorganic pyrophosphatase (PPase) in the reaction to degrade the + , NADH, ATP, ADP and AMP standards (>95% purity, 5 µL of 50 µM stocks) were resolved with thin layer chromatography (TLC) using PEI cellulose F plates and imaged following excitation at 254 nm.…”

“…This high yield reaction required lowered concentrations of reactants compared to previously published approaches. [13][14][15][16] Thus, there is opportunity for a broad range of reactants and more diverse set of possible products. Additionally, the labeled NAD + product can be directly used to generate labeled derivatives with an additional enzymatic reaction, such as NAADP, NADP(H), NADH or ADPR, without further processing.…”

“…To address this, we sought to improve the yield of the enzymatic synthesis of NAD + to facilitate the ability for individual labs to obtain specifically labeled NAD and NAD derivatives. [13][14][15][16] We surmised that a higher yield reaction would be amenable for labeling experiments and for generating labeled NADderivatives. Moreover, we sought a high yield reaction that could be performed using standard equipment as a useful and versatile resource for many labs.…”

“…For comparison, the combined enzymatic synthesis reactions used millimolar concentrations of reactants. [15][16] Notably, by simply adjusting the type of labeling on ATP or NMN, or using NMN derivatives (e.g. NMNH or nicotinic acid mononucleotide), this enzymatic synthesis could produce a variety of NAD-related nucleotides, highlighting the potential versatility of the approach.…”