47775-00-8

Upstream product

• 53-84-9 NAD 
• 865-05-4 nicotinamide adenine dinucleotide 
• 53-84-9 nicotiamide adenine dinucleotide 
• 67-56-1 methanol 

Downstream Products

• 148333-40-8 C₁₅H₂₃N₅O₁₄P₂ 

Relevant academic research and scientific papers

Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD+ Analogue

The synthesis, photophysics, and biochemical utility of a fluorescent NAD+ analogue based on an isothiazolo[4,3-d]pyrimidine core (NtzAD+) are described. Enzymatic reactions, photophysically monitored in real time, show NtzAD+ and NtzADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as NtzAD+ is converted to NtzADH, reflecting a complementary photophysical behavior to that of the native NAD+/NADH. NtzAD+ and NtzADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding tzADP-ribose. NtzAD+ also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer tzADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD+.

Clickable NAD analogues for labeling substrate proteins of poly(ADP-ribose) polymerases

Poly(ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple adenine diphosphate ribose (ADP-ribose) units from nicotinamide adenine dinucleotide (NAD) to substrate proteins. There are 17 PARPs in humans. Several PARPs, such as PARP-1 and Tankyrase-1, are known to play important roles in DNA repair, transcription, mitosis, and telomere length maintenance. To better understand the functions of PARPs at a molecular level, it is necessary to know what substrate proteins PARPs modify. Here we report clickable NAD analogues that can be used to label PARP substrate proteins. The clickable NAD analogues have a terminal alkyne group which allows the conjugation of fluorescent or affinity tags to the substrate proteins. Using this method, PARP-1 and tankyrase-1 substrate proteins were labeled by a fluorescent tag and visualized on SDS-PAGE gel. Using a biotin affinity tag, we were able to isolate and identify a total of 79 proteins as potential PARP-1 substrates. These include known PARP-1 substrate proteins, including histones and heterogeneous nuclear ribonucleoproteins. About 40% of the proteins were also identified in recent proteomic studies as potential PARP-1 substrates. Among the identified potential substrates, we further demonstrated that tubulin and three mitochondrial proteins, TRAP1 (TNF receptorassociated protein 1), citrate synthase, and GDH (glutamate dehydrogenase 1), are substrates of PARP-1 in vitro. These results demonstrate that the clickable NAD analogue is useful for labeling, in-gel detection, isolation, and identification of the substrate proteins of PARPs and will help to understand the biological functions of PARPs.

Biosynthesis of thiamin thiazole in eukaryotes: Conversion of NAD to an advanced intermediate

Thiazole synthase catalyzes the formation of the thiazole moiety of thiamin pyrophosphate. The enzyme from Saccharomyces cerevisiae (THI4) copurifies with a set of strongly bound adenylated metabolites. One of them has been characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2- carboxylic acid. Attempts toward yielding active wild-type THI4 by releasing protein-bound metabolites have failed so far. Here, we describe the identification and characterization of two partially active mutants (C204A and H200N) of THI4. Both mutants catalyzed the release of the nicotinamide moiety from NAD to produce ADP-ribose, which was further converted to ADP-ribulose. In the presence of glycine, both the mutants catalyzed the formation of an advanced intermediate. The intermediate was trapped with orthophenylenediamine, yielding a stable quinoxaline derivative, which was characterized by NMR spectroscopy and ESI-MS. These observations confirm NAD as the substrate for THI4 and elucidate the early steps of this unique biosynthesis of the thiazole moiety of thiamin in eukaryotes.

Transition state structure of the solvolytic hydrolysis of NAD+

The transition state structure has been determined for the pH- independent solvolytic hydrolysis of NAD+. The structure is based on kinetic isotope effects (KIEs) measured for NAD+'s labeled in various positions of the ribose ring and in the leaving group nitrogen. The KIEs for reactions performed at 100°C in 50 mM NaOAc (pH 4.0) were as follows: 1-15N, 1.020 ± 0.007; 1'-14C, 1.016 ± 0.002; [1-15N,1'-14C], 1.034 ± 0.002; 1'- 3H, 1.194 ± 0.005; 2'-3H, 1.114 ± 0.004; 4'-3H, 0.0997 ± 0.001; 5'3H, 1.000 ± 0.003; 4'-18O, 0.988 ± 0.007. The transition state structure was determined using bond energy/bond order vibrational analysis to predict KIEs for trial transition state models. The structure that most closely matches the experimental KIEs defines the transition state. A structure interpolation method was developed to generate trial transition state structures and thereby systematically search reaction coordinate space. Structures are generated by interpolation between reference structures, reactant NAD+ and a hypothetical {ribo-oxocarbenium ion plus nicotinamide} structure. The point in reaction coordinate space where all the predicted KIEs matched the measured ones was considered to locate the transition state structure. This occurred when the residual bond order to the leaving group nicotinamide, n(LG,TS), was 0.02 (bond length = 2.65 A?) and the bond order to the approaching nucleophile, n(Nu,TS), was 0.005 (3.00 A?). Thus, bond-breaking and bond-making in this A(N)D(N) reaction are asynchronous, and the transition state has a highly oxocarbenium ion-like character.

Transition state structure for the hydrolysis of NAD+ catalyzed by diphtheria toxin

Diphtheria toxin (DTA) uses NAD+ as an ADP-ribose donor to catalyze the ADP-ribosylation of eukaryotic elongation factor 2. This inhibits protein biosynthesis and ultimately leads to cell death. In the absence of its physiological acceptor, DTA catalyzes the slow hydrolysis of NAD+ to ADP- ribose and nicotinamide, a reaction that can be exploited to measure kinetic isotope effects (KIEs) of isotopically labeled NAD+s. Competitive KIEs were measured by the radiolabel method for NAD+ molecules labeled at the following positions: 1-15N = 1.030 ± 0.004, 1'-14C = 1.034 ± 0.004, (1- 15N, 1'-14C) = 1.062 ± 0.010, 1'-3H = 1.200 ± 0.005, 2'-3H = 1.142 ± 0.005, 4'-3H = 0.990 ± 0.002, 5'-3H = 1.032 ± 0.004, 4'-18O = 0.986 ± 0.003. The ring oxygen, 4'-18O, KIE was also measured by whole molecule mass spectrometry (0.991 ± 0.003) and found to be within experimental error of that measured by the radiolabel technique, giving an overall average of 0.988 ± 0.003. The transition state structure of NAD+ hydrolysis was determined using a structure interpolation method to generate trial transition state structures and bond-energy/bond-order vibrational analysis to predict the KIEs of the trial structures. The predicted KIEs matched the experimental ones for a concerted, highly oxocarbenium ion-like transition state. The residual bond order to the leaving group was 0.02 (bond length = 2.65 A?), while the bond order to the approaching nucleophile was 0.03 (2.46 A?). This is an A(N)D(N) mechanism, with both leaving group and nucleophilic participation in the reaction coordinate. Fitting the transition state structure into the active site cleft of the X-ray crystallographic structure of DTA highlighted the mechanisms of enzymatic stabilization of the transition state. Desolvation of the nicotinamide ring, stabilization of the oxocarbenium ion by apposition of the side chain carboxylate of Glu148 with the anomeric carbon of the ribosyl moiety, and the placement of the substrate phosphate near the positively charged side chain of His21 are all consistent with the transition state features from KIE analysis.

Pyridine Nucleotide Chemistry. A New Mechanism for the Hydroxide-Catalyzed Hydrolysis of the Nicotinamide-Glycosyl Bond

The mechanism of hydroxide-catalyzed hydrolysis of the glycosyl bond of β-NAD+ has been reinvestigated.The pH dependence of the rate of hydrolysis of β-NAD+ and related compounds has been determined over a range from pH 4.3 to 13.5.Between pH 8.5 and 11 the log of the rate constant is linearly dependent on pH with a slope of unity.Below pH 6.5 and above pH 12.5 the reaction becomes pH independent.The product of the reaction over the entire pH range is nicotinamide.A nonlinear least-squares fit of the data yields a pKeq corresponding to the pKa of ionization of the nicotinamide ribose diol, which has been determined independently by 13C NMR.Methanolysis of β-NAD+ yields a ratio of 3.7:1 for the β and α anomers of 1'-O-methyl-ADP ribose.Hydrolysis in a methanol/water mixture shows no selectivity for attack on the basis of the nucleophilicity of the attacking species.The importance of the ribose diol in the hydrolysis reaction was investigated with the isopropylidene derivative of β-nicotinamide riboside.Hydroxide-catalyzed decomposition of β-2',3'-O-isopropylidene nicotinamide riboside is pH independent below pH 7 and linearly dependent on hydroxide concentration above pH 10.In contrast to the results for β-NAD+, no pH-independent region is observed at high pH and the product of the pH-dependent reaction is 2-hydroxy-3-pyridinecarboxaldehyde; i.e., no detectable hydroxide-catalyzed release of nicotinamide is observed.On the basis of these data, as well as solvent isotope effects and data from previous investigations, we propose a new mechanism in which dissociative cleavage of nicotinamide-glycosyl bond is facilitated by the nicotinamide ribose diol anion through noncovalent stabilization of an oxo carbocation intermediate.