Co‐immobilized Phosphorylated Cofactors and Enzymes as Self‐Sufficient Heterogeneous Biocatalysts for Chemical Processes

Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co‐immobilizing phosphorylated cofactors (PLP, FAD⁺, and NAD⁺) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self‐sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD⁺‐dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.     

Co-immobilized Phosphorylated Cofactors and Enzymes as Self-Sufficient Heterogeneous Biocatalysts for Chemical Processes

Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co-immobilizing phosphorylated cofactors (PLP, FAD⁺, and NAD⁺) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self-sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD⁺-dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.     

Biological Nicotinamide Cofactor as a Redox‐Active Motif for Reversible Electrochemical Energy Storage

Nicotinamide adenine dinucleotide (NAD⁺) is one of the most well‐known redox cofactors carrying electrons. Now, it is reported that the intrinsically charged NAD⁺ motif can serve as an active electrode in electrochemical lithium cells. By anchoring the NAD⁺ motif by the anion incorporation, redox activity of the NAD⁺ is successfully implemented in conventional batteries, exhibiting the average voltage of 2.3 V. The operating voltage and capacity are tunable by altering the anchoring anion species without modifying the redox center itself. This work not only demonstrates the redox capability of NAD⁺, but also suggests that anchoring the charged molecules with anion incorporation is a viable new approach to exploit various charged biological cofactors in rechargeable battery systems.     

524—NAD/NADH as a model redox system: Mechanism,mediation, modification by the environment

The biologically important redox couple, β-nicotinamide adenine dinucleotide/1,4,β-dihydronicotinamide adenine dinucleotide, provides a grossly reversible prototype system for an overall electrode reaction consisting of two successive one-electron (1 e^?) transfer steps coupled with (a) dimerization of an intermediate free radical product, (b) protonation—deprotonation of an intermediate product, (c) other chemical reactions, (d) adsorption of reactant, intermediate and product species, and (e) mediation by electrode surface species. Cathodic reduction of NAD⁺ proceeds through two 1 e^? steps well separated in potential; protonation of the free radical produced on the first step occurs prior to the second electron-transfer; a first-order chemical reaction coupled to the latter may involve rearrangement of an initial dihydro product to 1,4-NADH (and some 1,6-NADH). In the apparently single stage 2 e^? anodic oxidation of NADH, the initial step is an irreversible heterogeneous electron transfer, which proceeds to at least some extent through mediator redox systems located close to the electrode surface; the resulting cation radical, NADH^+?, loses a proton (first order reaction) to form a neutral radical, NAD^?, which may participate in a second heterogeneous electron transfer (ECE mechanism) or may react with NAD^+? (disproportionation mechanism DISP 1 or half-regeneration mechanism) to yield NAD⁺.     

Analysis of NAD(P) and NAD(P)H cofactors by means of imprinted polymers associated with Au surfaces:: A surface plasmon resonance study

Crosslinked films consisting of the acrylamide-acrylamidophenylboronic acid copolymer that are imprinted with recognition sites for β-nicotinamide adenine dinucleotide (NAD⁺), β-nicotinamide adenine dinucleotide phosphate NADP⁺, and their reduced forms (NAD(P)H), are assembled on Au-coated glass supports. The binding of the oxidized cofactors NAD⁺ or NADP⁺ or the reduced cofactors NADH or NADPH to the respective imprinted sites results in the swelling of the polymer films through the uptake of water. Surface plasmon resonance (SPR) spectroscopy is employed to follow the binding of the different cofactors to the respective imprinted sites. The imprinted recognition sites reveal selectivity towards the association of the imprinted cofactors. The method enables the analysis of the NAD(P)⁺ and NAD(P)H cofactors in the concentration range of 1×10⁻⁶ to 1×10⁻³ M. The cofactor-imprinted films associated with the Au-coated glass supports act as active interfaces for the characterization of biocatalyzed transformations that involve the cofactor-dependent enzymes. This is exemplified with the characterization of the biocatalyzed oxidation of lactate to pyruvate in the presence of NAD⁺ and lactate dehydrogenase using the NADH-imprinted polymer film.     

Structures of Iridoid Synthase from Cantharanthus roseus with Bound NAD+, NADPH,or NAD+/10‐Oxogeranial: Reaction Mechanisms

Structures of the iridoid synthase nepetalactol synthase in the presence of NAD⁺, NADPH or NAD⁺/10‐oxogeranial were solved. The 10‐oxogeranial substrate binds in a transoid‐O1‐C3 conformation and can be reduced by hydride addition to form the byproduct S‐10‐oxo‐citronellal. Tyr178 Oζ is positioned 2.5 Å from the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1–C2 to form the cisoid isomer, leading to formation of the cis‐enolate, together with rotation about C4–C5, which enables cyclization and lactol production. The structure is similar to that of progesterone‐5β‐reductase, with almost identical positioning of NADP, Lys146(147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10‐oxogeranial structure also serves as a model for β‐face hydride attack in progesterone 5β‐reductases and is of general interest in the context of asymmetric synthesis.     

Structures of Iridoid Synthase from Cantharanthus roseus with Bound NAD+, NADPH,or NAD+/10‐Oxogeranial: Reaction Mechanisms

Structures of the iridoid synthase nepetalactol synthase in the presence of NAD⁺, NADPH or NAD⁺/10‐oxogeranial were solved. The 10‐oxogeranial substrate binds in a transoid‐O1‐C3 conformation and can be reduced by hydride addition to form the byproduct S‐10‐oxo‐citronellal. Tyr178 Oζ is positioned 2.5 Å from the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1–C2 to form the cisoid isomer, leading to formation of the cis‐enolate, together with rotation about C4–C5, which enables cyclization and lactol production. The structure is similar to that of progesterone‐5β‐reductase, with almost identical positioning of NADP, Lys146(147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10‐oxogeranial structure also serves as a model for β‐face hydride attack in progesterone 5β‐reductases and is of general interest in the context of asymmetric synthesis.     

Electrochemical detection of nicotinamide adenine dinucleotide based on molecular beacon-like DNA and E. coli DNA ligase

An electrochemical method for nicotinamide adenine dinucleotide (NAD⁺) detection with high sensitivity and selectivity has been developed by using molecular beacon (MB)-like DNA and Escherichia coli DNA ligase. In this method, MB-like DNA labeled with 5′-SH and 3′-biotin was self-assembled onto a gold electrode in its duplex form by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of NAD⁺, E. coli DNA ligase was activated, and the two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the NAD⁺-dependent E. coli DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a large conformational change in this surface-confined DNA structure, which in turn pushed the biotin away from the electrode surface and made the electrons exchange freely with the electrode. Then the generated electrochemical signals can be measured by differential pulse voltammetry (DPV). Under optimized conditions, a linear response to logarithmic concentration of NAD⁺ range from 3 nM to 5 μM and a detection limit of 1.8 nM were obtained. Furthermore, the proposed strategy had sufficient selectivity to discriminate NAD⁺ from its analogues.     

Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O2-tolerant NAD+-reducing [NiFe] hydrogenase

Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD⁺-reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H₂ conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function of the various metal cofactors present in the enzyme. Here, all iron-containing cofactors of the SH were investigated by ⁵⁷Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, which is consistent with the amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD⁺-reducing hydrogenases. For the first time, Fe–CO and Fe–CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective ¹³C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe–CO modes. The present approach explores the complex vibrational signature of the Fe–S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.     

Emissive Synthetic Cofactors: Enzymatic Interconversions of tzA Analogues of ATP,NAD+, NADH,NADP+, and NADPH

A series of enzymatic transformations, which generate visibly emissive isofunctional cofactors based on an isothiazolo[4,3‐d]pyrimidine analogue of adenosine ( ^tz A ), was developed. Nicotinamide adenylyl transferase condenses nicotinamide mononucleotide and ^tz ATP to yield N^tzAD⁺ , which can be enzymatically phosphorylated by NAD⁺ kinase and ATP or ^tz ATP to the corresponding N^tzADP⁺ . The latter can be engaged in NADP‐specific coupled enzymatic transformations involving conversion to N^tzADPH by glucose‐6‐phosphate dehydrogenase and reoxidation to N^tzADP⁺ by glutathione reductase. The N^tzADP⁺ / N^tzADPH cycle can be monitored in real time by fluorescence spectroscopy.     

Emissive Synthetic Cofactors: A Highly Responsive NAD+ Analogue Reveals Biomolecular Recognition Features

Apart from its vital function as a redox cofactor, nicotinamide adenine dinucleotide ( NAD⁺ ) has emerged as a crucial substrate for NAD⁺ -consuming enzymes, including poly(ADP-ribosyl)transferase 1 (PARP1) and CD38/CD157. Their association with severe diseases, such as cancer, Alzheimer's disease, and depressions, necessitates the development of new analytical tools based on traceable NAD⁺ surrogates. Here, the synthesis, photophysics and biochemical utilization of an emissive, thieno[3,4-d]pyrimidine-based NAD⁺ surrogate, termed N^thAD⁺ , are described. Its preparation was accomplished by enzymatic conversion of synthetic ^th ATP by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1). The new NAD⁺ analogue possesses useful photophysical features including redshifted absorption and emission maxima as well as a relatively high quantum yield. Serving as a versatile substrate, N^thAD⁺ was reduced by alcohol dehydrogenase (ADH) to N^thADH and afforded ^thADP-ribose ( ^th ADPr ) upon hydrolysis by NAD⁺ -nucleosidase (NADase). Furthermore, N^thAD⁺ was engaged in cholera toxin A (CTA)-catalyzed mono(^thADP-ribosyl)ation, but was found incapable in promoting PARP1-mediated poly(^thADP-ribosyl)ation. Due to its high photophysical responsiveness, N^thAD⁺ is suited for spectroscopic real-time monitoring. Intriguingly, and as an N7-lacking NAD⁺ surrogate, the thieno-based cofactor showed reduced compatibility (i.e., functional similarity compared to native NAD⁺ ) relative to its isothiazolo-based analogue. The distinct tolerance, displayed by diverse NAD⁺ producing and consuming enzymes, suggests unique biological recognition features and dependency on the purine N7 moiety, which is found to be of importance, if not essential, for PARP1-mediated reactions.     

Recycling of NAD+ using coimmobilized alcohol dehydrogenase andE. coli

The use of immobilized enzymes has opened the possibility of large scale utilization of NAD⁺-linked dehydrogenases, but the applications of this technique were limited by the necessity of providing the large amounts of NAD⁺ required by its stoichiometric consumption in the reaction. After immobilization of alcohol dehydrogenase and intactE. coli by glutaraldehyde in the presence of serum albumin, the respiratory chain was found to be capable of regenerating NAD⁺ from NADH. This NAD⁺ can be recycled at least 100 times, and thus the method is far more effective than any other, and, moreover, does not require NADH oxydase purification. The total NADH oxidase activity recovered was 10–30% of the initial activity. Although, NADH is unable to cross the cytoplasmic membrane, it was able to reach the active site of NADH dehydrogenase after immobilization. The best yield of NADH oxidase activity with immobilized bacteria was obtained without prior treatment of the bacteria to render them more permeable. The denaturation by heat of NADH oxidase in cells that are permeabilized was similar before and after immobilization. In contrast, the heat denaturation of soluble Β-galactosidase required either a higher temperature or a longer exposure after immobilization. The sensitivity of immobilized NADH oxidase to denaturation by methanol was decreased compared to permeabilized cells. As a result, it is clear that the system can function in the presence of methanol, which is necessary as a solvent for certain water insoluble substrates.     

The Preparation of Butyrylated NAD+ Type of Biological Molecules

Abstract

Butyrylated NAD⁺ and its fluorescent analog, 1,N ⁶-etheno NAD⁺ are prepared in good yields by employing two-phase system, i.e., water and CH₂Cl₂ containing dimethyaminopyridine and excess butyric anhydride. The reaction condition for this reaction is so specific that several other acylating conditions attempted were totally failed, and this developed methodology will be conveniently utilized for the further study of cyclic ADP-ribose (cADPR).     

Emissive Synthetic Cofactors: Enzymatic Interconversions of tzA Analogues of ATP,NAD+, NADH,NADP+, and NADPH

A series of enzymatic transformations, which generate visibly emissive isofunctional cofactors based on an isothiazolo[4,3‐d]pyrimidine analogue of adenosine ( ^tz A ), was developed. Nicotinamide adenylyl transferase condenses nicotinamide mononucleotide and ^tz ATP to yield N^tzAD⁺ , which can be enzymatically phosphorylated by NAD⁺ kinase and ATP or ^tz ATP to the corresponding N^tzADP⁺ . The latter can be engaged in NADP‐specific coupled enzymatic transformations involving conversion to N^tzADPH by glucose‐6‐phosphate dehydrogenase and reoxidation to N^tzADP⁺ by glutathione reductase. The N^tzADP⁺ / N^tzADPH cycle can be monitored in real time by fluorescence spectroscopy.     

NAD<Superscript>+</Superscript> metabolite levels as a function of vitamins and calorie restriction: evidence for different mechanisms of longevity

Background

NAD⁺ is a coenzyme for hydride transfer enzymes and a substrate for sirtuins and other NAD⁺-dependent ADPribose transfer enzymes. In wild-type Saccharomyces cerevisiae, calorie restriction accomplished by glucose limitation extends replicative lifespan in a manner that depends on Sir2 and the NAD⁺ salvage enzymes, nicotinic acid phosphoribosyl transferase and nicotinamidase. Though alterations in the NAD⁺ to nicotinamide ratio and the NAD⁺ to NADH ratio are anticipated by models to account for the effects of calorie restriction, the nature of a putative change in NAD⁺ metabolism requires analytical definition and quantification of the key metabolites.

Results

Hydrophilic interaction chromatography followed by tandem electrospray mass spectrometry were used to identify the 12 compounds that constitute the core NAD⁺ metabolome and 6 related nucleosides and nucleotides. Whereas yeast extract and nicotinic acid increase net NAD⁺ synthesis in a manner that can account for extended lifespan, glucose restriction does not alter NAD⁺ or nicotinamide levels in ways that would increase Sir2 activity.

Conclusions

The results constrain the possible mechanisms by which calorie restriction may regulate Sir2 and suggest that provision of vitamins and calorie restriction extend lifespan by different mechanisms.

     

Purification and characterization of a microbial dehydrogenase

Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native M _r value estimated by gelfiltration chromatography gave a value of 210,000. The enzyme made use of NAD⁺ less effectively than NADP⁺. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD⁺ or NADP⁺. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl₂. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD⁺ and NADP⁺ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD⁺ or NADP⁺. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.     

Analysis of metabolic transients in intact cells by rapid microfluorimetry

Summary The analysis of NAD⁺ (or NADP⁺) reduction-reoxidation transients in single living cells by rapid microfluorimetry provides a mean to screen the activity of various intracellular enzymes, the interplay of regulating cofactors as well as the influence of structural compartmentalization or membrane barriers. Examples of possible applications refer to the analysis of transient parameters, the pattern of enzymatic pathways in relation to cell growth, effects of cofactors or metabolic preloading, etc. Through the incorporation of a nitrogen chamber the method has been extended to cells (e. g. L cells, human astrocytoma) which require anaerobiosis for glycolytic reduction of NAD⁺. When glucose-6-phosphate is replaced by glucose-1-phosphate the lag which precedes NAD⁺ reduction is prolonged from 100–200 msec up to 500–1000 msec. This can be shortened in presence of glucose-1,6-phosphate (a coenzyme for phosphoglucomutase). Differences in the flux pattern of the forward reaction at the phosphoglucomutase are found in a pleiomorphic population of L cells: e. g. glucose-1-phosphate more easily channeled towards the Embden-Meyerhof sequence in the larger non-dividing individuals. Preincubation with glycerol or xylitol leads to a prolongation of all parameters in the fluorescence transients, while cyclic AMP and ethionine lead to the opposite. The pattern of fluorescence transients makes possible a differentiation between reversible and irreversible inhibitors of LDH. Thus, by rapid microfluorimetry it is possible to resolve the early and later phases of fluorescence transients into components corresponding to characteristic steps in the sequence of intracellular events or control states.

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Zusammenfassung Die Analyse von NAD⁺- (oder NADP⁺-)Reduktions-Reoxidationsübergängen in einzelnen lebenden Zellen durch schnelle Mikrofluorimetrie bietet eine Möglichkeit, die Aktivität verschiedener intrazellulärer Enzyme zu testen, das Zwischenspiel regulatorischer Faktoren zu studieren und den Einfluß struktureller Abteilungsbildung oder von Membranbarrieren zu untersuchen. Anwendungsbeispiele werden gebracht für die Analyse von Übergangsparametern, für die Untersuchung des Musters enzymatischer Stoffwechselschritte während des Zellwachstums und für die Untersuchung der Wirkung von Cofaktoren oder von Stoffwechselvorbelastungen. Durch Einbeziehung einer Stickstoffkammer konnte die Methode auf Zellen angewendet werden, die für die glykolytische Reduktion von NAD⁺ anaerobe Bedingungen verlangen (z. B. L-Zellen, menschliches Astrocytom). Wird Glucose-6-phosphat durch Glucose-1-phosphat ersetzt, so wird die NAD⁺-Reduktion von 100 bis 200 msek bis auf 500 bis 1000 msek verzögert. Diese Zeit kann bei Anwesenheit von Glucose-1,6-phosphat (einem Coenzym für Phosphoglucomutase) verkürzt werden. Differenzen im Fließmuster der vorgenannten Reaktion der Phosphoglucomutase werden in pleiomorphen Populationen von L-Zellen gefunden. So ging z. B. Glucose-1-phosphat leichter in die Embden-Meyerhof-Reaktionsfolge ein in den größeren, sich nicht teilenden Individuen.Vorinkubation mit Glycerin oder Xylit führt zu einer Verlängerung aller Parameter der Fluoreszenzübergänge, während cyklisches AMP und Äthionin das Gegenteil bewirken. Das Muster der Fluoreszenzübergänge ermöglicht eine Differenzierung zwischen reversiblen und irreversiblen Inhibitoren von LDH. So können durch schnelle Mikrofluorimetrie die frühen und späteren Phasen der Fluoreszenzübergänge in Teilschritte aufgelöst werden, die für den Ablauf intrazellulärer Vorgänge oder für Kontrollzustände charakteristisch sind.

     

Coenzyme Regeneration in Hexanol Oxidation Catalyzed by Alcohol Dehydrogenase

The enzymatic ways of coenzyme regeneration include the addition of a second enzyme to the system or the addition of the co-substrate. In the present study, both methods of enzymatic coenzyme (NAD⁺) regeneration were studied and compared in the reaction of hexanol oxidation catalyzed by alcohol dehydrogenase (ADH). As a source of ADH, commercial isolated enzyme and the whole baker??s yeast cells were used. First, coenzyme regeneration was employed in the reaction of acetaldehyde reduction catalyzed by the same enzyme that catalyzed the main reaction, and then NAD⁺ regeneration was applied in the reaction of pyruvate reduction catalyzed by l-lactate dehydrogenase (l-LDH). Hexanal was obtained as the product of hexanol oxidation catalyzed by isolated ADH while hexaonic acid was detected as a product of the same reaction catalyzed by baker??s yeast cells. All of the used biocatalysts were kinetically characterized. The mass reactions were described by the mathematical models. All models were validated in the batch reactor. One hundred percent hexanol conversion was obtained using permeabilized yeast cells using both methods of cofactor regeneration. By using isolated enzyme ADH, the higher conversion was achieved in a system with cofactor regeneration catalyzed by l-LDH.     

Chain‐Terminating and Clickable NAD+ Analogues for Labeling the Target Proteins of ADP‐Ribosyltransferases

ADP‐ribosyltransferases (ARTs) use NAD⁺ as a substrate and play important roles in numerous biological processes, such as the DNA damage response and cell cycle regulation, by transferring multiple ADP‐ribose units onto target proteins to form poly(ADP‐ribose) (PAR) chains of variable sizes. Efforts to identify direct targets of PARylation, as well as the specific ADP‐ribose acceptor sites, must all tackle the complexity of PAR. Herein, we report new NAD⁺ analogues that are efficiently processed by wild‐type ARTs and lead to chain termination owing to a lack of the required hydroxy group, thereby significantly reducing the complexity of the protein modification. Due to the presence of an alkyne group, these NAD⁺ analogues allow subsequent manipulations by click chemistry for labeling with dyes or affinity markers. This study provides insight into the substrate scope of ARTs and might pave the way for the further developments of chemical tools for investigating PAR metabolism.     

Alkylation of Adenine,Adenosine, and NAD+ with 1,3-Propanesultone. Synthesis of N6-(3-sulfonatopropyl)-NAD+, a new NAD+ derivative with substantial coenzyme activity

The reactivity of 1,3-propanesultone with adenine, adenosine, and NAD⁺ was studied in order to prepare N⁶-(3-sulfonatopropyl)-NAD⁺ ( 3b ), a new NAD⁺ derivative substituted at the purine moiety with sunstantial coanzyme activity for several dehydrogenases. The regiochemistry of the alkylation at the purione nucleus was investigated by UV, ¹H-NMR, and FAB-MS proved to be a powerful tool for determining the molecular weight of these polar and poorly volatile compounds. In addition, regular framgmentation of 3b and other NAD⁺ derivatives was observed.