Electrocatalytic Volleyball: Rapid Nanoconfined Nicotinamide Cycling for Organic Synthesis in Electrode Pores

In living cells, redox chains rely on nanoconfinement using tiny enclosures, such as the mitochondrial matrix or chloroplast stroma, to concentrate enzymes and limit distances that nicotinamide cofactors and other metabolites must diffuse. In a chemical analogue exploiting this principle, nicotinamide adenine dinucleotide phosphate (NADPH) and NADP⁺ are cycled rapidly between ferredoxin–NADP⁺ reductase and a second enzyme—the pairs being juxtaposed within the 5–100 nm scale pores of an indium tin oxide electrode. The resulting electrode material, denoted (FNR+E2)@ITO/support, can drive and exploit a potentially large number of enzyme‐catalysed reactions.     

Sensitive assay for nicotinamide adenine dinucleotide phosphate and its reduced form based on the bioluminescence method

An assay of reduced nicotinamide adenine dinucleotide phosphate (NADPH) by bioluminescence was investigated and applied for NADP⁺. The NADP⁺ is first reduced by glucose-6-phosphate dehydrogenase and then assayed in a mixture containing a NADPH/flavin mononucleotide oxidoreductase which in turn activates luciferase. Many interferences were observed and the method was modified accordingly. NADP⁺ and NADPH can be assayed separately or simultaneously within the range 1–100 pmol, which is sufficiently sensitive to be applied to biological materials. Many details and precautions must be taken into consideration.     

480—Redox and adsorption behaviour of some redox coenzymes involved in an electron-transport chain studied by the electro-optical technique

The redox and adsorption behaviour of some redox coenzymes involved in an electron-transport chain, i.e. ubiquinone-10 (CoQ), flavocoenzymes [flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)] and nicotinamide adenine dinucleotide (NAD⁺), has been studied at a gold electrode by cyclic voltammetry and specular reflectivity measurement. All the coenzymes investigated were found to participate in electron transport in adsorbed states on the electrode surface. Adsorbed CoQ and flavocoenzymes are reduced and the resulting products remain adsorbed at the surface. Contrary to them, adsorbed NAD⁺ is reduced and then desorbed immediately. Possible models for the surface orientation of adsorbed molecules were proposed based on the experimental data.Some analogies can be noted between the interfacial behaviour of these coenzymes at the electrode and that in mitochondria.     

Mechanostability of the Single‐Electron‐Transfer Complexes of Anabaena Ferredoxin–NADP+ Reductase

The complexes formed between the flavoenzyme ferredoxin–NADP⁺ reductase (FNR; NADP⁺=nicotinamide adenine dinucleotide phosphate) and its redox protein partners, ferredoxin (Fd) and flavodoxin (Fld), have been analysed by using dynamic force spectroscopy through AFM. A strategy is developed to immobilise proteins on a substrate and AFM tip to optimise the recognition ability. The differences in the recognition efficiency regarding a random attachment procedure, together with nanomechanical results, show two binding models for these systems. The interaction of the reductase with the natural electron donor, Fd, is threefold stronger and its lifetime is longer and more specific than that with the substitute under iron‐deficient conditions, Fld. The higher bond probability and two possible dissociation pathways in Fld binding to FNR are probably due to the nature of this complex, which is closer to a dynamic ensemble model. This is in contrast with the one‐step dissociation kinetics that has been observed and a specific interaction described for the FNR:Fd complex.     

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.     

Electrochemical enzymatic determinations of ethanol and -lactic acid with a carbon paste electrode modified chemically with nicotinamide adenine dinucleotide

The oxidized form of nicotinamide adenine dinucleotide (NAD⁺) is chemically immobilized at the surface of a carbon paste electrode containing n-octaldehyde. The NAD⁺ is converted to NADH by oxidation of ethanol and -lactic acid catalyzed by their respective dehydrogenases, and the NADH formed is oxidized electrochemically to the original NAD⁺, thus giving a well defined linear-sweep voltammetric peak. The peak area is linearly related to the amount of ethanol or -lactic acid in the range 0.05–2 × 10^-9 mol.     

Electrochemical enzymatic determinations of ethanol and l-lactic acid with a carbon paste electrode modified chemically with nicotinamide adenine dinucleotide

The oxidized form of nicotinamide adenine dinucleotide (NAD⁺) is chemically immobilized at the surface of a carbon paste electrode containing n-octaldehyde. The NAD⁺ is converted to NADH by oxidation of ethanol and l-lactic acid catalyzed by their respective dehydrogenases, and the NADH formed is oxidized electrochemically to the original NAD⁺, thus giving a well defined linear-sweep voltammetric peak. The peak area is linearly related to the amount of ethanol or l-lactic acid in the range 0.05–2 × 10^-9 mol.     

Equilibrium (NAD+/NADH) potential on poly(Neutral Red) modified electrode

The electrochemical regeneration of nicotinamide adenine dinucleotide (NAD⁺/NADH) has been one of the central subjects of bioelectrochemistry during past three decades. We report on the unique chemical electrocatalyst for NAD⁺/NADH regeneration based on electropolymerized Neutral Red. Using poly(Neutral Red) modified electrodes, the reversible polarographic waves of nicotinamide adenine dinucleotide reduction–oxidation and the equilibrium (NAD⁺/NADH) potential were observed. This was impossible using all known catalytic and mediator systems. The unique poly(Neutral Red) based electrocatalyst allowed us to determine the standard (NAD⁺/NADH) potential more precisely (E^′≅0.59 V SCE, pH 6.0).     

Amperometric Ethanol Biosensors Based on Chitosan‐NAD+‐Alcohol Dehydrogenase Films

The reagentless and oxygen‐independent biosensors for ethanol were developed based on the covalent immobilization of alcohol dehydrogenase (ADH) and its cofactor nicotinamide adenine dinucleotide (NAD⁺) on chitosan (CHIT) chains. The CHIT‐NAD⁺‐ADH structures were adsorbed onto carbon nanotubes (CNT) in order to provide a signal transduction based on the recycling of redox states of NAD cofactor at CNT (detection limit, 8–30 µM ethanol; dynamic range up to 20 mM). The CHIT‐NAD⁺‐dehydrogenase/CNT hybrid material represents a general approach to the development of dehydrogenases‐based electrochemical biosensors. Interestingly, the CHIT‐NAD⁺ solutions preserved their enzymatic activity even after five years of storage at 4 °C.     

Cathodic reduction of nicotinamide adenine dinucleotide and other adenine-containing compounds in acidic media

Both nicotinamide adenine dinucleotide (NAD⁺) and acid-hydrated NADH, as well as adenine, adenosine, adenosine mono-, di-, and tri-phosphate and adenosine diphosphoribose, undergo four-electron reductions of the protonated adenine ring in acidic media. The values of αn_a (transfer coefficient times the number of electrons involved in the rate-determining step), n (total number of electron transferred), and p (number of protons involved in the rate-determining step) agree well with values previously reported for adenine. Cathodic stripping voltammetry of an adsorbed film can be applied to these compounds. Rapid scan rates are required to eliminate the slow desorption step at ?1.1 V vs. SCE for some of these compounds. Hydration of the nicotinamide ring of NADH appears to inhibit this desorption step, but does not appear to be related directly to the electroactivity of the hydration product.     

Reverse Phase Liquid Chromatographic Analysis of the Impurities in Nicotinamide Adenine Dinucleotides

Abstract

Commercial preparations of several different nicotine adenine dinucleotides were examined by liquid chromatography on an octadecylsilane column (Margolis, S., et al., Clin. Chem., 22, 1322, 1976). Seven impurity peaks were detected in NADP⁺, eight in NADPH, and five in NAD⁺. The estimated purity of NADP⁺ from different commercial suppliers varied from 89 to 95 percent. For NADPH the purity ranged from 77.5 to 96 percent and for NAD⁺ from 90 to 93.5 percent. Preparations of NAD⁺ contained AMP, ADPR, nicotinamide, and two unidentified impurities. The impurities found in NADP⁺ and NADPH preparations did not correspond to compounds that we could identify. Four of the impurity peaks found in NADPH form under acidic storage conditions. Five of the impurity peaks observed in NADP⁺ and three of the impurity peaks in NAD⁺ form as products of alkali-catalyzed rearrangements.     

Mutation of Tyr-218 to Phe in <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> Secondary Alcohol Dehydrogenase: Effects on Bioelectronic Interface Performance

Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2° ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, β-nicotinamide adenine dinucleotide phosphate (NADP⁺), is more expensive and less stable than β-nicotinamide adenine dinucleotide (NAD⁺). PCR-based, site-directed mutagenesis was performed on 2° ADH in an attempt to adjust the cofactor specificity toward NAD⁺ by mutating Tyr²¹⁸ to Phe (Y218F 2° ADH). This mutation increased the K _m(app) for NADP⁺ 200-fold while decreasing the K _m(app) for NAD⁺ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD⁺, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2° ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD⁺. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD⁺ was electrically recycled 6.7 × 10⁴ times, suggesting that the Y218F 2° ADH-modified bioelectronic interface is stable.     

Recycling of NAD+ crosslinked to albumin or hemoglobin immobilized with multienzyme systems in artificial cells

A soluble immobilized NAD⁺ was prepared by creating a peptide binding between bovine serum albumin and N⁶-[(6-aminohexyl)]carbamoyl-methyl] nicotinamide adenine dinucleotide. When immobilized within semipermeable microcapsules with alcohol dehydrodgenase (EC 1.1.1.1) and malic dehydrogenase (EC 1.1.1.37) the albumin-NAD⁺ exhibited high cofactor recycling rates. NAD⁺ can also be similarly crosslinked to hemoglobin.     

An enzymatic-fluorimetric method for monitoring of ethanol in ambient air

A method is described for the continuous monitoring of ethanol in ambient air. The system consists of a scrubber coil for enrichment of the analyte from air in an aqueous solution and a directly connected fluorescence detector. Because of using a reagent solution containing alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD⁺) for absorption, ethanol can react directly with ADH and NAD⁺ during air sampling, producing NADH, which can be measured by fluorescence detection. The influence of reagent concentrations, gas flow rate and scrubber solution flow rate on the performance of the instrument was tested. Possible ozone interferences can be avoided by placing a KI coated filter in front of the scrubber inlet. The response time of the system was found to be 2.3 min and the detection limit about 1 ppb_V. The applicability of the developed method was demonstrated during a field campaign in Brazil.     

Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast†

The accurate quantification of the highly unstable intracellular cofactor nicotinamide adenine dinucleotide phosphate in its oxidized and reduced forms demands a thorough evaluation of the analytical workflow and dedicated methods reflecting their solution chemistry as well as the biological importance of their ratio. In this work, we present a workflow for the analysis of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in the yeast Pichia pastoris, including hot aqueous extraction, chromatographic separation in reversed‐phase conditions employing a 100% wettable stationary phase, and subsequent tandem mass spectrometric analysis. A thorough evaluation and optimization of the sample preparation procedure resulted in excellent biological repeatabilities (on average <10%, N = 3) without employing an internal standardization approach. As a consequence, the methodology proved to be appropriate for the relative assessment of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in different P. pastoris strains. The ratio of reduced versus oxidized nicotinamide adenine dinucleotide phosphate was significantly higher in an engineered strain overexpressing glucose‐6‐phosphate dehydrogenase than in the corresponding wildtype strain. Interestingly, a difference was also observed in the nicotinamide adenine dinucleotide phosphate pool size, which was significantly higher in the wildtype than in the modified strain.     

Towards understanding the origins of the different specificities of binding the reduced (NADPH) and oxidised (NADP+) forms of nicotinamide adenine dinucleotide phosphate coenzyme to dihydrofolate reductase

Lactobacillus casei dihydrofolate reductase (DHFR) binds more than a thousand times tighter to NADPH than to NADP⁺. The origins of the difference in binding affinity to DHFR between NADPH and NADP⁺ are investigated in the present study using experimental NMR data and hybrid density functional, B3LYP, calculations. Certain protein residues (Ala 6, Gln 7, Ile 13 and Gly 14) that are directly involved in hydrogen bonding with the nicotinamide carboxamide group show consistent differences in ¹H and ¹⁵N chemical shift between NADPH and NADP⁺ in a variety of ternary complexes. B3LYP calculations in model systems of protein-coenzyme interactions show differences in the H-bond geometry and differences in charge distribution between the oxidised and reduced forms of the nicotinamide ring. GIAO isotropic nuclear shieldings calculated for nuclei in these systems reproduce the experimentally observed trends in magnitudes and signs of the chemical shifts. The experimentally observed reduction in binding of NADP⁺ compared with NADPH results partly from NADP⁺ having to change its nicotinamide amide group from a cis- to a trans-conformation on binding and partly from the oxidised nicotinamide ring of NADP⁺ being unable to take up its optimal hydrogen bonding geometry in its interactions with protein residues.     

Novel nicotinamide adenine dinucleotide analogues as selective inhibitors of NAD-dependent enzymes

Three novel dinucleotide analogues of nicotinamide adenine dinucleotide (NAD⁺) have been synthesised from d-ribonolactone. These compounds incorporate a thiophene moiety in place of nicotinamide and are hydrolytically stable. They have been evaluated as inhibitors of adenosine diphosphate ribosyl cyclase, glutamate dehydrogenase and Sir2 acyltransferase activities. Enzyme specificity and a high level of inhibition was observed for the dehydrogenase.     

Solar Light Responsive Graphitic Carbon Nitride Coupled Porphyrin Photocatalyst that Uses for Solar Fine Chemical Production

Photocatalysis is a defendable manner for production of several organic chemicals, energy and its storage from solar energy. For the evolution of metal free, cost-effective catalyst a 2D composite has been appear as a photocatalyst. Here, we had reported the synthesis of a light harvesting composite as a photocatalyst which was assembled by a poly-condensation mechanism between graphitic carbon nitride and tetrakis(4-nitrophenyl) porphyrin and the resulting composite manifest the excellent light harvesting properties, suitable energy band and low charge recombination. The photocatalyst [(NO₂)₄TPP@g-C₃N₄] enables the efficient photocatalytic production of nicotinamide adenine dinucleotide (NADH) from consumed NAD⁺ also the production of organic chemicals like 4-methoxybenzylimines from 4-methoxybenzylamines. The photocatalytic efficiency of the photocatalyst was estimated by the percentage of NADH regeneration and the percentage yield of organic transformations. It shows the tetrakis(4-nitrophenyl) porphyrin could enhance the charge transfer capacity of graphitic carbon nitride which shows excellent photocatalysis activities and organic transformations.     

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.     

Synthesis of phosphodiester-type nicotinamide adenine dinucleotide analogs

Fourteen phosphodiester-type β-nicotinamide adenine dinucleotide (NAD⁺) analogs were prepared starting from nicotinamide. The phosphodiester linkage was effectively assembled in 69-93% yields via condensation reaction between 2′,3′-di-O-acetyl nicotinamide mononucleotide and alcohols in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride. The analog β-nicotinamide ribose-5-(2-phenylethyl) phosphate showed beneficial effects on cell growth of model microorganisms.