Nicotinamide isolation

The genus Amyris is known to produce alkaloids including nicotinamides and 2,5-diaryloxazoles. New nicotinamides isolated from this genus include (Z)-30, from A. plumieri [133], and 31, from A. texana [134], The structures of (Z)-30 and 31 were determined using spectroscopic methods [133,134], and the structure of 31 was confirmed by its synthesis from tyramine [134], The previously known ( )-30 was reported from A. sylvatica [135], and was synthesized from 2,4-dimethoxybenzaldehyde [136], The previously known 32 was isolated from A. texana [134]. 

Pyridine carboxamide [98-92-0] (nicotinamide) (1) and 3-pyridine carboxylic acid [59-67-6] (nicotinic acid) (2) have a rich history and their early significance stems not from their importance as a vitamin but rather as products derived from the oxidation of nicotine. In 1867, Huber prepared nicotinic acid from the potassium dichromate oxidation of nicotine. Many years later, Engler prepared nicotinamide. Workers at the turn of the twentieth century isolated nicotinic acid from several natural sources. In 1894, Su2uki isolated nicotinic acid from rice bran, and in 1912 Funk isolated the same substance from yeast (1). 

In 1913, Goldberger demonstrated that pellagra was due to a dietary deficiency. Pellagra had been eadier described by Thiery, who had coined the term mal de la rosa for this disease. Several decades later, Elvehjem and co-workers isolated nicotinamide from a Hver extract and identified it as a peUagra-preventing factor (1). 

In contrast to the nicotinamide nucleotide dehydrogenases, the prosthetic groups FMN and FAD are firmly associated with the proteins, and the flavin groups are usually only separated from the apoen2yme (protein) by acid treatment in water. However, in several covalently bound flavoproteins, the enzyme and flavin coen2ymes are covalently affixed. In these cases, the flavin groups are isolated after the proteolytic digestion of the flavoproteins. 

In the processes that require regeneration of cofactors such as nicotinamide adenine dinucleotide phosphate (NAD(P)H) and adenosine triphosphate (ATP), whole-cell biotransformations are more advantageous than enzymatic systems [12,15]. Whole cells also have a competitive edge over the isolated enzymes in complex conversions involving multiple enzymatic reactions [14]. 

In the Kohn-Sham Hamiltonian, the SVWN exchange-correlation functional was used. Equation 4.12 was applied to calculate the electron density of folate, dihydrofolate, and NADPH (reduced nicotinamide adenine dinucleotide phosphate) bound to the enzyme— dihydrofolate reductase. For each investigated molecule, the electron density was compared with that of the isolated molecule (i.e., with VcKt = 0). A very strong polarizing effect of the enzyme electric field was seen. The largest deformations of the bound molecule s electron density were localized. The calculations for folate and dihydrofolate helped to rationalize the role of some ionizable groups in the catalytic activity of this enzyme. The results are,... 

An isolated DNA molecule comprising DNA which encodes a group III alcohol dehydrogenase and DNA which encodes a BDS-active biocatalyst via nicotinamide adenosine dinucleotide-dependent manner. 

Most asymmetric reductions that can be enzymatically effected have been the reactions of ketones. These reactions can be conducted with whole cells as well as with isolated enzymes. In the latter case, of course, at least one equivalent of a cofactor such as NADH or NADPH (nicotinamide adenine dinucleotide) is required to serve as the actual reductant in the reaction system. 

The asymmetric reduction of prochiral functional groups is an extremely useful transformation in organic synthesis. There is an important difference between isolated enzyme-catalyzed reduction reactions and whole cell-catalyzed transformations in terms of the recycling of the essential nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] cofactor. For isolated enzyme-catalyzed reductions, a cofactor recycling system must be introduced to allow the addition of only a catalytic amount (5% mol) of NAD(P)H. For whole cell-catalyzed reductions, cofactor recycling is automatically achieved by the cell, and the addition of a cofactor to the reaction system is normally not required. 

It is possible to use isolated, partially purified enzymes (dehydrogenases) for the reduction of ketones to optically active secondary alcohols. However, a different set of complications arises. The new C H bond is formed by delivery of the hydrogen atom from an enzyme cofactor, nicotinamide adenine dinucleotide (phosphate) NAD(P) in its reduced form. The cofactor is too expensive to be used in a stoichiometric quantity and must be recycled in situ. Recycling methods are relatively simple, using a sacrificial alcohol, or a second enzyme (formate dehydrogenase is popular) but the real and apparent complexity of the ensuing process (Scheme 8)[331 provides too much of a disincentive to investigation by non-experts. 

This factor is particularly significant in OFBD since biological samples or isolates are used. In addition to background interference, fluorescence quenching has been demonstrated in a variety of biomolecules such as thiamine (vitamin Bi),(27) nicotinamide/28 nucleosides/nucleotides,(29) and pyruvate/30 To circumvent the obvious limitations associated with the use of UV or visible fluorophores in OFD, the potential... 

Oxidation of nicotine with chromic acid led to the isolation of pyridine-3-carboxylic acid, which was given the trivial name nicotinic acid. We now find that nicotinic acid derivatives, especially nicotinamide, are biochemically important. Nicotinic acid (niacin) is termed vitamin B3, though nicotinamide is also included under the umbrella term vitamin B3 and is the preferred material for dietary supplements. It is common practice to enrich many foodstuffs, including bread, flour, corn, and rice products. Deficiency in nicotinamide leads to pellagra, which manifests itself in diarrhoea, dermatitis, and dementia. 

Analogous to the KRED reductions they can be performed as whole-cell biotransformations [48, 49] (baker s yeast, for example, contains a number of EREDs) or with isolated enzymes [50-52]. In the latter case the nicotinamide cofactor can... 

Boron also appears to be involved in redox metabolism in cell membranes. Boron deficiency was shown to inhibit membrane H -ATPase isolated from plant roots, and H -ATPase-associated proton secretion is decreased in boron-deficient cell cultures [71]. Other studies show an effect of boron on membrane electron transport reactions and the stimulation of plasma reduced nicotinamide adenine dinucleotide (NADH) oxidase upon addition of boron to cell cultures [72, 73]. NADH oxidase in plasma membrane is believed to play a role in the reduction of ascorbate free radical to ascorbate [74]. One theory proposes that, by stimulating NADH oxidase to keep ascorbate reduced at the cell wall-membrane interface, the presence of boron is important in... 

The derivative (9) of 3,6-dideoxy-a-D-xyIo-hexopyranose (abequose) was isolated from a strain of Salmonella typhimurium,16 that (10) of 3,6-dideoxy-a-D-nfco-hexopyranose (paratose) from Salmonella paratyphi,54 and a mixture of 10 and the ester (11) of 3,6-dideoxy-a-D-arabino-hexopyranose (tyvelose) from Salmonella enteritidis.,6 It was shown that these derivatives are formed from cytidine 5 -(a-D-glu-copyranosyl pyrophosphate) by treatment with nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of cell extracts of the respective bacterial strain. For example, formation of 9 is characteristic of preparations from Salmonella, group B,55,56 or Pasteurella pseudotuberculosis, type II.56 The derivative 10 was obtained with extracts of Salmonella, group A,56 and Pasteurella pseudotuberculosis, type I and III,56 and a mixture of 10 and 11 with those of Salmonella, group D,55-60 or Pasteurella pseudotuberculosis, type IV 56.59,60 Under similar conditions, the ester (12) of cytidine 5 -pyro-... 

Kroncke, K.-D., Funda, J., Berschick, B., Kolb, H., and Kolb-Bachofen, V. (1991a). Macrophage cytocoxicity towards isolated rat islet cells Neither lysis nor its protection by nicotinamide are beta-cell specific. Diabetologia 34, 232-238. 

Why do we need vitamins Early clues came in 1935 when nicotinamide was found in NAD+ by H. von Euler and associates and in NADP+ by Warburg and Christian. Two years later, K. Lohman and P. Schuster isolated pure cocarboxylase, a dialyz-able material required for decarboxylation of pyruvate by an enzyme from yeast. It was shown to be thiamin diphosphate (Fig. 15-3). Most of the water-soluble vitamins are converted into coenzymes or are covalently bound into active sites of enzymes. Some lipid-soluble vitamins have similar functions but others, such as vitamin D and some metabolites of vitamin A, act more like hormones, binding to receptors that control gene expression or other aspects of metabolism. 

Pure NADP+ was isolated from red blood cells in 1934 by Otto Warburg and W. Christian, who had been studying the oxidation of glucose 6-phosphate by erythrocytes.13 They demonstrated a requirement for a dialyzable coenzyme which they characterized and named triphosphopyridine nucleotide (TPN+, but now officially NADP+ Fig. 15-1). Thus, even before its recognition as an important vitamin in human nutrition, nicotinamide was identified as a component of NADP+. 

Nicotinic acid was prepared in 1867 by oxidation of nicotine. Although it was later isolated by Funk and independently by Suzuki in 1911-1912 from yeast and rice polishings, it was not recognized as a vitamin. Its biological significance was established in 1935 when nicotinamide was identified as a component of NAD+ by von Euler and associates and of NADP+ by Warburg and Christian.3 Both forms of the vitamin are stable, colorless compounds highly soluble in water. 

The stability of enzyme-NADH was evidenced by its isolation after Sephadex chromatography and led to the direct spectrophotometric demonstration of enzyme-NADH. The formation of enzyme-NADH in this reaction was used to examine the stereospecificity of hydrogen transfer for acceptance from carbon 4 of the sugar nucleotide by enzyme-NAD+ and donation of the same hydrogen back to carbon 6. For both steps / -stereospecificity of hydrogen transfer to the nicotinamide moiety of the pyridine nucleotide was established. 

In the case of enzymes involved in biochemical pathways, the isolation is often based on activity assays. The nature of the activity assay depends on the enzymatic reaction and can involve, for example, the detection of a product on a thin-layer chromatography (TLC) plate (see Chapter 4, Section 1.2.1), the appearance or disappearance of a specific absorbance in a spectrophotometric assay, or a coupled assay involving the oxidation or reduction of a co-factor such as nicotinamide dinucleotide (NAD(H)), which can be measured by changes in fluorescence. 

Cognate preparation. 3-Aminopyridine. Prepare a cold sodium hypobromite solution from 32 g (10 ml, 0.2 mol) of bromine and 25 g (0.62 mol) of sodium hydroxide in 250 ml of water. Add in one portion 20 g (0.163 mol) of finely powdered nicotinamide (Expt 6.169) and stir vigorously for 15 minutes. Warm the solution in a water bath at 75 °C for 45 minutes. Isolate the crude product by continuous ether extraction (Section 2.22) of the cooled reaction mixture after saturation with sodium chloride. Dry the extract over potassium hydroxide pellets and remove the ether. Crystallise the dark residue from a 4 1 mixture of benzene-light petroleum (b.p. 60-80 °C) with the aid of decolourising charcoal. The yield of almost colourless product, m.p. 63 °C, is 9.3 g (61%). 

A purified D-glucose dehydrogenase, isolated from animal liver, catalyzes the oxidation of D-glucose to D-gluconic acid.202 A coenzyme (either nicotinamide adenine dinucleotide or its 2 -phosphate) is necessary for the oxidation. The reaction is reversible, and is specific for /J-D-glucopyranose it does not proceed with the a-D anomer, and it occurs only slowly with D-xylose. 

Isolated oxidoreductases always depend on cofactors for the transfer of electrons. Enzyme groups which are well characterized with respect to their biochemistry are those requiring the nicotinamide coenzymes NAD or NADP, the flavins FAD or FMN and the ortho-quinoids such as pyrroloquinoline quinone (PQQ) or trihydroxy-phenylalanine (TOPA). 

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