Regeneration of NADP

A ruthenium complex [RuCl2(TPPTS)2]2 was used for regeneration of NADP+ to NADPH withhydrogen. Thus, 2-heptanonewas reduced with alcohol dehydrogenase from Thermoanaerobacter brockii in the presence of the mthenium complex, NAD P, and hydrogen at 60°C to (S)-2-heptanol in 40 % ee. Turnover number was reported to be 18 (Figure 8.6) [5cj. 

The same system was applied to the HLADH catalyzed oxidation of a number of other meso-d o s, with similar results. Using the alcohol dehydrogenase from thermoanaer-obiuni brockii, it was possible to oxidize 2,4-pentanediol to give (5)-4-hydroxy-2-penta-none under indirect electrochemical regeneration of NADP". ... 

Intrasequential regeneration of NADP with TBADH and a Baeyer-Villiger monooxygenase (BVO) from Acinetobacter calcoaceti-... 

Figure 21. Photosensitized regeneration of NADP cofactors using 23 and 24 as photosensitizers.

Cytc mediation process is much more specific and is not affected by any inter-ferant that is not recognized by Cyt c. The specificity of this mediation process can be further utilized to provide regeneration of NADP+, which can be connected... 

The VAPOR enzymes are flavoenzymes and can be isolated from thermophilic bacilli. They are especially valuable because they allow the regeneration of all four forms of the pyridine nucleotides NADH, NAD+ NAD PH, NADP+ according to the following equations [55,61] ... 

An NADP(H)-dependent ADH of Lactobacillus brevis (LBADH) was identified as a suitable catalyst accepting a broad range of diketo esters A as substrate [8]. This stable enzyme is easily available in the form of a crude cell extract (recLBADH) from a recombinant E. coli strain [9]. The reaction with diketo esters la-lc was performed on a preparative scale, using substrate-coupled regeneration of NADPH (Scheme 2.2.7.2). 

The electron transfer between NADH and the anode may be accelerated by the use of a mediator. Synthetic applications have been described for the oxidation of primary and secondary alcohols to aldehydes and ketones catalyzed by yeast alcohol dehydrogenase (YADH) and the alcohol dehydrogenase from Thermoanaerobium brockii (TBADH) with indirect electrochemical regeneration of NAD+ and NADP+, respectively, using the tris(3,4,7,8-tetramethyl-l,10-phenanthroline) iron(II/III) complex as redox catalyst [59],... 

In fact, the a-ketoglutarate/glutamate dehydrogenase is a generally applicable method for the regeneration of NAD and NADP in laboratory scale productions. Both components involved are inexpensive and stable. Quite recently, a method for the oxidation of the reduced nicotinamide coenzymes based on bacterial NAD(P)H oxidase has been described [225], This enzyme oxidizes NADH as well as NADPH with low Km values. The product of this reaction is peroxide, which tends to deactivate enzymes, but it can be destroyed simultaneously by addition of catalase. The irreversible peroxide/catalase reaction favours the ADH catalyzed oxidation reaction, and complete conversions of this reaction type are described. 

The NADP-dependent TBADH was used for the laboratory-scale preparation of several chiral aliphatic and cyclic hydroxy compounds by reduction of the corresponding ketones. For the regeneration of NADPH, this reduction reaction can be coupled with the TBADH catalyzed oxidation of isopropanol. For the reduction of some ketones it was observed that the reaction rate was increased in the presence of the regenerating substrate isopropanol, for instance in the presence of 0.2 v/v isopropanol, the reduction rate of butanone or pentanone was increased 3-4-fold [57], In some cases, the enantiomeric excess of the reduction reaction is not very high, especially when small molecules are converted, but also for compounds such as acetophenone [138]. 

R)-alcohols in high enantiomeric excess can be obtained with the aid of the NADP-dependent ADH from Lactobacillus kefir. Due to the broad substrate specificity of this enzyme, aromatic, cyclic, polycyclic as well as aliphatic ketones can be reduced. A simple method for the regeneration of NADPH is given by the simultaneously coupled oxidation of isopropanol by the same enzyme. Several chiral alcohols (Table 8) were synthesized at a 2.5 mmol scale within a reaction time of 12-36 h [160]. 

The processes of electroreduction, and subsequent photochemical regeneration of the parent monomers, consequently form a closed cycle involving the transport of electrons and protons, as occurs in the reduction and oxidation of other systems, e.g. coenzymes such as NAD+ and NADP+ 13,U). 

ADHs catalyze the oxidation of alcohols to ketones with simultaneous reduction of NAD(P)+. Due to the reversibility of this reaction, ADH-catalyzed reactions can either be used for the synthesis of (chiral) compounds or for the regeneration of the coenzyme. The latter holds true, for example, in the case of substrate-coupled ADH-catalyzed reduction reactions using isopropanol or ethanol as the hydrogen donor. Several kinds of ADHs have already been described. ADHs of the EC 1.1.1.1 group are dependent on NAD+. They act on primary or secondary alcohols or on hemiacetals. In contrast, ADHs of the group EC 1.1.1.2 depend on NADP+. Some enzymes of this group oxidize only primary alcohols others act on secondary alcohols as well. 

Clostridium thermoaceticum contains the so-called AMAPOR (artificial-media-tor-accepting pyridine-nucleotide oxidoreductases), which are useful for electro-microbial regeneration of all four forms of pyridine nucleotides, too. An NADP(H) dependent AMAPOR from C. thermoaceticum has been purified and characterized [104]. It is able to react with rather different artificial mediators such as viologens or quinones, for example 1,4-benzoquinone, anthraquinone-2,6-disulfonate, or 2,6-dichloro-indophenol. 

Fig. 19 Regeneration of all four species of the pyridine nucleotides NADH, NAD+, NADPH, NADP+ by the VAPOR enzymes. f/++ viologen...

Fig. 22 Electroreduction of ketones or aldehydes using ADH as catalyst. Reduction system A shows the ADH-catalyzed reduction coupled with regeneration of NADPH or NADH by ferre-doxin-NADP+ reductase (FNR) or diaphorase (DP), respectively with assistance of methyl violo-gen as an electron mediator. In system B, ADH is used as sole enzyme which catalyzes both reduction of substrates and regeneration of cofactors...

For the asymmetric reduction of ketone and aldehyde derivates, two electrochemical reduction systems using ADH as catalyst were examined (Fig. 22) [108]. In system A, the reduced coenzymes are regenerated using either FNR for NADPH or DP for NADH. Methyl viologen serves as electron mediator between the electrode and FNR/DP. System B contains ADH as sole enzyme, which catalyzes both reduction of substrates and regeneration of cofactors. Phenylethanol is oxidized by ADH accompanied by reduction of NADP+ to NADPH and its oxidation product acetophenone is reduced electrochemically at a glassy carbon cathode. 

Recombinant E. coli cells coexpressing GDH from B. megaterium for regeneration of NADPH were applied in industrial scale to produce (R)-CHBE or (S)-CHBE in high optical purity in 300-350 g L-1 scale [163, 166,167]. Besides COBE, only glucose and a catalytic amount of NADP+ were fed as the substrates, the turnover numbers for NADPH were calculated to be 13,500 and 35,000 mol CHBE/mol, respectively [168]. 

In summary, current evidence [39-41] is thus consistent with the view that the ferredoxin/thioredoxin system functions in photosynthetically diverse types of plants as a master switch to restrict the activity of degradatory enzymes and activate biosynthetic enzymes in the light. It is significant that enzymes controlled by the ferredoxin/thioredoxin system (FBPase, SBPase, NADP-G3PDH, and PRK) function in the regenerative phase of the reductive pentose phosphate cycle that is needed to sustain its continued operation - i.e, to regenerate the carbon dioxide acceptor, Rbu-1,5-P2, from newly formed 3-PGA. It seems likely that one of these thioredoxin-linked enzymes limits the regeneration of Rbu-1,5-P2. 

Because the direct electrochemical oxidation of NAD(P)H has to take place at an anode potential of +900 mV vs. NHE or more, only rather oxidation-stable substrates can be transformed without loss of selectivity, thus limiting the applicability of this method. The electron transfer between NADH and the anode may be accelerated by the use of a mediator. At the same time, electrode fouling, which is often observed in the anodic oxidation of NADH, can be prevented. Synthetic applications have been described for the oxidation of 2-hexene-1 -ol and 2-butanol to 2-hexenal and 2-butanone catalyzed by yeast alcohol dehydrogenase (YADH) and the alcohol dehydrogenase from Thermoanaerobium brockii (TBADH), respectively, with indirect electrochemical regeneration of NAD" and NADP", respectively, using the tris(3,4,7,8-tetramethyl-l,10-phenan-throline) iron(II/III) complex as redox catalyst at an anode potential of 850 mV vs. NHE [106]. Under batch electrolysis conditions using a carbon felt anode, the turnover number per hour was 40. The current efficiency reached between 90 and 95%. 

A. In the RBC a deficiency of pyruvate kinase wonld tend to shunt glucose toward the hexose monophosphate pathway increasing ribu-lose 5-P levels, and the ratio of NADP+ to NADPH wonld decrease. NADH to NAD+ ratios wonld increase as a resnlt of lower pyruvate levels making more NADH available to rednce methemoglobin and regenerate NAD+. Becanse pyravate kinase is deficient, the last ATP formation site is compromised, and so is the formation of ATP in the RBC elevating the ADP to ATP ratio. 

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