HLADH

Figure 8.27 Reduction of aldehyde in SCCO2 by an isolated enzyme, horse liver alcohol dehydrogenase (HLADH) [20c] (a) Reaction scheme (b) fluorinated coenzyme soluble in CO2 and (c) effect of coenzyme on the reaction.

Organometallic aldehydes can be reduced enantioselectively with dehydrogenases. For example, optically active organometallic compounds having planar chiralities were obtained by biocatalytic reduction of racemic aldehydes with yeast [22c,d] or HLADH [22e] as shown in Figure 8.29. 

Rhin(bpy)3]3+ and its derivatives are able to reduce selectively NAD+ to 1,4-NADH in aqueous buffer.48-50 It is likely that a rhodium-hydride intermediate, e.g., [Rhni(bpy)2(H20)(H)]2+, acts as a hydride transfer agent in this catalytic process. This system has been coupled internally to the enzymatic reduction of carbonyl compounds using an alcohol dehydrogenase (HLADH) as an NADH-dependent enzyme (Scheme 4). The [Rhin(bpy)3]3+ derivative containing 2,2 -bipyridine-5-sulfonic acid as ligand gave the best results in terms of turnover number (46 turnovers for the metal catalyst, 101 for the cofactor), but was handicapped by slow reaction kinetics, with a maximum of five turnovers per day.50... 

Other mediators which have been used in combination with diaphorase for the regeneration of NAD+ are riboflavin and Vitamin K3, which is 2,3-dimethyl-1,4-naphthoquinone. However, especially riboflavin is not stable enough for synthetic applications [40]. Better stability is exhibited by phenanthrolindiones as mediators. In combination with diaphorase, Ohshiro [41] showed the indirect electrochemical oxidation of cyclohexanol to cyclohexanone using the NAD+ dependent HLADH with a turnover frequency of two per hour. For an effective enzymatic synthesis, this turnover frequency, however, would be too small. In our own studies, we were able to accelerate the NAD(P)+ regeneration considerably by lowering the electron density within the... 

Fig. 7. Horse-liver alcohol dehydrogenase (HLADH) catalyzed alcohol oxidation at a graphite felt anode modified by poly(acrylic acid) (PAA) under coimmobilization of ferrocene derivatives (Fc), diaphorase (Dp), and HLADH [39]...

We then coupled the regeneration system 1 to the horse liver alcohol dehydrogenase (HLADH) catalyzed oxidation of cyclohexanol to cyclohexanone as a model system (Fig. 9). 

Fig. 9. Schematic representation for the HLADH-catalysed enzymatic oxidation of cydohexanoi with indirect electrochemical NAD+ regeneration...

In a very special system, the photoelectrochemical regeneration of NAD(P)+ has been performed and applied to the oxidation of the model system cyclohexanol using the enzymes HLADH and TBADH. In this case, tris(2,2 -bipyridyl)ruthenium(II) is photochemically excited by visible light [43]. The excited Ru(II) complex acts as electron donor for AT,AT -dimethyl-4,4 -bipyridinium sulfate (MV2+) forming tris(2,2 -bipyridyl)ruthenium(III) and the MV-cation radical. The Ru(III) complex oxidizes NAD(P)H effectively thus... 

Fig. 10. HLADH-catalyzed conversion of cyclohexanol to cyclohexanone by anaerobic electrochemical regeneration of NAD4 mediated by complex 1 0.1 M phophate buffer of pH 8.0 containing 1 (lxl0 4M), NADH (5xl0 4M), cyclohexanol (lxlO 2M) and HLADH [25 U (EtOH)] at room temperature in an argon atmosphere. The working potential was 100 mV vs Ag/AgCl with a Sigraflex carbon foil anode of 23 cm2 surface area and a cell volume of 20 ml...

Recently, we adopted the same system for the reduction of 4-phenyl-2-butanone to (S)-4-phenyl-2-butanol using the NADH-dependent horse liver alcohol dehydrogenase (HLADH) and S-ADH from Rhodococcus sp [68] with high enantioselectivity (Fig. 17) [69]. As mediator, we applied the low-molecular... 

Fig. 17. Electroenzymatic reduction of 4-phenyI-2-butanone catalyzed by HLADH with in-situ indirect electrochemical regeneration of NADH using a Cp (2,2 -bipyridyl)aquo rhodium(III) complex as mediator...

With a good route to the key meso diol 128 in hand, the authors turned their attention to desymmetrization, using the known asymmetric hydrolysis of meso diacetates by Lipase AK (Scheme 23). The meso diol 128 was first converted to diacetate 140, and then hydrolyzed with Lipase AK to cleave selectively one of the two acetates, producing chiral hydroxyester 141. Oxidation, cleavage of the acetate, and lactonization yielded the (3S,4.R) lactone 129. The corresponding lactol (3S,4 )-130 was found to be the enantiomer of the compound produced in the HLADH synthesis. 

However, a pure enzyme, like horse liver alcohol dehydrogenase (HLADH), shows not only high stereoselectivity but regioselectivity as well, affording, for example, 89% yield of monoalcohol 5 from dione 4 with ee higher than 99% [14]. 

The latter were transformed into (+)-64, (-)-65 and (+)-66, respectively, in good yields. Lactone (+)-66 was also prepared in 37 % yield by stereospecific Horse Liver Alcohol Dehydrogenase (HLADH) catalyzed oxidation of the meso-diol 67. Lactones (-)-64 and (+)-64 are potentially interesting starting materials for the... 

Figure 9. Preparation of L- and D-lactaldehyde and L- and D-2-hydroxybutyraldehyde. HLADH, horse liver alcohol dehydrogenase (NADH was regenerated by glucose/glucose dehydrogenase).

HLADH oxidation of 3-methylpentane-l,3,5-triol yields, after silver oxide oxidation, (35)-(-l-)-mevalonolactone of 14% optical purity/" The synthesis of [4,5- C2]MVA using known procedures was omitted from last year s Report, " " and another synthesis of ( )-mevalonolactone has been reported. " ... 

Larsson KM, Adlercreutz P, Mattiasson B (1987) Study of horse Uver alcoholdehydroge-nase (HLADH) In AOT-cyclohexane reverse micelles. In Laane C, Tramper J, LUly MD (eds.) Biocatalysis In organic media. Elsevier, Amsterdam, p 355... 

Horse liver alcohol dehydrogenase (HLADH (E.C. 1.1.1.1), commercially available) is a well-documented enzyme capable of catalyzing the enantioselective oxidation of acyclic and cyclic meso-configurated dimethanol derivatives to chiral lactols and further to the corresponding chiral lactones with high enantioselectivity and in high yield (Table 11) 162 ,69. Incases where the two enantiomeric lactols are formed, a kinetic enantiomer separation can occur in the second oxidation step166. 

Table 11. Lactones by HLADH-Catalyzed Oxidation of mrao-Diols...

Thus, millimolar quantities of substrate 27a were converted using as little as 0.005 mol% cofactor and small amounts of enzyme (100 U recLBADH per g substrate 200 U HLADH per g substrate, respectively). The analytically pure products (S)- and (R)-28a were obtained easily by extraction in an isolated yield of >95%. HPLC analysis revealed >99% conversion and, additionally, only a single enantiomer could be detected (ee > 99%). 

Table 2.2.7.1 Relative enzymatic activities of HLADH, TBADH and recLBADH.

In the case of the (1 )-configured olefinic ketones high conversion rates and enantiomeric excesses were achieved on the analytical and on the semi-preparative scale. With TBADH, which requires a slightly higher reaction temperature, decomposition of the halogenated substrates was observed. In the case of CPCR, which was used as a partially purified enzyme preparation from the parental strain, the formation of up to 50% of the fully saturated alcohols was observed (Scheme 2.2.7.18). Substrate (l )-33 could be converted into enantiopure (S)- and (R)- E)-32, respectively, by recLBADH- and HLADH/CPCR-catalyzed reaction. 

Table 2.2.7.2 Coupled enzyme-substrate screening for olefinic ketones 32 and 33 with relative activities of recLBADH, HLADH, TBADH, and CPCR.

---