HLADH-catalyzed reduction

Using isolated enzymes instead of whole cells, similar problems are to be considered only in a few cases. ADH from Thermoanaerobium brockii shows varying enantiomeric excess of the product depending on the structure of the ketone to be reduced. Conversions with this enzyme yield in products with low (20% for the reduction of acetophenone) or high ee value (100% for the reduction of p-Cl-acetophenone). Predictions about the stereospecificity of HLADH catalyzed reductions can be made for simple acyclic substrates applying Prelog s rule [37] and for more complex compounds using the cubic-space model developed by Jones and Jakovac [38],... 

HLADH converts a wide range of substrates. For the predicition of the stereoselectivity of reduction reactions, originally Prelog s diamond lattice model was applied, which is based upon the characteristic properties of the ADH of Curvularia falcata [37]. This model describes the stereospecificity of HLADH catalyzed reductions of simple acyclic substrates such as aldehydes. Later on, for more complex acyclic and cyclic substrates, a cubic-space model of the active site was developed [38,121]. Other models are based upon symmetric properties [122-125] or upon a refined diamond lattice model [126-129]. 

The HLADH-catalyzed reduction of ( )-(97) is of interest since the alcohol product (98) is the thermodynamically less-preferred exo epimer. This emphasizes the fact that the geometry of an initial, kineti-cally controlled, product of an enzyme-catalyzed reduction reflects only the direction of attack of the hydride equivalent that is imposed by the orientation of substrate binding in the ES-complex. In this case, the reduction occurs on the re face of (15 )-(97), in accord with the predictions of the active site... 

In order to predict the stereochemical outcome of HLADH-catalyzed reductions, a number of models have been developed, each of which having its own merits. The first rationale emerged from the diamond lattice model of V. Prelog, which was originally developed for Curvularia falcata [798]. A more recently developed... 

The method of Jones uses sodium dithionite to regenerate NADH from catalytic NAD. It has been shown to be preparatively viable for HLADH-catalyzed reductions of a broad range of aldehydes and ketones in high yields. 

By fitting a cyclohexanone in the diamond lattice, Prelog has developed a step-by-step analysis of the HLADH catalyzed reduction. The positions marked (A to D) are forbidden oxidoreduction will not take place if binding of a potential substrate places a group in one of these locations. Positions marked (E to G) are undesirable, although their occupation by part of a substrate does not necessarily preclude the oxidoreduction. The rate of reaction will be very slow. The positions under the lattice (U) are also in this category. The location O (I) is a newly identified unsatisfactory position. Placement of a group here is to be avoided if possible, but slow reaction will still take place if it is occupied. 

Figure 7.3. Diamond lattice section analysis of the stereochemical course of HLADH-catalyzed reduction of the 2-norbornanone enantiomers. The orientations shown are considered to be those of the alcohol-like transition states which would be involved, with H being delivered from the e-re direction of Fig. 7.2. There are no unfavorable lattice interactions when ( + )- or (—)-isomers are positioned as shown in (a) and (b), respectively. Reductions to the endo-alcohols (+)- and ( —)- are thus permitted processes. The predominant formation of the ( + )-enantiomer is thought to be due to the preference of C-4 for its unhindered location in (a) over its position in (b) in which it approaches the forbidden A,I,C region of the lattice. The discrimination of HLADH against exo-alcohol formation is accounted for by the unfavorable interactions of C-6 with lattice positions J and I, as represented in (c) and (d), respectively (282). Reproduced with permission. Copyright 1978 by the American Chemical Society.

Reductions of symmetrical bicyclic diketones may also be effected selectively, as illustrated in Scheme 12. The stereospecificities of the HLADH-catalyzed transformations of the unsaturated decalin-diones (25) and (27) to the corresponding hydroxy ketones (26) and (28), and in fact all specificity aspects of this enzyme, are fully predictable using a simple to use, cubic-space model of the enzyme s... 

In addition to stereoselective metalation, other methods have been applied for the synthesis of enantiomerically pure planar chiral compounds. Many racemic planar chiral amines and acids can be resolved by both classical and chromatographic techniques (see Sect. 4.3.1.1 for references on resolution procedures). Some enzymes have the remarkable ability to differentiate planar chiral compounds. For example, horse liver alcohol dehydrogenase (HLADH) catalyzes the oxidation of achiral ferrocene-1,2-dimethanol by NAD to (S)-2-hydroxymethyl-ferrocenealdehyde with 86% ee (Fig. 4-2la) and the reduction of ferrocene-1,2-dialdehyde by NADH to (I )-2-hydroxymethyl-ferrocenealdehyde with 94% ee (Fig. 4-2lb) [14]. Fermenting baker s yeast also reduces ferrocene-1,2-dialdehyde to (I )-2-hydroxymethyl-ferro-cenealdehyde [17]. HLADH has been used for a kinetic resolution of 2-methyl-ferrocenemethanol, giving 64% ee in the product, (S)-2-methyl-ferrocenealdehyde... 

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...

Fig. 32 Reduction of cyclohexanone catalyzed by HLADH with simultaneous hydrogenase-driven regeneration of NADH in an organic-aqueous two-phase system...

Enantiomeric and diastereotopic face specific reductions are also readily effected on racemic bicyclic ketones. An illustration of the broad structural range that is amenable to enzyme-catalyzed transformation in this way is given in Scheme 38. While 2-decalones, such as ( )-(81)- 83), and the related heterocyclic analogs ( )-(85) are good substrates for HLADH, the 1-decalone ( )-(84) is not. However, by changing enzymes to MJADH, ( )-(84) becomes a good substrate.Similarly, TBADH is a highly satisfactory catalyst for stereospecific reduction of ( )-(86), but will not accept its dimethyl... 

HLADH is nicotinamide coenzyme (NADVNADH) dependent and catalyzes the redox equilibrium between a large number of alcohols and ketones or aldehydes (Figure 2). The equilibrium is overwhelmingly in favour of the reduction reaction. Phosphate analogs of the coenzymes, NADP+ and NADP may also serve as coenzymes in very limited situations. The oxidation-reduction takes place through a ternary complex in which the substrate and coenzyme are simultaneously bound in the active site of enzyme. 

Figure 2. Schematic view of the oxido-reduction catalyzed by HLADH.

Panza et al. synthesized a C02-philic amphiphile from the coenzyme nicatinamide adenine dinucleotide (MW 664) and a covalently attached perfluoropolyether (MW 2500) (Figure 7B) (73). The fluorofunctional coenzyme (FNAD) was soluble up to 5 mM in CO2 at room temperature and 1400 psi. The C02-soluble FNAD was able to participate in a cyclic oxidation/reduction reaction catalyzed by the enzyme horse liver alcohol dehydrogenase (HLADH) in CO2 at room temperature and 2600 psi. 

The opposite enantiomer selectivity towards these cage-shaped C2 ketones was demonstrated in oxidation-reduction mediated by horse liver alcohol dehydrogenase (HLADH) (170). Incubation of racemic C2 ketones with HLADH in a phosphate buffer (pH 7.0) containing coenzyme NADH afforded a mixture of the alcohols corresponding to the M C2 ketone and the recovered P C2 ketones, both with much higher optical purities than that found in the microbial reduction. In the oxidative direction (with NAD coenzyme), HLADH was found to preferentially catalyze oxidation of the alcohols corresponding to the M C2 ketone with excellent selectivity. 

Redox reactions catalyzed by alcohol dehydrogenases (e.g., from horse liver, HLADH) may be performed in organic solvents in both the reduction and oxidation mode, if the recycling system is appropriately modified (Sect. 2.2.1). Reduction of aldehydes/ketones and oxidation of alcohols is effected by NADH- or NAD" -recycling, using ethanol or wobutyraldehyde respectively. 

Thus, it has been proved that an ADH catalyzes the redox reaction between acetaldehyde and ethanol stereospecifically and that both YADH and HLADH exhibit the same specificity toward the coenzyme and the substrate. These two enzymes can also catalyze the reduction of other aldehydes. It has been ascertained that these reactions... 

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