Cofactor organic solvents

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

Metal ion-catalyzed hydrolytic processes have been studied for a long time, and many interesting systems have been explored which give valuable information about catalysis. However, with very few exceptions the catalysis afforded by these systems in water is disappointing when compared with enzymatic systems where a metal ion cofactor activates a substrate and a nucleophilic or basic group in an acyl or phos-phoryl transfer process. It has been noted that bulk water may not be a good medium to approximate the medium inside the active site of an enzyme where it is now known that the effective dielectric constants resemble those of organic solvents rather than water. 

Most kinetic resolutions of pharmaceutical intermediates that have been reported involve the use of hydrolases, particularly lipases and proteases. This is because many hydrolases are commercially available (in bulk and kit form), do not require cofactors and are active in many organic solvents (see Section 1.4). Processes can therefore, often be developed rapidly, using high substrate concentrations and without specialist knowledge. 

The use of water-miscible organic solvent-water mixtures is a particularly attractive method for use with cofactor-dependent enzymes due to its simphcity. The high water content can allow dissolution of both enzyme and cofactor, whilst the water-miscible solvent can provide a dual role in both substrate dissolution and as a cosubstrate for cofactor recycling (substrate-coupled cofactor recycling).The asymmetric reduction of a ketone intermediate of montelukast using an engineered ADH in the presence of 50 % v/v isopropanol offers a powerful demonstration of this methodology (Scheme 1.55). 

In addition to the retention of structural integrity in neat organic solvents, Klibanov and co-workers demonstrated that a diverse range of enzymes, from hydrolases and peroxidases to cofactor-dependent alcohol oxidases and ADHs, also retain activity. This pioneering work single-handedly led to the popularization of biocatalysis in neat organic solvent. 

Fig. 6. Outline of the stereospecific reduction of CAAE by aldehyde reductase (AR) with glucose dehydrogenase (GDH) as the cofactor regenerator in an organic solvent-water two-phase system...

The FeMo-co or M center of the FeMo protein has been identified spectroscoplcally(, 13,30) within the protein and has been extracted from the protein into N-methyl formamlde(31) and other organic solvents(32.33). Its biochemical authenticity can be assayed by its ability to activate FeMo protein from a mutant organism that produces protein that lacks the M center(31). The extracted cofactor resembles the M-center unit spectroscopically and structurally as shown in Table I. It seems reasonable to presume that the differences are due to variation in the ligation of the center between the protein and the organic solvent(34). 

Biocatalysts based on hydrolases (E.C. class 3, Table 5.2) ate mostly used as (purified) enzymes since they are cofactor independent, since these preparations are commercially available and because a number of hydrolases can be applied in organic solvents. Oxidoreductases (E.C. class 1) however, are relatively complex enzymes, which require cofactors and frequently consist of more than one protein component. Thus, despite the fact that efficient cofactor regeneration systems for NADH based on formate dehydrogenase (FDH) have been developed (Bradshaw et al, 1992 Chenault Whitesides, 1987 Wandrey Bossow, 1986, chapter 10) and that also an NADPH dependent FDH has been isolated (Klyushnichenko, Tishkov Kula, 1997), these enzymes are still mostly used as whole-cell biocatalysts. 

The chemical and spectroscopic properties of the cofactor F-430 have been reviewed [98,99], The structure of the macrocycle (Figure 4) was elucidated by x-ray crystallography and NMR spectroscopy [100], The free cofactor, which is present in substantial amounts in the cells, has an absorption maximum at 430 nm, hence its name. In the enzyme, the absorption maximum is blue shifted to 420 nm. The pentamethyl ester of F-430 is soluble in organic solvents and can be reduced to the Ni(I) state under aprotic conditions, resulting in an absorption peak shift to 382 nm [101], or can be oxidized to the Ni(III) state, giving an absorption peak at 368 nm [102],... 

Single-phase systems may also be based on the use of nearly anhydrous organic solvents. In this specific case, the incompatibility of enzymes and organic reaction media can be overcome in a number of ways, e.g., by enzyme immobilization or by modifying the enzyme to render it soluble in organic solvents [71, 72], However, the applicability of this system is limited, since many important biotransformations involve both hydrophobic and hydrophilic substrates, products and/or cofactors that are insoluble in the same reaction medium. 

A general method has been developed for utilization of cofactor-requiring enzymes in organic media [139]. ADH from horse liver as well as NADH were attached onto the surface of glass beads and afterwards suspended in a water-immiscible organic solvent containing the substrate. This method can be applied to other NAD+-dependent enzymes as well. Both NADH and NAD+ are efficiently regenerated with ADH-catalyzed oxidation of ethanol and reduction of isobutyr-aldehyde, respectively (Fig. 31). 

Lipases are of remarkable practical interest since they have been used in numerous biocatalytic applications, such as kinetic resolution of alcohols and carboxyl esters (both in water and in non-aqueous media) [1], regioselective acylations of poly-hydroxylated compounds, and the preparation of enantiopure amino acids and amides [2, 3]. Moreover, lipases are stable in organic solvents, do not require cofactors, possess broad substrate specificity, and exhibit, in general, a high enantioselectivity. All these features have contributed to make hpases the class of enzyme with the highest number of biocatalytic applications carried out in neat organic solvents. 

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