Enzymes with racemisation

Other methods of racemisation during resolution the Mannich reaction Resolution with racemisation in the manufacture of a drug Resolution with Enzymes... 

Kinetic resolution with racemisation using proteolytic enzymes... 

Pseudomonas is found in the section below on Dynamic Kinetic Resolutions. We will meet Pseudomonas fluorescens again in the next chapter where we also see enzymes in kinetic resolutions with racemisation of starting material (dynamic kinetic resolution). 

Kinetic resolution with racemisation Enzymes versus whole organisms Desymmetrisation with lipases Immobilised enzymes in desymmetrisation Polymer-supported reagents and enzymes Effects of amines on lipases and esterases Other acylating enzymes Enzymatic Oxidation... 

Since the demonstration of the compatibility of enzymes with metal complexes in one pot, this powerful concept has attracted much attention. Indeed, the use of transition metal nzyme combinations, independently highlighted by Reetz and Schimossek, ° Sturmer and BackvalP to effect tandem in situ racemisation and resolution has widely extended the scope of DKRs. In this powerful approach, the enzyme acts as an enantioselective resolving catalyst and the metal serves as a racemising catalyst for the efficient DKR. 

The enantiomorph of this deuteroamine, prepared by decarboxylation of a-deuteroglutamate in water, does not exchange deuterium with the solvent, under the same conditions. (a-Deuteroglutamate is prepared by enzyme-catalysed racemisation of d- or L-glutamate in deuterium oxide). Additional support for the stereospecificity of enzyme-catalysed decarboxylation of amino acids comes from experiments in which tyrosine was decarboxylated in DgO to give R-a-... 

The versatility of the combination of enzymes with metal catalysts is also well demonstrated by chemoenzymatic dynamic kinetic resolutions (DKRs). Indeed, to overcome the major drawback of kinetic resolution for which the maximum yield is limited to 50%, the combination of a metal-catalysed racemisation of the slow-reacting enantiomer with an enzyme-catalysed... 

Tyrosine phenol lyase, like the tryptophanase of Escherichia coli and the cystathionine synthetase of Salmonella typhimurium, is an enzyme with a broad substrate specificity and catalyses a whole series of related a, -elimination, -replacement and racemisation reactions In a reversal of the elimination reaction, it is capable... 

The ability of enzymes to achieve the selective esterification of one enantiomer of an alcohol over the other has been exploited by coupling this process with the in situ metal-catalysed racemisation of the unreactive enantiomer. Marr and co-workers have used the rhodium and iridium NHC complexes 44 and 45 to racemise the unreacted enantiomer of substrate 7 [17]. In combination with a lipase enzyme (Novozyme 435), excellent enantioselectivities were obtained in the acetylation of alcohol 7 to give the ester product 43 (Scheme 11.11). A related dynamic kinetic resolution has been reported by Corberdn and Peris [18]. hi their chemistry, the aldehyde 46 is readily racemised and the iridium NHC catalyst 35 catalyses the reversible reduction of aldehyde 46 to give an alcohol which is acylated by an enzyme to give the ester 47 in reasonable enantiomeric excess. 

An elegant way to avoid the low yields and the need for recycling half of the material in the case of kinetic resolutions is a dynamic kinetic resolution (DKR). The dynamic stands for the dynamic equilibrium between the two enantiomers that are kinetically resolved (Scheme 6.6A). This fast racemisation ensures that the enzyme is constantly confronted with an (almost) racemic substrate. At the end of the reaction an enantiopure compound is obtained in 100% yield from racemic starting material. Mathematical models describing this type of reaction have been published and applied to improve this important reaction [32, 33]. There are several examples, in which the reaction was performed in water (see below). In most cases the reaction is performed in organic solvents and the hydrolase-catalysed reaction is the irreversible formation of an ester (for example see Figs. 9.3, 9.4, 9.6, 9.12) or amide (for example see Figs. 9.13, 9.14, 9.16). 

For the enantiopure production of human rhinovirus protease inhibitors scientists from Pfizer developed a kinetic resolution and recycling sequence (Scheme 6.14 A). The undesired enantiomer of the ester is hydrolysed and can be racemised under mild conditions with DBU. This enzymatic kinetic resolution plus racemisation replaced a significantly more expensive chemical approach [52]. An enzymatic kinetic resolution, in combination with an efficient chemically catalysed racemisation, is the basis for a chiral building block for the synthesis of Talsaclidine and Revatropate, neuromodulators acting on cholinergic muscarinic receptors (Scheme 6.14B). In this case a protease was the key to success [53]. Recently a kinetic resolution based on a Burkholderia cepacia lipase-catalysed reaction leading to the fungicide Mefenoxam was described [54]. Immobilisation of the enzyme ensured >20 cycles of use without loss of activity (Scheme 6.14 C). 

The enzymes of the nucleic acid metabolism are used for several industrial processes. Related to the nucleobase metabolism is the breakdown of hydantoins. The application of these enzymes on a large scale has recently been reviewed [85]. The first step in the breakdown of hydantoins is the hydrolysis of the imide bond. Most of the hydantoinases that catalyse this step are D-selective and they accept many non-natural substrates [78, 86]. The removal of the carbamoyl group can also be catalysed by an enzyme a carbamoylase. The D-selective carbamoylases show wide substrate specificity [85] and their stereoselectivity helps improving the overall enantioselectivity of the process [34, 78, 85]. Genetic modifications have made them industrially applicable [87]. Fortunately hydantoins racemise readily at pH >8 and additionally several racemases are known that can catalyze this process [85, 88]. This means that the hydrolysis of hydantoins is always a dynamic kinetic resolution with yields of up to 100% (Scheme 6.25). Since most hydantoinases are D-selective the industrial application has so far concentrated on D-amino acids. Since 1995 Kaneka Corporation has produced 2000 tons/year of D-p-hydroxyphenylglycine with a D-hydantoinase, a d-carbamoylase [87] and a base-catalysed racemisation [85, 89]. 

The same enzyme catalyses the esterification of racemic 129 in non-aqueous solution with vinyl acetate. The released alcohol is CH2=CHOH, the enol of acetaldehyde it immediately forms acetaldehyde which self condenses and is removed from the equilibrium. The enzyme is filtered off, the enantiomerically pure alcohol (S) -129 and acetate (R)-130 separated by flash chromatography, and the ester hydrolysed to the alcohol without racemisation. Either method (esterification or hydrolysis) gives both enantiomers of a range of secondary alcohols.31... 

In the above example, an enzyme for the kinetic resolution was combined with silica to provide the dynamic part of a dynamic kinetic resolution. The role of the silica is to racemise the hemithioacetal that does not get acetylated. The racemisa-tion of most alcohols is unlikely to be so straightforward. Consider sec-phenethy-lalcohol 3 how would we racemise this Silica is unlikely to do the job. 

With the protease from Streptomyces griseus at pH 9.7 racemisation occurred at a reasonable rate and the enzyme still operated efficiently, giving (S) -23 in good yield and only slightly reduced ee. One crystallisation gave 94% ee, acceptable for an anti-inflammatory.10... 

Alternatively, the acetate 219 of the final product (+) -216 could be enantioselectively hydrolysed with another Amano lipase GC-20, chosen after a survey of fourteen enzymes. At this stage both enantiomers of the drug were needed for evaluation and the unreacted (S)-acetate could be hydrolysed to (5 ) -215 without racemisation. Again organic solvent was necessary E was 1 in pure water and maximum (>100) in toluene or CH2C12. 

Kellogg, Feringa and co-workers have achieved successful dynamic kinetic resolution reactions using cyclic hemiacetals as substrates[13, 14l The enzyme-catalyzed acetylation of 6-hydroxypyranone shown in Fig. 9-6 has been achieved with reasonable enantioselectivity with essentially complete conversion. The racemisation of the hemiacetal is presumed to proceed via reversible ring-opening of the pyranone1 1. The rate of reaction was found to greatly increase when the enzyme, lipase PS (Pseudomonas sp.) was immobilized on Hyflo Super Cell (HSC). 

Dynamic Kinetic Resolution. Another typical acid-catalysed reaction is the racemisation of chiral alcohols, due to inversion at the chiral carbon. This can actually be made use of in the formation of enantiopure compounds, by dynamic kinetic resolution using an enzyme, such as a lipase, that catalyses enantioseleetive esterification in an organic medium. By coupling zeolite Beta-catalysed intereonversion of benzylic alcohol enantiomers with enzyme-catalysed esterifieation of only one of the enantiomeric alcohols, almost complete eon version to enantiopure ester ean be achieved. ... 

Much of the world s cysteine is isolated from keratin, mostly obtained in the form of hair. The process, which is based on an acid hydrolysis of the protein, is smelly, and it produces waste products which are difficult to treat. It is progressively being replaced by an enzymatic synthesis which once again couples a stereospecific hydrolysis with a racemisation process. Methyl-2-chloroacrylate is converted into DL-amino-A2-thiazoline-4-carbo-xylate, and both enantiomers of this intermediate are then hydrolysed by an enzyme from Pseudomonas thiazolinophilum or Sarcina lutea directly to L-cysteine. The necessary racemisation occurs spontaneously during the reaction (Scheme 6.10). 

Base catalysed racemisation is a straightforward approach because enzymes typically operate at pH close to 7. Clearly the relevant substrates must possess a stereogenic center with an acidic proton, such as a-substituted carboxylic acid derivatives [53-55]. 

SaUcylaldehydes have been employed to assist in the racemization ofa-amino acid ethyl esters. Condensation of the amino acid ester with the salicylaldehyde derivative led to an increase in the acidity of the a-hydrogen atom (at the stereogenic centre) leading to rapid racemization. The DKR procedure employed an endoproteinase alcalase enzyme for the resolution step and various aldehydes for the in situ racemisation step. Hydrolysis of the imine liberated the amino acid ester and the aldehyde. The a-amino acids thus obtained in high yield (up to 94%) exhibited high enantiopurity (up to 98%) (Schemes 4.20 and 4.21) [56]. 

Enzymatic racemisation is an attractive option in DKR because the reactions catalysed by enzymes are performed under mild conditions. The Degussa group have recently described their successful commercialization of two DKR-based processes that employ racemases, namely (i) the DKR of 5-substituted hydantoins using whole cells coexpressing a L-carbamoylase, a hydantoin racemase and a hydantoinase and (ii) the DKR of N-acetyl amino acids using an acylase in combination with an N-acetyl amino acid racemase from Amycolatopsis orientalis. 

Racemisation via Sn2 displacement has been used for the DKR of a a-bromoester by enzymatic hydrolysis the product, a a-bromoacid, was less reactive in the Sn2 process. Investigations of the bromide source and the lipase employed led to an optimised system where an immobilised phosphonium bromide was used, together with cross-linked enzyme crystals from Candida rugosa lipase (CLEC-CRL), to afford the corresponding a-bromocarboxylic acid (Scheme 3.37). This procedure could be extended to a-chloroesters. 

Lutz and colleagues have reported a new process concept of DKR via preferential crystallization combined with enzymatic racemisation. This methodology was successfully applied to the DKR of asparagine by using an enzyme... 

But in order to utilise these reactions, a few conditions must be met. A selective enzyme is crucial and the organometallic catalyst must facilitate a fast racemisation of the substrate. Last, but not least, the catalyst should not influence the enzyme in terms of selectivity and reactivity. In the ideal case, the enzyme transforms one enantiomer of the substrate, giving rise to the corresponding product, which is not susceptible to metal-catalysed racemisation. Three major types of enzyme-metal combinations—lipase-ruthenium, sub-tilisin-ruthenium and lipase combined with a metal other than ruthenium—have been developed primarily as the catalysts for the DKRs of various secondary alcohols but also for diols, amines and esters. Meanwhile, the lipase-ruthenium combination has been the most used method up to the present time. 

The combination of a lipase and Shvo s catalyst has also been applied to the DKR of a- and jS-hydroxyphosphonates. Hydroxyphosphonates are an important class of substrates, with applications in medicinal chemistry (haptens of catalytic antibodies, phosphonic acid based antibiotics), biochemistry (enzyme inhibitors) and organic synthesis. Under typical conditions, Backvall s group has shown that the DKR of several dimethyl- and diethyl-a-hydr-oxyphosphonates proceeded with excellent enantioselectivities and moderate to good yields (Scheme 4.18). This was attributed to the coordination of the phosphonate moiety to the ruthenium catalyst at a low alcohol concentration. This DKR procedure was also applied to the de-racemisation of diethyl jS-hydroxyphosphonates. However, in contrast to the DKR results on the... 

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