Lipases secondary alcohols, resolution

Lipases from C. antarctica and P. cepacia showed higher enantioselectivity in the two ionic liquids l-ethyl-3-methylimidazolium tetrafluoroborate and l-butyl-3-methylimidazolium hexafluoroborate than in THE and toluene, in the kinetic resolution of several secondary alcohols [49]. Similarly, with lipases from Pseudomonas species and Alcaligenes species, increased enantioselectivity was observed in the resolution of 1 -phenylethanol in several ionic liquids as compared to methyl tert-butyl ether [50]. Another study has demonstrated that lipase from Candida rugosa is at least 100% more selective in l-butyl-3-methylimidazolium hexafluoroborate and l-octyl-3-nonylimidazolium hexafluorophosphate than in n-hexane, in the resolution of racemic 2-chloro-propanoic acid [51]. 

The wide substrate tolerance of lipases is demonstrated by the resolution of organometallic substrates [129-131]. The presence of tin, selenium, or tellurium in the structure of secondary alcohols does not inhibit the lipase activity and enantiopure organometallic alcohols were obtained by acylation in organic media (Figure 6.48). 

The low-temperature method has been applied to some primary and secondary alcohols (Fig. 1) For example, solketal, 2,2-dimethyl-1,3-dioxolane-4-methanol (3) had been known to show low enantioselectivity in the lipase-catalyzed resolution (lipase AK, Pseudomonas fluorescens, E = 16 at 23°C, 27 at 0oc) 2ia however, the E value was successfully raised up to 55 by lowering the temperature to —40°C (Table 1). Further lowering the temperature rather decreased the E value and the rate was markedly retarded. Interestingly, the loss of the enantioselectivity below —40°C is not caused by the irreversible structural damage of lipase because the lipase once cooled below —40°C could be reused by allowing it to warm higher than -40°C, showing that the lipase does not lose conformational flexibility at such low temperatures. 

The KR of secondary alcohols by some hydrolytic enzymes has been well known. The combinations of these hydrolytic enzymes with racemization catalysts have been explored as the catalysts for the efficient DKR of the secondary alcohols. Up to now, lipase and subtilisin have been employed, respectively, as the R- and 5-selective resolution enzymes in combination with metal catalysts (Scheme 2). 

The use of an enzyme in a cascade using nanoencapsulation has also been demonstrated [23]. In this case, the dynamic kinetic resolution (DKR) of secondary alcohols was achieved with an acidic zeolite and an incompatible enzyme, Candida antarctica lipase B (CALB) (Scheme 5.8). 

The one-pot dynamic kinetic resolution (DKR) of ( )-l-phenylethanol lipase esterification in the presence of zeolite beta followed by saponification leads to (R)-l phenylethanol in 70 % isolated yield at a multi-gram scale. The DKR consists of two parallel reactions kinetic resolution by transesterification with an immobilized biocatalyst (lipase B from Candida antarctica) and in situ racemization over a zeolite beta (Si/Al = 150). With vinyl octanoate as the acyl donor, the desired ester of (R)-l-phenylethanol was obtained with a yield of 80 % and an ee of 98 %. The chiral secondary alcohol can be regenerated from the ester without loss of optical purity. The advantages of this method are that it uses a single liquid phase and both catalysts are solids which can be easily removed by filtration. This makes the method suitable for scale-up. The examples given here describe the multi-gram synthesis of (R)-l-phenylethyl octanoate and the hydrolysis of the ester to obtain pure (R)-l-phenylethanol. 

The enzymatic resolution of racemic substrates now is a well-established approach for the synthesis of single enantiomers [1, 2]. A representative example is the kinetic resoluhon of secondary alcohols via lipase-catalyzed transesterification for the preparation of enantiomericaUy enriched alcohols and esters [3], The enzymatic resolution in general is straightforward and satisfactory in terms of optical purity, but it has an intrinsic Hmitation in that the theoretical maximum yield of a desirable enantiomer cannot exceed 50%. Accordingly, additional processes such as isolation, racemization and recycling of unwanted isomers are required to obtain the desirable isomer in a higher yield (Scheme 1.1). 

The lipase-catalyzed resolutions usually are performed with racemic secondary alcohols in the presence of an acyl donor in hydrophobic organic solvents such as toluene and tert-butyl methyl ether (Scheme 1.3). In case the enzyme is highly enantioselective E = 200 or greater), the resolution reaction in general is stopped at nearly 50% conversion to obtain both unreacted enantiomers and acylated enantiomers in enantiomerically enriched forms. With a moderately enantioselective enzyme E = 20-50), the reaction carries to well over 50% conversion to get unreacted enantiomer of high optical purity at the cost of acylated enantiomer of lower optical purity. The enantioselectivity of lipase is largely dependent on the structure of substrate as formulated by Kazlauskas [6] most lipases show... 

Scheme 1.3 Lipase-catalyzed resolution of secondary alcohols.

DKR of secondary alcohol is achieved by coupling enzyme-catalyzed resolution with metal-catalyzed racemization. For efficient DKR, these catalyhc reactions must be compatible with each other. In the case of DKR of secondary alcohol with the lipase-ruthenium combinahon, the use of a proper acyl donor (required for enzymatic reaction) is parhcularly crucial because metal catalyst can react with the acyl donor or its deacylated form. Popular vinyl acetate is incompatible with all the ruthenium complexes, while isopropenyl acetate can be used with most monomeric ruthenium complexes. p-Chlorophenyl acetate (PCPA) is the best acyl donor for use with dimeric ruthenium complex 1. On the other hand, reaction temperature is another crucial factor. Many enzymes lose their activities at elevated temperatures. Thus, the racemizahon catalyst should show good catalytic efficiency at room temperature to be combined with these enzymes. One representative example is subtilisin. This enzyme rapidly loses catalytic activities at elevated temperatures and gradually even at ambient temperature. It therefore is compatible with the racemization catalysts 6-9, showing good activities at ambient temperature. In case the racemization catalyst requires an elevated temperature, CALB is the best counterpart. 

Polymers derived from natural sources such as proteins, DNA, and polyhy-droxyalkanoates are optically pure, making the biocatalysts responsible for their synthesis highly appealing for the preparation of chiral synthetic polymers. In recent years, enzymes have been explored successfully as catalysts for the preparation of polymers from natural or synthetic monomers. Moreover, the extraordinary enantioselectivity of lipases is exploited on an industrial scale for kinetic resolutions of secondary alcohols and amines, affording chiral intermediates for the pharmaceutical and agrochemical industry. It is therefore not surprising that more recent research has focused on the use of lipases for synthesis of chiral polymers from racemic monomers. 

Although many publications have covered the enantioselectivity of lipases in the deacylation step, their enantioselectivity in the acylation step (i.e., towards the acyl donor) has received much less attention. Generally, the selectivity of lipases towards racemic esters or acids is low to moderate [75-77]. Directed evolution and site-directed mutagenesis lead to a significant increase in the selectivity of the wild-type enzymes [78-80]. However, the enantiomeric ratios attained are still well below those typically obtained in kinetic resolutions of secondary alcohols. 

A prominent example of chemoenzymatic catalysis in bio-organic chemistry is the dynamic kinetic resolution (DKR) of secondary alcohols (Scheme 9) [94, 95] and amines [96-99], In this process, a lipase is employed as an enantioselective acylation catalyst, and a metal-based catalyst ensures continuous racemization of the unreactive enantiomer. 

As described above, the resolution of many types of secondary alcohols by hydrolase-catalyzed acylation in an organic solvent is usually possible after screening for a selective lipase and optimization of the reaction conditions. 

A limitation on resolution is that the desired enantiomer is only half of the racemic starting material. Kurt Faber of the University of Graz has reported (Org. Lett. 2004,6,5009) a clever solution to this problem. On exposure of the sulfate 1 of a secondary alcohol to aerobically grown whole cells of Sulfolobus acidocaldarius DSM 639, one enantiomer of the sulfate was smoothly converted into the other enantiomer of the starting alcohol. The enzyme consumed the more reactive enantiomer > 200 times more rapidly than the less reactive enantiomer. For the last bit of conversion, the of the product alcohol will of course fall. One solution to this would be to run the reaction near 50% conversion, then hydrolyze the mixture to give high product alcohol 2. Exposure of the mixture to a lipase that selectively acetylated the minor enantiomer would then polish the of 2. 

The integration of a catalyzed kinetic enantiomer resolution and concurrent racemization is known as a dynamic kinetic resolution (DKR). This asymmetric transformation can provide a theoretical 100% yield without any requirement for enantiomer separation. Enzymes have been used most commonly as the resolving catalysts and precious metals as the racemizing catalysts. Most examples involve racemic secondary alcohols, but an increasing number of chiral amine enzyme DKRs are being reported. Reetz, in 1996, first reported the DKR of rac-2-methylbenzylamine using Candida antarctica lipase B and vinyl acetate with palladium on carbon as the racemization catalyst [20]. The reaction was carried out at 50°C over 8 days to give the (S)-amide in 99% ee and 64% yield. Rather surpris-... 

The stereoselectivity for substrates bearing a small and a large substituent (e.g. a secondary alcohol as shown in fig.6) is explained by assuming that when the secondary alcohol is subjected to resolution by a lipase, the fast reacting enantiomer binds to the active side in the manner shown in fig. 6a, however, when the other enantiomer reacts with the lipase, it is forced to accommodate its large substituent into the smallest pocket (fig. 6b). This rule works well for secondary alcohols. However for primary alcohols, the rule is only applicable if an oxygen atom is attached to the stereocenter. A similar rue was also proposed for the resolution of carboxylic acids. 

Secondary alcohols are by far the most frequently used targets in lipase-catalyzed resolutions. This is due to their importance in organic synthesis but also that lipases usually show much higher enantioselectivity in resolutions compared to primary and tertiary alcohols. 

Ghanem, A. Ginatta, C. Jian, Z. Schurig, V. Chirasil-/7dex with a new Cl 1-spacer for enantioselective gas chromatography application to the kinetic resolution secondary alcohols catalyzed by lipase. Chromatographia 2003, Vol. 57, S-275-281. 

Ghanem, A. Schurig, V. Entrapment of Pseudomonas cepacia lipase with cyclodextrin in sol-gel application to the kinetic resolution of secondary alcohols. Tetrahedron Asymmetry 2003, 14, 2547-2555. 

Ghanem, A. The utility of cyclodextrins, sol-gel procedure and gas chromatography in lipase-mediated enantioselective catalysis kinetic resolution of secondary alcohols. PhD Thesis, University of Tubingen, 2002. 

Dynamic kinetic resolutions of secondary alcohols and amines have been achieved by the combination of biocatalysts with metal catalysts.12 For example, a metal catalyst was used to racemize the substrate, phenylethanol, and a lipase was used for the enantioselective esterification as shown in Figure 12. The yield was improved from 50% in kinetic resolution without racemization of the substrate to 100% with metal catalyzed racemization. 

Subsequently the groups of Williams [7] and Backvall [8] showed, in 1996 and 1997, respectively, that lipase-catalyzed transesterification of alcohols could be combined with transition metal-catalyzed racemization to produce an efficient dynamic kinetic resolution of chiral secondary alcohols (Fig. 9.2). 

Figure 7.2 The structure of the faster reacting enantiomer in lipase-catalyzed esterification in kinetic resolution of racemic secondary alcohols or hydrolysis of the corresponding esters. Small and large refer to the relative size of the groups and not to the R/S notation.

Kinetic Resolution by Hydrolysis. Until very recently, kinetic resolution of racemic alcohols as ester derivatives was by far the most common type of asymmetric transformations involving lipases. There are number of examples involving acyclic secondary alcohols, such as the glyceraldehyde derivative in eq 1 and various related alkyl- and aryloxy substituted chloride and tosylate glycerol derivatives. - ... 

Kinetic Resolution by Transesterification. Asymmetric transformation involving acylation of chiral alcohols is by far the most common example of kinetic resolution by lipase-catalyzed transesterification, most commonly with irreversible vinyl esters. This field is now becoming the most widely applied technique involving lipases. Recent reports of the numerous secondary alcohol substrates include various monocyclic (eq 6) andacyclic compounds, cyanohydrins, sulfones, and glycals, to name a few. 

Examples of kinetic resolutions with lipases are numerous [9], Impressive enantioselectivities are often obtainable with secondary alcohols, e.g., in acetylations with vinyl acetate, or in hydrolysis of the racemic ester. Likewise, the corresponding amines can be resolved, e.g. by enantioselective acetylation with EtOAc as both acyl donor and solvent. This has been demonstrated by Gotor and coworkers using Novozym 435 [50]. The reaction (Scheme 13.3) follows Kazlauskas selectivity. In fact an impressive range of CALB (Novozym 435) catalyzed transformations on nitrogenated compounds have been collected in a recent review article [51]. 

The most widely used enzymes are the hydrolytic enzymes lipases, proteases, and nitrilases, probably because these enzymes do not require cofactors and are available commercially. They are particularly useful for resolution of esters, 5,7 and for organic synthesis." 9 Esterases can also catalyze esterification if the water concentration is low. Enzyme-catalyzed transcstcrification can be used for resolution of secondary alcohols and diols.10... 

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