Resolution with Lipase

Dynamic Kinetic Resolutions with Lipases and Esterases... 

Thiel and coworkers reacted imidazole with epoxycyclohexane to form the racemic hydroxycyclohexyl imidazole. Attempts to separate the enantiomers by kinetic resolution with lipase B of Candida antarctica and isopropenyl acetate as acylating agent [11,12] failed, but gave the racemic ester in high yields (see Figure 4.1). Alkylation was then... 

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]. 

Friedelr-Crafts reactions. A synthesis of a-hydroxyarylacetic esters is by reaction of arenes with glyoxalic esters. Such esters are amenable to resolution with lipase. 

Kamal, A. and Ramesh Khanna, G.B. (2001) A facile preparation of ( )-P-hydroxy nitriles and their enzymatic resolution with lipases. Tetrahedron Asymmetry, 12, 405 -410. 

Of course, the influence of organic solvents on enzyme enantioselectivity is not limited to proteases but it is a general phenomenon. Quite soon, different research groups described the results obtained with lipases [28]. For instance, the resolution of the mucolytic drug ( )-trans-sobrerol (11) was achieved by transesteriflcation with vinyl acetate catalyzed by the lipase from Pseudomonas cepacia adsorbed on celite in various solvents. As depicted in Scheme 1.3 and Table 1.5, it was found that t-amyl alcohol was the solvent of choice in this medium, the selectivity was so high ( >500) that the reaction stopped spontaneously at 50% conversion giving both +)4rans-sobrerol and (—)-trans-sobrerol monoacetate in 100% optical purity [29]. 

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]. 

We first examined the lipase-catalyzed resolution of azirine-2-methanol I, which we expected to have a versatile synthetic utility. As expected for primary alcohols, the enantioselectivity obtained in the transesterification with lipase PS in ether was low (E = 17 at best) at room temperature despite considerable efforts such as screening of lipases, solvents, additives, and acylating agents. 

The low-temperature method is effective not only in the kinetic resolution of alcohols but also in the enantioface-selective asymmetric protonation of enol acetate of 2-methylcyclohexanone (15) giving (f )-2-methylcyclohexanone (16). The reaction in H2O at 30°C gave 28% ee (98% conv.), which was improved up to 77% ee (82% conv.) by the reaction using hpase PS-C 11 in /-Pt20 and ethanol at 0°C. Acceleration of the reaction with lipase PS-C 11 made this reaction possible because this reaction required a long reaction time. The temperature effect is shown in Fig. 14. The regular temperature effect was not observed. The protons may be supplied from H2O, methanol, or ethanol, whose bulkiness is important. 

Lipase Amano PS-C II was also found to be useful for a high-temperature reaction in the resolution of a bulky substrate, l,l-diphenyl-2-propanol, which showed no reactivity under usual conditions with lipase PS. An enantiopure product was obtained at 40-120°C, and the highest conversion (39%) was obtained at 80-90°C. It is very interesting that a single enzyme is usable in the reaction at a very wide range of temperatures from —80°C to 120°C. 

Scheme 1.60 Resolution of a prochiral 1,4-dihydropyridine dicarboxylic ester with lipase AH in the presence of cyclohexane or DIPE...

Dynamic kinetic resolution enables the limit of 50 % theoretical yield of kinetic resolution to be overcome. The application of lipase-catalyzed enzymatic resolution with in situ thiyl radical-mediated racemization enables the dynamic kinetic resolution of non-benzylic amines to be obtained. This protocol leads to (/f)-amides with high enantioselectivities. It can be applied either to the conversion of racemic mixtures or to the inversion of (5)-enantiomers. 

Dynamic kinetic resolution (DKR) is a process in which the resolution process is coupled with in situ racemization of unreacted substrate. This has been shown to be a potential and feasible method to produce 100 % theoretical yield. We have developed a chemo-enzymatic DKR to obtain higher desired yield for (5)-ibuprofen. The combined base catalyst with lipase has resulted in high conversion and excellent ee of the product. 

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. 

Hoft reported about the kinetic resolution of THPO (16b) by acylation catalyzed by different lipases (equation 12) °. Using lipases from Pseudomonas fluorescens, only low ee values were obtained even at high conversions of the hydroperoxide (best result after 96 hours with lipase PS conversion of 83% and ee of 37%). Better results were achieved by the same authors using pancreatin as a catalyst. With this lipase an ee of 96% could be obtained but only at high conversions (85%), so that the enantiomerically enriched (5 )-16b was isolated in poor yields (<20%). Unfortunately, this procedure was limited to secondary hydroperoxides. With tertiary 1-methyl-1-phenylpropyl hydroperoxide (17a) or 1-cyclohexyl-1-phenylethyl hydroperoxide (17b) no reaction was observed. The kinetic resolution of racemic hydroperoxides can also be achieved by chloroperoxidase (CPO) or Coprinus peroxidase (CiP) catalyzed enantioselective sulfoxidation of prochiral sulfides 22 with a racemic mixmre of chiral hydroperoxides. In 1992, Wong and coworkers and later Hoft and coworkers in 1995 ° investigated the CPO-catalyzed sulfoxidation with several chiral racemic hydroperoxides while the CiP-catalyzed kinetic resolution of phenylethyl hydroperoxide 16a was reported by Adam and coworkers (equation 13). The results are summarized in Table 4. 

Anthonsen, T. and Hoff, B. (1998) Resolution of derivatives of 1,2-propanediol with lipase B from C antarctica. Effect of substrate structure, medium, water activity and acyl donor on enantiomeric ratio. Chem. Phys. Lipids, 93, 199-207. 

Normally, an equilibrium is formed, so that the ester serving as acyl source has to be employed in excess. The separation of the optically active esters, e.g., (S )-2 and (S)-4 from the unreacled alcohols (R)-l and (R)-3 may be effected by distillation or chromatography. Resolution of a-halocarboxylic acids can be achieved with lipase in butanol60. 

Some l-(2-furyl)-l-alkanols have also been resolved by hydrolase-catalyzed acylations (Scheme 4.20). Thus l-(2-furyl)-l-ethanol (46) is efficiently resolved by acylation with vinyl acetate catalyzed either by Lipozyme IM or PPL [77]. Resolution with a more complex acyl donor, ethoxyvinyl methyl fumarate, catalyzed by Lipase LIP (from Pseudomonas aeruginosa) has also been achieved [95]. The... 

Such resolution could be readily optimized by use of an appropriate acyl group which reacts efficiently with the enzyme employed [29]. For example, the acetate prepared from monofluorinated a-phenetyl alcohol was hydrolyzed with lipase MY at 34% conversion to afford the product only with 26% . Enhancement of optical purity to 73% was observed when the corresponding isobutyrate was hydrolyzed. The best results were obtained for hydrolysis of the isobutyrate by lipase PS, which afforded the product in 82% at 47% hydrolysis. Experience has shown (see Table 3) that one of the best combinations was hydrolysis of acetate with lipase MY or isobutyrate with lipase PS [30]. 

After optical resolution by lipase, the silylated furanol with a trifluoromethyl group, was converted into the corresponding butenolide by oxidation with magnesium monoperoxyphthalate (MMPP). Ring opening of this butenolide... 

Optically active aziridines have been prepared in high enantiomeric excess by the enzymatic resolution of meso diesters (94AG(E)599). For example, when the me o-bis(acetoxymethyl)aziridine (56) was subjected to enzymatic hydrolysis with lipase Amano P, the aziridine (57) was obtained in 98% ee (90TL6663). 

Two more examples in Table 5 include the hydrolysis of esters of trans-alcohols that proceed with high efficiency practically regardless of the nature of the substituents (72) and resolution of p-hydroxynitriles with lipase from Pseudomonas sp. In the latter case the enantioselectivity of the hydrolysis was improved by introducing sulfur into the acyl moiety (73). 

While kinetic resolution with the help of lipases or esterases has seen the greatest success for the synthesis of enantiomerically pure amines, the same target can be reached by employing co-transaminases (co-TA) to reductively transaminate ketones to either (S)- or (K)-amines, depending on the transaminase. The reaction is shown in Figure 7.22 with acetophenone and (S)-transaminase as an example (Shin, 1998, 1999). 

Racemic resolution of a-hydroxy esters was achieved with Pseudomonas cepacia lipase (PCL) and a ruthenium catalyst (for a list, see Figure 18.13) as well as 4-chlorophenyl acetate as an acyl donor in cyclohexane, with high yields and excellent enantiomeric excesses (Huerta, 2000) (Figure 18.14). Combining dynamic kinetic resolution with an aldol reaction yielded jS-hydroxy ester derivatives in very high enantiomeric excesses (< 99% e.e.) in a one-pot synthesis (Huerta, 2001). 

Asymmetric synthesis with lipases and esterases can basically be performed by two different approaches - the desymmetrization of prochiral or meso compounds and the enzymatic kinetic resolution of racemic mixtures. The main bottleneck of kinetic resolutions, product yields of maximum 50%, can be overcome if an in situ racemization of the starting material is possible. In this case all starting material can theoretically be converted to the desired product [34],... 

An example for the application of enzymatic kinetic resolutions with high E values in natural product synthesis is the chemoenzymatic synthesis of the northern half of epothilones (also see Sect. 4.1). Various lipases and esterases could be found with outstanding enantioselectivity (up to >100) among these were lipase B from Candida antarctica, a lipase from Burkholderia cepacia, a lipase from Pseudomonas sp., and a lipase from Streptomyces diastochromogenes, all affording the desired (S)-configurated alcohol with >99% enantiomeric excess (Fig. 4) [65],... 

Enzymatic DKRs have also been applied in domino one-pot processes [97]. The combination of a lipase-catalyzed resolution with an intramolecular Diels-Alder reaction led to interesting building blocks for the synthesis of natural products such as compactin [98,99] or forskolin [100-102], A ruthenium catalyst is employed for the racemization of the slow reacting enantiomer of the starting material. The DKR with lipase B from C. antarctica delivered high enantiomeric excesses which could mainly be contained through the Diels-Alder reaction (Fig. 12). 

For the resolution of cyanopentafluorophenylethanol with lipase, (he reaction temperature was decreased to improve the enantioselectivities (Figure 15(b)). The ameso comPounds were conducted to obtain fluorinated amino, . "UIC. 1 > e)). Esterification of meso alcohol gave the corresponding W-ammo acid, whereas the hydrolysis gave the corresponding(5)-product. 

In an enzymatic resolution approach, chiral (+)-tra .s-diol (60) was prepared by the stereoselective acetylation of racemic diol with lipases from Candida cylindraceae and P. cepacia. Both enzymes catalyzed the acetylation of the undesired enantiomer of racemic diol to yield monoacetylated product and unreacted desired (+)-trans-diol (60). A reaction yield of 40% and an e.e. of >90% were obtained using each lipase [104],... 

Finally, biocatalytic resolution was developed for more efficient production of D-pantolactone. Whereas the resolution of O-acyl pantolactone with lipases or esterases [12] did not lead to an industrially attractive process, the hydrolysis of rac-pantolactone by pantolactone hydrolases enabled development of a technically feasible and economic process. 

Scientists from Bristol-Myers Squibb developed a new side chain for Taxol, making it water-soluble. A kinetic resolution with Pseudomonas cepacia lipase (lipase PS-30 from Amano) was applied to obtain the desired material enantiopure (Scheme 6.10). After the lipase-catalysed hydrolysis of the wrong enantiomer (49% conversion) the ester was obtained with an ee of >99%. Separation and subsequent chemical cleavage of the ester yielded the desired enantiomer of the lactame, which could then be coupled to baccatin III [44]. 

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