Lipase from Pseudomonas

Regioselective hydrolysis of diesters is a challenging problem ia synthetic chemistry because the side reactions always reduce the yield of desired product. Some Upases are well suited to perform this task. Lipase OF-360 (Meito Sangyo) hydrolyzes diester (55) ia 74% theoretical yield and 93% ee (70). Lipase from Pseudomonas cepacia suspended ia diisopropyl ether saturated with water hydrolyzes triester (56) with a remarkable efficiency and regio- and stereoselectivity (71). 

The lipase from Pseudomonas sp. KIO has also been used to cleave the chloroacetate, resulting in resolution of a racemic mixture since only one enantiomer was cleaved. 

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]. 

Figure 2.11 CASTing of the lipase from Pseudomonas aeruginosa (PAL) leading to the construction of five libraries of mutants (A-E) produced by simultaneous randomization at sites composed of two amino acids. (For illustrative purposes, the binding of substrate (1) is shown) [25],...

Lipase from C.antarctica also catalyzes carbon-carbon bond formation through aldol condensation of hexanal. The reaction is believed to proceed according to the same mechanism as the Michael additions [113]. Lipase from Pseudomonas sp. 

Enzymes PPL, lipase from Pseudomonas fluorescens F-AP, lipase from Rhizopus orizae AP-6, lipase from Aspergillus niger, SP-254, lipase from Aspergillus oryzae P-2, Chirazyme WCPC, whole cell cultures of Penicillium citrinum WCPFL, whole cell cultures of Pseudomona fluorescens CAL-B, lipase from Candida antarctica B PS-C, lipase from Pseudomonas cepacia GCL, lipase from Geotrichum candidum. n.r. not reported. 

Lipase ANL, lipase from Aspergillus niger, BCL, lipase from Burkholderia cepacia (formerly Pseudomonas cepacia) CAL-B, lipase from Candida antarctica B PPL, lipase from Pseudomonas fluorescens PPL, pig pancreatic lipase. 

In turn, bis(2-hydroxyethyl)phenylphosphine P-borane 90 underwent acetylation only in the presence of the lipase from Pseudomonas fluorescens, but its stereochemical outcome depended on the solvent used (Equation 43). The absolute configuration of 91 was not determined. 

Organic solvent can affect the enzyme specificity [76]. Authors have indicated that transesterification of l,4-butyloxy-2-octylbenzene and butanol in presence of lipases from Pseudomonas can produce two different products when using hydrophilic (acetonitrile) or hydrophobic (toluene) solvents. Zaks and Klibanov [16], demonstrated that subtilisine and a-chymotrypsine specificites can be changed as a function of solvent types. This is true for a limited number of biocatalysts. 

Crude powder lipase from Pseudomonas cepecia Asymmetrical hydrolysis of ( + )l-chloro-2-acetoxy-3-(l-naphthyloxy)-propane T etrahydrofuran- phosphate buffer pH 7.1 (1 /3) No lag period observed for the product formation 8... 

Free lipase from Pseudomonas cepecia Acetone-phosphate buffer pH 7.1 (1.5/8.5) Asymmetrical hydrolysis of (+ )1 -chloro-2-acetoxyl-3-( 1 -naphthyloxy)-propane Not available 0.24 mM/min 13.563 mM 8... 

R. J. Kazlauskas, A. N. E. Weissfloch, A. T. Rappaport, L. A. Cuccia, A Rule to Predict Which Enantiomer of a Secondary Alcohol Reacts Faster in Reactions Catalyzed by Cholesterol Esterase, Lipase from Pseudomonas cepacia, and Lipase From Candida rugosa, J. Org. Chem. 1991, 56, 2655 - 2665. 

PCL lipase from Pseudomonas cepacia (now renamed to Burkholderia cepacia)... 

The first high-throughput ee assay used in the directed evolution of enantioselective enzymes was based on UV/Vis spectroscopy (16,74). It is a crude but useful screening system that is restricted to the hydrolytic kinetic resolution of racemic / -nitrophenyl esters catalyzed by lipases or esterases. The development of this assay arose from the desire to evolve highly enantioselective mutants of the lipase from Pseudomonas aeruginosa as potential biocatalysts in the hydrolytic kinetic resolution of the chiral ester rac-. The wild type leads to an E value of only 1.1 in slight... 

Finally, it is interesting to note that in most cases enhanced enantioselectivity was shown to be due to a reduced value of / cat/Am for the non-preferred enantiomer 143). This result is contrasted with the results of directed evolution of the lipase from Pseudomonas aeruginosa, in which case the value of for the preferred... 

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. 

A rule, similar to Prelog s rule, has been proposed for the enzyme-mediated hydrolysis of the esters of secondary alcohols. Esters of the enantiomers 31 usually react faster. This rule correctly predicted the configuration of 14 out of 15 substrates when cholesterol esterase was used, 63 out of 64 substrates with a lipase from Pseudomonas cepacia, and of 51 out of 55 cyclic substrates using a lipase from Candida rugosa24°. 

Although quite reliable empirical rules exist for the enantioselectivity of hydrolases for secondary alcohols (see Section 4.2.1.2), such rules are not as developed for primary alcohols, partly because many hydrolases often show low enantioselectivity. With some exceptions, lipases from Pseudomonas sp. and porcine pancreas lipase (PPL) often display sufficient selectivity for practical use. The model described in Figure 4.3 has been developed for Pseudomonas cepacia lipase (reclassified as Burkholderia cepacia), and, provided that no oxygen is attached to the stereogenic center, it works well for this lipase in many cases [41]. However, as soon as primary alcohols are resolved by enzyme catalysis, independent proof of configuration for a previously unknown product is recommended. 

Lipases from Pseudomonas sp. [Amano PS and Pseudomonas fluorescens lipase (PFL)] are useful. Provided that the conversion is high enough, the remaining (R)-2-methylalkanols R-2 can be obtained almost enantiomerically pure. The (S)-... 

Although lipases from Pseudomonas are usually the catalysts of choice for primary alcohols, 2-(2-furyl)-propan-l-ol (Scheme 4.8 7 n = 0 with instead of S) actually gives a higher E (E = 20) with Candida antarctica lipase (CALB) than it does with Pseudomonas sp. lipase (PSL) (E = 2) on acylation with vinyl acetate in pentane [78]. 

Oda et al. reported in 1992 a one-pot synthesis of optically active cyanohydrin acetates from aldehydes, which were converted to the corresponding racemic cyanohydrins through transhydrocyanation with acetone cyanohydrin, catalyzed by a strongly basic anion-exchange resin [21]. The racemic cyanohydrins were acety-lated by a lipase from Pseudomonas cepacia (Amano), with isopropenyl acetate as the acyl donor. The reversible nature of the base-catalyzed transhydrocyanation enabled continuous racemization of the unreacted cyanohydrins, thereby effecting the total conversion (Scheme 5.8). 

Nicolosi and coworkers have intensively investigated the exploitation of lipases for the selective deprotection of bioactive compounds [90]. For instance, as shown in Figure 6.8, the alternative use of the lipases from Candida antarctica (CalB) and Mucor miehei (Mml) enabled the preparation of different derivatives of the flavonoid quercetin (28) [91]. Similar results, this time exploiting the lipase from Pseudomonas cepacia, were obtained with the polyacetylated catechin 29 [92]. 

S)-N-(tert-butoxycarbonyl)-hydroxymethylpiperidine (8) is a key intermediate in the synthesis of a potent tryptase inhibitor (Scheme 7.5). It was synthesized from (R,.S)-3-hydroxyrnethylpiperidine via fractional crystallization of the corresponding L(-)dibenzoyl tartrate salt followed by hydrolysis and acylation [17]. The lipase from Pseudomonas cepacia (PS-30) immobilized on polypropylene accurel PP catalyzed the esterification of racemic 6 with succinic anhydride and toluene, giving the (S)-hemisuccinate ester (7). This was easily separated and hydrolyzed by base to the (S)-Boc-protected 3-hydroxymethylpiperidine (8). Using this repeated esterification procedure gave a 32% yield (maximum theoretical yield = 50%) and 98.9% . 

Residual activity of different enzymes after 1 hour treatment in supercritical CO2 at 150 bar (I, lipase from Candida cylindracea If, lipase Amano AY III, lipase from Pseudomonas sp. IV, esterase EP10 from Burkholderia gladioli) ... 

Conventional gas chromatography (GC) based on the use of chiral stationary phases can handle only a few dozen ee determinations per day. In some instances GC can be modified so that, in optimal situations, about 700 exact ee and E determinations are possible per day [29]. Such meclium-throughputmay suffice in certain applications. The example concerns the lipase-catalyzed kinetic resolution of the chiral alcohol (R)- and (S)-18 with formation of the acylated forms (R)- and (S )-19. Thousands of mutants of the lipase from Pseudomonas aeruginosa were created by error-prone PCR for use as catalysts in the model reaction and were then screened for enantioselectivity [29]. 

PPL and lipase from Pseudomonas sp. catalyze enantioselective hydrolysis of sulfinylalkanoates. For example, methyl sulfinylacetate (46) was resolved by Pseudomonas sp. lipase in good yield and excellent selectivity (62). This procedure was suitable for the preparation of sulfinylalkanoates where the ester and sulfoxide groups are separated by one or two methylene units. Compounds with three methylene groups were not substrates for the lipase (65). 

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