Esterases porcine liver

Porcine liver esterase (PLE) gives excellent enantioselectivity with both dimethyl 3-methylglutarate [19013-37-7] (lb) and malonate (2b) diester. It is apparent from Table 1 that the enzyme s selectivity strongly depends on the size of the alkyl group in the 2-position. The hydrolysis of ethyl derivative (2c) gives the S-enantiomer with 75% ee whereas the hydrolysis of heptyl derivative (2d) results in the R-monoester with 90% ee. Chymotrypsin [9004-07-3] (CT) does not discriminate glutarates that have small substituents in the 3-position well. However, when hydroxyl is replaced by the much bulkier benzyl derivative (Ic), enantioselectivity improves significantly. 

PPL = porcine pancreatic lipase PLE = porcine liver esterase. 

Levofloxacin (6) was enantioselectively obtained by the enzymatic hydrolysis of ofloxacin butyl ester by immobilization of porcine liver esterase (01MI25, 01MI32). 

The same urethane linker group that is a feature of conjugates 138 and 139 (Section 5.03.12) has also been used to provide SIN-1 13 conjugates of two vitamin E analogues, 6-tochopherol and Torolox , that undergo enzymatic bioactivation in the presence of porcine liver esterase to release nitric oxide <2006MI363>. 

Kinetic optical resolution of racemic alcohols and carboxylic acids by enzymatic acyl transfer reactions has received enormous attention in recent years56. The enzymes generally employed are commercially available lipases and esterases, preferentially porcine liver esterase (PLE) or porcine pancreatic lipase (PPL). Lipases from microorganisms, such as Candida cylindracea, Rhizopus arrhizus or Chromobacterium viscosum, are also fairly common. A list of suitable enzymes is found in reference 57. Standard procedures are described in reference 58. Some examples of the resolution of racemic alcohols are given39. 

If the bonded water is extracted by dry CO2 the enzyme is denaturated and loses its activity. Therefore a certain amount of water is necessary in the supercritical fluid because acting with water-saturated CO2 again causes an inhibition of the enzyme and consequent loss of activity. The optimal water concentration has to be determined for each enzyme separately. Table 9.2-1 shows the residual activity of lipase from Candida cylindracea, esterase from Mucor mihei, and esterase from Porcine liver after a incubation time of 22 hours in supercritical CO2 at 40°C. It is obvious that higher water concentrations cause a strong reduction in the residual activity compared to the activity of the untreated enzyme, which was set as 100 %. Further, the system-pressure has an influence because at higher pressures the activity-loss is lower with a larger amount of water in the system [7,8],... 

Residual activity at different CO2 humidity at 40°C, 22 hours incubation time and 170 mL total volume of the system (I, lipase from Candida cylindracea II, esterase from Mucor mihev, III, esterase from Porcine liver) ... 

Similar to the hydrolysis of malonates and glutarates, conversion of mesoderivatives of tartaric acid can be accomplished with Candida cyhndracea lipase (CCL) and porcine liver esterase in very good yield (23). Hydrolysis of (3) with esterase gives (+)-(4) with an ee 90.5% in >90% yield. Hydrolysis with lipase results in the formation of (—)-(4) (ee 92.5%, >90% yield). 

Stereoselective enzymatic hydrolyses of esters represent a further type of biotransformation that has been used for the synthesis of optically active organosilicon compounds. The first example of this particular type of bioconversion is illustrated in Scheme 15. Starting from the racemic (l-acetoxyethyl)silane rac-11, the optically active (l-hydroxyethyl)silane (5)-41 was obtained by a kinetic racemate resolution using porcine liver esterase (PLE E.C. 3.1.1.1) as the biocatalyst7. The silane (5)-41 (isolated with an enantiomeric purity of 60% ee bioconversion not optimized) is the antipode of compound (R)-41 which was obtained by an enantioselective microbial reduction of the acetylsilane 40 (see Scheme 8). 

The enantioselective hydrolysis of the racemic 2-acetoxy-l-silacyclohexane rac-78 represents a further example of an enzymatic kinetic racemate resolution (Scheme 15). Hydrolysis of this compound in the presence of porcine liver esterase (PLE E.C. 3.1.1.1) yielded the optically active 1-silacyclohexan-2-ol (S)-43 which was isolated with an enantiomeric purity of 93% ee7. Similar results were obtained when using a crude lipase preparation from Candida cylindracea (CCL E.C. 3.1.1.3) as the biocatalyst... 

Enantioselective enzymatic ester hydrolyses have also been used for the preparation of optically active silicon compounds with the silicon atom as the center of chirality. An example of this is the kinetic resolution of the racemic 2-acetoxy-l-silacyclohexane rac-(SiR,CR/SiS,CS)-79 with porcine liver esterase (PLE E.C. 3.1.1.1) (Scheme 16)65. Under preparative conditions, the optically active l-silacyclohexan-2-ol (SiS,CS)-80 was obtained as an almost enantiomerically pure product (enantiomeric purity >96% ee) in ca 60% yield [relative to (SiS,CS )-79 in the racemic substrate]. The biotransformation product could be easily separated from the nonhydrolyzed substrate by column chromatography on silica gel. 

Enantioselective enzymatic ester hydrolyses of prochiral trimethylsilyl-substituted diesters of the malonate type have been applied for the synthesis of the related optically active monoesters68. As an example of this particular type of biotransformation, the enantioselective conversion of the diester 82 is illustrated in Scheme 17. Hydrolysis of compound 82 in phosphate buffer, catalyzed by porcine liver esterase (PLE E.C. 3.1.1.1) or horse liver acetonic powder (HLAP), gave the optically active monoester 83 (absolute configuration not reported) in 86% and 49% yield, respectively. The enantiomeric purities... 

Chiral carbon framework of the monoterpenoid secologanin, leading to powerful chiral synthons, from readily available meso-forms. Moderate results were obtained with lipases such as porcine liver esterase (PLE), delivering the (15,2/ )-mono-acetate at a yield of 78% with 96% ee (Fig. 10), though preparation of the enzyme seemed to be crucial for the appropriate result. In contrast, pig pancreatic lipase (PPL) was significantly more efficient in forming the (—)-(l/ ,25) enantiomer at a yield of 96% and 98% ee [86, 87]. 

Fig. 21 Short chemo-enzymatic approach to enantiomerically pure (+)-pilocarpine from an easy available precursor using two lipase-catalysed reactions (PS (from AmanoEnzyme Inc.) PLE porcine liver esterase)...

A fluorescent complex [Ru(r 6-p-cym)Cl(L)]Cl (L = 2-[(2-aminoethyl)amino] ethyl-2-(methylamino)benzoate) has been synthesised by tagging a small fluoro-genic reporter onto the chelating ligand. The interaction of this complex with porcine liver esterase (PLE) showed that esterase-catalysed hydrolysis reactions can liberate methylisatoic acid (MIAH) from the ruthenium complex suggesting a possible use of similar derivatives in esterase-activated Ru-based prodrug delivery systems. The hydrolysis reaction appears to be slow [156]. 

Gana et al. developed and validated a reversed-phase high performance liquid chromatographic method for the kinetic investigation of the chemical and enzymatic hydrolysis of benazepril hydrochloride [37]. Kinetic studies on the acidic hydrolysis of benazepril hydrochloride were carried out in 0.1 M hydrochloric acid solution at 50, 53, 58 and 63°C. Benazepril hydrochloride appeared stable in pH 7.4 phosphate buffer at 37°C, and showed susceptibility to in vitro enzymatic hydrolysis with porcine liver esterase (PLE) in a pH 7.4 buffered solution at 37°C. 

Enzymatic hydrolysis of penam 158a with porcine liver esterase (PLE) followed by treatment with Eschenmoser s base (78HCA2851 82T2659) afforded salt 162, which was treated with potassium 2-ethylhexanoate in... 

A method similar in concept was described by Burd et al. (1977b) using haptens coupled via an ester bond to umbelliferone. Hydrolysis of the ester by porcine liver esterase is followed by the liberation of the fluorescent umbelliferone. This hydrolysis, however, is prevented or sharply reduced by the presence of antibody to the hapten free hapten present in the test sample competes for the available antibody and more hydrolysis is produced by the enzyme. 

Malathionase (ME) For measuring the inhibition of malathion esterase activity, general carboxylesterase from porcine liver (Sigma) was used at a final concentration of 16 jig protein/mL in 0.1M Tris HC1 buffer (pH-7.5). The procedure involves an indirect determination of the malathionase activity by coupling the hydrolysis of malathion to the reduction of a tetrazolium dye (42). An acetone solution of malathion was used as substrate to a final concentration of 3x10 4M. 

Ammonium sulfate (2 M) is a popular storage solution for commercial porcine liver esterase (PLE). Ammonium sulfate prevents microbial growth on the solution. Storage in 50% glycerol is also useful and this glycerol stock can be stored below 0°C. 

A further example of this kind of kinetic racemate resolution is the highly enantioselective hydrolysis of rac-(RyR/S,S)-235 using isolated porcine liver esterase (EC 3.1.l.l)287. 

---