Hydrolysis, enzymatic, solvents resolution

Fig. 2.8 Mechanism of concurrent sequential enzymatic kinetic resolution via hydrolysis-esterification in aqueous-organic solvent...

Another possibility to obtain 100% yield of the enantiopure product is to combine the kinetic resolution with an inversion reaction [25, 35, 36]. In this case an enzymatic hydrolysis is followed by a Mitsunobu inversion. It is, however, in fact a three-step reaction with solvent changes between the reactions. Similarly the sulfatase-catalysed enantios elective inversion of a racemic sulfate yields a homo-chiral mixture of alcohol and sulfate. This yields 100% enantiopure product after a second, acid-catalysed hydrolysis step, which is performed in organic solvent/water mixtures [26]. 

Proteins are probably more resistant toward proteolytic attacks in their native state and stabilizing factors (e.g., co-factor, correct parameter interval, co-solvent) are always considered optimized. Use of protein inhibitors is not recommended for safety reasons. The primary mechanism of proteolysis is the enzymatic hydrolysis of the peptide bond. The indicators of this reaction taking place in the system include loss of product or poor yield, lack of expected activity, changes in specific activity, change in MW, high background staining in ID SDS electrophoresis, smeared bands, and many lower MW bands of poor resolution, disappearance of bands, and discrepancies in MW. The preventive actions taken to prevent proteolysis are listed in Table 5. 

The enzymatic resolution of racemic amino acid derivatives in [BMIM][BF4] by papain was tested by Lou et al. Higher hydrolytic activity and enantioselectivity concerning asymmetric hydrolysis of D,L-p-hydroxyphenylglycine methyl ester was achieved in ionic liquid/buffer-solution compared to organic solvent solutions. Nearly no hydrolytic activity was observed in pure ionic liquid [42]. 

Roche D, Prasad K, Repic O (1999) Enantioselective acylation of 3-aminoesters using penicillin G acylase in organic solvents. Tetrahed Lett 40 3665-3668 Rolinson GN, Batchelor ER, Butterworth D et al. (1960) Formation of 6-aminopenicillanic acid from penicillin by enzymatic hydrolysis. Nat Lond 187 236-237 Rolinson GN, Geddes AM (2007) The 50th anniversary of the discovery of 6-aminopenicillanic acid (6-APA). Internatl J Antimicrob Agents 29 3-8 Resell CM, Ferndndez-Lafuente R, Guisdn JM (1993) Resolution of racemic mixtures by synthesis reactions catalyzed by immobilized derivatives of the enzyme peniciUin G acylase. J Mol Catal 84 365-371... 

BMY 14802 88 has also been prepared by lipase-catalyzed resolution of racemic BMY 14802 acetate ester 90 [148]. Lipase from Geotrichum candidum (GC-20 from Amano Enzyme Co.) catalyzed the hydrolysis of acetate 90 to / -(+)-BMY 14802 (Fig. 28) in a biphasic solvent system in 48% reaction yield (theoretical maximum yield is 50%) and 98% e.e. The rate and enantioselectivity of the hydrolytic reaction was dependent on the organic solvent used. The enantioselectivity E values) ranged from 1 in the absence of solvent to more than 100 in dichloromethane and toluene. S-(—)-BMY 14802 was also prepared by the chemical hydrolysis of undesired BMY 14802 acetate obtained during enzymatic resolution process. 

For the synthesis of p-lactam antibiotics, the presence of asymmetrical carbon at the 3 and 4 positions is critical to prepare optically active -lactams [197]. Nagai et al. [198] developed enzymatic synthesis of optically active p-lactams by lipase-catalyzed kinetic resolution using the enantioselective hydrolysis of iV-acyloxymethyl p-lactams 108 in an organic solvent (isopropyl ether saturated with water) and the transesterification of N-hydroxymethyl P-lactam 109 in organic solvent (metiiylene chloride) in tiie presence of vinyl acetate as acyl donor (Fig. 37). The reaction yield of 35-50% and e.e. s of 93 to more than 99% were obtained depending on the specific substrate used in the reaction mixture, Lipase B from Pseudomonas fragi and lipase PS-30 from Pseudomonas sp. were used in the reaction mixture. 

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