C. del Campo

Organic reactions catalysed by modified enzymes part I. Alteration of the substrate specificity of α-chymotrypsin by the modification process

Abstract The substrate specificity of some modified α-chymotrypsin derivatives in the synthesis of peptides, using natural and synthetic acyl donors and several nucleophiles, is studied. A systematic study of the alteration of active sites by the modification method is carried out. The method used in the present paper consists of covalent bonding of external lysines of the enzyme with a hydrophobic polymer. Enzymes with several degrees of modification have been obtained. These derivatives present different hydrophobic characteristics, depending on the degree of modification. All the modified enzymes are active in hydrophobic solvents such as benzene, toluene or 1,1,1-trichloroethane but need a small amount of water to be activated ( × > 60%). The modified enzyme has the same stereospecificity as the native enzyme, because only esters with L-configuration in the α-carbon are accepted as acyl donors. Nevertheless a greater number of acyl donors are accepted by the modified enzyme than by the native α-chymotrypsin, in the synthesis of peptides. On the other hand, the nucleophile subsite is more lipophilic in the modified enzyme than in the native α-chymotrypsin.

Hydrolysis of 2-substituted aryl and heteroaryl alkanoates by Candida rugosalipase

AbstractsThe hydrolysis of several esters catalyzed by lipase of Candida rugosa was used to analyse the interaction between the ester substrate and the active site and the geometry and steric restrictions of ‘alk‘, ‘ar‘ and ‘h‘ subsites. Using 4-R-C H -CH(R‘)-COOEt and ethyl trans-cinnamate, the presence of a low electronic density zone in the ‘ar‘ pocket is described, the dimensions of the pocket produced by Phe-296 and Leu-297 in the ‘alk‘ subsite and the size of the ‘h‘ subsite are described.

Structure—activity relationship in the hydrolysis of N-benzoylphenylalanine esters catalysed by α-chymotrypsin

Abstract The alteration by DMF of the active site of native α-chymotrypsin was analyzed by QSAR and molecular mechanics methodologies. The hydrolysis of several synthetic esters was used as the reaction model. In order to analyze the structural parameters of the substrates that influence enzymatic activity, classic monovalent isosters of phenylalanine were synthesized to be used as model substrates ( ortho, meta and para F, Cl, Me; m -Br and p -OH). The conformation of the ester that interacts with the enzyme has been determined. The dimensions of the ‘ar’ active subsite of α-chymotrypsin have been revised according to the results. The main variables that influence the stearase activity of α-chymotrypsin - expressed as log(1/ k ) - and the binding constant - log(1/ K m ) - have been analyzed. The active conformation of the ester (used in the binding process) was determined by QSAR analysis.

Organic reactions catalyzed by insolubilized enzymes: Part II. Influence of structure of the acyl donor in selectivity of α-chymotrypsin

Abstract The enzymatic activity of a new insolubilized α-chymotrypsin (α-CT) on agarose is analyzed. The esterase activity was tested versus several synthetic isosteres of L-phenylalanine methyl ester. The insolubilized derivative hydrolyzes, with similar initial rates, m-Cl, o-Cl and p-Cl and phenylalanine esters in several organic-aqueous media. 1,4-Dioxane, DMF and THF were used as organic water-miscible solvents at 50–70%. Acetonitrile poisons the enzyme in hydrolytic conditions, which could be related to the acidic character of this solvent. In kinetically controlled peptide synthesis, the enzyme can act only with L-Phe and m-Cl esters, using several amino acids as nucleophiles. These differences in enzymatic activity are analyzed and explained according to alterations in the active site of the enzyme. DOR experiments showed that the insolubilized α-CT used in this work was not stereoselective versus the D or L form of amino acid in the hydrolysis of esters at pH 6 and in the kinetically controlled peptide synthesis.

Design, synthesis and evaluation of a chiral propranolol selector

Abstract Synthesis of enantiomerically pure ( S )-(−)-propranolol, the most active of the widely used beta-blockers adrenergics, was achieved using a new chiral stationary phase (CSP) for the separation of a derivative. This simple methodology shows a significant improvement in the separation of the enantiomers of a propranolol derivative over previous methods.

A new route to amino-2-propanol structures with adrenergic β-blocker activity using low valent titanium

Abstract Amino-2-propanol structures can be obtained by addition to dibenzyl acetals of in situ generated dihalocarbenes using LVT (Low Valent Titanium). This methodology can be used to obtain adrenergic β-blockers with amino-2-propanol structure. Tetrahalomethanes are the best dihalocarbene precursors. The yields obtained using halofluoromethanes can be increased by addition of carbontetrachloride. A process that can imply halogen transfer may be proposed.

Enantiospecific hydrolysis of esters of nonsteroidal antiinflammatory drugs using lipase of Candida cylindracea

Abstract Enantiospecific hydrolysis of esters of 2-arylpropionic acids with antiinflammatory activity has been carried out. The racemic acids were synthetized by Willgerodt—Kindler synthesis in good yields. The enzymatic hydrolysis of the esters was carried out in a batch reactor with 0.2 M substrate concentration.