Resolution of Ring-Substituted Phenylalanines by the Action of α-Chymotrypsin on their Ethyl Esters

Abstract

TONG, J. H., PE~TCLERC, C., D'IORIO, A., and BENOITON, N. L. Resolution of ring-substituted phenylalanines by the action of a-chymotrypsin on their ethyl esters. Can. J. Blochem. 49, 877-881 (1971).The D-and L-isomers of the following ring-su bsti tu ted phenylalanines have k e n prepared from the racemates in $8-$0% yield: tyrosine, o-tyrosine, m-tyrosine, p-chlorophenylalanine, p-Wuorophenylalanine, and 3,4-dihydroxyphenylalanine. The resolutions were effected by digestion of the racemic ethyl esters with a-chymotrypsin at pH 5.0 (pH-stat). The L-isomers were crystallized from the concentrated digests; the D-isomers were obtained by saponification of the remaining D-esters after extraction into ethyl acetate. Analysis of the isomers by column chromatography indicated optical purities greater than 99.5%. The racemic esters were prepared with ethanol / hydrogen chloride and were used without isolation. TONG, J. H., PETITCLERC, C., D'IORPO, A., et BENOHTON, N. L. Resolution of ring-substituted phenylalanines by the action of a-chymotrypsin on their ethyl esters. Can. J. Biochem. 49,877-881 (1971). Du mtlange radmique, nous avons prkpart, avec des rendements variant de 60% & 80%, les isomeres D et L des phknylalanines suivantes substituQs dans le noyau: tyrosine, o-tyrosine, m-tyrosine, p-chlorophknylalanine, p-fluorophknylalanine, et 3,4-dihydroxyphknylalanine. Les rtsolutions sont effectuks par digestion des esters Cthyliques ractmiques avec I'a-chymotrypsine a pH 5.0 (pH-stat). Les L-isom&res sont cristallisks A partir des produits concentrb de la digestion les D-isom&res sont obtenus par saponification des D-esters qui restent apres extraction dam l'adtate d'tthyle. L'analyse des isomkres par chrsmatographie sur colonne indique des puretts optiques plus grandes que 99.5%. Nous avons prtpart les esters rackmiques avec I'tthanol / chlorure d'hydrogene et nous les avons utilkts sans isolation.A recent study by us of the metabolism of the optical isomers of o-hydroxyphenylalaninae (o-tyrosine) required the resolution of the "Clabelled racemate (1). This was accomplished by use of the stereospecific hydrolytic action of a-chyrnotrypsin on the racemic amino acid ester at pH 5 (2). The simplicity and other attractive features of this method have prompted us to apply it to the resolution of other ring-substituted phenylalanines. This paper describes the results of these experiments. A kinetic study of the interaction of a-chymotrypsin with carboxyl-substituted phenylalanines has already been published (3). ExprfmewtalMaterials DL-@-Tyrosine and 3,4-dihydroxy-DL-phenylalaniloe (DL-Dopa) were purchased from Mann Research Laboratories, Orangeburg, N.Y.; DL-m-tyrosine, from Moch-

Detection of Hepatic Phenylalanine 4-Hydroxylase in Classical Phenylketonuria

“…These results clearly rule out o-tyrosine as the reaction product. To determine if the product was p-or mtyrosine, the reaction mixture was subjected to column chromatography on an aminoacid analyzer under conditions reported to separate m-and p-tyrosine (17). A peak of radioactivity was recovered that cochromatographed with added, unlabeled, p-tyrosine (Fig.…”

Enzymatic resolution of amino acids via ester hydrolysis

“…N-Unprotected esters also have been used as substrates. Thus, ethyl esters of ring-substituted phenylalanines were resolved with a-chymotrypsin at pH 5 (to eliminate the danger of spontaneous hydrolysis of the esters), yielding the L-amino acids and unchanged D-esters in very high enantiomeric excess (Tong et al, 1971). The examples of a-chymotrypsin-catalyzed resolution of amino acids reported before 1980 are compiled in some review articles (Jones and Beck, 1976;Gais, 1995).…”

Purification, Characterization, and Molecular Cloning of A Novel Rat Liver Dopa/Tyrosine Sulfotransferase

“…Quantitative analysis showed that although L-p-tyrosine represents the predominant species, L-m-tyrosine constitutes a significant amount (2.8%) of the total tyrosine circulating in blood (46). Using bovine adrenal medulla extract or rat brain homogenate, it has been demonstrated that L-m-tyrosine was produced through the meta-hydroxylation of L-phenylalanine (48,49). Furthermore, in vivo studies have shown that L-mtyrosine could be converted to L-Dopa (50 -52) or m-tyramine (47,53), a decarboxylated product of L-m-tyrosine with neurotransmitter activity.…”