Phenylalanine ethyl ester, hydrolysis

Detailed kinetic studies revealed that glycine methyl ester and phenylalanine methyl ester in glycine buffer at pH 7.3 undergo a facile hydrolysis catalyzed by cupric ion (11). Under these conditions the reactions closely follow a first-order rate law in the substrate. Using these kinetic data it is possible to compare the rates of hydrolysis of DL-phenylalanine ethyl ester as catalyzed by hydronium, hydroxide, and cupric ion (see Table III). 

Table III. Acidic, Basic, and Cupric Ion—Catalyzed Hydrolysis of DL-Phenylalanine Ethyl Ester at pH 7.3 and 25° C.

The enhanced reactivity in the cupric ion-catalyzed hydrolysis cannot be due solely to the electrostatic effect of an attack of hydroxyl ion on a positively charged a -amino ester, since the introduction of a positive charge, two atoms from the carbonyl group of an ester, increases the rate constant of alkaline hydrolysis by a factor of 103 (10), whereas there is a difference of approximately 106 between the cupric ion-catalyzed and the alkaline hydrolyses of DL-phenylalanine ethyl ester. The effective charge on the cupric ion-glycine (buffer)-ester complex is +1, so that the factor of 106 cannot be explained by an increase in charge over that present in the case of betaine. Furthermore, the reaction cannot be due to attack by a water molecule on a positively charged a-amino acid ester, since the rate constant of the acidic hydrolysis of phenylalanine ethyl ester is very small. It thus seems... 

Carbonyl oxygen exchange was found during the cupric ion-catalyzed hydrolysis of DL-phenylalanine ethyl ester-carbonyl-O18 at pH 7.3 (11). This indicates that an additional intermediate is formed in this reaction. A mechanism (II) consistent with both the kinetic evidence and the oxygen-exchange evidence is given below. 

MIP catalyst 76 also performs enantioselective hydrolysis of non-activated d-and L-phenylalanine ethyl ester 78 at pH 7.4, with characteristic saturation kinetics (Scheme 13.19). While this catalyst does not have the precise shape complementarity to ester 78, a threefold enhancement compared to the blank reaction was observed. Furthermore, enantiodiscrimination was obtained, as compound 78 was hydrolyzed 1.44-fold faster than ent-78. Although the reported rate enhancement was modest compared to the standards in asymmetric synthesis, it can be considered as a first step in the development of imprinted polymers for enantioselective catalysts. 

Propose a mechanism for the acid-catalyzed hydrolysis of phenylalanine ethyl ester. 

Cross-linked crystals from subtilisin exhibited 27 times less activity than soluble subtilisin in the hydrolysis of benzoyl-L-phenylalanine ethyl ester. Denaturation of the enzyme and restrictions from substrate-dependent internal diffusion were ruled out. A shift in the pH-dependence of the maximum activity to higher pH-values was observed which was explained by inter molecular electrostatic... 

A lipase has been used to convert solid triolein (glycerol trioleate) to the monooleate by treatment with glycerol at 8°C.75 Other lipases have been used in the hydrolysis and transesterification of oils, as well as in the esterification of fatty acids without solvents.76 Peptides can be produced from eutectic mixtures of amino acid derivatives with the addition of a small amount of solvent.77 Immobilized sub-tilisin and thermolysin were used with 19-24% water or an alcohol to produce polypeptides. Subtilisin on celite (a di-atomaceous earth) was used to convert a mixture of L-phenylalanine ethyl ester and L-leucinamide containing 10% triethyleneglycol dimethyl ether to L-phenylala-nineleucinamide in 83% yield. Addition of 30% 2 1 ethanol/water reduced the time needed from 40 to 4 h. The enzyme could be used three more times. These reaction mixtures contained 0.13-0.75 g peptide per g reaction mixture compared with 0.015-0.035 when the reaction was... 

Several features of the above studies were reinvestigated in a detailed kinetic study of the copper(II) complexes of glycine methyl ester and phenylalanine ethyl ester in glycine buffer at pH 7.3 (26). Glycine was selected as a buffer in this study in order that a small increase in the glycine concentration caused by the hydrolysis reaction would not increase the concentration of copper(II) complexes to a significant extent. It was found that the rate constant for the hydrolysis of the copper(II) complex of DL-phenylalanine ethyl ester was 106 times greater than the rate constant obtained for the alkaline hydrolysis of the free ester (25). 

We have shown by a comparison of the pH dependence of the step characterized by ki that the hydrolysis of the enzyme-acyl compound is the rate-determining step for the enzymatic hydrolysis of the usual amino acid amide substrates. In the case of chymotrypsin, acetyl-L-phenylalanine ethyl ester is hydrolyzed 1,000 times faster than the corresponding amide and in the case of trypsin, benzoyl-L-arginine ethyl ester is hydrolyzed 300 times faster than the corresponding amide. This suggests that for the amide hydrolysis too the second step, the acylation of the enzyme, must be the rate-determining step, since the third step is obviously identical for esters and amides of the same amino acid derivatives. The pH dependence of the chymotrypsin-catalyzed hydrolysis of acetyl-L-tyrosine ethyl ester and acetyl-L-phenylalanine ethyl ester indicates that for these reactions ki and kz are of the same order of magnitude and both contribute to the over-all rate, as shown by Equation (4). 

L-N-Phthalimido-p-aminophenylalanine ethyl ester when reacted with ethylene oxide yields an intermediate which on treatment with phosphorus oxychloride gives rise to 4-5/5-(2-chloroethyl)-amino-L-phenylalanine ethyl ester. This on hydrolysis with hydrochloric acid offers the desired compound. 

Belokon YN, Kochetkov KA, Plieva FM, Ikonnikov NS, Maleev VI, Parmar VS, Kumar R, Lozinsky VI (2000) Enantioselective hydrolysis of a Shiff s based of DL-phenylalanine ethyl ester in water-poor media via the reaction catalyzed with a-chymofrypsin immobilized on hydrophilic macroporous gel support. Appl Biochem Biotechnol 84 97-106... 

Some examples of DKR based on racemization of secondary alkyl amine via Shiff base were shown in Scheme 5.9. Schiffbase formation of a-amino carboxylic esters significantly increases the acidity of the a-proton in comparison to that of the parent amino acid, thus enabling enantiomer-selective hydrolysis and ammonoly-sis through DKR [23]. For example, chymotrypsin catalyses the hydrolysis of a Schiff base of phenylalanine ethyl ester and an aromatic aldehyde (Scheme 5.9, Equation 5.6) [23b]. In this particular case, natural phenylalanine precipitates, leaving the aldehyde and unhydrolysed enantiomeric ester in solution. Addition of l,4-diazabicyclo[2.2.2]octane (DABCO) (as the Bronsted base) allows DKR to take place by promoting racemization of the Schiffbase. Similarly, Novozym 435... 

In 2006, Kragl s group developed the DKR of a-amino acid esters (e.g. phenylalanine ethyl ester) in a water/acetonitrile mixture, leading to the corresponding optically active a-amino acids in good yields and optical purities (Scheme 3.45). The Alcalase catalysed hydrolysis of the ester was combined with an in situ racemisation catalysed by 3,5-dinitrosalicylaldehyde. 

The preparation of iodothyronines labeled in different positions is necessary for metabolic studies which are required for the interpretation of the peripheral action of the thyroid hormone. 3,5-Diiodo-L-thyronine has been synthesized (34) from 3,5-diamino-p-methoxyphenosy-J r-acetyl-L-phenylalanine ethyl ester (diazotization, decomposition of the diazotization product by -f I, and hydrolysis of the 3,5-diiodo-p-methoxyphenoxy-iV>acetyl-L-phenylalanine ethyl ester) and labeled in the 3 and 5 positions. 3, 5 -DITh is necessary for the preparation of some of the labeled tri-and tetraiodothyronine (thyroxine). 

Pish protein concentrate and soy protein concentrate have been used to prepare a low phenylalanine, high tyrosine peptide for use with phenylketonuria patients (150). The process includes pepsin hydrolysis at pH 1.5 ptonase hydrolysis at pH 6.5 to Hberate aromatic amino acids gel filtration on Sephadex G-15 to remove aromatic amino acids incubation with papain and ethyl esters of L-tyrosine and L-tryptophan, ie, plastein synthesis and ultrafiltration (qv). The plastein has a bland taste and odor and does not contain free amino acids. Yields of 69.3 and 60.9% from PPG and soy protein concentrate, respectively, have been attained. 

Similarly, lysine has been incorporated into gluten hydrolyzate and lysine, threonine and tryptophan have been individually incorporated into zein hydrolyzates. Lysine, methionine, and tryptophan were incorporated simultaneously into hydrolyzates of protein from photosynthetic origin. A very interesting application of this procedure involved the preparation of low-phenylalanine plasteins from a combination of fish protein concentrate and soy protein isolate by a partial hydrolysis with pepsin then pronase to liberate mainly phenylalamine, tyrosine, and tryptophan, which were then removed on sephadex G-15. Desired amounts of tyrosine and tryptophan were added back in the form of ethyl esters and a plastein suitable for feeding to infants afflicted with phenylketonuria was produced. 

Melphalan and the racemic analog have been prepared by two general routes (Scheme I). In Approach (A) the amino acid function is protected, and the nitrogen mustard moiety is prepared by conventional methods from aromatic nitro-derivatives. Thus, the ethyl ester of N-phthaloyl-phenylalanine was nitrated and reduced catalytically to amine I. Compound I was reacted with ethylene oxide to form the corresponding bis(2-hydroxyethyl)amino derivative II, which was then treated with phosphorus oxychloride or thionyl chloride. The blocking groups were removed by acidic hydrolysis. Melphalan was precipitated by addition of sodium acetate and was recrystallized from methanol. No racemization was detected [10,28—30]. The hydrochloride was obtained in pure form from the final hydrolysis mixture by partial neutralization to pH 0.5 [31]. Variants of this approach, used for the preparation of the racemic compound, followed the same route via the a-acylamino-a-p-aminobenzyl malonic ester III [10,28—30,32,33] or the hydantoin IV [12]. 

The specificity of chymotrypsin for hydrolysis of peptide bonds formed by the carbo,xyl groups of tyrosine, phenylalanine, and tryptophan has been recognized for some time (Green and Neurath, 1954 Desnuelle, 1960). Action on synthetic substrates of leucine (Goldenberg et al., 1951) and methionine (Kaufman and Neurath, 1949) also has been noted although at much slower rates than observed with the aromatic amino acid derivatives. When protein substrates or synthetic ester substrates are examined, it is evident that a variety of bonds can be hydrolyzed by chymotrypsin. Inagami and Sturtevant (1960) observed that chymotryptic hydrolysis of a-benzoyl-L-arginine ethyl ester, a typical trypsin substrate, occurred at a maximum rate which was 20% of that observed with trypsin. Several ester substrates, such as p-nitrophenylacetate (Hartley and Kilby, 1954), are also hydrolyzed. 

The O-benzylated aldehyde 517 was also coiu erted into the a,p-unsaturated ester 522 through Wadsworth-Emmons reactioiT-- with ethyl 2-(diethoxyphosphono)acetate5 ° in excellent yield (Scheme 88). The intermediate alkene 522 was subjected to Sharpless asymmetric dihydroxylalion " to afford the diol ester 523 in excellent yield and with a diastereoselectivity in excess of 95 5. Subsequent to alkali-catalyzed hydrolysis of 523, the carboxylic acid obtained was condensed with the p-lolucncsulfonalc salt of glycine benzyl ester or phenylalanine benzyl ester, by the action of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), to afford the benzyl-protected amide derivatives catalytic... 

Enzymes other than CAL B have also been reported to operate under the biphasic conditions. CAL A and CAL B or a lipase from Mucor miehei were tested for the kinetic resolution of glycidol using vinyl acetate or vinyl butyrate. The enzymes were used either suspended in the free ILs or immobilized, when the reactions were carried out in [EMIM][NTf2] [71]. CALAwas inactive, butthe other two enzymes showed activity, albeit at 10-20% of that in the absence of COj whether they were free or immobilized. In general, the supported enzymes showed superior performance [71]. Chymotripsin, a specific protease for aromatic amino acids, was found to catalyze the hydrolysis or transesterification of the ethyl ester of N-acetyl-phenylalanine with propanol in scC02-[RMIM][PF5] (R = butyl or octyl) with or without added water [Eq. (15)]. 

From a toxicological point of view, the most important representative of this group of mycotoxins is ochratoxin A, 3R)-N-[(5-chloro-3,4-dihydro-8-hydroxy-3-methyl-1-0X0-lH-2-benzo-pyran-7-yl)carbonyl]-L-phenylalanine (12-92), the molecule of which phenylalanine JV-substituted with a derivative of 3R)-3,4-dihydro-3-methylisocoumarine that contains at C-5 a chlorine atom to which are attributed the toxic effects of ochratoxin A. The incorporation of the chlorine atom into the skeleton of ochratoxin A is done by the action of chloroperoxidase the chlorine donor is inorganic chloride. Ochratoxin B (12-92) differs from ochratoxin A only in the absence of the chlorine atom, while ochratoxin C (12-92) is an ethyl ester of ochratoxin A. Ochratoxins a, p and y resulting from from parent compounds by the loss of phenylalanine caused by peptide bond hydrolysis, are virtually non-toxic, in addition to ochratoxin B, and are not routinely monitored. 

Kawabata et al. [28] resolved methyl ( )-Ar-(Boc-Z,-phenylalanyl)-m-2-aminocyclopentanecarboxylate by fractional crystallization from ethyl acetate. The phenylalanine derivative was prepared from the methyl ester of racemic m-2-ACPC (11) by acetylation with JV-Boc-Z.-phenylalanine using l-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC). Edman degradation of the separated isomers, followed by deprotection, acid hydrolysis and desalting with anion-exchange resin, gave enantiomerically pure (15,2/ )- and (l/ ,2S)-2-ACPC. The absolute configurations were proved by X-ray diffraction of the /.-phenylalanine derivatives [28]. 

BOC-(D-Phe)2-OMe was synthesized with 78% yield by using isobutyl chloroformate and triethylamine, from BOC-D-Phe which had been prepared from D-phenylalanine, BOC-ON and D-phenylalanine methylester. BOC-(D-Phe)4-OMe was synthesized with water soluble carbodiimide (WSCI) with 84% yield from BOC-(D-Phe)2 prepared by alkaline hydrolysis of BOC-(D-Phe)2-OMe and (D-Phe)2-OMe prepared from BOC-(D-Phe)2-OMe by removing the BOC group in 4N-HCl/ethyl acetate. (D-Phe)4 HCl was synthesized similarly with 80% yield from BOC-(D-Phe)4-OMe, first by alkaline hydrolysis and then by removal of the BOC group. Similarly, the following peptide derivatives were synthesized to characterize the enzyme Boc-D-Phe, D-Phe tert-butyl ester, (D-Phe)2 HCl, Boc-(D-Phe)2, (D-Phe)2 methyl ester-HCl, Boc-(D-Phe)2 methyl ester, (D-Phe)3-HCl, Boc-(D-Phe)3, Boc-(D-Phe>3 methyl ester, Boc-(D-Phe>3 tert-butyl ester, (D-Phe)4-HCl, Boc-(D-Phe)4, Boc-(D-Phe>4 methyl ester, L-... 

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