Phenylalanine Methyl Ester Research Articles

Effects of diabetes on cardiac lysosomes and protein degradation.

Rates of proteolysis by hearts obtained from alloxan diabetic rats and perfused as working preparations with buffer simulating control sera were accelerated 30% above identically perfused control hearts. The total homogenate activities of cathepsin D and beta-N-acetylglucosaminidase, assayed in the presence of Triton X-100, decreased 15-35% in diabetic heart, but the activities of these lysosomal enzymes assayable in the absence of detergent were unchanged in the diabetic tissue. The effects of diabetes were examined further by centrifugation of particulate fractions from subtilisin-treated hearts of control and diabetic rats on polyvinylpyrrolidone-coated colloidal silica (Percoll) gradients. Two species of lysosomes were resolved on the basis of their densities. Both dense and buoyant lysosomes accumulated radioactivity when hearts were exposed to [14C]phenylalanine methyl ester. Dense lysosomes (1.06-1.09 g/ml) sedimented with mitochondria while buoyant lysosomes banded with Golgi and sarcolemmal particles (1.02-1.03 g/ml). When particulate fractions of hearts from diabetic animals were layered on the Percoll gradients, total activities of beta-N-acetylglucosaminidase and cathepsin D were decreased from control in buoyant lysosomes, but unchanged in dense lysosomes. These results demonstrated that the increase in proteolysis in the diabetic heart was associated with decreased total activity and latency of cathepsin D and beta-N-acetylglucosaminidase and an increased proportion of dense lysosomes in the particulate fraction.

Peptide bond formation by intermolecular aminolysis of d-glucopyranosyl esters of amino acids

The reaction of HO-protected and -unprotected d-glucopyranosyl esters of N-acylamino acids (Gly, Ala, Phe) with glycine and phenylalanine methyl esters in N, N-dimethylformamide at 38° and dichloromethane at 40°, respectively, led to rupture of the C-1 ester bond and formation of the corresponding N-acyldipeptide methyl ester. The relative reactivity of the C-1 ester bond toward aminolysis was greatly influenced by the structure of the amino acid nucleophile, the nature of the aglycon side-chain group, and the anomeric configuration of the d-glucopyranosyl ester involved. Evidence for a substantially lower acylating efficiency of the ester at C-2, as compared to that at C-1, was obtained by aminolysis of two fully acetylated 2- O-(acylaminoacyl)-β- d-glucopyranoses. Treatment of 1- O-(glycylglycylglycyl)-β- d-glucopyranose with phenylalanine methyl ester in N, N-dimethylformamide led to parallel hydrolysis and intermolecular aminolysis, to give the tripeptide and tetrapeptide methyl ester.

Inhibition of estrogen binding to rat alpha-fetoprotein by tryptophan p-nitrophenyl esters

Rat alpha-fetoprotein (AFP) contains a site that both binds the protease substrate tryptophan methyl ester (TrpOMe) and influences estrogen binding. We have studied the effect of changing the amino acid and ester portions of this compound on its binding to AFP, as measured by the ability of the ester to inhibit binding of [ 3H]-estrone to AFP. We find that: (1) AFP binds tryptophan esters better than phenylalanine or tyrosine esters, (2) substitution of butyl or benzyl for the methyl group in TrpOMe increases binding for AFP by 30- to 100-fold, (3) substitution of p-nitrophenol at the ester position increases binding for AFP by 10 5, (4) p-nitrophenyl substitution in the ester position of tyrosine or phenylalanine methyl ester increases the affinity for AFP by 10 3 to 10 4, (5) inhibition of estrogen binding to AFP by tryptophan p-nitrophenyl esters is reversible and competitive, and (6) the hydrolysis products of tryptophan p-nitrophenyl ester are ineffective in inhibiting estrogen binding to AFP. Based on these results, we suggest that AFP contains a tryptophan ester recognition site which binds p-nitrophenyl esters with high affinity and influences estrogen binding. The ester binding site may be located spatially near the estrogen binding site.

The Enantioface-differentiating Methylation of the N-Benzylidene-Dl-phenylalanine Methyl Ester in the Presence of Chiral Lithium Amides

Abstract The asymmetric methylation of the N-benzylidene-Dl-phenylalanine methyl ester was carried out in the presence of lithium salts of secondary amines derived from (S)-proline. The lithium amides of poly(imino-1-isobutylethylene) and its corresponding low-molecular-weight model compound, derived from (S)-leucine, were similarly used in order to examine the polymer effects with regard to the stereoselectivity.

Synthetic approaches to versatile hemoprotein model compounds built from porphyrins and peptides

Several strategies for the preparation of mono- and bi-functional porphyrins (amino and carboxylate) to which oligopeptides can be attached have been investigated. A porphyrinyl amino-acid derivative has been synthesised by coupling a porphyrin monopropionate with phenylalanine methyl ester. Routes to pyrroles bearing butoxycarbonylaminoethyl side-chains via Curtius-type degradations, are described, and their potential for elaboration into porphyrins is discussed. The most promising bifunctional porphyrin with differentially protected amino and carboxylate side-chains was found to be the butoxycarbonylhydrazido methyl ester derivative of mesoporphyrin-II.

A direct preparation of acetyl-(S)-phenylalanyl-(S)-phenylalanine methyl ester by a double asymmetric hydrogenation

Acetyl-(S)-phenylalanyl-(S)-phenylalamine methyl ester [Ac(S)Phe(S)PheOMe] is obtained with high diastereoselectivity and enantioselectivity by catalytic hydrogenation, using [Rh(dipamp)(cod)]+BF–4(dipamp =R,R-1,2-bis[(2-Methoxypheny) phenylphosphino] ethane, cod = cyclo-octa-1,5-diene) as chiral catalyst, of a substrate containing two prochiral doubly bonded carbon atoms, which is easily available from glycine and benzaldehyde.

Synthesis of L-aspartyl-.DELTA.z phenylalanine methyl ester

Synthesis of L-aspartyl-.DELTA.z phenylalanine methyl ester

Proteases as catalysts of enantioselective α-amino ester hydrolysis: 3. Pronase-catalysed hydrolysis of methyl esters of leucine, phenylalanine and valine

An investigation of the hydrolysis of various substrates ( l-Trp-OMe, l-Phe-OMe, l-Leu-OMe and l-Val-OMe) by pronase has shown that the highest activity is displayed with l-Leu-OMe substrate. Addition of Ca 2+ ions significantly enhances the rate of hydrolysis of l-Leu-OMe without affecting the hydrolysis of d-Leu-OMe. Thus, the enantioselectivity of the process is improved.

SIMPLE EVALUATION OF ENANTIOMER-SELECTIVITY OF CROWN ETHER USING MEMBRANE ELECTRODE

Abstract Enantiomer-selectivity of the crown ethers I through IV was evaluated by a membrane electrode method. Only chiral crown ether II showed some enantiomer-selectivity for both hydrochloride salts of phenylalanine methylester and phenylethylamine. This method was considered to be a convenient one for screening the enantiomer-selectivity of chiral crown ethers.

Determination of α‐L‐aspartyl‐L‐phenylalanine methylester hydrochloride in soft drinks

AbstractAnalytical evaluation of the artificial sweetener USAL (α‐L‐aspartyl‐L‐phenylalanine methylester hydrochloride) was carried out using a modified technique of amino acid analysis. The determination was performed using a functionally simplified semiautomatic amino acid analyser. The device was constructed using the accessories of the automatic amino acid analyser AAA 888 (Mikrotechna Praha).

The interaction of alpha-N-(p-toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester with thrombin and trypsin.

The syntheses are described of p-guanidino-L-phenylalanine and some of its derivatives. alpha-N-(p-Toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester is an excellent substrate of bovine trypsin (EC 3.4.21.4) (Km 57 micron; kcat. 320s-1 at pH 7.4-8.0) and a very poor substrate of human thrombin (EC 3.4.21.5) (Km 190 micron, kcat. 0.2s-1) and bovine chymotrypsin (EC 3.4.21.1). The ester inhibits thrombin clotting activity. It also inhibits the amidase and esterase activities of human thrombin, this inhibition being of the mixed type. The inhibition constant, K1, of the order of 1 micron, increases with increasing inhibitor concentration. This suggests that the enzyme binds the inhibitor at multiple sites. The importance of the residue at the P1 position [notation of Berger & Schechter (1970) Philos. Trans. R. Soc. London Ser. B 257, 249-264] in determining the selectivity of a substrate or quasi-substrate among trypsin-like enzymes is borne out. p-Guanidino-L-phenylalanine may have a use in the synthesis of selective peptide inhibitors of thrombin.

Carboxamidomethyl esters as reactive substrates for alpha-chymotrypsin. Orientational effects of hydrogen-bonding interactions.

Effects of hydrogen-bonding interactions of amide groups on reactivity of esters to alpha-chymotrypsin were studied. Of the methyl esters studied, only that from acetyl-L-phenylalanine has k3 rate-limiting. In methyl beta-phenylpropionates an alpha-acetamido substituent increased k2 greater than 550 times, k3 approximately 5 times; an alpha-acetylclycyloxy substituent increased k2 approximately 2 times, k3 approximately 6 times, both in comparison with the alpha-acetoxy esters. Essentially all carboxamidomethyl esters studied have k3 rate-limiting; reactivity to hydroxide is only 4 times that of methyl esters. In alpha-substituted beta-phenylpropionates, carboxamido-methyl esters show k2 values greater than 110 times greater than 280 times, greater than 26 times, and 7 times the k2 values of the methyl esters for the alpha substituents, acetoxy, acetylglycyloxy, hydroxy, and hydrogen, respectively. In esters of alpha-acetamido acids, carboxamidomethyl esters show k2 values 44 times, greater than 110 times, greater than 12 times, and approximately 33 times the k2 values of the methyl esters of glycine, alanine, leucine, and phenylalanine, respectively. Cyanomethyl esters also had k3 rate-limiting. Hydrogen-bonding to the enzyme of either an alpha-acetamido group or a carboxamidomethyl group combined with bonding of the beta-aryl group, orients the hydrolyzing groups properly, increasing k2. Hydrogen-bonding of both alpha-acetamido and carboxamido-methyl groups is effective to a lesser degree. The amide group appears to have small effects on Ks as hydrogen bonding is balanced by desolvation. It is proposed that desolvation during bonding increases k2 and Ks.

Acyl intermediates in penicillopepsin-catalysed reactions and a discussion of the mechanism of action of pepsins.

Penicillopepsin catalyses transpeptidation reactions involving the transfer of the N-terminal amino acids of suitable substrates via covalent acyl intermediates to acceptor peptides, usually the substrate. The major products obtained when Phe-Tyr-Thr-Pro-Lys-Ala and Met-Leu-Gly were used as substrates were Phe-Phe and Met-Met respectively. With Met-Leu-Gly the tetrapeptide Met-Met-Leu-Gly was observed as probable intermediate. Co-incubation of Leu-Tyr-Leu and Phe-Tyr-Thr-Pro-Lys-Ala led to the formation of Leu-Phe and Phe-Leu as well as Leu-Leu and Phe-Phe. No reaction was observed with tripeptides in which the first or second amino acid is glycine. It appears that two amino aicds with large hydrophobic residues are needed for the transpeptidation reaction. Nucleophilic compounds other than peptides, such as hydroxylamine, aliphatic alcohols and dinitrophenylhydrazine, were not acceptors for the acyl group. Leucine, phenylalanine and leucine methyl ester also had no effect on the reaction. The transpeptidation reaction proceeded readily at pH 3.6 and 4.7. At pH 6.0 the reaction was slow and at pH 1.9 little or no transpeptidation was observed. Porcine pepsin catalyses similar transpeptidation reactions. Sequence studies show that porcine pepsin and penicillopepsin are homologous. The present study also suggests that they have a very similar mechanism. Evidence available at this time indicates that the mechanism of these enzymes is complex and may be modulated by secondary substrate-enzyme interactions. A hypothesis is presented which proposes that pepsin-catalysed reactions proceed via different covalent intermediates (amino-intermediates or acylintermediates) depending on the nature of the substrate. The possibility that some reactions do not involve covalent intermediates is also discussed.

Distinct patterns of inhibitor-induced shift-down responses in mammalian cells.

Shift-down responses in mammalian cells have been brought about by moderate concentrations of azide, cyanide and phenylalanine methyl ester. Early events of the response in L cells and in Ehrlich ascites carcinoma included a rapid preferential inhibition of protein and a more gradual inhibition of DNA synthesis; RNA synthesis was uninhibited initially. In contrast, the response in freshly explanted rat bone marrow cells or secondary cultures of mouse embryo cells was characterized by a parallel rapid inhibition of protein and RNA syntheses, and an even more severe inhibition of DNA synthesis. The striking difference in shift-down response could not be correlated with changes in precursor pools and appeared to stem from differences in the regulation of synthetic rates. These shift-down systems are therefore suggested as useful systems for studying the regulatory mechanisms governing macromolecular syntheses in mammalian cells.

The Effect of N-Substitution on the Rates of Saponification of Some Amino Acid Esters

The saponification rates (measured at 25 ° by a titrimetric method) of the unprotonated forms of the methyl esters of glycine, alanine, leucine, valine, and phenylalanine were compared with those of the N-methyl, the N-acetyl, and the N-acetyl, N-methylamino acid analogues. N-Acetylation slightly increased or decreased the rate but N-methylation caused a reduction by as much as a factor of ten, depending on the complexity of the side-chain. The esters of the N-acetyl, N-methylamino acids, which exist as cis and trans isomers, were saponified at rates intermediate between those of the esters of the N-acetylamino acids and N-methylamino acids. Activation parameters were obtained for the phenylalanine and leucine derivatives. N-Methylation resulted in an increase in ΔH≠ and ΔS≠ which was attributed in part to solvation effects. The hydrolysis of the cationic esters of glycine and alanine was still evident at pH 11.0. N-Methylation had little effect on the rates of saponification of the charged forms.

Studies on the Use of Trityl Group in Peptide Synthesis

An industrial method for the synthesis of peptides using a trityl group as the protecting group has been investigated.The derivatives of the amino acids, containing a liophilic group in their side chain, such as γ-methyl, ethyl and benzyl glutamate, β-methyl, ethyl and benzyl aspartate and S-trityl cystein, were N-tritylated directly in good yields with 2-equiv. of trityl chloride in aprotic solvents in the presence of triethyl amine. In these reactions, it was found that the α-carboxyl group of amino acids derivatives was simultaneously tritylated during the N-tritylating process.Using these trityl derivatives, glutathione (GSH), α-aminobenzyl penicilline (ABP) and aspartyl phenylalanine methyl ester (APAM) were prepared in good yields.

Substrate specificity of ribosomal peptidyl transferase. II. 2′(3′)- O-Aminoacyl nucleosides as acceptors of the peptide chain in the fragment reaction

Transfer of the acetyl- l-leucine (AcLeu) residue from AcLeu-pentanucleotide to synthetic substrates under conditions of the fragment reaction was used to study the specificity of the acceptor site of ribosomal peptidyl transferase. 2′(3′)- O-Aminoacyl nucleosides are the simplest acceptor substrates. Their acceptor activity is dependent on the nature of the nucleoside to which the amino acid residue is bound. The acceptor activity decreased in the sequence 2′(3′)-O- l-phenylalanyladenosine (A-(Phe)) > I-(Phe) > C-(Phe); U-(Phe) was inactive. The presence of a free 2′-hydroxyl group in the ribose moiety of the aminoacyl adenosines was important for the acceptor activity, as shown by the low activity of dA-(Phe) in comparison with A-(Phe). Acceptor activity was influenced by the nature of the side chain of the amino acid residue bound to adenosine: A-(Phe) was a more active acceptor than puromycin, while the acceptor activity of 2′(3′)- O-glycyladenosine A-(Gly) was very low. Free phenylalanine, phenylalanine methyl ester, and adenosine did not act as acceptors. As terminal products of the reactions of AcLeu-pentanucleotide with puromycin, A-(Phe), I-(Phe) and C-(Phe), we isolated AcLeu-puromycin, 2′(3′)-O- acetyl- l-leucyl- l-phenylalanyladenosine (A-(AcLeu-Phe)), I-(AcLeu-Phe) and C-(AcLeu-Phe), respectively.

Chemistry of amino acids. VII. The synthesis and spectral studies of some 2-piperidones bearing beta-keto ester groups.

Some 2-piperidones (IX and X) bearing β-keto ester groups were synthesized from the methyl esters (V) of alanine and phenylalanine according to a route similar to that described in our previous paper. Spectral studies (infrared, ultraviolet and nuclear magnetic resonance) revealed that these β-keto esters (IX and X) mainly exist in the chelated enolic tautomer (E) both in the solid state and in solution. The NMR spectra of some indole derivatives (XI-XVI) were also investigated.

An Aminoacyltransferase Preparation from Beef Liver

Abstract An aminoacyltransferase preparation has been obtained from a beef liver homogenate, and a 44-fold purification of the activity has been achieved. The enzyme preparation catalyzes both peptide synthesis from suitable amino acid esters (e.g. phenylalanine methyl ester, tyrosine methyl ester, leucine methyl ester) and the hydrolysis of such esters, the former process predominating at pH values near 7. Both actions are completely inhibited by diisopropyl phosphorofluoridate. Evidence is presented to show that in the formation of dipeptides (e.g. phenylalanylphenylalanine) from amino acid esters (e.g. phenylalanine methyl ester), the corresponding dipeptide ester is an intermediate. In peptide synthesis, the enzyme preparation exhibits side chain specificity with respect to both the acyl donor and the amine acceptor, but the specificity with respect to the amine acceptor appears to be more restricted, and limited to esters of certain L-amino acids. Amino acid amides and peptide amides corresponding to susceptible esters are resistant to enzymic action.