Leucine Derivatives Research Articles

Inhibition of tyrosine protein kinases by halomethyl ketones.

A chloromethyl ketone derivative of lactic acid was shown to inhibit protein phosphorylation in plasma membranes of Ehrlich ascites tumor cells [Johnson, H. J., Zimniak, A., & Racker, E. (1982) Biochemistry 21, 2984-2989]. We now show that this inhibitor as well as three halomethyl ketone derivatives of amino acids and peptides specifically inhibits tyrosine protein kinase activity in intact plasma membranes and Triton extracts of plasma membrane of A-431 tumor cells. The most effective inhibitor is a bromomethyl ketone derivative of leucine that inhibits the phosphorylation of a protein that migrates to the same position as the EGF receptor in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of phosphorylation took place in the presence or absence of added EGF, and the inhibitor did not interfere with the binding of EGF to the receptor nor with the dephosphorylation of the EGF-stimulated phosphoprotein. EGF-dependent phosphorylation in a Triton extract of plasma membranes from normal placenta was considerably less sensitive to the bromomethyl ketone derivative of leucine. The tyrosine protein kinase activity of the transformation gene product of Fujinami virus was particularly sensitive to the bromomethyl ketone derivative of leucine, while the src gene product of Rous sarcoma virus was comparatively less sensitive. The bromomethyl ketone inhibitor interfered with the phosphorylation of the EGF receptor by [gamma-32P]-8-azido-ATP but much less with the light-sensitive binding. This observation and the lack of interference with EGF binding suggest that the inhibitor interacts with the protein kinase portion of the receptor complex.

13C NMR sequence analysis, 21. Stereoselectivity of oligopeptide syntheses

AbstractVarious diastereomeric N‐protected dipeptide methyl esters were synthesized from N‐protected racemic amino acids and racemic amino acid methyl esters. Derivatives of alanine, valine, leucine, and phenylalanine were condensed in various solvents including optically active solvents such as O‐acetyl lactic acid ethyl ester, N‐acetyl lactic acid ethyl ester, or N‐acetyl L‐alanine methyl ester. The N‐protected amino acids were activated by means of 2‐ethoxy‐1‐ethoxycarbonyl‐1,2‐dihydroquinoline (EEDQ), dicyclohexylcarbodiimide and 4‐chlorothiophenol, 1,1‐carbonyldiimidazole, 1‐diethoxyphosphino‐1,3,4‐triazole, chloroacetonitrile, and isobutyl chloroformate. The reaction mixtures containing four stereoisomers were analyzed by means of 13C NMR spectroscopy with respect to the mole ratios of the diastereomeric dipeptides. The stereoselectivity of all experiments was low. The number of experiments favoring the formation of L‐L (D‐D) sequences exceeded by far the number of those favoring L‐D (D‐L) sequences. Tripeptide syntheses, conducted under the reaction conditions of various dipeptide syntheses, revealed that the stereoselectivity is influenced by chiral neighboring groups. However, these “penultimate effects” are not stronger than solvent effects.

Purification, characterization and possible function of α- N-acylamino acid hydrolase from bovine liver

α- N-Acylamino acid hydrolase from bovine liver has been purified approx. 1700-fold with a yield of 28%. Based on the results of studies using polyacrylamide gel electrophoresis the purified enzyme is homogeneous. This enzyme catalyzes the hydrolysis of α- N-acylated amino acids to yield the acyl group and the corresponding amino acid, α-Acetylmethionine was the most rapidly hydrolyzed substrate tested for this enzyme. Most of the other α- N-acylated amino acids used as substrates were hydrolyzed. Some, however, were cleaved at less than 1% of the rate observed for N-acetylmethionine, α- N-Acylamino acid hydrolase did not catalyze the hydrolysis of N- acetyl- d-alanine , N-acetyl-β-alanine, ϵ- N-acetyllysine, N-acetylglycinamide or α- N-acetylated peptides. When incubated with this enzyme the chloroacetyl derivatives of leucine and valine were hydrolyzed approx. 8- and 3-times, respectively, more rapidly than the corresponding N-acetyl derivatives. On the other hand, N-formylmethionine was hydrolyzed at only 60% the rate of N-acetylmethionine. Other studies on α- N-acylamino acid hydrolase have shown that it is a dimer composed of two 40 000 molecular weight monomers, it is active over a broad pH range with maximal activity at approx. pH 8.5, and requires Zn 2+ for enzymatic activity. The substrate specificity and other properties of bovine liver α- N-acylamino acid hydrolase are very similar to those reported for acylase I isolated from porcine kidney [7]. We suggest that this enzyme functions in the catabolism of α- N-acetylamino acids resulting from the turnover of α- N-acetylated proteins.

Infrared spectra of N-acetyl-L-α-amino-acid N'-methylamides with aliphatic side chains in non-polar solvents

Infrared spectra of the L-alanine (I), L-leucine (II), L-valine (III) and L-tert-leucine (IV) N-acetyl N'-methylamides were measured. Amides I-IV are not self associated in tetrachlormethane in the concentration 2 . 10-5 mol l-1 at room temperature and in tetrachloroethylene in the concentration 1.5 . 10-4 mol l-1 at temperatures above 65° C. True conformational changes are observable only with the least flexible amide IV which exists at room temperature in a C5 conformation. This conformational type is also highly populated in the valine derivative III, but is less important in the alanine and leucine derivatives I and II in which the intramolecularly bonded C7 and the distorted hydrogen-nonbonded conformations contribute seriously.

Inhibition of enkephalin-degrading enzymes from rat brain and of thermolysin by amino acid hydroxamates

Benzyloxycarbonyl derivatives (Z) of amino acid hydroxamates have been found to inhibit the bacterial metalloendopeptidase thermolysin and enkephalin-degrading enzymes from rat brain. The hydroxamate derivatives of glycine, leucine, phenylalanine and D-phenylalanine inhibit thermolysin with K I values in the range of 3–23 μM. They also inhibit the enkephalin-degrading endopeptidase (enkephalinase) and aminopeptidase with different efficiencies, depending on the structure of the amino acid employed. Thus, Z-Gly-NHOH inhibits the enkephalinase and aminopeptidase with IC 50 values of 1 μM and 300 μM, respectively, whereas Z-D-Phe-NHOH inhibits the corresponding enzymes with IC 50 values of 0.2 μM and 1.5 μM.

Two-Step covalent immobilization of enzymes as a way for study of effects influencing catalytic activity

Isothiocyanatopropyl derivatives of trypsin and pepsin obtained by treatment with 3isothiocyanato-1-propyl isocyanate (in the first step) can be covalently bound to carriers having amino groups. Besides all the general principles valid for reactions of isothiocyanates with amines, prerequisite for the satisfactory coupling properties of isothiocyanatopropyl derivatives are (a) a sufficiently high modification degree with isothiocyanate, and (b) a concentration excess of NH2 carrier groups. With respect to the coupling properties, nonspecific sorptions and the catalytic properties of the immobilized trypsin isothiocyanatopropy l derivatives, the better carrier appears to be the cross-linked polyethy leneimine. Isothiocyanatopropyl, 3-isothiocyanato-bromopropyl, and isothiocyanatopenty l derivatives of leucine, insulin, and albumin also have good coupling properties. The mode in which the amidolytic and caseinolytic activity of isothiocyanatopropyl derivatives of trypsin was influenced after its immobilization on polyethyleneimine clearly indicated that the functional groups of the enzyme did not participate in the coupling to the carrier. Moreover, no intermolecular reactions were observed during the immobilization process. On the other hand, the conformational changes of the protein molecule are important, since they probably influence the changes in the catalytic properties of modified enzymes after their immobilization.

Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues.

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.

ChemInform Abstract: ANTINEOPLASTIC AGENTS. 2. STRUCTURE‐ACTIVITY STUDIES ON N‐PROTECTED VINYL, 1,2‐DIBROMOETHYL, AND CYANOMETHYL ESTERS OF SEVERAL AMINO ACIDS

AbstractDie analog der vorstehenden Arbeit (S. 1578 ff.) aus den Aminosäuren hergestellten Derivate (I) bis (IV) werden auf tumorhemmende Wirkung untersucht.

Antineoplastic agents. 2. Structure-activity studies on N-protected vinyl, 1,2-dibromoethyl, and cyanomethyl esters of several amino acids.

Previously reported work on N-protected activated esters of phenylalanine has been extended to include N-protected vinyl, dibromoethyl, and cyanomethyl esters of several other amino acids. These compounds have been synthesized and evaluated in Ehrlich ascites carcinoma, Walker 256 carcinosarcoma, and and P388 lymphocytic leukemia tests. Among compounds tested were derivatives of tyrosine, tryptophan, glycine, leucine, proline, aspartic acid, glutamic acid, 4-aminobutyric acid, and 6-aminocaproic acid. Compounds of greatest potential interest from this study are N-carbobenzoxyglycine 1,2-dibromoethyl ester and N-carbobenzoxy-L-leucine 1,2-dibromoethyl ester. Both compounds were highly active in Ehrlich ascites test systems (33 mg/kg/day). The glycine derivative was also active in the Walker 256 test (2.5 mg/kg/day. Values for LD50's in mice were 148 mg/kg (0.37 mmol/kg) and 225 mg/kg (0.50 mmol/kg) for glycine and leucine derivatives, respectively; therefore, these compounds do not appear to be toxic at effective dose levels.

On the derivatives for the ultramicrodetermination of amino acids by mass fragmentography

The fragmentation of eight volatile derivatives of alanine, valine, leucine, proline, and glutamic acid upon electron impact is studied for the concurrent ultramicrodetermination or the selective identification of amino acids by mass fragmentography. The relationship between the sensitivity of mass fragmentography and Σ 10% values (percentage of the total ionization over m e ) of N-trifluoroacetyl- l-prolyl, N-benzoyl, and N-pentafluorobenzoyl derivatives of alanine, valine, leucine, and proline is also investigated. The results obtained in this study suggest that N-trifluoroacetyl- l-prolyl n-butyl ester, N-benzoyl n-butyl ester, N-pentafluorobenzoyl n-butyl ester, and N-trifluoroacetyl l-menthyl ester derivatives can be used for the concurrent ultramicrodetermination or selective identification of amino acids by mass fragmentography because these four derivatives produce common intense fragment ions at m e 166, 105, 195, and 83 or diagnostic ions, respectively. Although N-trifluoroacetyl- l-4-thiazolidinecarbonyl n-butyl ester, N-trifluoroacetyl n-butyl ester, N-trimethylsilyl n-butyl ester, N-trimethylsilyl l-menthyl ester, and trimethylsilylated derivatives do not produce the common intense fragment ions, these derivatives are useful for the selective identification of ultra amounts of amino acids using mass fragmentography monitored the diagnostic ions, respectively. The general common rules between sensitivity and Σ 10% values were not adhered among the derivatives studied.

Reversible Inhibition of Carboxypeptidase A. IV. Inhibition of Specific Esterase Activity by Hippuric Acid and Related Species and other Amino Acid Derivatives and a Comparison with Substrate Inhibition

The anions of each of the following carboxylic acids exhibit uncompetitive inhibition of the hydrolysis of O-hippuryl-L-3-phenyllactic acid by bovine carboxypeptidase A at pH 7.5, 25°, ionic strength 0.2: hippuric acid, p-chloro- and p-nitrohippuric acids, hippurylglycine, carbobenzoxyglycine, phenaceturic acid, N'-(3-phenylpropanoyl)glycine, benzoxyacetic acid, 3-benzoylpropanoic acid, and O-hippuryl-D-mandelic acid. In each case, this uncompetitive inhibition is consistent with the ordered binding of substrate and inhibitor to the enzyme; i.e. the inhibitor binds to E.S but not to the free enzyme. Evidence is presented for the binding site for uncompetitive inhibitors being the same as for inhibitory ester substrate molecules. Comparison of the specificities of uncompetitive inhibitors and esters which display substrate inhibition provides evidence for a critical conformational change which controls the binding of uncompetitive inhibitors and inhibitory substrate molecules.D-Phenylalanine, D-leucine, D-p-nitrophenylalanine, glycyl-L-tyrosine, glycyl-L-phenylalanine, and glycyl-L-leucine are competitive inhibitors of the enzymic hydrolysis of O-hippuryl-L-3-phenyllactic acid, whereas the N-chloroacetyl derivatives of L-tyrosine, L-phenylalanine, and L-leucine are noncompetitive inhibitors. For the above D-amino acids, glycyl dipeptides, and N-chloroacetyl amino acids, the phenylalanine derivative in each case is a considerably stronger inhibitor than the corresponding leucine derivative. This preference is similar to that observed for the binding of peptide substrates but the reverse of that observed for ester substrates and simple mono- and dicarboxylate ion inhibitors.The peptide substrates carbobenzoxyglycylglycyl-L-phenylalanine and N-chloroacetyl-L-phenylalanine are noncompetitive inhibitors of the enzymic hydrolysis of O-hippuryl-L-3-phenyllactic acid. This clearly demonstrates the presence of different ester and peptide binding sites in this enzyme, which is consistent with conclusions from recent studies in other laboratories.

The essentiality of sulfhydryl groups to transport in Neurospora crassa

Three active transport systems for amino acids and another for glucose in germinated conidia of Neurospora were all inactivated during incubation with the sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, and iodoacetamide. Where tested, the inactivation by p-chloromercuribenzoate was reversed by the action of dithiothreitol. However, two other sulfhydryl reagents, the chloromethyl ketone derivatives of leucine and lysine, were found to have differential effects on the various transport systems. Inactivation by these two compounds did not occur at the active site of the permeases by affinity labeling, suggesting differential availability of sulfhydryl groups among the transport systems. Also, inactivation by these reagents was greatly enhanced in the presence of p-chloromercuribenzoate.

Substitution of Five Essential Amino Acids by their Alpha-keto Analogues in the Diet of Rats

Weanling male rats were fed diets containing essential amino acids as the sole source of nitrogen. Individual amino acids were replaced by equimolar quantities of their alpha-keto analogues ( as sodium or calcium salts ), with and without sufficient glutamate to make the diets isonitrogenous. Growth and feed efficiency in rats receiving the keto analogue of methionine were the same as in controls receiving L-methiOnine or DL-methionine with or without neomycin. Urea excretion increased compared with con trols fed L-methionine. Substitution of L-phenylalanine, L-leucine or L-valine by its keto analogue reduced feed efficiency by approximately 24%, 33%, and 50%, respectively; urea excretion rose correspondingly. In the case of the keto derivative of leucine, urea clearance doubled. When L-methionine, L-@phenylalanine, L-leucine, L-iSoleucine, and L-valine were simultaneously replaced by their keto analogues ( as calcium salts) iso nitrogenously, growth and feed efficiency were reduced 48% and 41%, respectively. Addition of neomycin to the diet did not alter these results significantly, suggesting that intestinal bacteria may not be important in ketoacid utilization. Although nitrogen excretion as a fraction of dietary nitrogen was increased, blood urea fell sharply; urea clearance again doubled. Plasma amino acid concentrations showed depression of valine and leucine, maintenance of phenylalanine, methionine, and isoleucine, and the ap pearance of alloisoleucine. The activities of urea cycle enzymes measured in liver homogenates were unchanged except for increased carbamyl phosphate synthetase and ornithine transcarbamylase in those rats given neomycin plus the keto analogues. The results show that these analogues can, with varying degrees of efficiency, replace the corresponding amino acids in the diet, even in the presence of intestinal bacteriostasis. The keto analogue of leucine appears to increase the renal clearance of urea. J. Nutr. 104: 1208â€1214, 1974.

Syntheses and Reactions of Optically Active Polymers. I. Syntheses and Polymerizations of N-Vinylbenzyl-l-amino Acid Derivatives

Abstract N-Vinylbenzyl-l-amino acids were synthesized by the reaction of l-amino acids with formylstyrenes and the subsequent reduction of the condensation products with sodium borohydride. The procedure was performed without protection of the various functional groups of amino acids and racemization was not accompanied by the above process. The method is useful for the synthesis of optically active vinyl monomers containing various amino acids with their intact functionality. As a representative l-amino acid, a variety of N-vinylbenzyl-l-leucine derivatives such as N-methyl-l-leucine, l-leucine ethyl ester, l-leucinol and N-formyl-l-leucine ethyl ester were prepared by means of chemical transformation. These optically active monomers of leucine derivatives were polymerized to optically active polymers.

Leucine and its Derivatives as Potential Elicitors of Insulin Secretion in Rats

This report describes the testing of a number of leucine derivatives for their ability to increase serum levels of glucose and immunoreactive insulin, Insulin levels increased in rats when L-leucine, DL-leucine, and L-leucineamide were administered. Esters of L-leucine caused no significant effect on glucose or insulin levels.

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.

Tetrazole analogues of amino acids and peptides IV : Resolution of racemic tetrazole analogues of N-benzyloxycarbonyl amino acids by means of hydrazide of L-tyrosine

Racemic tetrazole analogues of N-benzyloxycarbonyl derivatives of alanine, S-benzyl-cysteine, leucine, phenylalanine and valine were resolved by means of L-tyrosine hydrazide. The resolution of tetrazole analogue of N-benzyloxycarbonyl- DL-phenylalanine provided the L-enantiomer. From the other fractionation experiments tetrazole analogues of N-benzyloxycarbonyl- D-alanine, -S-benzyl- D-cysteine, - D-leucine, and - D-valine were obtained.

Isolation and properties of some reptilian and fish chymotrypsins

Chymotrypsinogens were isolated in highly purified form by a chromatographic procedure, from the pancreas of the turtles Chelydra serpentina and Pseudemys elegans. After activation with bovine trypsin, they both hydrolyze the substrate N- benzoyl- l- tyrosine ethyl ester about three times as rapidly as bovine chymotrypsin does. The rates of the inactivations by chloromethyl ketone derivative of l-phenylalanine and l-leucine were measured, on chymotrypsins from three reptiles and the tuna. From these, and from the relative activities on tyrosine and leucine substrates, a partial similarity to procine chymotrypsin C (which shows high rates with leucine derivatives), was discerned. It is therefore concluded that although the reactivity of the chymotrypsin active center is basically similar from the fish to the mammals, complex variations on the basic pattern in relation to the binding site are apparent in each species examined. A clear division into A and C types, as found in mammals, could not be made in the range of lower vertebrates investigated here. The alkylation and specificity evidence suggests that forms with combined features of both the A and C types have appeared in reptilian and fish species.

Deacylated Transfer Ribonucleic Acid as a Factor for Peptide Chain Initiation in Rabbit Reticulocyte Systems

Abstract Evidence is presented that is interpreted as reflecting a codon-directed specific requirement for deacylated transfer RNA in reactions associated in sequence with the initiation of polyphenylalanine synthesis in a fractionated transfer system derived from rabbit reticulocytes. The ability of deacylated transfer RNA to participate in these early reactions, as measured by a shift in the optimum Mg++ concentration for the synthesis of polyphenylalanine, is destroyed by mild oxidation with sodium periodate under conditions which destroy the ability of transfer RNA to accept amino acids. After periodate oxidation (to remove activity due to contaminating deacylated transfer RNA), preparations of transfer RNA containing phenylalanyl-tRNA or N-acetylphenylalanyl-tRNA are also inactive in these early reactions of chain initiation. Evidence is also presented that is interpreted as indicating that N-acylated amino acids may play no role in the initiation of α and β chains of globin. N-Formylmethionyl-tRNA was not detected in preparations containing methionyl-tRNA formed in intact reticulocytes. Similar results were obtained for the N-acetyl and N-formyl derivatives of phenylalanine, valine, alanine, leucine, and serine. Furthermore, very little formate, if any, is present as the NH2-terminal residues of incomplete globin peptide chains that are initiated in intact cells and that remain attached to polysomes during isolation. Most, if not all, of these nascent globin chains exist with valine with a free amino group as the NH2-terminal amino acid. It is suggested that the initiation of synthesis of globin peptides may involve a codon-directed requirement for deacylated transfer RNA, by a mechanism similar to that involved in the initiation of polyphenylalanine synthesis.

The hydrolysis of some N-benzoylamino acids in dilute mineral acid

The behavior of N-benzoylaspartic acid and N-benzoylglutamic acid has been investigated at 100° in dilute solution in water and aqueous hydrochloric acid within the pH range 3.1–0.5. Some data are presented concerning the hydrolysis of the N-benzoyl derivatives of alanine, β-alanine, leucine, glycine, serine, and β-ethanolamine in water, 0.1 N hydrochloric acid, and 2 N hydrochloric acid. Benzoylglutamic acid undergoes a pH-independent conversion into pyrrolid-2-one-5-carboxylic acid, which then hydrolyses in mineral acid to glutamic acid; however, N-benzoylaspartic acid, under similar conditions, hydrolyses much more rapidly by a route which does not involve the corresponding lactam as an intermediate. In anhydrous alcohols the solvolysis of N-benzoylaspartic acid gives mixtures of aspartic acid and the β ester.First-order rate constants have been obtained for the acid-catalyzed hydrolysis of pyrrolid-2-one-5-carboxylic acid (I) and azetidin-2-one-4-carboxylic acid (II) in water over this pH range.