Phenylalanine Amide Research Articles

Molecular variants of cholecystokinin after endogenous stimulation in humans: a time study

The time-dependent release of molecular variants of cholecystokinin (CCK) into the circulation was studied before and 1, 2, and 4 h after a test meal in six healthy volunteers. At each time period, 100 ml of blood were drawn in a manner to inhibit CCK degradation. Plasma was formed and CCK concentrated by Sep-Pak C18 cartridge chromatography. Molecular variants of CCK and gastrin were well separated from each other by high-performance liquid chromatography (HPLC). Molecular forms of CCK and gastrin were measured by radioimmunoassay using an antibody that requires the presence of the carboxyl-terminal phenylalanine amide for full recognition, implying that biologically active forms were detected. HPLC elution positions of gastrin forms were determined using a gastrin-specific antibody. Chromatographic separation of CCK from gastrin forms was complete, allowing separate integration of gastrin and CCK forms. Therefore no subtraction of gastrin-like immunoreactivity from CCK-like immunoreactivity (CCK-LI) was necessary and CCK-LI could be directly determined. Peaks of CCK-LI were integrated in the column eluates and the plasma concentrations were calculated. Total plasma CCK-LI rose from a value of 2.4 +/- 0.6 pM before the test meal to 6.4 +/- 0.8, 6.6 +/- 0.9, and 5.8 +/- 1.2 pM 1, 2, and 4 h postprandially. The major molecular forms released into the circulation eluted on HPLC in the position of CCK-58 and CCK-39 (which coelutes with CCK-33). Minor amounts were detected in the position of CCK-8. There was no significant difference in the relative proportions of the molecular forms released at the different time periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and biological activities of some cholecystokinin analogues substituted in position 29 by a β‐alanine

Syntheses of analogues of the C-terminal heptapeptide of cholecystokinin are described. These analogues were obtained by replacing glycine 29 by a beta-alanine. The C-terminal phenylalanine amide was in some cases substituted by 2-phenylethyl alcohol and/or residues of the C-terminal tetrapeptide by their D-enantiomers. These compounds were tested for their action on stimulation of amylase release from rat pancreatic acini and for their ability to inhibit binding of labeled CCK to rat pancreatic acini and guinea pig brain membranes. Some of these derivatives behaved as CCK receptor antagonists.

ChemInform Abstract: Asymmetric Synthesis of Amino Acids by Catalytic Reduction of Substituted Acylaminoacrylic Acid Azlactones. Part 21. Catalytic Asymmetric Synthesis of N‐Acyl‐p‐difluoromethoxyphenylalanine Phenylethylamides.

AbstractCatalytic reduction of the azlactones (I) in the presence of (S)‐(‐)‐α‐phenylethylamine (S)‐(‐)‐(II) predominantly gives the phenylalanine amides (III) with a diastereomeric excess of the (SS) diastereomers up to 47% depending on the solvent.

Asymmetric synthesis of amino acids by catalytic reduction of azlactones of substituted acylaminoacrylic acids. 23. Study of the reductive aminolysis of 2-methyl-4-benzyllidene-?2-oxazolin-5-one with the chiral catalytic system pd-s-aminoalcohol

1. Reductive aminolysis of 2-methyl-4-benzylidene-Δ2-oxazolin-5-one in the chiral catalytic system PdCl2-S-phenylglycinol results in the formation of phenylalanine amides with an excess of the SS-diastereomer. 2. Reductive aminolysis in the catalytic system PdCl2-S-phenylalaninol mainly gives phenylalanine amides with an excess of the RS-diasteromer.

Structure‐activity relationships of CCK26‐33‐related analogues modified in position 33

In the sequence of the C‐terminal octapeptide of cholecystokinin, the phenylalanine amide residue in position 33 is of primary importance for the biological activity. Indeed, removal of Phe33‐NH2 is a modification known to induce antagonist properties. The influence of the chemical nature of the Phe33‐NH2 side chain on the biological activity of CCK8 was investigated through replacement of this residue by several amino acids with different lipophilic properties in the sequence of Boc(Nle28, Nle31)CCK27‐33, an equipotent analogue of CCK8. The binding properties of these new CCK‐related analogues: Boc(Nle28,Nle31,X33)CCK27‐33 were measured on both mouse brain and guinea pig pancreatic membranes, and their peripheral activities on amylase secretion and contractions of guinea pig ileum. Among the various peptides modified in position 33, Boc(Nle28,Nle31,Naa33‐NH2)CCK27‐33(Naa = naphthylalanine) and Boc(Nle28,Nle31,Cha33‐NH2)CCK27‐33(Cha = cyclohexylalanine) displayed high affinities for central and peripheral CCK‐receptors and proved to be full agonists of CCK8 in the peripheral tests while Boc(Nle28,Nle31,Ala33‐NH2)CCK27‐33 was completely inactive. This suggests that, at the level of the Phe33‐NH2 subsite, the critical factor for optimal interaction with CCK‐receptors is not the aromatic nature of the side chain but its size and hydrophobicity.

Purification and characterization of peptides with corticotropin-releasing factor activity from porcine hypothalami.

Ten polypeptides that stimulated the release of corticotropin from superfused rat pituitary cells and that are structurally related to porcine corticotropin-releasing factor were isolated from porcine hypothalami. The purification was carried out by gel filtration followed by reversed-phase HPLC using trifluoroacetic acid or heptafluorobutyric acid as the ion-pairing agent in water/acetonitrile solvent systems. The purified peptides were homogeneous by chromatography and by sequence analysis. One major polypeptide was characterized. Its structure is -H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Gl u-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2 [Patthy, M., Horvath, J., Mason-Garcia, M., Szoke, B., Schlesinger, D. H. & Schally, A. V. (1985) Proc. Natl. Acad. Sci. USA 82, 8762-8766]. This 41-amino acid sequence is thought to represent porcine corticotropin-releasing factor. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptides, amino acid analysis, and carboxypeptidase Y digestion, the other nine polypeptides were found to be structurally similar to this 41-amino acid sequence. Modifications of this structure include deamidation of glutamine at position 26 or 29, oxidation of methionine at positions 21 and/or 38, a blocked N terminus, and deletion of phenylalanine amide at the C terminus. Eight of these nine modified peptides retained significant corticotropin-releasing factor activity as shown by the stimulation of corticotropin release from superfused rat and pig pituitary cells. Some of these peptides may be present in pig hypothalami, while the others could have been produced during the isolation.

Pronase-catalysed hydrolysis of amino acid amides

Pronase from Streptomyces griseus exhibits a high activity and enantioselectivity in the hydrolysis of three amino acid amides: leucine amide, phenylalanine amide and tryptophan amide. The influence of pH, temperature and metal ions on pronase (EC 3.4.24.4) amidase activity and enantioselectivity has been studied.

Nuclear magnetic resonance observation and dynamics of specific amide protons in T4 lysozyme.

We have produced T4 lysozyme using a bacterial expression system which allows efficient incorporation of isotopically labeled amino acids in lysozyme. By using conditions that repress the expression of various transaminases, we have incorporated 15N-labeled amino acid into the five phenylalanine residues of the protein. The relatively large spin--spin coupling (87 +/- 3 Hz) between the 15N nucleus and the phenylalanine amide protons may then be exploited in a variety of ways to selectively observe the five phenylalanine amide proton resonances. These include a simple "echo difference" technique which displays the amide proton resonances in one dimension and a "forbidden echo" technique [Bax, A., Griffey, R. H., & Hawkins, B.L. (1983) J. Magn. Reson. 55, 301-335] which gives two-dimensional information allowing the proton and 15N chemical shifts of each amide to be determined. With these approaches, all five phenylalanine amide protons give resolved resonances. Deuterium exchange experiments demonstrate that three of the five resonances are slow to exchange (half-times of about 1 week at pH 5.5 and 4 degrees C) while the other two are rapid with complete exchange in hours or less. These observations correlate well with the secondary structure of the protein which shows three residues in alpha-helical regions and two residues in surface-exposed environments. This approach of isotopic substitution on nitrogen or carbon atoms is of general utility and should allow virtually any proton on a protein of molecular weight 20 000 or thereabout to be selectively observed.

ChemInform Abstract: STUDIES ON ANGIOTENSIN‐CONVERTING ENZYME INHIBITORS. I. SYNTHESES AND ANGIOTENSIN‐CONVERTING ENZYME INHIBITORY ACTIVITY OF 2‐(3‐MERCAPTOPROPIONYL)‐1,2,3,4‐TETRAHYDROISOQUINOLINE‐3‐CARBOXYLIC ACID DERIVATIVES

AbstractNach Veresterung der S‐Tetrahydroisochinolincarbonsäure (In) mit tert.‐Butanol und Benzylalkohol werden auf dem im Formelschema skizzierten Weg die N‐Acylderivate (IV) ‐′‐ (VI) hergestellt.

COOH-terminal fragments of cholecystokinin a new class of cholecystokinin receptor antagonists

COOH-terminal fragments of cholecystokinin varying in length from 1 to 3 amino acids and their NH 2-terminal butyloxycarbonyl derivatives were investigated for their ability to interact with the cholecystokinin receptor on dispersed acini from guinea pig pancreas. No fragment stimulated amylase secretion when present alone, but each of the butyloxycarbonyl derivatives and the COOH-terminal tripeptide amide inhibited the stimulation of enzyme secretion by cholecystokinin. In each case the inhibition was surmounted by increasing the concentration of cholecystokinin. Each fragment also inhibited binding of 125I-labeled cholecystokinin, with significant inhibition occurring with 30 μM butyloxycarbonyl tripeptide amide, 0.3 mM butyloxycarbonyl dipeptide amide, 10 mM butyloxycarbonyl phenylalanine amide and 3 mM tripeptide amide of cholecystokinin. In each case, there was a close correlation between the ability of the fragment to inhibit binding of 125I-labeled cholecystokinin and its ability to inhibit cholecystokinin-stimulated amylase release, cholecystokinin-stimulated 45Ca outflux and cholecystokinin-stimulated residual stimulation of amylase secretion. The inhibition of amylase secretion caused by the butyloxycarbonyl tripeptide of cholecystokinin was reversible and specific for those peptides which interact with the cholecystokinin receptor (i.e., cholecystokinin, caerulein, gastrin); it did not inhibit the actions of bombesin, carbachol, physalaemin, vasoactive intestinal peptide, secretin, PHI, ionophore A23187 or 8-bromo cyclic AMP. These results demonstrate that COOH-terminal fragments of cholecystokinin comprise a new class of cholecystokinin receptor antagonists.

Studies on angiotensin-converting enzyme inhibitors. I. Syntheses and angiotensinconverting enzyme inhibitory activity of 2-(3-mercaptopropionyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives.

(3S)-2-[(2S)-3-Mercapto-2-methylpropionyl]-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid [(3S), (2S)-6a] was prepared by the reaction of (3S)-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid tert-butyl ester [(3S)-2a] or benzyl ester [(3S)-2b] with 3-benzoylthio-2-methylpropionyl chloride (3a), followed by fractional crystallization and removal of the protective group. The absolute configuration of (3S), (2S)-6a was confirmed by X-ray diffraction analysis of the thiazepino [4, 3-b] isoquinoline compound (7) derived from 6a. Resolution of 3-benzoylthio-2-methylpropionic acid (8) was completed by using optically active phenylalanine amide as a resolving agent. The other optical isomers of (3S), (2S)-6a were prepared by the reaction of (3S)- or (3R)-2b with optically active 3a. The in vitro ACE inhibitory activity of each isomer of 6a was evaluated. Among them, (3S), (2S)-6a was found to be the most potent inhibitor with an IC50 value of 8.6×10-9M. Compound (3S), (2S)-6a induced a dose-dependent inhibition of the pressor response to angiotensin I after oral administration to normotensive anesthetized rats. Moreover, (3S), (2S)-6a markedly reduced the systolic blood pressure in renal hypertensive rats (RHR) and spontaneously hypertensive rats (SHR). The in vivo ACE inhibitory activity and the hypotensive effects of (3S), (2S)-6a were comparable to those of captopril.

Characterization of multiple forms of porcine anterior pituitary proopiomelanocortin amino-terminal glycopeptide.

Chromatography on a molecular sieve column of a preparation of porcine proopiomelanocortin N-terminal glycopeptide purified from anterior pituitary resulted in the isolation of three forms of the peptide with respective apparent Mr 21 000, 17 500, and 13 500 on polyacrylamide/sodium dodecyl sulfate gel. Determination of the amino acid composition of each peptide revealed that the form with a molecular weight of 17 500 corresponds to the 80 amino acid residue porcine N-terminal glycopeptide (PNT 1-80) previously characterized [Larivière, N., Seidah, N.G., & Chrétien, M. (1981) Int. J. Pept. Protein Res. 18, 487-491]. The forms with molecular weight of 21 000 and 13 500 correspond respectively to longer and shorter forms of the N-terminal glycopeptide. The high molecular weight form contains 107 amino acid residues. Sequencing of the fragments obtained after cleavage of the molecule with cyanogen bromide and Myxobacter Lys-C protease indicated that an extension of 27 amino acid residues is linked to PNT 1-80 through a -Lys-Arg-sequence. The sequence of the extension is reported. The low molecular weight form corresponds to the first 61 residues of PNT 1-80. Pronase digestion of the peptide and dansylation of the digest revealed the presence of a residue of phenylalanine amide at position 61. A general model for the maturation of the N-terminal glycopeptide of proopiomelanocortin in porcine anterior pituitary is presented.

Molecular cloning and nucleotide sequence of full-length of cDNA coding for porcine gastrin.

We have cloned in EScherichia coli a cDNA copy of mRNA coding for the porcine antral mucosal hormone preprogastrin. Full-length double-stranded cDNA was synthesized and inserted into the Pst I endonuclease site in plasmid pBR322 by using a homopolymeric extension technique. A partial cDNA clone was used as a probe to identify a complete cDNA clone in a cDNA library by colony hybridization. Four positive clones were isolated, one of which corresponded to porcine preprogastrin mRNA. The nucleotide sequence of the cDNA insert (602 nucleotides) revealed 312 nucleotides in the entire mRNA coding region, 61 nucleotides in the 5' untranslated region, 86 nucleotides in the 3'untranslated region, and a poly(A) tail of 86 nucleotides. Gastrin is located near the carboxyl end of preprogastrin and is flanked at both its amino and carboxyl ends by a pair of basic amino acid residues. The presence of glycine and a pair of basic amino acid residues adjacent in the carboxyl-terminal phenylalanine of gastrin indicates that the glycine and a pair of basic amino acid residues may be required for the enzymatic amidation of phenylalanine to phenylalanine amide.

Physicochemical Property Modification Strategies Based on Enzyme Substrate Specificities II: α‐Chymotrypsin Hydrolysis of Aspirin Derivatives

Three aspirin derivatives, aspirin phenylalanine ethyl ester, aspirin phenylalanine amide, and aspirin phenyllactic ethyl ester, were investigated with respect to their hydrolysis by α‐chymotrypsin. Of the three compounds, aspirin phenylalanine ethyl ester was the best substrate, withkcat= 25sec−1andKm= 1.3 × 10−6Mat pH 8.0. The results for all substrates were in the range of expectation based on kinetic data for other substrates. The apparent latitude in the nature of the acylamide substituent of α‐chymotrypsin substrates makes this enzyme a good potential reconversion site for many drug derivatives.

Chemical determination of polypeptide hormones.

The presence or absence of peptide hormones in tissue extracts may in certain cases be demonstrated by exposing the extracts to conditions under which characteristic fragments of the polypeptide molecule in question are formed and then analyzing for such fragments. An approximate quantitation of the hormones may also be achieved thereby. In the present work the COOH-terminal fragments of polypeptides containing characteristic alpha-amide groups were released enzymatically and then converted into the fluorescent dansyl derivatives, which were identified by thin-layer chromatography. In this way the presence of secretin, cholecystokinin, and the vasoactive intestinal peptide in concentrates of porcine intestinal extracts were demonstrated by their COOH-terminal amide fragments: valine (or leucylvaline) amide, phenylalanine amide, and asparagine (or leucylasparagine) amide, respectively. The analytical methodology used in the present study may also be useful in devising simple and reliable chemical assay methods for the isolation of already known polypeptides and in the isolation of previously uncharacterized polypeptides from natural sources.

Enzymatic deamidation of the C-terminal tetrapeptide amide of gastrin by mammalian tissues

As the initial phase of a study of mammalian catabolism of the peptide hormone gastrin, we demonstrated in rat tissues the presence of an enzymatic activity catalyzing deamidation of the phenylalanine amide of the biologically active C-terminal tetrapeptide amide fragment of gastrin (Try-Met-Asp-Phe-NH 2) to produce the biologically inactive tetrapeptide free acid. Because an interfering aminopeptidase that acts on the C-terminal tetrapeptide amide was widespread in rat tissues, N-acylated BOC-tetrapeptide amide was used as the substrate for studying the amidase activity. Identification of BOC-tetrapeptide free acid as a product of the amidase reaction was achieved by thin-layer chromatography and by demonstrating that the reaction product was a substrate for carboxypeptidase A, which catalyzes liberation of phenylalanine from BOC-tetrapeptide free acid but not from BOC-tetrapeptide amide. A quantitative assay was developed to measure the generation of BOC-tetrapeptide free acid; this product and ammonia were shown to be produced in equivalent amounts during the reaction. The enzyme activity was concentrated in the microsomal cell fraction. The pH optimum for the reaction was found to be approximately 6.7. Liver was found to be a rich source of the amidase activity in the rat and this activity was also demonstrated to be present in small intestinal mucosa and several other tissues of the rat and of other mammalian species. Another enzymatic activity, most abundant in rat kidney, was found to catalyze hydrolysis of the peptide bond between methionine and aspartic acid of BOC-tetrapeptide amide. The amidase activity and the peptidase activity identified in the kidney may contribute to the inactivation of gastrin or of its biologically active degradation products in vivo.

Schiff base derivatives of peptide esters: Relative abundance of N‐terminal, C‐terminal and ‘internal’ fragments as a function of the blocking group

AbstractElectron‐impact (EI) mass spectrometry of peptide derivatives is usually interpreted in terms of fragmentation where the charge resides on the N‐terminal fragments and to a lesser degree on the less common, charged C‐terminal fragments. Substituted and unsubstituted benzylidene, cinnamylidene, α‐ and β‐naphthylidene derivatives of a reference tripeptide, valileala, gave both N‐ and C‐terminal fragments as well as molecular ions. The order of increasing ion current (normalized) in C‐terminal fragments was: acetylacetonyl, 4‐dimethylaminonaphthylidene, p‐dimethyl‐aminobenzylidene, 3‐pyridylmethylidene, p‐diethylaminocinnamylidene, benzylidene, 2‐hydroxy‐naphthylidene, 4‐pyridylmethylidene, p‐nitrobenzylidene, p‐methoxybenzylidene, p‐cyanobenzylidene, cinnamylidene, p‐dimethylaminocinnamylidene, β‐indolylmethylidene, β‐naphthylidene, 2‐pyridylmethylidene and α‐naphthylidene. The order for this value among the N‐terminal fragments is significantly different, however (Day, Falter, Lehman and Hamilton, J. Org. Chem. in press). In addition to N‐ and C‐terminal fragments, many spectra contain internal fragments, arising from loss of fragments from both ends, which provide sequence information. These fragments are found in the mass spectra of Schiff bases formed from various aromatic aldehydes with peptide esters. The interpretation of the latter pattern is facilitate in some cases by deuterium labeling at the α‐carbon of the N‐terminal amino acid residue of peptides. Such a pattern provides sequence information supplemental to that available involving N‐ and C‐terminal fragmentations. In derivatives of hexaglycine, tetraphenylalanine and tryptophylmethionylaspartyl (β‐OEt) phenylalanine amide, for example, substantial sequence information was contained in the internal fragments; in some cases the sequence could be deduced only if the internal fragments were utilized. The 4‐dimethylamino‐naphthylidene derivatives have proven to be the most useful to date in terms of volatility, tendency to maximize cleavage into N‐terminal fragments, intensity of molecular ions and generation of useful mass spectra of certain peptide esters refractory to mass spectrometry in the form of any other derivative investigated.

Inventaire des différentes activités peptidasiques intracellulaires de Streptococcus thermophilus: Purification et propriétés d'une dipeptide-hydrolase et d'une aminopeptidase

Summary Besides a proteolytic activity, four peptidases were isolated from S. thermophilus. The purified dipeptidase has a molecular weight of about 50 000 daltons. It was maximally active around pH 7.50 and 50°C, its apparent energy of activation was 4 500 cal./mole. The dipeptidase was most stable between pH 7.0 and 9.0, and for temperatures less than or equal to 50°C. E.D.T.A. or O-phenanthroline induced a very significant reduction in enzyme activity, but metal ions such as Co++, Zn++, Mn++, Mg++ or Ca++ can reactivate it partially after the inactivation by a chelating agent such as E.D.T.A. Iodoacetate, p-C.M.B. or D.F.P. induced a slight reduction in enzyme activity. Unsubstituted dipeptides only were hydrolysed by the enzyme. The dipeptidase exhibited a specificity toward dipeptides with a large and hydrophobic amino acid residue at the NH2-terminal end; the second amino acid residue (at the COOH-terminal end) had also an effect on enzymic activity. A homogeneous aminopeptidase — with regard to disc electrophoresis — was obtained. The molecular weight of this enzyme was found to be 62 000 daltons. It was maximally active around pH 6.40 and 35°C; its apparent energy of activation was 7 300 cal./mole. Aminopeptidase was most stable between pH 5.80 and 9.80 and at a temperature less than 40°C. E.D.T.A. or O-phenanthroline induced a complete inactivation of the enzyme, but metal ions such as Co++ and Mn++ can reactivate it completely; metal ions such as Ca++ and Zn++ or Mg++ partially after the inactivation by a chelating agent such as E.D.T.A. Zn++ 5.10−3 M and alcohols induced a significant reduction in enzyme activity. This enzyme can be precisely defined as an α-amino-acyl-peptide hydrolase EC.3.4.1. since only NH2-terminal end amino-acid residues were released from oligopeptides (comprising less than eleven amino-acid residues). Leucine-p-nitroanilide and amino-acid amides (except phenylalanine amide) were hydrolysed. Lys-Tyr and Leu-Leu were the best dipeptide substrates. Dipeptides with a glycyl or a histidyl residue at the NH2-terminal end were not hydrolysed as also dipeptides with a prolyl or phenylalanyl residue.

Enzymatic inactivation of peptide hormones possessing a C-terminal amide group.

A partially purified enzyme extracted from the bladder of the toad, Bufo marinus L., was found to cleave the glycine amide moiety from oxytocin, 8-lysine-vasopressin, 8-arginine-vasopressin, and other hormone analogs terminating in a primary carboxamide group; however, this enzyme does not attack hormone analogs terminating with a methylamide, dimethylamide, or carboxyl group. Preliminary experiments indicate that a functionally similar enzyme is also present in the mammalian kidney, the major target organ of neurohypophyseal antidiuretic hormones. This enzyme, besides inactivating oxytocin and 8-lysine-vasopressin, also cleaves the phenylalanine amide moiety from a tetrapeptide analog of gastrin, another hormone terminating in a primary carboxamide group. Attention is drawn to the possible general significance of "carboxamidopeptidases" for the termination of the action of peptide hormones in which the C-terminal amino acid residue bears a carboxamide group.