Ala NH Research Articles

Oxalamide-Bridged Ferrocenes: Conformational and Gelation Properties and In Vitro Antitumor Activity

The N-termini of the constitutional isomeric ferrocene peptides Boc–NH–Fn–CO–Ala–OMe (1) and Boc–Ala–NH–Fn–COOMe (2) were deprotected in the acidic milieu and subjected to oxalyl chloride-mediated dimerization to afford the oxalamides −(CO–NH–Fn–CO–Ala–OMe)2 (3) and −(CO–Ala–NH–Fn–COOMe)2 (4), respectively. The goal compounds were evaluated for their conformational properties, toluene gelation ability, and effect on cell viability of tumor (HeLa, MCF7, HepG2) and normal (HEK293T) cell lines. Detailed spectroscopic analysis in solution revealed the different conformational behavior of constitutional isomers 3 and 4, and oxalamide 3 was found to have a higher degree of chiral organization due to its involvement in stronger intramolecular hydrogen bonds. Crystallographic analysis showed the presence of two symmetry-independent conformers of compound 3 stabilized by stronger intramolecular hydrogen bonds. The tested compounds self-assemble in toluene to provide supramolecular nanostructures but were unable to form a stable gel. Interestingly, the formation of a stable gel was observed when the toluene solution of 4 was sonicated. Cytotoxic evaluation of 3 and 4 revealed a weaker growth-inhibitory effect on tumor cell lines compared to cisplatin. Oxalamide 3 appeared to be more cytotoxic to HeLa and MCF7 cancer cell lines than to normal HEK293T cells, indicating its potential for drug development as an anticancer agent.

Through the central V2, not V1 receptors influencing the endogenous opiate peptide system, arginine vasopressin, not oxytocin in the hypothalamic paraventricular nucleus involves in the antinociception in the rat

Our previous study has proven that hypothalamic paraventricular nucleus (PVN) played a role in the antinociception. The central bioactive substances involving in the PVN regulating antinociception were investigated in the rat. The results showed that electrical stimulation of the PVN increased the pain threshold, and l-glutamate sodium injection into the PVN elevated the pain threshold, but the PVN cauterization decreased the pain threshold; pain stimulation raised the arginine vasopressin (AVP), not oxytocin (OXT), leucine-enkephalin (L-Ek), β-endorphin (β-Ep) and DynorphinA 1–13 (DynA 1–13) concentrations in the PVN tissue using micropunch method, heightened AVP, L-Ek, β-Ep and DynA 1–13, not OXT concentrations in the PVN perfuse liquid, and reduced the number of AVP-, not OXT, L-Ek, β-Ep and DynA 1–13-immunoreactive neurons in the PVN especially in the posterior magnocellular part of the PVN using immunocytochemistry. There was a negative relationship between the PVN AVP concentration and the pain threshold; pain stimulation enhanced the AVP, not OXT mRNA expression in the PVN using in situ hybridization and RT-PCR; intraventricular injection of anti-AVP serum completely reversed l-glutamate sodium injection into the PVN-induced antinociception, and administration of naloxone — the opiate peptide antagonist, partly blocked this l-glutamate sodium effect, but anti-OXT serum pretreatment did not influence this l-glutamate sodium effect; l-glutamate sodium injection into the PVN-induced analgesia was inhibited by V2 receptor antagonist — d(CH 2) 5[D-Ile 2, Ile 4, Ala–NH 2 9]AVP, not V1 receptor antagonist — d(CH 2) 5Tyr(Me)AVP. The data suggested that the PVN was limited to the central AVP, not OXT, which was through V2, not V1 receptors influencing the endogenous opiate peptide system, to regulate antinociception.

Switching of turn conformation in an aspartate anion peptide fragment by NH · · · O− hydrogen bonds

Aspartic acid protease model peptides Z-Phe-Asp(COOH)-Thr-Gly-Ser-Ala-NHCy (1) and AdCO-Asp(COOH)-Val-Gly-NHBzl (3), and their aspartate anions (NEt4)[Z-Phe-Asp(COO-)-Thr-Gly-Ser-Ala-NHCy] (2) and (NEt4)[AdCO-Asp(COO-)-Val-Gly-NHBzl] (4), having an invariant primary sequence of the Asp-X(Thr,Ser)-Gly fragment, were synthesized and characterized by 1H-NMR, CD, and infrared (IR) spectroscopies. NMR structure analyses indicate that the Asp O(delta) atoms of the aspartate peptide 2 are intramolecularly hydrogen-bonded with Gly, Ser, Ala NH, and Ser OH, supporting the rigid beta-turn-like conformation in acetonitrile solution. The tripeptide in the aspartic acid 3 forms an inverse gamma-turn structure, which is converted to a beta-turn-like conformation because of the formation of the intramolecular NH . . . O- hydrogen bonds with the Asp O(delta) in 4. Such a conformational change is not detected between dipeptides AdCO-Asp(COOH)-Va-NHAd (5) and (NEt4)[AdCO-Asp(COO-)-Val-NHAd] (6). The pK(a) value of side-chain carboxylic acid (5.0) for 3 exhibits a lower shift (0.3 unit) from that of 5 in aqueous polyethyleneglycol lauryl ether micellar solution. NMR structure analyses for 3 in an aqueous micellar solution indicate that the preorganized turn structure, which readily forms the NH . . . O- hydrogen bonds, lowers the pK(a) value and that resulting hydrogen bonds stabilize the rigid conformation in the aspartate anion state. We found that the formation of the NH . . . O- hydrogen bonds involved in the hairpin turn is correlated with the protonation and deprotonation state of the Asp side chain in the conserved amino acid fragments.

On NMR isotropic chemical shift surfaces of peptide models

Nuclear magnetic resonance isotropic chemical shift (ICS) surfaces have been generated for all the atoms of the central Ala residue of the peptide models For–(L–Ala) n –NH 2, n=1, 3, and 5, at the GIAO-B3LYP/TZ2P level at a grid of 10° in the backbone dihedral angles φ and ψ for n=1 and at a grid of 30° for the larger models. The resulting periodic 3D ICS( φ, ψ) surfaces were fitted employing a number of suitable mathematical functions. A cosine expansion provided the most accurate and compact representation of the highly structured ICS surfaces. At 10th order, involving 66 coefficients, the mean errors in the 13C α and 1H α ICS predictions for the For–L–Ala–NH 2 model are 0.33 and 0.034 ppm, respectively. Similar fitting residues were found for other nuclei, as well. The computed surfaces are compared to results corresponding to an experimental database. The changes in chemical shifts due to the increase in the model size are investigated in detail.

Conformations of Gly nH + and Ala nH + Peptides in the Gas Phase

High-resolution ion mobility measurements and molecular dynamics simulations have been used to probe the conformations of protonated polyglycine and polyalanine (Gly nH + and Ala nH +, n = 3–20) in the gas phase. The measured collision integrals for both the polyglycine and the polyalanine peptides are consistent with a self-solvated globule conformation, where the peptide chain wraps around and solvates the charge located on the terminal amine. The conformations of the small peptides are governed entirely by self-solvation, whereas the larger ones have additional backbone hydrogen bonds. Helical conformations, which are stable for neutral Ala n peptides, were not observed in the experiments. Molecular dynamics simulations for Ala nH + peptides suggest that the charge destabilizes the helix, although several of the low energy conformations found in the simulations for the larger Ala nH + peptides have small helical regions.

Application of N-carboxyalkyl peptides to the inhibition and affinity purification of the porcine matrix metalloproteinases collagenase, gelatinase, and stromelysin

Several N-carboxyalkyl peptides were synthesized and tested as inhibitors of pig synovial collagenase, 72-kDa gelatinase and Stromelysin (matrix metalloproteinases MMP-1, MMP-2, and MMP-3). The most potent of the series, CH 3CH 2CH 2( R,S)CH(COOH)-NHLeuPheAlaNH 2, competitively inhibited cleavage of dinitrophenylProLeuGlyLeuTrpAla d ArgNH 2 at the GlyLeu bond by MMP-1 and MMP-2 ( K I = 30 and 40 μ m, respectively). A similar inhibitory potency was found for MMP-1 with soluble Type I collagen and MMP-3 with substance P as substrate. The inhibitor was coupled to EAH-Sepharose 4B through a C-terminal amide. In the presence of 2 m NaCl at pH 7.2, this matrix bound MMP-1, MMP-2, and MMP-3 from concentrated culture medium of pig synovial membranes. The enzymes coeluted at pH 4.1 and subsequently were resolved by chromatography on DEAE-Sephacel and heparin-Sepharose. Purified MMP-1 catalyzed the o-phenanthroline-sensitive cleavage of collagen into TC A and TC B fragments as well as slower hydrolysis of the α2 chain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of MMP-1 indicated a predominant polypeptide of approximately 44 kDa and minor species of approximately 24 and 21 kDa. The 44-kDa species and one of the smaller polypeptides reacted with an antiserum to residues 195–207 of human fibroblast MMP-1, indicating that porcine MMP-1 contains a similar sequence and that the smaller components were probably derived from MMP-1. Neither MMP-2 nor MMP-3 reacted with this antiserum. Purified porcine MMP-2 degraded gelatin but not collagen and exhibited an apparent M r of approximately 71 kDa. Additional smaller polypeptides were present, one of which may correspond to tissue inhibitor of metalloproteinases. MMP-3 showed doublets of approximately 47/46 and 26/25 kDa and cleaved substance P at the Gly 6Phe 7 bond. This procedure provides a rapid means of obtaining all three MMPs from one source in approximately 15% yield each.

Crystal structure and solution conformation of S,S′‐bis(Boc‐Cys‐Ala‐OMe): Intramolecular antiparallel β‐sheet conformation of an acyclic cystine peptide

The conformation of the acyclic biscystine peptide S,S'-bis(Boc-Cys-Ala-OMe) has been studied in the solid state by x-ray diffraction, and in solution by 1H- and 13C-nmr, ir, and CD methods. The peptide molecule has a twofold rotation symmetry and adopts an intramolecular antiparallel beta-sheet structure in the solid state. The two antiparallel extended strands are stabilized by two hydrogen bonds between the Boc CO and Ala NH groups [N...O 2.964 (3) A, O...HN 2.11 (3) A, and NH...O angle 162 (3) degrees]. The disulfide bridge has a right-handed conformation with the torsion angle C beta SSC beta = 95.8 (2) degrees. In solution the presence of a twofold rotation symmetry in the molecule is evident from the 1H- and 13C-nmr spectra. 1H-nmr studies, using solvent and temperature dependencies of NH chemical shifts, paramagnetic radical induced line broadening, and rate of deuterium-hydrogen exchange effects on NH resonances, suggest that Ala NH is solvent shielded and intramolecularly hydrogen bonded in CDCl3 and in (CD3)2SO. Nuclear Overhauser effects observed between Cys C alpha H and Ala NH protons and ir studies provide evidence of the occurrence of antiparallel beta-sheet structure in these solvents. The CD spectra of the peptide in organic solvents are characteristic of those observed for cystine peptides that have been shown to adopt antiparallel beta-sheet structures.

ChemInform Abstract: Solution Conformations of the Immunomodulator Muramyl Peptides

Abstract MDP, Murabutide and the MDP[D-Ala) analogue have been studied dissolved in dimethylsulfoxyde by means of 1 H-n.m.r., including the 2D.2Q 1 H- 1 H inadequate and NOESY (Nuclear Overhauser Effect Spectroscopy) experiments. The results confirm the “S” shaped conformation constituted by two adjacent β turns in MDP. The first turn is a tight “type II β turn” and takes place around the MurNac (N-acetylmuramic acid) moiety of MDP and Murabutide and is stabilized by a C 10 hydrogen bonding between the Ala NH (1 + 3) and the acetamido C = 0 (1). The second turn is centered around L-Ala-D-iGln and requires the α-carboxamide group of D-iGln for its stabilization and thus does not occur in Murabutide. The L-Ala → D-Ala Inversion in MDP[D-Ala] produces substantial conformational modifications leading to a cyclic structure with the iGln α-carboxamide protons and the acetamido methyl group in close spatial proximity. The dissociation of the adjuvant, antiinfection and pyrogenic activities in the 3 compounds is well depicted by these variations of conformations.

Solution conformations of the immunomodulator muramyl peptides

MDP, Murabutide and the MDP[D-Ala) analogue have been studied dissolved in dimethylsulfoxyde by means of 1H-n.m.r., including the 2D.2Q 1H- 1H inadequate and NOESY (Nuclear Overhauser Effect Spectroscopy) experiments. The results confirm the “S” shaped conformation constituted by two adjacent β turns in MDP. The first turn is a tight “type II β turn” and takes place around the MurNac (N-acetylmuramic acid) moiety of MDP and Murabutide and is stabilized by a C 10 hydrogen bonding between the Ala NH (1 + 3) and the acetamido C = 0 (1). The second turn is centered around L-Ala-D-iGln and requires the α-carboxamide group of D-iGln for its stabilization and thus does not occur in Murabutide. The L-Ala → D-Ala Inversion in MDP[D-Ala] produces substantial conformational modifications leading to a cyclic structure with the iGln α-carboxamide protons and the acetamido methyl group in close spatial proximity. The dissociation of the adjuvant, antiinfection and pyrogenic activities in the 3 compounds is well depicted by these variations of conformations.

N-(1-Ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinium–hydrogen maleate (1/1), enalapril (MK-421)

C20H29N2Os.C4H304, Mr=492.5 , mono- clinic, P21, a=11.224(4), b=6.645(2), c-- 17.824 (5)A, fl= 105.52 (3) ° , V= 1280.9 (7)A 3, Z =2, D x=1.27gcm -3, MoKa, 2=0.71069/~, g= 1.06 cm -1, F(000) = 524, T = 293 K, final R = 0.089 for 1187 observed reflections. The title compound (MK-421) is a potent orally active inhibitor of angiotensin-converting enzyme. The molecule is obser- ved with Ala NH + as a positively charged group. The backbone shows an extended conformation at Ala. The crystal-structure conformation is compared with the proposed biologically active conformation of enalapril

Characterization of the copper(II)- and nickel(II)-transport site of human serum albumin. Studies of copper(II) and nickel(II) binding to peptide 1-24 of human serum albumin by 13C and 1H NMR spectroscopy.

As a basis for understanding the role of albumin in the transport of metal ions, detailed investigations have been carried out to elucidate the structure of Ni(II)- and Cu(II)-binding site of the peptide residue corresponding to the NH2-terminal peptide fragment 1-24 of human serum albumin by 1H and 13C NMR spectroscopy. These studies have been conducted in aqueous medium at different pH values and at different ligand/metal ratios. The results show the following: (i) Diamagnetic Ni(II) complex and paramagnetic Cu(II) complex are in slow exchange NMR time scale. (ii) Titration results of Ni(II)-bound form of peptide 1-24 show the presence of a 1:1 complex in the wide pH range (6.0-11.0), and the same stoichiometry is proposed for Cu(II) as well. (iii) Analysis of the spectra suggests that both Ni(II) and Cu(II) have one specific binding site at the NH2-terminal tripeptide segment (Asp-Ala-His...) involving the Asp alpha-NH2, His N(1) imidazole, two deprotonated peptide nitrogens (Ala NH and His NH), and the Asp COO- group. (iv) Complexation of Ni(II) and Cu(II) causes conformational change near the metal-binding site of the polypeptide chain, but there is no other binding group involved besides those in the first three residues.

Kinetic studies on the chymotrypsin A α-catalyzed hydrolysis of a series of N-acetyl- l-phenylalanyl peptides

A series of N-acetyl- l-phenylalanyl peptides of general formula Ac-Phe-(Gly) n -NH 2 ( n = 0–2) has been synthesized to study the effect of leaving group chain length on the efficiency of chymotrypsin A α amidase and peptidase activities. The effect upon catalysis of hydrophobic side chains on the leaving group was investigated using similar substrates with one of the glycine residues selectively substituted by an alanine residue as in AcPheAlaNH 2, AcPheAlaGlyNH 2, and AcPheGlyAlaNH 2. Values of k cat and K m have been obtained from kinetic measurements at pH 8.00 and 25 °C. The results are shown to be consistent with binding schemes postulated from published model building studies. The catalytic reactions were studied over a range of temperature (15–35 °C) and in each case the Arrhenius law was obeyed. It was thus possible to obtain meaningful values for the thermodynamic functions of activation for the acylation step of the catalytic reaction. The results are shown to confirm the findings of postulated binding schemes but indicate that conclusions drawn from kinetic measurements at a single temperature may sometimes be misleading.

1H‐ and 13C‐NMR studies of N‐acetyl‐L‐alanine methylester and N‐acetyl‐L‐alanine methylamide. I. Self‐association

AbstractThe 1H‐ and 13C‐NMR spectra of N‐acetyl‐L‐alanine methylester and N‐acetyl‐L‐alanine methylamide were measured to examine the modes of self‐association of these molecules in solution. The different dilution shifts between these molecules seem to correspond to the difference in the associated state for each molecule. Consequently, for the former molecule, a dimer model forming the intermolecular hydrogen bond through Ala NH hydrogen atom in one molecule to Ala CO oxygen atom in another molecule was proposed. Another dimer model, which coincides with that proposed recently by Neel and coworkers, was proposed for the latter molecule. This second dimer model forms an intermolecular hydrogen bond through the NH of the N‐methylamide group in one molecule to the acetyl CO in another molecule.