Tripeptide Amide Research Articles

Cyclic Heterometallic Interactions formed from a Flexible Tripeptide Complex Showing Effective Antiferromagnetic Spin Coupling

Developing tunable motifs for heterometallic interactions should be beneficial for fabricating functional materials based on cooperative electronic communications between metal centers. Reported here is the efficient formation of cyclic heterometallic interactions from a complex containing an artificial tripeptide with metal binding sites on its main chain and side chains. X-ray structural analysis and X-ray absorption spectroscopy revealed that the cyclic metal-metal arrangements arise from the amide groups connecting four square-planar CuII centers and four octahedral NiII centers in a cyclic manner. UV/Vis spectral studies suggested that this efficient formation was achieved by the selective formation of the square-planar CuII centers and a crystallization process. Magnetic measurements using SQUID clarified that the cyclic complex represented the S=2 spin state at low temperatures due to effective antiferromagnetic interactions between the NiII and CuII centers.

Thyrotropin-Releasing Hormone Loaded and Chitosan Engineered Polymeric Nanoparticles: Towards Effective Delivery of Neuropeptides.

Thyrotropin-Releasing Hormone (TRH), a tripeptide amide with molecular formula L-pGlu-L-His-L- Pro-NH2, is used in the treatment of brain/spinal injury and certain central nervous system (CNS) disorders, including schizophrenia, Alzheimer's disease, epilepsy, depression, shock and ischemia due to its profound effects on the CNS. However, TRH's therapeutic activity is severely hampered because of instability and hydrophilicity owing to its peptidic nature which results into ineffective penetration into the blood brain barrier. In the present study, we report the synthesis and stability studies of novel chitosan engineered TRH encapsulated poly(lactide-co-glycolide) (PLGA) based nanoformulation. The aim of such an encapsulation is to allow effective delivery of TRH in biological systems as the peptidase degrade naked TRH. The synthesis of TRH was carried out manually in solution phase followed by its encapsulation using PLGA to form polymeric nanoparticles (NPs) via nanoprecipitation technique. Different parameters such as type of organic phase, concentration of stabilizer, ratio of organic phase and aqueous phase, rate of addition of organic phase were optimized, tested and evaluated for particle size, encapsulation efficiency, and stability of NPs. The TRH-PLGA NPs were then surface modified with chitosan to achieve positive surface charge rendering them potential membrane penetrating agents. PLGA, PLGA-TRH, Chitosan-PLGA and Chitosan-PLGA-TRH NPs were characterized and analyzed using Dynamic Light Scattering (DLS), Transmissiom Electron Microscopy (TEM) and Infra-red spectroscopic techniques.

Central regulation of hypothalamic-pituitary-thyroid axis under physiological and pathophysiological conditions.

TRH is a tripeptide amide that functions as a neurotransmitter but also serves as a neurohormone that has a critical role in the central regulation of the hypothalamic-pituitary-thyroid axis. Hypophysiotropic TRH neurons involved in this neuroendocrine process are located in the hypothalamic paraventricular nucleus and secrete TRH into the pericapillary space of the external zone of the median eminence for conveyance to anterior pituitary thyrotrophs. Under basal conditions, the activity of hypophysiotropic TRH neurons is regulated by the negative feedback effects of thyroid hormone to ensure stable, circulating, thyroid hormone concentrations, a mechanism that involves complex interactions between hypophysiotropic TRH neurons and the vascular system, cerebrospinal fluid, and specialized glial cells called tanycytes. Hypophysiotropic TRH neurons also integrate other humoral and neuronal inputs that can alter the setpoint for negative feedback regulation by thyroid hormone. This mechanism facilitates adaptation of the organism to changing environmental conditions, including the shortage of food and a cold environment. The thyroid axis is also affected by other adverse conditions such as infection, but the central mechanisms mediating suppression of hypophysiotropic TRH may be pathophysiological. In this review, we discuss current knowledge about the mechanisms that contribute to the regulation of hypophysiotropic TRH neurons under physiological and pathophysiological conditions.

Tripeptide Amide L-pyroglutamyl-Histidyl-L-Prolineamide (L-PHPThyrotropin- Releasing Hormone, TRH) Promotes Insulin-Producing Cell Proliferation

A very small tripeptide amide L-pyroglutamyl-L-histidyl-L-prolineamide (L-PHP, Thyrotropin-Releasing Hormone, TRH), was first identified in the brain hypothalamus area. Further studies found that L-PHP was expressed in pancreas. The biological role of pancreatic L-PHP is still not clear. Growing evidence indicates that L-PHP expression in the pancreas may play a pivotal role for pancreatic development in the early prenatal period. However, the role of L-PHP in adult pancreas still needs to be explored. L-PHP activation of pancreatic β cell Ca2+ flow and stimulation of β-cell insulin synthesis and release suggest that L-PHP involved in glucose metabolism may directly act on the β cell separate from any effects via the central nervous system (CNS). Knockout L-PHP animal models have shown that loss of L-PHP expression causes hyperglycemia, which cannot be reversed by administration of thyroid hormone, suggesting that the absence of L-PHP itself is the cause. L-PHP receptor type-1 has been identified in pancreas which provides a possibility for L-PHP autocrine and paracrine regulation in pancreatic function. During pancreatic damage in adult pancreas, L-PHP may protect beta cell from apoptosis and initiate its regeneration through signal pathways of growth hormone in β cells. L-PHP has recently been discovered to affect a broad array of gene expression in the pancreas including growth factor genes. Signal pathways linked between L-PHP and EGF receptor phosphorylation suggest that L-PHP may be an important factor for adult β-cell regeneration, which could involve adult stem cell differentiation. These effects suggest that L-PHP may benefit pancreatic β cells and diabetic therapy in clinic.

Synthesis of peptide derivatives of aspirin and their antibiogram

Peptide derivatives of Aspirin (1 to 8) were synthesized by using Ac2O/AcOH reaction with Salicyclic acid. Aspirin was coupled with amino acid amide and dipeptide amide and tripeptide amide using its p-nitrophenyl (NP) ester. The ester (Aspirin–ONP) was prepared using p-nitro phenol and DCC in EtOAc and was precipitated using EtOH. The synthesis of dipeptide amides were carried out in solution by stepwise elongationof the peptide chain from the C-terminal amino acid by coupling one amino acid at a time using DCC/HOBt method. Boc-group was used for Nα protection of all amino acids. The Boc-group cleavage was carried outusing 50% TFA/CH2Cl2. The amidation of C-terminal amino acid was carried out by treating the corresponding Boc–amino acid–ONp esters with dry NH3 in presence of DCC and HOBt. These compounds were subjected to antibiotic susceptibility studies against the Pseudomonas fluorescens, Escherichia coli and Staphylococcus aureus. Methionine amide of aspirin was active on S. aureus. Glycyl-glycyl-phenylalanyl-leucine amide ofaspirin was moderately active on all the three bacterial strains used. Glycyl-Phenylalanyl-leucine amide of aspirin and glycyl-glycyl-methionine amide of aspirin were active on P. fluorescens. Leucine amides of aspirinwas moderately active on S. aureus Methionine amides of aspirin was highly active on E. coli. Phenylalanylleucine amides of aspirin and glycyl-glycyl-phenylalanyl-methionine amide of aspirin were active on none ofthe organisms used in this study. In general the peptide derivatives of aspirin (1,3,4,5,6,7) were not active on all the three microbes used. Compounds 2 and 8 were sensitive to none of the organisms used in this study. By the major study, we observed that the derivative of aspirin showed a greater influence in inhibiting the test organisms used.Keywords: Aspirin–ONp, Boc–N3 (t-butyl azido formate), HOBt (1-Hydroxy benzotriazole), DCC (N, N1–Dicyclo hexyl carbodiimide) and DCU (N, N1–Dicyclohexyl urea).

Convenient synthesis of C-terminal di- and tri-peptide amides from N-protected dipeptidoylbenzotriazoles

N-Protected dipeptidoylbenzotriazoles react with aqueous ammonia to give dipeptide primary amides (77–98%) and with N-unprotected α-amino amides to afford tripeptide primary amides (82–86%).

Tetrahydrofuran Cα-Tetrasubstituted Amino Acids: Two Consecutive β-Turns in a Crystalline Linear Tripeptide

The synthesis of tetrahydrofuran Calpha-tetrasubstituted amino acids (TAAs) and their effect on the conformation in small peptides are reported. The synthesis starts from the protein amino acid methionine, which is protected at the C and N terminus and converted into the corresponding sulfonium salt by alkylation. Simple base treatment in the presence of an aryl aldehyde leads to the formation of tetrahydrofuran tetrasubstituted Calpha-amino acids in a highly diastereoselective (trans/cis ratio up to 97:3) reaction with moderate to good yields (35-78%) depending on the aldehyde used. Palladium-catalyzed coupling reactions allow a subsequent further functionalization of the TAA. The R,S,S-TAA-Ala dipeptide amide adopts a beta-turn type I conformation, whereas its S,R,S isomer does not. The R,S,S-Gly-TAA-Ala tripeptide amide shows in the solid state and in solution a conformation of two consecutive beta-turn type III structures, stabilized by i+3-->i intramolecular hydrogen bonds.

Kojic acid–tripeptide amide as a new tyrosinase inhibitor

Twenty two kojic acid-tripeptide amides were prepared using a solid-phase Fmoc/tBu strategy with Rink Amide SURE(R) resin. To effectively obtain kojic acid-tripeptide amide conjugates, the coupling conditions of kojic acid to the tripeptide on the resin were optimized. The tyrosinase inhibitory activity of kojic acid-tripeptide amides and the effect of the amino acid sequence on the activity were compared with those of kojic acid-tripeptide acids. The stability of kojic acid-tripeptide amides were then compared with those of kojic acid and kojic acid-tripeptides acids. As a consequence, kojic acid-FWY-NH(2) proved to be the best compound, with the highest inhibitory activity, which was maintained over different storage times under various temperatures and pHs.

Synthesis and utilization of 13C and 15N backbone-labeled proline: NMR study of synthesized oxytocin with backbone-labeled C-terminal tripeptide amide

The 13C and 15N backbone-labeled proline was prepared using Oppolzer's method based on application of a sultam as chiral auxiliary. This isotopomer was used in the synthesis of the 13C, 15N backbone-labeled C-terminal tripeptide amide fragment of neurohypophyseal hormone oxytocin. Finally, this tripeptide amide was coupled by segment condensation with N-Boc- or N-Fmoc-tocinoic acid, followed by N-deprotection with TFA or piperidine. The labeled oxytocin exhibited biological activity identical with that of natural oxytocin. A detailed 1H, 13C and 15N NMR study confirmed the assigned oxytocin conformation containing a beta-turn in the cyclic part of the molecule, stabilized by H-bond(s) that can be perturbed by the C-terminal tripeptide amide moiety as indicated by comparison of NMR data for both the tocine ring in oxytocin and tocinoic acid.

Orally Active Antiviral Tripeptide Glycyl-Prolyl-Glycinamide Is Activated by CD26 (Dipeptidyl Peptidase IV) before Transport across the Intestinal Epithelium

The tripeptide amide glycyl-prolyl-glycinamide (GPG-amide) is a new antiretroviral drug candidate, but its absorption mechanism is unknown. In this investigation, the transport and metabolism of GPG-amide were studied in a model of the human intestinal epithelium, Caco-2 cell monolayers. The results show that when the tripeptide amide came into contact with the apical enterocyte membrane, it was degraded by CD26 (dipeptidyl peptidase IV) to glycylproline and the antiretrovirally active metabolite glycinamide. Glycinamide retained antiretroviral activity in vitro after transport through the Caco-2 cell monolayers. The transport of glycinamide across Caco-2 cell monolayers occurred via passive diffusion with an apparent permeability coefficient of about 2 x 10(-6) cm s(-1), which suggests that it is absorbed by the oral route in sufficient amounts to be considered for oral administration. In conclusion, the tripeptide GPG-amide acts as a prodrug that is activated by CD26 to release the orally active antiretroviral compound glycinamide.

Proton inventory studies of alpha-thrombin-catalyzed reactions of substrates with selected P and P' sites.

Deuterium kinetic solvent isotope effects for the human alpha-thrombin-catalyzed hydrolysis of (1) substrates with selected P(1)-P(3) sites, Z-Pro-Arg-7-amido-4-methylcoumarin (7-AMC), N-t-Boc-Val-Pro-Arg-7-AMC, Bz-Phe-Val-Arg-4-nitroanilide (pNA), and H-D-Phe-L-Pip-Arg-pNA, are (DOD)k(cat) = (2.8-3.3) +/- 0.1 and (DOD)(k(cat)/K(m)) = (0.8-2.1) +/- 0.1 and (2) internally fluorescence-quenched substrates (a) (AB)Val-Phe-Pro-Arg-Ser-Phe-Arg-Leu-Lys(DNP)-Asp-OH, an optimal sequence, and (b) (AB)Val-Ser-Pro-Arg-Ser-Phe-Gln-Lys(DNP)-Asp-OH, recognition sequence for factor VIII, are (DOD)k(cat) = 2.2 +/- 0.2 and (DOD)(k(cat)/K(m)) = (0.8-0.9) +/- 0.1, at the pL (L = H, D) maximum, 8.4-9.0, and (25.0-26.0) +/- 0.1 degrees C. The most plausible models fitting the partial isotope effect (proton inventory) data have been selected on the basis of lowest values of the reduced chi squared and consistency of fractionation factors at all substrate concentrations, assuming rate-determining acylation. The data for Z-Pro-Arg-7-AMC are consistent with a single-proton bridge at the transition state phi(TS) = 0.39 +/- 0.05 and components for solvent reorganization phi(S) = 0.8 +/- 0.1 and phi(S) = 1.22 for k(cat) and k(cat)/K(m), respectively. The data for tripeptide amides fit bowl-shaped curves; an example is N-t-Boc-Val-Pro-Arg-7-AMC: phi(TS)(1) = phi(TS)(2) = 0.57 +/- 0.01 and phi(S) = 1 for k(cat) and 1.6 +/- 0.1 for k(cat)/K(m). Proton inventories for the nonapeptide (2b) are linear. The data for k(cat) for H-D-Phe-L-Pip-Arg-pNA and the decapeptide (2a) are most consistent with two identical fractionation factors for catalytic proton bridging, phi(TS)(1) = phi(TS)(2) = 0.68 +/- 0.02 and a large inverse component (phi(S) = 3.1 +/- 0.5) for the latter, indicative of substantial solvent reorganization upon leaving group departure. Proton inventory curves for k(cat)/K(m) for nearly all substrates are dome-shaped with an inverse isotope effect component (phi(S) = 1.2-2.4) originating from solvent reorganization during association of thrombin with substrate. These large contributions from medium effects are in full accord with the conformational adjustments required for the fulfillment of the dual, hemostatic and thrombolytic, functions of thrombin.

ACE inhibitors reduce fecundity in the mosquito, Anopheles stephensi

Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase, which cleaves dipeptides and, in some instances, dipeptide or tripeptide amides from the C-terminus of regulatory peptides (e.g. angiotensin I, bradykinin and substance P). The expression of ACE is highly regulated in insects, where it is thought to have a role in the metabolism of peptide hormones involved in regulating reproduction. After a blood meal, ACE activity in the female mosquito Anopheles stephensi, increases four-fold with much of the enzyme finally accumulating in the ovary. In the present study, we have studied the effect on reproduction of adding two selective inhibitors of ACE, captopril and lisinopril, to the blood meal. Both ACE inhibitors reduced the size of the batch of eggs laid by females in a dose-dependent manner, with no observable effects on the behaviour of the adult insect. The almost total failure to lay eggs after feeding on either 1 mM captopril or 1 mM lisinopril, did not result from interference with the development of the primary follicle, but was due to the inhibition of egg-laying. Since very similar effects on the size of the egg-batch were observed with two selective ACE inhibitors, belonging to different chemical classes, we suggest that these effects are mediated by the selective inhibition of the induced mosquito ACE, a peptidase probably involved in the activation/inactivation of a peptide regulating egg-laying activity in A. stephensi.

Tripeptide interference with human immunodeficiency virus type 1 morphogenesis.

Capsid assembly during virus replication is a potential target for antiviral therapy. The Gag polyprotein is the main structural component of retroviral particles, and in human immunodeficiency virus type 1 (HIV-1), it contains the sequences for the matrix, capsid, nucleocapsid, and several small polypeptides. Here, we report that at a concentration of 100 micro M, 7 of 83 tripeptide amides from the carboxyl-terminal sequence of the HIV-1 capsid protein p24 suppressed HIV-1 replication (>80%). The three most potent tripeptides, glycyl-prolyl-glycine-amide (GPG-NH(2)), alanyl-leucyl-glycine-amide (ALG-NH(2)), and arginyl-glutaminyl-glycine-amide (RQG-NH(2)), were found to interact with p24. With electron microscopy, disarranged core structures of HIV-1 progeny were extensively observed when the cells were treated with GPG-NH(2) and ALG-NH(2). Furthermore, nodular structures of approximately the same size as the broad end of HIV-1 conical capsids were observed at the plasma membranes of treated cells only, possibly indicating an arrest of the budding process. Corresponding tripeptides with nonamidated carboxyl termini were not biologically active and did not interact with p24.

Solid phase synthesis of partially protected tocinoic acid: Optimization with respect to resin and protecting groups

A few solid phase and solution approaches of good repute were applied in parallel with the aim to provide optimized routes to Boc- and Fmoc-tocinoic acid (3a and 3c) and the corresponding Tyr(Bu(t)) derivatives (3b and 3d). Boc-tocinoic acid is known to couple with tripeptide amides to give substituted oxytocin precursors in high yields, requiring only Boc-cleavage to furnish the corresponding hormone analogs with minimal loss of material. For comparison, two protected linear hexapeptides (2a and 2b) were prepared on three polystyrene supports, two with acid-labile handles and one a conventional chloromethylated resin, in yields of 62-82 and 58-76%, respectively. The intermediate 2a could be converted to 3a with physical data in agreement with those earlier reported. Similarly, the intermediate 2b was converted to 3b. The highest yields for both 2a and 2b were obtained with a 2-chlorotrityl chloride resin, which in addition provided advantages with respect to overall speed and convenience. Additional syntheses of 3c and 3d on this and of 3c on SASRIN resin, in conjunction with trityl instead of benzyl for side-chain protection of cysteine, were also elaborated.

Solid-phase synthesis of amino amides and peptide amides with unnatural side chains

Abstract Unnatural amino amides and peptide amides can be synthesized, using solid-phase chemistry, from glycine attached directly (or through an intervening peptide sequence) to a Rink resin. The glycine is converted to an activated benzophenone imine derivative, followed by C -alkylation and hydrolysis. This sequence provides a mild, high yielding route to Rink resin-bound racemic, unnatural, amino amides and di- and tripeptide amides. Cleavage with trifluoroacetic acid provides the final amide products in good yield and purity.

Exploratory pharmacokinetics and brain distribution study of a neuropeptide FF antagonist by liquid chromatography/atmospheric pressure ionization tandem mass spectrometry.

Dansyl-Pro-Gln-Arg-NH(2), an N-terminally modified tripeptide amide and a putative neuropeptide FF antagonist, was amenable to both positive-ion ESI and APCI. The protonated molecule yielded several fragment ions upon collision-induced dissociation in a quadrupole ion trap instrument for the development of LC/MS/MS assay methods. ESI clearly outperformed APCI in limits of detection, and was the method of choice for coupling with narrow-bore reversed-phase liquid chromatography to assess the pharmacokinetic profile and brain concentration of the neuropeptide FF antagonist in experimental animals. While plasma could be analyzed after rapid sample preparation, brain tissue required cleanup (solid phase extraction) and preconcentration before injection, and the assay was prone to matrix interference. This study indicated a rapid disappearance of dansyl-Pro-Gln-Arg-NH(2) from the plasma and the brain, and modest CNS bioavailability after intravenous administration to rats.

Synthesis of α-fluoroalkyl substituted peptides via enzymatic fragment condensation

A peptide library consisting of di- and tripeptide esters and tripeptide amides, respectively, containing α-Tfm amino acids in different positions was synthesized and tested for enzymatic fragment condensations catalyzed by the proteases α-chymotrypsin, trypsin and clostripain.

A study of a highly specific pyroglutamyl aminopeptidase type-II from the membrane fraction of bovine brain

Pyroglutamyl aminopeptidase type-II is reported to be a highly specific, membrane-bound neuropeptidase, which has the ability to hydrolytically remove the l-pyroglutamyl residue (pGlu) from the N-terminus of thyrotropin releasing hormone (TRH, pGlu-His-Pro-NH 2) and closely related tripeptides or tripeptide amides. The primary aim of this study was to purify this enzyme from bovine brain and to compare its characteristics with those previously reported. Following solubilization from the membrane fraction by limited proteolysis with trypsin, the enzyme was purified approximately 3000-fold with a 24% recovery of activity. A native molecular mass of 214 000 Da was determined for the purified enzyme by gel-filtration chromatography. A pH optimum of 6.8–7.6 was observed for the enzyme, with rapid inactivation occurring below pH 4.0 and above pH 9.2. Optimal enzyme activity was observed at 45°C. On the basis of its inhibition, in a time-dependent manner, by metal complexing agents and its subsequent reactivation in the presence of metal ions, the enzyme was identified as a metallopeptidase. Substrate specificity studies revealed that, with the exception of pGlu-Phe-Pro-NH 2, pGlu-7-amino-4-methyl-coumarin and pGlu- β-naphthylamide, the purified enzyme removes N-terminal pGlu from only tri- and tetrapeptides with a histidine residue in the penultimate position. A number of N-terminal pyroglutamyl peptides of varying length were shown to competitively inhibit the enzyme. Of these, luteinizing hormone releasing hormone (LHRH) and LHRH 1–5, although not substrates for the enzyme, were found to be potent inhibitors, with K i values of 8 and 11 μM, respectively. The study shows that while bovine brain PAPII shares many of the characteristics of PAPII from other mammalian tissues, its substrate specificity is not as narrow as previously reported.

Convergent synthesis of (−)-mirabazole B using a chloroimidazolidium coupling reagent, CIP

Convergent synthesis of (−)-mirabazole C (1), a tetra thiazoline/thiazole alkaloid isolated from blue-green alga, has been described. The successive thiazoline rings of (−)-mirabazole C were formed by a single-step cyclization mediated by TiCl4 treatment of tripeptide amide 4. Convergent synthesis of the key intermediate 33 derived from three 2-methylcysteine residues was first achieved using a newly developed coupling reagent, 2-chloro-1,3-dimethylimidazolidium hexafluorophosphate (CIP). The effectiveness of CIP for the coupling of α,α-dialkyl amino acids and the reaction pathway of the activation were clarified by the syntheses of model peptides containing an α,α-dimethylamino acid. A practical method of asymmetric synthesis of 2-methylcysteine by alkylation of 2,4-cis-oxazolidinone 23 has also been described.

Evaluation of the hydrophobic parameters of the amino acid side chains of peptides and their application in QSAR and conformational studies

We statistically analyzed the experimentally determined 1-octanol/water partition coefficient, log P, of a wide variety of N-acetyl-di- and tripeptide amides and unblocked di- and tripeptides to evaluate the hydrophobic parameters of the amino acid side chains of peptides. For blocked peptides, hydrophobic parameters were defined for amino acids having un-ionizable and ionizable side chains. For unblocked peptides, hydrophobic parameters were evaluated for 12 amino acids with un-ionizable side chains. In order to estimate the hydrophobic parameters for unnatural and ionizable amino acids, a procedure was developed based on the relationship between our hydrophobic parameters and the log P values of model amino acids calculated by the fragment constant method. Use of the hydrophobic parameters in quantitative structure-activity relationship (QSAR) studies of bioactive peptides and conformational studies of proteins have also been presented.