Cyanamide mediated syntheses of peptides containing histidine and hydrophobic amino acids

Summary Using the model of a primitive earth evaporation pond, the synthesis of three histidyl peptides in yields of up to 11% was demonstrated when aqueous solutions of histidine, leucine, ATP, cyanamide, and MgCl₂ were evaporated and heated for 24 h at 80°C. In addition, peptides were formed in yields of up to 56%, 35%, and 21%, respectively for phenylalanine, leucine, and alanine when aqueous solutions of the appropriate amino acid were evaporated and heated with cyanamide and one or more of the following components: ATP, AMP, 4-amino-5-imidazole carboxamide, or MgCl₂. The greatest peptide yield occurred at pH 3. But peptide formation was demonstrated for a system of Leu, cyanamide, and MgCl₂ adjusted to pH 7 with NH₄OH.Peptide synthesis was also studied in the presence of CaCl₂, ZnCl₂, different adenosine nucleotides, and UTP to compare their effects on peptide synthesis. The optimum conditions for cyanamide mediated peptide synthesis were also studied in terms of pH, reaction time, reaction temperature, and cyanamide concentration. The major side product in nearly all reactions studied appears to be an amino acid-cyanamide adduct. Peptides were analyzed and identified by thin layer chromatography, acid hydrolysis, and enzymatic degradation.     

The formation of peptides from glycine thioesters

Summary The condensation products obtained from 0.01M S-glycyl-N-acetyl-cysteamine at different pH's were investigated. The highest yields of diketo-piperazine (approx. 50%) were observed in phosphate buffers between pH 7.5 and 8.5. The highest yields of diglycine (46%), triglycine (10%) and tetraglycine (2%) were observed in carbonate buffers at pH 9.5. At pH 8.0, over 90% of the glycyl residues of 0.15M S-glycyl-N-acetylcysteamine were incorporated into condensation products, mainly DKP (60–70%). The yields of products from the condensation of S-glycyl-ethanethiol under similar conditions closely re-sembled those obtained with S-glycyl-N-acetylcysteamine.Abbreviations Boc-gly N-tert-butyloxycarbonylglycine - Ac-cys N-acetylcysteine - csa cysteamine - Ac-csa N-acetylcysteamine - DKP diketopiperazine - (gly) ₂ diglycine - (gly) ₃ triglycine - (gly) ₄ tetraglycine - glySEt S-glycyl-ethanethiol - glyS-(Ac-cys) S-glycyl-N-acetylcysteine - glyS-(Ac-csa) S-glycyl-N-acetylcysteamine - Boc-glyS-(Ac-cys) S-(Boc-glycyl)-N-acetylcysteine - Boc-glyS-(Ac-csa) S-(Boc-glycyl)-N-acetylcysteamine - Boc-glySEt S-(Boc-glycyl)-ethanethiol - gly-bydrox glycine hydroxamate     

Electrical excitability of proteinoid microspheres composed of basic and acidic proteinoids

Material flow equilibration, an endogenous interaction adjustment process underlying fulfillment of material flow continuity during material self-assembly, also underlies electrical excitability observed in the proteinoid microspheres as simulated models of the protocell. The spikings of the membrane potentials are attributed to a singular character of the interaction rate coefficients measuring the strengths of the coupling between basic and acidic proteinoids, in which the rates change singularly with time due to material flow equilibration.     

The effect of pyrophosphate, tripolyphosphate and ATP on the rate of the Fenton reaction

It has been recently reported that pyrophosphate, tri-polyphosphate, ATP and analogous ligands considerably decrease the yield of hydroxyl radicals by the Fenton reaction under conditions where [H₂O₂] > > [Fe(II)L_n]. It was suggested that this effect is due to the slowing down of the Fenton reaction by these ligands. This suggestion seemed surprising as polyphosphate ligands stabilize Fe(III). Indeed, a kinetic study points out that these ligands accelerate the rate of the Fenton reaction by several orders of magnitude. Thus it is suggested that the effect of the ligands on the yield of the hydroxyl radicals is due to the stabilization of the Fe(III) complexes which slows down, or inhibits, their reduction by the radicals formed in the system and thus decreases the overall yield of hydroxyl radicals.     

Characterization of glutathione conjugates of pyrrolylated amino acids and peptides by liquid chromatography-mass spectrometry and tandem mass spectrometry with electrospray ionization

High-performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ES-MS) and tandem mass spectrometry (MS-MS) was used to identify the products formed upon reaction of lysine-containing peptides with the neurotoxicant 2,5-hexanedione (2,5-HD). In addition, secondary autoxidative reaction products of the resultant alkylpyrroles with the biological thiol, glutathione, were characterized. ES mass spectra of the HPLC-separated conjugates showed intense [M+H]⁺ ions as well as several ions formed by amide and C-S bond cleavage. The glutathione conjugates of pyrrolylated amino acids and peptides were analyzed by ES ionization and MS-MS, and product-ion spectra showed fragmentation pathways typical of glutathione conjugates. ES-MS-MS analysis of a synthetic nonapeptide modeling a sequence found in neurofilament proteins showed pyrrole formation after incubation with 2,5-HD, and sequence ions were used to assign the position of the pyrrole adduct. Subsequent reaction of the pyrrolylated peptide with reduced glutathione was evidenced by a shift in m/z of the sequence ions of the reaction products with or without prior methylation. The results demonstrate the utility of ES-MS and ES-MS-MS in the characterization of xenobiotic-modified peptides and confirm that stable pyrrole-thiol conjugates are formed by the reaction of biological thils with pyrrolylated peptides.     

The first peptides: The evolutionary transition between prebiotic amino acids and early proteins

The issues we attempt to tackle here are what the first peptides did look like when they emerged on the primitive earth, and what simple catalytic activities they fulfilled. We conjecture that the early functional peptides were short (3-8 amino acids long), were made of those amino acids, Gly, Ala, Val and Asp, that are abundantly produced in many prebiotic synthesis experiments and observed in meteorites, and that the neutralization of Asp's negative charge is achieved by metal ions. We further assume that some traces of these prebiotic peptides still exist, in the form of active sites in present-day proteins. Searching these proteins for prebiotic peptide candidates led us to identify three main classes of motifs, bound mainly to Mg²⁺ ions: D(F/Y)DGD corresponding to the active site in RNA polymerases, DGD(G/A)D present in some kinds of mutases, and DAKVGDGD in dihydroxyacetone kinase. All three motifs contain a DGD submotif, which is suggested to be the common ancestor of all active peptides. Moreover, all three manipulate phosphate groups, which was probably a very important biological function in the very first stages of life. The statistical significance of our results is supported by the frequency of these motifs in today's proteins, which is three times higher than expected by chance, with a P-value of 3×10⁻². The implications of our findings in the context of the appearance of life and the possibility of an experimental validation are discussed.     

Cellular differentiation assessments by measuring the degree of cellular internalization and membrane adsorption using designed peptides

We demonstrate examples of cellular differentiation assessments, including cellular neurite outgrowth and fat cell maturation, by measuring the degree of membrane adsorption or cellular internalization using designed peptides. Because changes in the cellular membrane and cytosol during differentiation were shown to influence membrane adsorption and cellular internalization, we could successfully evaluate the extent of differentiation simply like stain indicators.     

Inductive effects in amino acids and peptides: Ionization constants and tryptophan fluorescence

Although inductive effects in organic compounds are known to influence chemical properties such as ionization constants, their specific contribution to the properties/behavior of amino acids and functional groups in peptides remains largely unexplored. In this study we developed a computationally economical algorithm for ab initio calculation of the magnitude of inductive effects for non-aromatic molecules. The value obtained by the algorithm is called the Inductive Index and we observed a high correlation (R² = 0.9427) between our calculations and the pK_a values of the alpha-amino groups of amino acids with non-aromatic side-chains. Using a series of modified amino acids, we also found similarly high correlations (R² > 0.9600) between Inductive Indexes and two wholly independent chemical properties: i) the pK_a values of ionizable side-chains and, ii) the fluorescence response of the indole group of tryptophan. After assessing the applicability of the method of calculation at the amino acid level, we extended our study to tryptophan-containing peptides and established that inductive contributions of neighboring side-chains are transmitted through peptide bonds. We discuss possible contributions to the study of proteins.     

ATP synthesis and storage

Since 1929, when it was discovered that ATP is a substrate for muscle contraction, the knowledge about this purine nucleotide has been greatly expanded. Many aspects of cell metabolism revolve around ATP production and consumption. It is important to understand the concepts of glucose and oxygen consumption in aerobic and anaerobic life and to link bioenergetics with the vast amount of reactions occurring within cells. ATP is universally seen as the energy exchange factor that connects anabolism and catabolism but also fuels processes such as motile contraction, phosphorylations, and active transport. It is also a signalling molecule in the purinergic signalling mechanisms. In this review, we will discuss all the main mechanisms of ATP production linked to ADP phosphorylation as well the regulation of these mechanisms during stress conditions and in connection with calcium signalling events. Recent advances regarding ATP storage and its special significance for purinergic signalling will also be reviewed.     

Are learning and attention related to the sequence of amino acids in ACTH/MSH peptides?

Learning and attention were examined in rats after injections of one of several molecules related to adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (MSH). The initial phase of the learning process was linearly related to the length of the peptide with the smallest fragment (MSH/ACTH 4–10) improving learning the most and the largest molecule (ACTH 1–24) exerting no effect. Later stages of the learning problem, which were sensitive to the attentional state of the organism, resulted in U-shaped relations with the length of the same peptides. Enhancement of attention was significant only for the MSH compounds. These data indicate that some behaviors may be influenced as a function of the redundant information contained in the molecule while other behaviors may be discretely related to the specific conformation of the molecule.     

The solid-state catalytic synthesis of tritium labeled amino acids,peptides and proteins

Summary New catalytic reaction between a solid bioorganic compound and activated spillover tritium (ST), based on High-temperature Solid-state Catalytic Isotopic Exchange (HSCIE) was examined. The HSCIE mechanism and determination of the reactivity of hydrogen atoms in amino acids, peptides and proteins was investigated. Quantum mechanical calculations of the reactivity of hydrogen atoms in amino acids in the HSCIE reaction were done. The carbon atom with a greater proton affinity undergoes a greater exchange of hydrogen for tritium in HSCIE. The electrofilic nature of spillover hydrogen in the reaction of HSCIE was revealed. The isotope exchange between ST and the hydrogen of the solid organic compound proceeds with a high degree of configuration retention at the carbon atoms. The HSCIE reaction enables to synthesize tritium labeled proteins with a specific activity of 20–30 mCi/mg and kept biological activity.Presented at the 3rd International Congress on Amino Acids, Peptides and Analogues. Vienna, August, 23–27, 1993     

ESR and optical absorption studies on the copper(II) interaction with small peptides containing aromatic amino acids

The interaction of Cu(II) with di- and tripeptides each containing phenylalanine, tryptophan or histidine in the amino acid chain has been investigated by means of electron spin resonance (ESR) and optical absorption spectroscopy. Cu(II) complexes of dipeptides and tripeptides exhibit different magnetic and optical parameters. Dipeptide complexes have larger g -values and smaller {A –values than tripeptide complexes. When compared to dipeptide complexes, the d-d band of the central metal ion is blue shifted for tripeptide complexes. There are no significant differences in the behavior of Cu(II) peptide complexes containing phenylalanine or tryptophan. Complexes of histidine containing peptides, however, show modified spectra caused by the participation of the imidazole nitrogen in the coordination to Cu(II). The imidazole nitrogen seems to coordinate in-plane with other coordinating atoms or in an axial position depending on the kind of peptide.Part of the Ph.D. thesis of L.S., D-26Dedicated to Prof. Dr. H. Glubrecht on the occasion of his 60th birthday     

Optimization of a free separation of 30 free amino acids and peptides by capillary zone electrophoresis with indirect absorbance detection: a potential for quantification in physiological fluids

This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2+/-0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm x 75 microm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 micromol/l (median 6.71 micromol/l). The method was linear within the 50-200 micromol/l concentration range (r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step.     

Arachidonoyl amino acids and arachidonoyl peptides: Synthesis and properties

N-Arachidonoyl (AA) derivatives of amino acids (glycine, phenylalanine, proline, valine, γ-aminobutyric acid (GABA), dihydroxyphenylalanine, tyrosine, tryptophan, and alanine) and peptides (Semax, MEHFPGP, and PGP) were synthesized in order to study the biological properties of acylamino acids. The mass spectra of all the compounds at atmospheric pressure electrospray ionization display the most intense peaks of protonated molecular ions; the detection limits for these compounds are 10 fmol per sample. AA-Gly showed the highest inhibitory activity toward fatty acid amide hydrolase from rat brain (IC₅₀ 6.5 μM) among all the acylamino acids studied. AA-Phe, AA-Tyr, and AA-GABA exhibited a weak but detectable inhibitory effect (IC₅₀ 55, 60, and 50 μM, respectively). The acylated amino acids themselves, except for AA-Glu, were stable to the hydrolysis by this enzyme. All the arachidonoylamino acids inhibited cabbage phospholipase D to various degrees; AA-GABA and AA-Phe proved to be the most active (IC₅₀ 20 and 27 μM, respectively). Attempts to detect the biosynthesis of AA-Tyr in homogenates of rat liver and nerve tissue in vitro were unsuccessful; however, AA-dopamine and AA-Phe, the products of its metabolism, were found. The highest contents of these metabolites were detected in liver homogenate and in the brain homogenate, respectively. Acylamino acids exert no cytotoxic effect toward the glioma C6 cells. It was shown that N-acylation of Semax with arachidonic acid results in enhancement of its hydrolytic stability and increases its affinity for the sites of specific binding in rat cerebellum membranes.     

NTS2-selective neurotensin mimetics with tetrahydrofuran amino acids

Stimulation of the NTS2 neurotensin receptor causes antipsychotic effects and leads to a promotion of the μ-opioid-independent antinociception, which is important in the modulation of tonic pain sensitivity. We report the synthesis and properties of a small library of peptidic agonists based on the active neurotensin fragment NT(8–13). Two tetrahydrofuran amino acid derivatives were synthesized to replace Tyr¹¹ in NT(8–13). Additionally, Arg⁸, Arg⁹, and Ile¹² of the lead peptide were exchanged by Lys, Lys, and Gly, respectively. The new compounds showed substantial NTS2 binding affinity and up to 1000-fold selectivity over NTS1. The highest selectivity (K_i(NTS2): 29 nM, K_i(NTS1): 35,000 nM) was observed for the peptide analog 17R^trans.     

Synthesis of peptides containing oxo amino acids and their crystallographic analysis

An isolated uncharged hydrogen bond acceptor such as the carbonyl functionality of an aldehyde or a keto group is absent in natural amino acids. Although glutamine and asparagine are known to hydrogen bond through the amide carbonyl group in their side chains, they also possess the amide ? NH₂ group, which can act as a hydrogen bond donor. This makes the structural study of peptides containing an oxo residue, with an isolated carbonyl group in the side chain, interesting. Here, we report the synthesis of δ‐ and ε‐oxo amino acids and their incorporation into oligopeptides as the N‐terminal residue. The resultant oxo peptides were extensively studied using X‐ray crystallography to understand the interactions offered by the oxo group in peptide crystals. We find that the oxo groups are capable of providing additional hydrogen bonding opportunities to the peptides, resulting in increased intermolecular interactions in crystals. The study thus offers avenues for the utilization of oxo residues to introduce intermolecular interactions in synthetic peptides.     

Nucleo amino acids as arginine mimetics in cyclic peptides

A general strategy for the synthesis of Fmoc protectednucleobase modified amino acids is presented. Fmoc protected nucleo amino acidsbearing a natural purine (guanine) as well as an artificial purine(isoadenine) in the side chain have been synthesized and incorporated into cyclicpentapeptides. The structure of the cyclic peptides is based on the well knownRGD peptides, which act as selective integrin antagonists. Thenucleo amino acids serve as conformationally constrained arginine mimeticswith a reduced basicity of the guanidino moiety.