Synthesis,conformation and biological activity of centrally modified pseudopeptidic analogues of For-Met-Leu-Phe-OMe

Summary. For-Met-βAlaψ[CSNH]-Phe-OMe (3), For-Met-βAlaψ[CH₂NH]-Phe-OMe (5), For-Met-NH-pC₆H₄-SO₂-Phe-OMe (8a), For-Met-NH-mC₆H₄-SO₂-Phe-OMe (8b) and the corresponding N-Boc precursors (2, 4, 7a, b) have been synthesized and their activity towards human neutrophils has been evaluated in comparison with that shown by the reference tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe). Chemotaxis, lysozyme release and superoxide anion production have been measured. ¹H NMR titration experiments and IR spectra have been discussed in order to ascertain the preferred solution conformation adopted by the tripeptide 3 with particular reference to the presence of a folded conformation centred at the centrally positioned thionated β-residue.     

Crystal structure and a twisted β-sheet conformation of the tripeptide L-leucyl-L-leucyl-L-leucine monohydrate trimethanol solvate: Conformation analysis of tripeptides

In order to test the helical preference of short oligo-L -leucines, we crystallized the tripeptide L -leucyl-L .-leucyl-L -leucine (LLL) and carried out x-ray diffraction studies of it (L -leucyl-L -leucyl-L leucine)₂. 3CH₃OH. H₂O, (C₃₉H₈₄N₆O₁₂). Crystallized in the monoclinic system, space group P2₁, cell parameters: a = 12.031(2), b = 15.578(3), c = 14.087(2) Å, α = 90°, β = 97.29(1)°, γ = 90°, V = 2618.6 ų. MW = 829.1, D_c = 1.051 gcm^?3. R index of 0.057 for 4213 reflections (λ_cukα = 1.5418 Å) > 2σ. LLL takes tip the β-sheet rather than a helical conformation in the crystalline stale. The three methanol molecules and the water molecule that constitute the solvent of crystallization form a network of hydrogen bonds to the LLL molecules and to one another. It is rather remarkable that though A and L have stronger helical preferences than G, neither AAA nor LLL form the crystalline helix but GAL does, indicating that the helical preferences depend on the sequence context. The residue L2 in molecule A and the residues L1 and L3 of molecule B do not show the preferred conformation for forming helices. Further, very remarkably. LLL exhibits a unique super secondary feature of the protein folding topology, namely the twisted β-sheet. Where as most short peptides show only the classical p-sheet conformation. Thai even the tripeptide LLL is able to exhibit the twisted β-sheet conformation, and with the correct left-handed twist this suggests that even very short peptide segments possess the ability to assume several of the characteristic topological features exhibited by proteins. An extensive review of tripeptide conformations has been carried out and some results of this study have been included here. © 1995 John Wiley & Sons, Inc.     

Influence of Mannosylation on Immunostimulating Activity of Adamant‐1‐yl Tripeptide

The mannosylated derivative of adamant‐1‐yl tripeptide (D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant‐1‐yl tripeptide (Man‐OCH₂CH(Me)CO‐D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) is a non‐pyrogenic, H₂O‐soluble, and non‐toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala), a well‐known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM.     

Inhibition of TRAP-induced angiogenesis by the tripeptide Phe-Pro-Arg, a thrombin-receptor-derived peptide analogue

Summary We have recently shown that thrombin promotes angiogenesis by a mechanism independent of fibrin formation. In the present paper, we investigated the effect of the thrombin-receptor-activating tetradecapeptide (TRAP_1–14, S₄₂FLLRNPNDKYEPF₅₅) for its effects on angiogenesis in the chick chorioallantoic membrane (CAM) system of angiogenesis. A dose-dependent promotion of angiogenesis is evident with TRAP. In contrast, a thrombin-receptor-derived tripeptide analogue H-Phe-Pro-Arg-OH (FPR), which was designed based on the S₄₂FLLR₄₆ sequence, caused an inhibition of angiogenesis in the CAM, and when it was combined with TRAP it caused a complete reversal of the angiogenesis-promoting effect of TRAP. These results indicate that the proteolytic exposure of the receptor N-terminal tetradecapeptide by thrombin can activate the post-thrombotic events related to angiogenesis. These events can be modulated by constrained peptide analogues such as FPR.     

Effect of prostaglandins E1, E2 and F2α on neutrophil aggregation

Recently we have found that chemotactic factors stimulate neutrophils in suspension to aggregate. Because of an obvious analogy to platelet aggregation, we examined the influence of three prostaglandins on this process. Prostaglandins E₁, E₂ and F_2α alone did not cause aggregation of the neutrophils but were able to partially inhibit the aggregation response induced by the synthetic chemotactic tripeptide, formly-methionyl-leucyl-phenylalanine. The minimal inhibitory concentrations for prostaglandins E₁, E₂ and F_2α were 10⁻⁷, 10⁻⁶ and 10⁻⁵M, respectively. These results are similar to those found for the prostaglandin-induced inhibition of platelet aggregation. It may be, therefore, that neutrophil aggregation, like platelet aggregation, is modulated by intracellular prostaglandins and other products of arachidonic acid metabolism.     

Estimating glutathione synthesis with deuterated water: a model for peptide biosynthesis

Glutathione (GSH), an intracellular tripeptide that combats oxidative stress, must be continually replaced due to loss through conjugation and destruction. Previous methods, estimating the synthesis of GSH in vivo, used constant infusions of labeled amino acid precursors. We developed a new method based on incorporation of ²H from orally supplied ²H₂O into stable C-H bonds on the tripeptide. The incorporation of ²H₂O into GSH was studied in rabbits over a 2-week period. The method estimated N, the maximum number of C-H bonds in GSH that equilibrate with ²H₂O as amino acids. GSH was analyzed by liquid chromatography/mass spectrometry after derivatization to yield GSH-N-ethylmaleimide (GSNEM). A model, which simulated the expected abundance at each mass isotopomer for the GSNEM ion at various values for N, was used to find the best fit to the data. The plateau labeling fit best a model with N = 6 of a possible 10 C-H bonds. Thus, the amino acid precursors do not completely equilibrate with ²H₂O prior to GSH synthesis. Advantages of this new method include replacing costly amino acid infusions with the oral administration of ²H₂O and a statistical basis for estimating N.     

An improved synthesis of thyrotropin releasing hormone (TRH) and crystallization of the tartrate

An improved synthesis of thyrotropin releasing hormone (TRH), pGlu-His-Pro-NH₂, is reported. Z-pGlu-ONB (N-hydroxy-5-norbornene-2,3-dicarboximide ester) was reacted with H-His-OH to yield a crystalline Z-pGlu-His-OH which was coupled with H-Pro-NH₂ by the $\text{HONB}\text{DCC}$ method to give Z-pGlu-His-Pro-NH₂ as a fine crystal. Hydrogenation of this protected tripeptide yielded pure TRH nearly quantitatively. The optical purity of TRH thus obtained was confirmed by the method L- and D- amino acid oxidase digestion. The crystallization of TRH was achieved as a tartrate, and the properties of the crystalline TRH-tartrate are described.     

Characterization of a peptidase fromDiplococcus pneumoniae

Some properties of a purified peptidase fromDiplococcus pneumoniae have been studied. The enzyme has a broad pH optimum between 6 and 8 and a K_m (onl-leucylglycylglycine) of 2.8mm. It is activated by low levels of Hg⁺⁺ and is inhibited by Mn⁺⁺, Co⁺⁺, β-mercaptoethanol and EDTA. Substrate specificity studies show that the enzyme is an exopeptidase of the aminopeptidase type, most active on tripeptide substrates bearing bulky substituents at the NH₂ ⁻ terminal end.     

Structure primaire du caséinomacropeptide de la caseine K caprine

The amino acid sequence of caprine CMP, the negatively charged C-terminal fragment released by chymosin (rennin EC 3.4.23.4) from goat K-casein at the initial stage of the milk-clotting process, has been investigated. The complete sequence has been determined by analysing chymotryptic and « thermolysinfragments of the CMP. Caprine CMP contains 66 amino acid residues, 2 being phosphorylated, Asp₂, Asn₅, Thr₁₁, Ser₆, SerP₂, Glu₇, Gln₂, Pro₆, Ala₉, Val₅, Met₁, Ile₆, Lys₃, His₁, and the carbohydrate-free polypeptide chain has a molecular weight of 6,998 daltons.The occurrence in caprine CMP of an additional phosphate group, linked to serine 168 in the C-terminal region Thr-Ser168-Thr-Glu170-Val.OH of the polypeptide chain, has given support to the phosphorylation code for caseins that we postulated earlier. According to this hypothesis, a specific phosphoryl kinase may recognize an anionic phosphorylation site corresponding to the tripeptide sequence Thr/Ser-X-Glu, X being any amino acid residue. Since the C-terminal sequence of bovine and caprine CMPs differ by the substitution Ala/Glu170 (caprine), phosphorylation of caprine serine 168 could be explained by the occurrence of the new phosphorylation site Ser168-Thr-Glu170.     

Differential Regulation of Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Signaling and Membrane Trafficking by Multiple P2 Receptors in Brown Adipocytes

Extracellular ATP triggers changes in intracellular Ca²⁺, ion channel function, and membrane trafficking in adipocytes. The aim of the present study was to determine which P2 receptors might mediate the Ca²⁺ signaling and membrane trafficking responses to ATP in brown fat cells. RT-PCR was used to determine which P2 receptors are expressed in brown fat cells. Responses to nucleotide agonists and antagonists were characterized using fura-2 fluorescence imaging of Ca²⁺ responses, and FM 1-43 fluorescence imaging and membrane capacitance measurements to assess membrane trafficking. The pharmacology of the Ca²⁺ responses fits the properties of the P2Y receptors for which mRNA is expressed, but the agonist and antagonist sensitivity of the membrane-trafficking response was not consistent with any P2 receptor described to date. Brown adipocytes expressed mRNA for P2Y₂, P2Y₆, and P2Y₁₂ metabotropic receptors and P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, and P2X₇ ionotropic receptors. The agonists ATP, ADP, UTP, UDP and 2′, 3′-(benzoylbenzoyl) ATP (BzATP) increased intracellular Ca²⁺, while 100 μM suramin, pyridoxal-phosphate-6-azophenyl-2′ 4′-disulfonic acid (PPADS), or Reactive Blue 2 partially blocked Ca²⁺ responses. ATP, but not ADP, UTP, UDP or BzATP activated membrane trafficking. The membrane response could be blocked completely with 1 μM PPADS but not by the antagonist MRS2179. We conclude that multiple P2 receptors mediate the ATP responses of brown fat cells, and that membrane trafficking is regulated by a P2 receptor showing unusual properties.     

A triple-helical model for (Gly-Pro-Hyp)n with cis peptide units

Synthetic regular polytripeptides of the type (Gly-R₂-R₃) where R₂, R₃, or both, are imino acids have been widely studied as model compounds for collagen. One such polytripeptide is poly(Gly-Pro-Hyp), since triplets with this sequence constitute about 10% of collagen. Recently, a new model has been proposed for this polytripeptide in which one of the three peptide bonds in the tripeptide unit is in the cis conformation, and the γ-hydroxyl group of hydroxyproline forms a direct interchain hydrogen bond within the triple helix. We have confirmed this structure by model building using computer techniques, and the helical parameters obtained by us are close to the experimentally observed values. The model is also found to be comparable in stability with other models from energy considerations.     

Analysis of peptide uptake in Pseudomonas aeruginosa: A fluorescamine labeling procedure

Intracellular products of peptide transport were analyzed after thin layer chromatography separation by a fluorescamine labeling procedure. Dipeptides penetrated Pseudomonas aeruginosa via 2 transport systems, one of high affinity with K_T values from 1–2 μM, and another one of low affinity (K_T about 30 μM). A single transport system assumed tripeptide uptake with K_T varying from 10–30 μM, depending on the tripeptide used. Peptides entering the cells were rapidly hydrolyzed into their constitutive amino acids.     

Experimental conformational study of two peptides containing α-aminoisobutyric acid. Crystal structure of N-acetyl-α-aminoisobutyric acid methylamide

Some theoretical studies have predicted that the conformational freedom of the α-aminoisobutyric acid (H-Aib-OH) residue is restricted to the α-helical region of the Ramachandran map. In order to obtain conformational experimental data, two model peptide derivatives, MeCO-Aib-NHMe 1 and Bu^tCO-LPro-Aib-NHMe 2 , have been investigated. The Aib dipeptide 1 crystallizes in the monoclinic system (a = 12.71 Å, b = 10.19 Å, c = 7.29 Å, β = 110.02°, Cc space group) and its crystal structure was elucidated by x-ray diffraction analysis. The azimuthal angles depicting the molecular conformation (? = ?55.5°, ψ = ?39.3°) fall in the α-helical region of the Ramachandran map and molecules are hydrogen-bonded in a three-dimensional network. In CCl₄ solution, ir spectroscopy provides evidence for the occurrence of the so-called ₅ and C₇ conformers stabilized by the intramolecular i → i and i + 2 → i hydrogen bonds, respectively. The tripeptide 2 was studied in various solvents [CCl₄, CD₂Cl₂, CDCl₃, (CD₃)₂SO, and D₂O] by ir and pmr spectroscopies. It was shown to accommodate predominantly the βII folded state stabilized by the i + 3 → i hydrogen bond. All these experimental findings indicate that the Aib residue displays the same conformational behavior as the other natural chiral amino acid residues.     

Antiproliferative Effect of the Tripeptide PyroGlu-Phe-GlyNH2on Murine Melanocytes: Transitory Delay of Cell Growthin Vitroand the Cell Cycle Specificity

Cell growth and differentiation in melanocyte cell populations are regulated by a wide range of bioactive substances. Recently, the tripeptide pyroGlu-Phe-GlyNH₂which inhibits melanocyte growthin vitrowas identified in both murine nontransformed melanocytes and malignant melanoma cells. The present study was undertaken to investigate the cell cycle specificity as well as the growth inhibitory profile of the tripeptide after a single or repeated administration to melanocyte cultures. Dose-related effects of the peptide were studied using three different bioassay systems: estimation of cell number, DNA synthesis, and cell flux into mitosis. Growth of melanocyte cultures as well as melanocyte mitotic activity were found to be reduced significantly by the tripeptide at two separate dose levels (10⁻¹¹and 10⁻¹⁴–10⁻¹⁵M). Growth inhibition of melanocyte population did not last long: less than 36 h after the first and less than 24 h after the second peptide addition to the cultures. The level of DNA synthesis in melanocytes remained unchanged after a single peptide administration. The findings indicate that the tripeptide pyroGlu-Phe-GlyNH₂causes transitory delay of cell growth in cultured melanocyte population resulting from a reversible inhibition of melanocyte transition from the G₂-phase of the cell cycle into mitosis.     

Triple helix–coil transition of covalently bridged collagenlike peptides

The thermal triple helix–coil transition of covalently bridged collagenlike peptides with repeating sequences of (Ala-Gly-Pro)_n, n = 5–15, was studied optically. The peptides were soluble in water/acetic acid (99:1) and were found to form triple-helical structures in this solvent system beginning with n = 8. The thermodynamic analysis of the transition equilibrium curves for n = 9–13 yielded the parameters ΔH°_s = ?7.0 kJ per tripeptide unit, ΔS°_s = ?23.1 J deg^?1 mol^?1 per tripeptide unit for the coil-to-helix transition, and the apparent nucleation parameter σ ? 5 × 10^?2. It was suggested through double-jump temperature experiments that the rate-limiting step during refolding is not only influenced by the difficulties of nucleation, but also by cis–trans isomerization of the Gly-Pro peptide bond.     

Effect of Oxygen-Derived Free Radicals and Oxidants on the Degradation in vitro of Membrane Phospholipids

The abilities of chemically generated hydroxyl radical (OH), superoxide anion (O^?) and hydrogen peroxide (H₂O₂) to degrade rat myocardial membrane phospholipids previously lableed with [1 -¹⁴C]arachidonic acid were studied. HO and H₂O₂ but not O₂^?_?, caused the degradation of phospha-tidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). With OH' and H₂O₂, the loss of radiolable in PC was accompanied by an increase in the radiolabel of lysophosphatidylcholine (LPC), but not in that of free fatty acid (FFA). These results suggest the hydrolysis of l-oxygen ester bond of PC by HO' and that H₂O₂ and that HO' and H₂O₂, but not O^?, are detrimental to the structure and function of membrane phospholipids. However, since μM amounts of HO' and mM amounts of H₂O₂ were necessary to affect the membrane phospholipids, it is likely that in the reprefused myocardium only HO', but not H₂O₂, may directly cause the breakdown of membrane phospholipids.     

A crystal structure with features of an antiparallel α-pleated sheet

A single-crystal x-ray diffraction analysis of Boc-L -Ala-D -aIle-L -Ile-OMe has been carried out. The analysis has shown (a) that the tripeptide molecules have in part an α-extended conformation, the torsion angles of the L -Ala and D -aIle residues being φ₁ = ?75.1° and ψ₁ = ?25.8° and φ₂ = 67.3° and ψ₂ = 44.1°, respectively, and (b) that the molecules are organized in rippled planes where they occur in relative antiparallel orientation linked together side by side by H bonds. This molecular organization of the tripeptide corresponds closely to that of an antiparallel α-pleated sheet, and likely constitutes the first example of a structure of this kind for which a characterization at the atomic level has been achieved. A molecular dynamics study has shown that the molecular conformation of the tripeptide in the crystalline state is determined primarily by intermolecular interactions. © 1994 John Wiley & Sons, Inc.     

Transport of ions across peritoneal membrane

The electrical conductance of ions across the peritoneal membrane of young buffalo (approximately 18-24 months old) has been recorded. Aqueous solutions of NaF, NaNO₃, NaCl, Na₂SO₄, KF, KNO₃, KCl, K₂SO₄, MgCl₂, CaCl₂, CrCl₃, MnCl₂, FeCl₃, CoCl₂, and CuCl₂ were used. The conductance values have been found to increase with increase in concentration as well as with temperature (15 to 35 °C) in these cases. The slope of plots of specific conductance, κ, versus concentration exhibits a decrease in its values at relatively higher concentrations compared to those in extremely dilute solutions. Also, such slopes keep on increasing with increase in temperature. In addition, the conductance also attains a maximum limiting value at higher concentrations in the said cases. This may be attributed to a progressive accumulation of ionic species within the membrane. The κ values of electrolytes follow the sequence for the anions: SO₄²⁻>Cl⁻>NO₃⁻>F⁻ while that for the cations: K⁺>Na⁺>Ca²⁺>Mn²⁺>Co²⁺>Cu²⁺>Mg²⁺>Cr³⁺>Fe³⁺. In addition, the diffusion of ions depends upon the charge on the membrane and its porosity. The membrane porosity in relation to the size of the hydrated species diffusing through the membrane appears to determine the above sequence. As the diffusional paths in the membrane become more difficult in aqueous solutions, the mobility of large hydrated ions gets impeded by the membrane framework and the interaction with the fixed charge groups on the membrane matrix. Consequently, the membrane pores reduce the conductance of small ions, which are much hydrated. An increase in conductance with increase in temperature may be due to the state of hydration, which implies that the energy of activation for the ionic transport across the membrane follows the sequence of crystallographic radii of ions accordingly. The Eyring's equation, κ=(RT/Nh)exp[−ΔH*/RT]exp[ΔS*/R], has been found suitable for explaining the temperature dependence of conductance in the said cases. This is apparent from the linear plots of log[κNh/RT] versus 1/T. The results indicate that the permeation of ions through the membrane giving negative values of ΔS* suggest that there may be formation of either covalent linkage between the penetrating ions and the membrane material or else the permeation may not be the rate-determining step. On the one hand, a high ΔS* value associated with the high value of energy of activation, E_a, for diffusion may suggest the existence of either a large zone of activation or loosening of more chain segments of the membrane. On the other hand, low value of ΔS* implies that converse is true in such cases, i.e., either a small zone of activation or no loosening of the membrane structure upon permeation.     

Effects of prostaglandin D2 on membrane potential in neuroblastoma X glioma hybrid cells as determined with a cyanine dye

A cyanine dye, diS-C₃-(5) was used to determine the effects of prostaglandins on the membrane potential in neuroblastoma X glioma cells (NG 108-15). The largest depolarization was seen with prostaglandin D₂ (ED₅₀ = 1.5 μM), and relative potencies of various prostaglandins (3 μM) were: D₂, 100; I₂, 41; E₁, 17; E₂, 7; and F_2α, 7. 5-Hydroxytryptamine in a dose over 100 μM also depolarized the membrane. The effect of prostaglandin D₂ was observed in a Na⁺-free medium or when Ca²⁺ was replaced by Sr²⁺. The addition of 3 mM ethylene-glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid or 5 mM Co²⁺ partially inhibited the effects. These observations suggest that the depolarization of membrane by prostaglandin D₂ may primarily be related to alteration of Ca²⁺ permeability in the cell membrane.     

Influenza virus H5N1 hemagglutinin (HA) T‐cell epitope conjugates: design,synthesis and immunogenicity

The influenza virus, major surface glycoprotein hemagglutinin (HA) is one of the principal targets for the development of protective immunity. Aiming at contributing to the development of a vaccine that remains the first choice for prophylactic intervention, a reconstituted model of HA, mimicking its antigenic properties was designed, synthesized and tested in mice for the induction of protective immunity. Four helper T lymphocyte [HTL (T₁, T₃, T₇ and T₈)] and four cytotoxic lymphocyte [CTL (T₂, T₄, T₅ and T₆)] epitopes were coupled in two copies each to an artificial carrier, SOC₄, which was formed by the repeating tripeptide Lys‐Aib‐Gly. The helical conformation of the SOC₄‐conjugates preserves the initial topology of the attached epitopes, which is critical for their immunogenic properties. Survival of immunized animals, ranged from 30 to 50%, points out the induction of protective immunity by using the SOC₄‐conjugates. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.