Radioimmunoassays for fish tail neuropeptides: II. Development of a specific and sensitive assay for and the occurrence of immunoreactive urotensin II in the central nervous system and blood of Catostomus commersoni

A sensitive radioimmunoassay (RIA) based on an antiserum to synthetic Gillichthys mirabilis urotensin II (UII) generated in rabbits, reacting with all known forms of the UII peptides, was developed. The UII was iodinated by either the chloramine-T or the lodogen method and was purified by high-pressure liquid chromatography. The antiserum, at a final dilution of 1:125,000, gave 50% binding of the iodinated UII. The sensitivity of the RIA was 1.8 ± 0.2 pg/assay tube (1.2 ± 0.2 fmol/tube; n = 7). Crossreactivity studies with various UII peptides, modified UII peptides, and fragments indicated that the immunoreactive recognition site of this antiserum is directed to the disulfide ring region (positions 6–11) of the UII peptides. Somatostatin 14, which has the part sequence 7–9 in common with UII peptides, did not crossreact. No detectable crossreactivity with urotensin I, urophysin B or D from Catostomus commersoni, arginine vasopressin, or arginine vasotocin could be shown. Displacement curves obtained with standard extracts of G. mirabilis urophyses were similar to those of synthetic G. mirabilis UII. comparison of RIA and bioassay of G. mirabilis urophysial extracts showed good correlation. Immunoreactive UII was detected in different parts of the brain, spinal cord, and blood of C. commersoni.     

Isolation and sequence determination of trichorzianines A antifungal peptides from Trichoderma harzianum

Trichorzianines A, membrane active peptides of the peptaibol class, were isolated from cultures of the mould Trichoderma harzianum. Trichorzianines A were separated into pure components by HPLC on octadecyl bonded and SiO₂ phases successively. Nine trichorzianines A (IIa, IIIa, IIIb, IIIc, IVb, Vb, VIa, VIb and VII) were isolated from the complex microheterogeneous mixture. Their N-terminal amino acid is acetylated, the C-terminal amino alcohol is either tryptophanol or phenylalaninol, 7 to 8 of the 19 residues are α-aminoisobutyric acid. Gas chromatography on a chiral phase showed isovaline to have the d -configuration and all the other optically active amino acids and amino alcohols to have the l -configuration. The amino acid sequences were determined from their positive ion FAB mass spectra which exhibited the preferential cleavage of the Aib 12-Pro 13 amide bond as a main fragmentation. The resulting fragments subsequently underwent amide bond ruptures that generated two series of abundant acylium ions which enabled direct determination of the 1–19 sequence. The relative position of the isomeric amino acids in the sequence of trichorzianine AVII was assigned from analysis of the N- and C-terminal oligopeptides yielded by its selective acidic hydrolysis. The microheterogeneity of trichorzianines A results mainly from single or multiple substitution of amino acids at the specific positions 5, 14, 16 and 19.     

Does cigarette smoking increase plasma urotensin II concentrations?

Objective Human urotensin II (UII) acts on the urotensin (UT) receptor and is the most potent mammalian vasoconstrictor identified to date. The role of UII in human cardiovascular regulation remains unclear, and the results of plasma measurements have been conflicting, perhaps because different measurement techniques have been used. The effects of cigarette smoking on plasma UII concentrations are unknown. The primary aim of our study was to demonstrate whether cigarette smoking had any effect on plasma UII concentrations in otherwise healthy volunteers. Our secondary aim was to compare the results obtained from assaying simultaneously using both radioimmunoassay (RIA) and immunoluminometric assay (ILMA). Methods Blood was taken from 20 healthy male non-smokers and 20 healthy male cigarette smokers. Plasma was separated and stored at −70°C. Samples were batch analysed simultaneously for UII using RIA and ILMA. Results Median (range) plasma UII concentrations were lower in non-smokers [1.67 (1.0–2.27) pg ml⁻¹] compared to smokers [2.62 (1.87–3.46) pg ml⁻¹] (P = 0.03) measured using RIA. Those who had smoked a cigarette in the 10 min before sampling had greater concentrations of UII [3.10 (1.87–4.60) pg ml⁻¹] compared to controls (P = 0.01). Plasma UII concentrations determined by ILMA were consistently low with no differences between groups. Conclusion The data obtained by RIA show that smoking may increase plasma concentrations of UII with a more pronounced increase when a cigarette has been smoked recently. There was a complete lack of correlation between RIA and ILMA for the whole data set, which suggests that some of the variability in plasma UII reported in the literature may result from differences between assays. Presented in part at the Anaesthetic Research Society meeting, Leicester, UK, 24 November 2005.     

Total synthesis of S-carbamoylmethyl bovine apocytochrome c by segment coupling

Three peptide segments corresponding to the complete sequence of the 104 amino acid protein bovine apocytochrome c were synthesized by the solid-phase method. The peptides Ac-[Cys(Cam)^14,17, GlyS²³]-apocytochrome c-(1–23) (I), CF₃CO-[GlyS⁶⁰]-apocytochrome c-(24–60) (II), and CF₃CO-apocytochrome c-(61–104) (III) were purified by chromatography on CM-cellulose, partition chromatography and/or HPLC. Each of the peptides was reacted with citraconic anhydride to block all of the lysine side chains, and the 61–104 peptide was treated with 10% hydrazine to remove the trifluoroacetyl group, to give the corresponding peptides Ia, IIa, and IIIa. Peptides IIa and IIIa were coupled together by reaction with silver nitrate/N-hydroxysuccinimide to give the 24–104 sequence. After removal of the trifluoroacetyl group from the amino terminus, peptide Ia was also coupled. Treatment of the peptide mixture with aqueous acetic acid removed the citraconyl groups, and purification by chromatography on CM-cellulose and HPLC gave a 0.6% yield of [Cys-(Cam)^14,17]-apocytochrome c. The synthetic product was shown to be identical to a sample derived from native bovine cytochrome c by paper or gel electrophoresis, HPLC and by chymotryptic or tryptic map.     

Enhancement of vascular smooth muscle cell migration by urotensin II

The effects of urotensin II (UII) on migration of human aortic smooth muscle cells (HASMCs) were investigated. UII (1–100 nM) significantly increased velocity of HASMC motility in a concentration-dependent manner. Stress-fiber formation and ERK (p44/p42) activity were also increased by UII. U0126 and PD 98059, MEK inhibitors, abolished the effects of UII on motility velocity and stress-fiber formation. These results suggest that UII enhances HASMC migration through activation of an ERK-dependent pathway.     

Opposite actions of urotensin II and relaxin‐2 on cellular expression of fibronectin in renal fibrosis: A preliminary experimental study

Our aim was to evaluate the role of urotensin II, urantide (urotensin II receptor antagonist) and relaxin‐2 on the cellular expression of fibronectin as a surrogate marker for renal fibrosis. We employed LLC‐PK1 renal tubular epithelial cells and assessed the influence on the fibrotic process of the above‐mentioned substances by using anti‐fibronectin antibodies in western blot analysis. The addition of urotensin II increased fibronectin expression. Urantide reduced the positivity for fibronectin caused by urotensin II (P<.05). The anti‐fibrotic action was more evident for relaxin‐2 (P<.01). Also in the model of TGF‐β1‐induced fibrosis, urantide and, to a greater extent, relaxin‐2 were able to significantly lessen fibronectin expression (respectively, P<.05 and P<.01). In conclusion, relaxin‐2 may reduce urotensin II‐induced renal fibrosis.     

Determination of stereochemistry stability coefficients of amino acid side‐chains in an amphipathic α‐helix

Abstract: We describe here a systematic study to determine the effect on secondary structure of d ‐amino acid substitutions in the nonpolar face of an amphipathic α‐helical peptide. The helix‐destabilizing ability of 19 d ‐amino acid residues in an amphipathic α‐helical model peptide was evaluated by reversed‐phase HPLC and CD spectroscopy. l ‐Amino acid and d ‐amino acid residues show a wide range of helix‐destabilizing effects relative to Gly, as evidenced in melting temperatures (ΔT_m) ranging from ?8.5°C to 30.5°C for the l ‐amino acids and ?9.5°C to 9.0°C for the d ‐amino acids. Helix stereochemistry stability coefficients defined as the difference in T_m values for the l ‐ and d ‐amino acid substitutions [(ΔT_m′ = T_mL and T_mD)] ranging from 1°C to 34.5°C. HPLC retention times [Δt_R(X_L?X_D)] also had values ranging from ?0.52 to 7.31 min at pH 7.0. The helix‐destabilizing ability of a specific d ‐amino acid is highly dependent on its side‐chain, with no clear relationship to the helical propensity of its corresponding l ‐enantiomers. In both CD and reversed‐phase HPLC studies, d ‐amino acids with β‐branched side‐chains destabilize α‐helical structure to the greatest extent. A series of helix stability coefficients was subsequently determined, which should prove valuable both for protein structure‐activity studies and de novo design of novel biologically active peptides.     

Fluorine‐18 labelling of PNAs functionalized at their pseudo‐peptidic backbone for imaging studies with PET

Peptide nucleic acids (PNAs) form a unique class of synthetic macromolecules, originally designed as ligands for the recognition of double‐stranded DNA, where the deoxyribose phosphate backbone of original DNA is replaced by a pseudo‐peptide N‐(2‐aminoethyl)glycyl backbone, while retaining the nucleobases of DNA. We have previously developed an original method to label oligonucleotide‐based macromolecules with the short‐lived positron‐emitter fluorine‐18 (t_1/2: 109.8 min) using the N‐(4‐[¹⁸F]fluorobenzyl)‐2‐bromoacetamide reagent. Using this method, we herein report the fluorine‐18‐labelling of 13 decameric PNAs ( OLP_1‐13 ), of the same sequence (CTCATACTCT), but presenting selected modification of the pseudo‐peptidic backbone at two or three of the thymine residues (positions 2, 5 and 8). Structural characteristics of these backbone modifications include either an amino acid side chain (L ‐Lys, L ‐Glu, L ‐Leu and L ‐Arg) or a glycosyl moiety (mannose, galactose, fucose, N‐Ac‐galactosamine and N‐Ac‐glucosamine) attached via an appropriate spacer. N‐(4‐[¹⁸F]fluorobenzyl)‐2‐bromoacetamide was synthesized in three radiochemical steps from 4‐cyano‐N,N,N‐trimethylanilinium trifluoromethanesulfonate and HPLC‐purified in 85–90 min (typical production: 3.7–4.8 GBq starting from a batch of 29.6–31.4 GBq of [¹⁸F]fluoride). Conjugation of the fluorine‐18‐labelled bromoacetamide reagent with the PNAs was performed in a mixture of acetonitrile and HEPES buffer (0.1 M, pH 7.9) for 10 min at 60°C and gave the corresponding pure labelled conjugated PNAs ([¹⁸F] c‐OLP_1‐13 ) after RP‐HPLC purification. The whole synthetic procedure, including the preparation of the fluorine‐18‐labelled reagent, provides up to 0.9 GBq (25 mCi) of HPLC‐purified [¹⁸F] c‐OLP_1‐13 in 160 min with a specific radioactivity of 45–65 GBq/µmol (1.2–1.7 Ci/µmol) at the end of synthesis starting from 29.6 to 31.4 GBq (800–850 mCi) of [¹⁸F]fluoride. Copyright © 2004 John Wiley & Sons, Ltd.     

Large fragments of nef-protein and gp110 envelope glycoprotein from HIV-1

Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and of nef-protein from HIV-1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4-(oxymethyl)-phenylacetamidomethyl resin using the N-α-butyloxycarbonyl amino acids with benzyl-based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc-amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc-amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C₁₈ reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H₂O) and in non-polar (TFE) environments. Immunoreactivity of anti-nef positive sera from HIV-1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions of nef-protein and mainly the immunodominance of the N- and C-termini of the molecule. Several of these peptides should prove to be useful for both diagnosis and vaccination purposes.     

Urotensin Ⅱ accelerates cardiac fibrosis and hypertrophy of rats induced by isoproterenol1

AIM: To study whether urotensin II (UII), a potent vasoconstrictive peptide, is involved in the development of cardiac hypertrophy and fibrogenesis of rats induced by isoproterenol (ISO). METHODS: Thirty male Wistar rats were randomly divided into 3 groups. Group 1 was the healthy control group, group 2 was the ISO group, and group 3 was the ISO+UII group. In groups 2 and 3, ISO (5 mg x kg(-1) x d(-1)) was given (sc) once daily for 7 d. Group 3 was also given UII in the first day [3 nmol/kg (5 microg/kg), iv], followed by sc (1.5 microg/kg) twice daily. Group 1 received 0.9% saline. UII receptor (UT) mRNA expression was determined by RT-PCR. The contents of UII and angiotensin II (Ang II) were determined by radioimmunoassay. In vitro, the effects of UII on DNA/collagen synthesis of cardiac fibroblasts were determined by [3H]thymidine/[3H]proline incorporation. RESULTS: The ratio of heart weight/body weight, plasma lactate dehydrogenase activity, myocardial malondialdehyde and hydroxyproline concentration increased significantly in the ISO group, as well as UT mRNA expression, plasma and cardiac UII and ventricular Ang II, compared with the control group (P< 0.01). ISO induced significant myocardial fibrogenesis. Moreover, UII+ISO co-treatment significantly increased the changes of biochemical markers of injury and the degree of cardiac hypertrophy and fibrosis. In vitro, 5 x 10(-9 )-5 x 10(-7 ) mol/L UII stimulated [3H]thymidine/[3H] proline incorporation into cardiac fibroblasts in a dose-dependent manner (P< 0.01). CONCLUSION: These results suggest that UII was involved in the development of cardiac fibrosis and hypertrophy by synergistic effects with ISO.     

Inhibitory Effect of an Urotensin II Receptor Antagonist on Proinflammatory Activation Induced by Urotensin II in Human Vascular Endothelial Cells

In this study, we investigated the effects of a selective urotensin II (UII) receptor antagonist, SB-657510, on the inflammatory response induced by UII in human umbilical vein endothelial cells (EA.hy926) and human monocytes (U937). UII induced inflammatory activation of endothelial cells through expression of proinflammatory cytokines (IL-1β and IL-6), adhesion molecules (VCAM-1), and tissue factor (TF), which facilitates the adhesion of monocytes to EA.hy926 cells. Treatment with SB-657510 significantly inhibited UII-induced expression of IL-1β, IL-6, and VCAM-1 in EA.hy926 cells. Further, SB-657510 dramatically blocked the UII-induced increase in adhesion between U937 and EA.hy926 cells. In addition, SB-657510 remarkably reduced UII-induced expression of TF in EA.hy926 cells. Taken together, our results demonstrate that the UII antagonist SB-657510 decreases the progression of inflammation induced by UII in endothelial cells.     

Solid phase synthesis of the retro viral nucleocapsid protein NCp10 of Moloney Murine Leukaemia virus and related “zinc-fingers” in free SH forms

The core of retroviruses contains a highly conserved, low molecular weight, basic protein that binds nucleic acids and is essential for genomic RNA packaging. The 56 amino acid protein, NCp10, of Moloney Murine Leukaemia virus (MoMuLV) has the CysX₂CysX⁴HisX⁴Cys zinc finger-like motif shared by all retrovirus nucleocapsid proteins. The native protein and five modified peptides containing the zinc binding domain were synthesized by solid phase in order to investigate the structural and biochemical role of Zn²⁺ chelation in MoMuLV NCp10 activity. The purity of the synthetic molecules was verified by HPLC and their sequences were confirmed by amino acid analysis and sequencing in the case of NCp10. Thiol dosage agreed with the theoretical value of free cysteine for all these molecules. Fluorescence measurements performed on synthetic NCp10 and zinc finger fragments showed that the tryptophan quantum yield was Zn²⁺ -dependent allowing a 1:1 stoichiometry for the complex to be determined. The apparent affinity constant of NCp10 for the metal was estimated to be superior to 10⁶ M^-1. The synthetic protein, in the presence of Zn²⁺ ions, possesses all the biological properties of NCp10 isolated from virions. It catalyzes both the MoMuLV RNA dimerization and the annealing of the replication primer tRNA^Pro onto MoMuLV RNA.     

Solid phase synthesis of a fully active analogue of cholecystokinin using the acid-stable Boc-Phe (p-CH2) SO3H as a substitute for Boc-Tyr(SO3H) in CCK8

Substitution of the -OSO₃H group in the sulfated-tyrosine by the non-hydrolyzable -CH₂SO₃H group was the first described modification of the sulfate ester that does not affect CCK₈ activity. In addition to its capacity to mimic the sulfated tyrosine residue, the amino acid Phe(p-CH₂SO₃Na) was shown to be stable in acidic media, including HF containing mixtures. The synthesis of Boc-Phe(p-CH₂SO₃Na)-OH in racemic and resolved forms and its introduction into the sequence of CCK₈ by solid phase using standard Boc/benzyl synthesis conditions and BOP as coupling reagent is now reported. The two CCK₈ analogues containing the l - or the d -Phe(p-CH₂SO₃Na) residue, obtained in satisfactory yields, were separated by HPLC and the stereochemistry of Phe(p-CH₂SO₃Na) residue in each peptide was established by NMR spectroscopy and confirmed by a separate solid phase synthesis in which the pure l isomer was used. Both CCK₈ analogues displayed high affinities for peripheral and central receptors (KI ～ 1 nm ) and proved to be full agonists in the stimulation of pancreatic amylase secretion. The ?stabilized-CCK₈ peptide”, easily prepared by solid phase, could replace the native peptide in biochemical and pharmacological studies. Moreover the modified amino acid Phe (p-CH₂SO₃Na) could also be used in solid phase synthesis to prepare a wide variety of CCK analogues and more generally, peptides analogues containing the acid-labile O-sulfated tyrosine.     

Synthesis of glycosylated tuftsins and tuftsin-containing IgG fragment undecapeptide

Syntheses are described of two new tuftsin derivatives containing a 2-acetamido-2-deoxy-D-galac-topyranosyl unit α- or β-glycosidically linked to the threonine's hydroxy side chain function and of the glycosylated undecapeptide corresponding to the tuftsin region of the heavy chain of IgG (amino acid sequence 289–299). The glycosylated tuftsins were synthesized by the solution procedure. Fmoc-[Gal NAc(Ac)₃α]Thr-OH and Fmoc-[GalNAc(Ac)₃β]Thr-OH were allowed to react with H-Lys(Z)-Pro-Arg(NO₂)-OBzl by the mixed anhydride procedure and the resulting glycosylated tetrapeptides were fully deblocked by catalytic hydrogenation followed by treatment with potassium cyanide, purified by ion exchange chromatography and characterized by analytical HPLC, elemental and amino acid analyses, optical rotation, and proton NMR spectroscopy. Synthesis of the glycosylated undecapeptide was achieved by the continuous flow solid phase procedure on 4-hydroxymethylphenoxyacetyl-norleucyl derivatized Kieselguhr-supported resin. Fmoc-amino acid symmetrical anhydrides or pentafluorophenyl esters, in the presence of N-hydroxybenzotriazole, were used as the acylating agents. To mimic the native sequence of the tuftsin region at the Fc-domain of immunoglobulin G a 2-acetam～do-2-deoxy-β-D-glucopyranosyl unit was N-glycosidically linked to the amide side chain of Asn 297. The glycosylated asparagine residue was introduced as N²-fluorenylmethyloxycarbonyl-N⁴-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopy-ranosyl)-asparagine pentafluorophenyl ester. After cleavage from the resin the glycopeptide was deprotect-ed, purified by ion exchange chromatography, and characterized by analytical HPLC, amino acid analysis, high voltage electrophoresis, and proton NMR. The conformational features of the glyco-undecapeptide were determined by circular dichroism measurements both in water and in 98% trifluoroethanol. Results of biological assays will be published elsewhere.     

Functional receptors for fish neuropeptide urotensin II in major rat arteries

Receptor binding of fish neuropeptide urotensin II (UII) was characterized in membranes isolated from major rat arteries. Monoiodinated UII radioligand (125I-UII) was prepared and purified using high-pressure liquid chromatography (HPLC). The contractile potency of iodinated UII (I-UII) on rat thoracic aorta strips was somewhat lower than that of native UII. The binding of 125I-UII to the membrane preparations of rat thoracic aorta was saturable, specific and time-dependent. Scatchard analysis indicated a single population of binding sites with an apparent dissociation constant of 5.9 x 10(-9) M. The calculated maximal number of binding sites was about 155 fmol/mg protein. The specific binding to the membrane preparations from the abdominal aorta and mesenteric artery was about 27 and 8%, respectively, of those in the thoracic aorta, which corresponds to the order of contractile potency of UII on rat blood vessels: thoracic aorta greater than abdominal aorta greater than mesenteric artery. The displacement of 125I-UII binding by the UII peptide or its fragments (UII-(5-12), UII-(6-12) and UII-(6-11] were also comparable to their contractile effects on rat thoracic aorta strips (UII greater than UII-(5-12) greater than UII-(6-12) much greater than UII-(6-11]. These results suggest that the fish neuropeptide, UII, can induce contraction of rat vascular tissue by interacting with its functional receptors.     

Urocontrin, a novel UT receptor ligand with a unique pharmacological profile

In recent years, several studies have demonstrated that urotensin II (UII) and urotensin II-related peptide (URP) can exhibit differential biological activity. So far, known antagonists of the urotensin II receptor (UT) are of limited usefulness for investigating the specific pathophysiological role of UII or URP. Therefore, identification of new compounds able to discriminate UII- and URP-associated biological activities is crucially needed. In the present study, we report preliminary data regarding the pharmacological properties of a novel UT ligand termed urocontrin, i.e. [Bip(4)]URP, that is able to reduce the ex vivo efficacy of hUII- but not URP-induced vasoconstriction in rat aortic rings. In vivo studies support the pharmacological profile described above. Although urocontrin exert some residual agonist activity, this compound should be useful for the rational design of potent molecules that would allow discriminating specific biological action mediated by UII or URP.     

Chemical synthesis of α-inhibin-92 by the thiocarboxyl segment coupling method

The 92 amino acid residue peptide, α-inhibin-92 (α-IB-92), has been synthesized by the thiocarboxyl segment strategy. Three segments were synthesized by the solid phase method, purified, and characterized: [GlyS³⁴]-α-IB-92-(l-34) (I), CF₃CO-[GlyS⁶⁵]-α-IB-92-(35–65) (II), and Msc-α-IB-92-(66–92) (III). All were reacted with citraconic anhydride followed by removal of the Msc group in III to give Ia, IIa, and IIIa, respectively. Peptide IIIa was coupled to IIa by the silver nitrate/N-hydroxysuccinimide procedure and, after removal of uncoupled segments and the trifluoroacetyl group, Ia was coupled followed again by removal of uncoupled segments. Final deblocking to remove citraconyl groups was accomplished under exceptionally mild conditions in aqueous acetic acid. The synthetic product was identical to natural α-IB-92 in amino acid analysis, HPLC, gel electrophoresis, and tryptic mapping. The synthetic peptide was indistinguishable from natural α-IB-92 in a radioimmunoassay and in an in vitro mouse pituitary assay for measuring suppression of FSH release in the presence of LHRH.     

Fmoe SPPS using Perloza™ beaded cellulose

Perloza? beaded cellulose was functionalised by a cyanoethylation reduction procedure to give aminopropyl Perloza. Fmoc-amino acids were anchored to aminopropyl Perloza beaded cellulose via the TFA labile 4-oxymethylphenoxyacetyl (HMPA) linker. Using Fmoc-aminoacyl-4-oxymethylphenoxyacetyl-2,4-dichloro-phenyl esters, all 20 amino acids were anchored at substitution levels ranging from 0.37 to 0.65 mmol/g. Fmoc-amino acids were also anchored using the peptide-amide linker 4-[(R,S)-1-[1-(9H-fluoren-9-yl)-methoxycarbonylamino]-(2′,4′dimethoxy-benzyl]phenoxyacetic acid. The Fmoc-aminoacyl resins were used for SPPS using Fmoc chemistry. SPPS was carried out using either an LKB Biolynx 4175 low-pressure pumped column continuous-flow peptide synthesiser or an ABI 430A automated vortexing batchwise instrument. Comparison of peptides made using each synthesiser showed little difference in quality of the crude peptides. Different Fmoc-amino acid activation methods (DIC/HOB_t/DMF, HBTU, DIC/HOB_t/DCM) were found to be equally useful with Perloza. Peptides were cleaved using TFA plus scavengers; however, the TFA-swollen resin was not readily separated from the TFA peptide solution by simple filtration. Therefore alternative cleavage workup procedures were used with Perloza. Peptides were purified by HPLC and characterised by HPLC and amino acid analysis, and in some cases by FAB-MS. Successful syntheses ranged from 5 to 34 amino acids in length. Some of the peptides were also synthesized using a polystyrene support and standardised (ABI Fastmoc?) SPPS protocols. The crude cleaved peptides from each synthesis were compared by HPLC analysis. The overall aim of our work with Perloza is synthesis of resin-bound peptide ligands for affinity chromatography and antibody generation. HPLC analysis of crude peptides showed that peptides up to 20 amino acids in length were of reasonable purity when synthesised using Perloza, thus encouraging us to continue with investigations of SPPS of resin-bound ligands.     

Solid phase synthesis of peptides containing the non-hydrolysable analog of (O)phosphotyrosine,P(CH2PO3H2)Phe

Studies about phosphorylation-dephosphorylation mechanisms require the development of probes capable of being used in in vitro and in vivo conditions. We show in this work that the chemically and enzymatically stable p(CH₂PO₃H₂) Phe analog of (O)phosphotyrosine can be easily introduced in peptides by the solid-phase method. It has been incorporated in the 344-357 sequence of the β₂ adrenergic receptor in place of the Tyr residue in position 350 and/or 354 in order to investigate the role of tyrosine phosphorylation in the receptor agonist-induced down-regulation. Since p(CH₂PO₃H₂)Phe is an ionized hydrophilic residue, peptides containing this amino acid do not easily permeate the cellular membranes. Therefore the modified amino acid was introduced in the synthetic pathway in its N-Boc- p (CH₂PO₃Et₂)Phe form, which could be partially or completely deprotected. Coupling steps, including that of the new amino acid, were performed with good yields (~60% total yield) and further deprotections provided both the p(CH₂PO₃H₂)Phe and p(CH₂PO₃HEt)Phe containing peptides with yields of around 20% each. The structure of the peptides was assessed by NMR, mass spectroscopy and amino acid analysis and the new amino acid was characterized under its phenyl-thiocarbamyl form (PTC).     

Bufokinin: a substance P-related peptide from the gut of the toad,Bufo marinus with high binding affinity but low selectivity for mammalian tachykinin receptors

A tachykinin peptide, termed bufokinin, was isolated in pure form from an extract of the intestine of the toad, Bufo marinus, and its primary structure was established as: Lys-Pro-Arg-Pro-Asp-Gln-Phe-Tyr-Gly-Leu-Met.NH₂. This sequence was confirmed by chemical synthesis and shows four amino acid substitutions (Arg → Lys,Lys³→ Arg,Gln⁵→ Asp and Phe⁸→ Tyr) compared with substance P. Binding parameters for synthetic bufokinin and mammalian tachykinins were compared using receptor-selective radioligands and crude membranes from rat tissues enriched in the NK-1 (submandibular gland), NK-2 (stomach fundus) and NK-3 (brain) receptors. In terms of inhibiting the binding of the selective radioligands, bufokinin (K_d= 0.3 nM) was 1.8-fold more potent than substance P at the rat NK-1 site, but it was only 2-fold less potent (K_d= 2.8 nM) than neurokinin A at the NK-2 site and only 2-fold less potent (K_d= 48 nM) than neurokinin B at the NK-3 site. Thus, bufokinin shows relatively high affinity but lack of selectivity for all three tachykinin binding sites in rat tissues.