Biomedical subjects
Publications and source records attributed to R Tomatis.
The synthesis of Leu-enkephalinamide, Leu-enkephalin-1-adamantylamide, Leu-[D Ala2]enkephalinamide and Leu-[D Ala2]enkephalin-1-adamantylamide are described. Preliminary results of pharmacological tests in vitro are reported.
Piperazinyl peptides related to Leu-enkephalin were obtained using as intermediates condensation products with iminodiacetic acid. The products are devoid of analgesic activity.
The synthesis of the protected duopentacontapeptide corresponding to the entire amino acid sequence I-52 of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The benzyloxycarbonyltetradecapeptide tert-butyloxycarbonylhydrazide (sequence 1-14) was selectively deblocked with trifluoroacetic acid and used to acylate, by the azide procedure, the peptide free base corresponding to the sequence 15-52. The isolated material was purified by ion exchange chromatography and the protecting groups were removed by successive treatments with anhydrous hydrogen fluoride, 1 M piperidine and mercuric acetate. F02M phosphate buffer, pH8. Determination of the inhibitory capacity indicated that the synthetic material is about 50% effective, at 30:1 inhibitor:trypsin molar ratio in inhibiting the tryptic hydrolysis of Nalpha-benzoyl-DL-arginine-4-nitroanilide. Full inhibition was achieved at a higher inhibitor:trypsin molar ratio. The stability constants and the standard free energy of binding of the complex between trypsin and the synthetic inhibitor have been determined.
The synthesis by fragment condensation of protected peptides corresponding to the amino acid sequences 15-35, 25-52 and 15-52 of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The Rudinger modification of the azide procedure was used in the fragment coupling steps. The tert-butyloxycarbonylheptapeptide hydrazide (sequence 22-28) was reacted with the heptapeptide methyl ester free base (sequence 29-35) and the resulting tert-butyloxycarbonyltetradecapeptide methyl ester after selective deprotection, coupled with the benzyloxycarbonylheptapeptide hydrazide (sequence 15-21) to give the protected peptide methyl ester corresponding to the 15-35 sequence which was then converted to the corresponding hydrazide. The synthesis of the 25-52 sequence was achieved by assembling the protected peptide hydrazide corresponding to the amino acid residues 25-35, with the C-terminal heptadecapeptide 36-52. The resulting protected octaeicosapeptide (sequence 25-52) was selectively deblocked with trifluoroacetic acid and acylated with the benzyloxycarbonyldecapeptide hydrazide 15-24 to give the desired octatriacontapeptide corresponding to sequence 15-52 of the inhibitor. An attempt to prepare the 15-52 sequence through the condensation of fragments corresponding to 15-35 and 36-52 sequences was unsuccessful. The identity and purity of the synthetized peptide derivatives wre established by elemental analysis (in some cases), amino acid analysis, optical rotation, and thin-layer chromatography in two solvent systems. The final products were also evaluated, after partial deprotection with anhydrous hydrogen fluoride or aqueous 90% trifluoroacetic acid, by paper electrophoresis at different pH values.
The synthesis of H-Tyr-Gly-Gly-Phe-Leu-OH (Leu-enkephalin), endogenus peptide with opiate-like activity, and two rearrangements concerning Leu-enkephalin N-terminus are described.
The synthesis, by fragment condensation, of protected peptides related to the N-terminal (positions 1-14) and C-terminal (positions 36-52) portions of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The alpha-carboxyl function of the lysyl residue in position 14 was used as the free acid, or protected by the tert-butyloxycarbonylhydrazide. Two different synthetic pathways were used in the preparation of the 36-52 sequence of the inhibitor. The identity and purity of the synthesized peptide derivatives were established by amino acid analysis of acid hydrolysates, optical rotation and this layer chromatography in two solvent sytems. The final products were also evaluated, after partial deprotection with anhydrous hydrogen fluoride or aqueous 90% trifluoroacetic acid, by paper electrophoresis at different pH values and enzymic digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.
The stability of native and reduced bovine pancreatic trypsin inhibitor (Kunitz inhibitor) in anhydrous hydrogen fluoride and their reaction with acetamidomethanol, in the same solvent, have been investigated. The bovine Kunitz inhibitor appears to be stable in liquid hydrogen fluoride but the reduced molecule loses about 50% of its ability to regain inhibitory power, upon air oxidation, by exposure to this solvent. Tyrosine residues appear to be affected by acetamidomethylation of the native protein to give a modified inhibitor which is still highly active in inhibiting trypsin. The extent of correct refolding, upon reoxidation, of the reduced tyrosine modified-inhibitor is greatly diminished. Tyrosine modification can be prevented by carrying out the acetamidomethylation reaction in the presence of excess anisole. The stability constants and the standard free energies of binding of the complexes between trypsin and the native and the tyrosine modified-inhibitor have been determined.
The general strategy for the synthesis, by conventional procedures, of the entire sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal) is discussed. The synthesis of two protected peptides corresponding to positions 2-10 and 1-10 of the proposed primary structure of the inhibitor is described. The heptapeptide free base threonyl-S-acetamidomethylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide (sequence 4-10) was acylated, by the azide procedure, with either the dipeptide benzyloxycarbonyl-gamma-tert-butylglutamylalanine hydrazide (sequence 2-3) or the tripeptide Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylala-nine hydrazide (sequence 1-3). The stereochemical homogeneity of the resulting peptides, benzyloxycarbonyl-gamma-tert-butylglutamylalanylthreonyl-S-acetamidomethyl-cysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide and Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylalanylthreonyl-S-acetamido-methylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonyl-hydrazide, was assessed, after partial deprotection with liquid hydrogen fluoride, by digestion with aminopeptidase M followed by quantitative amino acid analysis.
Synthesis is described of the protected tetrapeptide corresponding to positions 11-14 of the primary structure of the porcine pancreatic secretory trypsin inhibitor II (Kazal), in the form of free acid as well as protected hydrazide. The tetrapeptide tert-butyloxycarbonylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyl-lysine was prepared by stepwise elongation from the C-terminal Nepsilon-trifluoroacetyllysine using successively 1-succinimidyl benzyloxycarbonylprolinate, p-nitrophenyl N-tert-butyloxycarbonyl-S-acetamidomethylcysteinate and 1-phenyl-3-methyl-4-(tert-butyloxycarbonylglycyl)-oximinyl-5-(benzyloxycarbonylglycyl)-imino-2-pyrazoline as acylating agents. Alternately, the dipeptide benzyloxycarbonylprolyl-Nepsilon-trifluoroacetyllsine was transformed into the corresponding tert-butyloxycarbonylhydrazide which was reacted, after catalytic hydrogenolysis, with tritylglycyl-S-acetamido-methylcysteine to give the tetrapeptide tritylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyllsine tert-butyloxycarbonylhydrazide. The stereochemical homogeneity of the final products was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M, followed by quantitative amino acid analysis.
The synthesis of the amino-protected decapeptide tert-butyloxycarbonylhydrazide corresponding to positions 15-24 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal inhibitor) is described. The tripeptide free base threonyl-beta-tert-butylaspartylglycine tert-butyloxycarbonylhydrazide (sequence 22-24) was acylated with 1-succinimidyl o-nitrophenylsulfenylvalyl-S-acetamidomethylcysteinylglycinate (sequence 19-21). Removal of the amino protecting group from the resulting hexapeptide followed by acylation of the free base with either benzyloxycarbonylisoleucyl-O-tert-butyltyrosylasparaginylproline or O-nitrophenylsulfenylisoleucyl-O-tert-butyltyrosylasparaginylproline, via the pyrazoline active ester method, yielded the decapeptide tert-butyloxycarbonylhydrazide (sequence 15-24) in the form of Nalpha-benzyloxycarbonyl or Nalpha-O-nitrophenylsulfenyl derivative. The stereochemical homogeneity of the two decapeptides was assessed, after partial deprotection with liquid hydrogen fluoride, or thioacetamide and aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.
Synthesis is described of the partially protected octapeptide tert-butyloxycarbonyl-gamma-tert-butylglutamylasparaginyl-N-trifluoroacetyllysyl-N-trifluoracetyllysylarginyl-Ngamma-4,4'-dimethoxybenzhydryglutaminylthreonylproline corresponding to positions 36-43 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II. The tetrapeptide free base arginyl-Ngamma-4,4'-dimethoxybenzhydrylglutaminylthreonylproline was acylated, by the azide proceedure, with the tripeptide benzyloxycarbonyl-asparaginyl-N-trifluoroacetyllsyl-N-trifluoroacetyllysine hydrazide. The resulting protected heptapeptide was partially deblocked by catalytic hydrogenation and reacted with alpha-1-succinimidyl-gamma-tert-butyl tert-butyloxycarbonylglutamate. The stereochemical homogeneity of the ensuing octapeptide was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.
Synthesis is described of the partially protected nonapeptide tert-butyloxycarbonyl-valylleucylisoleucylglutaminyl-N-trifluoroacetyllsylserylglycylprolyl-S-acetamido-methylcysteine corresponding to positions 44-52 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II. The hexapeptide free base glutaminyl-N-trifluoroacetyllsylserylglycylprolyl-S-acetamidomethylcysteine, prepared by two alternative routes, was acylated by the azide procedure, with the tripeptide tert-butyloxycarbonylvalylleucylisoleucine hydrazide. The stereochemical homogeneity of the resulting nonapeptide was assessed, after partial deprotection by treatment with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.
Synthesis is described of the protected undecapeptide tert-butyloxycarbonylisoleucyl-threonyltyrosylserylasparaginyl-gamma-tert-butylglutamyl-S-acetamidomethylcysteinyl-valylleucyl-S-acetamidomethylcysteinylseriny hydrazide corresponding to positions 25-35 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal). The hepatapeptide free base methyl asparaginyl-gamma-tert-butylglutamyl-S-acetamidomethylcysteinylvalylleucyl-S-acetamidomethylcysteinylserinate (sequency 29-35) was acylated, by the azide procedure, with the tetrapeptide tert-butyloxycarbonl-isoleucylthreonyltyrosylserine hydrazide /sequence 25-28) and the resulting tert-butyloxycarbonylundecapeptide methyl ester was transformed into the corresponding hydrazide by hydrazinolysis. The stereochemical homogeneity of the final product was assessed, after partial deprotection with aqueous 90% trifuoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.
Since oximinyliminopyrrazoline ester (OPmp) present a peculiar characteristic in the omogenous peptide synthesis, the use of them, also in the synthesis of bradikinine by the solid phase technique, was duly taken into consideration and tried out. This synthesis was achieved by employing N-protected amino acid OPmp esters containing a methoxycarbonyl or a benzylaminocarbonyl as the acyl function in position 5 of the pyrazoline ring. No significant differences were found in the reactivity of such derivatives in respect of OPmp esters containing the benzyloxycarbonylglycyl moiety. The biological activity of the synthetized braikinine is similar to that of a sample of natural "standard".
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The purpose of this study was to evaluate the effects of dermorphin, a new synthetic powerful opiate-like heptapeptide, on plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels in fertile and postmenopausal women. In fertile subjects, dermorphin (5.5 micrograms/kg min for 30 min) decreases plasma LH (p less than 0.01 vs. baseline and placebo values), but not plasma FSH. The area under the curve during dermorphin infusion was significantly lower than during placebo infusion (p less than 0.01). Pretreatment with the opioid receptor antagonist naloxone, blocked the decrease of plasma LH levels. In postmenopausal women not subjected to any treatment, dermorphin infusion did not significantly modify plasma LH and FSH levels. On the contrary, its administration to postmenopausal subjects treated with conjugated estrogens and medroxyprogesterone acetate significantly decreased plasma LH levels (p less than 0.01, vs. baseline, placebo and area under the curve). Considering the modulatory role exerted by ovarian steroids on the activity of such receptors, these data also indicate that opioid systems play a very important part in the hypothalamus-pituitary-ovarian axis.