Chemical and enzymatic treatment of endothelin.

Endothelin-1 (ET), the most potent vasoconstrictor yet discovered, is a peptide containing 21 amino acids with two intrachain disulfide bridges. With the aim of obtaining two-chain derivatives, Et was submitted to chemical and enzymatic treatments. Reaction of ET with CNBr in 70% HCOOH gave, in addition to the expected [Hse7 lactone]-7,8-seco-ET and unreacted material, a by-product whose molecular weight was 25 m.u. greater than that of ET. When the reaction mixture, after lyophilisation, was immediately quenched with NH3-saturated dry MeOH, two products could be recovered in a 5:1 ratio, both obtained by nucleophilic attack of the homoserine lactone: the expected [Hse7-NH2]-7,8-seco-ET and [Hse7]ET, resulting from competitive intramolecular reaction of the deprotonated alpha-amino group of the Asp8 residue. The Lys9-Glu10 bond turned out to be very resistant to enzymatic attack both by Lys-C-endopeptidase and trypsin. The 9,10-seco-ET derivative could be obtained by treatment with Lys-C-endopeptidase only by using a high enzyme/ET ratio and after a prolonged incubation time. Cleavage of the Lys9-Glu10 bond could not be achieved by treatment with trypsin, even with a high enzyme/substrate ratio. The main product was 13,14-seco-ET, deriving from the action of chymotripsin (present as an impurity in the trypsin preparation) on Tyr13. The structure of these peptides was confirmed by amino-acid sequence analysis and fast atom bombardment mass spectrometry (FAB-MS). Nicking of the ET structure at different positions had different impact on the biological properties of the resulting derivatives.

Detection and mass spectrometric characterization of the major urinary and fecal metabolites of 9-methyl-1,2,3,4,6,7,12,12b-octahydroindolo[2,3-a]-quinolizine in the rat.

The metabolic fate of 9-methyl 1,2,3,4,6,7,12,12b-octahydroindolo[2,3-a]quinolizine (MIQ), a compound with promising pharmacological action on the CNS system, was investigated in the rat after an oral dose of 200 mg/kg, the maximal tolerated dose. Urine and feces were collected, exhaustively extracted with organic solvents and the metabolites detected by TLC analysis. The structures of the isolated metabolites were characterized by several mass spectrometry techniques (FD, EI, CI) and, in some cases, confirmed by synthesis. The major metabolic pathways of MIQ in the rat involve: C-oxidation of the methyl group in position 9 to a primary alcohol and to a carboxylic acid, N-oxidation of basic nitrogen and C-oxidation of the quinolizidine nucleus, probably at position 7.

Side-reactions in peptide synthesis. 2. Formation of by-products in the large-scale synthesis of THF-gamma 2 by solution synthesis.

The impurities which formed in the large-scale synthesis of THF-gamma 2, an immunomodulatory peptide of formula H-Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu-OH, were identified by a combination of analytical methods, and their structure confirmed by synthesis. Most impurities originated from side-reactions involving the aspartyl residue (cyclization, beta-aspartyl formation and cleavage). Based on this knowledge, modifications were introduced into the work up and the purification procedure which resulted in a very pure final product (> 99% by RP-HPLC).

Direct characterization of caffeoyl esters with antihyaluronidase activity in crude extracts from Echinacea angustifolia roots by fast atom bombardment tandem mass spectrometry.

Fast atom bombardment (FAB-MS) and fast atom bombardment tandem mass spectrometry (FAB-MS/MS) techniques (negative ions) have been successfully applied for identification of the constituents responsible for the antihyaluronidase activity of Echinacea angustifolia roots, whose extracts are widely employed for the adjuvant therapy of chronic inflammatory diseases. Crude extracts from different solvents were tested for antihyaluronidase activity, and those with the greatest inhibitory action (the ethylacetate, butylacetate and chloroform fractions, IC50 0.44, 0.50 e 0.62 mg/ml) were directly analyzed by MS. Full scan mass spectra produced intense molecular anions: collisional activation of these resulted in tandem mass spectra rich in significant product ions. Four main caffeoyl conjugates were detected and identified by tandem mass spectrometry (daughter and parent ion mode): 2,3-O-dicaffeoyltartaric acid (chicoric acid) and 5-O-dicaffeoylquinic acid (cynarine) and 2-O-caffeoyltartaric acid (caffaric acid) in the ethylacetate fraction. Among these caffeoyl conjugates, chicoric and caftaric acids had the greatest antihyaluronidase activity: IC50 = 0.42 and 0.61 mM, while the IC50 of cynarine and chlorogenic acid were 1.85 and 2.25 mM.

Bombesin receptor antagonists. 3. Irreversible alkylating analogues: melphalan derivatives.

Bombesin (BN)-like peptides (such as GRP, gastrin-releasing peptide) are autocrine growth factors for small cell lung carcinoma (SCLC). BN receptor antagonists can therefore find clinical application in the treatment of this highly malignant disease. The present paper deals with a new class of bombesin analogues carrying a nitrogen mustard at their N-terminus. Due to the irreversible binding to the BN receptor(s), these peptides eventually block the mitogenic effects of the natural ligand(s), regardless of their intrinsic "agonistic" or "antagonistic" structures. In Swiss 3T3 fibroblasts they inhibit [125I]GRP binding in the nanomolar/micromolar range. According to their "agonistic" or "antagonistic" structural features, they do or do not induce [3H]thymidine incorporation and p 115 phosphorylation. In competition experiments, alkylating "antagonists" selectively inhibit BN-induced thymidine incorporation either when given simultaneously with or 24 hours before the BN challenge. Alkylating "agonists" display antagonistic effects only in the sequential treatment.

New biosynthetic anthracyclines related to barminomycins incorporating barbiturates in their moiety.

Three new anthracyclines, FCE 21424 (2), FCE 24366 (3) and FCE 24367 (4), were isolated from culture broths of Streptomyces peucetius and its mutant strains after addition of sodium barbiturates during the fermentation. Structural assignment, achieved through spectroscopic and degradative studies, that the new anthracyclines had a common barminomycin-like structure incorporating different barbiturate moieties. The new anthracyclines were found to display outstanding cytotoxicity and remarkable potency "in vivo" against P388 ascitic leukemia.

Soft ionization techniques in mass spectrometry of distamycin and some analogues.

The results obtained using three different soft ionization techniques (field desorption (FD), fast atom bombardment (FAB) and desorption chemical ionization (DCI] on the antiviral antibiotic distamycin and on some analogues are reported. FAB mass spectra show more intense molecular ion and more regular fragmentation while FD and DCI mass spectra are dominated by thermally originated fragments. Information furnished by the three systems are different and complementary.

FD and FAB mass spectra of some oligopeptides of the tryptophyllin family.

Mass spectra of some natural thryptophyllins and synthetic analogues have been obtained by using field desorption and fast atom bombardment as soft ionization techniques to overcome the low volatility of these oligopeptides. Field desorption spectra generally show the molecular ion as base peak; some fragments of thermal origin are present at higher emitter heating current, thus providing additional structural information. Fast atom bombardment mass spectra show a more regular fragmentation of the peptide backbone, which allow the complete amino acid sequence to be checked. The presence of two consecutive proline residues in some of the examined molecules affects their fragmentation pattern in both field desorption and fast atom bombardment mass spectrometry giving rise to spectral features which are useful from the diagnostic point of view. The two ionization methods are compared and the advantage of the combined use of both techniques is pointed out.

Interaction of new derivatives of daunorubicin and adriamycin with DNA.

The interaction of a number of new derivatives of the antitumour antibiotics daunorubicin and adriamycin with calf-thymus DNA has been studied by equilibrium dialysis and low-shear viscosimetry. The results are interpreted in terms of structure of the compounds, also by examining molecular stereomodels.