Structure-activity relationships of neuromedin U. V. study on the stability of porcine neuromedin U-8 at the C-terminal asparagine amide under mild alkaline and acidic conditions

Porcine neuromedin U-8 (X-Asn-NH(2), X=H-Tyr-Phe-Leu-Phe-Arg-Pro-Arg) is occasionally unstable in the biological fluids used for bioassay as well as in the acidic solutions used for purification of synthetic peptides. In this study, HPLC examination of an incubate solution of X-Asn-NH(2) revealed that the main decomposition products in Tyrode's solution (pH 7.4) were either alpha- or beta-monocarboxylic acid analogs (X-Asn-OH or X-Asp-NH(2)), and that no dicarboxylic acid analog (X-Asp-OH) was produced. Further investigation, employing a model peptide (Y-Asn-NH(2), Y=Benzoyl-Pro-Arg) incubated in a 0.1 M sodium bicarbonate solution at 60 degrees C, revealed that the decomposition of C-terminal Asn-NH(2) occurred through the formation of an aminosuccinimide intermediate (Y-Asu), at a rate faster than that of Y-Asn-Ser peptide but slower than that of Y-Asn-Gly peptide. Mild acid hydrolysis of X-Asn-NH(2) examined in a 1 M HCl solution at 60 degrees C yielded X-Asn-OH and X-Asp-NH(2), which further decomposed to yield X-Asp-OH. The C-terminal degradation of X-Asn-NH(2) resulted in reduced biological and immunochemical binding activities.     

Structure-activity relationships of rat neuromedin U for smooth muscle contraction.

Rat neuromedin U (r-NMU) and its fragment peptide amides were synthesized by solid-phase methodology. Using a chicken crop smooth muscle contraction assay, the potency of r-NMU and its fragments relative to porcine neuromedin U-8 (p-NMU-8) was r-NMU: 10.25 +/- 2.88, r-NMU (6-23): 8.01 +/- 1.04, r-NMU (10-23): 2.76 +/- 0.46, r-NMU (13-23): 2.81 +/- 0.52, and r-NMU (16-23): 0.88 +/- 0.19, respectively. Two heptapeptides, r-NMU (17-23) and r-NMU (16-22), had a relative potency of 0.61 and 0.03 respectively, and elicited maximal contraction at a dose of 10 microM to a similar degree to p-NMU-8. The other shorter C-terminal fragments did not elicit the maximal contraction or any activity. In a rat uterus contraction assay, r-NMU (13-23), but not r-NMU (16-23), at a dose of 4 nM retained as high a stimulatory activity as r-NMU itself. r-NMU (17-22) was the smallest peptide fragment to elicit the maximal sustained contraction at 10 microM. These results indicate that the amino acid sequence Phe-Leu-Phe-Arg-Pro-Arg, corresponding to positions 17 to 22 of r-NMU, may be essential for contractile activity. N-terminal peptide segments Tyr-Gln-Gly-Pro corresponding to positions 6 to 9, and Ser-Gly-Gly corresponding to positions 13 to 15, appear to be of special importance for potent activity.     

Agonistic and antagonistic activities of neuromedin U-8 analogs substituted with glycine or D-amino acid on contractile activity of chicken crop smooth muscle preparations.

To study the structure-activity relationships of neuromedin U-8 (NMU-8) (H-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2) and to develop a NMU-8 antagonist, twenty-three NMU-8 analogs substituted with Gly or the corresponding D-amino acid(s) at positions 1-8 were synthesized by solid-phase techniques. On isolated chicken crop preparations, the contractile activity of the synthetic NMU-8 analogs was compared with that of NMU-8 and their antagonistic activity was assayed against NMU-8. The replacement of Phe2, Phe4, Arg5, Pro6, Arg7 or Asn8 with Gly brought about a drastic decrease of the agonistic activities. Substitution of the corresponding D-amino acid residue for Phe2, Phe4, Arg5, Pro6 or Asn8 caused a marked decrease of the agonistic activities, while the replacement of Tyr1 with D-form enhanced the activity. It was further revealed that [D-Pro6]-NMU-8 and [D-Leu3, D-Pro6]-NMU-8 exerted a non-competitive antagonistic activity against NMU-8 with x values of 5.22 +/- 0.12 and 5.34 +/- 0.09, respectively. [D-Phe2, D-Pro6]-NMU-8, [D-Arg5, D-Pro6]-NMU-8 and [D-Pro6, D-Asn8]-NMU-8 showed a very weak antagonism. The results indicated that 1) the side chain of each amino acid at positions 2, 4, 5, 6, 7 and 8 of NMU-8 is of relative importance for the expression of the contractile activity, and 2) [D-Pro6]-NMU-8 and its four analogs acted as an antagonist against NMU-8.     

Structure-activity relationships for non-congeneric structures. Application of substructural balance method for antiallergic activity

This paper presents a new research method of structure-activity relationships (SAR) based on the concept of substructural balance. By using antiallergic activity (PCA, rat, iv) of a non-congeneric set of 267 structures, the structural feature of active group is expressed in terms of substructural balance. Each structure was expressed with 100 new substructures and the number of each substructure in a molecule was counted. The substructural balance was expressed as their ratio. Structures were classified into three groups based on their potencies (ED50), active (44), median (33) and inactive (190) group. Using two substructural ratios, 80.53% of inactive and 57.58% of median structures were excluded from those that were active. Common features of active structures were shown as a zone indicating the optimal ranges of two substructural ratios. Two substructural ratios were determined out of 4950 substructural ratios, all possible combinations of 100 substructures (100C2), by selecting the greatest discriminatory power of inactive from active structures. The substructures used in this work include: the number of bonds comprising of the longest conjugate system, the number of skeletal atoms and the numbers of electron-donor pairs at certain distances in the molecule.     

A direct method for the alkylation of adenosine nucleosides at position 8.

Alkylation of adenosine derivatives at carbon 8 can be conveniently achieved by lithiation followed by reaction with an alkyl halide.     

Synthesis and structure-activity relationships of [MeTyr1, MeArg7]-dynorphin A(1-8)-OH analogues with substitution at position 8

A series of [MeTyr1, MeArg7]-Dynorphin A (Dyn)(1-8)-OH analogues, modified at position 8 with various amino acids, is described. Their biological activities were determined in the three bioassays [guinea pig ileum (GPI), mouse vas deferens (MVD), and rabbit vas deferens (RVD)] and in the mouse tail-pinch test after subcutaneous administration. None of the analogues tested displayed more potent kappa-opioid activity in the RVD than [MeTyr1, MeArg7, D-Leu8]-Dyn(1-8)-NHEt (1), which is a potent analgesic peptide with similar opioid receptor selectivity to that of Dyn. However, [MeTyr1, MeArg7, Melle8]-Dyn(1-8)-OH (11) showed about a twofold more potent analgesic effect than 1. Based on the obtained results it is conceivable that in the case of Dyn(1-8)-OH analogues both a lipophilic L-amino acid in position 8 and an unchanged 7-8 amide bond are essential to maintain potent kappa-opioid activity.     

A mechanism for the loss of 60 u from peptides containing an arginine residue at the C-terminus

The loss of 60 u from protonated peptide ions containing an arginine residue at the C-terminus has been investigated by means of low energy tandem mass spectrometry. The lowest energy conformation of singly charged bradykinin is thought to involve a salt-bridge structure, which may lead to the formation of two isomeric forms. It is thought that one isomer retains the ionizing proton at the C-terminal end of the peptide, leading to the formation of the [b _n?1 + H + OH]⁺ fragment ion, and the other isomer retains the charge at the N-terminus, leading to the formation of the [M + H ? 60]⁺ fragment ion. It was found that the formation of the [M + H ? 60]⁺ ion occurs only from singly charged precursor ions. In addition, the loss of 60 u occurs from peptides in which the charge is localized at the N-terminus. These results indicate that the mechanism of formation of the [M + H ? 60]⁺ ion may be driven by a charge-remote process.     

Structure-activity relationships in hydroxy-2,3-diarylxanthone antioxidants. Fast kinetics spectroscopy as a tool to evaluate the potential for antioxidant activity in biological systems

A structure-activity relationship has been established for eight hydroxy-2,3-diarylxanthones (XH) bearing hydroxy groups on the two aryl rings. One-electron oxidation by superoxide radical-anions (˙O(2)(-)) and ˙Trp radicals as well as reaction with ˙CCl(3)O(2) and ˙CHCl(2)O(2) radicals demonstrates that two OH groups are required for efficient antioxidant reactivity in cetyltrimethylammonium bromide micelles. Hydroxy groups at the meta and para positions on either of the two phenyl rings confer enhanced reactivity, but XH bearing an OH at the para position of either phenyl ring is unreactive. While oxidation is favoured by OH in both meta and para positions of 2-aryl xanthone substituents, addition of a third and/or fourth OH enhances electron-donating capacity. In Cu(2+)-induced lipid peroxidation of human LDL, the lag period preceding the commencement of lipid peroxidation in the presence of XH bearing OH at meta and para positions on the 3-phenyl ring is extended to twice that observed with a comparable concentration of quercetin, a reference antioxidant. These antioxidants are also superior to quercetin in protecting human skin keratinocytes against tert-butylhydroperoxide-induced oxidative stress. While XH antioxidant activity in model biological systems is consistent with the structure-activity relationship, their response is also modulated by the localization of XH and by structural factors.     

Application of photodiode array detection and fast atom bombardment mass spectrometry for the identification of the arginine residue in neuropeptides.

Chemical derivatization by phenylglyoxal (PGX) was applied to the identification of arginine in the neuropeptides dynorphin A (1-6) and substance P. The obtained products were separated on a short reversed phase C18 column and analysed on-line with the photodiode array UV technique. The selective attachment of a chromogenic molecule into the arginine residue resulted in significant change in the absorbance spectra around 250 nm, depending on the number of PGX molecules attracted. Further analysis employed fast atom bombardment mass spectrometry (FAB MS) and C-terminal sequencing for detailed verification of the derivatives formed during modification with PGX. The results clearly demonstrated that the photodiode array technique, when combined with chemical modification of certain amino acids, provides new possibilities for the analysis of peptide structures.     

Studies on the chemical modification of monensin. IV. Synthesis, sodium ion permeability, and biological activity of 7-O-acyl- and 7-O-alkylmonensins.

7-O-Acyl-(4a-e) and 7-O-alkylmonensins (5a-d) were prepared from monensin (1). Their lipophilicity, sodium ion permeability in human erythrocytes, antibacterial activity and effect on rat tail artery were examined. There was a correlation between lipophilicity and sodium ion permeability as well as between lipophilicity and antibacterial activity. We also found that the compound having larger sodium ion permeability, showed stronger contraction of rat tail artery. 7-O-Benzylmonensin (5c) exhibited higher lipophilicity and larger sodium ion permeability than monensin (1) among the tested monensin derivatives. In addition, antibacterial activity and contractile effect on rat tail artery of 5c were comparable to those of 1.     

Absolute stereochemistry of antifouling cembranoid epimers at C-8 from the Caribbean octocoral Pseudoplexaura flagellosa. Revised structures of plexaurolones

Eight cembranoid epimers at C-8 (1-8) were isolated from the organic extract of the Colombian Caribbean octocoral Pseudoplexaura flagellosa. Compounds 2, 4, and 6 are reported for the first time. Although compounds 1, 3, 5, 7, and 8 have been reported previously, their structures and NMR assignments are revised, completed or corrected. The structures of these compounds were established on the basis of detailed analysis of their spectroscopic data. Furthermore, the relative configurations of compounds 1-3 and 7 were confirmed by single-crystal X-ray diffraction. The absolute configurations of compounds 1-8 were determined using a combination of the modified Mosher method and unambiguous chemical interconversions. Evaluation of their antifouling properties using quorum sensing inhibition (QSI) and biofilm inhibition bioassays showed that compounds 3, 6, and 7 have excellent QSI activity against Chromobacterium violaceum, as measured by inhibition of the production of violacein pigment, without interfering with its growth. Furthermore, compounds 3, 5, 6, and 8 exhibited inhibition of biofilm maturation without interfering in the growth of Pseudomonas aeruginosa, Vibrio harveyi, and Staphylococcus aureus. This is the first report of cembranoids as inhibitors of bacterial biofilm and as compounds that interfere with QS in C. violaceum.     

Solid-phase peptide synthesis of analogues of the N-terminus A-ring fragment of the lantibiotic nisin: Replacements for the dehydroalanine (Dha) residue at position 5 and the first incorporation of a thioamide residue

A number of A-ring analogues of the lantibiotic nisin, containing replacements for the Dha residue at position 5, have been successfully prepared by solid-phase peptide synthesis. The Dha replacements include glycine, alanine, phenylalanine, serine and 1-aminocyclopropyl carboxylic acid (ACCa). The incorporation of a thioamide-isoleucine residue at position 4 is also described and represents the first reported preparation of a lantibiotic ring fragment containing a thioamide link.     

Chemistry of the phenoxathiins V. Correlation of the crystal and molecular structure with pharmacologic activity associated with 7- and 8-chloro-l-azaphenoxathiin

The crystal and molecular structure of the 7- and 8-chloro analogs of the 1-azaphenoxathiin system are described. In contrast to previous compounds possessing antipsychotic type activity, the title compounds are both nearly planar, and demonstrate significant substituent location sensitivity, with the 7-chloro analog exhibiting significant biologic activity while the 8-chloro isomer is nearly devoid of activity. The relationship of these compounds to the isosterically related phenothiazines in terms of design and molecular geometry considerations is also described.     

Structure-activity relationships in group 3 metal catalysts for asymmetric intramolecular alkene hydroamination. An investigation of ligands based on the axially chiral 1,1'-binaphthyl-2,2'-diamine motif

From a series of N,N'-disubstituted-1,1'-binaphthyl-2,2'-diamines, several group 3 metal complexes were synthesized via an in situ procedure. These chiral complexes were subsequently applied to catalysis of intramolecular alkene hydroamination. Significant structure-activity relationships were observed, most notably a reversal of stereoselectivity for cyclopentyl versus diphenylmethyl substituents.