HPLC separation and NMR structural elucidation of sn-1,2-, 2,3-, and 1,3-diacylglycerols from olive oil as naphthylethylurethane derivatives.

In this study, sn-1,2-, sn-2,3-, and sn-1,3-diacylglycerols were isolated from olive oil, and their urethane derivatives (urethanes) were prepared. Normal-phase high-performance liquid chromatography (NP-HPLC) separation of the urethane isomers was performed and the separate classes were studied by nuclear magnetic resonance (NMR). The use of 1H NMR and homo- and heteronuclear 2D techniques provided a great amount of information in a very short time, particularly when a high-field NMR instrument (700 MHz) was used. Particularly diagnostic for this kind of compound was the glyceridic moiety that presents typical chemical shifts both for carbon and hydrogen. These studies show the usefulness of NMR spectroscopy to recognize clearly the sn-1,3- and, moreover, sn-1,2- with respect to sn-2,3-diacylglycerols, although very minor differences occur between them.

A galpha(s) carboxyl-terminal peptide prevents G(s) activation by the A(2A) adenosine receptor.

The molecular mechanisms of interaction between G(s) and the A(2A) adenosine receptor were investigated using synthetic peptides corresponding to various segments of the Galpha(s) carboxyl terminus. Synthetic peptides were tested for their ability to modulate binding of a selective radiolabeled agonist, [(3)H]2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxam idoade nosine ([(3)H]CGS21680), to A(2A) adenosine receptors in rat striatal membranes. The Galpha(s) peptides stimulated specific binding both in the presence and absence of 100 microM guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS). Three peptides, Galpha(s)(378-394)C(379)A, Galpha(s)(376-394)C(379)A, and Galpha(s)(374-394)C(379)A, were the most effective. In the presence of GTPgammaS, peptide Galpha(s)(374-394)C(379)A increased specific binding in a dose-dependent fashion. However, the peptide did not stabilize the high-affinity state of the A(2A) adenosine receptor for [(3)H]CGS21680. Binding assays with a radiolabeled selective antagonist, [(3)H]5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4, 3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]SCH58261), showed that the addition of the Galpha(s) peptide modified the slope of the 5'-N-ethylcarboxamidoadenosine (NECA) competition curve, suggesting modulation of receptor affinity states. In the presence of GTPgammaS, the displacement curve was right-shifted, whereas the addition of Galpha(s)(374-394)C(379)A caused a partial left-shift. Both curves were fitted by one-site models. This same Galpha(s) peptide was also able to disrupt G(s)-coupled signal transduction as indicated by inhibition of the A(2A) receptor-stimulated adenylyl cyclase activity without affecting either basal or forskolin-stimulated enzymatic activity in the same membrane preparations. Shorter peptides from Galpha(s) and Galpha(i1/2) carboxyl termini were not effective. NMR spectroscopy showed the strong propensity of peptide Galpha(s)(374-394)C(379)A to assume a compact carboxyl-terminal alpha-helical conformation in solution. Overall, our results point out the conformation requirement of Galpha(s) carboxyl-terminal peptides to modulate agonist binding to rat A(2A) adenosine receptors and disrupt signal transduction.

Conformational studies on a synthetic C-terminal fragment of the alpha subunit of G(S) proteins.

It has recently been reported that synthetic peptides corresponding to the C-terminal sequence of G alpha, can be used to study the molecular mechanisms of interaction between this protein and G protein coupled receptors (Hamm et al., Science, 1988, Vol. 241, pp. 832-835). A conformational analysis on a 11 amino acids peptide from the G alpha(S) C-terminus, G alpha(S)(384-394) (H-QRMHLRQYELL-OH), was performed by nmr spectroscopy and molecular modeling methods. Two-dimensional nmr spectra, recorded in hexafluoroacetone/water, a mixture with structure stabilizing properties, showed an unusually high number of nuclear Overhauser effects, forming significative pattern to the drawing of a secondary structure. Conformations consistent with experimental NOE distances were obtained through molecular dynamics and energy minimization methods. These calculations yielded two stable conformers corresponding to an alpha-turn and a type III beta-turn involving the last five C-terminal residues. Interestingly, the alpha-turn conformation was found to overlap with good agreement the crystallographic structure of the same fragment in the G alpha(S) protein.

Solution conformation of a potent cyclic analogue of tuftsin: low- temperature nuclear magnetic resonance study in a cryoprotective mixture.

Tuftsin, a linear tetrapeptide (Thr-Lys-Pro-Arg), corresponding to the sequence 289-292 of the heavy chain of leukokinin, has been the object of intensive SAR studies during the past 30 years, owing to its numerous biological activities and to the possibility of generating a novel anticancer drug. A cyclic tuftsin analogue, c-[T-K-P-R-G], has biological activity 50 times higher than that of the parent linear peptide. Here we present a conformational study of c-[T-K-P-R-G] based on NMR data in a cryoprotective DMSO/water mixture. The preferred conformation is a type VIa turn centered on the K-P residues. The orientation of the side chains of the two basic residues (K and R) may represent the essential feature of the bioactive conformation of tuftsin. A possible role of tuftsin as a DNA binding motif is suggested by the similarity of the bioactive conformation of c-[T-K-P-R-G] and of the beta-turn conformation proposed by Suzuki for the [T,S]-P-K-R motif.

Synthesis and in vitro activities of NK-1 antagonists derived from L-tryptophan.

A study of structure-activity relationships of a series of L-tryptophan derivative NK-1 antagonist was performed using 3,5-bis(trifluoromethyl)benzyl ester of N-acetyl-L-Tryptophan (IV) as a starting point. The ester moiety was replaced with several amidic functions while the N-acetyl group (Ac-) was retained (compounds 1-8) or changed into a benzyloxycarbonyl group (Z-) (compounds 9-16). The compounds were tested on guinea pig ileum longitudinal muscle, rat colon muscolaris mucosae, and rat everted portal vein, representative of tachykinin NK-1, NK-2 and NK-3 receptors, respectively. Both, Ac- and Z-series showed generally moderate antagonist activity on tachykinin NK-1 receptors with respect to the reference drug IV. The most potent term was compound 2 (Ac-Trp-N(CH3)CH(CH3)Ph with S-configuration at the C-terminus) which exhibited pA2 values of 7.0, 4.2 and 4.4 on NK-1, NK-2 and NK-3 sites, respectively.

Synthesis and antinociceptive activity of peptides related to interleukin-1 beta 193-195 Lys-Pro-Thr.

To obtain information about the structure-activity relationships of analgesic peptides, we modified the previously reported tripeptide, H-Lys-Pro-Thr-OH (C). The proline part in C was replaced with various analogues of unconventional amino acids [(3aS, 7aS)-octahydroindole-2-carboxylic acid (Oic), (S,S,S,)-2-azabiciclo [3.3.0]octane-3-carboxylic acid (Aoc), D-Aoc, and (2S, 4R)-hydroxyproline (Hyp)] with varying lipophilic, steric, and conformational properties, and alternatively with Lys and Orn in the lysine part. Moreover, the threonine part was changed to various natural amino acids (Ser, Thr, Val, Leu). All the compound were screened in vivo for their analgesic effects in mouse writhing test. Compound 24 (H-Orn-Hyp-Val-OH), the most active compound within the series, showed an ED50 value of 10 mg/kg, which is comparable with the ED50 values exhibited by indometacin (4.1 mg/kg) and the dipeptide H-Lys-D-Pro-OH (6.9 mg/kg), both used as reference drugs.

Amphitoxin, a new high molecular weight antifeedant pyridinium salt from the Caribbean sponge Amphimedon compressa.

A new polymeric pyridinium alkaloid named amphitoxin [2] has been isolated from Amphimedon compressa, and its structure determined by spectroscopic analysis. In laboratory feeding experiments, crude extracts and purified amphitoxin [2] from A. compressa at lower than natural concentration levels effectively deterred feeding of a generalist predatory Caribbean reef fish, Thalassoma bifasciatum.

Linear diterpenes from the Caribbean sponge Myrmekioderma styx.

Four new oxygenated diterpenes 1-4 were isolated from the Caribbean sponge Myrmekioderma styx. The structures of these compounds were deduced by ms, uv, ir, 1H- and 13C-nmr, 1H-1H COSY, XHCORR, and COLOC experiments. Compounds 1-4 are lethal to brine shrimp (Artemia salina).