Dimitris Panagiotopoulos

13C NMR analysis of the triacylglycerol composition of Greek virgin olive oils

High‐resolution 13C NMR spectroscopy was used to study the potential of the method for the quantitative analysis of the most abundant fatty acids in the triacylglycerols of virgin olive oil samples harvested at different geographic areas of Greece. The analysis of the results was compared with data obtained with gas chromatography. Both methods showed results that differed only by 1–2 mol% of the fatty acids. NMR data provided the α/β ratio of oleic and linoleic acids. The results showed a preference for the β‐position in these unsaturated acids.

Structure elucidation and conformational analysis of gonadotropin releasing hormone and its novel synthetic analogue [Tyr(OMe)5, d-Lys6, Aze9NHEtGnRH: The importance of aromatic clustering in the receptor binding activity†

Summary The conformational properties of gonadotropin releasing hormone (GnRH) in dimethylsulfoxide- d 6 were investigated by nuclear Overhauser effect (nOe) enhancement studies and were compared with the conformational properties of its analogue [Tyr(OMe) 5 GnRH resulting after methylation of the tyrosine hydroxyl. Assignment of all backbone and side-chain protons was possible by combining information from intraresidue nOe studies with two-dimensional correlated spectroscopy (COSY/TOCSY) studies. Saturation of distinct proton resonances of the three aromatic residues Tyr, His, Trp, in clear areas of the NMR spectrum of GnRH resulted in interresidue enhancements of aromatic resonances indicating the proximity of the three aromatic rings. This spatial proximity is not observed in [Tyr(OMe) 5 ]GnRH and is correlated with a lower receptor binding affinity in the rat pituitary ( K d = 1.53 ± 0.35 × 10 −6 M) compared with that exerted by GnRH ( K d = 3.69 ± 0.89 × 10 −9 M). However, substitution of Gly at position 6 of [Tyr(OMe) 5 ]GnRH with d -Lys 6 and further replacement of Pro at position 9 with the more rigid Aze residue [Tyr(OMe) 5 , d -Lys 6 , Aze 9 NHEt]GnRH significantly improved the binding affinity ( K d = 0.689 ± 10.15 × 10 −9 ) and this may be due to the restoration of the ring cluster. Overall, the clustering of the aromatic rings observed in GnRH was not seen in [Tyr(OMe) 5 ]GnRH and this conformational difference may be responsible for receptor recognition and higher binding of the parent peptide.

Superimposition of potent non-peptide AT1 receptor antagonists with angiotensin II

The model of angiotensin II (ANG II) developed in our laboratory using a combination of NMR, fluorescence data and molecular graphics [Matsoukas, J.M. et al., J. Biol. Chem., 269 (1994) 5303] served as a template for a systematic superimposition of potent AT1 receptor antagonists with ANG II. The key amino acids in this model, tyrosine, phenylalanine and histidine, form a charge-relay system. The studied ANG II AT1 receptor antagonists were found to accommodate this relay system. The proposed model offers a motivation to synthetic chemists to develop ANG II antagonists that differ from the losartan prototype structure but possess an enhanced biological profile.

Interactions of angiotensin II with membranes using a combination of differential scanning calorimetry and 31P NMR spectroscopy

This study of angiotensin II (ANG II) membrane interactions uses a combination of31P NMR spectroscopy and differential scanning calorimetry (DSC), two valuable and complementary techniques which can provide useful information about the thermotropic and dynamic properties of peptide hormones in membranes. The major conclusion from the calorimetric experiments is that ANG II affects the phase properties of hydrated dipalmitoyl-phosphatidylcholine (DPPC) bilayers by mainly broadening the pretransition area. Preliminary31P NMR data seem to confirm the DSC results by showing that ANG II produces a lowering of the pretransition temperature but affects only minimally the main phase transition. In combination, the results from the two methods may indicate that the hormone produces its effects on the phospholipid head groups while its effects on the bilayer alkyl chains are not significant. Such results can be interpreted to mean that ANG II closely interacts with the phospholipid head groups perhaps up to the level of the interface, but does not enter deeper into the membrane bilayer.

Design and synthesis of a gonadotropin-releasing hormone (GnRH) analogue, [Tyr(OMe)5,d-Glu6,Aze9]GnRH: Receptor binding, gonadotropin release and ovulation studies

Gonadotropin-releasing hormone (GnRH) stimulates the release and synthesis of gonadotropin hormones (GtH) and is the key regulator of reproduction. The present study was carried out to design a potent GnRH analogue containing Tyr(OMe) at position 5 and ad-amino acid at position 6. This was based on a previous study in which [Tyr(OMe)5]GnRH was shown to have reduced potency compared to GnRH. A novel GnRH peptide containing Tyr(OMe)5 andd-Glu6 in combination with other substitutions at positions 9 and 10 was synthesized in the present study and tested for binding to the rat pituitary as well as potency in terms of gonadotropin (GtH) release in the goldfish pituitary and ovulation in sea bass. The results demonstrate that the replacement of the glycine residue at position 6 with ad-Glu in combination with the substitution of proline at position 9 with azetidine (Aze) increased the binding and biological activity of [Tyr(OMe)5]GnRH. The observed increased potency is likely to be related to the improved resistance to degradation. The present findings may lead to the development of a more potent GnRH agonist for inducing ovulation in fish.

Synthesis and activities of cyclic thrombin-receptor-derived peptide analogues of the Ser42-Phe-Leu-Leu-Arg46 motif sequence containing d-Phe and/or d-Arg

Cyclic analogues of the active thrombin receptor peptide SFLLR (TRP42–46) containingd-Phe and/ord-Arg have been prepared by the solid-phase method, purified by reversed-phase HPLC and bioassayed in a rat smooth muscle contractile assay. Cyclization was achieved by forming an amide linkage between the-NH2 and-COOH groups of the two leucine residues located at the N- and C-terminal positions of the linear protected precursor H2N-Leu-Arg(Pmc)-Y-Phe-Leu-OH (Y=Gly,Acp) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoroborate borate (HBTU) or 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) as coupling reagents andN,N′-diisopropylethylamine (DIPEA) in high dilution. Their structure was confirmed by fast by fast atom bombardment mass spectrometry and NMR methods. The cyclic peptides c-fLLrG, c-fLLRG, c-FLLrG and c-fLLrAcp, c-FLLrAcp so synthesized were assessed for their contractile activity in a rat gastric longitudinal muscle bioassay system which has been used previously to evaluate the biological activities of linear thrombin-receptor-derived polypeptides such as SFLLR (P5) and SFLLR-NH2 (P5-NH2).

Inhibition of TRAP-induced angiogenesis by the tripeptide Phe-Pro-Arg, a thrombin-receptor-derived peptide analogue

We have recently shown that thrombin promotes angiogenesis by a mechanism independent of fibrin formation. In the present paper, we investigated the effect of the thrombin-receptor-activating tetradecapeptide (TRAP1–14, S42FLLRNPNDKYEPF55) for its effects on angiogenesis in the chick chorioallantoic membrane (CAM) system of angiogenesis. A dose-dependent promotion of angiogenesis is evident with TRAP. In contrast, a thrombin-receptor-derived tripeptide analogue H-Phe-Pro-Arg-OH (FPR), which was designed based on the S42FLLR46 sequence, caused an inhibition of angiogenesis in the CAM, and when it was combined with TRAP it caused a complete reversal of the angiogenesis-promoting effect of TRAP. These results indicate that the proteolytic exposure of the receptor N-terminal tetradecapeptide by thrombin can activate the post-thrombotic events related to angiogenesis. These events can be modulated by constrained peptide analogues such as FPR.

Design and Synthesis of Potent Tyr(OMe)5-Gonadotropin-Releasing Hormone (GnRH) Analogues with Modifications at Positions 6, 9 and 10

We have recently reported the synthesis and the conformational properties of some Gonadotropin-releasing hormone (GnRH) analogues in which the tyrosine residue at position 5 is substituted with tyrosine-O-methyl (Keramida et al., Let. Pept. Sci., 3 (1996) 257/Matsoukas et al., Eur. J. Med. Chem., 32 (1997) 927). The analogue [Tyr-(OMe)5]-GnRH was found to exert a lower degree of desensitization than the native GnRH peptides in terms of pituitary gonadotropin (GTH) release in goldfish. Compared to GnRH, however, [Tyr-(OMe)5]-GnRH exerted a lower GTH-release potency in cultured goldfish pituitary fragments, and was bound with a lower binding affinity to the rat pituitary GnRH receptors. In order to increase the potency of [Tyr-(OMe)5]-GnRH, we have synthesized a group of GnRH peptides containing Tyr-(OMe)5 in combination with other substitutions at positions 6, 9 and 10 and we have estimated their binding affinity for the rat pituitary receptors and gonadotropin (GTH) release potency in the goldfish pituitary. A selected number of these analogues was also tested for their ability to induce ovulation in seabass. The important structural modifications that increased the binding and gonadotropic activity of [Tyr(OMe)5]-GnRH in vitro and in vivo were found to include the replacement of the proline at position 9 with azetidine, glycine amide terminus with an alkyl amide group and Gly6 residue with hydrophilicd-amino acids such asd-Arg6. Overall, the findings provide SAR information on a group of novel GnRH peptides that can be also used to induce ovulation in teleosts.

Structural elucidation and conformational properties of the immunomodulator linomide

Linomide is a new synthetic immunomodulator which exerts prominent anti-autoimmune effects in various experimental models. Recently, it was tested in clinical trials to patients suffering from multiple sclerosis and showed to inhibit the activity of the disease. Therefore, due to its pharmacological importance, we attempted elucidate its structure using one-dimensional and two-dimensional nuclear magnetic resonance (NMR) techniques and study its conformational properties using a combination of two-dimensional NMR spectroscopy and molecular modeling. The conformational analysis of linomide was based on the measurement of interproton nuclear Overhauser enhancement (NOE) values obtained from a two-dimensional NMR spectrum and a number of molecular modeling techniques used to calculate the low energy conformers of this compound. This information will serve as an aid to synthetic chemists whom their research activity is focused on developing linomide analogs with better biological profiles.