Improved proton affinity measurements for proline and modified prolines using triple quadrupole and ion trap mass spectrometers.

Proton affinity (PA) of compounds such as proline, cis-3-methylproline, cis-3-ethylproline, cis-3-isopropylproline and cis-3-isopentanylproline was determined by kinetic method with amines as the reference bases. The effective temperatures determined using ion trap and triple quadrupole mass spectrometers were found to be significantly different. In the case of the triple quadrupole instruments, the effective temperature depends significantly on the collision energy. The influence of the apparent basicity (GBapp) on the effective temperature may be used to estimate the difference in protonation entropy (DeltaDeltaS degrees) between the sample and reference compounds. In case of the ion trap mass spectrometer, the variation of the effective temperature as a function of the excitation amplitude is small, so it is difficult to account for the contribution of the entropy effects to the proton affinity value. A better estimation of the PA and DeltaDeltaS degrees values for the investigated molecules is obtained by combining the GBapp and Teff data pairs that are obtained from both the mass spectrometers.

Proton affinity of diastereoisomers of modified prolines using the kinetic method and density functional theory calculations: role of the cis/trans substituent on the endo/exo ring conformation.

The proton affinity (PA) of cis/trans-3-prolinoleucines and cis/trans-3-prolinoglutamic acids have been studied by the kinetic method and density functional theory (DFT) calculations. Several conformations of the neutral and the protonated modified prolines, in particular the endo and exo ring conformations, were analyzed with respect to their contribution to the PA values. When the substituent is an alkyl, both the diastereoisomers have the same PA value. However, the PA values for the diastereoisomers are different when the substituted chain contains functional groups (e.g. a carboxyl group). This variation in PA values could be attributed to the existence of intramolecular hydrogen bonds.

Proton affinity of proline and modified prolines using the kinetic method: role of the conformation investigated by ab initio calculations.

The proton affinities of proline, cis-3-methylproline and cis-3-ethylproline have been measured by the kinetic method using an ion trap instrument; the values obtained are 936, 940.5, and 943 kJ mol(-1), respectively. The experimental values are consistent with those obtained by high-level ab initio calculations (B3LYP/6-31+G*//B3LYP/6-31G* and B3P86/6-31+G*//B3LYP/6-31G*). Several conformations of neutral and protonated proline were considered, in particular the endo and exo ring structure and the position of the carboxyl group. These results show the importance of the position of the hydrogen atom of the carboxyl group in determining the most stable protonated proline structure.

Met174 side chain is the site of photoinsertion of a substance P competitive peptide antagonist photoreactive in position 8.

Numerous photoaffinity studies of the NK-1 receptor have been carried out with peptide agonist analogues of substance P (SP). However, no information is available with regard to the domain interaction of peptide antagonists within this receptor. We describe herein the photoaffinity labelling of the SP receptor with a peptide antagonist analogue, Bapa(0)[(pBzl)Phe(8),DPro(9),MePhe(10),Trp(CHO)(11)]SP. Photolabelling, enzymatic or chemical cleavage of the covalent complex, purification via streptavidin-coated beads and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis led us to show that the methyl of Met174 side chain, within the receptor's second extracellular loop, is covalently linked to the antagonist photoreactive at position 8.

Further delineation of the two binding sites (R*(n)) associated with tachykinin neurokinin-1 receptors using [3-Prolinomethionine(11)]SP analogues.

Two binding sites are associated with neurokinin-1 substance P receptors in both transfected cells and mammalian tissues. To further delineate the interactions between the crucial C-terminal methionine of substance P and these two binding sites, we have incorporated newly designed constrained methionines, i.e. (2S, 3S)- and (2S,3R)-prolinomethionines. The potencies of these C terminus-modified SP analogues to bind both sites and to activate phosphatidylinositol hydrolysis and cAMP formation have been measured, together with those of their corresponding sulfoxides and sulfones. The molecular nature of these two binding sites and their selective coupling to effector signaling pathways are discussed in the light of current models of receptor activation. The less abundant binding site is coupled to G(q/11) proteins, whereas the most abundant one interacts with G(s) proteins in Chinese hamster ovary cells transfected with human neurokinin-1 receptors. The specific orientation of the C-terminal methionine side chain imposed by these constraints shows that macroscopically chi(1) and chi(2) angles of this crucial C-terminal residue are similar in both binding sites. However, slight but significant variations in the rotation around the Cgamma-S bond yield different either stabilizing or destabilizing interactions in the two binding sites. These results highlight the need of such constrained amino acids to probe subtle interactions in ligand-receptor complexes.

Asymmetric synthesis of Boc-N-methyl-p-benzoyl-phenylalanine. Preparation of a photoreactive antagonist of substance P.

The asymmetric synthesis of (S)-Boc-N-methyl-p-benzoyl-phenylalanine was performed by alkylation of sultam Boc-sarcosinate. The levorotatory sultam led to (S)-Boc-N-methyl amino acids with high optical purity. This photoreactive amino acid was incorporated into the sequence of a Substance P peptide antagonist. Comparison of the affinity and antagonistic properties of Biotinyl-apa-[D-Pro9, MePhe(pBz)10, Trp11]SP for human tachykinin NK-1 receptor demonstrated that this photoreactive antagonist should be a suitable tool for photolabelling studies.

Tachykinin NK-1 receptor probed with constrained analogues of substance P.

The action of rotameric probes introduced either in position 7 or 8 in the sequence of substance P (SP) was investigated, i.e. L-tetrahydroisoquinoleic acid (Tic), L-fluorenylglycine (Flg), L-diphenylalanine (Dip), the diastereoisomers of L-1-Indanylglycine (Ing) and L-benz[f]indanylglycine (Bfi), the Z- and E-isomers of dehydrophenylalanine and dehydronaphthylalanine (delta ZPhe, delta EPhe, delta ZNal, ENal) and L-O,O'-dimethylphenylalanine (Dmp). The aim of this study was the topographical characterization of the binding subsites of human NK-1 receptor expressed in CHO cells, especially the S7 and S8 subsites, corresponding to residues Phe7 and Phe8 of substance P. According to the binding potencies of these substituted-SP analogues, the S7 binding subsite is smaller than the S8 subsite: the S7 subsite accepts only one aromatic nucleus, while the S8 can accommodate three coplanar nuclei altogether. These findings are compatible with the idea that the S8 binding subsite may reside in the extracellular loops of the hNK-1 receptor. NK-1 agonists bind to human NK-1 receptor and activate the production of both inositol phosphates and cyclic AMP. As already quoted for septide, [pGlu6, Pro9]SP(6-11), discrepancies are observed between affinity (K1) and activity (EC50) values for IPs production. While a weak correlation between K1 and EC50 values for IPs production could be found (r = 0.70), an excellent correlation could be demonstrated between their affinities (K1) and their potencies (EC50) for cAMP production (r = 0.97). The high potency (EC50) observed for "septide-like' molecules on PI hydrolysis, compared to their affinity is not an artefact related to the high level of NK-1 receptors expressed on CHO cells since a good correlation was found between EC50 values obtained for PI hydrolysis and those measured for spasmogenic activity in guinea pig ileum bioassay (r = 0.94).