Location of the three disulfide bonds in an antimicrobial peptide from Amaranthus caudatus using mass spectrometry.

The disulfide bridge pairing of Ac-AMP2, a 30-residue peptide isolated from the seeds of Amaranthus caudatus, is determined using a fast method involving enzymatic fragmentation followed by identification of the fragments with FAB mass spectrometry. The results confirm the location of the three disulfide bridges as previously established by NMR spectroscopy and molecular modelling.

Conformational restriction of Tyr and Phe side chains in opioid peptides: information about preferred and bioactive side-chain topology.

The side chain of Tyr and Phe was fixed into the gauche(-) or gauche(+) conformation by using the Tic Htc structures, and into the trans conformation by using an aminobenzazepine-type (Aba) structure. When incorporated into dermorphin or deltorphin II, the Tic and Htc analogues all showed a large decrease in both mu and delta affinities and activities. Fixation of Phe(3) in the trans rotamer resulted in a large increase in delta affinity in the dermorphin analogue, whereas in the [Aba(3)-Gly(4)] deltorphin II analogue, good delta affinity is maintained despite the removal of the Glu side chain. Whereas several authors propose a gauche(-) preferred conformation for the Phe(3) side chain, these results suggest a trans conformation at the delta receptor. The use of these conformationally constrained residues for evaluating the preferred solution conformation in the flexible N-terminal tripeptide Tyr-D-Ala-Phe is illustrated. The (1)H-nmr parameters--chemical shift, temperature dependence, and nuclear Overhauser effects to the D-Ala(2) methyl protons in the different analogues--provide direct evidence to confirm the proposed sandwich conformation in the native peptides.

Comparing conformations at low temperature and at high viscosity. Conformational study of somatostatin and two of its analogues in methanol and in ethylene glycol.

The influence of low temperature and high viscosity on the conformation of somatostatin and two of its analogues was investigated by 1H NMR in solution. The conformation of native somatostatin, a cyclic octapeptide agonist DC13-116 and a linear octapeptide agonist were compared in ethylene glycol at 303 K and in methanol at low temperature. The first goal of this study was to investigate if either low temperature or high viscosity is the more important for the reduction of the conformational freedom. Secondly we wanted to compare the amount of information concerning the conformation present in both solvents. A larger amount of NOESY cross-peaks is observed in ethylene glycol at room temperature compared to methanol at low temperature. This indicates that the raising of the viscosity is a more important factor in reducing the flexibility of peptides than the lowering of the temperature.

Conformational study of cyclosporin A in acetone at low temperature.

The conformation of cyclosporin A (CsA), an undecapeptide with seven N-methylated amino acids, was studied in acetone at 193 K. Previous studies of the conformation of CsA in different solvents, in the cyclosporin-cyclophilin complex and in complexes with LiCl showed that the conformation of the free and the bound CsA are different. Differences were observed at the conformation of the MeLeu9-MeLeu10 peptide bond, which is cis in solution and trans in the complex, and in the orientation of the amide protons and the N-Me groups. By using acetone, which is a proton acceptor, we wanted to influence the orientation of the amide protons. In the conditions used in this study a new conformation is found, which differs as well from the one previously observed in solution as from the conformation observed in the complex. This conformation has a cis peptide bond between MeLeu9 and MeLeu10. The trans conformation of the peptide bond MeLeu9-MeLeu10, which is necessary for biological activity, was not induced. One of the amide protons is involved in an intramolecular H-bridge stabilising a beta-turn around Sar3MeLeu4, and three of the seven NMe groups are oriented to the centre of the molecule.

Conformational study of three endothelin antagonists with 1H NMR at low temperature and molecular dynamics.

The conformations of three endothelin antagonists, a cyclic pentapeptide, a linear tripeptide and a linear hexapeptide, are compared by 1H NMR and molecular dynamics. The three analogues have a Leu and a DTrp side chain which are oriented parallel, and an acidic group next to the DTrp residue.

Conformational study of a series of somatostatin analogues with antitumor and/or GH inhibitory activity.

A series of somatostatin analogues with varying activities have been studied by 1H NMR in CD3OH at low temperature in order to find a possible structural explanation for the differentiation of biological activities. In somatostatin analogues with GH release inhibitory activity a beta-turn/beta-sheet backbone conformation is present, which is shown to be characteristic of somatostatin-derived peptides exhibiting this biological activity. On the other hand, among the analogues with antitumor activity, a deviation from these typical structural features is clearly observed, but not general conformational model can be proposed.

Synthesis and biological activities of psi (CH2NH) pseudopeptide analogues of the C-terminal hexapeptide of neurotensin.

Each peptide CO-NH function in the biologically important C-terminal 8-13 sequence of neurotensin was replaced by the reduced CH2-NH isostere using the rapid in situ solid phase procedure developed by Sasaki & Coy. In general this modification resulted in a drop in receptor affinity except for the [Arg psi(CH2NH) Arg]-NT8-13 analogue (PIC50 9.23 vs. NT8-13 pIC50 8.03). This analogue also showed enhanced enzymatic stability, but acted as a full agonist as shown by the observation of relaxations of guinea-pig colon ascendens.

Synthesis, biological activity and conformational study of a somatostatin hexapeptide analogue containing a reduced peptide bond.

The cyclic hexapeptide analogue of somatostatin, c[Phe psi(CH2-N)Pro-Phe-D-Trp-Lys-Thr], was prepared by solid-phase synthesis of the linear precursor, followed by cyclization using diphenylphosphoryl azide. The inhibition of GH release, as well as receptor affinity, is greatly decreased. Conformational analysis by NMR in DMSO/H2O (1/1) revealed the presence of a type II' beta-turn in the core tetrapeptide region and a delta-turn over the reduced peptide bond.

An evaluation of microwave heating for the rapid hydrolysis of peptide samples for chiral amino acid analysis.

The hydrolysis of peptide samples in 6 N deuterium chloride by microwave heating has been investigated. The influence of the power and time of heating on the recovery of the amino acids and on their racemization was studied. A high recovery of sensitive amino acids and minimal racemization, as determined by the amount of deuterium incorporation, is obtained by using an intermediate irradiation power and 30 min reaction time.

Morphiceptin and beta-casomorphin-5 analogues containing a reduced peptide bond: selective mu-receptor agonists and a novel mu antagonist, H-Tyr-Pro psi (CH2-NH)Phe-Pro-Gly-OH.

In order to prevent enzymatic degradation of beta-casomorphin-5 (1) and morphiceptin, reduced peptide bonds were incorporated at the 2-3 and 3-4 bonds, respectively. The analogues were synthesized by a combination of solid phase methodology and reductive alkylation of resin-bound peptide amines with Boc-amino acid aldehydes (Boc: tert-butyloxycarbonyl) in the presence of NaBH3CN. During reversed phase high pressure liquid chromatography purification, peak shape distortions could be observed. Epimerization was excluded, based on gas chromatography/mass spectroscopy analysis, which indicated acceptable levels of racemization (less than 3%) in the crude product. Instead, the phenomena could be attributed to slow cis/trans isomerizations originating from the Xxx-Pro bonds in the sequence. The presence of different conformational isomers was also established by 1H-nmr spectroscopy in DMSO-d6. All analogues showed high stability in blood plasma, enhanced binding affinity for the mu receptor, and very low binding to the delta receptor. While the Phe 3 psi(CH2-N)Pro4 analogues (3) and (5) displayed agonist activity, the Pro 2 psi(CH2-NH)Phe3 modified analogue (2) showed antagonist activity comparable to D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2.

Dermorphin tetra- and heptapeptide analogues containing a [3,4] amide bond replacement by a carbon-carbon double and single bond.

Seven dermorphin hepta- and tetrapeptide analogues containing [3,4] amide bond replacement by a carbon-carbon double and single bond were prepared. 1H NMR studies of the pseudoheptapeptide in DMSO indicate the presence of extended conformations with stacking of the side chains in the N-terminal part and an inverse gamma-turn around Ser7 in the conformational equilibrium. The binding data show that the affinity of the analogues for the mu-receptor is only slightly diminished in the D-Ala2 series and is more affected in the D-Arg2 series. Since the Gly4NH is not present in these compounds we conclude that this NH is not required to stabilize the bioactive conformation nor is it directly involved in binding to the receptor.

Backbone modifications in somatostatin analogues: relation between conformation and activity.

Twenty cyclic and linear analogues of somatostatin have been compared with respect to their conformational behavior and biological activity. It appears that all active compounds have in common a well defined, predominant backbone conformation. For linear peptides, this conformation can only be detected at low (-80 degrees C) temperature by NMR measurements. Selectivity is suggested to be determined by the nature and topology of the side chains linked to this common backbone conformation. The side chain conformation is also only accessible for NOE measurements in the low temperature range.

Assignment of the 1H-NMR resonances of the four rotamers of beta-casomorphin-5 in DMSO.

We report the complete assignment of the 1H-nmr spectrum of beta-casomorphin-5 in DMSO-d6 solution. With a combination of one-dimensional, double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) spectra, we were able to differentiate the four conformers originating from two Xxx-Pro bonds present in the sequence. Exchange peaks in the ROESY spectra confirmed the presence of four interchanging conformational isomers. Based on integrations, the relative populations of the four species were estimated, while characteristic sequential nuclear Overhauser enhancements (NOEs) were used to determine the orientation of the Xxx-Pro bonds. This orientation was also shown to correlate with the chemical shift changes for the alpha protons of both the Xxx and Pro residues. Finally, interresidue NOEs indicate conformational preferences for the aromatic side chains, especially in the all-trans conformer.

Conformation of two somatostatin analogues in aqueous solution. Study by NMR methods and circular dichroism.

Cyclic-disulfide-containing analogues of somatostatin, Xaa1-Cys2-Xaa3-DTrp4-Lys6-Thr5-Xaa7- Xaa8 [Xaa1 = H or DPhe; Xaa3 = Phe or Tyr; Xaa7 = Cys, Me2Cys or Me2DCys; Xaa8 = OH, Thr8 (OH) or Thr8NH2], were examined in aqueous solution by 1H-NMR spectroscopy and circular dichroism. The influence of the helical nature of the disulfide bridge and the presence of exocyclic residues on biological activity were investigated with particular care.

Synthesis and conformational analysis of a series of galactosyl enkephalin analogues showing high analgesic activity.

Two galactosyl derivatives of [DMet2,Pro5] enkephalin-amide (compound 1), namely [DMet2,Pro5] enkephalin [N1.5-beta-D-galactopyranosyl] amide (compound 2) and O1.5-(beta-D-galactopyranosyl) [DMet2,Hyp5] enkephalin-amide (compound 3) have been synthesized. Such glycosylpeptides have been shown to be extremely potent analgesic agonists. The conformational analysis of these three compounds in DMSO-d6 solution has been carried out using two-dimensional NMR methods. Both the parent compound (1) and the beta N-galactosyl derivative (2) show similar NMR parameters which are consistent with fairly rigid beta-strands at both the N-terminus and C-terminus, connected by a glycine residue that displays a mixture between multiple conformational states. Thus, although the beta N-galactosyl derivative (2) has been shown to be significantly more potent than the parent compound (1) in the tail immersion and paw pressure tests of analgesia, no correlation can be established between the conformation of (1) and (2) in DMSO and the difference in analgesic activity. In contrast, important conformational differences with respect to (1) and (2) have been detected in the beta O-galactosyl derivative (3). In this case, only one of the likely conformations for (1) and (2) are consistent with the experimental data. These data show that the position of the galactose residue in compound (3) causes Gly3 to loose flexibility leading to a more rigid folded conformation. Such a change in conformation could be related to the difference in analgesic activity between (2) and (3).

Psi [CH2NH] backbone-modified peptides: first unequivocal observation of a C7 structure in a linear peptide.

We have elucidated the X-ray diffraction structures of the psi[CH2NH] backbone-modified analogs of Z-Pro-Leu-Gly-NH2 and t-Boc-Pro-Leu-Gly-NH2 (N alpha-protected derivatives of the tripeptide amide representing the C-terminal tail of oxytocin) with the "reduced peptide bond" located at the Pro-Leu sequence. The comparative results of these pseudopeptides show that conformational properties are similar (i.e., C7 structure at the Pro), whereas the unmodified peptides diverge substantially (i.e., t-Boc-Pro-Leu-Gly-NH2 and H-Pro-Leu-Gly-NH2 each show type-II beta-bend at the Leu-Gly; and Z-Pro-Leu-Gly-NH2 shows an open folded structure). The results for t-Boc-Pro psi[CH2NH]Leu-Gly-NH2 represent the first unequivocal proof for the existence of a C7 structure in a linear peptide.

Cyclic lactam analogues of Ac-[Nle4]alpha-MSH4-11-NH2.

Two side-chain cyclic lactam analogues of the 4-11 fragment of alpha-melanocyte-stimulating hormone (alpha-MSH), Ac-[Nle4,D-Orn5,Glu8]alpha-MSH4-11-NH2 and Ac-[Nle4,D-Orn5,D-Phe7,Glu8]alpha-MSH4-11-NH2, were prepared on p-methylbenzhydrylamine resin by using a combination of N alpha-Boc and N alpha-Fmoc synthetic strategies with diphenyl phosphorazidate mediated cyclization. The melanotropin activities of these two analogues were examined and compared relative to those of alpha-MSH, Ac-[Nle4]alpha-MSH4-11-NH2, and Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the frog (Rana pipiens) skin bioassay, the L-Phe7 17-membered ring cyclic analogue was slightly more potent than the linear Ac-[Nle4]alpha-MSH4-11-NH2 and exhibited prolonged melanotropic bioactivity (greater than or equal to 4 h). In this same assay, the D-Phe7 cyclic analogue was more than 100-fold less potent than the L-Phe cyclic analogue and was 10,000 times less potent than linear Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the lizard skin (Anolis carolinensis) bioassay, the L-Phe7 cyclic analogue was 100-fold less potent than Ac-[Nle4]alpha-MSH4-11-NH2, while the D-Phe7 cyclic analogue was 10,000-fold less potent than both Ac-[Nle4]alpha-MSH4-11-NH2 and the D-Phe7 linear derivative Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. The solution conformation of these two cyclic analogues in dimethyl sulfoxide-d6 was examined by 1D and 2D 500-MHz 1H NMR spectroscopy. Our analysis suggests an H bond stabilized C10 (or C13) turn for the D-Phe7 cyclic structure while the L-Phe7 analogue is more conformationally flexible.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the bioactive conformation in a cyclic hexapeptide analogue of somatostatin containing a cis-peptide bond mimic.

N-methyl- alpha -benzyl-o-aminomethylphenylacetic acid was incorporated into a cyclic somatostatin analogue in order to mimic a cis-peptide bond configuration. The high biological potency of one of the isomers of the cyclic peptide strongly argues in favour of the proposed cis-configuration of the peptide bond at that position in the parent peptide. This represents the first cis-peptide bond mimic which has high biological activity.

Determination of the chiral purity of dipeptide isosteres containing a reduced peptide bond by gas chromatographic analysis.

The stereochemical purity of psi (CH2-NH) dipeptides has been determined using gas chromatography-mass spectrometry. Different structures were found due to the derivatization procedures. A selective preparation of the linear bistrifluoroacetylated derivative and the monotrifluoroacetylated lactam makes it possible to monitor the chiral purity of the pseudodipeptides synthesized. Racemization occurring during peptide hydrolysis can be differentiated from racemization during the synthesis by using deuterium labelling. The method allows the optimization of the synthesis protocols and will be useful for further monitoring of the chiral purity of the pseudopeptides synthesized.