Aza-peptides. III. Experimental structural analysis of aza-alanme and aza-asparagine-containing peptides

To determine the structural perturbations induced by the CαH→Nα exchange in aza-peptides, we have examined by H NMR and IR spectroscopy various derivatives of the aza-analogues of alanine, aspartic acid and asparagine in different organic solvents with increasing polarity. Their general formulas are: R'-AzXaa-NR²R³, R'-Pro-AzXaa-NR²R³ and R-AzXaa-Pro-NR²R³ (where AzXaa denotes the aza-analogue of the amino acid residue Xaa = Ala, Asp, Asn; R = Boc, Z; R², R³= H, Me, iPr). The aza-analogue of an amino acid residue appears to be a strong p-turn-inducing motif, and the AzAsn carboxamide side-chain is capable of interacting, as a proton donor, with the preceding peptide carbonyl group.     

Reduced amide pseudopeptide analogues of a malaria peptide possess secondary structural elements responsible for induction of functional antibodies which react with native proteins expressed in Plasmodium falciparum erythrocyte stages

A Ψ[CH₂NH] isoster bond was introduced by replacing one peptide bond at a time within the 1513 malaria peptide KEKMV motif to obtain a set of five pseudopeptides. The motif belongs to a Plasmodium falciparum malarial peptide coded 1513, derived from the MSP-1 protein. This high-binding motif included in the 1513 peptide is involved in the attachment of the malarial parasite to human erythrocytes. The novel malaria 1513 Ψ[CH₂NH] surrogates were analyzed using RP-HPLC and MALDI-TOF mass spectrometry techniques. Nuclear magnetic resonance experiments allowed definition of the five pseudopeptide analogues’ secondary structural features. Such structures are present in only a very few molecules in the 1513 parent peptide. A molecular model demonstrating the solution of the three-dimensional structure of the 1513 peptide Pse-437 analogue was constructed on the basis of ¹H-NMR spectral parameters. Monoclonal antibodies were generated to the five 1513 malaria peptide pseudopeptide analogues. These antibodies not only recognize the native MSP-1 (195 kDa) and its 83 kDa and 42 kDa proteolytic processing proteins but also different SPf(66)_n malaria vaccine batches containing the native sequence. In addition, the mAbs were able to modify the kinetics of Plasmodium falciparum parasites’ intraerythrocytic development and their ability to invade new RBCs. The presented evidence suggests that peptide bond-modified peptides could reproduce a transient state in 1513's native sequence and represent useful candidates in the development of a second generation of effective malarial vaccines.     

Ψ[CH2O] pseudodipeptide synthesis An improved approach which allows absolute configuration determination

An improved way to obtain Ψ[CH₂O] pseudodipeptide units is proposed, involving an intramolecular Williamson's reaction, with displacement of bromine by an alkoxide, instead of the classical intermolecular one. Until now, Ψ[CH₂O] pseudodipeptide synthesis done by this new method, has used a protected form of the amino alcohol hydroxyl group to prepare the acyclic precursor. In the present paper, the use of an active ester of the brominated carboxylic acid avoids this protection step. The pseudodipeptides Ac-GlyΨ[CH₂O]-d,l -Ala-OH and Ac-Ser(Bzl)Ψ[CH₂O]-d,l -Ala-OH were obtained in high yields, through a delta-lactam intermediate, which furthermore allows the determination of the absolute configuration of compounds using HPLC and appropriate nuclear magnetic resonance (NMR) techniques.     

Synthesis and in vitro activities of new tryptophan-modified and thiomethylene-containing pseudopeptide antagonists of the neurokinins

A series of pseudopeptide analogs of the substance P-like hexapeptide Ava-Phe-Phe-Gly-Leu-Met-NH₂ was produced by N_α-protection, introduction of the thiomethylene bond, of d - and non-proteinogenic amino acids, and alteration of the side chain of tryptophan. Synthesis of the pseudopeptides on a solid phase was successfully improved by direct formation of the CH₂—S bond on the resin. However, while thiomethylene formation between leucine and norleucine led to the expected SS diastereoisomer, the major product of the similar coupling between two phenylalanines was the SR isomer. An improved resistance of the analogs to proteolysis was observed, which could be related to the structural changes. Interestingly, these modifications led to three water-soluble and potent neurokinin antagonists on classical in vitro bioassays.     

An active pseudopeptide analog of the leucokinin insect neuropeptide family

A pseudopeptide analog of the active core of the leucokinin insect neuropeptide family was synthesized and found to retain myotropic activity. No reports of active pseudopeptide analogs of an insect or other invertebrate neuropeptide have previously appeared in the literature. The pseudopeptide (Pheψ[CH₂-NH] Phe-Ser-Trp-Gly-NH₂) contains a reduced-amide linkage between the two N-terminal Phe residues. Unlike its amide-bond containing counterpart, the activity of the pseudopeptide was not destroyed upon exposure to aminopeptidase M.     

Replacement of the Disulfide Bridge in a KLK3-Stimulating Peptide Using Orthogonally Protected Building Blocks

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H-Gly-Hisψ(NHCO)Lys-OH,partially modified retro-inverso analogue of the growth factor glycyl-L-histidyl-L-lysine with enhanced enzymatic stability

A partially retro-inverso analogue of the natural growth factor GHK was synthesized, in which the —CONH— bond between histidine and lysine was modified as —NHCO—. This modification is not expected to perturb the spatial distribution of the side-chains, and therefore the binding processes, compared to the native peptide. In the synthesis of the analogue two possible systems for deblocking of N^π-Born group of histidine have been applied and compared. An alternative method is also described for the incorporation of malonyllysine into the peptide chain. When evaluated with respect to resistance toward degradation by human plasma in vitro, the new peptide analogue showed approximately a ten-fold increase in stability versus the parent peptide.     

Synthesis and protease-catalyzed hydrolysis of a novel hydrazinopeptide

In order to explore the potentiality of hydrazinopeptides as protease inhibitors, the resistance of the hydrazinopeptidic bond toward proteolysis was investigated. To this end, the novel hydrazinohexapeptide Z-Ala₂-Pro-Val-hIle-Leu-OMe ( 1 ), where hIle represents hydrazinoisoleucine, was designed and synthesized together with the parent peptide Z-Ala₂-Pro-Val-Ile-Leu-OMe ( 2 ). The interactions of 1 and 2 with human leukocyte elastase (HLE) and porcine pancreatic elastase (PPE) were analyzed comparatively. We observed that 1 behaved as a substrate for both elastases, without the formation of a stable acyl-enzyme as in the case of azapeptides. Compounds 1 and 2 were cleaved at the same site (-Val-/-NH-) with a slight delay of hydrolysis for 1 compared to 2 (k_cat/K_M for 1 vs. 2 decreased by a factor of 2.7 for the HLE-catalyzed hydrolysis at pH 8.0 and 25°C). The presence of the hydrazinopeptidic bond (-CONHNH-) in 1 reduced by a factor of 4.7 the apparent enzyme affinity without abolishing it. These results indicate that suitably designed hydrazinopeptides may represent interesting targets in the search for protease resisting pseudopeptides.     

Peptides and Pseudopeptides as SIRT6 Deacetylation Inhibitors

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A new approach to obtaining Nα -t-Boc-amino acid aldehydes from asparagine and glutamine for reduced amide pseudopeptide solid-phase synthesis

Reduced amide pseudopeptides have been proposed as structural probes that could be useful as potential malarial vaccine components. However, designing determined pseudopeptide sequences containing isoster peptide bonds, either on an asparagine (Asn) or on a glutamine (Gln) residues, can become difficult because these precursor amino acid aldehydes are obtained in yields lower than 0.5%. This work presents a new strategy for obtaining both Asn and Gln aldehydes based on a controlled side-chain protection approach as well as a suitable solvent partition procedure. FT-IR, (1) H-NMR and (13) C-NMR were used for molecule characterization and identification. Amino acid aldehydes were successfully incorporated into a 20-mer peptide from a malarial-relevant sequence, and their impact on the molecule's conformational properties was assessed.