Microwave-enhanced solid-phase synthesis of N,N'-linked aliphatic oligoureas and related hybrids.

A practical and efficient microwave-assisted solid-phase method for the synthesis of N,N'-linked oligoureas and related amide/urea hybrid oligomers, featuring the use of succinimidyl (2-azido-2-substituted ethyl) carbamate monomers, is reported. The rate enhancement of urea formation under microwave irradiation combined with the mild conditions of the phosphine-based azide reduction makes this approach very effective for routine synthesis of oligoureas and possibly for library production.

Plant polyphenols: chemical properties, biological activities, and synthesis.

Eating five servings of fruits and vegetables per day! This is what is highly recommended and heavily advertised nowadays to the general public to stay fit and healthy! Drinking green tea on a regular basis, eating chocolate from time to time, as well as savoring a couple of glasses of red wine per day have been claimed to increase life expectancy even further! Why? The answer is in fact still under scientific scrutiny, but a particular class of compounds naturally occurring in fruits and vegetables is considered to be crucial for the expression of such human health benefits: the polyphenols! What are these plant products really? What are their physicochemical properties? How do they express their biological activity? Are they really valuable for disease prevention? Can they be used to develop new pharmaceutical drugs? What recent progress has been made toward their preparation by organic synthesis? This Review gives answers from a chemical perspective, summarizes the state of the art, and highlights the most significant advances in the field of polyphenol research.

Solid phase synthesis of aromatic oligoamides: application to helical water-soluble foldamers.

Synthetic helical aromatic amide foldamers and in particular those based on quinolines have recently attracted much interest due to their capacity to adopt bioinspired folded conformations that are highly stable and predictable. Additionally, the introduction of water-solubilizing side chains has allowed to evidence promising biological activities. It has also created the need for methods that may allow the parallel synthesis and screening of oligomers. Here, we describe the application of solid phase synthesis to speed up oligomer preparation and allow the introduction of various side chains. The synthesis of quinoline-based monomers bearing protected side chains is described along with conditions for activation, coupling, and deprotection on solid phase, followed by resin cleavage, side-chain deprotection, and HPLC purification. Oligomers having up to 8 units were thus synthesized. We found that solid phase synthesis is notably improved upon reducing resin loading and by applying microwave irradiation. We also demonstrate that the introduction of monomers bearing benzylic amines such as 6-aminomethyl-2-pyridinecarboxylic acid within the sequences of oligoquinolines make it possible to achieve couplings using a standard peptide coupling agent and constitute an interesting alternative to the use of acid chloride activation required by quinoline residues. The synthesis of a tetradecameric sequence was thus smoothly carried out. NMR solution structural studies show that these alternate aminomethyl-pyridine residues do not perturb the canonical helix folding of quinoline monomers in protic solvents, contrary to what was previously observed in nonprotic solvents.

Design, synthesis and evaluation of ß-lactam antigenic peptide hybrids; unusual opening of the ß-lactam ring in acidic media.

ß-Lactam peptides were envisioned as conformational constraints in antigenic peptides (APs). Three different ß-lactam tripeptides of varying flexibility were prepared in solution and incorporated in place of the central part of the altered melanoma associated antigenic peptide Leu(27)-Melan-A(26-35) using solid phase synthesis techniques. Upon TFA cleavage from the solid support, an unexpected opening of the ß-lactam ring occurred with conservation of the amide bond. After adaptation of the solid phase synthesis strategy, ß-lactam peptides were successfully obtained and both opened and closed forms were evaluated for their capacity to bind to the antigen-presenting class-I MHC HLA-A2 protein system. None of the closed ß-lactam peptides bound to HLA-A2, but their opened variants were shown to be moderate to good HLA-A2 ligands, one of them being even capable of stimulating a Melan-A-specific T cell line.

Crystal structures of HLA-A*0201 complexed with Melan-A/MART-1(26(27L)-35) peptidomimetics reveal conformational heterogeneity and highlight degeneracy of T cell recognition.

There is growing interest in using tumor associated antigens presented by class I major histocompatibility complex (MHC-I) proteins as cancer vaccines. As native peptides are poorly stable in biological fluids, researchers have sought to engineer synthetic peptidomimetics with greater biostability. Here, we demonstrate that antigenic peptidomimetics of the Melan-A/MART-1(26(27L)-35) melanoma antigen adopt strikingly different conformations when bound to MHC-I, highlighting the degeneracy of T cell recognition and revealing the challenges associated with mimicking native peptide conformation.

Synthetic anticancer vaccine candidates: rational design of antigenic peptide mimetics that activate tumor-specific T-cells.

A rational design approach was followed to develop peptidomimetic analogues of a cytotoxic T-cell epitope capable of stimulating T-cell responses as strong as or stronger (heteroclytic) than those of parental antigenic peptides. The work described herein focused on structural alterations of the central amino acids of the melanoma tumor-associated antigenic peptide Melan-A/MART-1(26-35) using nonpeptidic units. A screening was first realized in silico to select altered peptides potentially capable of fitting at the interface between the major histocompatibilty complex (MHC) class-I HLA-A2 molecule and T-cell receptors (TCRs). Two compounds appeared to be high-affinity ligands to the HLA-A2 molecule and stimulated several Melan-A/MART-1 specific T-cell clones. Most remarkably, one of them even managed to amplify the response of one clone. Together, these results indicate that central TCR-contact residues of antigenic peptides can be replaced by nonpeptidic motifs without loss of binding affinity to MHC class-I molecules and T-cell triggering capacity.

Inhibition of topoisomerase I cleavage activity by thiol-reactive compounds: importance of vicinal cysteines 504 and 505.

DNA topoisomerase I (Top1) is a nuclear enzyme that plays a crucial role in the removal of DNA supercoiling associated with replication and transcription. It is also the target of the anticancer agent, camptothecin (CPT). Top1 contains eight cysteines, including two vicinal residues (504 and 505), which are highly conserved across species. In this study, we show that thiol-reactive compounds such as N-ethylmaleimide and phenylarsine oxide can impair Top1 catalytic activity. We demonstrate that in contrast to CPT, which inhibits Top1-catalyzed religation, thiolation of Top1 inhibited the DNA cleavage step of the reaction. This inhibition was more pronounced when Top1 was preincubated with the thiol-reactive compound and could be reversed in the presence of dithiothreitol. We also established that phenylarsine oxide-mediated inhibition of Top1 cleavage involved the two vicinal cysteines 504 and 505, as this effect was suppressed when cysteines were mutated to alanines. Interestingly, mutation of Cys-505 also altered Top1 sensitivity to CPT, even in the context of the double Cys-504 to Cys-505 mutant, which relaxed supercoiled DNA with a comparable efficiency to that of wild-type Top1. This indicates that cysteine 505, which is located in the lower Lip domain of human Top1, is critical for optimal poisoning of the enzyme by CPT and its analogs. Altogether, our results suggest that conserved vicinal cysteines 504 and 505 of human Top1 play a critical role in enzyme catalytic activity and are the target of thiol-reactive compounds, which may be developed as efficient Top1 catalytic inhibitors.

Covalent modification of a melanoma-derived antigenic peptide with a natural quinone methide. Preliminary chemical, molecular modelling and immunological evaluation studies.

A LigandFit shape-directed docking methodology was used to identify the best position at which the melanoma-derived MHC class-I HLA-A2-binding antigenic peptide ELAGIGILTV could be modified by attaching a small molecule capable of fitting at the interface of complementary determining regional (CDR) loops of a T-cell receptor (TCR) while triggering T-cell responses. The small molecule selected here for determining the feasibility of this alternative track to chemical alteration of antigenic peptides was the electrophilic quinone methide (+)-puupehenone (), a natural product that belongs to a family of marine metabolites capable of expressing immunomodulatory activities. A preliminary chemical reactivity model study revealed the efficacy of the thiol group of a cysteine (C) side-chain in its nucleophilic addition reaction with in a regio- and diastereoselective manner. The best TCR/HLA-A2 ligand [i.e., ELAGCGILTV-S-puupehenol ()] then identified by the LigandFit docking procedure was synthesized and used to pulse HLA-A2(+) T2 cells for T-cell stimulation. Among the ELAGIGILTV-specific T-cell clones we tested, five of them recognized the conjugate in spite of its low binding affinity for the HLA-A2 molecules. The resulting T-cell stimulation was determined through the intracytoplasmic secretion of IFN-gamma and the percentage of T-cells thus activated. These highly encouraging results indicate that small non-peptidic natural product-derived molecules attached onto the central part of an antigenic peptide can fit at the TCR/HLA-A2 interface with induction of T-cell responses.

Selective catalysis with peptide dendrimers.

Peptide dendrimers incorporating 3,5-diaminobenzoic acid 1 as a branching unit (B) were prepared by solid-phase synthesis of ((Ac-A(3))(2)B-A(2))(2)B-Cys-A(1)-NH(2) followed by disulfide bridge formation. Twenty-one homo- and heterodimeric dendrimers were obtained by permutations of aspartate, histidine, and serine at positions A(1), A(2), and A(3). Two dendrimers catalyzed the hydrolysis of 7-hydroxy-N-methyl-quinolinium esters (2-5), and two other dendrimers catalyzed the hydrolysis of 8-hydroxy-pyrene-1,3,6-trisulfonate esters (10-12). Enzyme-like kinetics was observed in aqueous buffer pH 6.0 with multiple turnover, substrate binding (K(M) = 0.1-0.5 mM), rate acceleration (k(cat)/k(uncat) > 10(3)), and chiral discrimination (E = 2.8 for 2-phenylpropionate ester 5). The role of individual amino acids in catalysis was investigated by amino acid exchanges, highlighting the key role of histidine as a catalytic residue, and the importance of electrostatic and hydrophobic interactions in modulating substrate binding. These experiments demonstrate for the first time selective catalysis in peptide dendrimers.

Synthesis and esterolytic activity of catalytic peptide dendrimers.

Peptide dendrimers were prepared by solid-phase peptide synthesis. Monomeric dendrimers were first obtained by assembly of a hexapeptide sequence containing alternate standard alpha-amino acids with diamino acids as branching units. The monomeric dendrimers were then dimerized by disulfide-bridge formation at the core cysteine. The synthetic strategy is compatible with functional amino acids and different diamino acid branching units. Peptide dendrimers composed of the catalytic triad amino acids histidine, aspartate, and serine catalyzed the hydrolysis of N-methylquinolinium salts when the histidine residues were placed at the outermost position. The dendrimer-catalyzed hydrolysis of 7-isobutyryl-N-methylquinolinium followed saturation kinetics with a rate constant of catalysis/rate constant without catalysis (k(cat)/k(uncat)) value of 3350 and a rate constant of catalysis/Michaelis constant (k(cat)/K(M)) value 350-fold larger than the second-order rate constant of the 4-methylimidazole-catalyzed reaction; this corresponds to a 40-fold rate enhancement per histidine side chain. Catalysis can be attributed to the presence of histidine residues at the surface of the dendrimers.