Exploring privileged structures: the combinatorial synthesis of cyclic peptides

Head-to-tail cyclic peptides have been reported to bind to multiple, unrelated classes of receptor with high affinity. They may therefore be considered to be privileged structures. This review outlines the strategies by which both macrocyclic cyclic peptides and cyclic dipeptides or diketopiperazines have been synthesised in combinatorial libraries. It also briefly outlines some of the biological applications of these molecules, thereby justifying their inclusion as privileged structures.

Combinatorial Synthesis and Biological Evaluation of Inhibitors of D-Ala-D-Ala-Ligase

Report for the scientific sojourn carried out at the Max Planck Institut of Molecular Phisiology, Germany, from 2006 to 2008.The work carried out during this postdoctoral stage was focused on two different projects. Firstly, identification of D-Ala D-Ala Inhibitors and the development of new synthethic approaches to obtain lipidated peptides and proteins and the use of these lipidated proteins in biological and biophysical studies. In the first project, new D-Ala D-Ala inhibitors were identified by using structural alignments of the ATP binding sites of the bacterial ligase DDl and protein and lipid kinases in complex with ATP analogs. We tested a series of commercially available kinase inhibitors and found LFM-A13 and Tyrphostine derivatives to inhibit DDl enzyme activity. Based on the initial screening results we synthesized a series of malononitrilamide and salicylamide derivatives and were able to confirm the validity of these scaffolds as inhibitors of DDl. From this investigation we gained a better understanding of the structural requirements and limitations necessary for the preparation of ATP competitive DDl inhibitors. The compounds in this study may serve as starting points for the development of bi-substrate inhibitors that incorporate both, an ATP competitive and a substrate competitive moiety. Bisubstrate inhibitors that block the ATP and D-Ala binding sites should exhibit enhanced selectivity and potency profiles by preferentially inhibiting DDl over kinases. In the second project, an optimized synthesis for tha alkylation of cysteins using the thiol ene reaction was establisehd. This new protocol allowed us to obtain large amounts of hexadecylated cysteine that was required for the synthesis of differently lipidated peptides. Afterwards the synthesis of various N-ras peptides bearing different lipid anchors was performed and the peptides were ligated to a truncated N-ras protein. The influence of this differently lipidated N-ras proteins on the partioning and association of N-Ras in model membrane subdomains was studied using Atomic Force Microscopy.

Combinatorial synthesis and directed evolution applied to the production of alpha-helix forming antimicrobial peptides analogues

Antimicrobial peptides (AMPs) are effector molecules of innate immune systems found in different groups of organisms, including microorganisms, plants, insects, amphibians and humans. These peptides exhibit several structural motifs but the most abundant AMPs assume an amphipathic alpha-helical structure. The alpha-helix forming antimicrobial peptides are excellent candidates for protein engineering leading to an optimization of their biological activity and target specificity. Nowadays several approaches are available and this review deals with the use of combinatorial synthesis and directed evolution in order to provide a high-throughput source of antimicrobial peptides analogues with enhanced lytic activity and specificity.

Combinatorial synthesis and biological evaluation of isoxazole-based libraries as antithrombotic agents

The 3-substitutedphenyl-5-isoxazolecarboxaldehydes have been identified as activated aldehydes for the generation of isoxazole-based combinatorial libraries on solid phase through automation. Three highly functionalized isoxazole-based libraries comprising of 32, 96 and 45 compounds each have been synthesized in parallel format using Baylis Hillman reaction, Michael addition, reductive amination and alkylation reactions. With an objective of lead generation all the three libraries were evaluated for their antithrombin activity in vivo.

Liquid-phase combinatorial synthesis.

A concept termed liquid-phase combinatorial synthesis (LPCS) is described. The central feature of this methodology is that it combines the advantages that classic organic synthesis in solution offers with those that solid-phase synthesis can provide, through the application of a linear homogeneous polymer. To validate this concept two libraries were prepared, one of peptide and the second of nonpeptide origin. The peptide-based library was synthesized by a recursive deconvolution strategy [Erb, E., Janda, K. D. & Brenner, S. (1994) Proc. Natl. Acad. Sci. USA 91, 11422-11426] and several ligands were found within this library to bind a monoclonal antibody elicited against beta-endorphin. The non-peptide molecules synthesized were arylsulfonamides, a class of compounds of known clinical bactericidal efficacy. The results indicate that the reaction scope of LPCS should be general, and its value to multiple, high-throughput screening assays could be of particular merit, since multimilligram quantities of each library member can readily be attained.

Solid-phase combinatorial chemistry and scintillation proximity assays

The Scintillation Proximity Assay (SPA) is a method that is frequently used to detect and quantify the strength of intermolecular interactions between a biological receptor and ligand molecule in aqueous media. This thesis describes the synthesis of scintillant-tagged-compounds for application in a novel cell-based SPA. A series of 4-functianlised-2,5-diphenyloxazole molecules were synthesised. These 4-functionalised-2,5-diphenyloxazoles were evaluated by Sense Proteomic Ltd. Accordingly, the molecules were evaluated for the ability to scintillate in the presence of ionising radiation. In addition, the molecules were incorporated into liposomal preparations which were subsequently evaluated for the ability to scintillate in the presence of ionising radiation. The optimal liposomal preparation was introduced into the membrane of HeLa cells that were used successfully in a cell-based SPA to detect and quantify the uptake of [14C]methionine. This thesis also describes the synthesis and subsequent polymerisation of novel poly(oxyethylene glycol)-based monomers to form a series of new polymer supports. These Poly(oxyethylene glycol)-polymer (POP) supports were evaluated for the ability to swell and mass-uptake in a variety of solvents, demonstrating that POP-supports exhibit enhanced solvent compatibilities over several commercial resins. The utility of POP-supports in solid-phase synthesis was also demonstrated successfully. The incorporation of (4’-vinyl)-4-benzyl-2,5-diphenyloxazole in varying mole percentage into the monomer composition resulted in the production of chemically functionalised scintillant-containing poly(oxyethylene glycol) polymer (POP-Sc) supports. These materials are compatible with both aqueous and organic solvents and scintillate efficiently in the presence of ionising radiation. The utility of POP-Sc supports in solid-phase synthesis and subsequent in-situ SPA to detect and quantify, in real-time, the kinetic progress of a solid-phase reaction was exemplified successfully.In addition, POP-Sc supports were used successfully both in solid-phase combinatorial synthesis of a peptide nucleic acid (PNA)-library and subsequent screening of this library for the ability to hybridise with DNA, which was labelled with a suitable radio-isotape. This data was used to identify the dependence of the number and position of complimentary codon pairs upon the extent of hybridisation. Finally, a further SPA was used to demonstrate the excellent compatibility of POP-Sc supports for use in the detection and quantification of enzyme assays conducted within the matrix of the POP-Sc support.

Sobre o uso de métodos quimiométricos em química combinatória

Combinatorial chemistry has emerged as a tool to circumvent a major problem of pharmaceutical industries to discover new lead compounds. A rapid and massive evaluation of a myriad of newly synthesised compounds can be carried out. Combinatorial synthesis leads to high throughput screening en masse towards another myriad of biological targets. The design of a set of compounds based upon combinatorial chemistry may be envisaged by using of QSPR-SIMCA and QSAR-SIMCA as tools for classification purposes. This work deals with the definition and establishment of a spanned substituent space (SSS) that reduces the analogue numbers with no exclusion of global content. The chemical diversity may be set properly within a specified pharmacological field. This allows a better use of its potentiality without loosing information.

Aplicações da química combinatória no desenvolvimento de fármacos

Combinatorial Chemistry has become a very efficient methodology in drug research. Recent progress in combinatorial synthesis performed both in solid and solution phase have led to a change in the paradigm for the identification and optimization of lead compounds. This article gives an overview of the principal characteristics of combinatorial libraries and some examples of the application of this methodology in the identification of test compounds and lead compound optimization, either from synthetic or natural sources.

Méthodologie pour la synthèse combinatoire d’azapeptides: application à la synthèse d’analogues aza-GHRP-6 en tant que ligands du récepteur CD36

Les azapeptides sont des mimes peptidiques où le carbone alpha d’un ou de plusieurs acides aminés est remplacé par un atome d’azote. Cette modification tend à stabiliser une conformation en repliement beta en raison de la répulsion électronique entre les paires d’électrons libres des atomes d’azote adjacents et de la géométrie plane de l’urée. De plus, le résidu semicarbazide a une meilleure résistance face aux protéases en plus d’être chimiquement plus stable qu’une liaison amide. Bien que les propriétés des azapeptides en fassent des mimes peptidiques intéressants, leurs méthodes de synthèses font appel à la synthèse laborieuse d’hydrazines substituées en solution. Le peptide sécréteur d’hormone de croissance 6 (GHRP-6, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) est un hexapeptide synthétique qui possède une affinité pour deux récepteurs distincts: les récepteurs GHS-R1a et CD36. Les travaux effectués au cours de mon doctorat qui seront détaillés dans cet ouvrage visent à atteindre deux objectifs: (1) le développement d’analogues du peptide GHRP-6 sélectif à un seul récepteur et (2) la mise au point d’une nouvelle méthodologie pour la synthèse combinatoire d’azapeptides. En réponse au premier objectif, la synthèse parallèle de 49 analogues aza-GHRP-6 a été effectuée et certains candidats sélectifs au récepteur CD36 ont été identifiés. L’étude de leurs propriétés anti-angiogéniques, effectuée par nos collaborateurs, a également permis d’identifier des candidats intéressants pour le traitement potentiel de la dégénérescence maculaire liée à l’âge. Une nouvelle approche pour la synthèse combinatoire d’azapeptides, faisant appel à l’alkylation et la déprotection chimiosélective d’une sous-unité semicarbazone ancrée sur support solide, a ensuite été développée. La portée de cette méthodologie a été augmentée par la découverte de conditions permettant l’arylation régiosélective de cette sous-unité semicarbazone, donnant accès à treize nouveaux dérivés aza-GHRP-6 possédant des résidus aza-arylglycines aux positions D-Trp2 et Trp4. L’élaboration de conditions propices à l’alkylation et la déprotection chimiosélective de la semicarbazone a donné accès à une variété de chaînes latérales sur l’acide aminé « aza » préalablement inaccessibles. Nous avons, entre autres, démontré qu’une chaîne latérale propargyl pouvait être incorporée sur l’acide aminé « aza ». Tenant compte de la réactivité des alcynes, nous avons ensuite élaboré des conditions réactionnelles permettant la formation in situ d’azotures aromatiques, suivie d’une réaction de cycloaddition 1,3-dipolaire sur support solide, dans le but d’obtenir des mimes de tryptophane. Sept analogues du GHRP-6 ont été synthétisés et testés pour affinité au récepteur CD36 par nos collaborateurs. De plus, nous avons effectué une réaction de couplage en solution entre un dipeptide possédant un résidu aza-propargylglycine, du paraformaldehyde et une variété d’amines secondaires (couplage A3) afin d’accéder à des mimes rigides d’aza-lysine. Ces sous-unités ont ensuite été incorporées sur support solide afin de générer sept nouveaux azapeptides avec des dérivés aza-lysine à la position Trp4 du GHRP-6. Enfin, une réaction de cyclisation 5-exo-dig a été développée pour la synthèse de N-amino imidazolin-2-ones en tant que nouveaux mimes peptidiques. Leur fonctionnalisation par une série de groupements benzyliques à la position 4 de l’hétérocycle a été rendue possible grâce à un couplage Sonogashira précédant la réaction de cyclisation. Les propriétés conformationnelles de cette nouvelle famille de composés ont été étudiées par cristallographie aux rayons X et spectroscopie RMN d’un tétrapeptide modèle. L’activité biologique de deux mimes peptidiques, possédant un résidu N-amino-4-méthyl- et 4-benzyl-imidazolin-2-one à la position Trp4 du GHRP-6, a aussi été examinée. L’ensemble de ces travaux devrait contribuer à l’avancement des connaissances au niveau des facteurs structurels et conformationnels requis pour le développement d’azapeptides en tant que ligands du récepteur CD36. De plus, les résultats obtenus devraient encourager davantage l’utilisation d’azapeptides comme peptidomimétiques grâce à leur nouvelle facilité de synthèse et la diversité grandissante au niveau de la chaîne latérale des acides aminés « aza ».

Difficult macrocyclizations: New strategies for synthesizing highly strained cyclic tetrapeptides

Cyclic tetrapeptides are an intriguing class of natural products. To synthesize highly strained cyclic tetrapeptides; we developed a macrocyclization strategy that involves the inclusion of 2-hydroxy-6-nitrobenzyl (HnB) group at the N-terminus and in the middle of the sequence. The N-terminal auxiliary performs a ring closure/ring contraction role, and the backbone auxiliary promotes cis amide bonds to facilitate the otherwise difficult ring contraction. Following this route, the all-L cyclic tetrapeptide cyclo-[Tyr-Arg-Phe-Ala] was successfully prepared.

A safety catch linker for Fmoc-based assembly of constrained cyclic peptides

A new safety-catch linker for Fmoc solid-phase peptide synthesis of cyclic peptides is reported. The linear precursors were assembled on a tert-butyl protected catechol derivative using optimized conditions for Fmoc-removal. After activation of the linker using TFA, neutralization of the N-terminal amine induced cyclization with concomitant cleavage from the resin yielding the cyclic peptides in DMF solution. Several constrained cyclic peptides were synthesized in excellent yields and purities. Copyright (c) 2005 European Peptide Society and John Wiley & Sons, Ltd.

A versatile synthetic approach to peptidyl privileged structures using a "safety-catch" linker

Peptidyl privileged structures have been widely used by many groups to discover biologically active molecules. In this context, privileged substructures are used as hydrophobic anchors, to which peptide functionality is appended to gain specificity. Utilization of this concept has led to the discovery of many different active compounds at a wide range of biological receptors. A synthetic approach to these compounds has been developed on a safety-catch linker that allows rapid preparation of large libraries of these molecules. Importantly, amide bond formation/cleavage through treatment with amines is the final step; it is a linker strategy that allows significant diversification to be easily incorporated, and it only requires the inclusion of an amide bond. In addition, chemistry has been developed that permits the urea moiety to be inserted at the N-terminus of the peptide, allowing the same set of amines (either privileged substructures or amino acid analogues) to be used at both the N- and C-termini of the molecule. To show the robustness of this approach, a small library of peptidyl privileged structures were synthesized, illustrating that large combinatorial libraries can be synthesized using these technologies.

Carbohydrate-based scaffolds in drug discovery

Carbohydrates have been proven as valuable scaffolds to display pharmocophores and the resulting molecules have demonstrated useful biological activity towards various targets including the somatostatin receptors (SSTR), integrins, HIV-1 protease, matrix metalloproteinases (MMP), multidrug resistance-associated protein (MRP), and as RNA binders. Carbohydrate-based compounds have also shown antibacterial and herbicidal activity.