Javier Ruiz-Rodriguez

Postsynthetic modification of peptides: chemoselective C-arylation of tryptophan residues.

In recent years the model on which the pharmaceutical industry is based has undergone dramatic changes. Every year, more biological entities are being accepted by the FDA and other regulatory agencies. Furthermore, the most recent biological drugs contain non-natural building blocks, as it has been shown in peptides, but also in antibodies and proteins. [1] Thus, the development of new methodologies for the selective and straightforward chemical modification of biomolecules is a scientific challenge that has tremendous implications in drug discovery, as well as in chemical biology and proteomics. The chemical methods to perform these modifications in native peptides or proteins are normally restricted to a reduced set of reactions which nearly always involve the nucleophilic side chains of amino acids such as Lys, Cys, Asp or Glu. [2]

Amide-to-ester substitution allows fine-tuning of the cyclopeptide conformational ensemble.

Natural or designed peptide ligands rarely bind to cognate receptors in their most stable conformation when in solution. The receptor, through reciprocal induced fitting, applies pressure on the conformational ensemble of the peptide to select its complementary conformation. Given the flexible nature of the peptides’ modular architecture, a bioactive sequence must often be primed for recognition of its target to achieve high binding affinity or specificity. For rational peptide design, cyclization or structure-inducing residues have been successfully used to accomplish ligand preorganization. However, deconvolution of the averaged NMR spectra of strained peptides or other macrocycles had shown that the bound-state conformations are poorly populated in the free uncomplexed state. In small cyclic peptides, the stereochemistry of the backbone, rather than interactions with or among side chains, determines the conformational ensemble by establishing a defined pattern of local torsional preferences. Intramolecular H bonds act on the equilibrium distribution of the conformational ensemble and favor specific conformations. Thus, we envisioned amide-to-ester substitution or “Ester Scan” as an interesting modification for the peptide backbone, which influences the conformational ensemble as well as its equilibrium distribution; this influence is achieved through modulation of the backbone torsional preferences and H-bonding pattern, respectively. As a model we choose cilengitide (CIL), which is an ArgGly-Asp (RGD) peptide of sequence cyclo[RGDfNMeV] (see Scheme 1), with well-characterized biological and conformational properties. CIL displays nanomolar inhibition of vitronectin binding to the isolated avb3 and avb5 integrin receptors, and it blocks integrin-dependent adhesion of tumor and endothelial cells to immobilized extracellular matrix (ECM) proteins and reduces angiogenesis and tumor growth in vivo. Modulation of the internal H-bond pattern of nonmethylated CIL precursors—achieved by changing the flexibility or the chirality of the backbone—was found to influence the antagonist activity on the vitronectin (VN) and fibrinogen (FB) receptors, and has been proposed to control laminin P1 vs. vitronectin receptor specificity. We synthesized all five depsi-analogues of the depsipeptide CIL (D1–D5) by stepwise assembling of the linear precursors on 2-CTC resin (Fmoc/tBu strategy) and cyclization in solution. For the introduction of the a-hydroxy acid residues onto the growing peptide chain, their HFA-activated/protected derivatives were used (Scheme 1). For the acylation of the free hydroxy group, DIPDCI/DMAP-activation was used. For conventional peptide cyclization, the optimal site for macrocycle formation is between the Gly (acting as the C terminus) and Asp (acting as the N terminus) residues because Gly cannot epimerize. This strategy was used for the synthesis of parent CIL, D1, and D2 depsipeptides with PyBOP/HOAt. The macrolactonization required for the preparation of D3 was successful with MSNT activation and NMI as the base. For the depsipeptides D4 and D5, certain particularities of the ester bond had to be taken into account. Our attempts to synthesize the linear precursor of D4 (OGly analogue) starting from OGly as the C terminus, resulted in low yields, and was likely because of cleavage of the ester bond that was mediated by base during repeated treatment with piperidine. Therefore, NMeVal was chosen as the C terminus. For the preparation of D5, the cyclization was performed at the (apparently) less attractive position between (d)Phe and NMeVal. Macrolactamization at less hindered sites was impeded by intramolecular nucleophilic attack on the ester bond, which occurred during peptide chain elongation, and eliminated the (D)Phe–NMeVal couple as dioxopiperazine. Given the increased steric demand of the N-terminal NMeVal, we chose the more reactive PyAOP as the coupling reagent and HOAt as the additive. After cleavage from the solid support, all linear peptide and depsipeptide precursors were obtained in over 85% yield. Head-to-tail cyclization was performed in solution and gave good yields in all cases and, finally, the protecting groups on the side chains were removed using [Pd(PPh3)4]/phenylsilane [*] T. Cupido, Dr. J. Spengler, Dr. J. Ruiz-Rodriguez, Prof. F. Albericio Institute for Research in Biomedicine (IRB), Barcelona Science Park (PCB) and CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine PCB Baldiri Reixac 10, 08028 Barcelona (Spain) Fax: (+34)93-403-7126 E-mail: tommaso.cupido@irbbarcelona.org albericio@irbbarcelona.org

Polythiazole linkers as functional rigid connectors: a new RGD cyclopeptide with enhanced integrin selectivity

Polythiazole amino acids clasp linear peptides to generate cyclic derivatives, however, the resulting species are not merely stapled peptides but bear a complex heterocyclic moiety displaying its intrinsic set of interactions. As a proof of concept, a bisthiazole moiety has been grafted onto an RGD sequence to deliver a new cilengitide analogue with improved integrin selectivity and remarkable in vivo antiangiogenic activity.

Siamese Depsipeptides: Constrained Bicyclic Architectures**

has been designed. Cyclic depsipeptide dimers connected by a CC bond were constructed. Two kind of possible connections can be envisaged, those that share a side chain bond or those that share a backbone bond. Owing to the resulting proximity of both structurally identical cycles, they can be called Siamese depsipeptides. For Siamese depsipeptides, the hydroxy acid of the natural compound was replaced with tartaric acid and the second cycle was constructed on its additional OH and CO2H function. Both modes of connection prevent steric interaction between the two cycles, and H-bond interactions are expected to be minimal because ester bonds are not H-bond donors and are weak H-bond acceptors. [5] The two Siamese depsipeptides proposed are shown in Scheme 1. The cyclic depsipeptide sansalvamide A (SA), which is produced by a marine fungus and shows cancer cell cytotoxicity, was selected as a model. SA is composed of four amino acids (2 � Leu, Phe, Val) and one hydroxy acid (leucic acid). [6] More than 80 peptidic analogues, wherein the hydroxy acid and the amino acid constituents were substituted for d-amino acids and N-methyl amino acids with preserved or altered side chains, are already reported. Some of them exhibit greater activity and better selectivity than the natural SA. [7] However, although SA is a relatively small and structurally simple depsipeptide, an active sequence has not been disclosed to date.

A Novel Protecting/Activating Strategy for β-Hydroxy Acids and Its Use in Convergent Peptide Synthesis

beta-Hydroxy acids were reacted with hexafluoroacetone and carbodiimides to give carboxy-activated six-membered lactones in good yields. On reaction with amines, the corresponding amides were obtained. We demonstrate the following applications of this protecting/activating strategy: preparation of carboxamides in solution and on solid phase (both normal and reverse mode); recovery and reuse of the excess material in solid-phase synthesis; and convergent solid-phase peptide synthesis (CSPPS) with peptide segments bearing C-terminal Ser or Thr with very low levels of epimerization (<1%, HPLC).

Total regioselective control of tartaric acid.

An efficient strategy to synthesize tartaric acid building blocks for totally regioselective transformations or derivatizations was disclosed. Starting from l-tartaric acid or l-dimethyl tartrate, respectively, we obtained type I and II building blocks with orthogonal sets of protecing groups (4-8 steps, 38-56% overall yield).