Hypervalent Iodine-Mediated Late-Stage Peptide and Protein Functionalization

Hypervalent iodine compounds are powerful reagents for the development of novel transformations. As they exhibit low toxicity, high functional group tolerance, and stability in biocompatible media, they have been used for the functionalization of biomolecules. Herein, we report recent advances up to June 2021 in peptide and protein modification using hypervalent iodine reagents. Their use as group transfer or oxidizing reagents is discussed in this Minireview, including methods targeting polar, aromatic, or aliphatic amino acids and peptide termini.

Development of New Bicyclic Peptide Formats and their Application in Directed Evolution

Shiyu Chen

Cyclic peptides have a range of properties that make them attractive as therapeutics such as high binding affinity, good target selectivity and low toxicity. While most peptides in the clinic are derived from nature, various powerful in vitro evolution techniques allow now the de novo development of cyclic peptide ligands to targets of interest. Our group is specialized in the development of bicyclic peptide ligands by phage display. A current limitation of phage-selected bicyclic peptides is their conformational flexibility limiting the binding affinity due to entropic effects or even preventing the generation of binders to challenging targets. The main goal of my thesis was the development of new bicyclic peptide formats that are conformationally more rigid or even have a stable fold in solution. The proposed bicyclic peptide formats are based on a rigid chemical structure containing multiple polar groups and peptides that are anchored to the structure and fold tightly around by forming non-covalent interactions. I also envisioned to cyclise peptide phage display libraries with several such chemical structures in parallel to generate and screen structurally highly diverse bicyclic peptide libraries. In a first project, I designed and synthesized three new compounds that have i) three thiol-reactive groups to react with peptides and ii) several polar groups. Two of these compounds efficiently and selectively cyclised linear peptides containing three cysteine residues. Functional and structural analysis of the bicyclic peptides showed that the relatively small compounds at the centre of bicyclic peptides significantly influence the activity and conformation of the peptides. These results demonstrated the prominent structural role of chemical linkers in peptide macrocycles. In a second project, I applied the compounds developed in the first project in phage selections. The sequences of isolted bicyclic peptide ligands were strongly influenced by the small chemical structures, suggesting that the peptides fold closely around them. X-ray structure analysis revealed that one of the chemical structures indeed nucleated peptides by forming H-bond interactions. This result showed that small rigid chemicals can serve as nucleating scaffolds and directs the way towards the generation of peptide ligands being folded in solution. A third project is based on an unexpected observation in the second project, namely that panning peptide phage libraries without prior alkylation with compounds yielded mostly peptides with four cysteines. The peptides bound with good affinity and characterization revealed that they contain two disulphide bridges and thus formed bicyclic peptide structures. An interesting feature of disulfide-based bicyclic peptide libraries was the high topological diversity: oxidation of peptide libraries of the form Xm CXn CXo CXp yielded 3 × (m+n+o+p) different bicyclic peptide topologies because the fourth cysteine could appear in any of the (m+n+o+p) randomized amino acid positions (X) and the four peptides in such peptides could pair in three different ways. X-ray structure analysis revealed an important structural role of the disulfide bridges. The new method offers an easy and robust way to generate bicyclic peptide ligands.

EPFL2013

Development of Stapling Methods by Selective Side Chain Modifications of Bioactive Peptides

Christopher Matthew Bandeli Kourra

Cyclic peptide therapeutics fill the gap between small molecules (5000 Da) as a separate class of drugs, combining the advantages of both in terms of high selectivity, bioavailability, synthetic accessibility and low toxicity. However, their conformational flexibility and proteolytic instability results in fast metabolism. Therefore, modification of their chemical and structural properties at the proteolytically vulnerable sites can circumvent these deficiencies. Several strategies exist to stabilise peptides through local or global constraints, side chain functionalisation or amide bond surrogates. This thesis describes the development and application of two chemical transformations to peptides with the aim of greatly enhancing their metabolic stability either via side chain to side chain cyclisation or disulfide modification. Following the functionalisation, a comparative evaluation of the pharmacological properties of the modified peptides was conducted with respect to the parent peptides. The first cyclisation strategy aimed to apply the redox-neutral chemistry developed in the burgeoning field of borrowing hydrogen technology to peptides, as a new class of tolerable substrates. These would contain the requisite amine and alcohol functional groups enabling their direct coupling to form the lactam or N-alkylated cycle. Numerous investigations into this transformation with a plethora of substrates and catalysts did not lead to the desired cyclisation. For the second approach, a simple one-pot procedure for the 'stapling' of disulfide bonds in native peptides was developed. Tris(2-carboxyethyl)phosphine mediated reduction of the disulfide bond was followed by concomitant insertion of a 'doubly electrophilic' carbon bridge to form a physiologically stable dithioacetal bond. The reaction was performed under mild, biocompatible reaction conditions, allowing for the facile conversion of native peptides into more stable analogues. The protocol was applicable to a range of bioactive peptides with a multitude of reactive functional groups demonstrating the high versatility of this approach. The importance and utility of the modified analogues were verified by testing their binding affinity and biological activity against the corresponding parent peptides. The impact of the disulfide modification on these properties for each peptide were analysed on a case-by-case basis. The SCS modified analogues of Oxytocin and Vasopressin maintained binding affinities in the same order of magnitude as their respective parent compounds across all corresponding receptors. Nevertheless, this was not the case for SCS-Octreotide and SCS-Somatostatin. Those peptides which maintained comparable binding affinities were then tested for their functional activity at the receptors of biological interest. SCS-Oxytocin retained its agonist potency displaying sub-nanomolar activity at the Oxytocin receptor, whilst SCS-Vasopressin exhibited sub micromolar functional response at the Vasopressin 1A receptor. Then the stability of the modified peptides in human serum was evaluated. In some cases, the enhancement in serum stability was vastly increased. In particular our modified SCS-Oxytocin analogue clearly demonstrated a significant improvement in serum half-life, as well as heat and pH stability over its native parent form. This disulfide stapling method could have wide-reaching application for the development of more stable therapeutic analogues.

EPFL2016

Application of Hypervalent Iodine Reagents for Novel Cysteine Labeling

Romain Gaëtan Tessier

Selective functionalization of peptides and proteins has always been a valuable tool for the study and manipulation of biological processes. In this context, cysteines are of particular interest. Their intrinsic high nucleophilicity makes these amino acids excellent targets for peptides and proteins modification. As a result, cysteine labeling techniques have been extensively investigated. Nevertheless, while bioconjugation requires high efficiency, selectivity and kinetic under mild and aqueous conditions, most of the reported methodologies exhibit significant limitations. For instance, lack of chemoselectivity, as well as low or uncontrolled reactivity, are frequently reported. Furthermore, many of these methods rely on reagents challenging to prepare, store and use. In this regard, hypervalent iodine reagents are attractive. Heterocyclic Î»3-iodanes demonstrated high reactivity and selectivity, together with great stability and low toxicity. Nevertheless, applications to peptides and proteins modification remain rare. Employing ethynylbenziodoxolone reagents (EBXs), we previously described an efficient and chemoselective alkynylation of organosulfur compounds. The process was performed under âopen-flaskâ conditions, at room temperature, without any metal- or additive-assistance. In collaboration with Dr. Adibekian and co-workers, we then employed this methodology to intracellular proteomic profiling of cysteine residues. Nevertheless, only the most reactive cysteines were efficiently labeled. Accordingly, the goal of my thesis was to investigate the reactivity between any cysteine residues and EBX reagents, under mild and aqueous conditions. We started our investigations with treatment of glutathione, a naturally occurring tripeptide, with an azide-containing EBX reagent. Although a complete conversion of the starting material was observed, the corresponding thioalkyne product was not formed. Instead, we observed thiol-yne reactivity and selective thiol trans-addition in Î²-position of the EBX reagent, leading to vinylbenziodoxolone product (VBX). This efficient cysteine labeling exhibited high selectivity, fast kinetic and great robustness. Various cysteine-containing peptides and proteins, along with diverse EBX reagents, were well tolerated. When an azide-containing EBX reagent was employed, strain-promoted azide-alkyne cycloadditions (SPAAC) were successfully achieved. Alternatively, the hypervalent iodine function was engaged in aqueous Suzuki-Miyaura cross-couplings. Various boronic acids, containing electron-rich and âdeficient aryls, heteroaryls and vinyls, were successfully subjected to this palladium-promoted process. Remarkably, both SPAAC and cross-coupling were orthogonal to each other. We then investigated the singular reactivity of the TMS-EBX reagent. Under basic aqueous conditions, we observed fast desilylation of the hypervalent iodine reagent. Upon glutathione treatment, the resulting EBX substrate was engaged in a thiol-yne reaction. Nevertheless, the corresponding VBX product exhibited a low stability and led to alkynylated glutathione. Further optimization furnished an efficient, selective and rapid ethynylation of numerous cysteine-containing peptides.

EPFL2019