Ionic Liquid-Mediated Approach for the Synthesis of Site-Specific Thioether Conjugates.

Site-specific conjugation approaches are of great importance in drug discovery, notably for the synthesis of biochemical probes or molecular conjugates for targeted delivery. Herein, we report a mild ionic liquid (IL)-mediated thiolation technique that relies on the use of 1,3-ethyl-methyl imidazolium acetate, [C2 mim][OAc] as a solvent and precursor to generate activated IL, as well as a solvent for the conjugation reaction. First, a focused library of active ILs was prepared for functionalizing/conjugating cysteine-containing small molecules and unprotected peptides. Interestingly, a bifunctional active IL could also be successfully employed as a linker for the conjugation of peptides lacking Cys. This study sets the ground for further investigation of the use of active ILs for modifying, labeling or conjugating larger and more complex therapeutic modalities such as proteins and antibodies.

Late-Stage Functionalization of Histidine in Unprotected Peptides.

The late-stage functionalization (LSF) of peptides represents a valuable strategy for the design of potent peptide pharmaceuticals by enabling rapid exploration of chemical diversity and offering novel opportunities for peptide conjugation. While the C(sp2 )-H activation of tryptophan (Trp) is well documented, the resurgence of radical chemistry is opening new avenues for the C-H functionalization of other aromatic side-chains. Herein, we report the first example of LSF at C2 of histidine (His) utilizing a broad scope of aliphatic sulfinate salts as radical precursors. In this work, the exquisite selectivity for histidine functionalization was demonstrated through the alkylation of complex unprotected peptides in aqueous media. Finally, this methodology was extended for the installation of a ketone handle, providing an unprecedented anchor for selective oxime/hydrazone conjugation at histidine.

Stapled Peptides by Late-Stage C(sp3 )-H Activation.

Despite the importance of stapled peptides for drug discovery, only few practical processes to prepare cross-linked peptides have been described; thus the structural diversity of available staple motifs is currently limited. At the same time, C-H activation has emerged as an efficient approach to functionalize complex molecules. Although there are many reports on the C-H functionalization of amino acids, examples of post-synthetic peptide C-H modification are rare and comprise almost only C(sp2 )-H activation. Herein, we report the development of a palladium-catalyzed late-stage C(sp3 )-H activation method for peptide stapling, affording an unprecedented hydrocarbon cross-link. This method was first employed to prepare a library of stapled peptides in solution. The compatibility with various amino acids as well as the influence of the size (i,i+3 and i,i+4) and length of the staple were investigated. Finally, a simple solid-phase procedure was also established.

Synthesis and biological evaluation of analogues of the potent ADAM8 inhibitor cyclo(RLsKDK) for the treatment of inflammatory diseases and cancer metastasis.

The metalloproteinase ADAM8 serves as a pivotal catalyst in the development of inflammatory diseases and cancer metastasis. The cyclic peptide cyclo(RLsKDK) has been shown to inhibit the enzymatic activity of ADAM8 with high specificity and potency. Herein we report a structure-activity relationship (SAR) study of cyclo(RLsKDK) that involves the synthesis and biological evaluation of the lead compound and structural analogues thereof. This study provides insight into the ligand-receptor interactions that govern the binding of cyclo(RLsKDK) to the ADAM8 disintegrin domain and represents a stepping stone for the development of new treatments for inflammatory diseases and cancer metastasis.

Synthesis and biological evaluation of tyrosine modified analogues of the α4ß7 integrin inhibitor biotin-R8ERY.

The α4ß7 integrin is a well-known target for the development of drugs against various inflammatory disease states including inflammatory bowel disease, type 1 diabetes and multiple sclerosis. The synthesis of a small library of cell-permeable ß7 integrin inhibitors based on the peptide biotin-R(8)ERY is reported, in which the tyrosine residue has been modified by using the Suzuki-Miyaura cross-coupling reaction. The synthesised peptidomimetics were evaluated in a cell adhesion assay and shown to inhibit Mn(2+)-activated adhesion of mouse TK-1 T cells to mouse MAdCAM-1. All of the synthesised peptidomimetics are more active than our previously reported lead compound biotin-R(8)ERY with two of the analogues, 6 and 7, exhibiting IC(50) values of <15 µM.

Oxime/Hydrazone Conjugation at Histidine: Late-Stage Functionalization Approach of Unprotected Peptides.

Synthetic molecular probes have recently been in focus for their potential use in target deconvolution, target engagement studies, and imaging. With the field expanding, new strategies to develop such tools are in high demand. While traditional conjugation techniques relying on inherently nucleophilic amino acids such as cysteine (Cys) and lysine (Lys) or pre-incorporated non-natural amino acids are still heavily used, novel methodologies for the direct and site-selective modification of peptides are attracting increasing attention. Of particular interest are Late-Stage Functionalization (LSF) approaches based on radical chemistry as they afford mild and biocompatible alternatives to transition-metal catalysis. A recent synthetic method, which leverages the unique reactivity of histidine (His), has proven to be a promising new strategy for LSF and site-selective conjugation of unprotected peptides. In this chapter, detailed step-by-step protocols depicting the C2-alkylation of His-containing peptides, the unveiling of a ketone as handle for hydrazone conjugation, and its use to site-selectively introduce a fluorophore at this residue are discussed. In addition to its application toward the synthesis of molecular probes, this methodology can be employed in peptide-based drug discovery programs, offering the possibility to rapidly explore the chemical space surrounding peptide hits. Finally, this strategy is also amenable to the preparation of novel peptide-ASO/small molecule drug conjugates.

NOXA upregulation by the prohibitin-binding compound fluorizoline is transcriptionally regulated by integrated stress response-induced ATF3 and ATF4.

Fluorizoline is a new synthetic molecule that induces p53-independent apoptosis, in several tumor cell lines and in primary leukemia cells, by selectively targeting prohibitins (PHBs). In this study, we describe how fluorizoline induces BCL-2 homology 3-only protein NOXA, without modulating the protein levels of anti-apoptotic B-cell lymphoma-2 (BCL-2) family members prior to caspase activation, as well as how it synergizes with the BCL-2 and BCL-XL inhibitor ABT-737 to induce apoptosis. Interestingly, fluorizolinetreatment triggers the activation of the integrated stress response (ISR) in HeLa and HAP1 cells, with increased eukaryotic translation initiation factor 2α phosphorylation, and induction of ATF3, ATF4, and CHOP. Moreover, PHB downregulation induces similar ISR activation and apoptosis as with fluorizoline treatment. In addition, we studied the essential role of the pro-apoptotic protein NOXA in fluorizoline-induced apoptosis and we describe its mechanism of induction in HeLa and HAP1 cells. Moreover, we identified ATF3 and ATF4 as the transcription factors that bind to NOXA promoter upon fluorizoline treatment. Furthermore, using ATF3 and ATF4 CRISPR HeLa and HAP1 cells, we confirmed that both factors mediate the induction of NOXA and apoptosis by fluorizoline. In conclusion, fluorizoline treatment triggers the activation of the ISR that results in the induction of ATF3 and ATF4, important regulators of NOXA transcription in fluorizoline-induced apoptosis.

Novel preparation of chiral α-amino acids using the Mitsunobu-Tsunoda reaction.

An efficient synthesis of racemic or optically active α-amino acids by modified-Mitsunobu alkylation of a racemic or chiral glycine template from alcohols was developed. Libraries of amino acids were prepared in moderate to good yield with good to high enantioselectivity. This simple method widens the scope for preparation of structurally diverse amino acids.