Fluorescent Isoindole Crosslink (FlICk) Chemistry: A Rapid, User‐friendly Stapling Reaction

Abstract

The stabilization of peptide secondary structure via stapling is a ubiquitous goal for creating new probes, imaging agents, and drugs. Inspired by indole‐derived crosslinks found in natural peptide toxins, we employed ortho‐phthalaldehydes to create isoindole staples, thus transforming inactive linear and monocyclic precursors into bioactive monocyclic and bicyclic products. Mild, metal‐free conditions give an array of macrocyclic α‐melanocyte‐stimulating hormone (α‐MSH) derivatives, of which several isoindole‐stapled α‐MSH analogues (Ki≈1 nm) are found to be as potent as α‐MSH. Analogously, late‐stage intra‐annular isoindole stapling furnished a bicyclic peptide mimic of α‐amanitin that is cytotoxic to CHO cells (IC50=70 μm). Given its user‐friendliness, we have termed this approach FlICk (fluorescent isoindole crosslink) chemistry.

Cooperative Stapling of Native Peptides at Lysine and Tyrosine or Arginine with Formaldehyde

“…Inspired by the widespread involvement of lysine (Lys, K) residues in posttranslational modifications of proteins, we envisioned that the intramolecular crosslinking of Lys with a suitable neighboring nucleophilic residue using an aldehyde "glue" molecule could offer a new method for stapling native peptides. [9,10] Formaldehyde (HCHO) has long been used as a fixation reagent for investigating proteinprotein and protein-DNA reactions. [11] Bioanalytical studies using mass spectrometry (MS) and NMR spectroscopy have shown that nucleophilic amino acid residues such as Lys, Cys, and arginine (Arg) can be crosslinked by HCHO via various methylene linkages.…”

Fluorescent cyclic peptides for cell imaging

“…An illustrative example was shown by Perrin et al ., who prepared bicyclic fluorescent peptides via the condensation of an ortho‐phthalaldehyde unit with the amine and thiol reactive groups of a cyclic peptide precursor to yield a fluorescent isoindole staple. [ 62 ] Other examples include the coordination of an iridium complex to the imidazole groups of histidine side chains of an RGD‐containing peptide for simultaneous cyclisation and luminescence formation [ 63 ] and a double “click” cyclisation reaction from two biscyclooctynylated units of a linear precursor to yield cyclopeptide adducts bearing a bisazide fluorogenic dye. [ 64 ] Despite the simplicity of inherent fluorescent cyclic peptides, they often show suboptimal characteristics (e.g., wavelengths, quantum yields) for widespread application in molecular imaging.…”

Designing stapled peptides to inhibit protein‐protein interactions: An analysis of successes in a rapidly changing field

“…[151] Todorovic et al have developed a one-pot stapling strategy which couples a cysteine and an amine-terminated amino acid in the presence of ortho-phthalaldehydes forming an isoindole-bridged peptide which is inherently fluorescent ( Figure 9A). [20] Functionalisation is not limited to fluorescence, and Wu et al,…”

“…In parallel, the chemistry landscape for stapled peptides is continuously diversifying with a dozen "novel" linker chemistries reported in publications in 2019. [17][18][19][20][21][22][23] In some instances, only very subtle differences in the staple chemistry, or stereochemistry, trigger a significant change in the binding affinity, the pharmaceutical profile of the peptide, or give added functionality. [21,[24][25][26][27][28][29] Despite multiple publications and reviews describing reliable methods for the synthesis of stapled peptides [30][31][32][33][34][35] 35 ] and the commercialisation of the most common unnatural amino acids, their synthesis remains costly and until a few years ago, nonautomated.…”