5-Hydroxypyrroloindoline Affords Tryptathionine and 2,2'-bis-Indole Peptide Staples: Application to Melanotan-II.

With peptides increasingly favored as drugs, natural product motifs, namely the tryptathionine staple, found in amatoxins and phallotoxins, and the 2,2'-bis-indole found in staurosporine represent unexplored staples for unnatural peptide macrocycles. We disclose the efficient condensation of a 5-hydroxypyrroloindoline with either a cysteine-thiol or a tryptophan-indole to form a tryptathionine or 2-2'-bis-indole staple. Judicious use of protecting groups provides for chemoselective stapling using α-MSH, which provides a basis for investigating both chemoselectivity and affinity. Both classes of stapled peptides show nanomolar Ki's, with one showing a sub-nanomolar Ki value.

Toward tryptathionine-stapled one-bead-one-compound (OBOC) libraries: solid phase synthesis of a bioactive octretoate analog.

New somatostatin analogs are highly desirable for diagnosing and treating neuroendocrine tumors (NETs). Here we describe the solid-phase synthesis of a new octreotate (TATE) analog where the disulfide bond is replaced with a tryptathionine (Ttn) staple as part of an effort to prototyping a one-bead-one-compound (OBOC) library of Ttn-stapled peptides. Library design provides the potential for on- and off-bead screening. To validate our method, we labelled Ttn-TATE with a fluorescent dye to demonstrate binding to soluble somatostatin receptor subtype-2 and staining of Ar42J rat prostate cancer cells. By exploring this staple in the context of a ligand of known affinity, this method paves the way for an OBOC library construction of bioactive octreotate analogs and, more broadly speaking, tryptathionine-staped peptide macrocycles.

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

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.

Synthesis of the Death-Cap Mushroom Toxin α-Amanitin.

α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, Amanita phalloides. As a potent inhibitor of RNA polymerase II, α-amanitin is toxic to eukaryotic cells. Recent interest in α-amanitin arises from its promise as a payload for antibody-drug conjugates. For over 60 years, A. phalloides has been the only source of α-amanitin. Here we report a synthesis of α-amanitin, which surmounts the key challenges for installing the 6-hydroxy-tryptathionine sulfoxide bridge, enantioselective synthesis of (2 S,3 R,4 R)-4,5-dihydroxy-isoleucine, and diastereoselective sulfoxidation.

Efficient oxidation of N-protected tryptophan and tryptophanyl-dipeptides by in situ generated dimethyldioxirane provides hexahydropyrroloindoline-containing synthons suitable for peptide synthesis and subsequent tryptathionylation.

A series of hydroxypyrroloindoline (Hpi) containing dipeptides along with the corresponding monomeric Hpi-α-amino acid (Hpi-2-carboxylate), were prepared by reacting a series of N α-protected-tryptophans in aqueous or biphasic [water/cyclopentyl methyl ether (CPME)] solutions containing Oxone® (potassium peroxymonosulfate) and acetone. This procedure avoids the tedious distillation of unstable dimethyldioxirane (DMDO), which is commonly used to oxidize indoles. Monomers N α-Boc-Hpi-OH and N α-Fmoc-Hpi-OH were readily incorporated by solid-phase peptide synthesis (SPPS) into a peptide containing a cysteine; in trifluoroacetic acid (TFA), the Hpi underwent intramolecular dehydrative condensation with the cysteine thiol to afford the anticipated tryptathionine crosslink. This eco- and user-friendly oxidative methodology greatly simplifies the synthesis of Hpi derivatives while enabling the synthesis of tryptathionine crosslinks characteristic of phalloidin and amanitin, two potent peptide toxins of present interest.

Rationally Designed Amanitins Achieve Enhanced Cytotoxicity.

For 70 years, α-amanitin, the most cytotoxic peptide in its class, has been without a synthetic rival; through synthesis, we address the structure-activity relationships to inform the design of new amatoxins and disclose analogues that are more cytotoxic than the natural product when evaluated on CHO, HEK293, and HeLa cells, whereas on liver-derived HepG2 cells, the same toxins show diminished cytotoxicity.

FlICk (fluorescent isoindole crosslinking) for peptide stapling.

The rigidification of peptide secondary structure via stapling is an important and enduring goal in the development of functional peptides for biochemical and pharmaceutical applications. In addition, the incorporation of fluorophores and chromophores has been a sought-after application for creating peptidic probes of cellular function and localization. The combined application of peptide stapling and fluorescent-readout is featured by the reaction of ortho-phthalaldehydes to create isoindole staples, thus transforming inactive linear and monocyclic precursors into fluorescent or visibly colored monocyclic and bicyclic products with noted biological activity. Given its user-friendliness, we have termed this approach FlICk (fluorescent isoindole crosslink) chemistry and we have featured this application on an array of high-affinity macrocyclic α-MSH derivatives as well as for late-stage intra-annular isoindole stapling furnished a bicyclic peptide mimic of α-amanitin that is cytotoxic to CHO cells. The synthetic methods for preparing substituted ortho-phthalaldehydes along with subsequent applications to FlICk stapling are detailed herein.

Solid-phase synthesis of a novel phalloidin analog with on-bead and off-bead actin-binding activity.

Specific effectors of actin polymerization have found use as dynamic probes of cellular morphology that may be used to gauge cellular response to stimuli and drugs. Of various natural products that target actin, phalloidin is one of the most potent and selective inhibitors of actin depolymerization. Phalloidin and related members of the phallotoxin family are macrocyclic heptapeptides bearing a characteristic and rigidifying transannular tryptathionine bridge. Here we describe a solid-phase synthesis of a new phalloidin analog as a prototype for library development with the potential for on- and off-bead screening. To validate our method, we labelled the phalloidin derivative with a fluorescent dye which stained actin in CHO cells. Furthermore, a bioassay was developed allowing actin polymerization on beads carrying a phalloidin derivative.