Ethynyl benziodoxolones: functional terminators for cell-penetrating poly(disulfide)s

Abstract

Outperforming cell-penetrating peptides (CPPs), cell-penetrating poly(disulfide)s (CPDs) are attracting increasing interest. CPDs are accessible by ring-opening disulfide-exchange polymerization under mild conditions in neutral water. Initiation of the polymerization with thiols results in quantitative labeling of one CPD terminus with initiators of free choice. In contrast, labeling of the other terminus with iodoacetamides has so far been ineffective because of poor yields and the high excess of reagents needed. In this report, we introduce hypervalent iodine reagents as operational terminators of the synthesis of CPDs, also at high dilution. The power of the approach is exemplified with green-fluorescent initiators and ethynyl benziodoxolone terminators containing additional azides for CuAAC with red-fluorescent alkynes. The absorption spectra of the resulting CPDs demonstrate that stoichiometric application of ethynyl benziodoxolone terminators results in 46% incorporation. FRET between green-fluorescent initiators and red-fluorescent terminators demonstrates significant folding of CPDs in solution; it disappears upon reductive depolymerization. Substrates attached to the new termini are shown to enter into HeLa cells. Moreover, disappearance of FRET in the cytosol corroborated the reductive cleavage of CPDs upon internalization. Beyond the introduction of enthynyl benziodoxolones as operational terminators, these findings thus demonstrate also the compatibility of CuAAC with poly(disulfide)s and the usefulness of doubly-labeled CPDs for structural and mechanistic studies

Sidechain Engineering in Cell‐Penetrating Poly(disulfide)s

“…However, the results were not convincing (not shown). CuAAC (copper(I)‐catalyzed alkyne‐azide cycloaddition),[5][21][22] compatible with CPDs,[23] was chosen next. In monomers 9 , azides were added without changing the arginine motif in original CPD 2 ( Scheme ; monomers with alkynes instead of azides gave poorly soluble CPDs, not shown).…”

Cell-Penetrating Peptides: Design Strategies beyond Primary Structure and Amphipathicity

“…A conceptually innovative way for improving cell penetration is the use of cell-penetrating polydisulfides (CPDs) [ 93 ]. Polydisulfides, obtained by ring-opening disulfide-exchange polymerization, are guanidinium-rich compounds in which the peptide backbone is replaced by a polydisulfide one [ 94 ]. The topic has been reviewed in detail elsewhere [ 13 ].…”

“…The topic has been reviewed in detail elsewhere [ 13 ]. Briefly, CPDs are interesting as they are involved in dynamic covalent chemistry on the cell surface and enter cells by thiol-mediated uptake ( Figure 3 c) [ 94 ]. The most promising CPDs are those shown in Figure 3 a,b.…”

“…This system was further developed to obtain double-labeled CPDs with a FRET pair at opposite ends of the polymer. These CPDs were shown to enter cells, although they were split into two fragments at the outer leaflet of the membrane during uptake [ 94 ]. For a comprehensive overview of this topic, please refer to the review by Matile and co-workers [ 13 ].…”

“… ( a , b ) structures of the most promising examples of cell-penetrating polydisulfides containing guanidinium groups and a polydisulfide backbone, taken in part, with permission from reference [ 13 ] (Copyright © 2015, Royal Society of Chemistry). ( c ) Schematic representation of the polydisulfide dynamic covalent chemistry on the cell surface and their cell entry by thiol-mediated uptake, followed by reductive depolymerization by glutathione ( k e = rate of endocytosis, k t = rate of translocation, k d = rate of depolymerization, k n = rate of nuclear uptake), taken from reference [ 94 ]. …”