Modular Diazo Compound for the Bioreversible Late-Stage Modification of Proteins

Abstract

We introduce a versatile strategy for the bioreversible modification of proteins. Our strategy is based on a tricomponent molecule, synthesized in three steps, that incorporates a diazo moiety for chemoselective esterification of carboxyl groups, a pyridyl disulfide group for late-stage functionalization with thiolated ligands, and a self-immolative carbonate group for esterase-mediated cleavage. Using cytochrome c (Cyt c) and the green fluorescent protein (GFP) as models, we generated protein conjugates modified with diverse domains for cellular delivery that include a small molecule, targeting and cell-penetrating peptides (CPPs), and a large polysaccharide. As a proof of concept, we used our strategy to effect the delivery of proteins into the cytosol of live mammalian cells in the presence of serum. The cellular delivery of functional Cyt c, which induces apoptosis, highlighted the advantage of bioreversible conjugation on a carboxyl group versus irreversible conjugation on an amino group. The ease and utility of this traceless modification provide new opportunities for chemical biologists.

Simultaneous Covalent Modification of K-Ras(G12D) and K-Ras(G12C) with Tunable Oxirane Electrophiles

“…Recently, Shokat et al developed an α,β-diketoamide and a β-lactone ligand that covalently target the K-Ras­(G12R) and K-Ras­(G12S) mutants, respectively, demonstrating the feasibility of targeting weakly nucleophilic amino acids. , Carboxyl residues, which comprise up to 12% of the whole proteome, offer greater opportunities than cysteine and lysine residues (with abundances of 2.3% and 5.9% respectively) for accessing and modulating the biological functions of diverse proteins, including those defined as “undruggable” targets . Reactive groups such as diazo, oxirane, and Woodward’s reagent K have been developed for bioconjugation with Glu/Asp residues. − For example, Waldmann et al have developed a novel class of covalent probes based on Woodward’s reagent K that allow selective covalent labeling of Glu residues inside PDE6δ binding pockets . Useugi et al found serendipitously that an oxirane-based ligand forms a covalent adduct with an aspartic acid residue within the active site of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) .…”

Toward a Traceless Tag for the Thiol‐Mediated Uptake of Proteins

“…Such ideal small‐molecule TMU tags could be attached to the POI in situ under bioorthogonal conditions prior to delivery and detached after uptake in the cytosol to release the POI in its native form (Figure 1c). Many elegant bioconjugation strategies are available to react with protein surface lysines, glutamates, aspartates, cysteines, methionines, tyrosines and arginines [39–58] . Similarly diverse strategies have been reported as well to release the attached motifs in the cytosol [39–47] …”

“…Many elegant bioconjugation strategies are available to react with protein surface lysines, glutamates, aspartates, cysteines, methionines, tyrosines and arginines [39–58] . Similarly diverse strategies have been reported as well to release the attached motifs in the cytosol [39–47] …”

“…Many elegant bioconjugation strategies are available to react with protein surface lysines, glutamates, aspartates, cysteines, methionines, tyrosines and arginines. [39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58] Similarly diverse strategies have been reported as well to release the attached motifs in the cytosol. [39][40][41][42][43][44][45][46][47] For the first attempt toward a clickable TMU tool for traceless delivery in practice, we selected a bioreversible esterification method that has been introduced by Raines and coworkers (Figure 1b).…”

Iron-sensitive protein conjugates formed with a Wittig reaction precursor in ionic liquid

“…The ionic liquid-based condensation reaction can provide a protein conjugate bearing a unique enamine linkage that could be of potential use for studying iron species in biological systems. While genetic engineering produced a number of useful caging/uncaging strategies to interrogate biomolecular or cellular activities, 3 chemical protein modification approaches are increasingly studied as an alternative approach, [27][28][29] and the present study represents the utility of the nonaqueous bioconjugation method to directly install the iron-sensitive moiety. Beyond the protein bioconjugation application, our discovery of the iron-mediated hydrolysis of the enamine linkage would also give a new design paradigm for the design of small molecule-based activity-based imaging reporters, as there are still limited selections of the iron-responsive warheads [30][31][32] with a potential synthetic challenge.…”