Chemoselective Caging of Carboxyl Groups for On‐Demand Protein Activation with Small Molecules

Abstract

Tools for on-demand protein activation enable impactful gain-of-function studies in biological settings. Thus far, however, proteins have been chemically caged at primarily Lys, Tyr, and Sec, typically through the genetic encoding of unnatural amino acids. Herein, we report that the preferential reactivity of diazo compounds with protonated acids can be used to expand this toolbox to solvent-accessible carboxyl groups with an elevated pK a value. As a model protein, we employed lysozyme (Lyz), which has an active-site Glu35 residue with a pK a value of 6.2. A diazo compound with a bioorthogonal self-immolative handle esterified Glu35 selectively, inactivating Lyz. The hydrolytic activity of the caged Lyz on bacterial cell walls was restored with two small-molecule triggers. The decaging was more efficient by small molecules than by esterases. This simple chemical strategy was also applied to a hemeprotein and an aspartyl protease, setting the stage for broad applicability.

Pinpointing Acidic Residues in Proteins

“…Particularly, the diazo scaffold 2 (Figure 1A) can be easily derivatized for further reactivity modulation or multi-functionalization. With these advances, Raines et al has demonstrated the valuable bio-reversibility of protein esterification by intracellular esterases and extended the use of this innovative technique in protein cytosolic delivery, [19] on-demand activation of chemically caged proteins, [20] and late-stage multi-functionalization. [21] Diazo-bearing natural products have been identified as bacterial metabolites such as azaserine and kinamycins and exhibit diazo-dependent activities including anti-tumor properties.…”

Enantioselective insertion of vinyl diazoacetates into O–H bonds of carboxylic acids

“…10 However, only moderate enantioselectivities were observed for vinyl diazoacetates in their report. In continuation with our research interests in metal-carbene chemistry, especially in enantioselective X–H 11 and Csp 2 –H bond 12 insertion reactions, herein we wish to report the highly enantioselective O–H insertion reaction between vinyl diazoacetates and carboxylic acids 13 in the presence of an achiral rhodium complex and a chiral phosphoric acid in which the enantioselective addition occurs at the carbenic rather than the vinylogous position (Scheme 1d).…”

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

“…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).…”

“…[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). [39][40][41][42] In this method, the carboxylic acids of glutamate and aspartate residues in POI A react with α-arylα-diazoamides such as 1-3 to generate POI conjugate B. Ester hydrolysis catalyzed by cytosolic esterases would then reconvert POI conjugate B back to the native POI A, releasing the respective alcohols 4-6 as side products.…”

“…Early studies have shown that α-diazoamide 1 enabled the delivery of GFP and RNase A in cancer cells, where ester cleavage revealed the native proteins (Figure 1c). [40] Whilst this manuscript was in preparation, Raines and coworkers have further extended this strategy by modifying α-diazoamide tags in 2 and 3 [39,42] to enable the faster release of POIs, and the late-stage labeling of esterified proteins with a thiolated substrate, including CPPs [1][2][3][4][5][6] for promoting cellular uptake. In this study, we explore the potential of combining the Raines esterification approach and TMU for traceless protein delivery (Figure 1).…”