Chemoselective and photocleavable cysteine modification of peptides and proteins using isoxazoliniums

Abstract

It is of ongoing interest to develop new approaches for efficient and selective modification of cysteine residues on biomolecules. Here we present a comprehensive study on a newly developed isoxazolinium-mediated cysteine modification of peptides and proteins. Using a stoichiometric amount of isoxazolinium reagents generated in situ from a catalytic amount of silver salts, cysteine-containing peptides can be efficiently modified to afford products in nearly complete conversions. With the optimized conditions, free cysteine containing proteins HSA and BSA, as well as a site-directed mutated therapeutic protein (BCArg) can be efficiently and selectively labelled using small amounts of the isoxazolinium reagents. We find that the phenylacyl thioether linkage bearing an alkyne moiety can be rapidly cleaved under irradiation of UV-A light, giving the formation of a thioaldehyde moiety, which can be converted back to cysteine by reduction.

Mono-PEGylation of a Thermostable Arginine-Depleting Enzyme for the Treatment of Lung Cancer

“…Recombinant Bacillus caldovelox arginase mutant (BCA-M) was obtained via over-expression of the BCA-M enzyme in E. coli and purification using heat treatment and nickel chelation affinity chromatography [ 42 ]. One to six molar equivalents of 20 kDa PEG were added into purified the BCA-M (3 mg/mL) and incubated at 4 °C for overnight.…”

Tunable heteroaromatic azoline thioethers (HATs) for cysteine profiling

“…1b). 55 Despite all these advances, only ~17% of cysteine in the entire proteome has been identified so far.…”

“…1b ). 55 Despite all these advances, only ∼17% of cysteine in the entire proteome has been identified so far. 56,57 Moreover, the resulting bioconjugates exhibit poor mass detection sensitivity limiting their applications in the identification of low abundant cysteine in the proteome.…”

Protein Modifications: From Chemoselective Probes to Novel Biocatalysts

“…Sulfur containing electrophiles (e.g., sulfones and sulfonate esters) have also been widely used as cysteine selective probes, whilst activated thiols (e.g., thiosulfates, thiosulfonates and TNBthiols) provide good chemoselectivity and offer a handle for further modifications through thiol-disulfide exchange reactions [30,50,54]. In recent years a panel of structurally diverse electrophiles has emerged for cysteine selective labeling and functionalization, including: bromo-oxetanes [58]; quaternized viny/alkynil pyridines [59]; dinitroimidazoles [60]; isoxazoliniums [61]; cyclopropenyl ketones [62]; 3-bromo-5-methylene pyrrolones [63]; phosphonamidates [64,65]; vinylphosphonothiolates [66].…”