Revisiting Disulfide–Yne and Disulfide–Diazonium Reactions for Potential Direct Modification of Disulfide Bonds in Proteins

Abstract

To find their potential use in protein research, direct addition of a disulfide compound to alkyne (namely disulfide–yne reaction) and S-arylation with arenediazonium salt (namely disulfide–diazonium reaction) were investigated in aqueous or protic solutions. The reaction of dimethyl disulfide with 5-hexynol performed best under 300 nm irradiation in the presence of sodium acetate to afford 5,6-bis­(methylthio)-5-hexenol in 60% yield. Without the prior reduction of a disulfide bond to thiols, the disulfide–yne reactions have the advantage of 100% atom economy. Disulfide–diazonium reaction was triggered by sodium formate and accelerated by photoirradiation with a 450 nm LED lamp (5 W). The reaction of 3,4-dihydroxy-1,2-dithiane with 2-(prop-2-yn-1-yloxy)­benzene-1-diazonium tetrafluoroborate (8b) afforded 2-(benzofuran-3-yl)-1,3-dithiepane-5,6-diol (13), confirming that both S substituents originate from the same disulfide molecule. The trastuzumab antibody was incubated with diazonium 8b, followed by α-lytic protease digestion, LC-ESI-MS/MS analysis, and Mascot search, to verify that the proximal C229 and C232 residues on the same heavy chain were reconnected with a (benzofuranyl)­methine moiety that originated from 8b, unlike the expected disulfide rebridging across two heavy chains. Nonetheless, disulfide–diazonium reactions still have potential for rebridging disulfide bonds if appropriate proteins and diazonium agents are chosen.

Recent advances in the dichalcogenation reactions of unsaturated compounds via double functionalization

“…More recently, Fang and coworkers developed a similar HCO 2 Na-promoted strategy for the synthesis of 2-(benzofuran-3-yl)-1,3-dithiepane-5,6-diol 4c starting from 3,4-dihydroxy-1,2dithiane 4a and 2-( propargyloxy)benzenediazonium salt 4b under 450 nm LED irradiation (Scheme 4a). 18 It was confirmed that both sulfur substituents of the product originated from the same disulfide molecule. The protocol was also employed for the modification of trastuzumab antibody through disulfide re-bridging reactions (Scheme 4b).…”

Trifluoroacetic Acid Mediated Additive-Free Late-Stage Native Peptide Cyclization to Form Disulfide Mimetics via Thioketalization with Ketones

“…Recently, Poulsen et al reported an oxSTEF reagent which generated the bis-thio ketene acetal linked products via double thiol exchange, 19 while Fang et al developed a diazonium salt reagent to generate thioacetal-bridged peptides directly from a disulfide via the photoinduced radical process. 20 All of the above methods were conducted in aqueous buffer systems, which required good water solubility of peptides. Unfortunately, this requirement may not be fulfilled when hydrophobic or aggregation-prone peptides were used as substrates.…”

“…Unfortunately, this requirement may not be fulfilled when hydrophobic or aggregation-prone peptides were used as substrates . Moreover, the generation of a new chiral center without stereoselectivity at the thioacetal carbon (in Loh’s and Fang’s cases) , or mixture of Z / E isomers (in Poulsen’s case) led to the heterogeneity of the products and may affect the biological evaluation. Developing robust single carbon bis-cysteine rebridging methods without sequence bias and product structural heterogeneity for bioactive peptide cyclization is of great interest.…”

“…A similar process was developed in 2020 by Loh et al, where the α,α-dichloroketone reagents bearing functional payloads underwent double nucleophilic substitutions with two thiol groups to give rise to thioacetal linkages. Recently, Poulsen et al reported an oxSTEF reagent which generated the bis-thio ketene acetal linked products via double thiol exchange, while Fang et al developed a diazonium salt reagent to generate thioacetal-bridged peptides directly from a disulfide via the photoinduced radical process . All of the above methods were conducted in aqueous buffer systems, which required good water solubility of peptides.…”