Grafting Alkyne Handles on Cysteines

One of the most common ways to study biomolecules is to label specific amino acids, mostly cysteine, with chemical handles for further modification. Azide-alkyne click reaction is a very popular strategy due to its high reactivity and reaction rate. Therefore, numerous reagents have been developed to specifically label proteins with terminal alkynes, including iodoacetamide alkynes and maleimide derivatives. However, limitations like insufficient selectivity and low stability remain as long-lasting concerns. To address these problems, one common strategy is introducing a linker between the targeting residue and the alkyne moiety, which potentially brings in unwanted structural changes. A reagent that can introduce small-sized terminal alkyne with one step under mild conditions is in demand.

Scheme 1. Cysteine-labeling study in this work

In this study, the Waser group presented a series of reagents that selectively ethynylate cysteine in a one-pot manner (Scheme 1). The reagents were designed based on the structures of hypervalent iodine compounds, which are popular in chemical biology applications for their high selectivity, sufficient stability, and low toxicity. A new series of trimethyllsilylethynylbenziodoxolone (TMS-EBX) compounds were developed that can be applied to both simple peptides and complex proteins under physical conditions.

Derivatives with electron-withdrawing or electron-donating groups were prepared and tested with glutathione (5a) to investigate the rearrangement rate and efficiency, among which the nitro-substituted analog (6b) achieved highest conversion at 99 % yield (pH 8.2, 37^oC, 15 min) as shown in Scheme 2. Different pH conditions and buffers were also evaluated. Notably, 6b exhibited moderate to high reactivities within the widely used pH range (pH 6.2-8.4) and in most common buffers, including Tris, HEPES, and TAPS.

Scheme 2. Evaluation of different derivatives with various substitution group.

Next, the authors researched into the impact of steric hindrance and the different cysteine locations (Scheme 3). They synthesized small peptides with different amino acids besides cysteine and also with cysteine at either N-terminal or C-terminal. The results demonstrated that the labeling efficiency was not affected by steric hindrance or the location of cysteine residues. Furthermore, they tested 6b with more complex peptides, including fragments from HSA (97 % conversion), GAP26 (68 % conversion) and Hepaticis C virus glycoprotein E2 (94 % conversion).

Scheme 3. A) Scope of the ethynylation with respect to tetrapeptides. B) Application to larger peptides. [a] Reactions were carried out on a 1.00 mmol scale. [b] Reactions were carried out on a 0.50 mmol scale. Yields were determined by relative integration based on HPLC–MS.

With the fact that most cysteine residues in natural environment are in disulfide form, the authors demonstrated a one-pot method with TCEP as reducing reagent and 6b as labeling reagent. 86% conversion was obtained when testing with natural bioactive peptides oxytocin. However, they did observe significant degradation of 6b at the presence of excess TCEP when only increasing the equivalents of TCEP.

The reactivities of terminal thiol-alkyne with the azide-contained fluorescent probe (Cy7-azide) were evaluated as follows (Scheme 4). The reactions were performed under mild conditions and relatively high yields were obtained. Additionally, compound 6b can be employed for further modifications such as thiol-ester formation and Sonogashira coupling.

Scheme 4. A) CuAAC between terminal thioalkynes and Cy7-N₃. Cy7 = Cyanine7 dye. B) Alkyne hydration in acidic media. C) One-pot thiol alkynylation and subsequent Sonogashira cross-coupling. TPPTS = Triphenylphosphine-3,3’,3’’-trisulfonic acid trisodium salt.

However, the highly reactive 6b brought up the concern of side reactivity when applying to trastuzumab, an HER2 antibody for breast cancer treatment. Partial decomposition of the antibody was observed when treating with 6b. The Waser group then optimized the reaction conditions by switching to less reactive compounds (6a and 6c). The yield was eventually optimized to 97 % by altering pH, temperature, and buffers. The results suggested that, even with higher reactivity, 6b might not be the best choice when applying to complicated biological environment.

The application of these reagents in proteome-wide labeling was also explored (Figure 1). Hela cell lysates were treated with compound 6a-e, respectively. Compound 6d displayed highest labeling ability, which was consistent with the results mentioned above. 6d also has high chemoselectivity on cysteine over other amino acids as shown in Figure 1. In addition, due to the strong hydrophobicity of 6d, it exhibited better labeling capability when applying directly to living Hela cells.

Figure 1. Left: Fluorescence image of HeLa lysate treated with 10 mm TMS-EBX reagents. Right: Chemoselectivity of 6d towards nucleophilic amino acids in HeLa lysate (n = 6). Error bars are S.E.M.

Overall, this study demonstrated a series of hypervalent iodine reagents that can selectively label cysteine residues with a one-pot strategy. Those compounds are applicable under various conditions that are widely used in biological experiments. Although with the concern of side reactivity and degradation, these reagents still serve as good additions for cysteine labeling methods, which accommodate the limitations of some existing reagents.

Reference

1.     Tessier, Romain, et al. "Ethynylation of Cysteines from Peptides to Proteins in Living Cells." Angewandte Chemie International Edition (2020).