An efficient and practical new strategy for desulfurization of peptide proteins

On August 8, 2022, the team of Professor Li Xuechen of the University of Hong Kong published a research paper entitled “Superfast desulfurization for protein chemical synthesis and modification” at Chem.

The results report an add-and-done desulfurization, ADD-based peptide protein desulfurization method based on sodium tetraethylborate. This method is simple and efficient, has a wide range of substrates, and can be used for a variety of polypeptide/protein thiol removal containing unprotected cysteine (Cys) or penicillamine (Pen) residues, providing a practical desulfurization strategy for the accurate chemical synthesis and chemical semisynthesis of complex biological macromolecules.

The corresponding author of the paper is Li Xuechen; The first author is Sun Zhenquan.

The chemical synthesis of polypeptides and proteins and their fixed-point modification can provide biological macromolecules with precise chemical structures, especially for the study of the role of post-translational modification of proteins on various life activities, the preparation of D-configuration polypeptide proteins that cannot be obtained by recombinant expression technology from scratch, and the development of new polypeptide protein drugs. In the field of chemical protein synthesis, the application of desulfurization chemistry has greatly expanded the polypeptide protein ligation technology based on cysteine (Cys). This makes the site of polypeptide protein fragment coupling no longer limited to cysteine residues with a natural abundance of only about 1.8%, but instead enables the desulfurization of temporarily introduced thiol-carrying mutant amino acids (e.g., Cys, Pen, etc.) into natural amino acid residues with higher abundance (e.g., Cys→ Ala, Pen→Val, etc.) after the completion of chemical coupling. However, with the development of chemical protein total synthesis/semisynthesis, the increasingly complex structure of biological macromolecules has put forward higher requirements for the reported peptide protein desulfurization methods in terms of functional group compatibility, side reaction control, and reaction yield. We are still pursuing gentler, more efficient, and simpler desulfurization strategies to better achieve the chemical synthesis of peptide proteins and their precise modifications.

Recently, Professor Li Xuechen’s team at the University of Hong Kong has developed an efficient and practical peptide protein desulfurization strategy (Add-and-done desulfurization, ADD), which can achieve chemically selective ultra-rapid desulfurization of polypeptide/protein molecules containing unprote Chinese cted thiol groups. This desulfurization method based on sodium tetraethylborate is simple to operate (only need to add reagents, mix well), react quickly (t1/2 ~ 5s), and do not require the complex conditions and equipment required by conventional desulfurization methods, such as inert atmosphere protection, ultraviolet radiation, metal catalysts or important exogenous thiol additives.

Figure 1: Comparison of traditional desulfurization methods with ultra-rapid desulfurization methods (ADD).

Figure 2: Discovery of ultra-rapid desulfurization method (ADD) and optimization of its conditions.

Figure 3: Application of ultra-rapid desulfurization method (ADD) on random mercapto-containing polypeptides.

This method has been successfully applied in a variety of different peptides/protein substrates, demonstrating compatibility with different amino acid residues (Met, Trp, Tyr), protein post-translational modifications (N-glycosylation, Q-serotoninization) and universal mercaptolides (-Acm, -Thz, -Succinimide). At room temperature, a certain amount of aqueous solution of sodium tetraethylborate can be converted to the cysteine (Cys) or penicillamine (Pen) residues in the substrate into the corresponding alanine (Ala) or valine (Val) within tens of seconds to the three (2-carboxyethyl) phosphine (TCEP) solution containing the substrate. It can also be used for sulfur removal from commercial proteins, providing a practical tool for potential protein spatial structure analysis by quickly constructing proteins without disulfide bonds without the need for recombinant technology.

Figure 4: Application of ultra-rapid desulfurization method (ADD) on thiol-containing proteins.

Figure 5: Application of ultra-rapid desulfurization (ADD) to protein chemical synthesis.

ADD can even overcome a certain concentration of free radical quenching agent for desulfurization, which can be easily combined with existing natural chemical ligation (NCL), thus achieving chemical total synthesis of the leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) membrane part and the chemical semisynthesis of the fixed-point serotonin-modified histone H3 (H3Q5ser).

Figure 6: Mechanism study of ultra-rapid desulfurization method (ADD).

Through related mechanism research experiments, the authors proposed a mechanism based on the oxidation of sodium tetraethylborate by oxygen in the air in aqueous solution, releasing ethyl radicals and thus triggering a radical desulfurization chain reaction. (Source: Science Network)

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