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The mystery of the inversion ligation reaction: Why can NCL achieve efficient selectivity and chemoselectivity?
In the field of modern biochemistry, native chemical ligation (NCL) is actually an important extension of the concept of chemical ligation. It is a method of covalently condensing two or more unprotected peptides to construct a larger Polypeptide chain. NCL is the most efficient method for synthesizing native or modified proteins of typical sizes, especially those with less than about 300 amino acids.
In the NCL reaction, the ionized thiol group of the N-terminal cysteine residue of an unprotected peptide attacks the C-terminal thioester of a second unprotected peptide in an aqueous buffer solution at pH 7.0. In liquid.
In the NCL process, the initial transthioesterification step is a reversible process, which makes the reaction both chemoselective and regioselective, ultimately forming a linked intermediate. This intermediate rapidly rearranges via an intramolecular S,N-acyl transfer, resulting in a native amide (or "peptide") bond at the point of attachment.
Reaction mechanism and its characteristics
In the NCL reaction, the most effective and commonly used thiol catalyst is 4-mercaptophenylacetic acid. The reversible nature of the reaction makes NCL highly regioselective during its synthesis. For example, in the presence of an internal cysteine residue, the yield of the final product was still very high, which was attributed to the irreversibility of the second step S-N acyl transfer under the reaction conditions.
During the reaction, almost no by-products that combine with other functional groups are generated. This feature makes NCL a highly precise chemical synthesis method.
NCL's history dates back to 1992, when the concept of "chemical bonding" was developed by Steven Kent and Martina Schnoelzer at the Scripps Research Institute. This innovation not only opened the precedent for the covalent condensation of unprotected peptides, but was also further extended to NCL technology in 1994, allowing oxalic acid diester bonds to form between peptides and ultimately convert into native amide bonds.
Contemporary Applications and Future Prospects
NCL technology forms the basis for today's chemical protein synthesis and has been widely used to prepare a variety of proteins and enzymes. Its main advantage is that the efficiency of connecting long peptides by this technology is often close to quantitative, thus allowing the synthesis of many proteins that cannot be synthesized by traditional methods due to their size, modifications and other chemical structures.
NCL's method is inherently green chemistry due to its atom economy and the use of non-hazardous solvents.
NCL is usually carried out in 6 M guanidine hydrochloride in aqueous solution in the presence of an aromatic thiol catalyst, and the yields of the resulting peptides are usually near quantitative. However, for light-sensitive peptides, contact with ketones should be avoided because this may affect peptide synthesis and reaction efficiency.
ConclusionIn addition, NCL technology can flexibly use different sulfur-containing amino acids or peptides containing N-terminal selenoamino acids for synthesis, demonstrating its strong potential in synthetic biology.
In summary, the reversibility of the NCL reaction and its excellent selectivity make it an important technology for protein synthesis. As experts explore the potential applications of these reactions, NCL will undoubtedly continue to play an important role in future biomedical research. Conversely, we must also think: How many possible new technologies are waiting to be discovered for future protein engineering?
