Chemical ligation is a set of techniques used for creating long

peptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. A ...

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

chains. It is the second step of a convergent approach. First, smaller peptides containing 30-50

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...

s are prepared by conventional chemical

peptide synthesis In organic chemistry, peptide synthesis is the production of peptides, compounds where multiple amino acids are linked via amide bonds, also known as peptide bonds. Peptides are chemically synthesized by the condensation reaction of the carboxyl ...

. Then, they are completely deprotected. Chemical ligation is the technique of coupling these peptides by chemoselective reaction to give a unique reaction product, usually in aqueous solution. With several coupling steps, proteins of up to 200-300

s can be produced.

Methods of chemical ligation

There are various techniques described in literature.

Native chemical ligation

The most practical and robust method for the chemoselective reaction of unprotected peptides is

native chemical ligation Native Chemical Ligation (NCL) is an important extension of the chemical ligation concept for constructing a larger polypeptide chain by the covalent condensation of two or more unprotected peptides segments. Native chemical ligation is the most e ...

. Native chemical ligation has overcome the limitations of the classical synthetic organic chemistry approach to the total synthesis of proteins, and enables the routine total or semi- synthesis of protein molecules. The original chemical ligation methods involved the formation of a non-native bond at the ligation site. Subsequently, native chemical ligation was developed. In native chemical ligation, an unprotected peptide-thioester reacts with a Cys-peptide to give a ligation product with a native amide ('peptide') bond at the ligation site. In this method, the initial thioester-linked ligation product intermediate rearranges to form an

amide bond In organic chemistry, an amide, also known as an organic amide or a carboxamide, is a compound with the general formula , where R, R', and R″ represent organic groups or hydrogen atoms. The amide group is called a peptide bond when it is p ...

. Native chemical ligation relies on the presence of a

cysteine Cysteine (symbol Cys or C; ) is a semiessential proteinogenic amino acid with the formula . The thiol side chain in cysteine often participates in enzymatic reactions as a nucleophile. When present as a deprotonated catalytic residue, sometime ...

residue at the ligation site. Methods using removable auxiliary groups can in some instances extend the use of native chemical ligation to non-cysteine residues, as can the use of desulfurization subsequent to the ligation (e.g. converting a Cys to an Ala).

Expressed protein ligation

By exploiting naturally occurring inteins it is possible to prepare a recombinant polypeptide C-terminal

thioester In organic chemistry, thioesters are organosulfur compounds with the functional group . They are analogous to carboxylate esters () with the sulfur in the thioester playing the role of the linking oxygen in the carboxylate ester, as implied by t ...

. This enables the use of large recombinant protein-derived thioesters in native chemical ligation. The recombinant thioester can be ligated to a synthetic peptide bearing an N-terminal cysteine. Native chemical ligation of this kind using recombinant C-terminal thioesters is known as

expressed protein ligation Expression may refer to: Linguistics * Expression (linguistics), a word, phrase, or sentence * Fixed expression, a form of words with a specific meaning * Idiom, a type of fixed expression * Metaphorical expression, a particular word, phrase, ...

. Recombinant expression can also be used to give a Cys-polypeptide for use in native chemical ligation.

Staudinger ligation

The Staudinger ligation, first reported in 2000, in principle enables the ligation of peptide segments independent of the terminal amino acids. The method is based on the

Staudinger reaction The Staudinger reaction is a chemical reaction of an organic azide with a phosphine or phosphite produces an iminophosphorane. The reaction was discovered by and named after Hermann Staudinger. The reaction follows this stoichiometry: :R3P + ...

. The Staudinger ligation continues to be developed and has not yet found widespread use.

Ser/Thr ligation

Ser/Thr ligation (STL) was introduced into protein chemical synthesis as an alternative ligation strategy. ''

Serine Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − form un ...

Threonine Threonine (symbol Thr or T) is an amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), a carboxyl group (which is in the deprotonated −COO� ...

-ligation'' involves merger of a side-chain unprotected peptide segment containing a C-terminal salicylaldehyde (SAL) ester and another peptide segment with an N-terminal Ser or Thr residue. The chemoselective reaction between the peptide SAL ester and 1,2-hydroxylamine group of Ser or Thr leads to the formation of an N,O-benzylidene acetal linked intermediate, which undergoes acidolysis to afford a natural peptidic Xaa-Ser/Thr linkage. This method does not involve the use of unnatural amino acids, and it is simple to operate. Ser/Thr ligation provides a complementary method for protein chemical synthesis and semisynthesis.

References

* Schnölzer M, Kent SB. 1992, "Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease." ''Science''. 256:221-5 * Dawson PE, Muir TW, Clark-Lewis I, Kent SB. 1994, "Synthesis of proteins by native chemical ligation." ''Science''. 266:776-9. * Muir TW. 2003, "Semisynthesis of proteins by expressed protein ligation." ''Annu Rev Biochem''. 72:249-89. * Nilsson BL, Soellner MB, Raines RT. 2005, "Chemical Synthesis of Proteins." ''Annu. Rev. Biophys. Biomol. Struct.'' 34:91-118 * Bang D, Pentelute BL, Kent SB. 2006, "Kinetically controlled ligation for the convergent chemical synthesis of proteins." ''Angew Chem Int Ed Engl.'' 45:3985-8. * Kent SB. 2009, "Total chemical synthesis of proteins." ''Chem.Soc.Rev.'' 38, 338–351. {{doi, 10.1039/b700141j. * Zhang Y, Xu C, Kam HY, Lee CL, Li X. 2013, "Protein chemical synthesis by serine/threonine ligation." ''Proc. Natl. Acad. Sci. USA.'' 17:6657-6662

External links

* Aldrich
Chemical Ligation
''ChemFiles'' 2008 Vol. 8, No. 1, giving an overview on modern Chemical Ligation methods and literature. Chemical synthesis