Chemical ligation
Encyclopedia
Chemical ligation is a set of techniques used for creating long peptide
or protein
chains. It is the second step of a convergent approach
. First, smaller peptides containing 30-50 amino acid
s are prepared by conventional chemical peptide synthesis
. 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 amino acid
s can be produced.
. 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.
Native chemical ligation relies on the presence of a cysteine
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).
s it is possible to prepare a recombinant
polypeptide C-terminal thioester
. 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. Recombinant expression can also be used to give a Cys-polypeptide for use in native chemical ligation.
. The Staudinger ligation continues to be developed and has not yet found widespread use.
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
or protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
chains. It is the second step of a convergent approach
Convergent synthesis
In chemistry a convergent synthesis is a strategy that aims to improve the efficiency of multi-step chemical synthesis, most often in organic synthesis...
. First, smaller peptides containing 30-50 amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s are prepared by conventional chemical peptide synthesis
Peptide synthesis
In organic chemistry, peptide synthesis is the production of peptides, which are organic compounds in which multiple amino acids are linked via amide bonds which are also known as peptide bonds...
. 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 amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s can be produced.
Methods of chemical ligation
There are various techniques described in literature. The most practical and robust method for the chemoselective reaction of unprotected peptides is native chemical ligationNative chemical ligation
Native chemical ligation or NCL is the most widely used form of chemical ligation, a technique for constructing a large polypeptide from two or more unprotected peptides. In native chemical ligation a peptide containing a C-terminal thioester reacts with another peptide containing an N-terminal...
. 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.
Native chemical ligation relies on the presence of a cysteine
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
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 inteinIntein
An intein is a segment of a protein that is able to excise itself and rejoin the remaining portions with a peptide bond. Inteins have also been called "protein introns"....
s it is possible to prepare a recombinant
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
polypeptide C-terminal thioester
Thioester
Thioesters are compounds with the functional group C-S-CO-C. They are the product of esterification between a carboxylic acid and a thiol. Thioesters are widespread in biochemistry, the best-known derivative being acetyl-CoA.-Synthesis:...
. 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. 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 reactionStaudinger reaction
The Staudinger reaction or Staudinger reduction is a chemical reaction in which the combination of an azide with a phosphine or phosphite produces an iminophosphorane intermediate. Combined with the hydrolysis of the aza-ylide to produce a phosphine oxide and an amine, this reaction is a mild...
. The Staudinger ligation continues to be developed and has not yet found widespread use.
External links
- Instant insight outlining chemical ligation from the Royal Society of ChemistryRoyal Society of ChemistryThe Royal Society of Chemistry is a learned society in the United Kingdom with the goal of "advancing the chemical sciences." It was formed in 1980 from the merger of the Chemical Society, the Royal Institute of Chemistry, the Faraday Society and the Society for Analytical Chemistry with a new...
- Chemical Ligation Aldrich ChemFiles 2008 Vol. 8, No. 1, giving an overview on modern Chemical Ligation methods and literature.