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Peptide synthesis and it application

Peptides are naturally occurring biological molecules found in all organisms. It is a short chain of about 30-40 amino acid carry secondary structure of protein stabilized by peptide bond between two amino acid. The shortest peptides are dipeptides, consisting of 2 amino acids joined by a single peptide bond, followed by tripeptides, tetrapeptides, etc. The natural process of producing long peptides (proteins) in living organism is known as protein biosynthesis. There are various peptides required for the normal life of an organism, but sometimes they do not synthesized in required amount, or if synthesized, it is in altered form. Therefore, it is required to synthesize these essential proteins ex vivo.

Peptide synthesis and it application

Peptides

Peptides are naturally occurring biological molecules found in all organisms. It is a short chain of about 30-40 amino acid carry secondary structure of protein stabilized by peptide bond between two amino acid. The shortest peptides are dipeptides, consisting of 2 amino acids joined by a single peptide bond, followed by tripeptides, tetrapeptides, etc. The natural process of producing long peptides (proteins) in living organism is known as protein biosynthesis. There are various peptides required for the normal life of an organism, but sometimes they do not synthesized in required amount, or if synthesized, it is in altered form. Therefore, it is required to synthesize these essential proteins ex vivo. The chemical synthesis of peptide is a tedious job and synthesis of insulin and oxytocin justify the difficult task of chemical synthesis of peptides. But now days, the artificial or chemical synthesis of these biologically active and essential peptides are carried out by several methods such as liquid-phase synthesis and solid-phase synthesis (1). The artificially or chemically synthesized peptides are frequently used for studies in immunotherapy, vaccination, and many other areas of advanced research and are mostly synthesized by solid-phase synthesis in a laboratory scale and also on an industrial scale.

Solid-phase synthesis of peptides

Solid-phase peptide synthesis of peptide is most common method of peptide synthesis today. In this process, C-terminal of an amino acid is not protected with a chemical group as happen in natural process, but it is bound to an activated solid support, such as polystyrene or polyacrylamide which facilitates rapid separation of growing peptide chain from different reaction mixtures during peptide synthesis. The synthesis of artificial peptide occurs by coupling the C-terminus of the incoming amino acid to the N-terminus of the other amino acid. Therefore, the artificial synthesis of peptides occurred in C-to-N direction which is opposite to the natural process of protein synthesis during which occur in N-to-C direction. There are several steps involved in the synthesis of peptides such as peptide deprotection, amino acid coupling, peptide cleavage and peptide purification

Peptide protection

The functional groups present in the side chain of several amino acids are reactive in nature. They interact with functional groups of amino acids during peptide synthesis and negatively influence production and purity of newly formed peptides. To minimize the effect of these reactive functional groups during peptide synthesis, several chemicals have been developed to bind and protect them from nonspecific reactions.

Amino acid coupling

For amino acid coupling, activation of the carboxylic acid present on incoming amino acid is carried out with the help of coupling reagents such as diisopropylcarbodiimide (DIC) or dicyclohexylcarbodiimide (DCC). These coupling reagents react with the C-terminus carboxyl group of an incoming amino acid to make a nucleophilic attack on the N-terminus of deprotected primary amino group of growing peptide chain.

Peptide cleavage

Removal of all remaining protecting groups after completion of deprotection and coupling steps is known as peptide cleavage. Cleavage can be carried out by acidolysis with the help of several strong acids listed below.

 Hydrogen fluoride (HF)

 hydrogen bromide (HBr)

 Trifluoromethane sulfonic acid (TFMSA)

These chemicals are used to cleave Boc and Bzl groups from peptide chain, while cleavage of Fmoc and tBut required TFA, a relatively milder acid. The proper execution of the process removes protecting group at N-terminal of the last amino acid added, protecting group at C-terminal from the first amino acid and other side-chain protecting groups if any present.

Peptide purification

Peptide purification is usually based on a combination of several separation methods that exploit the physiochemical characteristics such as size, charge and hydrophobicity of peptides. Purification techniques include:

 size-exclusion chromatography

 ion exchange chromatography (IEC)

 partition chromatography

 high-performance liquid chromatography (HPLC)

References

1. Merrifield, R. B. et al. (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149-54.

2. Carpino, L. A. et al. (1957) Oxidative reactions of hydrazines. Iv. Elimination of nitrogen from 1, 1-disubstituted-2-arenesulfonhydrazides1-4. J. Am. Chem. Soc. 79, 4427-31.

3. McKay, F. C., Albertson, N. F. (1957) New amine-masking groups for peptide synthesis. J. Am. Chem. Soc. 79, 4686-90.

4. Anderson, G. W., McGregor, A. C. (1957) T-butyloxycarbonylamino acids and their use in peptide synthesis. J. Am. Chem. Soc. 79, 6180-3.

5. Carpino, L. A., Han, G. Y. (1972) 9-fluorenylmethoxycarbonyl amino-protecting group. J. Org. Chem. 37, 3404-9.

For more information visit on- http://www.biosyn.com/peptide-synthesis-services.aspx



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published by biosynthesis011 2015-05-16

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