BioTherapi: Bioinformatics for Therapeutic Peptides and Proteins
|G.P.S. Raghava | Bioinformatics Centre | IMTECH | CRDD | Team Members | Contact | FAQ|
Facility to Community
Techniques in peptide synthesis
1. Solid-Phase Peptide Synthesis: Solid phase peptide synthesis (SPPS), developed by R. B. Merrifield, was a major breakthrough allowing for the chemical synthesis of peptides and small proteins.
The first stage of the technique consists of peptide chain assembly with protected amino acid derivatives on a polymeric support. The second stage of the technique is the cleavage of the peptide from the resin support with the concurrent cleavage of all side chain protecting groups to give the crude free peptide.
The general principle of SPPS is one of repeated cycles of coupling-deprotection. The free N-terminal amine of a solid-phase attached peptide is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached.
There are two major used forms of solid phase peptide synthesis – Fmoc (base labile alpha-amino protecting group) and t-Boc (acid labile protecting group). Each method involves different resins and amino acid side-chain protection and consequent cleavage/deprotection steps. Fmoc chemistry is known for generating peptides of higher quality and in greater yield than t-Boc chemistry. Impurities in t-Boc-synthesized peptides are mostly attributed to cleavage problems, dehydration and t-butylation.
After cleavage from the resin, peptides are usually purified by reverse phase HPLC using columns such as C-18, C-8, and C-4.
The primary advantage of SPPS is its high yield. As peptides consists of many amino acids, if the yield for each amino acid addition is much less than 100%, overall peptide yields are negligible. For example, if each amino acid addition has a 90% yield then the overall yield of a 50 amino acid peptide is only 0.5%. Modern SPPS instrumentation pushes coupling and deprotection yields to greater than 99.99%, giving an overall yield of greater than 99% for a 50 amino acid peptide.
2. Liquid-phase synthesis: Liquid-phase peptide synthesis is a classical approach to peptide synthesis. It has been replaced in most labs by solid-phase synthesis (see below). However, it retains usefulness in large-scale production of peptides for industrial purposes.
3. Microwave assisted peptide synthesis: Although microwave irradiation has been around since the late 1940s, it was not until 1986 that microwave energy was used in organic chemistry. During the end of the 1980s and 1990s, microwave energy was an obvious source for completing chemical reactions in minutes that would otherwise take several hours to days. Through several technical improvements at the end of the 1990s and beginning of the 2000s, microwave synthesizers have been designed to provide both low and high energy pockets of microwave energy so that the temperature of the reaction mixture could be controlled. The microwave energy used in peptide synthesis is of a single frequency providing maximum penetration depth of the sample which is in contrast to conventional kitchen microwaves.
In peptide synthesis, microwave irradiation has been used to complete long peptide sequences with high degrees of yield and low degrees of racemization. Microwave irradiation during the coupling of amino acids to a growing polypeptide chain is not only catalyzed through the increase in temperature, but also due to the alternating electromagnetic radiation to which the polar backbone of the polypeptide continuously aligns to. Due to the this phenomenon, the microwave energy can prevent aggregation and thus increases yields of the final peptide product.
Despite the main advantages of microwave irradiation of peptide synthesis, the main disadvantage is the racemization which may occur with the coupling of cysteine and histidine. A typical coupling reaction with these amino acids are performed at lower temperatures than the other 18 natural amino acids.
As of January 2009, over 200 microwave peptide synthesizers are in use with the rate of acceptance increasing. 4. Custom Peptide Synthesis: AnaSpec