Peptide Synhtesis Overview
Peptides are synthesized by coupling the carboxyl group or C-terminus of one amino acid to the amino group or N-terminus of another. Due to the possibility of unintended reactions, protecting groups are usually necessary. Chemical peptide synthesis starts at the C-terminal end of the peptide and ends at the N-terminus. This is the opposite of protein biosynthesis, which starts at the N-terminal end.
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.
Solid-phase peptide synthesis (SPPS), pioneered by Robert Bruce Merrifield, resulted in a paradigm shift within the peptide synthesis community. It is now the accepted method for creating peptides and proteins in the lab in a synthetic manner. SPPS allows the synthesis of natural peptides which are difficult to express in bacteria, the incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins, which consist of D-amino acids.
Small solid beads, insoluble yet porous, are treated with functional units ('linkers') on which peptide chains can be built. The peptide will remain covalently attached to the bead until cleaved from it by a reagent such as anhydrous hydrogen fluoride or trifluoroacetic acid. The peptide is thus 'immobilized' on the solid-phase and can be retained during a filtration process, whereas liquid-phase reagents and by-products of synthesis are flushed away.
The general principle of SPPS is one of repeated cycles of coupling-wash-deprotection-wash. The free N-terminal amine of a solid-phase attached peptide is coupled (see below) 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. The superiority of this technique partially lies in the ability to perform wash cycles after each reaction, removing excess reagent with all of the growing peptide of interest remaining covalently attached to the insoluble resin.
The overwhelmingly important consideration is to generate extremely high yield in each step. For example, if each coupling step were to have 99% yield, a 26-amino acid peptide would be synthesized in 77% final yield (assuming 100% yield in each deprotection); if each step were 95%, it would be synthesized in 25% yield. Thus each amino acid is added in major excess (2~10x) and coupling amino acids together is highly optimized by a series of well-characterized agents.
There are two majorly used forms of SPPS -- Fmoc and Boc. Unlike ribosome protein synthesis, solid-phase peptide synthesis proceeds in a C-terminal to N-terminal fashion. The N-termini of amino acid monomers is protected by these two groups and added onto a deprotected amino acid chain.
Automated synthesizers are available for both techniques, though many research groups continue to perform SPPS manually.
SPPS is limited by yields, and typically peptides and proteins in the range of 70 amino acids are pushing the limits of synthetic accessibility. Synthetic difficulty also is sequence dependent; typically amyloid peptides and proteins are difficult to make. Longer lengths can be accessed by using native chemical ligation to couple two peptides together with quantitative yields.
Since its introduction over 40 years ago, SPPS has been significantly optimized. First, the resins themselves have been optimized.Furthermore, the linkers between the C-terminal amino acid and polystyrene resin have improved attachment and cleavage to the point of mostly quantitative yields. The evolution of side chain protecting groups has limited the frequency of unwanted side reactions. In addition, the evolution of new activating groups on the carboxyl group of the incoming amino acid have improved coupling and decreased epimerization. Finally, the process itself has been optimized. In Merrifield initial report, the deprotection of the amino group resulted in the formation of a peptide-resin salt, which required neutralization with base prior to coupling. The time between neutralization of the amino group and coupling of the next amino acid allowed for aggregation of peptides, primarily through the formation of secondary structures, and adversely affected coupling. The Kent group showed that concomitant neutralization of the amino group and coupling of the next amino acid led to improved coupling. Each of these improvements has helped SPPS become the robust technique that it is today.
Synthesizing long peptides:
Stepwise elongation, in which the amino acids are connected step-by-step in turn, is ideal for small peptides containing between 2 and 100 amino acid residues. Another method is fragment condensation, in which peptide fragments are coupled. Although the former can elongate the peptide chain without racemization, the yield drops if only it is used in the creation of long or highly polar peptides. Fragment condensation is better than stepwise elongation for synthesizing sophisticated long peptides, but its use must be restricted in order to protect against racemization. Fragment condensation is also undesirable since the coupled fragment must be in gross excess, which may be a limitation depending on the length of the fragment.
A new development for producing longer peptide chains is chemical ligation: Unprotected peptide chains react chemoselectively in aqueous solution. A first kinetically controlled product rearranges to form the amide bond. The most common form of native chemical ligation uses a peptide thioester that reacts with a terminal cysteine residue.
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 this phenomenon, the microwave energy can prevent aggregation and thus increases yields of the final peptide product. There is however no clear evidence that microwave is better than simple heating and some peptide laboratories regard microwave just as a convenient method for rapid heating of the peptidyl resin. Heating to above 50-55 degrees Celsius also prevents aggregation and accelerates the coupling.
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. A number of peptides does not survive microwave synthesis or heating in general. One of the more serious side effects is dehydration (loss of water) which for certain peptides can be almost quantitative like pancreatic polypeptide (PP). This side effect is also seen by simple heating without the use of microwave.
As of January 2009, over 200 microwave peptide synthesizers are in use with the rate of acceptance increasing.
This is a relatively straightforward operation, where two cysteines are oxidised under carefully controlled conditions to form a disulphide bridge. These peptides are stable at neutral to low pH but can be reversibly reduced at higher pH.
These are formed when an activated amino function reacts with the side chain of a Cys residue to create a thioether. Advantages are the ease of formation and their pH stability.
These are highly suited to making structurally restrained peptide libraries.
Cyclic through a peptide bond.
Much depends on the natural folding of the peptide in the solvent used for the cyclisation reaction.This process is more problematic than the disulphide or thioether route.
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