Peptide synthesis is the production of peptides, which are organic compounds in which multiple amino acids bind via peptide bonds which are also known as amide bonds. The biological process of producing long peptides (proteins) is known as protein biosynthesis.
Chemistry
Peptides are synthesized by coupling the carboxyl group or C-terminus of one amino acid to the amino group or N-terminus of another.
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.
Solid-phase synthesis
Coupling step in solid-phase peptide synthesis
Solid-phase peptide synthesis (SPPS), pioneered by Robert Bruce Merrifield,[1] 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 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-deprotection. 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 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 70~100 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.
t-Boc solid-phase peptide synthesis
When Merrifield invented SPPS in 1963, it was according to the t-Boc method. t-Boc (or Boc) stands for (t)ert-(B)ut(o)xy(c)arbonyl. To remove Boc from a growing peptide chain, acidic conditions are used (usually neat TFA). Removal of side-chain protecting groups and the peptide from the resin at the end of the synthesis is achieved by incubating in hydrofluoric acid (which can be dangerous or even deadly); for this reason Boc chemistry is generally disfavored. Also, HF cleavage needs to be done in special fume hoods using specialized equipment. However for complex syntheses Boc is favourable. When synthesizing nonnatural peptide analogs which are base-sensitive (such as depsipeptides), Boc is necessary.
Fmoc solid-phase peptide synthesis
This method was introduced by Carpino in 1972 and further applied by Atherton in 1978. Fmoc stands for 9H-(f)luoren-9-yl(m)eth(o)xy(c)arbonyl which describes the Fmoc protecting group, first described as a protecting group by Carpino in 1970. To remove an Fmoc from a growing peptide chain, basic conditions (usually 20% piperidine in DMF) are used. Removal of side-chain protecting groups and peptide from the resin is achieved by incubating in trifluoroacetic acid (TFA), deionized water, and triisopropylsilane. Fmoc deprotection is usually slow because the anionic nitrogen produced at the end is not a particularly favorable product, although the whole process is thermodynamically driven by the evolution of carbon dioxide. The main advantage of Fmoc chemistry is that no hydrofluoric acid is needed. It is therefore used for most routine synthesis.
The use of BOP reagent was first described by Castro et al in 1975.
Solid supports
The physical properties of the solid support, and the applications to which it can be utilized, vary with the material from which the support is constructed, the amount of crosslinking, as well as the linker and handle being used.
Polystyrene resin
Polystyrene resin is a versatile resin and it is quite useful in multi-well, automated peptide synthesis, due to its minimal swelling in dichloromethane.
Polyamide resin
Polyamide resin is also a useful and versatile resin. It seems to swell much more than polystyrene, in which case it may not be suitable for some automated synthesizers, if the wells are too small.
PEG based Resin
ChemMatrix(R) is a new type of resin which is based on PEG that is crosslinked. ChemMatrix(R) has claimed a high chemical and thermal stability (is compatible with Microwave synthesis) and has shown higher degrees of swellings in acetonitrile, dichloromethane, DMF, N-methyl pyrollidone, TFA and water compared to the polystyrene based resins. ChemMatrix has shown significant improvements to the synthesis of hydrophobic sequences. ChemMatrix is recommended for the synthesis of difficult and long peptides.
Protecting groups
Due to amino acid excesses used to ensure complete coupling during each synthesis step, polymerization of amino acids is common in reactions where each amino acid is not protected. In order to prevent this polymerization, protecting groups are used. This adds additional deprotection phases to the synthesis reaction, creating a repeating design flow as follows:
- Protective group is removed from trailing amino acids in a deprotection reaction
- Deprotection reagents washed away to provide clean coupling environment
- Protected amino acids dissolved in a solvent such as dimethylformamide (DMF) are combined with coupling reagents are pumped through the synthesis column
- Coupling reagents washed away to provide clean deprotection environment
Currently, two protective groups (t-Boc, Fmoc) are commonly used in solid-phase peptide synthesis. Their lability is caused by the carbamate group which readily releases CO2 for an irreversible decoupling step.
t-Boc protective group
Main article: Di-tert-butyl dicarbonate
The t-Boc group was commonly used for protecting the terminal amine of the peptide, requiring the use of more acid stable groups for side chain protection in orthogonal strategies. It retains usefulness in reducing aggregation of peptides during synthesis. Boc groups can be added to amino acids with t-Boc anhydride and a suitable base.
Boc cleavage
Fmoc protective group
Fmoc cleavage
The Fmoc (9H-fluoren-9-ylmethoxycarbonyl) is currently a widely used protective group that is generally removed from the N terminus of a peptide in the iterative synthesis of a peptide from amino acid units. The advantage of Fmoc is that it is cleaved under very mild basic conditions (e.g. piperidine), but stable under acidic conditions. This allows mild acid labile protecting groups that are stable under basic conditions, such as Boc and benzyl groups, to be used on the side-chains of amino acid residues of the target peptide. This orthogonal protecting group strategy is common in the art of organic synthesis.
