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 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.
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.
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.
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.
FMOC is preferred over BOC due to ease of cleavage; however it is less atom-economical, as the fluorenyl group is much larger than the tert-butyl group. Accordingly, prices for FMOC amino acids were high until the large-scale piloting of one of the first synthesized peptide drugs, enfuvirtide, began in the 1990s, when market demand adjusted the relative prices of the two sets of amino acids.
Because the liberated Fluorenyl group is a chromophore, deprotection by FMOC can be monitored by UV absorbance of the runoff, a strategy which is employed in automated synthesizers.
Another carbamate based group is the benzyloxy-carbonyl (Z) group. It is removed in harsher conditions: HBr/acetic acid or catalytic hydrogenation. Today it is almost exclusively used for side chain protection.
These activating agents were first developed. Most common are dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC). Reaction with a carboxylic acid yields a highly reactive O-acyl-urea. During artificial protein synthesis (such as Fmoc solid-state synthesizers), the C-terminus is often used as the attachment site on which the amino acid monomers are added. To enhance the electrophilicity of carboxylate group, the negatively charged oxygen must first be "activated" into a better leaving group. DCC is used for this purpose. The negatively charged oxygen will act as a nucleophile, attacking the central carbon in DCC. DCC is temporarily attached to the former carboxylate group (which is now an ester group), making nucleophilic attack by an amino group (on the attaching amino acid) to the former C-terminus (carbonyl group) more efficient. The problem with carbodiimides is that they are too reactive and that they can therefore cause racemization of the amino acid.
To solve the problem of racemization, triazolols were introduced. The most important ones are 1-hydroxy-benzotriazole (HOBt) and 1-hydroxy-7-aza-benzotriazole (HOAt). Others have been developed. These substances can react with the O-acylurea to form an active ester which is less reactive and less in danger of racemization. HOAt is especially favourable because of a neighbouring group effect. Recently, HOBt has been removed from many chemical vendor calalogues; although almost always found as a hydrate, HOBt may be explosive when allowed to fully dehydrate and shipment by air or sea is heavily restricted.
Newer developments omit the carbodiimides totally. The active ester is introduced as a uronium or phosphonium salt of a non-nucleophilic anion (tetrafluoroborate or hexafluorophosphate): HBTU, HATU, PyBOP.
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 cystein residue.
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. Another disadvantage is that allyl containing amino acid derivatives cannot be coupled to amino acids using microwave irradiation due to uncontrolled polymerization.
The eficciency of microwave energy in enancing the solid phase synthesis of peptides is still a matter of debate. Recent work has shown that there is no "microwave effect" that enhances the solid phase synthesis of peptides. The increased reaction rates observed when coupling amino acids in a microwave reactor are only a result of the high temperatures used in this reactor.
Setting up glassware for manual peptide synthesis
Manual peptide synthesis can be accomplished in a fritted-filter reaction vessel with a three-way valve fitted onto a 1 L side arm vacuum flask by way of a 1-hole stopper. One valve is used to bubble nitrogen, which is first passed through a small column of Drierite, and then into the reaction mixture to agitate the solution and mix reagents. The other valve is used to evacuate excess reaction solutions and wash solvent using a vacuum flask. All glass pieces to be used in Solid-phase synthesis should be treated with a silanizing agent (such as 1-5% dimethyldichlorosilane in DCM) prior to use, to avoid accumulation of static charge, which makes the resin very difficult to handle.
Preparation of polyamide-Rink resin
Polyamide (PL-DMA) resin (1g) is treated with ethylene diamine (40 ml) in a 50 ml Falcon tube overnight on a rocker, then filtered, washed with 5x10 ml of 1:1 dimethylformamide (DMF):dichloromethane (DCM) solution, 5x10 ml of 1:1 DCM, and loaded with Fmoc-Rink using Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (3 eq), 1-Hydroxybenzotriazole Hydrate (HOBt) (3 eq), and Diisopropylethylamine (DIPEA) (6 eq) in 1:1 DCM:DMF. It can then be dried under vacuum and stored at −15 °C until needed.
Handling the resin before, and during synthesis
The resin is first swelled for 15 minutes in 10 ml of 1:1 DCM:DMF and drained. The resin is also washed with 5x10 ml of 1:1 DCM and DMF after each completed amino acid coupling.
Fmoc deprotection after each amino acid coupling is accomplished using 2x10 ml of 20% piperidine in DMF, with N2 agitation for 10 minutes each treatment. The resin is then washed with 5x10 ml DMF, followed by 5x10 ml of 1:1 DCM:DMF. An alternate treatment is 1% DBU in DMF; this gentler treatment allows removal of Fmoc groups in the presence of other base-labile moieties.
Adding amino acids
An amino acid is coupled to the deprotected N-terminal amine of the resin, or previously coupled amino acid, using a coupling mixture such as the protected amino acid (3 eq), PyBOP (3 eq), HOBt (3 eq), and DIPEA (6 eq) in 1:1 DCM:DMF until the resin is negative to ninhydrin. Another popular set of coupling conditions is amino acid (4.4 eq), HBTU (4 eq), and DIPEA (8 eq) in DMF. Amino acids can also be purchased as the pre-activated ester, in which case coupling agents such as HBTU and PyBOP are unnecessary.
Monitoring the progress of amino acid couplings
The progress of amino acid couplings can be followed using ninhydrin, or p-chloranil. The ninhydrin solution turns dark blue (positive result) in the presence of a free primary amine but is otherwise colorless (negative result). The p-chloranil solution will turn the resin beads dark black or blue in the presence of a primary amine if acetaldehyde is used as the solvent or in the presence of a secondary amine, if acetone is used instead; the beads remain colorless or pale yellow otherwise. (The tests are outlined below)
Testing by ninhydrin (1)
Add 2 drops of 40% phenol in ethanol, 2 drops of 0.014 mol/L KCN in pyridine, and 4 drops of 5% ninhydrin in ethanol to a microcentrifuge tube along with a spatula tip size sample of resin, then vortex the mixture and heat for 5 minutes at 100 °C.
Testing by chloranil (2)
Add 5 drops of acetone or acetaldehyde, 5 drops of a saturated solution of p-chloranil in toluene, plus a small spatula-tip-size sample of resin to a microcentrifuge tube, then vortex the mixture and allow to stand at room temperature for 5 minutes. Acetone is used for the detection of secondary amines, where acetaldehyde is used for primary amines.
Continuing peptide extension
Once the coupling of the amino acid is complete, the resin is washed, the Fmoc group deprotected with piperidine, and the resin washed again to prepare it for the next coupling. This process is repeated until all necessary amino acids have been added.
Acetylating the N-terminus
After the peptide sequence is completed, the N-terminal amine can be acetylated with 2 ml of 1:1 acetic anhydride and triethylamine in 10 ml of 1:1 DCM:DMF for 1 hour or until negative to ninhydrin, and the resin then washed with 5x10 ml of 1:1 DCM:DMF, before the peptide is cleaved from the resin. The N-terminus can also be left as the free amine if required.
Cleaving the peptide from the resin
The resin is treated with a cocktail of trifluoroacetic acid (TFA) and cleavage scavenger reagents, such as triisopropylsilane (TIPS), 1,2-ethanedithiol, and water (H2O). The choice of scavengers is dependent on the amino acid sequence. The resin is then filtered away, and the combined filtrates allowed to stand for 1 hour to ensure removal of the acid labile protecting groups.
Workup of peptides after cleavage from the resin
The TFA is evaporated to dryness (or a heavy oil or glass if it does not solidify) on the rotary evaporator, followed by the addition of 5 ml of diethyl ether to the flask to precipitate the peptide, and remove the bulk of the by-products. Once the peptide is precipitated with the ether, filter through a sintered glass funnel and redissolve peptide into 60% AcN/ 0.1 %TFA. The peptide solution is then frozen in a dry ice/ethanol bath and lyophilised (dried). The result is a crude peptide which is stable for a number of years. The peptide can then be purified by Ion-exhange and Reverse Phase chromatography.