The most widely used coupling method in Fmoc SPPS is the activated ester method either pre-formed (pre-activated species) or in situ (without pre-activation).
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.
Usually, Fmoc is split off by a short treatment (3 to 5 minutes) with piperidine/DMF (1:4). In general this treatment is repeated and slightly prolonged (7 to 10 minutes). Under those conditions complete deblocking is attained in most cases. Thus, deviations are restricted to cases of sluggish cleavage or base-sensitive sequences.
Two major chemistries for solid phase peptide synthesis are Fmoc (base labile protecting group) and t-Boc (acid labile a-amino protecting group). Each method involves fundamentally different amino acid side-chain protection and consequent cleavage/deprotection methods, and resins; t-Boc method requires use of stronger HF containing anisole alone or anisole plus other scavengers, where peptide-resins assembled by Fmoc chemistry usually cleaved by less harsh Reagents K or R. Fmoc chemistry is known for peptide synthesis of higher quality and in greater yield than t-Boc chemistry. Impurities in t-Boc-synthesized peptides mostly attributed to cleavage problems, dehydration and t-butylation. For peptide assembly HBTU/HOBt, carbodiimide-mediated coupling and PyBOP/HOBt are the most popular routines. Peptides usually purified by reversed-phase HPLC (high performance liquid chromatography) using columns such as C-18, C-8, and C-4.
Solid-phase peptide synthesis consists of three distinct sets of operations: 1) chain assembly on a resin; 2) simultaneous or sequential cleavage and deprotection of the resin-bound, fully protected chain; and 3) purification and characterisation of the target peptide. Various chemical strategies exist for the chain assembly and cleavage/deprotection operations, but purification and characterisation methods are more or less invariant to the methods used to generate the crude peptide product.
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.
The following is an outline of the synthetic steps for peptide synthesis on polyamide or polystyrene resin, using the base labile 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group. Using the techniques outlined below, one will obtain a peptide which is capped on the N-terminus with and acetyl group, and on the C-terminus with a primary amide (CONH2).
A new developement 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 native chemical ligation uses a peptide thioester that reacts with a terminal cystein residue.
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 attachement 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(carbonil group) more efficient. The problem with carbodiimides is that they are too reactive and that they can therefore cause racemization of the amino acid.
In addition, ONSu esters of Fmoc amino acids were prone to the formation of the side product succinimido-carbonyl--alanine-N-hydroxysuccinimide ester (3-4).
A special paragraph will be dedicated to the problems caused by peptide aggregation in the course of the synthesis. This phenomenon is a major cause of trouble as it is difficult to predict, is sequence dependent and no universal solution has been found up to now.
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 -15oC until needed.
Better results will be obtained by repeating a coupling with fresh reagents (and changing coupling parameters if a low conversion was obtained) rather than by prolonging the reaction. Generally, coupling protocols may be changed in the course of a synthesis, especially when optimizing an SPPS.