Detailed description of Fmoc solid phase peptide synthesis

Resins for peptide synthesis
The most commonly used resins in Fmoc solid phase peptide synthesis are Wang resin, Rink resin and in some cases CTC resin. Rink resin is used to make the C- terminal amidated peptides, while the other two are used to make the C-terminal free peptides.

Peptide synthesis and choice of resin
There exist several different resins for peptide synthesis but most often are there are used Wang resin, Rink resin or CTC resin. Wang resin gives the most stable bond between between the peptide chain and the resin, which gives much more consistent yield of the crude peptide. CTC resin is very sensitive, the temperature and humidity during the reaction, as well as the swelling and shrinking of the resin between washes, can affect the stability of the bond between the peptide chain and the CTC resin, which can give variation of the yield of the crude peptide. CTC resin is however, still frequently used for peptide synthesis because CTC resin has its own unique advantages, especially for peptides that have cysteine, histidine or proline in the C-terminal position. The reactivity of the functional group on CTC resin is very high. The first amino acid can be coupled to the resin without activation of the carboxyl group, which minimize the risk for racemization of the first amino acid. This property of the CTC resin is very helpful when for example making peptides that have cysteine or histidine as the C-terminal amino acid, because these two amino acids are prone to racemization. Furthermore, there are also a range of other advantages of CTC resin.

rink resin

 

Protection of amino acid side chains and N-terminus for Fmoc solid phase peptide synthesis
During peptide synthesis, the N-terminal amino group needs to be protected by Fmoc protection group. It is however, also necessary to protect the active functional groups on the side chains of many amino acids. These active side chain groups will not only interfere with the peptide bond formation during the coupling, but also cause side reactions during the final cleavage. Therefore, they must be protected. The side-chain protecting groups have to be stable during the deprotection of the N-terminal Fmoc protecting group, and they must be cleaved off in the final cleavage step.

Peptide synthesis and choice of protection groups
In Fmoc peptide chemistry, the deprotection of Fmoc is accomplished in mild basic solution (piperidine), and the side-chain protecting groups have to be stable under this condition. They must however, be easily cleaved under the final acid resin cleavage condition. The most commonly used side-chain protecting groups in Fmoc peptide chemistry are the following: Arg(Pbf),Asn(Trt),Asp(OtBu),Cys(Trt),Glu(OtBu),Gln(Trt),His(Trt),Lys(Boc),Ser(tBu),Thr(tBu),Trp(Boc), Tyr(tBu). When making modified peptides, some protecting groups need to be removed individually without affecting others. Some of these special protection groups are: Lys(Dde),Lys(Mmt),Asp(ODmab),Cys(Acm).
The N-terminal amino protecting group Fmoc can be easily removed by 20-30% Piperidine in DMF. The reaction is very fast, usually reaches completion within 4-10 min. Adding 0.1M HOBt to the deprotecting mixture will suppress the side reaction of the aspartimide formation when Asp is in the peptide sequence. The Fmoc deprotection can be sluggish when the secondary structure formation of the peptide is high. In this case, the first choice would be to extend the reaction time. When such action is not sufficient, the stronger base DBU can be used together with piperidine.

fmoc

Formation of the peptide bond during peptide synthesis
The N-terminal amino protecting group Fmoc is removed by adding 20-30% piperidine diluted in DMF. The coupling mechanism in solid phase peptide synthesis is basically the same as in solution, the carboxyl group of the incoming amino protected amino acid will be activated first and then it will react with the amino group of the previous amino acid in the peptide sequence after the Fmoc group has been removed to form the peptide bond. In solid phase peptide synthesis the excess (2 to 10 folds) of incoming amino acid is used to drive the peptide formation to completion. The excess materials, together with the side products in reaction mixture, are removed simply by wash, once the formation reaction is completed, because the peptide is bound to resin via the C-terminus.
The carboxyl group of the incoming amino acid in the peptide sequence needs to be activated. The activating reagent must be highly reactive and at the same time be able to suppress the racemization of the amino acid.

Cleavage and deprotection of peptides from peptide synthesis
In Fmoc solid phase peptide chemistry, the cleavage of a peptide from resin and deprotection of all the side chain protecting groups are carried out at the same time in trifluoroacetic acid (TFA) solution. During this process, there are many reactions going on simultaneously. The by-products from the cleaved side chain protecting groups are highly active cations, which can react with the peptide again forming stable covalent bonds to produce unwanted impurities, which in turn can cause serious problems for peptide purification. These side reactions can be suppressed by adding nucleophilic scavengers that will "trap" the cations before they react with the peptide. Most commonly used scavengers in peptide chemistry include water, EDT, TA , TIPS , phenol etc.

Purification and characterization of peptides made by peptide synthesis
The separation principle of the HPLC when purifying peptides is illustrated in Figure 4. Basically, each peptide molecule has certain binding affinity towards the column packing material (called solid phase) and certain distribution (partition) between solid phase and mobile phase. The differences in physical properties between the target peptide and the by-products like sequences with mising amino acids are used for purification and characterization. The stronger the binding interaction between the peptide and the packing material, the longer it takes for the peptide to elute from the column (red dots).
Before and after HPLC purification the peptide is controlled by mass spectrometry to confirm the correct peptide sequence.

mobile phase

Schematic illustration of the principle of HPLC.

The major HPLC method used in peptide production are reverse phase HPLC (RP-HPLC). In RP-HPLC, peptides bind to the column packing material through hydrophobic interactions. Most of the RP-HPLC column packing materials are silicon based polymers with hydrocarbon alkyl chains on the surface. The numbers of carbons in the hydrocarbon alkyl chains vary and the longer the alkyl chain, the more hydrophobic the packing material is. As the hydrophobicity of the mobile phase increases, the peptide molecules will be eluted off from the column. The most hydrophobic peptides will be the last peptides that are released from the column. The larger the hydrophobicity difference is between the peptides, the better the separation will be.