Peptide Synthesis Techniques

Peptides

Peptides are short sequences of amino acids, either fragments of proteins, or biological active sequences. Peptides can be biological active, for example several hormones are peptides, but they can also be copies of smaller parts of proteins. Synthetic peptides are peptides that are synthesized in a laboratory.

The three peptide synthesis methods

Peptide synthesis can be categorized into three different types: 1) Recombinant biological peptide synthesis, 2) Chemical solution phase peptide synthesis and 3) Solid phase peptide synthesis.

A peptide made by Recombinant biological peptide synthesis (recombinat technique) uses living cells as the factory to produce the peptide, and the peptide is then extracted from the cells. Peptides made by recombinant technique are superior for peptides longer than 60-80 amino acids. The disadvantage is that it is expensive to make these peptides, and very time consuming. It is only possible to make very few modifications on peptides made by recombinant techniques. CASLO does not offer peptides made by recombinant technique.

Peptides made by chemical solution phase peptide synthesis and solid phase peptide synthesis are produced in a laboratory through the condensation between amino acids. Chemical solution phase peptide synthesis is superior for peptides 2-6 amino acids long in large quantities (10 grams up to kg). The technique is too expensive to be used for smaller quantities, and can not be used for longer peptides. CASLO does not offer peptides made by chemical solution phase peptide synthesis.

Laboratories nearly only use one special variant of solid phase peptide synthesis named Fmoc solid phase peptide synthesis. CASLO only offers peptides made by Fmoc solid phase peptide synthesis. The technique can be used for peptides from 2-3 amino acids up to 60-70 amino acids long. Fmoc solid phase peptide synthesis can be used for quantities from 1 mg up to multiple grams.

Fmoc Solid phase peptide synthesis

In Fmoc solid phase peptide synthesis the peptide chain is made from the C-terminus to the N-terminus of the peptide (please note that peptides in cells are made from N- to C-terminus, and peptide sequences are also always written from N- to C-terminus).

In general Fmoc solid phase peptide synthesis involves the following steps: The first amino acid at the C-terminus of the peptide is attached to an insoluble polymeric support (called resin) through a covalent bond between the carboxyl group of the amino acid and the resin. The amino group at the N-terminus is protected by a Fmoc protection group and the side-chains, that have functional groups, will be protected by side-chain protection groups. The second step is then to remove the Fmoc protection on the N-terminus of the resin bound peptide, without removing the protection groups on the side-chains. The third step is to add excess of a protected amino acid which is the next amino acid in the peptide sequence, plus the necessary coupling reagents and solvents to carry out the coupling reaction. 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 peptide-resin will be thoroughly washed after each step so that all the excess reagents and by-products will be removed and only the peptide attached to the resin will stay in the reaction container. Repetition of the cycle is carried on by N-terminal deprotection, wash, coupling, wash for each amino acid until the peptide sequence has been achieved. Then, the peptide chain is released from the resin, and the side-chain protecting groups are removed in the same cleavage procedure, when the peptide is released from the resin.

Each coupling step is rarely 100%. This means that a number of peptides with amino acids missing from the correct sequence, are build up during the synthesis. The amount of peptide on the resin, that has not reacted with the amino acid in the coupling step, will have the N-terminus blocked by a capping procedure and therefore, only a very low amount of the wrong peptide, will participate in the next coupling step. The peptide that is released from the resin is a raw peptide, and it has typically a relatively low purity. In order to get the desired purity it is therefore, necessary to purify the peptide by high pressure liquid chromatography (HPLC). The peptides that have amino acids missing from the correct sequence will have different retention times in the HPLC procedure compared with the correct peptide, and will be removed during purification. It is therefore possible to obtain a very high purity by HPLC purification of the raw peptide. The purified peptide is then analyzed by mass spectrometry to verify the sequence.

Resins for peptide synthesis

There exist several different resins for peptide synthesis but 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

Choice of resin for peptide synthesis

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 choice of protection groups for Fmoc 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.

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) and 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) and Cys(Acm).

The N-terminal amino protecting group Fmoc can easily be 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.

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 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 below. 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.

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.

Before and after HPLC purification the peptide is controlled by mass spectrometry to confirm the correct peptide sequence.