Peptide Synthesis Techniques | FMOC & SPPS Explained

Peptides

Peptides are short sequences of amino acids, either fragments of proteins, or biological active sequences. These 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.

Example of a peptide structure used in CASLO peptide synthesis

The three peptide synthesis methods

Scientists can categorize peptide synthesis 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 (recombinant technique) uses living cells as the factory to produce the peptide, and then scientists extract the peptide 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.

CASLO peptide bond formation reaction diagram

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: CASLO attaches the first amino acid at the C-terminus of the peptide to an insoluble polymeric support, called resin, through a covalent bond between the carboxyl group of the amino acid and the resin. A Fmoc protection group protects the amino group at the N-terminus, and side-chain protection groups protect the side-chains that have functional groups.

The second step is then to remove the Fmoc protection on the N-terminus of the resin bound peptide. This is done without removing the protection groups on the side-chains. The third step is to add excess of a protected amino acid. This amino acid is the next one in the peptide sequence. This step also adds the necessary coupling reagents and solvents to carry out the coupling reaction.

CASLO first activates the carboxyl group of the incoming amino protected amino acid. This amino acid then reacts with the amino group of the previous amino acid in the peptide sequence. This happens after CASLO removes the Fmoc group, to form the peptide bond. In solid phase peptide synthesis, CASLO uses excess (2 to 10 folds) of incoming amino acid. This drives the peptide formation to completion.

CASLO thoroughly washes the peptide-resin after each step. This removes all the excess reagents and by-products. Only the peptide attached to the resin stays in the reaction container. CASLO carries on repetition of the cycle by N-terminal deprotection, wash, coupling, wash for each amino acid.

This continues until the peptide sequence has been achieved. Then, CASLO releases the peptide chain from the resin. At the same time, CASLO removes the side-chain protecting groups in the same cleavage procedure. This happens when CASLO releases the peptide 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. This happens during the synthesis. Some peptide remains on the resin without reacting with the amino acid in the coupling step. This peptide will have its N-terminus blocked by a capping procedure. Therefore, only a very low amount of the wrong peptide will participate in the next coupling step.

The peptide that CASLO releases from the resin is a raw peptide, and it typically has a relatively low purity. To get the desired purity, CASLO must therefore 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 CASLO removes them during purification. CASLO can therefore obtain a very high purity by HPLC purification of the raw peptide. CASLO then analyzes the purified peptide by mass spectrometry to verify the sequence.

Diagram of CASLO Fmoc solid-phase peptide synthesis steps

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

Chemical structure of an amidated C-terminus in CASLO peptide synthesis

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.

Chemical structures of resins used in CASLO Fmoc peptide synthesis

The choice of protection groups for Fmoc peptide synthesis

During peptide synthesis, CASLO needs to protect the N-terminal amino group with a 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, CASLO must protect them. The side-chain protecting groups have to remain stable during the deprotection of the N-terminal Fmoc protecting group, and CASLO must cleave them off in the final cleavage step.

In Fmoc peptide chemistry, CASLO accomplishes the deprotection of Fmoc in mild basic solution (piperidine), and the side-chain protecting groups have to remain stable under this condition. They must however, easily cleave 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, CASLO needs to remove some protecting groups individually without affecting others. Some of these special protection groups are: Lys(Dde), Lys(Mmt), Asp(ODmab) and Cys(Acm).

CASLO can easily remove the N-terminal amino protecting group Fmoc by using 20-30% Piperidine in DMF. The reaction is very fast, usually reaching completion within 4-10 min. Adding 0.1M HOBt to the deprotecting mixture suppresses the side reaction of the aspartimide formation when Asp is in the peptide sequence.

The Fmoc deprotection can become sluggish when the secondary structure formation of the peptide is high. In this case, CASLO would first choose to extend the reaction time. When such action is not sufficient, CASLO can use the stronger base DBU together with piperidine.

Chemical structures of Hobt

Cleavage and deprotection of peptides from peptide synthesis

In Fmoc solid phase peptide chemistry, CASLO carries out the cleavage of a peptide from resin and deprotection of all the side chain protecting groups 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. These cations can react with the peptide again, forming stable covalent bonds. This produces unwanted impurities, which in turn can cause problems for peptide purification. CASLO can suppress these side reactions by adding nucleophilic scavengers. These scavengers “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.

Each peptide molecule has a certain binding affinity towards the column packing material, called the solid phase. It also has a certain distribution, or partition, between the solid phase and the mobile phase.

Scientists use the differences in physical properties between the target peptide and the by-products, like sequences with missing amino acids, 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.

Principle of HPLC Diagram

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 elute off from the column. The most hydrophobic peptides are the last peptides that the column releases. The larger the hydrophobicity difference is between the peptides, the better the separation will be.

CASLO controls the peptide by mass spectrometry before and after HPLC purification, to confirm the correct peptide sequence.