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CASLO ApS is a Danish company specialized in peptide synthesis technologies. CASLO offers reliable customer designed peptides at low prices. Products from CASLO are delivered worldwide, and we have regular customers in more than ten countries. Please contact CASLO with the information necessary for the peptide synthesis and you will receive a very competitive price quotation within 24 hours. CASLO gives fast and professional support and products are delivered with money back guarantee.

Peptide Synthesis

Peptides are short sequences of amino acids, either fragments of proteins, or independend biological active sequences. Peptides can be biological active, for example several hormones are peptides, but they can also be copies of inactive or active parts of proteins. Synthetic peptides are peptides that are synthesized in a laboratory.
Peptide synthesis can be categorized into two types: Recombinant biological peptide synthesis and chemical peptide synthesis. Recombinant biological peptide synthesis uses living cells as the factory to produce the peptide and the peptide is then extracted from the cells. Synthetic peptides are produced in a laboratory through the condensation between amino acids. The recombinant biological peptide synthesis is superior for peptides longer than 70-80 amino acids whereas the chemical peptide synthesis is superior for shorter peptides or modified peptides. CASLO offer only peptides made by chemical peptide synthesis.

peptide bond
Figure-1: Formation of peptide bond

Peptide synthesis by chemical method
Peptide synthesis is made by binding amino acids together via amide bonds, according to specific sequence information. When there is more than one functional group in an amino acid, which may interfere with the specific peptide bond formation, those groups that shall not participate will be protected temporarily to ensure that the peptide synthesis will proceed in the right direction.

Solid phase chemical peptide synthesis
Solid phase peptide synthesis is applicable to almost all peptides regardless of the number and the arrangement of amino acids in the sequence. In general it is however, complicated to make peptides much longer than 70-80 amino acids by solid phase peptide synthesis. Peptides much longer than 70-80 amino acids should be made by biological recombinant peptide synthesis. A unique advantage of solid phase peptide synthesis method is that it has made the automation of the synthesis possible, which leads to the possibility to obtain large number of peptides within a short period of time. CASLO only offers peptides made by solid phase peptide synthesis.
When peptides and proteins are made in living cells, it starts from the N-terminus and the biological synthesis is then made from N- to C-terminus. This is also the reason why peptides are written from N- to C-terminus. In solid phase peptide synthesis however, the peptide chain is made from the C-terminus to the N-terminus of the peptide. In general, 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 solid phase support or resin) through the covalent bond between the carboxyl group of the amino acid and the resin. The amino group and the side-chain that has a functional group will be temporarily protected. The second step is then to remove the protection on the N-terminus of the resin bound peptide, without removing the protection on the side chain. The third step is to add excess of a protected amino acid which is next in the peptide sequence, plus the necessary coupling reagents and solvents to carry out the coupling reaction. The peptide-resin will be thoroughly washed in 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 of 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 called 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. There are two major solid phase peptide synthesis methods based on the N-terminal amino protecting group, Boc peptide synthesis and Fmoc peptide synthesis. Fmoc-chemistry is almost always used in modern peptide synthesis.

peptide synthesis cycle
Figure 2. Schematic representation of solid phase peptide synthesis process.

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. The following link describes resins for peptide synthesis in details:

Peptide synthesis and choice of resin

Protection of amino acid side chains and N-terminus for Fmoc solid phase peptide synthesis
During the 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.

repeat the cycle
Figure-3: Illustration of solid phase peptide synthesis

Please see the following link for more information about protection groups:

Peptide synthesis and choice of protection groups

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
Even though it is one of the strong advantages of solid phase peptide synthesis that no intermediate separation is needed, it also cause difficulties in the HPLC purification of peptides. Any by-product on the resin will be accumulated and mixed with the target peptide in the crude product after cleavage. Some of them have very similar physical and chemical properties to the target peptide, for example, with only one or few amino acids being deleted (deletion sequence), or with one or few protecting groups being re-attached to the target peptide. The traditional methods of separation in solution phase peptide synthesis, such as extraction, re-crystallization, etc. can not be used to obtain high purity of target peptides from solid phase peptide synthesis. HPLC is now nearly always used for peptide purification and characterization of the purity. The separation principle of the HPLC 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 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).

mobile phase
Figure 4. Schematic illustration of the principle of HPLC.

The two major HPLC methods used in peptide production are reverse phase HPLC (RP-HPLC) and ionic exchange HPLC (IE-HPLC). CASLO peptides are always purified and characterized by RP-HPLC so this method will be described here. 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.