Chemical Synthesis Services
Starting with protected amino acid building blocks and the appropriate resin CSS decides on a synthetic strategy to reach the target sequence.
Peptides can be made linearly from a polymeric support (shown below) starting from commercially available, protected amino acid building blocks or segments of the peptide can be made then coupled together until the target sequence is obtained (shown below).
Peptide segment ligation
Whatever the synthetic strategy each synthesis must start with the appropriate resin. This will depend on the C-terminus of your peptide whether it be acid, amide, hydrazide or some other functionality. Each amino acid must be protected at the N-terminus and any side-chain functionalities must also be protected to avoid unambiguous couplings. CSS uses commercially available Fmoc or Nsc protected amino acids. These groups are cleaved in mild base and use orthogonal acid labile side-chain protection which are removed on completion of the synthesis when the peptide is cleaved from the resin.
Before an amino acid can be coupled to the free amino of another amino acid its carboxyl terminus must first be activated with a coupling reagent. CSS has developed its very own coupling reagent for this purpose. The reagent has proven to be superior to other popular methods for activation with our technologies facilitating the synthesis of longer chain peptides and proteins, and indeed those peptides with difficult sequences to make.
During synthesis we monitor each coupling to ensure that the synthesis is proceeding as efficiently as possible and, if not, steps are taken to improve this by performing different coupling techniques or to restart the synthesis if it appears poor. This not only ensures that we keep the cost of peptides low but it also allows us to inform the customer of any changes to delivery time early on in the contract.
The first step in the synthesis is to attach the first protected amino acid at the C-terminus to the appropriate resin. This is then placed on the synthesiser where it is deprotected so that it can be coupled to the next amino acid in the sequence.
The deprotection solution is passed through a UV monitor which is specific for the protecting group used. The monitor is connected to a chart recorder which gives a peak according to the amount of protecting group present. By comparing the area under this initial deprotection peak with each subsequent deprotection in the synthesis we can calculate the percentage coupling for each amino acid and obtain a deprotection profile.
Once a peptide synthesis is complete the most difficult process in obtaining the target sequence must be addressed - its purification. HPLC, gel filtration, dialysis are very reputable methods for purifying proteins. However, the impurities from a peptide synthesis can be so similar in molecular weight and structure to the target sequence that it can be difficult, sometimes even impossible, to pull out the correct material. CSS has overcome this problem by developing a tag which, once bound to the N-terminus of the completed sequence on the resin, can either act as a hydrophobic chromatographic probe or can be bound to an insoluble support to aid purification after cleavage of the peptide from the resin.
Below is an example of a peptide cleaved with and without the purification tag.
Trace A is guinea pig eotaxin with the purification tag attached, the difference in retention time between the tag peptide and its impurities is 3-4min. Trace B is guinea pig eotaxin without the tag; the target peptide elutes exactly with the impurities thus HPLC purification is virtually impossible. Gel filtration is also difficult due to the nature of the impurities. The tag aided purification of this peptide dramatically i.e single step.