Service - Peptide custom synthesis - Technical Information

Technical Information

GeneCust peptides are obtained through solid phase synthesis using Fmoc chemistry and we can obtain several hundred peptides with the help of automatic synthesis. GeneCust offers a large variety of purity levels, quantities and modifications of the peptides, according to the clients’ request.

How does this work ?

The peptides are chemically synthesised through the Fmoc strategy. Developed in the late 1970’s and reviewed in 2000, Fmoc chemistry is the preferred method for solid phase peptide synthesis. It implies assembling the peptide chain stepwise, one amino acid at a time, while attached to an insoluble resin support. Like this, the reaction by-products can be washed at each step. It uses orthogonal protector groups like Fmoc (9-fluorenylmethoxycarbonyl) group that protects the amino terminus of the amino acids.

Synthesis Cycle

During the solid phase synthesis, the amino acid at the C-terminal of the peptide of interest has to be connected to an insoluble support via its carboxyl group. After this, the protective group from the amino group is eliminated. An excess of the second amino acid is introduced. After the coupling, the excess reagent is eliminated by washing and the protective groups are removed from the N-terminal extremity of the peptide, in order to permit the addition of the third amino acid. This process is repeated until the desired sequence is obtained. Bellow, the details of the 4 steps :

  • Step 1 Raisin charge: The first step consists of adding the first amino acid to the solid support through its C-terminal extremity through an ester or amide bond, depending on the type of the C-terminal (acid or amide). After the coupling, the unreacted hydroxyl groups of the resin are protected by benzoic anhydride or acetic anhydride.
  • Step 2 Release of the amine (Fmoc deprotection): The protective group Fmoc is eliminated by applying a short treatment with 20% piperidine in DMF (dimethylformamide). The reaction is complete in 10 minutes.
  • Step 3 Coupling reaction: A large excess of amino acids it’s used (around 2-10 times the capacity of the resin).The extremities of these amino acids are activated via their carboxyl groups in order to form amide or ester bonds. Often this is made possible by using coupling reagents (PyBOP, TBTU or HBTU).The excess of amino acids imply an excess of reagents (in general 60-200 mM) to ensure the efficient diffusion. The necessary time for a complete acylation depends on the nature of activated residue, the peptide sequence bonded to the resin and the concentration of reagents.
  • Step 4 Final cleavage: The trifluoroacetic acid (TFA) is widely used for the cleavage of the peptide from the resin and for elimination of the protective groups of the side chains.
TFA

Purification

A wide range of purity degrees are available :

  • • Crude peptides
  • • Immunograde peptide (purity >75%): the rude peptide is precipitated and washed several times for a complete extraction of organic impurities and for elimination of secondary products. Also a standard HPLC step is applied. This purity degree is usually enough for testing or producing antibodies.
  • • Purified peptides (purity >85%): supplementary purifications by HPLC allow to eliminate the peptides resulted during the incomplete coupling reactions, peptides which lack one or more amino acids. The purity above 85% can be used for the production of antiserum, titration of antibodies by ELISA and for producing monoclonal antibodies.
  • • Highly-purified peptides (purity > 95%): this purification degree is needed for most of the biological or enzymatic studies.

For a purity level higher than 98%, please send us an inquiry.

Quality Control

GeneCust proposes high-quality peptides, produced by experts and delivered with all the required documents:

  • • HPLC Chromatogram
  • • Mass spectrometry
  • • Synthesis report