Peptide synthesis and purification services are available at lab to small scale pilot production scales. In addition a range of post-synthesis labelling chemistries are available to modify peptides for subsequent detection.
a) Peptides synthesis will be carried out by solid state peptide chemistry either manually or using an automated peptide synthesizer. Typically, fluorenylmethoxycarbonyl (Fmoc) will be employed to block amino groups. Reactive side chains will be blocked by a variety of acid-labile protecting groups. Coupling of protected amino acids to a nascent peptide will be accomplished by converting their terminal carboxyl groups to active benzotriazole esters using coupling reagents such as N-hydroxybenzotriazole / 2-(1H-Benzotriazole-1-yl)-1,1,3,3-teteramethyluronium hexafluorophosphate (HOBt / HBTU). A variety of alternative coupling strategies are available in the case of a difficult peptide sequence. Fmoc removal and coupling efficiency may be monitored during synthesis by performing a ninhydrin (Kaiser) test on resin samples to detect the presence of free amines.
b) Labeling will be carried out while the peptide is attached to the resin, allowing monitoring of labeling and facilitates the elimination of excess, unbound label. Available sites for labeling include the N-terminal Alpha-amine group or Epsilon-amino groups of lysine or cysteine thiols. When the N-terminal alpha-amino group is labeled, a spacer group such as Beta-alanine is typically used to separate the label from the peptide. Labels can include various fluorophors, peptides with donor-acceptor fluorophors and/or chromophors for fluorescence resonance energy transfer (FRET) studies, biotin, polyethylene glycol. Peptides can be cyclized by forming dsulfide bridges, by head-to-tail coupling of by coupling to and internal amn acid side chain.
c) Cleavage and post-synthesis workup: The completed peptide will be cleaved from the resin and deprotected using a 95% trifluoroacetic acid (TFS) cocktail containing various scavenger molecules: viz. phenol, thioanisole, ethanedithiol, etc. The cleaved peptide will be precipitated in cold diethylether, sedimented, and washed several times to remove scavenger complexes with cleaved side-chain protecting groups. Final purification is accomplished by preparative reversed phase HPLC on C8 or C18 columns. The quality and identity of the purified product will be ascertained by: i) MALDI-TOF mass spectrometry to verify the peptide mass ii) analytical rpHPLC to check purity iii) quantitative amino acid analysis will be employed to determine the peptide content when required.
For more details on synthesis scale and pricing contact PLeavis@analyticalbiotechservices.com for consultation and a quotation of pricing and availability.
