A special paragraph will be dedicated to the problems caused by peptide aggregation in the course of the synthesis. This phenomenon is a major cause of trouble as it is difficult to predict, is sequence dependent and no universal solution has been found up to now.
The introns are removed in an enzymic reaction called splicing and the
exaons joined or spliced together in order to produce a continuous, uninterrupted polypeptide code.
Study of the ability of the ribosome to synthesize a number of fMetinitiated dipeptides from CCA amino acyl acceptors suggests that EFP acts to promote synthesis with acceptors that are poor donors for the the reconstituted peptidyl transferase.">
Study of the ability of the ribosome to synthesize a number of fMetinitiated dipeptides from CCA amino acyl acceptors suggests that EFP acts to promote synthesis with acceptors that are poor donors for the the reconstituted peptidyl transferase.
Reliable methods detecting minute amounts of unreacted amino groups are essential for the monitoring of the coupling reaction. On the other hand the ability of detecting small quantities of Fmoc peptide allows the control of the completion of the Fmoc cleavage.
Two major chemistries for solid phase peptide synthesis are Fmoc (base labile protecting group) and t-Boc (acid labile a-amino protecting group). Each method involves fundamentally different amino acid side-chain protection and consequent cleavage/deprotection methods, and resins; t-Boc method requires use of stronger HF containing anisole alone or anisole plus other scavengers, where peptide-resins assembled by Fmoc chemistry usually cleaved by less harsh Reagents K or R. Fmoc chemistry is known for peptide synthesis of higher quality and in greater yield than t-Boc chemistry. Impurities in t-Boc-synthesized peptides mostly attributed to cleavage problems, dehydration and t-butylation. For peptide assembly HBTU/HOBt, carbodiimide-mediated coupling and PyBOP/HOBt are the most popular routines. Peptides usually purified by reversed-phase HPLC (high performance liquid chromatography) using columns such as C-18, C-8, and C-4.
Since the purity of the peptide obtained from the synthesis is sequence dependent, purification to >95% will be performed upon request. Major impurities can range from 10% to 50% by weight. They consist of small water-soluble molecules, salts and protecting groups from the cleavage reaction, deletion peptides created due to incomplete coupling during synthesis, and modified peptides created during the cleavage. These species can be removed using reversed phase HPLC. We recommend that, under most circumstances, all peptides be purified prior to employing them in research studies.
For large scale synthesis of well known peptides solution or liquid phase peptide synthesis can be applied. These "classical" methods for synthesis in solution are labour, time, and skill intensive largely due to the unpredictable solubility characteristics of intermediates.
Since the peptides synthesized by the PRC often provide the basis for months and years of research and publications, we strongly believe that stringent quality control is an essential component of the synthesis process. To ensure the highest quality of products we regularly and frequently perform preventive maintenance on our synthesizers and ancillary equipment. Additionally, prior to each synthesis run a full calibration check is performed on the synthesizer to make sure that the instrument is running within specifications.
These modifications can have stringent requirements. Please call to make an appointment with a Peptide Synthesis Specialist to discuss your particular application and requirements.
Here we review the strategies for the solid-phase synthesis of peptides starting from the side chain of the C-terminal amino acid. Furthermore, we provide experimental data to support that C-terminal and side-chain syntheses give similar results in terms of purity. However, the stability of the two bonds that anchor the peptide to the polymer may determine the overall yield and this should be considered for the large-scale production of peptides. In addition, resins/linkers which do not subject to side reactions can be preferred for some peptides.
Post-cleavage adduct formation, salt complexing, deletion peptides, and incomplete deprotection during cleavage, continue to be problematic for all peptide synthesis labs. As a measure of the quality of the peptides produced the Peptide Synthesis lab provides the following to investigators:
Hydrolysis of a peptide bond is slow but does occur. Store peptides as dry as possible at -20C or -80C. Desiccate if possible. Please note that hydrophobic peptides bind avidly to glass. Cysteine-containing peptides will oxidize over time, even when stored at -20C. Long term storage of peptides in solution is not recommended regardless of the temperature.