Would you ever consider an alternative to reversed- phase HPLC to purify your synthetic peptides? It seems like a silly question, right. And like many of you, I literally laughed at my Product Manager when he asked me this same question in my first days at Biotage.
Fast forward a few years and my answer to that question is now very different. For those of you that have followed this blog, you’ll know that I have switched to reversed-phase flash chromatography almost exclusively for my peptide purification. In today’s post, I’ll highlight some of the critical reasons that have influenced my change in mindset.
Continue reading How to purify synthetic peptides: what are the options?
Since the development of Fmoc-based solid phase peptide synthesis, a wide variety of cleavage cocktails have emerged. Each cleavage cocktail contains a unique combination of scavengers designed to prevent either side reactions mediated by the released protecting groups or the side chains themselves, or both during the peptide cleavage reaction. As the number of scientists performing peptide synthesis grows, the question “which cleavage cocktail should I use?” comes up more often than not.
In today’s post, I’ll highlight the role of of scavengers for peptides containing cysteine residues.
Continue reading Peptides containing cysteine: the role of scavengers in cleavage cocktail
Orthogonal side chain protecting groups, particularly for Fmoc-based solid phase peptide synthesis, are growing not only in diversity, but also in popularity. These protecting groups enable post-synthesis chemistry while the peptide is still on resin, often times increasing efficiency, decreasing side reactions, and generally simplifying the overall process.
I’ve already done some work with many of the commercially available orthogonally protected amino acids including allyl and alloc, Acm, and ivDde for a variety of downstream applications. In today’s post, I’ll discuss some work optimizing the removal of a 4-methoxytrityl (Mmt) group from cysteine side chains.
Continue reading How to: Measure and optimize the removal of Mmt protecting groups
Disulfide rich peptides have gained significant attention recently due to their incredible biological stability and tolerance to epitope grafting. This class of peptides is often folded in solution, assuming the desired disulfide bond pattern correlates with the most thermodynamically stable structure. Sometimes though, especially for chemically synthesized cysteine rich peptides, this is not the case. The result is a complex mixture of peptides with varying disulfide bonding patterns and identical mass.
Using pairs of cysteine residues with matched orthogonal side chain protecting groups during chemical synthesis allows for precise regioselective control of the disulfide bond pattern on-resin, simplifying final purification steps. In today’s post, I’ll explore conditions for removing acetamidomethyl (Acm) protecting groups with simultaneous disulfide bond formation.
Continue reading Optimizing the removal of an Acm protecting group