Optimizing the removal of an STmp protecting group

Disulfide rich peptides are unique in both their incredibly high cysteine content, but also in the stability imbued by the multiple disulfide bonds.  These peptides, stable under extreme conditions that would either denature or degrade a similar linear peptide, make disulfide rich peptides attractive as both therapeutics or as scaffolds upon which to construct non-native functionality.  Synthesizing these compounds, however, still remains a challenge.

I have discussed previously strategies that enable on-resin chemistry via orthogonal protecting groups.  These groups can be removed under mildly acidic, metal catalyzed, or even oxidizing conditions.  In today’s post, I’ll demonstrate the utility of using disulfide shuffling as a cysteine protection strategy. Continue reading Optimizing the removal of an STmp protecting group

Peptides containing cysteine: the role of scavengers in cleavage cocktail

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

How to: Measure and optimize the removal of Mmt protecting groups

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

Optimizing the removal of an Acm protecting group

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

How to load the first amino acid onto Wang resin

While resins loaded with the natural 20 amino acids are commercially available these days, there may be times when loading the first amino acid onto the resin in house may be necessary.  And unlike loading the first amino acid onto amide-leaving resins, the first coupling reaction for C-terminal acids can be chemically more challenging.

There are several protocols published both in the literature as well as in technical notes from many peptide reagent and instrument suppliers, but they typically occur at room temperature over extended periods of time (3-24 hours and repeated).  In today’s post, I’ll evaluate several conditions suitable for efficiently loading the first amino acid onto Wang-type resin. Continue reading How to load the first amino acid onto Wang resin

Preventing aspartimide rearrangements during Fmoc-based solid phase peptide synthesis

Aspartimide rearrangements are a particularly nasty side reaction that can occur during fmoc-based solid phase peptide synthesis.  Not only is this a mass-neutral side reaction, chromatographically resolving the undesired, rearranged product can be particularly difficult.  To make matters worse, this side reaction can occur at any point during the synthesis after the Asp has been incorporated into the peptide.

In a prevous post, I described method that I have found useful for identifying whether or not an aspartimide rearrangment as occured during synthesis of a peptide that contains an aspartimide-susceptible sequence.  In today’s post, I’ll discuss some strategies that can be used to suppress, or even eliminate this side reaction. Continue reading Preventing aspartimide rearrangements during Fmoc-based solid phase peptide synthesis