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.

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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.

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Disulfide rich peptides – in which order should the disulfide bonds be formed during on-resin oxidation?

Disulfide rich peptides are being identified in species of both plants and animals at increasing rates. As new molecules are discovered and disulfide bonding patterns characterized, the need for simplified chemical synthesis strategies is also increasing.

I have previously written about optimizing removal of several orthogonal side chain protecting groups including allyl, alloc, ivDde and acetamidomethyl (Acm) groups. The question that I’ll address today, though, is does the order in which the disulfide bonds are formed matter for cleaning up reactions to produce chemically synthesized disulfide rich peptides?

Continue reading Disulfide rich peptides – in which order should the disulfide bonds be formed during on-resin oxidation?

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.

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How to choose the right resin functionality for solid phase peptide synthesis

As a chemist new to the peptide community, there are many choices that have to be made.  Which coupling reagents to use? Heat or no heat to promote chemistry? And most importantly, which resin?  I have talked previously about resin choices, from loading levels to swelling capacity and how they affect the synthesis outcome.  But I haven’t addressed yet a fundamental feature of commercially available resins, and that’s the functional handle to which the peptide chain is conjugated.

In today’s post, I’ll describe some, and I mean only some, of the most commonly used chemical functionalities for Fmoc-based solid phase peptide synthesis and some scenarios in which you would choose one resin type over another.

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How to use a scouting column for your peptide purification

Whenever I synthesized a new peptide, I always ran a “scout run” – a small scale injection, usually with an analytical HPLC column – to both get an idea of the crude purity and also to identify a shorter, more optimal gradient for the actual purification.  This strategy is still probably fine when you want to use reversed phase flash chromatography for your purification strategy, but is there a better way?

In today’s post, I’ll discuss using a scouting column to screen gradient conditions prior to peptide purification with reversed phase flash chromatography.

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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

How to choose an ion pairing agent to improve your peptide purification

Ion pairing agents are used in a variety of strategies to improve overall purification efficiency. In a previous post, I utilized ion pairing agents to increase the peptide’s hydrophobicity, improving retention by the stationary phase and enabling purification.  But what other strategies can be improved by using ion pairing agents?

In this post, I’ll utilize ion pairing agents to enable rapid peptide purification by flash chromatography.  The use of ion pairing agents can in fact alter the peptide’s apparent hydrophobicity sufficiently that the desired peptide and it’s closely eluting impurities can be resolved.  The question is, which one to choose?

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How does methanol as a mobile phase solvent impact peptide purification by reversed-phase flash chromatography?

Recently there has been substantial motivation to consider and evaluate alternative, more environmentally friendly solvents.  Some countries have even gone so far as to ban some of the more toxic, yet commonly used solvents.  In addition to general toxicity, additional consideration in the green chemistry movement is the volume of solvent used in any particular application.  In this regard, purification solvent selection is closely monitored as they are often used in large quantities.

One alternative that is growing in popularity is the use of methanol in place of acetonitrile for reversed phase purification of small molecules.  Methanol is certainly less expensive, but is also a more environmentally-friendly solvent for use in purification applications.  But it’s use for peptide purification has not been widely adopted to date.  In today’s post, I’ll compare the purification efficiency of methanol when compared to acetonitrile for peptide purification by reversed phase flash chromatography.

Continue reading How does methanol as a mobile phase solvent impact peptide purification by reversed-phase flash chromatography?