How does my mass spectrometer carrier solvent impact mass-directed purification of peptides?

Mass-directed purification, whether with a preparative HPLC or a bench-top flash system, is quickly gaining interest in the peptide purification space.  The simple fact is that using a specific mass, rather that UV absorbance, to trigger fraction collection allows for greater confidence in the identity of the collected fraction.  Importantly though, this technique can also reduce your time required for purification, by significantly reducing or even eliminating the need for secondary mass analysis of each collected fraction.

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Increasing your peptide purity using a focused gradient with flash chromatography

Reversed phase flash chromatography is increasingly being utilized by peptide chemists to decrease purification time and efforts. The larger particles used in flash columns enable large crude sample loads and can lead to highly pure peptide samples despite lower resolution when compared to traditional HPLC methods. However, there are some situations where the purity achieved isn’t sufficient. Then what can you do?

In today’s post, I’ll describe using a focused gradient to achieve higher purity peptides than is possible with a more traditional linear gradient.

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Synthesis of peptides containing three disulfide bonds: can it be fully automated?

I have experimented a lot with disulfide rich peptides lately, finding conditions that work well for not only the linear synthesis, but also for on-resin cysteine oxidations.  Although simple scaffolds are useful for determining orthogonal protecting group removal and cysteine oxidation conditions, many of the peptides of interest today are much more complex – three or more disulfide bonds, and often head-to-tail cyclization.

In today’s post, I put to use three orthogonal protection strategies to optimize a fully automated synthesis of linaclotide.

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How to purify synthetic peptides: what are the options?

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.

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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.

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

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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