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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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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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?
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 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?
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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