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

Has my peptide undergone an aspartimide rearrangement?

Side reactions.  Words that cause a little shiver to run down every chemists’ spine.  As peptide chemists, we worry about both chemical side reactions like diketopiperazine or aspartimide rearrangements, and secondary structure formation as causes for failed peptide syntheses.  But how do you know what to look for?  What is a susceptible sequence and how can you confirm if one of these structural rearrangements has occurred?

In today’s post, I’ll discuss a couple strategies that have been published that illustrate how to identify if an aspartimide rearrangement has in fact occurred during your peptide synthesis. Continue reading Has my peptide undergone an aspartimide rearrangement?

Celebrating one year of the peptide synthesis and purification blog!

Wow! I can’t believe it’s been a year since this whole blogging adventure started.  This started as a technical resource for me and my colleagues within the peptide synthesis and purification space.  The idea was to enable easy access to some of the hard-to-find answers for common questions that I have encountered working with different peptide groups.

The blog has grown steadily this year and for that I thank you.  In the following post, I’ll highlight a few of the most popular posts from the past year as well a few of my favorites.  I hope you will consider providing feedback using the survey link so that we can continue to grow, keeping the content interesting and relevant.

Continue reading Celebrating one year of the peptide synthesis and purification blog!

What mobile phase flow rate should I use for my peptide purification with flash chromatography?

I’ve recently worked with several peptide groups that are trying out flash purification with their peptides for the first time.  And it never fails, every single interaction includes the question “what flow rate should I use for these cartridges?”

There is a lot of information available highlighting optimal flow rates for HPLC method development, but very little information for larger particle stationary phases.  I personally have used a wide range of flow rates for my peptide purification with differing outcomes.  So in today’s post I’d like to have a more thorough discussion about mobile phase flow rate and it’s impact on your chromatography.

Continue reading What mobile phase flow rate should I use for my peptide purification with flash chromatography?

Using mixed stationary phases to improve your peptide purification with flash chromatography

One common technique in HPLC for improving difficult peptide separations is to extend the column length, a topic I explored for flash chromatography in a previous post.  However, alternative purification strategies are sometimes necessary as the purification bottleneck grows with increasing peptide library size, both in number and scale.

In this post, I explore using two identical size cartridges in series with each packed with a different stationary phase.  I wanted to try this to see if I could improve peptide purity with the ultimate goal of reducing the time demand of peptide purification. Continue reading Using mixed stationary phases to improve your peptide purification with flash chromatography

Can I improve my peptide purification by increasing the column length?

There are several strategies often employed to improve peptide purity achieved using reversed phase HPLC.  These strategies can include, changing column length, particle size, particle functionality (C4 vs C18).  I have experimented a bit with some of these criteria while purifying peptides using reversed phase flash chromatography but one obvious change that I have not yet explored is the length of column.

In today’s post, I’ll explore how the length of the cartridge affects the overall resolution and purification efficiency using reversed phase flash column chromatography.

Continue reading Can I improve my peptide purification by increasing the column length?

Does loading method influence my peptide recovery after purification?

In peptide purification, sample loading onto the column is rarely considered.  Most, if not all, HPLC instruments come equipped with a sample injection loop which demands a liquid injection of the sample for purification.  If you decide to use flash chromatography to purify your peptides though, liquid injection is no longer the exclusive method for sample introduction to the column.  Alternatively, dry loading crude material is a strategy often used in small molecule purifications, particularly when sample solubility concerns arise.

The first question I asked myself when considering a new sample loading strategy is whether or not the purification efficiency will be maintained.  A close second though is whether or not the loading method will cause significant differences in peptide recovery.

In today’s post, I’ll compare recovery efficiencies for peptides purified using reversed phase flash chromatography but loaded onto the cartridge using either direct liquid injection or dry loaded onto reversed phase material. Continue reading Does loading method influence my peptide recovery after purification?

How much peptide is recovered from a reversed phase C18 cartridge during flash purification?

Whether it’s the bonded stationary phase, particle size, or even particle pore size, scientists today are offered a plethora of choices when it comes to reversed phase HPLC columns.  An often acknowledged concern in the peptide community though is peptide recovery from reversed phase purification efforts, particularly for precious peptide mixtures.  But how is peptide recovery impacted when you use reversed phase flash chromatography for purification?

In today’s post, I’ll compare recovery levels for two peptides that differ in length as well as crude purity using reversed phase flash chromatography.  In addition to comparing two peptides, I’ll also evaluate how recovery is impacted by altering the mobile phase pH.

Continue reading How much peptide is recovered from a reversed phase C18 cartridge during flash purification?

How many amino acid equivalents should I use for room temperature synthesis?

Big pharmaceutical companies have begun to refocus their efforts towards peptide discovery projects with the hopes of identifying the next big peptide drug.  There are often hundreds to thousands of peptides synthesized as part of these efforts, demanding parallel synthesis platforms and room temperature peptide synthesis protocols.

Previously, I identified a minimum number of amino acids equivalents required to ensure a high quality microwave synthesis.  Conducting synthesis at room temperature will certainly require different conditions than microwave heating.  Let’s explore how the number of equivalents will impact the synthesis results.

Continue reading How many amino acid equivalents should I use for room temperature synthesis?

Room temperature allyl ester and alloc deprotections – what is the lifetime of Palladium?

In a previous post, I did some work evaluating the efficiency of alloc removal with tetrakis palladium using microwave assistance and atmospheric conditions, which worked beautifully.  Given the known sensitivity of palladium catalysts (see Derek Lowe’s post for a humorous dialogue), I sought to further explore the sensitivity of palladium towards the alloc removal in the context of a peptide.

In this post, I’ll explore a variety of atmospheric, room temperature alloc deprotection conditions aimed at evaluating the catalytic lifetime of palladium tetrakis for effective alloc removal.

Continue reading Room temperature allyl ester and alloc deprotections – what is the lifetime of Palladium?