Red Glead Discovery (RGD) is a pre-clinical drug discovery CRO offering a broad range of services to Life Science clients. With a focus on small molecules and peptides, their drug discovery platform ranges from medicinal chemistry and synthesis to ADME and biology. In addition to the CRO business, they also perform research collaborations with various biotech and academic partners.
Peptides, by nature, are composed of amino acids with potentially ionizable chemical moieties. The ionization state of any of these moieties can significantly impact the peptide’s chromatographic behavior, both in terms of peak shape and retention by the solid support. Peptide purification by reversed-phase chromatography, however, almost exclusively includes an acidic additive to the mobile phase solvents, maintaining the solution at a pH of 2-3 throughout the purification cycle. But have you ever considered trying an alternative additive in the mobile phase to improve your purification results?
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.
Peptides, while exhibiting potential for unique specificity and affinity, still suffer from stability issues when introduced to a biological system. One strategy to overcome these stability issues is include a covalent bond, creating a peptide macrocycle.
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?
Almost all the peptides I have synthesized were subsequently purified using a reversed-phase C18 column. Sometimes this worked, but sometimes it didn’t work so well. When my C18 purifications failed, I questioned whether or not I could have predicted this outcome prior to extensive HPLC efforts. Since then, I have learned that the amino acid composition of the peptide may give some clues to the peptide’s chromatographic behavior.
While there are numerous stationary phase functionalization types for reversed-phase chromatography, in today’s post I will describe some differences I have observed when purifying peptides using C18- or C4- functionalized stationary phases for peptide purification.
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 previous post, I evaluated how flow rate can impact my purification efficiency using flash chromatography. I noticed though, that my peptide eluted significantly later with high mobile phase flow rates. I hypothesized that the increased pressure (caused by higher flow rates) was driving the compound further into the pores, increasing the overall interaction with the stationary phase and causing the increased retention. We know that the particle size and particle pore size impact resolution and purification efficiency, so how does flow rate play a role with a different stationary phase?
Hydrocarbon stapling as a strategy to stabilize secondary structures of peptides, while introduced by Miller, Blackwell and Grubbs in the mid 1990s, really grew to the forefront with seminal work by Schaffmeister and Verdine in early 2000s. Protocols have been developed that enable this post-synthesis modification while the peptide is still on resin, but often these metathesis reactions are performed manually, and at room temperature.
In today's post, I'll compare several different sets of reaction conditions using microwave heating with the goal of expediting the olefin metathesis reaction, without compromising reaction efficiency, and towards automating the entire synthesis.
There are many techniques available to analyze and identify synthetic compounds that we are taught in the first few years of our chemistry education. While tools like NMR spectroscopy, IR spectroscopy, and others, are extremely useful for determining or confirming the structure of synthetic small molecules, these strategies are not as well suited for quick characterization of peptides. As a result, peptide chemists rely heavily on peak shape observed during a liquid chromatography step and mass spectrometry for mass confirmation.
In today's post, I'll discuss several of the mass spectrometry techniques that are used for analyzing crude or purified peptide samples.
More and more we are seeing groups that would historically undertake only traditional organic chemistry or possibly biochemistry/biology, incorporate peptides into their research programs. While this is good for expanding the application scope and diversity in the peptide space, bringing synthesis in house can be a daunting undertaking.
In today's post, I'll talk about what it takes to get a peptide synthesis operation up and running, with a few considerations along the way.
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.
Resins for solid phase peptide synthesis can vary significantly in both functionalization and composition, leading to mixed results at the end of a synthesis. Previously, I demonstrated how the resin loading level affects the success or failure of your peptide synthesis.
In today’s post, I’ll highlight how both the hydrophilicity and swelling capacity of your resin can influence your peptide synthesis.
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.
When it comes to synthesizing a peptide, the first thing that comes to mind is the number of stoichiometric equivalents to use. Sometimes that number is as few as 1.5, sometimes it’s as high as 20!
But have you ever thought about the liquid volume that contains those molecules and how that might affect the success of your coupling reaction? In this post I will discuss the impact of amino acid concentration in the overall success of solid phase peptide synthesis.
It used to be easy with only polystyrene based resin types, but nowadays there is a broad choice of types to choose from, including everything from the C-terminal functionality (Rink vs Wang) to the polymer from which the resin itself is synthesized.
All resins have one thing in common, and that’s the reactive site loading level. In this post, I will share my experiences with how this important factor impacts the success of peptide synthesis.