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?
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?
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?
More often than not and as a peptide chemist, I am asking myself in which solvent should I dissolve my peptide prior to purification by flash chromatography. I have rarely considered an alternative to the standard liquid injection. Dry loading is a common technique used by organic chemists prior to their normal-phase purification efforts, especially if the compound isn’t particularly soluble in the mobile phase solvents. To the best of my knowledge, dry loading is not commonly used for peptide purifications.
Immediately, questions come to mind as I attempt this new loading technique. Will my peak shape change if load additional material? Does the stationary phase need to be equilibrated before use? What solvent should I use to load my crude sample? How will my sample recovery be affected? I will address a few of these questions in today’s discussion.
Continue reading Impact of wetting an in-line guard column for reversed-phase peptide purification with flash column chromatography
Orthogonal amino acid protecting groups effectively expand the chemical tool kit available to peptide chemists allowing for synthesis of much more complex molecules. Often times, orthogonal protecting groups are used in Fmoc-based chemistry to facilitate post-synthesis modifications of peptides, like the addition of small molecule fluorophores and more commonly now, peptide cyclization efforts.
In a previous post, I discussed optimizing the removal of an ivDde protecting group. In today’s post, I’ll explore the removal of an alloc protecting group from a lysine residue.
Continue reading Using microwave heating to expedite your allyl ester or alloc deprotection
Every now and then I work with new groups as they embark on a journey incorporating peptides and peptide synthesis into their research. More often than not, no one in the group has experience with peptide synthesis as they are getting operations off the ground. As a result, one of the most common questions I receive from these groups is how much amino acid should be used during synthesis.
In a previous post, I discussed the importance of amino acid concentration particularly for synthesis of long peptides. In today’s post though I will address the number of equivalents of amino acid. Large numbers of amino acid equivalents can often be used to drive coupling reactions to near completion, but the question today is how few equivalents can be used to successfully synthesize your peptide.
Continue reading how many amino acid equivalents should I use for my microwave assisted synthesis?
While many of the standard amino acids can be purchased pre-loaded onto Wang type resins, there are still cases where coupling the first amino acid onto Wang resin manually is necessary. In my case, an unnatural amino acid was required on the C-terminus so there was not a commercially available source.
This coupling reaction comes with it’s own set of challenges, which is why many people perform a large scale batch preparation of the pre-loaded resin. But that’s for a later discussion. In today’s post I’ll address a different question. How do you quantify the amount of amino acid loaded onto the resin?
Continue reading How to quantify your first amino acid loading onto Wang resins
As the complexity of peptides continues to grow, so does the use of amino acids with side chain protecting groups that can be selectively removed, leaving the peptide on resin and the remaining side chain protecting groups intact. While there are protocols to be found in the literature, they may not work to the highest level of efficiency every single time. This can lead to disasterous results for any subsequent chemistry.
In today’s post, I’ll evaluate a variety of conditions for removing an ivDde protecting group.
Continue reading Optimizing the removal of an ivDde protecting group
Chemical synthesis of peptides, and even proteins, offers the possibility to expand the functionality and stability imbued by nature. However, chemical synthesis of very long peptides and small proteins remains today an exceedingly difficult task. Several ligation strategies have been developed that help to alleviate this challenge. These strategies though, require a purified, yet fully protected peptide fragment.
Purification of a fully protected peptide species can be challenging by standard reversed-phase techniques, primarily due to the limited solubility of protected peptides in aqueous solutions. In today’s post, I will discuss using normal-phase chromatography for purification of protected peptides.
Continue reading Can you use normal-phase chromatography to purify protected peptides?
Every now and again I hear the question “which solvent do you recommend for my solid phase peptide synthesis?” Historically, dichloromethane (DCM) was used as a solvent for solid phase synthesis as the kinetics of amino acid activation and amine coupling were much more favorable. However, solubility concerns, particularly for Fmoc-protected amino acids limited the utility of the solvent. Nowadays, DMF and NMP are the two principle solvents for both microwave assisted and room temperature solid phase peptide synthesis. But the question remains, which one is better?
In today’s post, I will compare how the choice of dimethylformamide (DMF) or N-methylpyrolidone (NMP) effects the synthesis of a short yet very hydrophobic peptide.
Continue reading Synthesizing hydrophobic peptides – choosing the right solvent