how many amino acid equivalents should I use for my microwave assisted synthesis?

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.

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How to quantify your first amino acid loading onto Wang resins

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?

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Optimizing the removal of an ivDde protecting group

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.

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Can you use normal-phase chromatography to purify protected peptides?

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.

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Synthesizing hydrophobic peptides – choosing the right solvent

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.

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Green solvents for solid phase peptide synthesis?

As peptide therapeutics continue to gain interest from the medical community and pharmaceutical companies, concerns regarding the cost of manufacturing also grow.  Cost  includes the expense of reagents and solvents, including DMF and NMP, used in the synthesis but also subsequent disposal.  Combining this fact along with growing conversations about strategies to make chemistry green(er) and more of interest to this blog, greener solvents in peptide chemistry.

The looming question is, what replacement solvents should we use and is Fmoc-based solid phase peptide synthesis still efficient?  In this post, I will highlight a few solvents mentioned during a green chemistry presentation at TIDES 2017, a conference specifically geared towards peptide and oligonucleotide therapeutic development Continue reading Green solvents for solid phase peptide synthesis?

Which stationary phase should I chose for my peptide purification?

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 functionalizations 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 purifications.

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Why won’t my peptide stick to my column?

Conversations are routinely held regarding handling hydrophobic peptides, but hydrophilic peptides offer their own challenges when it comes to purification.  In a previous post, I synthesized Octa-Arg, an extremely hydrophilic peptide. I used  ion pairing reagents to increase the peptide’s overall retention by the stationary phase, but choosing the solvent should to use for solubilizing the peptide for purification by flash column chromatography was no easy task.

In today’s post, I’ll investigate several solvents commonly used to inject peptide samples for purification and evaluate their impact in peptide retention by the stationary phase.

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How to purify hydrophilic peptides with flash column chromatography

Synthesizing hydrophilic peptides can be relatively straightforward, especially when compared to the trials and tribulations encountered when synthesizing hydrophobic peptides.  However, purifying hydrophilic peptides using standard reversed-phase chromatography can be a challenge.    In a previous post, I encountered this problem during my analytical HPLC analysis of crude peptide mixtures.

In this post, I will discuss the use of ion pairing agents to increase the perceived hydrophobicity of your peptide, increasing the column retention and enabling a smooth purification of your hydrophilic peptide.

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Microwave heating – a route to better quality crude peptides

Historically, solid phase peptide synthesis has been conducted at room temperature, demanding long reaction times and often double coupling to ensure a quality crude peptide product.  More recently however, different strategies have been identified to heat the reactor vial, increasing the overall reaction rate and potentially the crude purity of your peptide.

In today’s post I will demonstrate that microwave heating can improve the crude purity of your desired peptide.

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