Welcome to the Biotage Peptide Synthesis Blogs.

      How To: Measure and Optimize the Removal of MMT Protecting Groups

      Sep 22, 2020 2:41:38 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides

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      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.

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      How to synthesize hydrophobic peptides - Choosing the Right Solvent

      Sep 1, 2020 5:39:23 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Troubleshooting and Optimization

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      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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      How to purify hydrophilic peptides - Why won't my peptide stick to my column?

      Jul 27, 2020 7:58:04 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase, Loading Techniques

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

      Jul 20, 2020 7:42:04 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides

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      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.

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      Can I use a guard column for peptide purification with reversed-phase flash column chromatography?

      Jul 13, 2020 2:55:39 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Loading Techniques

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      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.  However, 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.

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

      Jul 13, 2020 2:53:13 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides

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      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 just 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 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 purify synthetic peptides - what are the options?

      Jul 7, 2020 2:15:52 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides

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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.

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      Peptide Workflow in action at Red Glead Discovery

      May 28, 2020 3:33:10 PM / by Amit Mehrotra posted in Peptides, Workflow, Alstra

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      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.

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      How to improve peptide purification by altering the mobile phase pH

      Apr 17, 2020 11:45:00 AM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase, Solvents

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      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?

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      Using Mixed Stationary Phases to Improve Your Peptide Purification with Flash Chromatography

      Apr 15, 2020 6:00:00 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Sfär

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      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.

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      Cyclic Peptides Webinar Recap

      Apr 15, 2020 2:40:31 PM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, Webinar, Automation, Alstra

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      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.  

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      Disulfide Rich Peptides - which order should the disulfide bonds be formed?

      Apr 15, 2020 2:38:56 PM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, Synthesis, Alstra

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      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?

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      Which Stationary Phase Should I Chose For My Peptide Purification?

      Apr 12, 2020 6:45:00 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Sfär, Media and Resin

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      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.

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      Preventing Aspartimide Rearrangements During Fmoc-based Solid Phase Peptide Synthesis

      Apr 9, 2020 1:15:00 PM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, Synthesis

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      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.

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      How does flow rate affect my peptide purification efficiency when using a small pore stationary phase

      Apr 6, 2020 6:08:00 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase, Troubleshooting and Optimization, Sfär, Selekt, Media and Resin

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      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?

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      Using microwave heating for your stapled peptide synthesis

      Jan 24, 2020 10:27:08 PM / by Elizabeth Denton posted in Peptides, Synthesis, Alstra

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      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.

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