Welcome to the Biotage Peptide Synthesis Blogs.

      How to decrease your time for peptide purification optimization

      Feb 16, 2021 9:00:00 AM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, Peptide Purification, Method development

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      Let's be honest.  Optimizing a purification protocol for a single peptide is not something that one wants to spend a lot of time thinking about, let alone actually pursuing.  There are times though, that a little bit of effort in this arena can very impactful when considering the time required to fully synthesize, purify and deliver a peptide. 

      In today's post, I'll discuss an approach combining the use of a focused gradient with a step gradient that minimizes the steps necessary to identify a more optimal flash purification gradient for peptide purification. 

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      Optimizing the removal of an STmp protecting group

      Dec 29, 2020 3:29:00 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Troubleshooting and Optimization

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      Disulfide rich peptides are unique in both their incredibly high cysteine content, but also in the stability imbued by the multiple disulfide bonds.  These peptides, stable under extreme conditions that would either denature or degrade a similar linear peptide, make disulfide rich peptides attractive as both therapeutics or as scaffolds upon which to construct non-native functionality.  Synthesizing these compounds, however, still remains a challenge.

      I have discussed previously strategies that enable on-resin chemistry via orthogonal protecting groups.  These groups can be removed under mildly acidic, metal catalyzed, or even oxidizing conditions.  In today’s post, I’ll demonstrate the utility of using disulfide shuffling as a cysteine protection strategy.

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      Getting started with Flash Chromatography for peptide purification - Tips and Tricks

      Oct 29, 2020 4:00:58 PM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, Peptide Purification

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      Have you ever tried a new-to-you technique, only to find yourself frustrated by the lack of results?  Frustration that only seems to compound when you search for resources that may help you get started and only to come up empty-handed?  Me too!!  And often for me, this effort ends with me back to using whatever the previous strategy may have been, because whether it's efficient or not, it's the devil I already know.  

      I've been working with flash chromatography as my primary tool for peptide purification for a couple of years now and honestly encountered some of these similar experiences.  But rather than give up, I pushed on and with the help of some other scientists, have identified some strategies that can help make this technique much easier.

      In today's post, I've compiled a few of the most helpful strategies into a tips and tricks list that should help you to be more successful using flash chromatography for your peptide purification.

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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 choose your acidic mobile phase modifier for peptide purification using Reversed-Phase flash chromatography

      Jun 17, 2020 1:32:27 PM / by Elizabeth Denton posted in Troubleshooting and Optimization, Sfär, Peptide Purification

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      When purifying crude peptides, we always use a mobile phase modifier. The modifier simplifies a potentially complex purification by driving the desired peptide, and any impurities in the sample, into a single protonation state. That way, you're only trying to purify a single peptide, rather than many variants of the same peptide that only differ by the presence or absence of a couple protons - from many peaks to one!

      Acids are commonly used as mobile phase modifiers. Some groups choose trifluoracetic acid (TFA), while others choose formic acid (FA). But are there others that should be considered as well? In today's post, I'll explore the use of an acidic buffer as a mobile phase modifier and compare the purification efficiency to that observed with TFA.

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      How does mobile phase modifier concentration impact peptide purity with flash chromatography?

      Jun 2, 2020 6:44:01 PM / by Elizabeth Denton posted in Reversed-phase, Troubleshooting and Optimization, Sfär, Peptide Purification

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      We all use mobile phase modifiers, regardless of purification strategy, for purification of our synthetic peptides.  With ionizable side chains, something that affects the pH of the mobile phase is a must to ensure that the desired peptide (and any impurities) elute from the column in a single, well defined peak.  

      While a 0.1% modifier concentration is basically standard these days (sometimes you'll see different),  I started to wonder if this was really enough modifier to fully protonate all possible side chains when using flash chromatography to purify peptides.  In today's post I'll look into the effects of different mobile phase modifier concentrations with respect to general peak shape and final sample purity.

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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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      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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      Automating stapled peptide synthesis: overcoming DMF poisoning

      Apr 1, 2020 9:16:05 PM / by Elizabeth Denton posted in Troubleshooting and Optimization

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      Covalent stapling strategies that stabilize a particular secondary structure have garnered much attention as interest in peptide therapeutics continues to grow.  One such strategy - using olefin-bearing unnatural amino acids covalently bonded using ring-closing metathesis - has been exploited to the greatest extent thusfar.

      In today's post, I'll discuss some strategies to overcome DMF poisoning of the Grubbs catalyst used during the metathesis reaction towards fully automating the synthesis and secondary chemistry required for stapled peptides.

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      Analyzing crude peptide samples by Mass Spectrometry: what are the options

      Jan 10, 2020 3:30:02 PM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, HPLC

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      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.

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      Optimizing the removal of an ACM protecting group

      Nov 14, 2019 3:40:28 PM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, Synthesis, Alstra

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      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.

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      Does amino acid concentration really matter during peptide synthesis?

      Nov 8, 2019 4:15:44 PM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, Media and Resin, Automation, Alstra

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      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.

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      How long should I let my cleavage reaction stir at room temperature?

      Sep 27, 2019 3:30:52 PM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization

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      As the rules for cell permeability continue to be elucidated, peptides are increasingly being used to deliver either themselves or cargo to the cell’s interior.  One thing is clear, increasing the overall cationic charge of the peptide enhances it’s delivery to not only the cytoplasm, but also the nucleus or other subcellular compartments.  To achieve the positive charge, large numbers of arginine residues are most often incorporated into the peptide sequence.

      This begs the question though, should I change my cleavage protocol?  In today’s post, I’ll evaluate several lengths of time used to cleave and fully deprotect an Arg-rich peptide sequence.

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      Five Tips and Tricks for Success in Solid Phase Peptide Synthesis

      Sep 27, 2019 3:26:17 PM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, Synthesis, Alstra

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      In my role as a peptide application scientist, I have had the pleasure of working with many groups that are venturing into the world of peptides for the first time.  Although it seems rather  straightforward to experienced synthetic chemists, producing acceptable yield and purity certainly comes with unique challenges in solid phase peptide synthesis .

      In this post I would like to present some of the tips and tricks that I have picked up along the way.

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