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      Elizabeth Denton


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      Post synthesis workup: What steps are necessary and what aren't?

      Jun 19, 2019 5:35:32 PM / by Elizabeth Denton posted in Developments, Peptides, workflow, peptide workflow, solid phase peptide synthesis, cleavage

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      You’ve just finished a peptide synthesis and now it’s time to cleave the peptide from the resin. You’ve selected a specific cleavage cocktail, performed the reaction and now what? The vast majority of peptide chemists will precipitate their peptide using an ether solution, lyophilize, and move on to purification. But is that the only option?

      In today’s post I’ll highlight an alternative strategy that saves both processing time, potentially dangerous reagents, all without compromising the integrity of the recently synthesized peptide.

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      Room temperature allyl ester and alloc deprotections - what is the lifetime of palladium?

      Jun 11, 2019 8:30:33 PM / by Elizabeth Denton posted in solid phase peptide synthesis, orthogonal protecting groups, selecting deprotection, synthesis optimization

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      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. 

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      How to choose the right resin functionality for solid phase peptide synthesis

      Jun 11, 2019 8:30:01 PM / by Elizabeth Denton posted in Peptides, solid phase peptide synthesis, synthesis tips, synthesis optimization

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      As a chemist new to the peptide community, there are many choices that have to be made.  Which coupling reagents to use? Heat or no heat to promote chemistry? And most importantly, which resin?  I have talked previously about resin choices, from loading levels to swelling capacity and how they affect the synthesis outcome.  But I haven't addressed yet a fundamental feature of commercially available resins, and that's the functional handle to which the peptide chain is conjugated.

      In today's post, I'll describe some, and I mean only some, of the most commonly used chemical functionalities for Fmoc-based solid phase peptide synthesis and some scenarios in which you would choose one resin type over another.

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      Can you use normal phase chromatography to purify protected peptides?

      Jun 11, 2019 8:29:38 PM / by Elizabeth Denton posted in normal phase, reversed-phase, flash purification, peptide synthesis, solid phase peptide synthesis, peptides and flash chromatography

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      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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      Using microwave heating to expedite your allyl ester or alloc deprotection

      Jun 11, 2019 8:28:51 PM / by Elizabeth Denton posted in peptide, peptide synthesis, solid phase peptide synthesis, orthogonal protecting groups, synthesis optimization

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      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.

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      Can I improve my peptide purification by increasing the column length?

      May 23, 2019 4:41:39 PM / by Elizabeth Denton posted in reversed-phase, peptide workflow, v-10 touch, solid phase peptide synthesis, peptide purification, peptides and flash chromatography

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      There are several strategies often employed to improve peptide purity achieved using reversed phase HPLC.  These strategies can include, changing column length, particle size, particle functionality (C4 vs C18).  I have experimented a bit with some of these criteria while purifying peptides using reversed phase flash chromatography but one obvious change that I have not yet explored is the length of column.

      In today's post, I'll explore how the length of the cartridge affects the overall resolution and purification efficiency using reversed phase flash column chromatography.

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      Using double coupling to improve your peptide synthesis

      Apr 29, 2019 6:06:48 PM / by Elizabeth Denton posted in peptide workflow, solid phase peptide synthesis, synthesis tips

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      There are several strategies employed when a peptide synthesis requires optimization.  Typically, the first thing considered is whether or not to double couple specific amino acids within the sequence.  This is somewhat of a change in mentality from traditional room temperature synthesis strategies where double coupling is frequently used for the entire peptide sequence.

      In a previous post, I briefly described several scenarios in which doubling coupling can be used in conjunction with microwave heating to improve the overall crude peptide purity.  In today’s post, I will delve more deeply into the question of whether or not double coupling is necessary to improve your peptide synthesis.

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      Optimizing the removal of an ivDde protecting group

      Apr 29, 2019 6:05:57 PM / by Elizabeth Denton posted in peptide workflow, solid phase peptide synthesis, synthesis tips

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      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 from the lysine side chain amine.

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      How To Load The First Amino Acid Onto Wang Resin

      Apr 29, 2019 6:05:23 PM / by Elizabeth Denton posted in peptide workflow, solid phase peptide synthesis, first amino acid loading, resin loading, synthesis tips

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      While resins loaded with the natural 20 amino acids are commercially available these days, there may be times when loading the first amino acid onto the resin in house may be necessary.  And unlike loading the first amino acid onto amide-leaving resins, the first coupling reaction for C-terminal acids can be chemically more challenging.

      There are several protocols published both in the literature as well as in technical notes from many peptide reagent and instrument suppliers, but they typically occur at room temperature over extended periods of time (3-24 hours and repeated).  In today’s post, I’ll evaluate several conditions suitable for efficiently loading the first amino acid onto Wang-type resin.

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      How to quantify your first amino acid loading onto Wang resins

      Apr 29, 2019 6:04:33 PM / by Elizabeth Denton posted in solid phase peptide synthesis, quantifying resin loading, first amino acid loading, resin loading, synthesis tips

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      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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      How long are amino acid stock solution stable for successful solid phase peptide synthesis?

      Apr 29, 2019 6:03:52 PM / by Elizabeth Denton posted in peptide, peptide workflow, solid phase peptide synthesis, synthesis tips

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      In today’s post I’ll answer the above question by comparing the crude purity of peptides synthesized using amino acid stock solutions or freshly dissolved amino acids.

      In previous posts I have described using high concentrations of amino acids to improve your peptide synthesis among some other tips and tricks.  But there is a particularly handy tip that was left off the list.

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      Peptide Workflow by Biotage

      Mar 28, 2019 3:47:33 PM / by Elizabeth Denton posted in peptide, peptide synthesis, peptide workflow, remove solvents

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      What is the main goal of a peptide chemist? Elizabeth Denton, Ph.D., explains how Biotage sees the peptide synthesis workflow and how we focus on shortening the process time for scientists.

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      How to handle peptides that contain methionine

      Mar 28, 2019 2:24:34 PM / by Elizabeth Denton posted in Peptides, solid phase peptide synthesis, side reactions, methionine oxidation

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      The diversity of amino acid side chain functionalities, coupled with secondary structure, gives peptides and proteins their unique properties and activities. However, when it comes to chemically synthesizing peptides or even small proteins, the side chain functionalities can do more harm than good.

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      What do you do when your peptide synthesis fails?

      Feb 14, 2019 2:40:48 PM / by Elizabeth Denton posted in peptide synthesis

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       It never fails. A sequence looks relatively straightforward on paper and it's of moderate length so you start synthesizing without too much thought into the protocol. Your synthesis is finished and you run the analytical HPLC but your product is nowhere to be found (or only very small quantities). So what went wrong? And what can you do differently to increase your crude yield and purity?

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      Webinar: Synthesizing Disulfide-rich Peptides

      Feb 1, 2019 4:46:25 PM / by Elizabeth Denton posted in Disulfide Bonds, peptide synthesis, webinar

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      Elizabeth Denton, PhD, Senior Scientist Peptide Chemistry, recorded a webinar titled Disulfide Rich Peptides: Optimizing and Automating Syntheses with Regioselective Formation of Disulfide Bonds. To learn more, read the description below as well as watch the recording!

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      Synthesis of peptides containing three disulfide bonds: can it be fully automated?

      Jan 4, 2019 3:56:10 PM / by Elizabeth Denton posted in Peptides, Disulfide Bonds

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      I have experimented a lot with disulfide rich peptides lately, finding conditions that work well for not only the linear synthesis, but also for on-resin cysteine oxidations.  Although simple scaffolds are useful for determining orthogonal protecting group removal and cysteine oxidation conditions, many of the peptides of interest today are much more complex – three or more disulfide bonds, and often head-to-tail cyclization.

      In today’s post, I put to use three orthogonal protection strategies to optimize a fully automated synthesis of linaclotide.

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