Welcome to the Biotage Peptide Synthesis Blogs.

      Peptide purification with flash column chromatography - a beginner's experience

      Jul 18, 2019 8:59:34 PM / by Elizabeth Denton posted in Developments, Peptides, reversed-phase, flash chromatography, solid phase peptide synthesis, peptide purification

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      As a peptide chemist, I was trained to purify my peptides with reversed-phase HPLC, just as many a peptide chemist before me. Despite the hundreds of hours I’ve logged in front of an HPLC, injecting samples and collecting peak fractions, I can’t imagine using any other method to purify my freshly synthesized and cleaved peptides.  In fact, you’d be hard pressed to convince me to try something else.  But here I am, trying something new.  Wish me luck!

      In this post, I’ll describe my experiences using flash chromatography to purify a new peptide sample.

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      How does methanol as a mobile phase solvent impact peptide purification by reversed-phase flash chromatography?

      Jul 18, 2019 2:10:15 PM / by Elizabeth Denton posted in Peptides, reversed-phase, selectivity, solvent strength, solid phase peptide synthesis, peptide purification

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      Recently there has been substantial motivation to consider and evaluate alternative, more environmentally friendly solvents.  Some countries have even gone so far as to ban some of the more toxic, yet commonly used solvents.  In addition to general toxicity, additional consideration in the green chemistry movement is the volume of solvent used in any particular application.  In this regard, purification solvent selection is closely monitored as they are often used in large quantities.

      One alternative that is growing in popularity is the use of methanol in place of acetonitrile for reversed phase purification of small molecules.  Methanol is certainly less expensive, but is also a more environmentally-friendly solvent for use in purification applications.  But it’s use for peptide purification has not been widely adopted to date.  In today’s post, I’ll compare the purification efficiency of methanol when compared to acetonitrile for peptide purification by reversed phase flash chromatography.

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      Does loading method influence my peptide recovery after purification?

      Jul 17, 2019 6:25:29 PM / by Elizabeth Denton posted in Peptides, reversed-phase, solid phase peptide synthesis, peptide purification, peptides and flash chromatography, loading method

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      In peptide purification, sample loading onto the column is rarely considered.  Most, if not all, HPLC instruments come equipped with a sample injection loop which demands a liquid injection of the sample for purification.  If you decide to use flash chromatography to purify your peptides though, liquid injection is no longer the exclusive method for sample introduction to the column.  Alternatively, dry loading crude material is a strategy often used in small molecule purification, particularly when sample solubility concerns arise.

      The first question I asked myself when considering a new sample loading strategy is whether or not the purification efficiency will be maintained.  A close second though is whether or not the loading method will cause significant differences in peptide recovery.

      In today's post, I'll compare recovery efficiencies for peptides purified using reversed phase flash chromatography but loaded onto the cartridge using either direct liquid injection or dry loaded onto reversed phase material.

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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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      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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      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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      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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