Coxon, Christopher

D’Aloisio, Vera

English

Working with Teledyne Isco, we developed a purification method that was able to separate a 'deletion' peptide form the parent peptide-peptoid hybrid using flash chromatography. The efficiency was equivalent to preparative HPLC but achieved in a shorter time

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     Edinburgh Research Explorer                                      Reversed Phase Flash Chromatography Purification of Peptide-Peptoid HybridsCitation for published version:D’Aloisio, V & Coxon, C 2021, Reversed Phase Flash Chromatography Purification of Peptide-PeptoidHybrids..Link:Link to publication record in Edinburgh Research ExplorerGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 23. Feb. 2022Reversed Phase Flash Chromatography Purification of Peptide-Peptoid Hybrids Chromatography Application NoteAN115, Jan 15, 2021Application OverviewPeptide-peptoid hybrids or ‘peptomers’ are oligomeric synthetic polymers, in which one or more amino acids is replaced with a peptoid submonomer. The  key structural feature is the relocation of the amino acid side chain from the α-carbon to the adjacent nitrogen atom of the peptide bond (see scheme in  Fig. 1). Generally, these species exhibit physicochemical properties similar  to the parent peptides.Purification of peptide-peptoid hybrids can be obtained by reversed phase chromatography with excellent recovery and resolution. In general, for optimising the separation of a mixture of peptides and similar compounds,  it is best to consider parameters, such as sample loading and stationary  phase particle size. In this application note, the separation of a peptomer  from a peptide side-product using Teledyne ISCO’s RediSep® C18 reversed  phase columns as the stationary phase will be shown.Experimental SectionThe separation of a mixture of one peptomer and one close-running peptide (retention times 9.34 and 8.06 min, respectively over a 20 min gradient H2O/MeCN as shown in Fig. 3) was achieved with both RediSep® Rf C18 4.3 g and RediSep Rf C18 Gold® 5.5 g, using  the Teledyne ISCO CombiFlash® NextGen 300+. Table 1 shows the sequences of the peptomer and its side-product, which appears as a deletion of the parent compound. Parameters kept constant in this study are listed in Table 2, while the parameters that have been modified are showed in Table 3.Figure 1. Peptides and peptomersTable 2. Fixed parameters used in Flash  chromatography separationInstrumentation Teledyne ISCO CombiFlash NextGen 300+Wavelengths 214 nm (red) 254 nm (purple)Mobile phases Solvent A: Purified H2O Solvent B: MethanolFlow Rate 13 mL/minEquilibration Volume28.7 mLGradient % Solvent B 10 100 100 90 90Minute Initial 5 7.6 8 8.8Run Length 8.8 min, not including equilibration timeTable 1. Peptomer sequence and side-product sequence.  The letter F highlighted in bold is the amino acid  replaced with its peptoid equivalent.Entry   SequencePeptomer   VTPDGVPFAF-NH2Side-product   VTPDGVPFA-NH2Vera D’Aloisio1 and Christopher R. Coxon2*Chromatography Application Note AN115Characterization of crude and purified peptomer was performed with a low resolution LCMS Waters TQD mass spectrometer equipped with an Agilent ZORBAX SB-C18 Stable Bond Analytical column (5 µm particle size, 4.6 x 150 mm) with a binary eluent system comprising MeCN/H2O (20 min gradient: from 90% H2O/10% MeCN to 100% MeCN with 0.1 % formic acid) as mobile phases. Electrospray ionisation mass spectrometry was conducted in positive ion mode (m/z range: 600–1900) using a cone voltage of 50 V, desolvation temperature of 350 °C and source temperature of 100 °C. ResultsWhen a low amount of peptide-peptomer mixture (8 mg) was loaded on C18 reversed phase 4.3 g RediSep column (Run 1), flash chromatography successfully separated the product using water/methanol as the mobile phase (Figure 1). No ion pairing reagent (e.g. TFA) was required for the separation. Higher peak resolution was obtained when the same amount of initial mixture (8 mg) was loaded on a C18 reversed phase 5.5 g RediSep Gold column (Figure 2, Run 2).Loading a higher amount of crude mixture (16 mg) incurred a loss of resolution when a normal RediSep C18 4.3 g was used (Run 3). In contrast, the successful separation at this scale when using a RediSep Gold C18 5.5 g (Run 4)  highlights the importance of the use of a column with lower particle size when the separation is scaled up. The gold column stationary phase composition exhibits a patented spherical Flash media, which affords the benefits of tighter packing without increasing back pressure. The resolving power is doubled compared with a standard flash chromatography column, allowing the separation of difficult compounds with low ∆Rf, such as a parent peptomer and truncation side-product and, at the same time, allowing the loading of twice as much compound without losing resolution of the peaks.Table 3. Parameters changed in different runs.Run N. Column Amount loaded (liquid loading)1 RediSep Rf C18  4.3 g 0.5 mL (8 mg/mL)2 RediSep Rf C18  5.5 g GOLD 0.5 mL (8 mg/mL)3 RediSep Rf C18  4.3 g 1 mL (8 mg/mL)4 RediSep Rf C18  5.5 g GOLD 1 mL (8 mg/mL)Sample: ljmu019-8mg-8min-gold CombiFlash NEXTGEN : LJMU Thursday 08 October 2020 02:40PMPage 1 of 1Column: C18 4-6gFlow Rate: 18 ml/minEquilibration Volume: 28.7 mlInitial Waste: 0.0 mlAir Purge: 0.0 minSolvent A: WaterSolvent B: MethanolPeak Tube Volume: 15 mlNon-Peak Tube Volume: 15 mlLoading Type: LiquidWavelength 1 (red): 214nm    Peak Width: 30 sec    Threshold: 0.20 AUWavelength 2 (purple): 254nmRun Notes:0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.001020304050607080901000.000.050.100.150.200.250.300.350.400.450.50Absorbance Percent B12356Run Length 8.8 MinDuration %B Solvent A Solvent B0.0 10.0 Water Methanol5.0 100.0 Water Methanol2.5 100.0 Water Methanol0.0 50.0 Water Methanol1.3 50.0 Water MethanolPeak # Start Tube End Tube1 A:2 A:32 A:5 A:51 2 3 4 567891011 12 13 14 15161718192021 22 23 24 25262728293031 32 33 34 35363738394041 42 43 44 45464748495051 52 53 54 55565758596061 62 63 64 65666768697071 72 73 74 75Rack A16 mm x 150 mm TubesSample: 201008@13-59-1 CombiFlash NEXTGEN : LJMU Thursday 08 October 2020 01:59PMPage 1 of 1Column: C18 4-6gSN: E00401004804E4A5 Lot: 301116369WFlow Rate: 18 ml/minEquilibration Volume: 28.7 mlInitial Waste: 0.0 mlAir Purge: 0.0 minSolvent A: WaterSolvent B: MethanolPeak Tube Volume: 18 mlNon-Peak Tube Volume: 18 mlLoading Type: L quidWavelength 1 (red): 214nm    Peak Width: 30 sec    Threshold: 0.20 AUWavelength 2 (purple): 260nmAll Wavelength (orange): 200-300nm    Peak Width: 30 sec    Threshold: 0.20 AURun Notes:0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.001020304050607080901000.000.050.100.150.200.250.300.350.400.450.50Absorbance Percent B123456Run Length 8.8 MinDuration %B Solvent A Solvent B0.0 10.0 Water Methanol5.0 100.0 Water Methanol2.5 100.0 Water Methanol0.0 50.0 Water Methanol1.3 50.0 Water MethanolPeak # Start Tube End Tube1 A:2 A:22 A:3 A:33 A:4 A:51 2 3 4 567891011 12 13 14 15161718192021 22 23 24 25262728293031 32 33 34 35363738394041 42 43 44 45464748495051 52 53 54 55565758596061 62 63 64 65666768697071 72 73 74 75Rack A16 mm x 150 mm TubesSample: ljmu019-15mg-normal_redisep CombiFlash NEXTGEN : LJMU Thursday 08 October 2020 06:10PMPage 1 of 2Column: C18 4-6gFlow Rate: 13 ml/minEquilibration Volume: 28.7 mlInitial Waste: 0.0 mlAir Purge: 0.0 minSolvent A: WaterSolvent B: MethanolPeak Tube Volume: Max.Non-Peak Tube Volume: Max.Loading Type: LiquidWavelength 1 (red): 214nm    Peak Width: 30 sec    Threshold: 0.20 AUWavelength 2 (purple): 254nmRun Notes:0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.001020304050607080901000.000.050.100.150.200.250.300.350.400.450.50Absorbance Percent B1234Run Length 8.8 MinDuration %B Solvent A Solvent B0.0 10.0 Water Methanol5.0 100.0 Water Methanol2.5 100.0 Water Methanol0.4 90.0 Water Methanol0.8 90.0 Water MethanolPeak # Start Tube End Tube1 A:2 A:22 A:3 A:41 2 3 4 567891011 12 13 14 15161718192021 22 23 24 25262728293031 32 33 34 35363738394041 42 43 44 45464748495051 52 53 54 55565758596061 62 63 64 65666768697071 72 73 74 75Rack A16 mm x 150 mm TubesSample: ljmu019-16mg-gold CombiFlash NEXTGEN : LJMU Thursday 08 October 2020 03:00PMPage 1 of 1Column: C18 4-6gFlow Rate: 18 ml/minEquilibration Volume: 28.7 mlInitial Waste: 0.0 mlAir Purge: 0.0 minSolvent A: WaterSolvent B: MethanolPeak Tube Volume: 15 mlNon-Peak Tube Volume: 15 mlLoading Type: LiquidWavelength 1 (red): 214nm    Peak Width: 30 sec    Threshold: 0.20 AUWavelength 2 (purple): 254nmRun Notes:0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.001020304050607080901000.000.050.100.150.200.250.300.350.400.450.50Absorbance Percent B123456Run Length 8.8 MinDuration %B Solvent A Solvent B0.0 10.0 Water Methanol5.0 100.0 Water Methanol2.5 100.0 Water Methanol0.0 50.0 Water Methanol1.3 50.0 Water MethanolPeak # Start Tube End Tube1 A:2 A:32 A:5 A:51 2 3 4 567891011 12 13 14 15161718192021 22 23 24 25262728293031 32 33 34 35363738394041 42 43 44 45464748495051 52 53 54 55565758596061 62 63 64 65666768697071 72 73 74 75Rack A16 mm x 150 mm TubesRediSep Rf C18 4.3 g8 mg16 mgRediSep Rf Gold C18 5.5 gFigure 2. Chromatograms from separations obtained by varying the sample amount and the column media.Chromatography Application Note AN115Teledyne ISCOP.O. Box 82531, Lincoln, Nebraska, 68501 USAToll-free: (800) 228-4373 • Phone: (402) 464-0231 • Fax: (402) 465-3091Teledyne ISCO is continually improving its products and reserves the right to change product  specifications, replacement parts, schematics, and instructions without notice. v1.0Figure 3. HPLC chromatograms before and after separation. Fraction 1 contains the side-product  of the parent sequence, while Fraction 2 contains the desired purified peptomer.ConclusionsThe separation of polar compounds such as peptides and peptidomimetics is usually achieved by reversed phase preparative-HPLC due to the typically higher resolving power compared to traditional flash chromatography. Separating closely related peptides, differing by one or two amino acids, can be challenging even when using reversed-phase liquid chromatography. The use of preparative-HPLC is also time-consuming, requires expensive columns, with the possibility of loading only small amounts of sample in each run, meaning that multiple injections are required. Flash chromatography using pre-packed C18 reversed-phase columns provides convenient and comparable preparative separations with automated flash chromatography instrumentation.In this work, separation of a peptomer from its side product was successfully achieved using flash chromatography pre-packed columns. This allowed the introduction of higher crude sample amounts and achieved purity comparable with preparative-HPLC but requiring a shorter run time (8.8 min). The peak resolution was clearly influenced by the column particle size and the loading, suggesting that for higher loading and difficult separation, the extra resolution of the RediSep Gold C18 technology is key for successful separation.1  School of Pharmacy and Biomolecular Sciences, Faculty of Science, Liverpool John Moores University,    Liverpool, United Kingdom, L3 3AF2*Corresponding author, Institute of Chemical Sciences, School of Engineering and Physical Sciences,     Heriot-Watt University, Edinburgh, United Kingdom, EH14 4ASCrude SampleFraction 1 (deletion)Fraction 2 (pure peptomer)

Reversed Phase Flash Chromatography Purification of Peptide-Peptoid Hybrids

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Reversed Phase Flash Chromatography Purification of Peptide-Peptoid Hybrids

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