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Integrating Countercurrent Separations into Natural Product Purification Workflow

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Presentation from Dr. Guy Harris at the annual meeting of the American Society of Pharmacognosy (ASP) in Lexington Kentucky (2018) during the CENAPT/Gilson Workshop on Countercurrent Chromatography.

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Integrating Countercurrent Separations into Natural Product Purification Workflow

  1. 1. Integrating Countercurrent Separations into Natural Product Purification Workflows Countercurrent Chromatography Workshop Amercian Society of Pharmacognosy Annual Meeting Lexington, Kentucky July 21, 2018 Guy H. Harris, PhD Independent Consultant Asbury, New Jersey 1
  2. 2. Integrating Countercurrent Separations into Natural Product Purification Workflows or… “How and Why to use CS when you don’t have too!” Countercurrent Chromatography Workshop Amercian Society of Pharmacognosy Annual Meeting Lexington, Kentucky July 21, 2018 Guy H. Harris, PhD Independent Consultant Asbury, New Jersey 2
  3. 3. Systematic Solvent System Selection Process Incorporation “Almost is good enough” G,Harris ASP CS Workshop, July 21, 2018 3
  4. 4. Getting Started Quickly = Solvent Systems - Kd - Sf 1) The most critical parameters are distribution ratio, Kd and stationary phase retention (stationary phase fraction), Sf 2) Chemistry not instrumentation! 3) Liquid-liquid distribution! General rules to get started quickly: 1) Almost all literature natural product CS have been accomplished with HEMWat or CMWat SS series => Great starting (and likely ending) point o Don’t get too creative to start => remove yourself as a variable J 2) Be systematic in search for SS => HEMWat and CMWat series are predictable o pH control 3) Minimize SS development by exploiting elution-extrusion (EECCC) using “almost is good enough” systems developed for particular lab process problems 4) Query scientific literature** for target or related compounds ** The CS scientific literature describing natural products separations is extremely complicated and inconsistent to sort through (acronyms, terminology, reliability,…) BUT - the principle behind all is simple => liquid-liquid distribution => the take home from any paper is the SS G,Harris ASP CS Workshop, July 21, 2018 4
  5. 5. CS Chromatogram in Terms of Kd 0 0.5 1 1.5 2 2.5 0 10 20 30 40 50 60 70 80 Vr (mL) Column Volume: 17.89mL Stationary Phase Retention: 70% Theoretical Plates, N: 750 Vo Kd=1 Kd=2 Kd=4 Vm Vc Calculated for: Kd = [stationary phase] / [mobile phase] where Kd = distribution ratio Vr = Vm + KdVs Kd = (Vr – Vm)/Vs where: Vr = retention volume Vm = mobile phase volume Vs = stationary phase volume Sf = Vs / Vc where Sf = stationary phase fraction G,Harris ASP CS Workshop, July 21, 2018 5
  6. 6. Two Fundamental Types of Modern CCS Instrumentation Distinguished by the mechanism of phase retention and mixing Hydrodynamic “CCC” Hydrostatic “CPC” Ito coil, MLCPC, HPCCC,… “Sanki”, FCPC,… G,Harris ASP CS Workshop, July 21, 2018 6
  7. 7. CCS Instrumentation Considerations • Huge overlap in applicability of CCC and CPC instrumentation to most natural product problems • “Black box” whose purpose is to retain a liquid stationary phase (= THE COLUMN) – Purpose of hardware, pumps and CS instrument, is simply to put the correct liquid in the correct place at the correct time! – Stationary phase retention • Primary factors to consider: – Stationary phase retention, stationary phase retention, stationary phase retention….. • Dependent on sample, solvent system, flow rate – Reliability G,Harris ASP CS Workshop, July 21, 2018 7
  8. 8. HEMWat SS Series Letter Number Heptane Ethyl Acetate Methanol Water %Water LP A 6 0 1 0 1 B 7 1 19 1 19 C 8 1 9 1 9 D 9 1 6 1 6 F 10 1 5 1 5 G 11 1 4 1 4 H 12 1 3 1 3 J 13 2 5 2 5 40 K 14 1 2 1 2 L 15 2 3 2 3 51 M 16 5 6 5 6 N 17 1 1 1 1 64 P 18 6 5 6 5 Q 19 3 2 3 2 R 20 2 1 2 1 S 21 5 2 5 2 T 22 3 1 3 1 U 23 4 1 4 1 V 24 5 1 5 1 W 25 6 1 6 1 X 26 9 1 9 1 Y 27 19 1 19 1 Z 28 1 0 1 0 Composition of phases by numbered system: I.J.Garrard. L.Janaway, D.Fisher, J. Liq. Chrom. Rel. Tech., 30 (2007), 151-163 Solvent systems by letter: ARIZONA G,Harris ASP CS Workshop, July 21, 2018 8
  9. 9. Correlation of Kd and CCS Chromatogram – GUESS Mix Standards Relative Compound Polarity Polar Nonpolar RelativeSSPolarity Polar Non- polar C C C A A A F F F N N N 40% H2O Kd=1 logKd=0 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 30 40 50 60 70 logKd % Water O OH OOH OH O O OH O OH O OH O N N N N H O O Caffeine (C) Ferulic acid (F) Aspirin (A) Naringenin (N) “test tube” Kd CS Chromatogram 51% H2O 64% H2O G,Harris ASP CS Workshop, July 21, 2018 9
  10. 10. Bioassay-Guided Natural Product Isolation as a 3-Step Process Step Purpose Common Methodology 1 Resolution & Recovery • Resolution of multiple activities • Recovery from biomatrix • Localize assay interferences • Macroporous resins • Liquid-liquid extraction (LLE) • CS 2 Purification • Separation of active from inactive components • Orthogonal separation mechanism to steps 1 and 3 • Association of activity with a specific component(s) • Silica gel / diol • IEX • Low pressure RP • CS 3 Polishing • Final purification • Highest resolution • RP/NP HPLC • Crystallization • CS Harris, G.H., in The Handbook of Industrial Mycology, ed. Z. An, Marcel Dekker, New York, 2004 G,Harris ASP CS Workshop, July 21, 2018 10
  11. 11. P-504062-001X 0 50 100 %MobilePhase 0 500 mVolts 0 10 20 30 Minutes Zero96_B:196_B:296_B:396_B:496_B:596_B:696_B:796_B:896_B:996_B:1096_B:1196_B:1296_B:1396_B:1496_B:1596_B:1696_B:1796_B:1896_B:1996_B:2096_B:2196_B:2296_B:2396_B:2496_B:2596_B:2696_B:2796_B:2896_B:29 c:hplcpreptamara96wellf.110prokchp2.gdt : WL_1 : P-504062-001X: Inj. Number: 1 c:hplcpreptamara96wellf.110prokchp2.gdt : WL_2 : P-504062-001X: Inj. Number: 1 Aqueous Organic * * * Wild type C. albicans hetEF31 – PSDVB Reverse Phase 2 – Ion Exchange 3 – RP HPLC BioassayChromatographic Separation Example - Yefafungin Bioassay-Guided Isolation Roemer, et al., Chem Biol. 18 (2011) 148-164G,Harris ASP CS Workshop, July 21, 2018 11
  12. 12. Yefafungin HN N O O O OP OCH3 O H NP O OH O H NO O HO OH Roemer, et al., Chem Biol. 18 (2011) 148-164 G,Harris ASP CS Workshop, July 21, 2018 12
  13. 13. 504094- 001G 0 50 100 -500 0 500 m Vol ts 0 20 40 Minute s Zero F:1 F:2 F:3 F:4 F:5 F:6 F:7 F:8 F:9 F:10 F:11 F:12 F:13 F:14 F:15 F:16 F:17 F:18 F:19 F:20 Example - Colletofungins Bioactivity-Guided Isolation 0 100 200 300 400 500 600 0 20 40 60 80 L-472723 L-472724 min0 5 10 15 20 25 30 mAU 0 250 500 750 1000 1250 1500 1750 DAD1 C, Sig=210,16 Ref=360,100 (04_16_0204_16B.D) 0.338 1.068 2.031 2.388 3.1503.363 4.465 4.893 5.215 5.562 5.979 6.324 6.7316.914 7.319 7.962 8.394 8.862 9.244 9.631 10.042 10.441 10.676 10.98611.183 11.423 11.65711.847 12.117 12.47812.635 13.102 13.534 13.90714.09714.293 14.897 15.185 15.59315.81016.003 16.303 16.87017.04217.255 17.61417.825 18.069 18.854 19.32719.500 19.841 20.115 20.670 21.383 22.062 22.310 22.743 23.54023.749 25.591 26.419 26.927 27.890 DAD1 E, Sig=280,16 Ref=360,100 (04_16_0204_16B.D) 2.426 3.513 5.210 5.630 7.520 7.962 8.858 9.515 10.212 10.685 11.432 11.867 12.116 12.654 13.092 13.538 13.896 14.293 14.86515.027 15.544 16.272 16.69616.81417.000 17.529 18.279 18.507 18.85019.055 19.408 19.729 19.955 20.670 21.44121.61221.804 22.189 22.404 22.761 23.182 23.539 24.04424.243 24.714 25.118 25.746 26.132 26.836 27.151 PMP1, Solvent B min0 5 10 15 20 25 30 mAU 0 500 1000 1500 2000 DAD1 C, Sig=210,16 Ref=360,100 (04_16_0204_16C.D) 1.225 1.6041.795 2.046 2.409 3.1543.366 4.8345.0445.221 5.848 6.268 6.804 7.0507.255 7.728 8.075 8.379 8.9529.166 9.523 10.047 10.331 10.702 10.98911.164 11.691 12.130 12.623 13.02513.161 13.548 13.91314.059 14.34514.53314.705 15.073 15.284 15.525 15.956 16.265 16.665 17.315 17.747 18.322 18.614 18.905 19.358 19.691 20.176 20.811 21.197 21.743 22.352 22.781 23.546 23.787 24.059 24.856 25.202 25.586 26.408 26.940 27.871 28.448 30.636 DAD1 E, Sig=280,16 Ref=360,100 (04_16_0204_16C.D) 2.432 3.427 8.189 8.656 8.894 9.521 10.435 10.712 10.95111.16111.366 11.708 12.135 12.636 12.867 13.238 13.57813.72613.922 14.34814.53414.722 15.019 15.288 15.504 15.856 16.07616.263 16.663 17.004 17.359 17.624 17.905 18.29218.493 19.198 19.691 20.354 20.795 21.447 21.797 22.493 22.791 23.166 23.542 24.016 24.234 24.723 25.108 25.830 26.049 26.296 26.673 27.918 28.440 29.059 PMP1, Solvent B 1 – PSDVB Reverse Phase 2 – CCC 3 – RP HPLC G.Harris, et al. (Merck Research Labs) unpublishedG,Harris ASP CS Workshop, July 21, 2018 13
  14. 14. Colletofungins CH3 OH OH OH CH3 OH O CH3 CH3 CH3 CH3 CH3 O CH3 OH O O O R 1 G.Harris, et al. (Merck Research Labs) unpublishedG,Harris ASP CS Workshop, July 21, 2018 14
  15. 15. Step 1 – Resolution and Recovery • Recovery from biomatrix – Method choice depends upon extraction solvent • Water miscible or immiscible – Commonly used methodologies • Solid phase adsorption (SPE) • Liquid-liquid partition • CS • Resolution of multiple biologically active components – Low resolution methods sufficient – Must be chemically “gentle” – Reproducible • pH control G,Harris ASP CS Workshop, July 21, 2018 15
  16. 16. Elution-Extrusion (EECCC) 0 10 20 30 40 50 elution extrusion Kd=1 Kd=0.01 Kd=0.2 Kd=5 Kd=100 System Parameters CCC Vc=135mL F=6mL/min Sf=0.8 N=1000 Time(min) Berthod, A., Ruiz-Angel, M.J., Carda-Broch, S., Anal. Chem. 75 (2003), 5886-5894 Berthod, A., Hassoun, M. Harris, G.H., J. Liq. Chrom. Rel. Tech. 28 (2005), 1851-1866 Berthod, A., Friesen, B., Inui, T., Pauli, G. Anal. Chem. 79 (2007), 3371-3382 Kd=1 logKd=0 Exploiting the “support-free liquid stationary phase” G,Harris ASP CS Workshop, July 21, 2018 16
  17. 17. 0.01 0.2 1 5 100 MP SP 0.01 0.2 1 5 Berthod, A., Ruiz-Angel, M.J., Carda-Broch, S., Anal. Chem. 75 (2003), 5886-5894 Berthod, A., Hassoun, M. Harris, G.H., J. Liq. Chrom. Rel. Tech. 28 (2005), 1851-1866 Berthod, A., Friesen, B., Inui, T., Pauli, G. Anal. Chem. 79 (2007), 3371-3382 Classical mode Elution-Extrusion MP SP after one column volume of SP after SP extrusion Elution – Extrusion (EECCC) No band broadening of SP retained components 0.01 0.2 1 5 100 G,Harris ASP CS Workshop, July 21, 2018 17
  18. 18. Distribution of Example Plant Natural Products In Standardized Solvent System Armbruster, et al. J. Liq. Chrom. & Rel. Tech., 24 (2001) 1827-1840 Kd Stationary PhaseMobile Phase 1 0.1 10 100 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 LogKd CCC Fraction Number Glycyrhizzic Acid Glycyrrhetinic Acid Secologanin Stigmasterol Berberine Brucine Sparteine Sanguinarine Catechin Umbelliferone Esculetin Genistein Ellagic Acid fatty acids polyphenolics Kd=1 SS = CH2Cl2 : CH3OH : H2O (5:6:4); LP=MP G,Harris ASP CS Workshop, July 21, 2018 18
  19. 19. HSCCC Fraction Number Weight(mg) 200 180 160 140 120 100 80 60 40 20 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Weight Distribution of the 64 Fraction Sets nonpolar polar CH2Cl2 xt aqueous xt “dual-mode” = EECCC CH2Cl2 : CH3OH : H2O (5:6:4) 500 mg MeOH extract (hexane partitioned, PVP treated) Armbruster, et al. J. Liq. Chrom. & Rel. Tech., 24 (2001) 1827-1840G,Harris ASP CS Workshop, July 21, 2018 19
  20. 20. Generic Strategy for Purification of Crude Reaction Mixtures Removal of Triphenylphosphine Oxide from Mitsunobu Reaction Products – Purification of Reaction Products • Purification of synthetic products from crude reaction mixtures containing triphenylphosphine oxide can be problematic using typical separation methods • A standard CCC strategy was developed based upon the one column volume elution – extrusion methodology • TPPO elutes at D=1 using HEMWat SS16 • Products more polar than TPPO elute early earlier; products more non- polar than TPPO elute in column extrusion • Advantages: • Directly applicable to crude reaction mixtures – no sample preparation • No method development for CCC • Recovery and purity of products equivalent to that obtained using preparative RP HPLC • Complete recovery of entire reaction mixture • TPPO retention independent of solvent system pH Edwards, N.A., et al., J.Chrom. A, 1323 (2014) 49-56 G,Harris ASP CS Workshop, July 21, 2018 20
  21. 21. Mitsunobu Reaction Product Purification – Method and Theory 0 10 20 30 40 Time (min) Absorbance254nm 1 2 0 1 2 3 4 5 6 0 0.5 1 1.5 2 intensity Column Volumes elution extrusion A B D=0.01 D=0.2 D=1 D=5 D=100 elution extrusion Dynamic Extractions Spectrum HPCCC Semi-preparative Column, Vc=135mL 1600rpm, 30 oC Hexanes:EtOAc:MeOH:Water (5:6:5:6, v:v:v:v) Reverse Phase Mode (upper phase stationary) Stationary Phase Retention = 80% Elution, LP @ F=6mL/min, 0 - 30min Extrusion UP @ F=10mL/min, 30.1 – 46min TPPO tr~23’, D=1.02 Method Theory polar non-polar Edwards, N.A., et al., J.Chrom. A, 1323 (2014) 49-56 G,Harris ASP CS Workshop, July 21, 2018 21
  22. 22. Mitsunobu Reaction Product Purification – Example 5 0 10 20 30 40 Time (min) Absorbance254nm * * D=9.00 * TPPO Dynamic Extractions Spectrum HPCCC Semi-preparative Column, Vc=135mL 1600rpm, 30 oC Hex:EtOAc:MeOH:Water (5:6:5:6, v:v:v:v) Reverse Phase Mode (upper phase stationary) Stationary Phase Retention = 80% Elution, LP @ F=6mL/min, 0 - 30min Extrusion UP @ F=10mL/min, 30.1 – 46min Crude Sample 229mg @ 48% HPLC area purity Pure Product 26.5mg @ >99% HPLC area purity HPLC HPCCC HPLC yielded 9mg product @ 93% HPLC area purity Edwards, N.A., et al., J.Chrom. A, 1323 (2014) 49-56 G,Harris ASP CS Workshop, July 21, 2018 22
  23. 23. Mitsunobu Reaction Product Purification – Example 11 0 10 20 30 40 Time (min) Absorbance254nm * * D=0.49 * TPPO Dynamic Extractions Spectrum HPCCC Semi-preparative Column, Vc=135mL 1600rpm, 30 oC Hex:EtOAc:MeOH:Water (5:6:5:6, v:v:v:v) Reverse Phase Mode (upper phase stationary) Stationary Phase Retention = 80% Elution, LP @ F=6mL/min, 0 - 30min Extrusion UP @ F=10mL/min, 30.1 – 46min Crude Sample 182mg @ 51% HPLC area purity Pure Product 24.1mg @ 92% HPLC area purity HPLC yielded 5mg product @ >99% HPLC area purity HPLC HPCCC Edwards, N.A., et al., J.Chrom. A, 1323 (2014) 49-56 G,Harris ASP CS Workshop, July 21, 2018 23
  24. 24. Mitsunobu Reaction Product Purification – Example 10 0 10 20 30 40 Time (min) Absorbance254nm * * * TPPO D=1.27 Crude Sample 219mg @ 31% HPLC area purity Pure Product 17.6mg @ 98% HPLC area purity HPLC yielded 18mg product @ >99% HPLC area purity Dynamic Extractions Spectrum HPCCC Semi-preparative Column, Vc=135mL 1600rpm, 30 oC Hex:EtOAc:MeOH:Water (5:6:5:6, v:v:v:v) Reverse Phase Mode (upper phase stationary) Stationary Phase Retention = 80% Elution, LP @ F=6mL/min, 0 - 30min Extrusion UP @ F=10mL/min, 30.1 – 46min HPLC HPCCC Edwards, N.A., et al., J.Chrom. A, 1323 (2014) 49-56 G,Harris ASP CS Workshop, July 21, 2018 24
  25. 25. Step 2 – Purification • Chemical resolution of desired bioactive component(s) from inactive components of extract • Wide choice of methodology – Moderate resolution sufficient if orthogonal – Ideally one separation but can be a combination of two – Common methods • Normal phase chromatography • Ion exchange • CS G,Harris ASP CS Workshop, July 21, 2018 25
  26. 26. 0 5 10 15 20 25 30 35 0 5 10 15 20 Time (min) Adsorbance 0 5 10 15 20 25 30 35 0 5 10 15 20 Time (min) Adsorbance A B C D E F G H C D E 0 5 10 15 20 25 30 35 0 5 10 15 20 Time (min) Adsorbance C D E Non-Orthogonal Separations RP HPLC - A RPHPLC-B More or less equivalent separation mechanisms Harris, G.H., in The Handbook of Industrial Mycology, ed. Z. An, Marcel Dekker, New York, 2004 G,Harris ASP CS Workshop, July 21, 2018 26
  27. 27. 0 5 10 15 20 25 30 35 0 5 10 15 20 Time (min) Adsorbance 0 5 10 15 20 25 30 35 0 5 10 15 20 Fraction (min) Adsorbance 0 5 10 15 20 25 30 35 0 5 10 15 20 Time (min) Adsorbance A B C D E F G H CC E B A Orthogonal Separations Polymeric RP CCC(NP) Reverse phase adsorption followed by normal phase partition Harris, G.H., in The Handbook of Industrial Mycology, ed. Z. An, Marcel Dekker, New York, 2004 G,Harris ASP CS Workshop, July 21, 2018 27
  28. 28. -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Time (min.) AreaCounts MEK extract EECCC Rich Cut, 2.6 mg Example - Discovery Isolation of Moriniafungin MEK extract (40mg) EECCC solvent system: hexane:EtOAc:MeOH:H2O (5:5:5:5);UP=MP @ 3.0 mL/min., tail -> head Conway Centrichrome DP1000, Vc=175mL, 800rpm Preparative RP HPLC Moriniafungin (~1.5 mg) CO2H CHO OO OH CH3O O O O HO2C moriniafungin novel sordarin analog Basilio, A., et al. Bioorganic and Medicinal Chemistry 14 (2006), 560-566G,Harris ASP CS Workshop, July 21, 2018 28
  29. 29. EECCC Based Strategy for Antifungal Fermentation Extract Analysis and Isolation • Two-step process consisting of two moderate resolution but orthogonal separation methods 1) CHP20P - reverse phase adsorption, gradient elution increases generality 2) EECCC – normal phase liquid-liquid partition, EECCC mode increases generality • Standardization yields chromatographic retention information useful for dereplication and allows semi-automation – Kd standardization allows for sample dependent variation of Sf – LCMS analysis of fractions for early recognition of knowns G,Harris ASP CS Workshop, July 21, 2018 29
  30. 30. EECCC Separation Conditions • Instrumentation: – Conway CentriChrome DP-1000 containing 2 – 175 mL bicoils (4 total coils) – Control with in-house automated unit • Operating Parameters: – elution-extrusion mode, upper phase = mobile phase at 2.0mL/min (175mL total); extrusion with lower phase = stationary phase (175.1mL to 367.5mL total elution volume); tail->head, both processes at 800 RPM – “Ito” type injection – solvent system: hexane:EtOAc:MeOH:H2O (3:5:3:5) – Sf routinely 0.75 – 0.85 An In-House Built Semiautomated Countercurrent Chromatography Workstation”, Napolitano, C., Wismer, M., Furlano, E., Harris, G., Uhrig, B., Blake, K., Kath, G., Dufresne, C., JALA, 2009, 27-35 G,Harris ASP CS Workshop, July 21, 2018 30
  31. 31. Hydrodynamic EECCC Separation of Antifungal Standard Compounds WF22210 pneumocandin Bo enfumafungin arundifungin ascosteroside extrusion elution Kd~1 200nm CCC = Conway Centrichrome DP1000 (Vc=175mL) N O NH O HO NH OH HO HO O N OH O HN OH O OH NH HO O NH OH H H H O H2N O N O NH HO O H2N O NH OH HO HO OSO3H O N OH O HN OH O OH N H HOOOH N H O O AcO H H HO2C OH O OH HO OH HO O OH OH OH OH O HO O O OH H CO2H O OH HO CH3O HO G,Harris ASP CS Workshop, July 21, 2018 31
  32. 32. Hydrostatic EECCC Separation of Antifungal Standard Compounds extrusion elution Kd~1 WF22210 pneumocandin Bo enfumafungin arundifungin ascosteroside 200nmCPC = Kromaton (Vc=1L) N O NH O HO NH OH HO HO O N OH O HN OH O OH NH HO O NH OH H H H O H2N O N O NH HO O H2N O NH OH HO HO OSO3H O N OH O HN OH O OH N H HOOOH N H O O AcO H H HO2C OH O OH HO OH HO O OH OH OH OH O HO O O OH H CO2H O OH HO CH3O HO G,Harris ASP CS Workshop, July 21, 2018 32
  33. 33. Distribution of Antifungal Activity in Routine Extracts E-000002378 E-000002726 E-000900085 E-000504402 E-000504403 E-000504405 E-000504406 E-000504413 E-000504415 E-000504426 E-000504427 E-000504429 E-000504441 E-000504444 E-000504445 E-000504446 E-000504450 E-000504455 E-000504461 E-000504471 E-000504504 E-000504519 E-000504521 E-000504521 E-000504525 E-000504526 E-000504531 E-000504537 E-000504541 E-000504607 E-000504608 P-000502922 P-000502924 10 20 30 ascosteroside ascosteroside analogs unidenified triterpenes arundifungin enfumafungin novel papulacandins lipopeptides Kd 0 0.5 1 1.5 2 4 8 16 90 ∞ fraction polarity G,Harris ASP CS Workshop, July 21, 2018 33
  34. 34. Step 3 – Polishing • Final cleanup of desired compound • Target compound identified • Limited choice of methodology – High resolution desired – Common methodology • Preparative RP HPLC • Crystallization • SFC • CS G,Harris ASP CS Workshop, July 21, 2018 34
  35. 35. Example - Antifungal Cyclodepsipeptide Isolation N O O O NHO N OCH3 O NHO NO O NH N N O O N O CO2H J. Med. Chem. 1994, 37, 1908-1917 Discovery Isolation Mitsubishi CHP20Y Chromatography Acetone Culture Extract Standardized EECCC H:E:M:W (3:5:3:5) RP HPLC Preparative Isolation Kd < 0.1 Mitsubishi CHP20Y Chromatography Acetone Culture Extract Optimized EECCC H:E:M:W (5:4:5:4) Kd ~ 0.6 G,Harris ASP CS Workshop, July 21, 2018 35
  36. 36. EECCC Solvent System Optimization for Cyclodepsipeptide -100 100 300 500 700 900 1100 0 5 10 15 20 Time (min) A280nm(AU*s) 3:5:3:5 5:5:5:5 5:4:5:4 5:3:5:3 extrusionelution MiniDE Vc=5.4mL, 2200rpm UP=MP, 0.5mL/min, T->H Control using Agilent 1100 HPLC 0 500 1000 1500 2000 0 10 20 30 40 Fraction mAU*s(280nm) extrusionelution Conway CentriChrome “auto” Vc=175mL, 800rpm 5:4:5:4, UP=MP, 3mL/min 9.67mL fractions (38) Feed – CHP20P rich cut 74mg in 5mL UP + 5mL LP for each of 2 coils G,Harris ASP CS Workshop, July 21, 2018 36
  37. 37. Example - HSCCC Separation of Australifungin Phenomenex Ultracarb 30 ODS, 9.4x250mm 55% CH3CN / 45% 25mM K2HPO4 pH6.8 55 oC, A280 nm P.C. Inc. MLCPC, coil volume = 385 mL hexane:EtOAc:MeOH: 25mM K2HPO4 pH6.8 (7:3:5:5), 3 mL/min, 800 rpm, T->H A280 nm - O.D. Hensens, et al. J. Org. Chem., 60 (1995) 1772-1776. - S.M. Mandala, et al. J. of Antibiot., 48 (1995) 349-356. Solid stationary phase interaction problems G,Harris ASP CS Workshop, July 21, 2018 37
  38. 38. Example - CCC Separation of Viridiofungins P.C., Inc. MLCPC, coil volume 385 mL, 800 rpm, T->H, upper phase = MP, hexane:EtOAc:MeOH:H2O+0.1%H3PO4 (5:5:5:5), 950 mg of crude tricarboxylic acid fraction obtained from MEK extract viridiofungin A (558 mg) recovered from extruded stationary phase 95 mg 30 mg K=0.48 K=0.30 HO2C HO CO2H NHO RHO2C O OH N H B R = A R = C R = Viridiofungins: Harris, G.H., Jones, E.T.T., Meinz, M.S., Nallin-Omstead, M., Helms, G.L., Bills, G.F., Zink, D., Wilson, K.E. Tetr. Lett., 1993, 34, 5235-5238 Solvent System: Oka, F., Oka, H., Ito, Y, J. Chromatog. A., 1991, 538, 99-108 G,Harris ASP CS Workshop, July 21, 2018 38
  39. 39. Natural Product CS Literature • There is a lot, many reviews for ideas,….. • Is there a natural product that can’t be separated using CS? • Focus on solvent system and mode of operation – Useful recent review of elution mode operational modes: X.-Y. Huang, Tr. Anal. Chem. (2016) 77, 214-225 • Useful recent compilations: – Terpenes: K. Skalicka-Wozniak, I. Garrard, Phytochem. Rev. (2014) 13, 547-572 – General: J.B. Friesen, et al., J. Nat. Prod. (2015) 78, 1765-1796 – General: K. Skalicka-Wozniak, I. Garrard, Nat. Prod. Rep. (2015) 32, 1556-1561 G,Harris ASP CS Workshop, July 21, 2018 39
  40. 40. Systematic Solvent System Selection GUESS methodology F.Oka, H. Oka, Y. Ito, J. Chrom. A (1991), 538, 99-108 Process Incorporation Workflow section in latest CCS Review Friesen et al., J. Nat. Prod. (2015), 78, 1765-1796 “Almost is good enough” Elution – Extrusion CCC Range of other CS operational modes available G,Harris ASP CS Workshop, July 21, 2018 40
  41. 41. Acknowledgements Gilson Purification and Gregoire Audo American Society of Pharmacognosy and Laura Stoll G,Harris ASP CS Workshop, July 21, 2018 41

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