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K based-chromatography: future and potential of Countercurrent Chromatography

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Future and potential of Countercurrent Chromatography (CCC) from preparative isolation of compounds to the production of Knock-out Extracts.
Can CCC become a mainstream technique?

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K based-chromatography: future and potential of Countercurrent Chromatography

  1. 1. The Future of K-Based Chromatography The 9th International CCC Event in Chicago/USA Conference: August 1-3, Workshop: July 30-31, 2016 Dominican University, River Forest, IL (U.S.A.)
  2. 2. CCS as a “niche” technique • Preparative isolation of known compounds HO HO C O OH HO HO H C C H C O OH HO HO H C C H C O O OH OH HO C O OH
  3. 3. CCS as a “niche” technique • Specialized method of performing separations already done with column chromatography methods. HO HO H C C H C O OH HO HO H C C H C O O OH OH HO C O OH
  4. 4. Countercurrent Separations Into the Mainstream A wok in every kitchen and http://lh4.googleusercontent.com/public/l33bGiAu5pLcwwuK9O68cynPISlfGvpyWbYWUj-Vrqqt70OIrBK8in8xcMUa0lP40mZij6vDSPa381L7sOO16GqzZbCWcmmKdDu_Mr7FQR9JBT0O_sr0kSiZZEjlq3cQ__Xq9qheN3hSTFbC-wLR0qmzthl4JxQvlb0PX9XUQz7m29bUW1E a CCS instrument in every laboratory
  5. 5. What is necessary to make CCS a “mainstream” technique?
  6. 6. What is necessary to make CCS a “mainstream” technique? A. Purify and characterize low concentration metabolites 3. Multiple step isolation schemes that integrate CCS 2. Sequential CCS runs B. Innovations that push the limits of the technology C. Emphasize the importance of K 1. Enrichment Strategies D. Applications of CCS to Discovery
  7. 7. http://www.interfaces.com/blog/wp-content/uploads/2013/05/Divergent-Road-with-low-hanging-fruit.jpg A. Isolation of low concentration compounds will lead to new compounds
  8. 8. new chalcones 4, 11, & 12 previously unreported in hops: 5, 6, 9b, & 13 Estrogens and Congeners from Spent Hops (Humulus lupulus) Lucas R. Chadwick, Dejan Nikolic, Joanna E. Burdette, Cassia R. Overk, Judy L. Bolton, Richard B. van Breemen, Roland Frohlich, Harry H. S. Fong, Norman R. Farnsworth, and Guido F. Pauli J. Nat. Prod. 2004, 67, 2024-2032 http://www.aspca.org/~/media/Files/pet-care/poison-control/plants/large-images/hops-1.ashx A. Purify and characterize unknown analytes
  9. 9. A. Purify and characterize low concentration analytes Enrichment and identification of ginkgotoxin Quantitative (400 MHz) 1H-NMR analysis (a) the extract of a single seed; (b) the targeted HSCCC fraction corresponding to a K value of 1.62 (ChMWat, 10:5:5); (c) synthetic Liu, Y.; Chen, S. N.; McAlpine, J. B.; Klein, L. L.; Friesen, J. B.; Lankin, D. C.; Pauli, G. F., Quantification of a Botanical Negative Marker without an Identical Standard: Ginkgotoxin in Ginkgo biloba. Journal of Natural Products 2014, 77, 611-617 59 µg of ginkgotoxin per seed
  10. 10. Alpinia combined fraction in hexane / tert-butylmethylether / methanol / water 5:5:5:5 0 0.25 0.5 0.75 1 1.33 2 4 ¥K'(1) A OH O Alpinia DCM extract in hexane / ethyl acetate / methanol / water 5:5:5:5 0 0.25 0.5 0.75 1 1.33 2 4 ¥K'(1) A 0 5 10 15 mg 280nm 230nm mg OH O OO HO OO O O OH O Orthogonality
  11. 11. A. Purify and characterize low concentration metabolites : 2. Sequential CCS runs CCC Sample Cutting for Isolation of Prenylated Phenolics from Hops Lucas R. Chadwick, Harry H. S. Fong, Norman R. Farnsworth, and Guido F. Pauli Journal of Liquid Chromatography & Related Technologiesw, 28: 1959–1969, 2005
  12. 12. 3. Multiple step isolation schemes that integrate CCS Almost a third of the surveyed articles perform a preliminary column chromatography step prior to the CCS experiment. column media: • silica gel • C-18 functionalized silica gel • D101 • XAD-7, XAD-2, XAD-4 • Toyopearl TSK HW-50(F) • AB-8 • Sephadex LH-20 • polyamide preparative steps: § solid-liquid extraction § liquid-liquid extraction § precipitation § flash chromatography A. Purify and characterize low concentration metabolites : Friesen, J. B.; McAlpine, J. B.; Chen, S.-N.; Pauli, G. F., Countercurrent Separation of Natural Products: An Update. Journal of Natural Products 2015, 78, 1765-1796.
  13. 13. What is necessary to make CCS a “mainstream” technique? A. Purify and characterize low concentration metabolites B. Innovations that push the limits of the technology C. Emphasize the importance of K 1. Off/online Detection: UV, ELSD, MS, & NMR 2. Knockout strategies for biological testing. D. Applications of CCS to Discovery
  14. 14. B.1. GC shows phytochemical complexity crude 2 major 16 minor compounds fraction #3 out of 6 60 compounds refractionation of #3 600 compounds
  15. 15. Silymarin HSCCC separation, HChMWat/AcOH (1:20:22:12:12) 15 Isosilybins A+B Silybins A+B α,β–cis Sily-compounds Silydianin Silychristins Isosilychristins Taxifolin Silydianin-like compound Aromadendrin 1 H NMR spectra (600 MHz, DMSO-d6), 3.4–5.5 ppm. B.1.
  16. 16. B.2. Innovations that push the limits of the technology http://heartcurrents.files.wordpress.com/2010/03/wp_heart_fresh-cranberry-juice-12614054901.jpg http://www.chemistry-blog.com/wp-content/uploads/2008/10/column.jpg active fractions Diaion HP-20 active fractions - benzoic acid An experimental implementation of chemical subtraction Journal of Pharmaceutical and Biomedical Analysis 46 (2008) 692–698 Shao-Nong Chen, Allison Turner, Birgit U. Jaki, Dejan Nikolic, Richard B. van Breemena, J. Brent Friesen, Guido F. Pauli
  17. 17. Total Extract (TE) K-Targeted Metabolites (Ts) DESIGNER Extract (Ts-DE) Difference ExtractTotal Extract K-Targeted Metabolites Enriched Depleted Extract Extract Ramos Alvarenga, R. F.; Friesen, J. B.; Nikolic, D.; Simmler, C.; Napolitano, J. G.; vanBreemen, R.; Lankin, D. C.; McAlpine, J. B.; Pauli, G. F.; Chen, S. N., K-Targeted Metabolomic Analysis Extends Chemical Subtraction to DESIGNER Extracts: Selective Depletion of Extracts of Hops (Humulus lupulus). Journal of Natural Products 2014, 77, 2595-2604.
  18. 18. Total Extract HEMWat 0 IX IX-PDE HterAcWat +3 IX IX-PDE IX-DE Total Extract HEMWat 0 8-PN 6-PN XH HEMWat -3 8-PN/6-PN 8-PN/6-PN-PDE 8-PN/6-PN-DE8-PN/6-PN-PDE Total Extract HEMWat 0 XH XH-PDE HEMWat -3 XH XH-PDE IX-DE IX HterAcWat +3Total Extract HEMWat 0 8-PN 6-PN XH HEMWat -3 8-PN/6-PN MultiT-PDE XH MultiT-DE MultiT-PDE MultiT-PDE IX Knockout Extraction Schemes Ramos Alvarenga, R. F.; Friesen, J. B.; Nikolic, D.; Simmler, C.; Napolitano, J. G.; vanBreemen, R.; Lankin, D. C.; McAlpine, J. B.; Pauli, G. F.; Chen, S. N., K- Targeted Metabolomic Analysis Extends Chemical Subtraction to DESIGNER Extracts: Selective Depletion of Extracts of Hops (Humulus lupulus). Journal of Natural Products 2014, 77, 2595-2604. B.2. Innovations that push the limits of the technology
  19. 19. What is necessary to make CCS a “mainstream” technique? A. Purify and characterize low concentration metabolites B. Innovations that push the limits of the technology C. Emphasize the importance of K D. Applications of CCS to Discovery 1. Reciprocal Symmetry Plots 2. Monitoring Eluent Phase Composition
  20. 20. 2 3 20 69 184 247 99 26 16 0 50 100 150 200 250 300 K value distribution #ofcompounds 1 1 11 19 3 1 0 5 10 15 20 # compounds K value K values of compounds for articles isolating only one compoundC. Emphasize the importance of K Friesen, J. B.; McAlpine, J. B.; Chen, S.-N.; Pauli, G. F., Countercurrent Separation of Natural Products: An Update. Journal of Natural Products 2015, 78, 1765-1796.
  21. 21. Model Compounds: HO H H H H O O OH OH O O O HO H H H CH3 OH OH O OHO OH N O OH O OH O HO O O OH O OHO O H HO H HO H H OHH O OH OH O OH O O OH OH OH O O OH OH HO O O H HO H HO H H OHH O OH OH N N N N O O N H O OH NH2 N N OH S O O O SO O O S O O O 3Na N H N O OH H H O O O O O O O O OH OH OH OOH HO The GUESSmix Friesen J.B, Pauli G.F. Journal of Liquid Chromatography and Related Technologies, 28: 2777-2806, 2005 b O Q r R U F Y C I E MZ V G T X H D N A
  22. 22. C. Emphasize the importance of K Pauli, G.; Pro, S.; Friesen, J. B. Countercurrent Separation of Natural Products. J. Nat. Prod. 2008, 71, 1489-1508 Using the GUESSmix for method development
  23. 23. C. Emphasize the importance of K Using GUESSmix to explore solvent system families. Friesen, J.B. Pauli, G.F. Analytical Chemistry 79: 2320-2324 (2007) Symmetry Midline 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ¥K A M Q V U F N Z E
  24. 24. C.1. K-based chromatograms • The “sweet spot” present in CCS separations that is related to the distribution constant (K = Cstat/Cmob) of a certain compound in a particular solvent system. • The key to CCS separations is putting the target compound(s) in the sweet spot where optimal separation can take place. GUESSmix in HEMWat 4646 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 34 5.33 8 16 ¥K A 280nm 230nm Reciprocal Symmetry Plots
  25. 25. The GUESSmix in HEMWat 4646 0 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425mL A 280nm 230nm Reciprocal Symmetry (ReS and ReSS) Plots have 0 £ K £ ¥ on the x axis. Friesen, J.B. Pauli, G.F. Analytical Chemistry 79: 2320-2324 (2007) M Q V U F N Z E Symmetry Midline 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ¥K A M Q V U F N Z E
  26. 26. HEMWat +5 3737 -0.01 0 0.5 1 1.5 2 2.67 4 8 ¥K'(2) A 280nm 230nm HEMWat +3 4646 -0.01 0 0.5 1 1.5 2 2.67 4 8K'(2) A 280nm 230nm HEMWat +4 3746 -0.01 0 0.5 1 1.5 2 2.67 4 8K'(2) A 280nm 230nm HEMWat Systems I M M M Q Q Q Z Z Z U U U F F F ¥ ¥ C.1. ReS and ReSS plots Friesen, J. B.; Pauli, G. F., Journal of Agricultural and Food Chemistry 2008, 56, 19-28
  27. 27. HEMWat 0 5555 -0.01 0 0.5 1 1.5 2 2.67 4 8K'(2) A 280nm 230nm HEMWat +2 3755 -0.01 0 0.5 1 1.5 2 2.67 4 8 ¥K'(2) A 280nm 230nm HEMWat +1 4655 -0.01 0 0.5 1 1.5 2 2.67 4 8K'(2) A 280nm 230nm HEMWat Systems II M Z F F F U U U M M Q Q Q Z Z ¥ ¥ C.1. ReS and ReSS plots Friesen, J. B.; Pauli, G. F., Journal of Agricultural and Food Chemistry 2008, 56, 19-28
  28. 28. C.1. ReS and ReSS plots 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ∞ KD A 280nm 230nm I II III r C F U V M Q N Z E O b 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ∞ KD A I II III MSrun time = 7.2 hours run time = 6.3 hours Friesen, J.B. Pauli, G.F. Analytical Chemistry 79: 2320-2324 (2007)
  29. 29. 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ∞ KD A 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ∞ KD A I II III I II III run time = 4.8 hours run time = 5.8 hours C.1. ReS and ReSS plots Friesen, J.B. Pauli, G.F. Analytical Chemistry 79: 2320-2324 (2007)
  30. 30. C. Emphasize the importance of K CCC Sample Cutting for Isolation of Prenylated Phenolics from Hops Lucas R. Chadwick, Harry H. S. Fong, Norman R. Farnsworth, and Guido F. Pauli Journal of Liquid Chromatography & Related Technologiesw, 28: 1959–1969, 2005
  31. 31. C.2. Monitoring Eluent Phase Composition 0 0.5 1 0 50 100 25 50 75 100 125 Sf value K value minutes pma value K value Sf value 0 0.2 0.4 0.6 0.8 1 299,500 300,500 301,500 302,500 303,500 0 20 40 60 80 100 120 PMA values time in min PMA UV Sf K/10 Pauli, G. F.; Pro, S. M.; Chadwick, L. R.; Burdick, T.; Pro, L.; Friedl, W.; Novak, N.; Maltby, J.; Qiu, F.; Friesen, J. B., Real-Time Volumetric Phase Monitoring: Advancing Chemical Analysis by Countercurrent Separation. Analytical Chemistry 2015, 87, 7418-7425. ChMWat LP mobile EECCC Automated Separation of Green Tea Catechins . terAcWat 5:5:10 solvent system with lower phase mobile.0.5 mL/mn flow rate, 2,000 rpm, 0.65 Sf , 20 oC.
  32. 32. D. Applications of CCS to discovery 1. Isolation of target compounds 2. bioassy guided fractionation 3. metabolomic studies Find a single fish in the sea Identify compounds with certain biological activity Identify as many compounds as possible from a single source
  33. 33. D. Applications of CCS to discovery 2. bioassay guided fractionation 0 0.5 1 1.5 2 2.5 3 3.5 4 4.57 5.33 6.4 8 10.67 16 32 ¥ Oplopanax crude extract in hexane / DCM / methanol / water 7:3:7:3 hexane / ethyl acetate 8:2 hexane / ethyl acetate 7:3 (1) neroplomacrol (novel) (2) neroplofurol (novel) (3) oplopandiol (4) falcarindiol (5) sesamin Sesquiterpenes from Oplopanax horridus Taichi Inui,Yuehong Wang, Dejan Nikolic, David C. Smith, Scott G. Franzblau, and Guido F. Pauli J. Nat. Prod. 2010, 73, 563–567 http://www.wnps.org/plants/oplopanax_horridus.html
  34. 34. Advanced applications of counter-current chromatography in the isolation of anti-tuberculosis constituents from Dracaena angustifolia Ryan J. Case, Yuehong Wang, Scott G. Franzblau, D. Doel Soejarto, Lohi Matainaho, Pius Piskaut, Guido F. Pauli Journal of Chromatography A, 1151 (2007) 169–174 D. Applications of CCS to discovery 2. bioassay guided fractionation http://210.240.49.194/~wonder/plant/plant91/plant91-11/plt91-11-3-7.htm
  35. 35. Biochromatogram Alpinia DCM extract in HEMWat 5/5/5/5 0 30 40 50 60 70 80 90 100 110 120Tube A 0 20 40 60 80 100 280nm 230nm % inhibition at 64 ug/ml % Alpinia DCM extract in HEMWat 5/5/5/5 0 30 40 50 60 70 80 90 100 110 120Tube A 0 5 10 15 mg 280nm 230nm mg MIC 1.8-0.9 307.5 O O O O ACA
  36. 36. redistribution of anti-TB activity Alpinia DCM extract in HEMWat 5/5/5/5 0 40 42 44 46 48 50 52 54Tube A 0 20 40 60 80 100 280nm 230nm % inhibition at 64 ug/ml % Alpinia Fraction 3, H/tBME/M/Wat 5:5:5:5 0 15 25 35 45 55 65 75 85 95 105 115tube A 0 20 40 60 80 100 % 230nm 280nm % inhibition
  37. 37. D.2. Bioassay-Guided Fractionation Fig. 5. Purification of the subfractions 24.4 and 24.5 using analytical HSCCC. Instrument: analytical HSCCC TBE-20; coil column volume: 16 ml; flow rate: 0.3 ml/min; sample loaded: 18.12mg in 200l diphase solvent; diphase solvent system:HEMW (5.25:5:5.25:5); stationary phase: upper phase; mobile phase: lower phase. Talanta. 2010 Sep 15;82(4):1521-7. doi: 10.1016/j.talanta.2010.07.036. Quick identification of apoptosis inducer from Isodon eriocalyx by a drug discovery platform composed of analytical high-speed counter-current chromatography and the fluorescence-based caspase-3 biosensor detection. Han QB, Yu T, Lai F, Zhou Y, Feng C, Wang WN, Fu XH, Lau CB, Luo KQ, Xu HX, Sun HD, Fung KP, Leung PC.
  38. 38. Fig. 3. Effects of VOAS fractions from solvent system 8 (heptane–ethyl acetate–methanol–water at a volume ratio of 27:23:27:23%) on HUVEC viability. HUVECs were treated with (A) VOAS fraction groups (three consecutive fractions combined into one group) at 1:100 dilution and (B) individual fractions at 1:100 dilution from active fraction groups. MTS assay was performed after 48 h of incubation. Data are expressed as % of medium control and are mean ± SD. *Significant inhibitory effects compared to vehicle control (Veh, DMSO 1%) (P < 0.05, unpaired Student’s t- test). VOAS at 10, 33 and 100 g/mL was used as an inhibitory control. The results demonstrated that fraction groups III–VI and fractions 7–15 exhibited significant anti-endothelial properties. J Chromatogr A. 2012 May 4;1236:132-138. doi: 10.1016/j.chroma.2012.03.013. Bioactivity- guided fractionation of the volatile oil of Angelica sinensis radix designed to preserve the synergistic effects of the mixture followed by identification of the active principles. Yeh JC, Garrard IJ, Cho CW, Annie Bligh SW, Lu GH, Fan TP, Fisher D. D.2. BAGF
  39. 39. D. Applications of CCS to discovery Phytoconstituents from Vitex agnus-castus Fruits Chen SN, Friesen JB, Webster D, Nikolic D, van Breemen RB, Wang ZJ, Fong HH, Farnsworth NR, Pauli GF. Fitotherapia, in press 2011, 70 kg Fruit defat with petroleum ether and extract with methanol (879 g) Diaion HP-20 flash chromatography (5 fractions) Silica Gel Vacuum-Liquid Chromatography (13 fractions) Sephadex LH-20 CC (4 fractions) HSCCC (11 fractions) HPLC YMC ODS-AQ HPLC YMC C-18 (21) p-coumaric acid (22) 4-hydroxybenzoic acid (23) ficusal (24) vladirol (25) balanophonin 24 compounds total 1 new compound 3. metabolomic studies
  40. 40. Fig. 2. Deconvolved biochromatogram of O. horridus crude extract. The x-axis represents the K values of the CCC fractions, and the y-axis indicates the anti-TB activities of the fractions. The bioactivities for all fractions observed at 50 μg/ml (black line) were deconvolved into Gaussian peaks to produce 19 biopeaks (colored), which are representative of the individual active principles and thus were used for Pearson's correlation. Each of the 19 biopeaks contains multiple compounds which were further analyzed by GC–MS. Fig. 6. Distribution of the 24 anti-TB active metabolites in the 3D CCC–GC– Pearson matrix. The assigned peaks are color coded in accord to the structural classes: pink for polyketides, green for cadinol type sesquiterpenes, blue for ledol type sesquiterpenes, and brown for others. Fitoterapia. 2012 Oct;83(7):1218-25. doi: 10.1016/j.fitote.2012.06.012. Unbiased evaluation of bioactive secondary metabolites in complex matrices. Inui T1, Wang Y, Pro SM, Franzblau SG, Pauli GF. D.3. metabolomic studies

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