Recently there has been a resurgence of interest in cyclic peptides due to their therapeutic advantages in terms of potency, permeability, proteolytic stability, and unique selectivity relative to traditional smaller drug molecules. Cyclosporin is a family of cyclic peptides widely used as autoimmune suppression agents. Cyclosporin analogs consist of eleven amino acids with the main difference lying at the side chain of its amino acid residues. In this study, a single step separation method was developed utilizing Supercritical Fluid Chromatography (SFC) to resolve five naturally occurring cyclosporin analogs (Cyclosporin A, B, C, D, and H) on a bare silica-packed column. The optimized method involved use of ethanol-modified carbon dioxide as mobile phase on a bare silica column at 80 °C and UV detection at 220 nm. Although column temperature and back pressure generally had insignificant effect on SFC separation, it was found in our study that increasing temperature and pressure greatly improved peak shape and resolution.
References
[1]
Guarracino, D. (2015) Wheel of Fortune—Cyclic Peptides Hit the Mimetic Jackpot: Current Syntheses, Uses and Roles for Cyclic Peptide Mimetics. Current Chemical Biology, 9, 36-52.
http://dx.doi.org/10.2174/2212796809666150911201950
[2]
Vorherr, T. (2015) Modifying Peptides to Enhance Permeability. Future Medicinal Chemistry, 7, 1009-1021.
http://dx.doi.org/10.4155/fmc.15.43
[3]
Ahlbach, C.L., Lexa, K.W., Bockus, A.T., Chen, V., Crews, P., Jacobson, M.P. and Lokey, R.S. (2015) Macrocycles in New Drug Discovery. Future Medicinal Chemistry, 7, 2121-2130. http://dx.doi.org/10.4155/fmc.15.78
[4]
Watson, G.M., Gunzburg, M.J., Ambaye, N.D., Lucas, W.H., Traore, D.A., Kulkarni, K., Cergol, K.M., Payne, R.J., Panjikar, S. and Pero, S.C. (2015) Cyclic Peptides Incorporating Phosphotyrosine Mimetics as Potent and Specific Inhibitors of the Grb7 Breast Cancer Target. Journal of Medicinal Chemistry, 58, 7707-7718.
http://dx.doi.org/10.1021/acs.jmedchem.5b00609
[5]
Rashad, A.A., Kalyana S., Ramalingam V., Aneja, R., Duffy, C. and Chaiken, I. (2015) Macrocyclic Envelope Glycoprotein Antagonists That Irreversibly Inactivate HIV-1 before Host Cell Encounter. Journal of Medicinal Chemistry, 58, 7603-7608. http://dx.doi.org/10.1021/acs.jmedchem.5b00935
[6]
Cai, M., Marelli, U.K., Bao, J., Beck, J.G., Opperer, F., Rechenmacher, F., McLeod, K.R., Zingsheim, M.R., Doedens, L. and Kessler, H.(2015) Systematic Backbone Conformational Constraints on a Cyclic Melanotropin Ligand Leads to Highly Selective Ligands for Multiple Melanocortin Receptors. Journal of Medicinal Chemistry, 58, 6359-6367.
http://dx.doi.org/10.1021/acs.jmedchem.5b00102
[7]
Dreyfuss, M., Harri, H., Hofmann, H., Kobel, H., Pache, W. and Tscherter, H. (1976) Cyclosporin A and C New metabolites from Trichoderma polysporum. European Journal of Applied Microbiology, 3, 125-133.
http://dx.doi.org/10.1007/BF00928431
[8]
Ahn, E.Y., Shrestha, A., Nguyen, H.H., Nguyen, L.H., Yoon, Y.J. and Park, J.W. (2014) Structural Characterization of Cyclosporin A, C and Microbial Bio-Transformed Cyclosporin A Analog AM6 Using HPLC-ESI-Ion Trap-Mass Spectrometry. Talanta, 123, 89-94. http://dx.doi.org/10.1016/j.talanta.2014.01.067
[9]
Carruthers, S.G., Freeman, D.J., Koegler, J.C., Howson, W., Keown, P.A., Laupacis, A. and Stiller, C.R. (1983) Cyclosporin A and C. Clinical Chemistry, 29, 180-183.
[10]
Lensmeyer, G.L., Wiebe, D.A. and Carlson, I.H. (1987) Identification and Analysis of Nine Metabolites of Cyclosporine in Whole Blood by Liquid Chromatography. Clinical Chemistry, 33, 1841-1850.
[11]
Thurbide, K.B. and Zhang, J. (2005) Separation of Linear Gramicidins Using Carbon Dioxide-Containing Mobile Phases. Analytical and Bioanalytical Chemistry, 382, 1227-1233. http://dx.doi.org/10.1007/s00216-005-3270-9
[12]
Patel, M.A., Riley, F., Ashraf, K.M., and Taylor, L.T. (2012) Supercritical Fluid Chromatographic Resolution of Water Soluble Isomeric Carboxyl/Amine Terminated Peptides Facilitated via Mobile Phase Water and Ion Pair Formation. Journal of Chromatography A, 1233, 85-90. http://dx.doi.org/10.1016/j.chroma.2012.02.024
[13]
Zheng, J., Pinkston, J.D., Zoutendam, P.H. and Taylor L.T. (2006) Feasibility of Supercritical Fluid Chromatography/Mass Spectrometry of Polypeptides with Up to 40-Mers. Analytical Chemistry, 78, 1535-1545.
http://dx.doi.org/10.1021/ac052025s
[14]
Kalinoski, H.T., Wright, B.W. and Smith, R.D. (1988) Chemical Ionization Mass Spectra of High Molecular Weight, Biologically Active Compounds Produced Following Supercritical Fluid Chromatography. Biomedical & Environmental Mass Spectrometry, 15, 239-242. http://dx.doi.org/10.1002/bms.1200150409
[15]
Aaltonen, O., Alkio, M., Lundell, J., Ruohonen, S., Parvinen, L. and Suoninen, V. (1988) Purification of Pharmaceuticals and Nutraceutical Compounds by Sub and Supercritical Chromatography and Extraction. Pharmaceutical Technology Europe, 10, A42-A54.
[16]
Viswanadhan, V.N., Ghose, A.K., Revankar, G.R. and Robins, R.K. (1989) Atomic Physicochemical Parameters for Three Dimensional Structure Directed Quantitative Structure-Activity Relationships. Journal of Chemical Information and Computational Science, 29, 163-172. http://dx.doi.org/10.1021/ci00063a006
[17]
Potter, B., Palmer, R.A., Withnall, R. and Chowdhry, B. (2003) Two New Cyclosporin Folds Observed in the Structures of the Immunosuppressant Cyclosporin G and the Formyl Peptide Receptor Antagonist Cyclosporin H at Ultra-High Resolution. Organic & Biomolecular Chemistry, 1, 1466-1474. http://dx.doi.org/10.1039/b210086j
[18]
Zheng, W. (2008) Separation of Cyclosporins and Other Antibiotics by HSCCC. Journal of Chromatographic Science, 47, 354-358. http://dx.doi.org/10.1093/chromsci/47.5.354
[19]
Enmark, M., Asberg, D., Samuelsson, J. and Fornstedt, T. (2014) The Effect of Temperature Pressure and Co-Solvent on a Chiral Supercritical Fluid Chromatography Separation. Chromatography Today, 8, 14-17.
[20]
Ibhfiez, E., Tabera, J., Herraiz, M. and Reglero, G. (1994) Effect of Temperature and Density on the Performance of Micropacked Columns in Supercritical Fluid Chromatography. Journal of Chromatography A, 667, 249-255.
http://dx.doi.org/10.1016/0021-9673(94)89074-9
[21]
Caldwell, J. and Webster, G.K. (2014) Super Critical Fluid Chromatography. CRC Press, Taylor & Francis Group LLC, Boca Raton.
[22]
Gasparrini, F., Misiti, D. and Villani, C. (1990) Direct Resolution of Racemic Compounds on Chiral Microbore Columns by Sub- and Supercritical Fluid Chromatography, Journal of High Resolution Chromatography, 13, 182-184.
http://dx.doi.org/10.1002/jhrc.1240130309
[23]
West, C., Bouet, A, Routier, S. and Lesellier, E. (2012) Effects of Mobile Phase Composition and Temperature on the Supercritical Fluid Chromatography Enantioseparation of Chiral Fluoro-Oxoindole-Type Compounds with Chlorinated Polysaccharide Stationary Phases. Journal of Chromatography A, 1269, 325-335.
http://dx.doi.org/10.1016/j.chroma.2012.09.078
[24]
Thurbide, K. and Zhang, J. (2005) Separation of Linear Gramicidins Using Carbon Dioxide-Containing Mobile Phases. Analytical and Bioanalytical Chemistry, 382, 1227-1233. http://dx.doi.org/10.1007/s00216-005-3270-9
[25]
Bowers, L.D. and Mathews, S.E. (1985) Investigation of the Mechanism of Peak Broadening Observed in the High- Performance Liquid Chromatographic Analysis of Cyclosporine. Journal of Chromatography A, 333, 231-238.
http://dx.doi.org/10.1016/S0021-9673(01)87348-9
[26]
Augustijns, P.F., Brown, S.C., Willard, D.H., Consler, T.G., Annaert, P.P, Hendren, R.W. and Bradshaw, T.P. (2000) Hydration Changes Implicated in the Remarkable Temperature-Dependent Membrane Permeation of Cyclosporin A. Biochemistry, 39, 7621-7630. http://dx.doi.org/10.1021/bi9929709
[27]
Wang, C. and Zhang, Y. (2013) Effects of Column Back Pressure on Supercritical Fluid Chromatography Separations of Enantiomers Using Binary Mobile Phases on 10 Chiral Stationary Phases. Journal of Chromatography A, 1281, 127-134. http://dx.doi.org/10.1016/j.chroma.2013.01.050
[28]
Wang, C., Tymiak, A.A. and Zhang, Y. (2014) Optimization and Simulation of Tandem Column Supercritical Fluid Chromatography Separations Using Column Back Pressure as a Unique Parameter. Analytical Chemistry, 86, 4033- 4040. http://dx.doi.org/10.1021/ac500530n
[29]
Lou, X., Janssen, H.G., Snijders, H. and Cramers, C.A. (1996) Pressure Drop Effects on Selectivity and Resolution in Packed Column Supercritical Fluid Chromatography. Journal of High Resolution Chromatography, 19, 449-456.
http://dx.doi.org/10.1002/jhrc.1240190806
[30]
Brunelli, C., Zhao, Y., Brown, M.H. and Sandra, P. (2008) Development of a Supercritical Fluid Chromatography High-Resolution Separation Method Suitable for Pharmaceuticals Using Cyanopropyl Silica. Journal of Chromatography A, 1185, 263-272. http://dx.doi.org/10.1016/j.chroma.2008.01.050
