Preparation of cyclodextrin chiral stationary phases by organic soluble catalytic 'click' chemistry


We describe an effective and simple protocol that uses click chemistry to attach native β-cyclodextrin (β-CD) to silica particles, resulting in a chiral stationary phase (CCNCSP) that can be used for the enantioseparation of chiral drugs by high-performance liquid chromatography (HPLC). Starting from β-CD, the CCNCSP is prepared in several steps: (i) reaction of β-CD with 1-(p-toluenesulfonyl)-imidazole to afford mono-6-toluenesulfonyl-β-CD; (ii) azidolysis of mono-6-toluenesulfonyl-β-CD in dimethylformamide to give mono-6-azido-β-CD (N3-CD); (iii) reaction of cuprous iodide with triphenylphosphine to form an organic soluble catalyst CuI(PPh3); (iv) preparation of alkynyl-modified silica particles; and (v) click chemistry immobilization of N3-CD onto alkynyl-modified silica to afford the desired chiral stationary phase. Synthesis of the stationary phase and column packing takes 1 week.

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Figure 5: HPLC enantioseparation of chiral drugs using 5- and 3-μm CCNCSP with diode array detector detection at 254 nm.


  1. 1

    Tang, W.H. & Ng, S.C. Synthesis of cationic single-isomer cyclodextrins for the chiral separation of amino acids and anionic pharmaceuticals. Nat. Protoc. 2, 3195–3199 (2007).

    CAS  Article  Google Scholar 

  2. 2

    Tang, W.H. & Ng, S.C. Facile synthesis of mono-6-amino-6-deoxy-α-, β-, γ-cyclodextrin hydrochlorides for molecular recognition, chiral separation and drug delivery. Nat. Protoc. 3, 691–697 (2008).

    CAS  Article  Google Scholar 

  3. 3

    Armstrong, D.W., Ward, T.J., Armstrong, R.D. & Beesley, T.E. Separation of drug stereoisomers by the formation of β-cyclodextrin inclusion complexes. Science 232, 1132–1135 (1986).

    CAS  Article  Google Scholar 

  4. 4

    Easton, C.J. & Lincoln, S.F. Chiral discrimination by modified cyclodextrins. Chem. Soc. Rev. 25, 163–170 (1996).

    CAS  Article  Google Scholar 

  5. 5

    Xiao, Y., Ong, T.T., Tan, T.T.Y. & Ng, S.C. Synthesis and application of a novel single-isomer mono-6-deoxy-6-(3R,4R-dihydroxypyrrolidine)-beta-cyclodextrin chloride as a chiral selector in capillary electrophoresis. J. Chromatogr. A 1216, 994–999 (2009).

    CAS  Article  Google Scholar 

  6. 6

    Wang, Y., Ong, T.T., Li, L.S., Tan, T.T.Y . & Ng, S.C. Enantioseparation of novel 'click' chemistry derived native β-cyclodextrin chiral stationary phase for high-performance liquid chromatography. J. Chromatogr. A 1216, 2388–2393 (2009).

    CAS  Article  Google Scholar 

  7. 7

    Wang, Y., Young, D.J., Tan, T.T.Y . & Ng, S.C. 'Click' immobilized perphenylcarbamated and permethylated cyclodextrin stationary phases for chiral high-performance liquid chromatography. J. Chromatogr. A 1217, 5103–5108 (2010).

    CAS  Article  Google Scholar 

  8. 8

    Muderawan, I.W., Ong, T.T. & Ng, S.C. Urea bonded cyclodextrin derivatives onto silica for chiral HPLC. J. Sep. Sci. 29, 1849–1871 (2006).

    CAS  Article  Google Scholar 

  9. 9

    Pirkle, W.H. & Welch, C.J. Use of simultaneous face to face and face to edge π-π interactions to facilitate chiral recognition. Tetrahedron: Asymmetry 5, 777–780 (1994).

    CAS  Article  Google Scholar 

  10. 10

    Zhang, T., Nguyen, D. & Franco, P. Enantiomer resolution screening strategy using multiple immobilised polysaccharide-based chiral stationary phases. J. Chromatogr. A 1191, 214–222 (2008).

    CAS  Article  Google Scholar 

  11. 11

    Zhang, T. et al. Solvent versatility of immobilized 3,5-dimethylphenylcarbamate of amylose in enantiomeric separations by HPLC. J. Chromatogr. A 1075, 65–75 (2005).

    CAS  Article  Google Scholar 

  12. 12

    Armstrong, D.W. et al. Macrocyclic antibiotics as a new class of chiral selectors for liquid chromatography. Anal. Chem. 66, 1473–1484 (1994).

    CAS  Article  Google Scholar 

  13. 13

    Cho, H.S., Choi, H.J. & Hyun, M.H. Preparation of a new crown ether-based chiral stationary phase containing thioester linkage for the liquid chromatographic separation of enantiomers. J. Chromatogr. A 1216, 7446–7449 (2009).

    CAS  Article  Google Scholar 

  14. 14

    Lloyd, D.K., Li, S. & Ryan, P. Protein chiral selectors in free-solution capillary electrophoresis and packed-capillary electrochromatography. J. Chromatogr. A 694, 285–296 (1995).

    CAS  Article  Google Scholar 

  15. 15

    Wisbuba, D. & Schurig, V. Enantiomer separation by pressure-supported electrochromatography using capillaries packed with Chirasil-Dex polymer-coated silica. Electrophoresis 20, 2779–2785 (1999).

    Article  Google Scholar 

  16. 16

    Fujimura, K., Ueda, T. & Ando, T. Retention behavior of some aromatic compounds on chemically bonded cyclodextrin silica stationary phase in liquid chromatography. Anal. Chem. 55, 446–450 (1983).

    CAS  Article  Google Scholar 

  17. 17

    Fujimura, K., Suzuki, S., Hayashi, K. & Masuda, S. Retention behavior and chiral recognition mechanism of several cyclodextrin-bonded stationary phases for dansyl amino acids. Anal. Chem. 62, 2198–2205 (1990).

    CAS  Article  Google Scholar 

  18. 18

    Kawaguchi, Y., Tanaka, M., Nakae, M., Funaso, K. & Shone, T. Chemically bonded cyclodextrin stationary phases for liquid chromatographic separation of aromatic compounds. Anal. Chem. 55, 1852–1857 (1983).

    CAS  Article  Google Scholar 

  19. 19

    Han, X.X., Yao, T.L., Liu, Y., Larock, R.C. & Armstrong, D.W. Separation of chiral furan derivatives by liquid chromatography using cyclodextrin-based chiral stationary phases. J. Chromatogr. A 1063, 111–120 (2005).

    CAS  Article  Google Scholar 

  20. 20

    Wang, C.L., Jiang, C.X. & Armstrong, D.W. Considerations on HILIC and polar organic solvent-based separations: use of cyclodextrin and macrocyclic glycopetide stationary phases. J. Sep. Sci. 31, 1980–1990 (2008).

    CAS  Article  Google Scholar 

  21. 21

    Ng, S.C., Chen, L., Zhang, L.F. & Ching, C.B. Facile preparative HPLC enantioseparation of racemic drugs using chiral stationary phases based on mono-6-azido-6-deoxy-perphenylcarbamoylated β-cyclodextrinimmobilized on silica gel. Tetrahedron Lett. 43, 677–681 (2002).

    CAS  Article  Google Scholar 

  22. 22

    Lai, X.H., Bai, Z.W., Ng, S.C. & Ching, C.B. Preparation and enantioseparation characteristics of two chiral stationary phases based on mono(6A-azido-6A-deoxy)-perphenylcarbamoylated α- and β-cyclodextrin. Chirality 16, 592–597 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Muderawan, I.W., Ong, T.T. & Ng, S.C. Urea bonded cyclodextrin derivatives onto silica for chiral HPLC. J. Sep. Sci. 29, 1849–1871 (2006).

    CAS  Article  Google Scholar 

  24. 24

    Bock, V.D., Hiemstra, H. & van Maarseveen, J.H. CuI-catalyzed alkyne–azide 'click' cycloadditions from a mechanistic and synthetic perspective. Eur. J. Org. Chem. 2006, 51–68 (2006).

    Article  Google Scholar 

  25. 25

    Straub, B.F. μ-Acetylide and μ-alkenylidene ligands in 'click' triazole syntheses. Chem. Commun. 37, 3868–3870 (2007).

    Article  Google Scholar 

  26. 26

    Rostovtsev, V., Green, L.G., Fokin, V.V. & Sharpless, K.B. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective ligation of azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596–2599 (2002).

    CAS  Article  Google Scholar 

  27. 27

    Kamijo, S., Tienan, J. & Yamamoto, Y. Four-component coupling reactions of silylacetylenes, allyl carbonates, and trimethylsilyl azide catalyzed by a Pd(0)–Cu(I) bimetallic catalyst. Fully substituted triazole synthesis from seemingly internal alkynes. Tetrahedron Lett. 45, 689–691 (2004).

    CAS  Article  Google Scholar 

  28. 28

    Meng, J.C., Fokin, V.F. & Finn, M.G. Kinetic resolution by copper-catalyzed azide–alkyne cycloaddition. Tetrahedron Lett. 46, 4543–4546 (2005).

    CAS  Article  Google Scholar 

  29. 29

    Alix, A., Chassaing, S., Pale, P. & Sommer, J. 'Click chemistry' in CuI-zeolites: a convenient access to glycoconjugates. Tetrahedron 64, 8922–8929 (2008).

    CAS  Article  Google Scholar 

  30. 30

    Santoyo-Gonzalez, F. Multivalent neoglycoconjugates by regiospecific cycloaddition of alkynes and azides using organic-soluble copper catalysts. Org. Lett. 5, 1951–1954 (2003).

    Article  Google Scholar 

  31. 31

    Lummerstorfer, T. & Hoffmann, H. Click chemistry on surfaces: 1,3-dipolar cycloaddition reactions of azide-terminated monolayers on silica. J. Phys. Chem. B 108, 3963–3966 (2004).

    CAS  Article  Google Scholar 

  32. 32

    Moses, J.E. & Moorhouse, A.D. The growing applications of click chemistry. Chem. Soc. Rev. 36, 1249–1262 (2007).

    CAS  Article  Google Scholar 

  33. 33

    Hein, C.D., Liu, X.-M. & Wang, D. Click chemistry, a powerful tool for pharmaceutical sciences. Pharm. Res. 25, 2216–2230 (2008).

    CAS  Article  Google Scholar 

  34. 34

    Binder, W.H. & Sachsenhofer, R. 'Click' chemistry in polymer and material science: an update. Macromol. Rap. Commun. 29, 952–981 (2008).

    CAS  Article  Google Scholar 

  35. 35

    Binder, W.H. & Sachsenhofer, R. 'Click' chemistry in polymer and materials science. Macromol. Rap. Commun. 28, 15 (2008).

    Article  Google Scholar 

  36. 36

    Zhang, Y., Guo, Z., Ye, J. & Xu, Q. Preparation of novel β-cyclodextrin chiral stationary phases based on click chemistry. J. Chromatogr. A 1191, 188–192 (2008).

    CAS  Article  Google Scholar 

  37. 37

    Wang, Y., Xiao, Y., Tan, T.T.Y. & Ng, S.C. Click chemistry for facile immobilization of cyclodextrin derivatives onto silica as chiral stationary phases. Tetrahedron Lett. 49, 5190–5191 (2008).

    CAS  Article  Google Scholar 

  38. 38

    Wang, Y., Xiao, Y., Tan, T.T.Y. & Ng, S.C. Application of click-chemistry-based perphenylcarbamated β-CD chiral stationary phase in CEC. Electrophoresis 30, 705–711 (2009).

    CAS  Article  Google Scholar 

  39. 39

    Tan, T., Ng, S.-C., Wang, Y. & Xiao, Y. Synthesis of mono-6-tosyl-β-cyclodextrin, a key intermediate for the functional cyclodextrin derivatives. Protoc. Exchange doi:10.1038/protex.2011.214.

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Funding from the Singapore Ministry of Education Academic Research Fund Tier 2 (project number ARC 9/06) and an Agency for Science, Technology and Research SERC grant (grant no: 092 101 0056) in support of this project is gratefully acknowledged. Y.W. is grateful to Nanyang Technological University for the award of a Ph.D. scholarship.

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Y.W. designed the research; Y.W., H.C., Y.X., C.H.N. and T.S.O. carried out the synthesis experiments; Y.W. and H.C. performed the analytical experiments; Y.W., T.T.Y.T. and S.C.N. wrote the paper. All authors have discussed the results and approved the final manuscript.

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Correspondence to Timothy Thatt Yang Tan or Siu Choon Ng.

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The authors declare no competing financial interests.

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Wang, Y., Chen, H., Xiao, Y. et al. Preparation of cyclodextrin chiral stationary phases by organic soluble catalytic 'click' chemistry. Nat Protoc 6, 935–942 (2011).

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