Synthesis 2016; 48(11): 1622-1629
DOI: 10.1055/s-0035-1561603
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© Georg Thieme Verlag Stuttgart · New York

A Practical Synthesis of 6,8-Difluoro-7-hydroxycoumarin Derivatives for Fluorescence Applications

Jeff K. Kerkovius
Department of Chemistry and Chemical Biology, University of British Columbia, Okanagan, Kelowna, BC, V1V 1V7, Canada   Email: frederic.menard@ubc.ca
,
Frederic Menard*
Department of Chemistry and Chemical Biology, University of British Columbia, Okanagan, Kelowna, BC, V1V 1V7, Canada   Email: frederic.menard@ubc.ca
› Author Affiliations
Further Information

Publication History

Received: 03 February 2016

Accepted after revision: 08 March 2016

Publication Date:
19 April 2016 (eFirst)

Abstract

A practical synthesis for 6,8-difluoro-7-hydroxycoumarin-3-carboxylic acid – one of the most widely used coumarin fluorescence imaging dye for bioconjugation – is reported. The synthesis was optimized for a preparative scale to obtain 14 g of the fluorescent coumaryl acid, 6,8-difluoro-7-hydroxycoumarin-3-carboxylic acid. Using this sequence, the preparation of its NHS ester requires a single chromatographic separation for a total of eight synthetic steps. Coupling of the key coumaryl NHS ester is also demonstrated with an unprotected hydroxyamine linker chain for further derivatization. This report provides a convenient access to the fluorinated dye for research labs as an alternative to currently cost-prohibitive commercial sources.

Supporting Information

 
  • References

    • 1a Kushon SA, Ley KD, Bradford K, Jones RM, McBranch D, Whitten D. Langmuir 2002; 18: 7245
    • 1b Huang Z.-M, Zhang Y.-Z, Kotaki M, Ramakrishna M. Composites Sci. Tech. 2003; 63: 2223
    • 1c Kumaraswamy S, Bergstedt T, Shi X, Rininsland F, Kushon S, Xia W, Ley K, Achyuthan K, McBranch D, Whitten D. Proc. Nat. Acad. Sci. U.S.A. 2004; 101: 7511
    • 1d Didenko VV, Moore VC, Baskin DS, Smalley RE. Nano Lett. 2005; 5: 1563
    • 1e Fan L.-J, Zhang Y, Murphy CB, Angell SE, Parker MF. L, Flynn BR, Jones WE. Coord. Chem. Rev. 2009; 253: 410
    • 1f Gather MC, Meerholz K, Danz N, Leosson K. Nat. Photonics 2010; 4: 457
    • 1g Kim HN, Guo Z, Zhu W, Yoon J, Tian H. Chem. Soc. Rev. 2011; 40: 79
    • 1h Wallikewitz BH, Kabra D, Gélinas S, Friend RH. Phys. Rev. B 2012; 85: 045209
    • 3a Pollard TD. Mol. Biol. Cell 2010; 21: 4061
    • 3b Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G. Nat. Rev. Drug Discovery 2011; 10: 221
    • 3c Pritz S, Doering K, Woelcke J, Hassiepen U. Expert Opin. Drug Discovery 2011; 6: 663
    • 3d Bodor DL, Rodríguez MG, Moreno N, Jansen LE. Curr. Protoc. Cell Biol. 2012; 55: Chap. 8, Unit 8.8
    • 3e Janzen WP. Chem. Biol. 2014; 21: 1162
    • 4a Chang C.-W, Mycek M.-A In Reviews in Fluorescence . Vol. 7. Geddes C. Springer; Berlin: 2010: 173
    • 4b Lacoste J, Vining C, Zuo D, Spurmanis A, Brown CM In Reviews in Fluorescence . Vol. 7. Geddes C. Springer; Berlin: 2010: 269
    • 4c Dean KM, Palmer AE. Nat. Chem. Biol. 2014; 10: 512
    • 5a Rust MJ, Bates M, Zhuang X. Nat. Methods 2006; 3: 793
    • 5b Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF. Science 2006; 313: 1642
    • 5c Donnert G, Keller J, Medda R, Andrei MA, Rizzoli SO, Luehrmann R, Jahn R, Eggeling C, Hell SW. Proc. Natl Acad. Sci. U.S.A. 2006; 103: 11440
    • 5d Fernández-Suárez M, Ting AY. Nat. Rev. Mol. Cell Biol. 2008; 9: 929
  • 6 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (1), is commercialized under the trademark name Pacific BlueTM at a cost that is often prohibitive for academic research, who are arguably the most ardent users of such dyes as a molecular tool. For example, at the time of writing, the price of coumaryl NHS ester 10 was listed at 245 USD for 5 mg (cat. # P10163, ThermoFisher Scientific).
  • 7 Gee KR, Haugland RP, Sun W.-C. Bioorg. Med. Chem. Lett. 1998; 8: 3107
  • 8 At pH 10, the quantum yield for ethyl ester 8 was determined to be 0.78, compared to 0.70 for the nonfluorinated analogue 1 (see ref. 7).
    • 9a Cohen JD, Thompson S, Ting AY. Biochemistry 2011; 50: 8221
    • 9b Drake CR, Miller DC, Jones EF. Curr. Org Synth. 2011; 4: 498
    • 9c Beatty KE, Williams M, Carlson BL, Swarts BM, Warren RM, van Helden PD, Bertozzi CR. Proc. Nat. Acad. Sci. U.S.A. 2013; 110: 12911
    • 9d Meinig JM, Peterson BR. ACS Chem Biol. 2015; 10: 570
    • 10a Kikuchi K, Takakusa H, Nagano T. Trends Anal. Chem. 2004; 23: 407
    • 10b Marras SA. E, Kramer FR, Tyagi S. Nucleic Acids Res. 2002; 30: e122
    • 10c Yuan L, Lin W, Zheng K, Zhu S. Acc. Chem. Res. 2013; 46: 1462
    • 10d Li S.-Y, Liu L.-H, Rong L, Qiu W.-X, Jia HZ, Li B, Li F, Zhang XZ. Adv. Funct. Mater. 2015; 25: 7317
  • 11 Gee KR, Haugland RP, Sun W.-C. J. Org. Chem. 1997; 62: 6469
  • 12 Gee KR, Haugland RP, Sun W.-C. Patent PCT US 5830912, 1998
  • 13 Coleman DJ, Naleway JJ. Patent PCT US 8460862, 2013 . Contains a preparation for 9, but no characterization data.
  • 14 Weidner-Wells MA, Fraga-Spano SA. Synth. Commun. 1996; 26: 2775
  • 15 Full characterization of 1 has never appeared in the literature, despite its wide use for close to 20 years. Prior to this report, the only spectral data available for any intermeditate presented here were 1H and 19F NMR chemical shifts (without coupling constants).
  • 16 While the transformations described herein are inspired by previous reports, several modifications have been made to enable a reproducible, large-scale synthesis of 1.
  • 17 High pressure hydrogenation was used on a small scale (see ref. 13).
  • 18 We found that the AcOH-solvated crystals of 10 keep the aromatic alcohol protonated, making for more stable long-term storage of the compound (the acid-free crystals of 10 are light green).