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

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.

Key words

fluorinated coumarin dyes - fluorinated coumaric acid - fluo­rescent dye - imaging probe - NHS ester

• 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
• 2a Czarnik AW. Acc. Chem. Res. 1994; 27: 302
• 2b Topics in Fluo­rescence Spectroscopy Vol. 9: Advanced Concepts in Fluorescence Sensing, Pt. A: Small Molecule Sensing . Geddes CD, Lakowicz JR. Springer; Berlin: 2005: 218
• 2c Topics in Fluorescence Spectroscopy Vol. 10: Advanced Concepts in Fluorescence Sensing, Pt. B: Macromolecular Sensing. Geddes CD, Lakowicz JR. Springer; Berlin: 2005: 274
• 2d Wang L, Zhao W, O’Donoghue MB, Tan W. Bioconjugate Chem. 2007; 18: 297
• 2e Kim HN, Lee MH, Kim HJ, Kim JS, Yoon J. Chem. Soc. Rev. 2008; 37: 1465
• 2f Petryayeva E, Krull UJ. Anal. Chim. Acta 2011; 706: 8
• 2g Chan J, Dodani SH, Chang CJ. Nat. Chem. 2012; 4: 973
• 2h Krumova K, Cosa G. Photochemistry 2013; 41: 279
• 2i Zhou S, Yuan L, Hua X, Xu L, Liu S. Anal. Chim. Acta 2015; 877: 19
• 2j Peltomaa R, López-Perolio I, Benito-Peña E, Barderas R, Moreno-Bondi MC. Anal. Bioanal. Chem. 2016; 408: 1805
• 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 Blue^TM 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).