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. 2024 Apr 15;15(5):677–683. doi: 10.1021/acsmedchemlett.4c00085

Synthesis of Monofluorinated 7-Hydroxycoumarin-3-Carboxamides as Cell-Permeable Fluorescent Molecular Probes

Digamber Rane 1, Anver Basha Shaik 1, Szu Lee 1, Xiaojun Hu 1, Blake R Peterson 1,*
PMCID: PMC11089655  PMID: 38746887

Abstract

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To facilitate studies of engagement of protein targets by small molecules in living cells, we synthesized fluorinated derivatives of the fluorophore 7-hydroxycoumarin-3-carboxylic acid (7OHCCA). Compared to the related difluorinated coumarin Pacific Blue (PB), amide derivatives of 6-fluoro-7-hydroxycoumarin-3-carboxylic acid (6FC) exhibited substantially brighter fluorescence. When linked to the anticancer drug paclitaxel (Taxol) via gamma-aminobutyric acid (GABA), the acidity of the phenol of these coumarins profoundly affected cellular efflux and binding to microtubules in living cells. In contrast to the known fluorescent taxoid PB-GABA-Taxol, the less acidic 6FC-GABA-Taxol was more cell-permeable due to a lower susceptibility to active efflux. In living cells, this facilitated the imaging of microtubules by confocal microscopy and enabled quantification of binding to microtubules by flow cytometry without added efflux inhibitors. The photophysical, chemical, and biological properties of 6FC derivatives make these compounds particularly attractive for the construction of fluorescent molecular probes suitable for quantitative analysis of intracellular small molecule–protein interactions.

Keywords: Anticancer agents, Fluorescent probes, Target engagement, Microtubules, Flow cytometry, Confocal microscopy

Quantification of engagement of protein targets by small molecules in physiologically relevant environments such as living cells is important for drug discovery and development.1 One of the most widely used methods for these types of studies of small molecule–protein interactions in living cells is NanoBRET,2,3 where protein targets of interest are expressed in cells fused to nanoluciferase. The binding of ligands linked to red fluorophores to these targets can be detected through bioluminescence resonance energy transfer (BRET). This approach can be used to measure interactions of these probes with proteins in cells as well as their displacement by nonfluorescent competitors.4

As an alternative method for studies of target engagement, we recently reported5 the fluorescent probe cellular binding assay (FPCBA). This new method uses flow cytometry for quantitative studies of the binding of cell-permeable fluorescent probes and competitors to specific native (untagged) proteins that are overexpressed in living cells. Although FPCBA was previously validated using allosteric activators of Protein Kinase C isozymes,5 our previous studies6−8 of interactions of fluorescent derivatives of the anticancer drug paclitaxel (Taxol) with microtubules in living cells were instrumental in developing this method. Key to this approach is the use of cell-permeable fluorophores that can be efficiently detected by flow cytometry when linked to ligands of protein targets.

Derivatives of 7-hydroxycoumarin-3-carboxylic acid (7OHCCA, 1, Figure 1) are particularly promising as cell-permeable fluorophores compatible with flow cytometry.9 When the phenol of these coumarins is deprotonated, these compounds generally absorb strongly near 400 nm, allowing for efficient excitation with a 405 nm violet laser. This facilitates the detection of these compounds in biological systems by both flow cytometry and confocal microscopy. Because the pKa of the phenol of 7OHCCA is 7.0–7.5,10−12 its brightness under physiological conditions can be enhanced by fluorine and other electron-withdrawing groups that increase its acidity.13 In the fluorophore Pacific Blue (PB, 2),11 fluorination at the 6- and 8-positions reduces the pKa of the phenol to 4.7 (3.7 for PB methyl ester),12 providing a superior low-molecular-weight fluorophore for studies of labeled proteins14 and small molecules.15 However, when used to study interactions of small molecules with intracellular proteins in live cells, we have found that PB derivatives are often efficient substrates of efflux transporters such as p-glycoprotein (MDR1).6−8 This high susceptibility to active cellular efflux mechanisms can reduce the ability of related molecular probes to engage intracellular targets in the absence of efflux inhibitors such as verapamil.16

Figure 1.

Figure 1

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Structures of 7-hydroxycoumarin-3-carboxylic acids (1–5) and related blue-fluorescent derivatives of paclitaxel (Taxol, 6–10).

As an approach to improve the photophysical properties and biological activities of blue-fluorescent molecular probes, we synthesized a series of monofluorinated analogues of 7OHCCA. We modified 7OHCCA with fluorine at the 5-, 6-, and 8-positions to afford 5FC (3), 6FC (4), and 8FC (5, Figure 1). The synthesis of 6FC (4)17 and its ethyl ester18 from 4-fluororesorcinol has been described, but the fluorescence properties of these compounds were not reported. 8FC (5) is a known compound,12 previously termed Jericho Blue, and has been studied as ester and ether derivatives, but amide derivatives of 3–5 have not been published. To compare the chemical and photophysical properties of amide derivatives of coumarin 1–5 as spectroscopic standards, we measured pKa values, molar extinction coefficients, and relative quantum yields of N-hexyl amide derivatives. These studies revealed that in aqueous solution at pH 7.4, amides derived from 6FC (4) are substantially brighter than amides derived from 7OHCCA (1), PB (2), 5FC (3), and 8FC (5). To investigate how substitution with fluorine might affect their biological activities, we additionally linked these coumarins to the anticancer agent paclitaxel (Taxol) via γ-aminobutyric acid (GABA). This afforded the novel molecular probes 6 and 8–10. In contrast to the known6−8 molecular probe PB-GABA-Taxol (7), these less acidic unsubstituted and monofluorinated coumarin derivatives (6, 8–10) were substantially less susceptible to active cellular efflux. This facilitated cellular imaging and enabled binding assays of microtubules in living HeLa cells in the absence of efflux inhibitors.

To synthesize coumarin acids 3–5, we modified our previously reported15 route used to prepare PB (2) on gram scale. This route to PB (2) beneficially avoids the need to purchase or synthesize costly 2,4-difluororesorcinol by starting with the relatively inexpensive 2,3,4,5-tetrafluorobenzoic acid or the corresponding benzonitrile.11,19 As shown in Scheme 1, monofluorinated benzaldehydes bearing benzyl ether substituents (17–19) were prepared by nucleophilic aromatic substitution of three trifluorinated benzonitriles (11–13) with benzyl alcohol, followed by conversion to the benzaldehydes with DIBAL. For our previously reported synthesis of PB (2) using similar methodology,15 we hydrogenized 2,4-bis(benzyloxy)-3,5-difluorobenzaldehyde to 3,5-difluoro-2,4-dihydroxybenzaldehyde with palladium on carbon. However, for 17–19, we found that palladium diacetate provided a more selective catalyst for the hydrogenolysis of benzyl groups without any reduction of the aldehyde. Similarly, when palladium diacetate was used for hydrogenolysis of the corresponding precursor to PB (2), 2,4-bis(benzyloxy)-3,5-difluorobenzaldehyde,15 this catalyst afforded a 90% yield (0.3 g scale), without any over-reduction. This over-reduction was occasionally observed when the aldehyde starting material was not sufficiently pure. Cyclization of 20–22 to coumarins 3–5 with Meldrum’s acid was followed by synthesis of the amine-reactive NHS esters (23–25). These esters were treated with 1-aminohexane to afford N-hexyl amides (26–28) for subsequent analysis as standards by optical spectroscopy. We previously reported5 the analogous N-hexyl amides of 7OHCCA (29) and PB (30) for comparison.

Scheme 1. (A) Synthesis of Monofluorinated Coumarins 3–5, Amine Reactive NHS Esters 23–25, and Hexyl Amides 26–28 as Spectroscopic Probes for Comparison with the Known Probes 29 and 30 and (B) Synthesis of Novel Fluorescent Taxoids 6 and 8–10.

Scheme 1

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ND: compound was isolated as a mixture of regioisomers that were separated after the next step.

Absorbance and fluorescence emission spectra of probes 26–28 in aqueous buffer compared with those of 29 and 30 are shown in Figure 2. Changes in these spectra as a function of concentration were used to determine the molar extinction coefficients and relative quantum yields of these fluorophores, as listed in Table 1 (data shown in Figure S1, Supporting Information). These studies revealed a remarkably high molar extinction coefficient of 37,000 M–1 cm–1 and quantum yield of 0.84 for 6FC-hexanamide (27), making it the brightest fluorophore in the series (1.45-fold brighter than PB-hexanamide (30), based on the products of ε405 nm and Φ (pH 7.4)). To quantify the acidities of 26–30, the effects of pH on the absorbance spectra of these compounds were analyzed by nonlinear regression as shown in Figure 2 (measured pKa values are listed in Table 1).

Figure 2.

Figure 2

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(A) Absorbance (10 μM) and fluorescence emission spectra (100 nM) of coumarin amides in aqueous PBS (pH 7.4 for 26–28 and 30, pH 10 for 29, and 1% DMSO). Values for λmax are listed in Table 1. (B) Measurements of the pKa of 26–30 (10 μM) by absorbance spectroscopy in aqueous buffer (1% DMSO). pKa values were calculated by nonlinear regression (3-parameter fit, GraphPad Prism).

Table 1. Photophysical and Chemical Properties of Coumarin Amides 26–30 in Aqueous Buffera.

Compound Abs./Em. λmax (nm, pH 7.4) ε405 nm (M–1 cm–1, 10% DMSO, pH) Φ (pH) pKa
5FC-hexanamide (26) 400/449 25,000 (7.4) 0.73 (7.4) 6.9
6FC-hexanamide (27) 401/451 37,000 (7.4) 0.84 (7.4) 6.5
8FC-hexanamide (28) 402/457 28,000 (7.4) 0.69 (7.4) 6.0
7OHCCA-hexanamide (29) 401/447 22,000 (10) 0.35 (7.4) 0.62 (10) 7.3
PB-hexanamide (30) 403/447 29,000 (7.4) 0.74 (7.4) 4.1
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a

Molar extinction coefficients were measured at 405 nm in PBS containing 10% DMSO to facilitate determination of concentrations of DMSO stock solutions by 10-fold dilution into PBS. Absorbance, emission, and quantum yields were measured in PBS (1% DMSO). Molar extinction coefficients for 29 and 30 were previously described.5 The QY shown for 30 is based on a previously reported15 PB-biotin amide. pKa and other values were measured with at least two replicates, and 95% confidence intervals for pKa values from curve fitting are ±0.1 for 26–29 and ±0.2 for 30.

To investigate how monofluorinated coumarins might affect the biological properties of molecular probes, we linked these fluorophores and 7OHCCA to paclitaxel by amidation of H2N-GABA-Taxol-TBS8 (31, Scheme 1) followed by removal of the TBS group. Other fluorescent analogues of paclitaxel are of significant interest as probes of mechanisms of action of this drug and related tubulin-binding anticancer agents.20−26 Examination of living HeLa cells treated with fluorescent taxoids 6–10 by confocal microscopy revealed substantial differences in cellular uptake in the absence and presence of verapamil (see Figure 3 and the expanded fields shown in Figure S2, Supporting Information). When efflux was inhibited by cotreatment with verapamil (100 μM), treatment with these probes at a concentration of 100 nM resulted in strong association with intracellular microtubules. However, under these conditions, PB-GABA-Taxol (7) exhibited the lowest cellular fluorescence, presumably due to its high sensitivity to residual active efflux, as previously described.8 In contrast, in the absence of verapamil, PB-GABA-Taxol (7) showed very little cellular uptake due to active efflux, 7OHCCA-GABA-Taxol (6) exhibited high cellular uptake, and the monofluorinated coumarins (8–10) showed intermediate levels of uptake, as evidenced by their labeling of cellular microtubules.

Figure 3.

Figure 3

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Confocal laser scanning micrographs of living HeLa cells treated with probes 6–10 (100 nM, 3 h, 37 °C). Images on the right are of HeLa cells that were additionally treated with the efflux inhibitor verapamil (100 μM). Laser power and gain settings are identical in each image to allow for accurate comparisons. The confluence of cells in each image was ca. 100%.

In the absence of verapamil, the cellular permeabilities of taxoids 6–10 were observed by microscopy to correlate with the pKa values of cognate fluorescent amides 26–30. Correspondingly, these changes in pKa affected the calculated hydrophobicities (cLogD values) of these taxoid probes (ChemAxon Marvin software, v. 23.3.0). For this analysis, the ChemAxon method was used with ChemAxon-calculated pKa values that were within one pKa unit of the experimental values measured for 26–30 (Table 1). The most acidic probe (7) was calculated to exhibit the highest predicted polarity (cLogDpH7.4 = 2.2; calculated pKa (ChemAxon) = 5.1) and the least acidic probe (6) the lowest predicted polarity (cLogDpH7.4 = 6.7; calculated pKa (ChemAxon) = 7.7). Intermediate polarities were calculated for monofluorinated coumarins 8 (cLogDpH7.4 = 3.0; calculated pKa (ChemAxon) = 6.2), 9 (cLogDpH7.4 = 3.0; calculated pKa (ChemAxon) = 6.2, and 10 (cLogDpH7.4 = 3.1; calculated pKa (ChemAxon) = 6.4). For comparison, the drug paclitaxel was calculated with the same method to exhibit cLogP = 3.3 (experimental27,28 LogP = 3.66). These results suggest that at pH 7.4, the monofluorinated coumarin probes most closely mimic the polarity of paclitaxel.

In biochemical assays, PB-GABA-Taxol (7) binds cross-linked microtubules with Kd = 265 nM.8 In living HeLa cells, this probe can also be used to quantify interactions of small molecules with microtubules by flow cytometry in the presence of verapamil.6 To evaluate the cellular affinities of the less acidic fluorescent taxoids (6, 8–10) for microtubules, we treated living HeLa cells with these compounds and 7 as a control and used previously described6 saturation binding assays to quantify binding to microtubules by flow cytometry. In these assays, total binding was measured by varying the probe concentration, and nonspecific binding was measured by the addition of excess paclitaxel (100 μM) to block specific binding of the fluorescent probe. For curve fitting, the lowest maximal concentration that achieved ca. 50% saturation was used to minimize the effect of these probes on living cells. This approach provided the most consistent measurements of both cellular affinities and Bmax values, a measure of binding sites in cells. Bmax is additionally affected by the brightness of the specific fluorophore of these probes in the intracellular environment. Cellular dissociation constants (Kd) were measured both in the presence and absence of verapamil using nonlinear regression with a one-site total and nonspecific binding model (GraphPad Prism) as shown in Figure 4.

Figure 4.

Figure 4

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Saturation binding of fluorescent taxoids 6–10 to microtubules of living HeLa cells. Cells in suspensions were treated with the compounds for 3 h at 37 °C and analyzed by flow cytometry. Cells were treated with probes in the presence (A) and absence (B) of verapamil (100 μM) to modulate cellular efflux. To measure nonspecific binding, paclitaxel (100 μM) was additionally added as a specific competitor (N = 3). For all data sets, 95% confidence intervals from curve fitting were < 2-fold. Concentrations of DMSO stock solutions of fluorescent taxoids 6–10 were measured by absorbance spectroscopy using the molar extinction coefficients of standards 26–30. S/B: Maximal signal-to-background ratio (fold change). [FBS] = 4%. [DMSO] = 1%.

Consistent with confocal microscopy (Figure 3 and Figure S2, Supporting Information), the efflux inhibitor verapamil (100 μM) enhanced the apparent affinities of all fluorescent taxoids for cellular microtubules. As shown in Figure 4A, in the presence of verapamil, the monofluorinated coumarin probes (8–10) exhibited similar affinities, with Kd values within 2-fold of each other (cellular Kd (8) = 0.15 μM; cellular Kd (9) = 0.15 μM; cellular Kd (10) = 0.23 μM). These affinities were substantially higher than the more polar PB-GABA-Taxol (cellular Kd (7) = 1.4 μM). Correspondingly, the most hydrophobic taxoid (6) exhibited the highest affinity for microtubules in cells (cellular Kd (6) = 0.08 μM) when efflux was inhibited. This value is within ca. 5-fold or less of the affinity of paclitaxel itself for purified microtubules (biochemical Kd = 15 nM,29Ki = 19 nM,30Ki = 27 nM,31Ki = 31 nM,31Kd = 50 nM,32 and Kd = 70 nM33).

In the absence of verapamil, active efflux prevented quantification of the affinity of PB-GABA-Taxol (7) for cellular microtubules (Figure 4B), as previously reported.6 However, the greater cellular permeability and intracellular accumulation of three of the less acidic taxoids enabled quantification of these interactions. The least active of these compounds was probe 8, which exhibited a substantially higher signal-to-background (S/B0.4 μM = 7-fold) than 7 (S/B3 μM = 2-fold), but its low affinity prevented accurate determination of cellular Kd. In contrast, probes 6, 9, and 10 bound more tightly to microtubules, allowing quantification of affinity in cells (cellular Kd (6) = 1.2 μM; cellular Kd (9) = 2.4 μM; cellular Kd (10) = 3.5 μM). Additionally, 7OHCCA-derived probe 6 exhibited the greatest signal-to-background ratio (S/B0.4 μM = 8-fold), likely due to its higher affinity and lower susceptibility to efflux.

In summary, we synthesized and investigated a series of monofluorinated coumarins (3–5) and related fluorescent taxoids as molecular probes (6–10) of cellular microtubules. We found that the 6FC (4) fluorophore, derivatized as a hexyl amide (27), exhibited a remarkably high molar extinction coefficient of 37,000 M–1 cm–1 and quantum yield of 0.84. The brightness of this fluorophore, calculated as the product of the molar extinction coefficient and quantum yield, was 1.45-fold greater than that of a hexanamide derivative of Pacific Blue (2), one of the brightest commercially available coumarins. Measurements of the pKa values of these monofluorinated fluorophores indicate that they will be predominantly anionic under physiological conditions, which can beneficially reduce nonspecific binding to biomolecules. To investigate effects on biological activity, we synthesized four novel fluorescent analogues of paclitaxel. Properties calculations predicted that taxoids linked to monofluorinated coumarin amides will exhibit hydrophobicities (cLogD (8–10)pH 7.4 = 3.0–3.1) that are comparable to the parent drug paclitaxel (cLogP (paclitaxel) = 3.3, experimental27,28 LogP (paclitaxel) = 3.66). Studies of the differential sensitivities of fluorescent taxoids 6–10 to active cellular efflux revealed that monofluorinated coumarins are substantially less susceptible to efflux compared to difluorinated fluorophore Pacific Blue, enabling higher cellular permeability and engagement of cellular microtubules in the absence of efflux inhibitors. Taxoid 6, derived from the nonfluorinated 7OHCCA fluorophore, exhibited the lowest sensitivity to active efflux and the greatest affinity for microtubules in living cells both in the presence (cellular Kd (6) = 0.08 μM) and absence (cellular Kd (6) = 1.2 μM) of the efflux inhibitor verapamil. However, this probe (6) is predicted to be over 1000-fold more hydrophobic (cLogD (6)pH 7.4 = 6.7) than paclitaxel. This high hydrophobicity may enhance nonspecific interactions with other biomolecules and might affect the ability of this probe to faithfully mimic some biological properties of paclitaxel. The exceptionally high fluorescence brightness of derivatives of 6FC (4), and its decreased susceptibility to efflux compared to PB, make this monofluorinated fluorophore particularly attractive for the synthesis of cell-permeable molecular probes that engage intracellular target proteins.

Acknowledgments

We thank the OSU Comprehensive Cancer Center (NIH P30-CA016058), the NIH (R01 CA272257), and Maryann Z. and Larry Kennedy for financial support. We thank the OSU College of Pharmacy Instrumentation Facility and the OSU Campus Chemical Instrument Center (CCIC) for technical support.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.4c00085.

  • Additional figures showing optical spectroscopy data, confocal micrographs, and NMR spectra. Synthetic procedures, compound characterization data, and biological methods are provided. (PDF)

Author Contributions

‡ D. R. and A. B. S. contributed equally.

The authors declare the following competing financial interest(s): A patent application has been filed related to the research reported in this manuscript.

Supplementary Material

ml4c00085_si_001.pdf (5.9MB, pdf)

References

  1. Stefaniak J.; Huber K. V. M. Importance of Quantifying Drug-Target Engagement in Cells. ACS Med. Chem. Lett. 2020, 11 (4), 403–406. 10.1021/acsmedchemlett.9b00570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Machleidt T.; Woodroofe C. C.; Schwinn M. K.; Mendez J.; Robers M. B.; Zimmerman K.; Otto P.; Daniels D. L.; Kirkland T. A.; Wood K. V. NanoBRET-A Novel BRET Platform for the Analysis of Protein-Protein Interactions. ACS Chem. Biol. 2015, 10 (8), 1797–1804. 10.1021/acschembio.5b00143. [DOI] [PubMed] [Google Scholar]
  3. Robers M. B.; Dart M. L.; Woodroofe C. C.; Zimprich C. A.; Kirkland T. A.; Machleidt T.; Kupcho K. R.; Levin S.; Hartnett J. R.; Zimmerman K.; Niles A. L.; Ohana R. F.; Daniels D. L.; Slater M.; Wood M. G.; Cong M.; Cheng Y. Q.; Wood K. V. Target engagement and drug residence time can be observed in living cells with BRET. Nat. Commun. 2015, 6, 10091. 10.1038/ncomms10091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Robers M. B.; Friedman-Ohana R.; Huber K. V. M.; Kilpatrick L.; Vasta J. D.; Berger B. T.; Chaudhry C.; Hill S.; Muller S.; Knapp S.; Wood K. V. Quantifying Target Occupancy of Small Molecules Within Living Cells. Annu. Rev. Biochem. 2020, 89, 557–581. 10.1146/annurev-biochem-011420-092302. [DOI] [PubMed] [Google Scholar]
  5. Yin Y.; Zhao S. L.; Rane D.; Lin Z.; Wu M.; Peterson B. R. Quantification of Binding of Small Molecules to Native Proteins Overexpressed in Living Cells. J. Am. Chem. Soc. 2024, 146 (1), 187–200. 10.1021/jacs.3c07488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Andres A. E.; Mariano A.; Rane D.; Peterson B. R. Quantification of Engagement of Microtubules by Small Molecules in Living Cells by Flow Cytometry. ACS Bio & Med. Chem. Au 2022, 2 (5), 529–537. 10.1021/acsbiomedchemau.2c00031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Andres A. E.; Rane D.; Peterson B. R. Pacific Blue-Taxoids as Fluorescent Molecular Probes of Microtubules. Methods Mol. Biol. 2022, 2430, 449–466. 10.1007/978-1-0716-1983-4_28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lee M. M.; Gao Z.; Peterson B. R. Synthesis of a Fluorescent Analogue of Paclitaxel That Selectively Binds Microtubules and Sensitively Detects Efflux by P-Glycoprotein. Angew. Chem., Int. Ed. Engl. 2017, 56 (24), 6927–6931. 10.1002/anie.201703298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lavis L. D.; Raines R. T. Bright building blocks for chemical biology. ACS Chem. Biol. 2014, 9 (4), 855–866. 10.1021/cb500078u. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Offenbacher H.; Wolfbeis O. S.; Furlinger E. Fluorescence Optical Sensors for Continuous Determination of near-Neutral Ph Values. Sens. Actuators 1986, 9 (1), 73–84. 10.1016/0250-6874(86)80008-7. [DOI] [Google Scholar]
  11. Sun W.-C.; Gee K. R.; Haugland R. P. Synthesis of novel fluorinated coumarins: Excellent UV-light excitable fluorescent dyes. Bioorg. Med. Chem. Lett. 1998, 8 (22), 3107–3110. 10.1016/S0960-894X(98)00578-2. [DOI] [PubMed] [Google Scholar]
  12. Chen H. M.; Nasseri S. A.; Rahfeld P.; Wardman J. F.; Kohsiek M.; Withers S. G. Synthesis and evaluation of sensitive coumarin-based fluorogenic substrates for discovery of α-acetyl galactosaminidases through droplet-based screening. Org. Biomol. Chem. 2021, 19 (4), 789–793. 10.1039/D0OB02484H. [DOI] [PubMed] [Google Scholar]
  13. Abrams B.; Diwu Z.; Guryev O.; Aleshkov S.; Hingorani R.; Edinger M.; Lee R.; Link J.; Dubrovsky T. 3-Carboxy-6-chloro-7-hydroxycoumarin: a highly fluorescent, water-soluble violet-excitable dye for cell analysis. Anal. Biochem. 2009, 386 (2), 262–269. 10.1016/j.ab.2008.12.018. [DOI] [PubMed] [Google Scholar]
  14. Cohen J. D.; Thompson S.; Ting A. Y. Structure-guided engineering of a Pacific Blue fluorophore ligase for specific protein imaging in living cells. Biochemistry 2011, 50 (38), 8221–8225. 10.1021/bi201037r. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lee M. M.; Peterson B. R. Quantification of Small Molecule-Protein Interactions using FRET between Tryptophan and the Pacific Blue Fluorophore. ACS Omega 2016, 1 (6), 1266–1276. 10.1021/acsomega.6b00356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wu C. P.; Calcagno A. M.; Ambudkar S. V. Reversal of ABC drug transporter-mediated multidrug resistance in cancer cells: evaluation of current strategies. Curr. Mol. Pharmacol. 2008, 1 (2), 93–105. 10.2174/1874467210801020093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Song D. K.; Guo Y.; Lu Z.; Shi Z. Synthesis and characterization of coumarin fluorescent compounds. Jingxi Huagong 2009, 26 (6), 616–619. [Google Scholar]
  18. Chen K. Y.; Guo Y.; Lu Z. H.; Yang B. Q.; Shi Z. Novel Coumarin-based Fluorescent Probe for Selective Detection of Bisulfite Anion in Water. Chin. J. Chem. 2010, 28 (1), 55–60. 10.1002/cjoc.201090035. [DOI] [Google Scholar]
  19. Kerkovius J. K.; Menard F. A Practical Synthesis of 6,8-Difluoro-7-hydroxycoumarin Derivatives for Fluorescence Applications. Synthesis 2016, 48 (11), 1622–1629. 10.1055/s-0035-1561603. [DOI] [Google Scholar]
  20. Barasoain I.; Diaz J. F.; Andreu J. M. Fluorescent taxoid probes for microtubule research. Methods Cell Biol. 2010, 95, 353–372. 10.1016/S0091-679X(10)95019-X. [DOI] [PubMed] [Google Scholar]
  21. Bhattacharyya B.; Kapoor S.; Panda D. Fluorescence spectroscopic methods to analyze drug-tubulin interactions. Methods Cell Biol. 2010, 95, 301–329. 10.1016/S0091-679X(10)95017-6. [DOI] [PubMed] [Google Scholar]
  22. Díaz J. F.; Barasoain I.; Andreu J. M. Fast kinetics of Taxol binding to microtubules. Effects of solution variables and microtubule-associated proteins. J. Biol. Chem. 2003, 278 (10), 8407–8419. 10.1074/jbc.M211163200. [DOI] [PubMed] [Google Scholar]
  23. Buey R. M.; Calvo E.; Barasoain I.; Pineda O.; Edler M. C.; Matesanz R.; Cerezo G.; Vanderwal C. D.; Day B. W.; Sorensen E. J.; Lopez J. A.; Andreu J. M.; Hamel E.; Diaz J. F. Cyclostreptin binds covalently to microtubule pores and lumenal taxoid binding sites. Nat. Chem. Biol. 2007, 3 (2), 117–125. 10.1038/nchembio853. [DOI] [PubMed] [Google Scholar]
  24. Lukinavicius G.; Reymond L.; D’Este E.; Masharina A.; Gottfert F.; Ta H.; Guther A.; Fournier M.; Rizzo S.; Waldmann H.; Blaukopf C.; Sommer C.; Gerlich D. W.; Arndt H.-D.; Hell S. W.; Johnsson K. Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat. Methods 2014, 11 (7), 731–733. 10.1038/nmeth.2972. [DOI] [PubMed] [Google Scholar]
  25. Pineda J. J.; Miller M. A.; Song Y.; Kuhn H.; Mikula H.; Tallapragada N.; Weissleder R.; Mitchison T. J. Site occupancy calibration of taxane pharmacology in live cells and tissues. Proc. Natl. Acad. Sci. U.S.A. 2018, 115 (48), E11406-E11414 10.1073/pnas.1800047115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ross J. L.; Santangelo C. D.; Makrides V.; Fygenson D. K. Tau induces cooperative Taxol binding to microtubules. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (35), 12910–12915. 10.1073/pnas.0402928101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Dash A. K. The dark side of paclitaxel. Oncol. Rev. 2010, 4 (2), 71. 10.1007/s12156-010-0052-1. [DOI] [Google Scholar]
  28. Singh S.; Dash A. K. Paclitaxel in cancer treatment: perspectives and prospects of its delivery challenges. Crit. Rev. Ther. Drug Carrier Syst. 2009, 26 (4), 333–372. 10.1615/CritRevTherDrugCarrierSyst.v26.i4.10. [DOI] [PubMed] [Google Scholar]
  29. Caplow M.; Shanks J.; Ruhlen R. How taxol modulates microtubule disassembly. J. Biol. Chem. 1994, 269 (38), 23399–23402. 10.1016/S0021-9258(17)31528-4. [DOI] [PubMed] [Google Scholar]
  30. Sharma S.; Lagisetti C.; Poliks B.; Coates R. M.; Kingston D. G.; Bane S. Dissecting paclitaxel-microtubule association: quantitative assessment of the 2’-OH group. Biochemistry 2013, 52 (13), 2328–2336. 10.1021/bi400014t. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Andreu J. M.; Barasoain I. The interaction of baccatin III with the taxol binding site of microtubules determined by a homogeneous assay with fluorescent taxoid. Biochemistry 2001, 40 (40), 11975–11984. 10.1021/bi010869+. [DOI] [PubMed] [Google Scholar]
  32. Li Y.; Edsall R. Jr; Jagtap P. G.; Kingston D. G.; Bane S. Equilibrium studies of a fluorescent paclitaxel derivative binding to microtubules. Biochemistry 2000, 39 (3), 616–623. 10.1021/bi992044u. [DOI] [PubMed] [Google Scholar]
  33. Matesanz R.; Barasoain I.; Yang C.-G.; Wang L.; Li X.; de Inés C.; Coderch C.; Gago F.; Barbero J. J.; Andreu J. M.; Fang W.-S.; Díaz J. F. Optimization of Taxane Binding to Microtubules: Binding Affinity Dissection and Incremental Construction of a High-Affinity Analog of Paclitaxel. Chem. Biol. 2008, 15 (6), 573–585. 10.1016/j.chembiol.2008.05.008. [DOI] [PubMed] [Google Scholar]

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ml4c00085_si_001.pdf (5.9MB, pdf)

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