Fluorogenic Sydnone-Modified Coumarins Switched-On by Copper

Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

pubs.acs.org/OrgLett

Fluorogenic Sydnone-Modified Coumarins Switched-On by CopperFree Click Chemistry Camille Favre†,‡ and Fred́ eŕ ic Friscourt*,†,‡ †

Institut Européen de Chimie et Biologie, Université de Bordeaux, 2 rue Robert Escarpit, 33607 Pessac, France Institut de Neurosciences Cognitives et Intégratives d’Aquitaine, CNRS UMR5287, Bordeaux, France

‡

Downloaded via KAOHSIUNG MEDICAL UNIV on July 11, 2018 at 21:42:15 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: The synthesis, photophysical characterization, and biochemical application of sydnone-modified coumarins, a novel class of fluorogenic clickable reagents, are reported. The sydnone moiety, a stable aromatic 1,3-dipole, efficiently quenched the fluorescence of coumarin, which could be restored, with a 132-fold enhancement, upon cycloadditions with cyclooctynes, thereby expanding the fluorogenic click toolbox. TD-DFT calculations suggest that the fluorescence quenching of the sydnone-modified coumarins is likely due to the presence of an energetically low-lying nonemissive chargeseparated state.

(ROS).11 Consequently, fluorogenic tetrazines have recently been developed to react in a metal-free fashion with transcyclooctene via inverse-electron-demand Diels−Alder cycloaddition;12 however, the challenging preparation of transcyclooctene and its sensitivity to isomerize quickly into unreactive cis-cyclooctene have precluded its widespread biological utilization. Recent reports have established that strained alkynes can be incorporated into proteins using site-specific mutagenesis with artificial amino acids,13 and into glycans by hijacking the cell’s biosynthetic machinery with unnatural metabolic precursors,14 suggesting that the use of fluorogenic azido probes could open the exciting possibility of tracking biomolecules in real time in living organisms. However, azides have been shown to be prone to reduction into amines by endogenous thiols,15 which consequently may activate the fluorogenic probe leading to unspecific fluorescence. To address this limitation, we envisaged using sydnones, highly stable aromatic mesoionic 1,3-dipoles, as a replacement for azides. Sydnones can undergo thermal [3 + 2] cycloadditions with alkynes to afford pyrazoles, after elimination of carbon dioxide via retrocycloaddition.16 Furthermore, we and others have recently shown that sydnones can react with cyclooctyne probes in a metal-free click manner17,18 for the bioconjugation and detection of proteins. Herein, we describe the synthesis, photophysical properties, and biochemical evaluation of the two very first fluorogenic sydnones. The two compounds were designed based on the coumarin dye 1, since its fluorescence properties have been shown to be modulated by the electron density of the attached

C

opper-catalyzed cycloadditions between azides and linear alkynes (CuAAC), more commonly known as click chemistry,1 have revolutionized, over the past decade, our way of generating and studying small organic molecules, complex materials, and biomacromolecules.2 To expand the click methodology applicability in living systems, highly strained cyclooctynes have been devised to react efficiently with azidobiomolecules without the need for a metal catalyst, and were elegantly employed for the challenging labeling of posttranslational modifications in living cells.3 In this context, the recent development of fluorogenic click reagents, which become fluorescent upon chemical reactions or target binding, has considerably extended our chemical toolbox,4 especially in situations where sensitive direct visualization is necessary. The rational chemical design of a fluorogenic probe, activated by click chemistry, stems from the concept that modification of a known chromophore with a clickable moiety should alter its photophysical properties leading to fluorescence quenching. Upon click reaction, the quenching handle is transformed into a new functionality, restoring in the process the initial fluorescence of the dye. While the development of fluorogenic cyclooctynes, by following this conceptual approach, has experienced rather limited success,5 azides, conversely, have proven to be efficient quenchers of known fluorophores such as coumarin,6 benzothiazole,7 BODIPY,8 fluorescein,9 and rhodamine.10 Importantly, the fluorogenic azido reagents were efficiently turned-on with various degrees of fluorescence enhancement upon CuAAC with linear alkynes (the cyclooctyne functionality being often considered too bulky for its inconspicuous introduction into biomolecules) and were therefore mostly employed for bioimaging in fixed cells due to the presence of the copper(I) catalyst, known to be cytotoxic via the production of reactive oxygen species © XXXX American Chemical Society

Received: May 19, 2018

A

DOI: 10.1021/acs.orglett.8b01587 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters substituents (Figure 1).19 For instance, previous reports have elegantly described the fluorescence quenching of coumarin by

unmodified 7-methoxycoumarin (11) (Table 1). Gratifyingly, the mesoionic ring significantly quenched the coumarin dye, Table 1. Photophysical Properties of Methoxycoumarin Derivatives 4, 5, 11, 13, and 14 Determined in Methanol at 25 °C coumarin derivative 11 4 5 13 14

Figure 1. Coumarin-based fluorogenic click reagents.

λabs [nm] 320 350 343 351 337

λema [nm] 391 417 414 438 421

d

Stokes shift [nm]

ΦFb

F.E.c

71 67 71 87 84

0.03 0.005 0.004 0.66 0.12

− − − 132 30

Determined at λexc = 350 nm. bFluorescence quantum yield, quinine sulfate in aqueous 1.0 N H2SO4 as standard. cFluorescence enhancement calculated from the respective sydnone quantum yield. dDetermined at λexc = 320 nm. a

introducing either an azide6 (coumarin 2) or a terminal alkyne20 (compound 3) at its 3- or 7-position, respectively. Consequently, we reasoned that the change in electron density initiated from the conversion of a sydnone moiety, present at the 3-position of the coumarin scaffold, into a pyrazole ring upon click reaction should significantly alter the fluorescence properties of the chromophore, providing a novel fluorogenic system. As depicted in Scheme 1, sydnone-modified coumarins 4 and 5 were easily prepared from commercially available 2-

with sydnone 4 and chlorosydnone 5 exhibiting very low fluorescence quantum yields (4, ΦF = 0.005; 5, ΦF = 0.004), as compared to fluorescent 7-methoxycoumarin (11) (ΦF = 0.03). Next, we reacted sydnones 4 and 5 with the cyclooctyne BCN (12), and to our delight, the resulting pyrazoles 13 and 14 were generated efficiently with a second-order rate constant of 0.13 and 0.37 M−1·s−1, respectively (determined by monitoring product formation by 1H NMR spectroscopy in a mixture of CD3OD and CDCl3 (Figures S1−S2 in the Supporting Information (SI))), a rate approximatively three times faster than azides.22 More importantly, pyrazoles 13 and 14 exhibited strong fluorescence when excited around 340 nm with a maximum emission of 438 nm for 13 and 421 nm for the chloropyrazole 14 (Table 1, Figure 2, and Figures S3−S7 in the SI). In addition, both pyrazoles were characterized with particularly high fluorescence quantum yields (13, ΦF = 0.66; 14, ΦF = 0.12), noting that 7-methoxycoumarin fluorophores exhibit usually weak fluorescence as compared to their hydroxy or amino analogues.23 To gain insight into the fluorescence quenching mechanism of coumarin by the sydnone moiety, we examined the structural and electronic properties of compounds 4 and 5. X-ray crystallographic analysis of both sydnone-modified coumarins 4 and 5 (Figure 3) revealed a nonplanar connection, in the solid state, between the coumarin scaffold and the sydnone moiety with a dihedral angle of 56°, which prompted us to examine the electronic properties of their excited states for the presence of an energetically low-lying nonemissive Twisted Intramolecular Charge Transfer (TICT) state.24 Accordingly, we calculated the vertical excitation energies, representative of the first four excited singlet states, of both sydnone-modified coumarins 4 and 5 by time-dependent density functional theory (TD-DFT) at the B3LYP/6311+G(d) level using a polarized continuum model (PCM) of methanol to take into account the potential solvent effect, and characterized the nature of each transition by plotting their electron density difference (Figure 4A). Supported by the obtained high oscillator strength and matching energy values, the calculated π−π* transition between the ground state (GS) and the second excited singlet state S2 is in good agreement with the experimentally observed blue-shifted maximum of

Scheme 1. Synthesis of Sydnone-Modified Coumarins 4 and 5

hydroxy-4-methoxybenzaldehyde (6). First, the 3-amino coumarin scaffold was generated in a single step by refluxing aldehyde 6 with anhydride 7 in the presence of sodium acetate. Then, removal of the N-acetyl protecting group of 8 under acidic conditions, followed by alkylation of the free amine with bromoacetic acid, led to the desired glycine 10. Finally, the sydnone ring was built in a two-step protocol by nitrosation of the amino group with tert-butyl nitrite, followed by trifluoroacetic anhydride induced cyclization, providing the targeted 3sydnone methoxycoumarin 4 in 68% yield. Substitution of the 4-position of the mesoionic ring with a chlorine atom has been shown to significantly enhance the reactivity of the sydnone with alkynes by lowering its distortion energy.18,21 Consequently, we also prepared coumarin chlorosydnone 5 in 62% yield by electrophilic chlorination of the parent sydnone 4. The photophysical properties of the sydnone-modified coumarins 4 and 5 were then determined and compared to B

DOI: 10.1021/acs.orglett.8b01587 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

Figure 2. (A) Fluorogenic strain-promoted cycloaddition between BCN (12) and sydnone-modified coumarins 4 and 5. (B) Fluorescence emission spectra of sydnones 4 and 5 (dashed lines; λexc = 350 nm; OD350 = 1), and the corresponding pyrazole products 13 (solid line; λexc = 350 nm; OD350 = 1) and 14 (solid line; λexc = 340 nm; OD340 = 1) in MeOH. Inset: visual comparison of the fluorescence intensity of methoxycoumarin derivatives 4, 5, 13, and 14 in MeOH with excitation at 365 nm.

Figure 4. Energy diagram with their oscillator strength values (f) and total electron density differences plots between the ground state (GS) and the relevant excited singlet state (S1, S2) (decreasing intensity shown in blue, increasing intensity in red) of sydnones 4 and 5 (A) and pyrazoles 15 and 16 (B).

electronic coupling between the GS and S1, consequently restoring the fluorescence of the cycloadducts. Taken together, these TD-DFT computed results suggest that the fluorescence quenching observed for the sydnone-modified coumarins 4 and 5 is likely due to the presence of an energetically low-lying nonemissive charge-separated state. Finally, we evaluated the ability of fluorogenic sydnones 4 and 5 to fluorescently label proteins in no-wash conditions. As model proteins, native bovine serum albumin (BSA, negative control) and BSA conjugated with BCN cyclooctynes (BSABCN; for preparation, see SI) were incubated with either 4 or 5 in phosphate buffer saline (PBS, pH 7.4) at 37 °C for 3 h and immediately analyzed, without any washing steps, by in-gel fluorescence imaging (Figure 5). Importantly, a robust fluorescent signal was detectable when BSA-BCN conjugate was employed in the presence of the fluorogenic probes 4 (lane 5) and 5 (lane 7) or azido-fluorescein (Azide-FITC 17,25 lane 3) for a positive control, whereas no labeling was observed in control experiments using native BSA (lanes 4 and 6), highlighting the signal specificity of 4 and 5 in a biochemical context. In summary, sydnone-modified coumarins represent a new class of fluorogenic click reagents. The presence of the sydnone functionality efficiently quenches, likely due to the presence of an energetically low-lying nonemissive charge-separated state, the fluorescence of the methoxycoumarin dye, with a fluorescence quantum yield as low as 0.004. Importantly, upon fast strain-promoted cycloaddition with the cyclooctyne bicyclo[6.1.0]nonyne (BCN), the fluorogenic sydnones generated pyrazole cycloadducts that were found to be strongly fluorescent, displaying photophysical characteristics of parent 7-methoxycoumarin, namely a maximum of fluorescence emission around 430 nm, a large Stokes shift (∼85 nm), but with significantly higher fluorescence quantum

Figure 3. Crystal structures of sydnone-modified methoxycoumarins 4 and 5. (A) Top view. (B) Side view. θ, dihedral angle between the coumarin and the sydnone ring. Key: C, light gray; H, white; N, blue; O, red; Cl, green.

absorbance of 5 as compared to 4 (Table 1 and Figures S4−S5 in the SI), validating the level of computation chosen. Interestingly, the TD-DFT calculations indicate that the lowest energy transition (GS → S1) for both sydnones 4 and 5 results into the formation of a charge-separated state (with an increase in electron density onto the coumarin dye and a decrease density on the sydnone moiety), with almost negligible electronic coupling corresponding to a Franck−Condon forbidden transition, thus quenching the fluorescence of sydnone-modified coumarins 4 and 5. In addition, we also analyzed the electronic properties of the coumarin-pyrazole products following the aforementioned TDDFT approach. Compellingly, transformation of the sydnone rings into simplified model dimethyl-pyrazoles 15 and 16, upon hypothetic cycloaddition with dimethylacetylene, significantly destabilized the excited charge-separated state (S2, Figure 4B), making the first excited singlet state S1 a π−π* transition with high oscillator strength, indicating a good C

DOI: 10.1021/acs.orglett.8b01587 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Prof. Christoph J. Fahrni for helpful discussions. F.F. acknowledges financial support from the CNRS (ATIP-Avenir program), the “Investments for the Future” Programme IdEx Bordeaux (ANR-10-IDEX-03-02), the Laboratory of Excellence TRAIL (ANR-10-LABX-57), and the INCa-Cancéropôle GSO. This work has benefited from the facilities and expertise of the Biophysical and Structural Chemistry platform (BPCS) at IECB, CNRS UMS3033, Inserm US001, Bordeaux University (http://www.iecb.u-bordeaux.fr/index.php/fr/ plateformestechnologiques).

■

(1) (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596. (b) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057. (2) (a) Binder, W. H.; Sachsenhofer, R. Macromol. Rapid Commun. 2008, 29, 952. (b) Thirumurugan, P.; Matosiuk, D.; Jozwiak, K. Chem. Rev. 2013, 113, 4905. (3) (a) Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2011, 44, 666. (b) Debets, M. F.; van Hest, J. C. M.; Rutjes, F. Org. Biomol. Chem. 2013, 11, 6439. (c) Friscourt, F.; Boons, G. J. Bioorthogonal Reactions for Labeling Glycoconjugates. In Click Chemistry in Glycoscience: New Developments and Strategies; Bielski, R., Witczak, Z. J., Eds; Wiley: 2013; p 211. (4) (a) Lavis, L. D.; Chao, T. Y.; Raines, R. T. ACS Chem. Biol. 2006, 1, 252. (b) Shieh, P.; Bertozzi, C. R. Org. Biomol. Chem. 2014, 12, 9307. (5) (a) Jewett, J. C.; Bertozzi, C. R. Org. Lett. 2011, 13, 5937. (b) Friscourt, F.; Fahrni, C. J.; Boons, G. J. J. Am. Chem. Soc. 2012, 134, 18809. (c) Shie, J. J.; Liu, Y. C.; Hsiao, J. C.; Fang, J. M.; Wong, C. H. Chem. Commun. 2017, 53, 1490. (6) Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill, H. N.; Wang, Q. Org. Lett. 2004, 6, 4603. (7) Herner, A.; Girona, G. E.; Nikic, I.; Kallay, M.; Lemke, E. A.; Kele, P. Bioconjugate Chem. 2014, 25, 1370. (8) Shie, J. J.; Liu, Y. C.; Lee, Y. M.; Lim, C.; Fang, J. M.; Wong, C. H. J. Am. Chem. Soc. 2014, 136, 9953. (9) Shieh, P.; Hangauer, M. J.; Bertozzi, C. R. J. Am. Chem. Soc. 2012, 134, 17428. (10) Shieh, P.; Dien, V. T.; Beahm, B. J.; Castellano, J. M.; WyssCoray, T.; Bertozzi, C. R. J. Am. Chem. Soc. 2015, 137, 7145. (11) Gaetke, L. M.; Chow, C. K. Toxicology 2003, 189, 147. (12) (a) Devaraj, N. K.; Hilderbrand, S.; Upadhyay, R.; Mazitschek, R.; Weissleder, R. Angew. Chem., Int. Ed. 2010, 49, 2869. (b) Meimetis, L. G.; Carlson, J. C. T.; Giedt, R. J.; Kohler, R. H.; Weissleder, R. Angew. Chem., Int. Ed. 2014, 53, 7531. (c) Kozma, E.; Girona, G. E.; Paci, G.; Lemke, E. A.; Kele, P. Chem. Commun. 2017, 53, 6696. (13) Borrmann, A.; Milles, S.; Plass, T.; Dommerholt, J.; Verkade, J. M. M.; Wiessler, M.; Schultz, C.; van Hest, J. C. M.; van Delft, F. L.; Lemke, E. A. ChemBioChem 2012, 13, 2094. (14) Agarwal, P.; Beahm, B. J.; Shieh, P.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2015, 54, 11504. (15) Lin, V. S.; Chen, W.; Xian, M.; Chang, C. J. Chem. Soc. Rev. 2015, 44, 4596. (16) Browne, D. L.; Harrity, J. P. A. Tetrahedron 2010, 66, 553. (17) (a) Plougastel, L.; Koniev, O.; Specklin, S.; Decuypere, E.; Creminon, C.; Buisson, D. A.; Wagner, A.; Kolodych, S.; Taran, F. Chem. Commun. 2014, 50, 9376. (b) Liu, H.; Audisio, D.; Plougastel, L.; Decuypere, E.; Buisson, D. A.; Koniev, O.; Kolodych, S.; Wagner, A.; Elhabiri, M.; Krzyczmonik, A.; Forsback, S.; Solin, O.; Gouverneur, V.; Taran, F. Angew. Chem., Int. Ed. 2016, 55, 12073. (c) Narayanam, M. K.; Liang, Y.; Houk, K. N.; Murphy, J. M. Chem. Sci. 2016, 7, 1257.

Figure 5. (A) Fluorogenic labeling of BSA−BCN conjugate with sydnone-modified coumarins 4 and 5. (B) In-gel visualization of BSA−BCN conjugate or native BSA incubated with sydnone 4, 5, or azide 17 (250 μM) or no reagent (−) for 3 h, by fluorescence imaging (top row; λexc: trans UV) and by Coomassie Blue stain to reveal total protein content (bottom row).

yields (up to 0.66), implying notable fluorescence enhancements up to 132-fold. In addition, the fluorogenic sydnones were successfully applied, in a biochemical context, for the highly specific labeling of proteins in no-wash conditions. Interestingly, the cyclooctyne BCN was recently installed as a chemical reporter into the cell walls of Gram-positive bacteria26 as well as in complex glycans of zebrafish,14 paving the way for the utilization of these fluorogenic sydnones for the specific visualization of biomolecules in living organisms.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b01587. Experimental procedures, kinetics data (Figures S1−S2), absorption and fluorescence spectra (Figures S3−S7), computational data (Tables S1−S4), crystal structures of 4 (CCDC 1844074) and 5 (CCDC 1844087), and copies of the 1H and 13C NMR spectra for all new products (PDF) Accession Codes

CCDC 1844074 and 1844087 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Frédéric Friscourt: 0000-0002-0749-0861 D

DOI: 10.1021/acs.orglett.8b01587 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters (18) Favre, C.; de Cremoux, L.; Badaut, J.; Friscourt, F. J. Org. Chem. 2018, 83, 2058. (19) Schiedel, M. S.; Briehn, C. A.; Bauerle, P. Angew. Chem., Int. Ed. 2001, 40, 4677. (20) Zhou, Z.; Fahrni, C. J. J. Am. Chem. Soc. 2004, 126, 8862. (21) Tao, H.; Liu, F.; Zeng, R.; Shao, Z.; Zou, L.; Cao, Y.; Murphy, J. M.; Houk, K. N.; Liang, Y. Chem. Commun. 2018, 54, 5082. (22) Dommerholt, J.; Schmidt, S.; Temming, R.; Hendriks, L. J. A.; Rutjes, F.; van Hest, J. C. M.; Lefeber, D. J.; Friedl, P.; van Delft, F. L. Angew. Chem., Int. Ed. 2010, 49, 9422. (23) Heldt, J. R.; Heldt, J.; Stori, M.; Diehl, H. A. Spectrochim. Acta, Part A 1995, 51, 1549. (24) Grabowski, Z. R.; Rotkiewicz, K.; Rettig, W. Chem. Rev. 2003, 103, 3899. (25) Kii, I.; Shiraishi, A.; Hiramatsu, T.; Matsushita, T.; Uekusa, H.; Yoshida, S.; Yamamoto, M.; Kudo, A.; Hagiwara, M.; Hosoya, T. Org. Biomol. Chem. 2010, 8, 4051. (26) Shieh, P.; Siegrist, M. S.; Cullen, A. J.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 5456.

E

DOI: 10.1021/acs.orglett.8b01587 Org. Lett. XXXX, XXX, XXX−XXX