Europium-based responsive luminescent probes for reactive oxygen species

Csongor Szjjrt, Elias Pershagen,

a a,b

K. Eszter Borbas

*a

a Department of Chemistry - BMC, Uppsala University, SE-75123 Uppsala, Sweden. email: csongor.szijjarto@kemi.uu.se b Department of Organic Chemistry, The Arrhenius Laboratory, Stockholm University, SE-10691 Stockholm, Sweden

Intensity / a.u.

Responsive luminescent probes based on the trivalent lanthanides have longer emissive lifetimes, narrower peaks, and larger Stokes shifts than well-established 1 organic fluorophores. Due to the poor absorption of the lanthanides a lightharvesting antenna is usually incorporated into the complex. The antennae can be used to create analyte-responsive probes by the modulation of either the antenna 2 photophysics, or the antenna-to-metal energy transfer efficiency. Here we investigate the suitability of 7-substituted coumarins as antennae for responsive lanthanide probe construction.

Photophysical characterization
140000 120000 100000 80000 60000 40000 20000 0 315 330 345 360 375 390 405 Wavelength / nm 420 435 450 EuOMe EuBpin Eu12 EuOH

The excitation spectra of the Eu-complexes were recorded in HEPES buffer. The spectra are similar to the UV absorption spectra, consisting of a single peak around 360 nm for Eu12, EuBpin and EuOMe. The excitation spectrum of EuOH consists of peaks at 356 and 408 nm, suggesting sensitization via the coumarin phenol and coumarin phenolate species, respectively.

Synthesis

Adding H2O2 to a 10 M solution of Eu12 and EuBpin yielded a gradual decrease in Eu-emission when excitation was performed at 340 nm, and an increase upon 400 nm-excitation. Exposure of EuBpin and Eu12 to H2O2 affords EuOH. The latter possesses a strong excitation peak extending well into the visible (up to 430 nm), while the caged complexes are excitable only up to ~360 nm. ([H2O2] = 0-1000 M, arrow indicates change with increasing [H2O2 ], excitation at 340 nm, peak with asterisk is due to scattered light, left: exc. at 340 nm, right: exc. at 400 nm)

Cyclen derivatives 1Et and 1tBu were chosen as scaffolds for the complexes. The protected carboxylic acids can stabilize the lanthanide complex after deprotection, and the primary amine serves as the point of attachment for the antennae. 1Et or 1tBu and the coumarin acid (2) were reacted with HATU as the coupling reagent in the presence of DIPEA. The coupled products were isolated in good yields. Exposure of 3tBu to TFA cleaved the tert-butyl ester groups quantitatively. 3Et was functionalized at the 7-OH position with 4-pinacolatoboron benzyl bromide or 4-methoxybenzyl chloride. Cleavage of the ethyl esters was possible with excess NaOH. Treatment of 6Bpin or 6OMe with EuCl3 yielded EuBpin or EuOMe in high yield.

The complexes with boronic acid cages reacted with both H2O2 and ONOO, but much faster with the latter one. EuOMe showed good selectivity toward peroxynitrite. (left: changes in the time resolved emission spectra of the Eu-complexes with ROS/RNS, 10 M complex, 200 M ROS/RNS). The changes in the emission intensity of Eu-complexes upon treatment with increasing concentrations of peroxynitrite. (right: 30 min incubation time, 10 M complex, 200 M ONOO-, exc. at 340 nm, em. at 615 nm)

Conclusions
Emissive europium complexes sensitized by coumarin antennae were prepared in short, high-yielding syntheses. The excitation properties of the complexes are dependent on the antenna electronics. The latter is readily tuned by varying the substituent in the 7-position of the coumarin. 7-Boronate or 7-(4-pinacolatoboronbenzyl) cages were rapidly cleaved by peroxynitrite to the corresponding phenol, furnishing the first ratiometric single-component luminescent probes for this ROS. Furthermore, the design is not limited to the analytes used in this work, since many other biologically or environmentally relevant species are known to induce chemoselective reactions that install phenolic oxygens.

We have also prepared Eu12 to find out what effect the 7-Bpin group has on the photophysical properties of the coumarin antenna and the Eu complex. Coumarin 10 was coupled to 1tBu with the same procedure used before with other coumarins. In order to cleave all the tBu ester groups of 11 quantitatively we had to expose it to TFA overnight, which resulted in complete hydrolysis of the pinacol ester to the boronic acid. Treatment of ligand 12 with Eu(OTf)3 provided Eu12 in high yield.

Acknowledgements
This work was supported by the Swedish Research Council, Carl Tryggers Stiftelse, and the Departments of Chemistry, Uppsala University and Organic Chemistry, Stockholm University.

References
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