Tuning the Spectroscopic Properties of Ratiometric Fluorescent Metal

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Tuning the Spectroscopic Properties of Ratiometric Fluorescent Metal Indicators: Experimental and Computational Studies on Mag-Fura-2 and Analogues Guangqian Zhang, Denis Jacquemin, and Daniela Buccella J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b11045 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 6, 2017

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The Journal of Physical Chemistry

Tuning the Spectroscopic Properties of Ratiometric Fluorescent Metal Indicators: Experimental and Computational Studies on Mag-fura-2 and Analogues Guangqian Zhang,a Denis Jacquemin*,b,c and Daniela Buccella*,a a

Department of Chemistry, New York University, New York, New York, 10003. bLaboratoire CEISAM-UMR CNRS 6230, Université de Nantes, 2 Rue de la Houssinière, BP 92208, 44322 Nantes Cedex 3, France. cInstitut Universitaire de France, 1 rue Descartes, F-75231 Paris Cedex 05, France. ABSTRACT: In this joint theoretical and experimental work, we investigate the properties of Mag-fura-2 and seven structurally related fluorescent sensors designed for ratiometric detection of Mg2+ cations. The synthesis of three new compounds is described, and the absorption and emission spectra of all sensors in both their free and metal-bound forms are reported. A Time-Dependent Density Functional Theory approach accounting for hydration effects using a hybrid implicit/explicit model is employed to calculate the absorption and fluorescence emission wavelengths, and to study the origins of the hypsochromic shift caused by metal binding for all the sensors in this family, as well as to investigate the auxochromic effects of various modification of the “fura” core. The metal-free forms of the sensors are shown to undergo strong intramolecular charge-transfer upon light absorption, which is largely suppressed by metal complexation resulting in predominantly locally excited states upon excitation of the metal complexes. Our computational protocol might aid in the design of new generations of fluorescent sensors with low energy excitation and enhanced properties for ratiometric imaging of metal cations in biological samples.

INTRODUCTION Metal-responsive small molecule fluorescent indicators have become essential tools in the study of the cell biology of metals, allowing the detection of metal cations in cells with outstanding sensitivity and selectivity.1-2 Wavelength ratiometric sensors are a class of fluorescent indicators that undergo a shift in excitation or emission wavelength upon binding their target analyte. This shift enables the use of a ratio of two signals, i.e., emission intensities at two different wavelengths or emission intensities upon excitation at two wavelengths, as the sensing readout.3 Compared to an increase or decrease of a single fluorescence signal, a ratiometric readout is less sensitive to sensor concentration, to the effects of photobleaching, uneven illumination and other external factors that affect the fluorescence output.4 Ratiometric metal-responsive sensors are, therefore, preferred for the analysis of non-homogeneous samples and for fluorescence microscopy studies where quantitative, rather than qualitative information is sought about the target metal. Despite their superior performance, ratiometric metal sensors are far less common and more difficult to design than their intensity-based, turn-on and turn-off counterparts. Many ratiometric indicators display typical donor-π-acceptor (D-π-A) structures (often referred to as “push-pull” structures) and undergo photoinduced intramolecular charge transfer (ICT). Interaction of the positively charged metal with either donor or acceptor groups could stabilize or destabilize the ICT state, thus significantly shifting the absorption and emission wavelengths.5 Unfortunately, the relative scarcity of computational

models fully addressing the spectroscopic properties and the effect of the metal binding in this type of systems has contributed to make the design and development of ratiometric metal indicators a mostly trial-and-error exercise. Two factors can explain this scarcity. First, it is only quite recently that theoretical models providing an accurate description of ICT in large compounds have emerged, notably due to the improvements in the exchange-correlation functionals used in Density Functional Theory (DFT). Second, to be of interest, the theoretical models have to be able to explore emission, and hence (partly) describe the potential energy surface of the excited state, meaning that analytical gradients are, in practice, necessary. For these reasons, Time-Dependent DFT (TD-DFT) has become the de facto standard in modeling fluorescent dyes.6 Ratiometric fluorescent sensors of the “fura” family, including Fura-27 and Furaptra8 (a.k.a Mag-fura-2, Figure 1), were developed in the late 1980s and have since become ubiquitous tools in the study of cellular Ca2+ and Mg2+, respectively, shedding light on changes in cation concentration involved in a large number of processes ranging from signaling to regulation of cellular metabolic activity. The 6-amino-2-oxazolyl benzofuran fluorophore core that characterizes the fura sensors is also found in compounds designed for fluorescence-based detection of other biologically relevant ions such as Na+ (SBFO9 and compound 1210) and Zn2+ (FuraZin11 and ZnAFR212). Most sensors in this popular family display large Stokes shifts and undergo hypsochromic shifts in their absorption spectra upon metal binding, which make them suitable for

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al-free and -bound states.

METHODS

Figure 1. Representative examples of ratiometric metal ion indicators based on the 6-amino-2-oxazolyl benzofuran fluorophore core.

excitation-ratiometric detection modalities and great tools for fluorescence microscopy applications. However, they require high-energy excitation that can lead to significant interference from autofluorescence of biological samples, increased scattering, as well as potential photodamage to the sample. In an effort to obtain better sensors for the study of Mg2+ in biological samples, the Buccella laboratory developed a series of enhanced Mag-fura-2 analogues in which replacement of the oxygen atom in the oxazole group of the fluorophore by either sulfur or selenium induced a significant redshift of both the excitation and emission maxima of the sensors, as well as an increase in their Stokes shift.13 In this work, we expand upon those initial efforts by building a small library of structurally related sensors incorporating systematic modifications in the fluorophore structure. We employ this family of analogues to benchmark spectroscopic properties calculated by TD-DFT, with the goal of building a computational model to aid in the future design of ratiometric fluorescent sensors with enhanced properties. To our knowledge, there is only one previous report of a theoretical study of a related ratiometric metal sensor, but it focused on the ground state properties only.14 The TDDFT protocol used here accounts for solvation effects with a refined model and relies on one of the most robust functionals developed to date (see computational details). Our studies provide insights into the nature of the transitions that characterize Mag-fura-2 and, more generally, the fura sensors, illustrating how metal binding affects the nature of the excited state and how chemical modifications influence the absorption and emission wavelengths of the compounds in both the met-

1. EXPERIMENTAL METHODS General Synthetic Protocols 2-Amino-4-benzyloxyphenol-N,N,O-triacetic acid trimethyl ester (1),8 ethyl 2-(bromomethyl)thiazole-5-carboxylate (5),13 ethyl 2(chloromethyl)oxazole-5-carboxylate (4),7 and 2-iodobenzothiazole 1015 were prepared according to literature procedures. All other reagents were purchased from commercial sources and used as received. Solvents were purified and degassed by standard procedures. NMR spectra were acquired on Bruker Avance 400 or Bruker Avance 600 spectrometers. 1H NMR chemical shifts are reported in ppm relative to SiMe4 (δ = 0) and were referenced internally with respect to residual protio impurity in the solvent (δ = 7.26 for CHCl3, 3.31 for CHD2OD). 13C NMR chemical shifts are reported in ppm relative to SiMe4 (δ = 0) and were referenced internally with respect to the solvent signal (δ 77.16 for CDCl3, 49.00 for CD3OD). Coupling constants are reported in Hz. Melting points were collected on a BUCHI 510 melting point apparatus and are reported uncorrected. Lowresolution mass spectra were acquired on an Agilent 1100 series LC/MSD trap spectrometer, using electrospray (ES) ionization. Highresolution mass spectrometry (HRMS) analyses were conducted on an Agilent 6224 TOF LC/MS Mass Spectrometer using ES ionization. Analytical thin layer chromatography (TLC) was performed on polyester-backed 200 μm silica gel sheets. Preparative TLC was performed on 1000 μm silica gel plates. Reversed-phase HPLC analyses were conducted on an Agilent 1260 system with UV-Vis absorption and fluorescence detection, using a Poroshell C18 reversed phase column (4.6×50 mm, 2.7 μm particle size) and eluting with a gradient of 10% to 100% acetonitrile/water (+ 0.1% trifluoroacetic acid). Synthesis of 2-amino-4-benzyloxyphenol-N,N,O-triacetic acid trimethyl ester, compound 2. A solution of 2-amino-4benzyloxyphenol-N,N,O-triacetic acid trimethyl ester (compound 1, 1.7 g, 3.94 mmol) in acetic acid (8 mL) under nitrogen atmosphere was treated with Pd on carbon (10%, 419 mg, 0.394 mmol) and stirred under H2 (1 atm). After 2.5 h, the reaction mixture was filtered through a pad of Celite, washing with EtOAc. The combined filtrate was concentrated and the residue was purified by column chromatography on silica gel (1:3 EtOAc/hexanes) to yield compound 2 as a light brown oil (1.15 g, 86%, Rf = 0.27 in 1:2 EtOAc/hexanes). 1H NMR (400 MHz, CDCl3, δ): 6.72 (d, J = 8.6 Hz, 1H), 6.41 (d, J = 2.8 Hz, 1H), 6.33 (dd, J = 8.6, 2.8 Hz, 1H), 4.57 (s, 2H), 4.17 (s, 4H), 3.77 (s, 3H), 3.71 (s, 6H). 13C{1H} NMR (100 MHz, CDCl3, δ): 171.8, 170.0, 151.7, 143.8, 141.0, 117.9, 108.8, 107.5, 67.7, 53.7, 52.2, 52.0. ESI-MS (m/z) [M+Na]+ calcd for C15H19NO8, 364.1; found 363.8. Synthesis of 2-amino-4-hydroxy-5-nitrosophenol-N,N,Otriacetic acid trimethyl ester, compound 3. To a solution of compound 2 (1.15 g, 3.37 mmol) in aqueous HCl (4 mL, 23%) was added NaNO2 (256 mg, 3.71 mmol) in water (2 mL) at 0 °C over 15 min, and the reaction was stirred at 0 °C for 45 min. The reaction mixture was diluted with water (30 mL) and extracted with EtOAc (3×20 mL). The combined organics were dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (EtOAc) to yield nitroso aminophenol 3 as a brown solid (550 mg, 44%, Rf = 0.46 in EtOAc). M.p. 140-141 °C. 1H NMR (600 MHz, CD3OD, δ): 6.43 (s, 1H), 5.38 (s, 1H), 4.71 (s, 2H), 4.40 (s, 4H), 3.80 (s, 3H), 3.78 (s, 6H). 13C{1H} NMR (150 MHz, CD3OD, δ): 182.4, 171.2, 169.0, 157.0, 152.3, 148.3, 105.5, 95.6, 66.2, 56.5, 52.88, 52.85. ESI-MS (m/z) [M+Na]+ calcd for C15H18N2O9, 393.1; found 392.9. Synthesis of compound 6. A suspension of compound 3 (56 mg, 0.15 mmol), ethyl 2-(chloromethyl)oxazole-5-carboxylate (compound 4, 28 mg, 0.15 mmol), and K2CO3 (41 mg, 0.30 mmol) in dry DMF (3.0 mL) was stirred at 100 °C for 30 min. After cooling down to room temperature, the reaction mixture was diluted with water (30 mL) and extracted with EtOAc (3×20 mL). The combined organics were dried over Na2SO4 and concentrated in vacuo. The residue was purified by


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column chromatography on silica gel (2:1 EtOAc/hexanes) to give compound 6 as a yellow solid (30 mg, 40%, Rf = 0.26 in 1:1 EtOAc/hexanes). M.p. 113-114 °C. 1H NMR (400 MHz, CD3OD, δ): 8.07 (s, 1H), 7.34 (s, 1H), 7.26 (s, 1H), 4.81 (s, 2H), 4.44 (q, J = 7.1 Hz, 2H), 4.29 (s, 4H), 3.80 (s, 3H), 3.74 (s, 6H), 1.41 (t, J = 7.1 Hz, 3H). 13 C{1H} NMR (150 MHz, CDCl3, δ): 171.4, 168.8, 157.2, 153.0, 150.4, 149.3, 147.0, 144.0, 141.2, 135.8, 135.6, 105.5, 101.8, 66.6, 62.2, 54.1, 52.5, 52.2, 14.4. HR-TOF-MS (m/z): [M+H]+ calcd for C22H23N3O11, 506.14054; found 506.14305. Synthesis of compound 7. A suspension of compound 3 (56 mg, 0.15 mmol), ethyl 2-(bromomethyl)thiazole-5-carboxylate (compound 5, 38 mg, 0.15 mmol), and K2CO3 (41 mg, 0.30 mmol) in dry DMF (3.0 mL) was stirred at 100 °C for 30 min. After cooling down to room temperature, the reaction mixture was diluted with water (20 mL) and extracted with EtOAc (3×10 mL). The combined organics were dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (2:3 EtOAc/hexanes) to give compound 7 as a yellow solid (30 mg, 38%, Rf = 0.49 in 1:1 EtOAc/hexanes). M.p. 119-120 °C. 1H NMR (400 MHz, CDCl3, δ): 8.53 (s, 1H), 7.20 (s, 1H), 7.17 (s, 1H), 4.72 (s, 2H), 4.42 (q, J = 7.1 Hz, 2H), 4.28 (s, 4H), 3.80 (s, 3H), 3.75 (s, 6H), 1.41 (t, J = 7.1 Hz, 3H). 13 C{1H} NMR (100 MHz, CDCl3, δ): 171.4, 168.9, 160.9, 158.9, 156.0, 149.7, 149.3, 147.0, 140.9, 136.1, 132.3, 105.4, 102.0, 66.7, 62.3, 54.1, 52.4, 52.1, 14.4. HR-TOF-MS (m/z): [M+H]+ calcd for C22H23N3O10S, 522.11769; found 522.11835. Synthesis of ethyl 2-iodothiazole-5-carboxylate, 9. To a solution of ethyl 2-aminothiazole-5-carboxylate 8 (86 mg, 0.5 mmol) and ptoluenesulfonic acid monohydrate (333 mg, 1.75 mmol) in MeCN (2 mL) was added a solution of NaNO2 (69 mg, 1.0 mmol) and KI (216 mg, 1.3 mmol) in water (0.5 mL) at 0 °C over 5 minutes. The reaction was stirred at room temperature overnight and then diluted with aqueous NaHCO3 (15 mL) and extracted with EtOAc (3×10 mL). The combined organics were dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (1:15 EtOAc/hexanes) to give thiazole 9 as a white solid (120 mg, 85%, Rf = 0.39 in 1:9 EtOAc/hexanes). M.p. 40-41 °C. 1H NMR (400 MHz, CDCl3, δ): 8.10 (s, 1H), 4.36 (q, J = 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H). 13C{1H} NMR (150 MHz, CDCl3, δ): 160.0, 149.4, 135.8, 107.9, 62.1, 14.4. ESI-MS (m/z): [M+H]+ calcd for C6H6INO2S, 283.9; found 283.9. Synthesis of compound 11. A mixture of compound 10 (40 mg, 0.079 mmol), compound 9 (33 mg, 0.118 mmol), and CuI (37 mg, 0.197 mmol) in 1:5 Et3N/DMSO (1.8 mL) was stirred under inert atmosphere at 65 °C overnight. After cooling down to room temperature, the reaction mixture was diluted with water (15 mL) and extracted with EtOAc (3×10 mL). The combined organics were dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (2:3 EtOAc/hexanes) to give compound 11 as a yellow solid (16 mg, 38%, Rf = 0.35 in 1:1 EtOAc/hexanes). M.p. 109-110 °C. 1H NMR (600 MHz, CD3OD, δ): 8.45 (s, 1H), 7.51 (s, 1H), 7.46 (s, 1H), 4.85 (s, 2H), 4.41 (q, J = 7.1 Hz, 2H), 4.30 (s, 4H), 3.81 (s, 3H), 3.75 (s, 6H), 1.40 (t, J = 7.1 Hz, 3H). 13 C{1H} NMR (150 MHz, CD3OD, δ): 173.3, 170.6, 167.4, 162.3, 159.6, 151.8, 150.1, 149.9, 142.1, 132.5, 131.5, 111.3, 108.2, 66.8, 63.2, 54.9, 52.7, 52.4, 14.5. HR-TOF-MS (m/z): [M+H]+ calcd for C22H23N3O9S2, 538.09485; found 538.09503. General procedure for quantitative hydrolysis of esters 6, 7 and 11 to yield B1, B2 and B3. A sample of ester (0.0100 mmol) in methanol (100 μL) was treated with aqueous potassium hydroxide (100 μL, 1.2 M, 0.12 mmol) and stirred at room temperature for 24 h. The solution was transferred quantitatively to a volumetric flask and diluted with buffer at pH 7.0 (50 mM PIPES, 100 mM KCl) to a final concentration of 2.00 mM. Quantitative hydrolysis of the ester was verified by HPLC, and the identity of the product was verified by MS analysis: Compound B1: ESI-MS (m/z) [M-H]- calcd for C17H12N3O11, 434.0; found 434.0.

Compound B2: ESI-MS (m/z) [M-H]- calcd for C17H13N3O10S, 450.0; found 449.6. Compound B3: ESI-MS (m/z) [M-H]- calcd for C17H13N3O9S2, 466.0; found 465.8. The stock solution was divided into small aliquots, flash frozen in liquid nitrogen, and stored below -20 °C. Spectroscopic methods All aqueous solutions were prepared using de-ionized water having a resistivity of 18 MΩ/cm. Other solvents were supplied by commercial vendors and used as received. Piperazine-N,N-bis(2ethanesulfonic acid) (PIPES), 99.999% KCl, 99.999% MgCl2, and highpurity acids and bases were purchased from Sigma Aldrich. Stock solutions of the sensors in their acid form were stored at -20 °C in 100-200 μL aliquots, and thawed immediately before each experiment. Measurements at pH 7.0 were conducted in aqueous buffer containing 50 mM PIPES and 100 mM KCl. Buffers were treated with Chelex resin (Bio-Rad) according to the manufacturer’s protocol, to remove adventitious metal ions unless otherwise noted. Measurements of pH were conducted using a Mettler Toledo FE20 with glass electrode. UV-visible absorption spectra were acquired with a Cary 100 spectrophotometer using quartz cuvettes from Starna (1 cm path length). Fluorescence spectra were acquired with a QuantaMaster 40 Photon Technology International spectrofluorometer equipped with xenon lamp source, emission and excitation monochromators, excitation correction unit, and PMT detector. Emission spectra were corrected for the detector wavelength-dependent response. Measurements were conducted at 25.0 ± 0.1 °C. Fluorescence quantum yields were determined using 0.5-5.0 μM solutions of the sensors in aqueous buffer at pH 7.0, exciting at the reported absorption maxima for each compound. Solutions of quinine in 0.5 M aqueous sulfuric acid, with a reported quantum yield of 0.546 upon excitation at 347 nm,16 were used as standards. 2. COMPUTATIONAL METHODS DFT and TD-DFT calculations on all molecules were carried out with the Gaussian 09 program package,17 applying both a tightened selfconsistent field convergence criterion (10−9-10−10 au) and an improved optimization threshold (10−5 au on average forces). For each molecule, we have optimized the geometry of the ground electronic state and computed its vibrational spectra to ascertain the nature of the optimized structure. Next, the geometry and vibrational frequencies of the lowest excited state were determined with TD-DFT using analytical gradients. The same DFT integration grid, namely the so-called ultrafine pruned (99,590) grid, was used for all calculations. All DFT and TD-DFT calculations were performed with the M06-2X hybrid exchange-correlation functional18 that has been shown to be an adequate choice for investigating structures and excited states of many classes of molecules.19-22 While structural parameters were determined with the 6-31G(d) atomic basis set, transition energies have been obtained with the more extended 6-311+G(2d,p) atomic basis set. Bulk solvation effects (here water) have been quantified using the Polarizable Continuum Model (PCM)23 that was systematically applied to all computational steps (geometry, vibration and optical spectra). The structural and vibrational parameters of the excited state were obtained in the linear-response (LR) PCM model,24-25 considering the socalled equilibrium limit. To determine the absorption and emission energies, we have applied the corrected LR (cLR) PCM approach26 in its non-equilibrium limit as this protocol is suited to investigate rapid transitions between two electronic states.

RESULTS AND DISCUSSION Design and synthesis of sensors: introducing systematic variations in the “fura” scaffold Starting with the basic Mag-fura-2 platform, we designed a small library of fluorescent compounds (Chart 1) introducing systematic modifications in the benzofuran and/or oxazole moieties, to explore the role of the different components of the


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Chart 1. Ratiometric Mg2+ fluorescent sensors included in this study

core of the fluorophore on the optical properties of the resulting sensors. In compounds Mag-S and Mag-Se, the oxygen atom of the oxazole moiety is replaced by heavier chalcogens sulfur and selenium, respectively.13 Previously reported amide derivative C127 and triazole compound C215 were also included in our study to assess the role of the carboxylate group that is typically invoked as the accepting group in these push-pull dyes. Finally, to help establish the effects of modifications on the benzofuran fragment, compounds B1 to B3 were designed. In these compounds, the benzofuran moiety was replaced by benzoxazole or benzothiazole groups. These fragments are often found in other luminescent compounds with biological applications, such as luciferin analogues as well as in a variety of DNA intercalators used as nuclear stains.28 The assembly of benzoxazole-based sensors B1 and B2 Scheme 1. Synthesis of sensors B1 and B2

hinged on the synthesis of nitrosophenol 3, which was obtained in moderate yield by hydrogenolysis of benzylprotected aminophenol 1 followed by nitrosation (Scheme 1). Condensation of the nitrosophenol with halomethyl azoles 4 or 5 followed by ester hydrolysis led to compounds B1 and B2, respectively. Sensor B3 was obtained by copper-mediated cross coupling between iodides 9 and 10 followed by hydrolysis of the ester protecting groups (Scheme 2). Spectroscopic properties of the sensors Photophysical characterization of the new sensors B1-B3 was conducted in aqueous buffer at pH 7.0 and 25 °C to facilitate comparisons with previously reported analogues. Normalized absorption and emission profiles both in the absence and presence of Mg2+ are shown in Figure 2, and the absorption and

Scheme 2. Synthesis of sensor B3


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Figure 2. Absorption and emission spectra of Mag-fura-2 analogues in aqueous buffer at pH 7.0, 25 °C, in the absence (dashed line) or presence (solid line) of 200 mM Mg2+ in solution. (A) Mag-fura-2, (B) Mag-S, (C) Mag-Se, (D) C1, (E) B1, (F) B2, (G) B3, and (H) C2. The * denotes scattered light from the excitation beam.

emission maxima are listed in Tables 1 and 2, respectively. For ease of comparison, the properties of Mag-fura-28,28 and the previously reported Mag-S, Mag-Se,13 amide derivative C127 and triazole-functionalized compound C215 are also included. All the compounds show large Stokes shifts in aqueous solution (greater than 7000 cm-1) and undergo a blue shift in their absorption spectra upon Mg2+ binding. They are, therefore, potentially suitable for excitation ratiometric sensing applications of this cation. To characterize the optical properties of the Mg2+-bound species, we employed a high concentration of Mg2+ in solution (200 mM), in large excess with respect to the concentration of the sensor, to quantitatively shift the binding equilibrium toward the formation of the complex. As discussed previously,13 single atom replacement of oxygen by sulfur or selenium in the azole group of Mag-fura-2

affords a significant bathochromic shift of both absorption and emission spectra. Similar effects were observed in going from compound B1 to B2; the replacement of the oxazole by a thiazole induces a 26 nm bathochromic shift in the absorption and a 59 nm bathochromic shift in the emission of the sensor in its metal-free form. Comparable bathochromic shifts are seen for the Mg2+-bound forms. Conversion of the carboxylic acid group of Mag-S into an amide, as in compound C1, results in further shift to longer absorption and emission wavelengths, which are desirable changes in the search for sensors with low excitation and emission energies for bioimaging applications. Modifications in the benzofuran fragment, on the other hand, have a main impact on the emission but only moderate effect on the absorption wavelength. Compound B1, with a benzoxazole fragment, displays a longer emission wavelength (ca. 50 nm shift) than benzofuran-based Mag-fura-2 in both Mg2+-


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Table 1. Comparison between theoretical and experimental absorption wavelengths for the compounds in Chart 1. Δλ is the wavelength shift elicited by metal of complexation. The theoretical values correspond to vertical cLR-M06-2X/6-311+G(2d,p) transition energies using the IE model. Experimental λmax (nm)a 2+

Theoretical λmax (nm)

Sensor

Mg Complex

Δλ

Sensor

Mg2+ Complex

Δλ

Mag-fura-2

369

330

-39

341

296

-45

Mag-S

396

350

-46

370

313

-57

Mag-Se

412

360

-52

380

320

-60

B1

380

334

-46

347

291

-56

B2

406

357

-49

374

306

-68

B3

420

361

-59

382

318

-64

C1

412

356

-56

395

321

-74

C2

354

326

-28

326

283

-43

a

Measurements conducted in aqueous buffer at pH 7.0, 25 °C, in the absence or presence of 200 mM Mg2+ in solution.

Table 2. Comparison between theoretical and experimental emission wavelengths for the compounds in Chart 1. See caption of Table 1 for more details. Experimental λmax (nm),a ϕb

Mag-fura-2 Mag-S Mag-Se

Theoretical λmax (nm)

Sensor

Mg2+ Complex

Δλ

Sensor

Mg2+ Complex

Δλ

511, 0.24c

491, 0.30c

-20

419

357

-62

572, 0.17

d

547, 0.30

d

-25

466

384

-82

584, 0.09

d

562, 0.18

d

-22

479

395

-84

B1

555, 0.086(2)

548, 0.032(1)

-7

445

354

-91

B2

614, 0.107(5)

605, 0.085(3)

-9

502

383

-119

B3

613, 0.182(7)

610, 0.085(4)

C1

595, 0.053

C2

f

493, 0.42

e

565, 0.164

-3

506

396

-110

e

-30

501

395

-106

f

-10

412

333

483, 0.235

a

-79 2+

Measurements conducted in aqueous buffer at pH 7.0, 25 °C, in the absence or presence of 200 mM Mg in solution. bQuinine sulfate in 0.5 M H2SO4 (ϕ = 0.546) was employed as fluorescence standard. Numbers in parentheses correspond to the uncertainty in the last significant digit. cFrom reference 28. dFrom reference 13. eFrom reference 27. f From reference 15.

free and -bound forms. The absorption maximum shifts much less to the red (ca. 10 nm change). Similarly, in comparison to benzofuran-based Mag-S, the emission wavelength of benzoxazole-based analogue B2 is bathochromically shifted by 51 nm, whereas the absorption spectrum only shifts by 10 nm in the metal-free form. Computational studies At pH 7.0, a fraction greater than 90% of the total sensor concentration in solution should be in the fully deprotonated form, estimated from reported pKa values for o-aminophenolN,N,O-triacetic acid (APTRA).29 We, therefore, focused our computational studies on fully deprotonated ter- or quadrianions for the free compounds and mono- or di-anions for the dyes complexed with Mg2+. A crystal structure of the complex of APTRA with Mg2+ was employed to build the initial geometry used as starting point in the optimization of the metal-

bound sensors.30 As negatively charged species are known to be challenging for theory, we first investigated the importance of the solvation model considering bulk solvation only, as given by the polarizable continuum model (PCM), and a hybrid implicit/explicit approach (IE) in which all carboxylate groups were solvated with one explicit water molecule. In both models, the magnesium coordination sphere was completed with one water to reach a pseudo-octahedral geometry, as in the crystal structure.30 The starting configuration of these water molecules, with the two hydrogen atoms pointing towards the two oxygen atoms of the carboxylate groups, was chosen following literature on microhydration of this anion.31-33 Optimized structures for Mag-fura-2 are shown in Figure 3. For this compound, the computed vertical transition wavelengths are 345 nm (free) and 296 nm (Mg2+ complex) with the PCM approach, and 341 nm (free) and 296 nm (Mg2+ complex) with the IE model. Likewise, for C2, the PCM absorption wave-


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Figure 3. Representation of the optimal geometries of Mag-fura-2 obtained with the PCM (left) and IE (right) solvation models. The top structures correspond to the free sensor, whereas the bottom structures represent the Mg2+ complexes.

lengths are 333.7 nm (free) and 283.3 nm (Mg2+ complex) whereas the IE values are 326.0 nm (free) and 282.8 nm (Mg2+ complex). Similar results are obtained for the other compounds. In short, the addition of explicit water molecules in the model principally impacts the transition wavelengths determined for the free complex, though the obtained blue shifts compared to the PCM results remain small. In the following, we discuss only the results obtained with the IE approach. The absorption wavelengths calculated for all compounds are listed in Table 1 along with the experimental values. For all dyes, a significant hypsochromic shift is predicted by TD-DFT upon complexation with Mg2+, which fits experimental trends. It is obvious that theory undershoots the experimental transition energies by ca. 30-40 nm, and this error can be explained, on the one hand, by the selection of the M06-2X functional19-22 and, on the other hand, by the use of the vertical approximation that neglects vibronic couplings.34 Nevertheless, the trends are nicely reproduced and this statement holds for both the auxochromic effects and the difference between the free and complexed forms. Indeed, the Δλ values calculated with theory (see Table 1) are typically in reasonable agreement with experiment. This agreement can be illustrated by considering the linear correlation coefficient, R, between theoretical and experimental values that reaches 0.98 (see Figure 4). To rationalize the hypsochromic shifts, we have determined density difference (Δρ) plots and the associated charge transfer (CT) distance following a well-known procedure (see the Computational Methods Section as well as the Supporting Information, SI).35-36 As an illustration, the Δρ plots obtained for free and complexed Mag-fura-2 are given in Figure 5. In the metal-free structure, one notices a clear CT from the amine and the vicinal phenyl ring (mostly in blue) towards the oxazole ring (mostly in red). The computed CT distance is 3.14 Å. In contrast, after complexation with the Mg2+ cation, the lone pair of the amine is no longer available (as proposed by Tsien in his original work with fura-27) and the excited state presents a typical π−π* nature with no significant CT involved (the CT distance is indeed as small as 0.45 Å). Similar trends are ob-

tained for other compounds, as shown in Table 1 and S1 in the SI. In summary, the computational analysis supports the notion that the observed hypsochromic shifts upon complexation are due to a change in the nature of the underlying electronic transitions, from CT in the metal-free form to a more localized in the metal bound form. The computed and experimental fluorescence wavelengths are listed in Table 2. As for the absorption, one notices a hypsochromic shift upon complexation with Mg2+, but the agreement between experiment and theory is much less satisfying, with absolute Δλ values predicted by TD-DFT that are much larger than their experimental counterparts. We attribute this discrepancy to possible dissociation of the metal in the excited state of some compounds, such that the observed emission may originate from a mixture of the metal-bound and free form. Indeed, such dissociation has been studied for Magfura-237 and reported in other fura systems.7,38 The computa400 380

Theory (nm)

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360 340 320 300 280 320

340

360

380

400

420

440

Experiment (nm) Figure 4. Comparison between experimental and theoretical absorption wavelength (nm) for both free (blue circles) and Mg2+complexed (red circles) sensors. The central straight line is the linear correlation line.


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sonable charge separation in the excited state. The plots of Figures 5 and S1 also support this statement. Indeed, very small electron density change is observed on the two oxygen atoms of the carboxylate in Mag-fura-2, indicating that the actual acceptor in this molecule is the azole moiety. In turn, this observation suggests that other groups can be incorporated at that position to improve other properties, e.g., solubility and ease of synthesis, without significantly affecting the optical signatures. Influence of the modifications on the benzofuran fragment

Figure 5. Representation of the difference between total electron densities of the ground and excited states for Mag-fura-2 in its free (top) and Mg2+-complexed (bottom) forms. The red (blue) regions represent increase (decrease) of electron density upon photon absorption. The selected contour threshold is 1.2 x10-3 a.u.

tional analysis suggests that this possible dissociation is not caused by a buildup of positive charge on the nitrogen atom in the excited state as one may suspect. On the contrary, the Merz-Kollman charges determined on the nitrogen atom bound to the magnesium atom do not change significantly in going from the ground state to the excited state (e.g. +0.02 e for the unbound form). As an exercise, we computed the enthalpy associated with a model complexation reaction of Mg2+ with Mag-fura-2 in the ground and excited states, and obtained a ca. 10 kcal/mol lower enthalpy of association for the excited state reaction compared to the ground state one.39 These results are in agreement with a greater dissociation constant in the excited state, as measured experimentally.40 Influence of the modifications on the azole charge acceptor fragment In going from Mag-fura-2 to Mag-S and next Mag-Se, significant bathochromic shifts of the spectra are observed in both free and Mg2+-complexed forms. These come with increasing CT distances, computed to be 3.14 Å, 3.61 Å and 3.66 Å for the three compounds in their free forms (see Table S1 in the SI); as well as with greater photo-induced changes of the total charges of the azole group, which vary by -0.18 e, -0.30 e and 0.32 e for Mag-fura-2, Mag-S and Mag-Se, respectively, when going from the ground state to the excited state. In other words, the accepting power of the azole moiety increases when replacing O by S and then Se, a notion that is consistent with the trends in electron deficiencies of the heterocycles,41 and has a strong influence on the optical properties of the sensors. An interesting case is the triazole-based compound C2 because it does not present a carboxylate group on the fluorophore. The computed CT distance for this compound is 3.19 Å, similar to the CT distance of Mag-fura-2 (3.14 Å), which reveals that the putative pulling effect of the carboxylate bound to the azole ring is not necessary for obtaining a rea-

As summarized above, modification of the benzofuran fragment, replacing a carbon (or, more strictly speaking, a methine unit) by a nitrogen atom in the heterocycle to turn the fluorophores into bezoxazoles, has greater impact on the emission than on the absorption wavelengths of the sensors, increasing their Stokes shift. To explain this, we provide key geometrical parameters of the ground and excited state of Mag-fura-2 and B1 in Figure 6, focusing on the modified heterocycle. First, we notice that the largest variations on the bond lengths between the two electronic states are localized on the two chemical bonds around the methine unit (Magfura-2) or nitrogen atom (B1), the three other bonds of the five-member cycles being less affected by the electronic transition. This indicates that changing the methine unit will significantly impact the optical properties, in particular the Stokes shift, whereas the substitution of the oxygen atom will induce smaller variations (see also below). Second, in the case of B1, the two C-N bonds in the bezothiazole present almost equal bond lengths in the excited state geometry (1.345 and 1.351 Å, δ=0.006 Å) suggesting large electronic delocalization leading to stabilization of the excited state and a significant red shift of the emission. The ground state geometry of B1, on the other hand, shows much more limited delocalization (δ=0.098 Å). In contrast, in the parent compound Mag-fura-2, the two bonds around the methine unit remain significantly different after absorption of light (δ=0.076 Å and 0.023 Å, in the ground and the excited states, respectively), hence the increase of delocalization in the excited state structure is smaller than in B1, and the emission less red shifted.

Figure 6. Selected bond lengths in the bezofuran core of Magfura-2 (left) and benzoxazole in B1 (right). All values are in Å. The blue and red values correspond to the ground and excited state values, respectively. Only the central part of the dyes is displayed.

An additional modification that was investigated involved replacing the oxygen of the benzoxazole by sulfur. In this case, only a minor redshift in either absorption or emission was observed when comparing analogues B2 and B3, a much smaller impact than observed when similar modification is introduced in the acceptor oxazole group. The small change in properties between B2 and B3 suggests that the identity of the


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chalcogen in the benzoxazole fragment does not have a large impact on the relative energies of the ground and excited states. As seen in the density difference plot of Figure S1, this position is only moderately involved in the calculated electron density changes, and the CT parameters determined for both B2 and B3 are also similar in both free and bound forms (Table S1).

CONCLUSIONS In summary, we have synthesized and characterized a new set of Mag-fura-2 analogues incorporating various modifications in the 2-oxazolyl bezofuran core of the sensors, which led to significant red shifts of both absorption and emission wavelengths. Significantly, the combined effect of turning the benzofuran moiety into a benzoxazole or benzothiazole, and the use of electron deficient thiazole as acceptor, led to emission wavelengths above 600 nm in water for the resulting compounds (from 491 nm emission for the metal-bound form of the parent Mag-fura-2). We have employed a first-principle method to probe the excited states, namely, TD-DFT and we have accounted for solvation in a hybrid implicit/explicit approach. Though our model did not account for vibronic couplings, the optical properties of this small library of compounds was reproduced with good correlations with experimental values for the absorption wavelengths. This costeffective approach can therefore be used in future work to design new sensors. Our computational model provided insight into the nature of the electronic transitions that characterize these ratiometric sensors, and the effect of the metal binding on them. All compounds are characterized by ICT excited states with large CT distances in their free form. Metal coordination reduces the availability of the lone pair on the nitrogen atom of the fluorophores, causing a change to a locally excited state of π-π* character and a hypsochromic shift in the absorption and emission wavelengths. Our analysis revealed that the greatest impact on the optical properties is obtained with modifications on the acceptor groups, the more electron deficient azoles leading to greater charge transfer in the excited state and lower energy emission, as desired. In addition, the TD-DFT results indicate that the carboxylate group attached to the azole moiety is not required as the “pull” group in these pushpull dyes, but that the azoles are the effective acceptors. Modifications on the benzofuran moiety had a more modest impact on absorption but a more significant influence on the emission. Because of the different natures of the electronic transitions involved in each form, changes in the fluorophore structure that result in increased charge delocalization have a greater impact on the optical properties of the metal-free sensors and a smaller impact on those of the metal-bound species. This observation has some important implications on the design of wavelength-shifting compounds for ratiometric sensing and bioimaging applications. First, modifications that lead to stabilization of the ICT state with respect to the locally excited state will lead to greater separation of the absorption/emission wavelengths of the metal-free sensor with respect to the metal-bound form. Just like large Stokes shifts are desirable in general fluorescence applications, greater separation and reduced overlap between the absorption and/or emission bands of the metal-free and metal-bound species are

beneficial in ratiometric imaging of metals, as they facilitate the distinction of the two forms by fluorescence microscopy. Second, it becomes apparent that tuning the locally excited state is crucial for shifting the absorption wavelength of the metal-bound form (which displays the highest-energy absorption band in the system) and thus achieve excitation with lower energy sources desirable for live cell imaging. With a suitable computational model to guide our efforts, we are now exploring compounds in which the bezofuran moiety is replaced by more extended conjugated systems, seeking to develop enhanced tools for ratiometric detection of Mg2+ and other biologically relevant ions.

ASSOCIATED CONTENT Supporting Information Representation of density difference plots, list of CT distances and charges for all compounds, coordinates of optimized geometries, and characterization data for all new sensors. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (D.B.) *E-mail: [email protected] (D.J.)

ACKNOWLEDGMENT D.J. acknowledges the European Research Council (ERC) and the Région des Pays de la Loire for financial support in the framework of a Starting Grant (Marches-278845) and the LUMOMAT RFI project, respectively. This research used resources of (i) the GENCI-CINES/IDRIS, (ii) the CCIPL (Centre de Calcul Intensif des Pays de Loire), and (iii) the Troy cluster in Nantes. D.B. acknowledges the National Science Foundation for support of the research through grant No. CHE-1555116.

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(27) Gruskos, J. J.; Zhang, G.; Buccella, D. Visualizing Compartmentalized Cellular Mg2+ on Demand with SmallMolecule Fluorescent Sensors. J. Am. Chem. Soc. 2016, 138, 14639-14649. (28) Haugland, R. P. Handbook of Fluorescent Probes and Research Products; 9th ed.; Molecular Probes Inc.: Eugene, Oregon, 2002. (29) Peiyi, L.; Hualin, Z. Kinetics of the Complex Formation between Fe(III) and o-Hydroxybenzylamine-N,N,OTriacetic Acid (HBATA) and o-Aminophenyl-N,N,O-Triacetic Acid (APTA). Inorg. Chim. Acta 1990, 173, 255-259. (30) Brady, M.; Piombo, S. D.; Hu, C.; Buccella, D. Structural and Spectroscopic Insight into the Metal Binding Properties of the o-Aminophenol-N,N,O-Triacetic Acid (APTRA) Chelator: Implications for Design of Metal Indicators. Dalton Trans. 2016, 45, 12458-12464. (31) Gao, J.; Garner, D. S.; Jorgensen, W. L. Ab Initio Study of Structures and Binding Energies for Anion-Water Complexes. J. Am. Chem. Soc. 1986, 108, 4784-4790. (32) Michaux, C.; Wouters, J.; Perpète, E. A.; Jacquemin, D. Ab Initio Investigation of the Hydration of Deprotonated Amino Acids. J. Am. Soc. Mass Spectrom. 2009, 20, 632-638. (33) Liu, D.; Wyttenbach, T.; Carpenter, C. J.; Bowers, M. T. Investigation of Noncovalent Interactions in Deprotonated Peptides:   Structural and Energetic Competition between Aggregation and Hydration. J. Am. Chem. Soc. 2004, 126, 32613270. (34) Santoro, F.; Jacquemin, D. Going Beyond the Vertical Approximation with Time-Dependent Density Functional Theory. WIREs Comput. Mol. Sci. 2016, 6, 460-486. (35) Le Bahers, T.; Adamo, C.; Ciofini, I. A Qualitative Index of Spatial Extent in Charge-Transfer Excitations. J. Chem. Theory Comput. 2011, 7, 2498-2506. (36) Jacquemin, D.; Bahers, T. L.; Adamo, C.; Ciofini, I. What Is the "Best" Atomic Charge Model to Describe throughSpace Charge-Transfer Excitations? Phys. Chem. Chem. Phys. 2012, 14, 5383-5388. (37) Meuwis, K.; Boens, N.; Gallay, J.; Vincent, M. Photophysics of Mag-Fura-2: A Fluorescent Indicator for Intracellular Mg2+. Chem. Phys. Lett. 1998, 287, 412-420. (38) Van den Bergh, V.; Boens, N.; De Schryver, F. C.; Ameloot, M.; Steels, P.; Gallay, J.; Vincent, M.; Kowalczyk, A. Photophysics of the Fluorescent Ca2+ Indicator Fura-2. Biophys. J. 1995, 68, 1110-1119. (39) We estimated with DFT the enthalpy of the idealized complexation reaction,

[Mag-fura-2]4-4H2O + [Mg(H2O)6]2+ → {[Mag-fura-2]Mg(H2O)}2-4H2O + 5 H2O using the structures displayed in Figure 3 for the hydrated free and Mg2+-bound Mag-fura-2. This very rough approximation of the actual mechanisms leads to an enthalpy of -21.8 kcal/mol for the ground state and a significantly smaller enthalpy of -11.2 kcal/mol in the excited state. In addition, in B3 the exctited state enthalpy for the corresponding reaction is smaller (-7.4 kcal/mol) which is qualitatively consistent with the very small shift in the emission spectra, hinting at more efficient decomplexation in the excited state. (40) Meuwis, K.; Boens, N.; Gallay, J.; Vincent, M. Photophysics of Mag-Fura-2: A Fluorescent Indicator for Intracellular Mg2+. Chem. Phys. Lett. 1998, 287, 412-420. (41) Gilchrist, T. L. Heterocyclic Chemistry; 3rd ed.; Pearson Education, 2007.


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Tuning the Spectroscopic Properties of Ratiometric Fluorescent Metal

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