A Molecular Chameleon with Fluorescein and Rhodamine

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A Molecular Chameleon with Fluorescein and Rhodamine Spectroscopic Behaviors Ling Li,†,‡ Chunyan Wang,†,‡ Jianjian Wu,§ Yu Chung Tse,*,§ Yue-Peng Cai,⊥ and Keith Man-Chung Wong*,† †

Department of Chemistry and §Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of Science and Technology of China, No. 1088, Tangchang Boulevard, Nanshan District, Shenzhen 518055, P. R. China ⊥ School of Chemistry and Environment, South China Normal University, Guangzhou 510006, P. R. China S Supporting Information *

ABSTRACT: A new class of fluorescein/rhodamine hybrids with two spirolactone rings was reported to exhibit dual-output fluorescent behaviors independently. Isolation and characterization for two diastereomers, trans-RhOH and cis-RhOH, have been made and their X-ray crystal structures determined. In a basic environment, the spirolactone ring on the hydroxyl side will be opened to give a fluorescein-like optical output with the lowest absorptions at 485 and 530 nm emission. On the other hand, a rhodamine-like optical output, i.e., 528 nm absorption and 575 nm emission, will be switched on by a H+ or a Hg2+ ion, attributed to the spirolactone ring opening on the amino side. In a methanol−buffer system with different pH values, the corresponding pKa values for the hydroxyl and amino groups were determined as 5.7 and 2.3, respectively. Selective Hg2+-sensing properties have also been discussed, and log Ks values of about 3.60 and 3.73 were determined. Confocal microscopic images of Caenorhabditis elegans incubated with RhOH were found to show enhanced fluorescent intensity with a Hg2+ ion, demonstrating the potential application of RhOH for in vivo biological imaging.

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INTRODUCTION Rhodamine and fluorescein are both the most commonly used organic dyes, in particular of the development and application of molecular probes for biological imaging.1−4 Rhodol, also known as rhodaflour, is another kind of organic dye with the structural hybrid of rhodamine and fluorescein.1−3 The combination of the merits from rhodamine and fluorescein, including high water solubility, photostability, strong fluorescence, low excitation wavelength, and less sensitivity to pH changes, renders rhodol as another suitable candidate for fluorescent probing. On the other hand, rhodamine derivatives were found to exist in ring-opened and ring-closed forms with very distinguished colors and emission behaviors. The chemosensing studies of rhodamine derivatives, with high selectivity and sensitivity, have blossomed for over a decade for the detection of various analytes, such as transition-metal cations of Hg2+, Fe3+, Cu2+, Cr3+, and Au3+ as well as biologically related compounds of nucleic acid, hydrogen peroxide, glucose, peroxynitrite, nitric oxide, and hypochlorous acid.5,6 However, the use of the same concept on the related studies of fluorescein7 and rhodol3 is relatively less explored. The combination of a single molecule tethered with individual fluorescein and rhodamine by means of a linker or a spacer has been shown to exhibit fluorescent Hg2+ sensing via a FRET mechanism8a,b and a two-color fluorescent probe for intracellular pH.8c An interesting class of rhodamine derivatives with two spirolactam moieties from resorcinol (1,3-dihydroxylbenzene) © 2015 American Chemical Society

with two hydroxyl groups has recently been reported by us and others.9 Such a class of compounds have been demonstrated to show various spectroscopic behaviors arising from two successive ring-opening processes, and their use as chemodosimeters has been studied.9a Herein, we report a new class of dual-output fluorescent probes based on a fluorescein/rhodamine hybrid, RhOH (Scheme 1), with two spirolactone rings. The scaffold of this single molecule is from the fused structure of fluorescein and rhodamine subunits, and the spectroscopic outputs from the subunits would be independently switched on, Scheme 1. Synthesis of RhOHa

a

(i) 98% H2SO4, 100 °C, 3 h.

Received: September 17, 2015 Published: December 16, 2015 205

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Inorganic Chemistry

Table 1. Crystal and Structure Determination Data of cisRhOH and trans-RhOH

depending on the position of ring opening. Detailed spectroscopic responses in an acidic or a basic environment, selective Hg2+-sensing behaviors, and related in vivo biological imaging have also been studied.

cis-RhOH·2H2O

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empirical formula fw temperature, K wavelength, Å cryst syst space group a, Å b, Å c, Å α, deg β, deg γ, deg volume, Å3 Z density (calcd), g cm−3 cryst size, mm × mm × mm index ranges

RESULTS AND DISCUSSION Synthesis and Characterizations. According to our previous work on the rhodamine derivatives with two spirocyclic rings,9a the presence of two hydroxyl groups in fluorescein was anticipated to construct a linear array with three (X) or two (Y) spirocyclic rings (Scheme 1). Neither X nor Y could be obtained under various reaction conditions and stoichiometries. To our surprise, reaction of S1 and fluorescein afforded an interesting compound, RhOH, with the structural combination of fluorescein and rhodamine subunits. In order to analyze and estimate the reaction site of fluorescein for the electrophilic attack, natural bond orbital (NBO) analysis was performed at the density functional theory (DFT)//B3LYP/631G(d) level. Natural population analysis was used to determine the electron density of an individual atom on fluorescein. The electron density of the carbon atom at the 4′ (or 5′) position was found to be higher (Figure S1, upper) relative to that at the 2′ (or 7′) position, indicative of a higher susceptibility for electrophilic attack. On the other hand, the results of the same analysis on rhodol suggested that the electron density of the corresponding carbon atom is lower (Figure S1, bottom), and therefore the corresponding linear structure of a rhodamine derivative with two spirocyclic rings was obtained.9a Such a theoretical calculation is supportive of the experimental results in the synthesis of RhOH. In principle, there are altogether four isomers, including two diastereomers, trans-RhOH and cis-RhOH, and two enantiomers for each diasteromer. Only one pair of diastereomers has been isolated and characterized, while no attempt has been made to separate the enantiomers. The identities of trans-RhOH and cis-RhOH have been confirmed by 1H and 13C NMR (Figures S2−S5), as well as low-resolution and high-resolution mass spectrometry (MS). Their molecular structures have also been determined by Xray crystallography, and all of the experimental details are given in Table 1. The perspective drawing is shown in Figure 1, and the selected bond distances (Å) and angles (deg) are tabulated in Table 2. Both of them clearly reveal that they are in two ringclosed conformations. The main backbone of RhOH consists of five six-membered rings in an arrangement in which two xanthene motifs share the same benzene ring with a turning angle of 60°. Slightly distorted coplanarities are observed for such five six-membered rings with interplanar angles of 4.64 and 9.58° between two xanthene moieties in trans-RhOH and cis-RhOH, respectively. In trans-RhOH, the planes of two 3Hisobenzofuran-1-ones are pointing in opposite directions and are orthogonal to the xanthene plane with dihedral angles of 84.68−87.67°, while dihedral angles of 89.43−88.72° are observed in cis-RhOH with the two 3H-isobenzofuran-1-one planes aligned in the same direction. The C−C−O bond angles and C−O bond distances about the spirolactone ring are in the ranges of 101.0−102.2° and 1.494−1.513 Å, respectively, which are comparable to those of rhodamine derivatives. Despite the coplanarity along such five six-membered rings, no close π−π stacking was observed probably because of the presence of orthogonal 3H-isobenzofuran-1-one planes. Dual-Output Responses. It is worth noting that dual ouput of fluorescein-like or rhodamine-like optical properties

trans-RhOH·1.5C4H10O

C38H31NO9 645.64 298(2) 0.71073 orthorhombic P2(1)2(1)2(1) 8.3836(12) 18.751(3) 23.344(3) 90 90 90 3669.8(9) 4 1.169

C44H41NO8.50 719.78 113(2) 1.54187 triclinic P1̅ 8.2867(2) 9.9403(3) 23.3563(16) 89.016(6) 86.264(6) 73.891(5) 1844.44(15) 2 1.296

0.21 × 0.17 × 0.12

0.30 × 0.10 × 0.10

−10 ≤ h ≤ 9, −15 ≤ k ≤ 22, −27 ≤ l ≤ 28 reflns collected/ 19012/6627 [R(int) = unique 0.0721] completeness (2θ = 100.0 25.242°), % data/restraints/ 6627/2/431 param GOF on F2 1.089 final R indices [I > R1 = 0.0504, wR2 = 2σ(I)] 0.1273 largest diff peak 1.008 and −0.455 and hole, e Å−3

−8 ≤ h ≤ 9, −11 ≤ k ≤ 11, −28 ≤ l ≤ 27 22873/6518 [R(int) = 0.0992] 98.8 6518/3/502 1.068 R1 = 0.1426, wR2 = 0.3483 0.701 and −0.545

could be swtiched on independently in this single molecule of RhOH. Spectroscopic studies of RhOH were performed with various stimuli, i.e., in the presence of a H+, a OH−, or a Hg2+ ion. Figure 2 shows the electronic absorption and emission spectra in two distinguished outputs. Identical spectroscopic responses were observed for trans-RhOH and cis-RhOH, indicating that the ring-opening behaviors of two stereoisomers are almost the same. Under basic conditions, the solution color of RhOH in methanol (MeOH) was changed from colorless to yellow. Fluorescein-like optical properties were switched on, leading to the emergence of vibronic-structured absorption bands at 405, 431, 457, and 485 nm and an emission band at 530 nm. On the basis of the rich spectroscopic characteristics of fluorescein derivatives, such absorption and emission are attributed to the spirolactone ring opening as a result of deprontonation on the hydroxyl group of RhOH in a basic environment. Extended π conjugation over a xanthene moiety with a ketone group was then formed. On the other hand, the addition of a H+ or a Hg2+ ion would result in the absorption bands at 433, 464, 495, and 528 nm, which is responsible for the red color solution. At the same time, an emission at 575 nm, which resembles that of the rhodamine derivatives, was observed. Similar to other rhodamine derivatives, which will undergo a ring-opening process upon protonation or the addition of certain transition-metal cations, this rhodamine-like optical output is presumably due to the spirolactone ring opening to give another extended π conjugation over a xanthene moiety with an ammonium group. 206

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Figure 1. Perspective drawings of cis-RhOH (left) and trans-RhOH (right) with an atomic numbering scheme. Hydrogen atoms and solvent molecules are omitted for clarity. Thermal ellipsoids are drawn at the 25% probability level.

different wavelengths, which are characteristics of fluoresceinlike or rhodamine-like optical output depending on the nature of the inputs (Figures S6−S9). The observation of fluoresceinlike absorption and emission bands in relatively low intensity in MeOH in the absence of acid or base is ascribed to the presence of a small amount of fluorescein ring-opened species in such “neutral” conditions. For the formation of fluoresceinlike ring-opened species, about 10−15 equiv of OH− would be required to reach saturation for the optical signals, whereas 60 equiv of H+ are required for rhodamine-like species. The reversibility of such acid−base dual-output responses has been tested by electronic absorption spectroscopy. Within five alternating cycles upon the addition of p-toluenesulfonic acid and triethylamine into cis-RhOH (concentration = 10−5 M) in MeOH, a satisfactory reversible responsive output could be obtained (Figure S10). By monitoring of the fluorescein-like and rhodamine-like absorbances at 457 and 537 nm, respectively, in a MeOH−buffer solution (1:1, v/v) with different pH values (Figure 3), the corresponding pKa values of

Table 2. Selected Bond Lengths (Å) and Angles (deg) for cisRhOH and trans-RhOH with Estimated Standard Deviations Given in Parentheses cis-RhOH C8−O2 C7−C8 C8−C9 C8−C16 O5−C21 C20−C21 C21−C22 C21−C29 O2−C8−C7 O2−C8−C9 O2−C9−C16 C9−C8−C16 O5−C21−C20 O5−C21−C22 O5−C21−C29 C20−C21−C29

trans-RhOH Bond Lengths (Å) 1.511(6) C8−O2 1.514(6) C7−C8 1.499(6) C8−C9 1.507(6) C8−C16 1.513(5) O5−C21 1.515(5) C20−C21 1.475(6) C21−C22 1.504(6) C21−C29 Bond Angles (deg) 101.0(3) O2−C8−C7 109.2(4) O2−C8−C9 106.9(4) O2−C8−C16 110.5(3) C9−C8−C16 106.4(3) O5−C21−C20 101.2(3) O5−C21−C22 109.0(3) O5−C21−C29 109.7(3) C20−C21−C29

1.500(10) 1.515(11) 1.491(11) 1.517(11) 1.494(10) 1.516(11) 1.514(11) 1.503(11) 102.2(6) 107.9(7) 107.2(7) 111.7(7) 107.4(6) 102.0(6) 107.8(6) 110.0(7)

Figure 3. (Left) Electronic absorption spectra of trans-RhOH (50 μM) in different pH values and a MeOH−buffer solution (1:1, v/v). (Right) Plots of the pH values against log[(Amax − A)/(A − Amin)] at 457 and 537 nm. Figure 2. Schematic diagram (top) and electronic absorption and emission spectra (bottom) for the dual outputs of cis-RhOH with different inputs in MeOH. Concentration = 3 × 10−5 M.

RhOH for the hydroxyl and amino groups were determined as 5.7 and 2.3. The emission spectra of trans-RhOH (50 μM) in different pH values in a MeOH−Buffer solution are depicted in Figure S11. The fluorescein-like emission is relatively weak at high pH value while the rhodamine-like emission is higher at low pH value, suggesting that the emission quantum yield of rhodamine-like species is higher than that of fluorescein-like species. Such pKa values are comparable to those of rhodol green (pKa = 5.6) and rhodamine derivatives (pKa = 3.1).10

Electronic absorption and emission titration studies of RhOH in MeOH (in both the trans and cis forms) with various concentrations of base or acid were investigated. In general, the profiles of the spectroscopic changes showed a gradual emergence of new absorption and emission bands at 207

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opening process with a Hg2+ ion to give rhodamine-like spectroscopic behavior. The log Ks values for Hg2+ ion binding were found to be 3.60 (±0.12) and 3.73 (±0.02) by electronic absorption and emission spectroscopies, respectively. The similar binding constant values are indicative of the same origin for the new absorption and emission bands arising from the formation of rhodamine-like ring-opened species. Detection limits in the range of (2.1−5.7) × 10−5 M were also estimated from the electronic absorption and emission titration results. Compared to the related rhodamine derivatives with spirolactam rings, the higher detection limits in RhOH are attributed to the lack of an additional binding site to coordinate a Hg2+ ion. The close resemblance of the experimental data to the theoretical fit suggested the binding mode of 1:1 stoichiometry (Figure 5, insets). This 1:1 complexation mode has been further confirmed by the method of continuous variation showing a break point at a [RhOH]/([RhOH] + [Hg2+]) mole fraction of 0.5 (Figure S13) and the observation of a peak of m/z 910 for the adduct of [RhOH + Hg2+ + ClO4−]+ in the MS spectrum (Figure S14). The very similar IR spectra of RhOH and RhOH + Hg2+ ion (Figure S15) indicate the absence of the C···O bond involved in the binding process. The proposed binding mode of RhOH with a Hg2+ ion is depicted in Scheme 2.

Carboxyseminaphthorhodafluor-1 (carboxy-SNARF-1) is a related compound with a structural combination of fluorescein and rhodamine containing only one spirocyclic ring,4b which has been widely used as a intracelluar pH indicator by using flow cytometry, microplate readers, confocal imaging, or microspectrofluorometry. However, the relatively high pKa (ca. 7.5) value limits application in an acidic or a cytosolic environment. The occurrence of two pKa values for RhOH arising from independent ring-opening processes leading to distinguished fluorescein-like or rhodamine-like spectroscopic properties provides a wider working pH range and is beneficial for the development of an intracelluar pH indicator. Selective Hg2+ Ion Sensing. In view of the spectral changes from the effect of a Hg2+ ion, the potential use of RhOH as a molecular probe was explored. Selective sensing properties of RhOH were investigated for a series of metal cations. A solution of trans-RhOH only with a Hg2+ ion could exhibit a drastic color change from colorless to red (Figure 4)

Scheme 2. Proposed Binding Mode of RhOH with a Hg2+ Ion Figure 4. Selectivity study of trans-RhOH (concentration = 5 × 10−5 M) in MeOH upon the addition of various metal ions (18 equiv). Inset: Corresponding color change of trans-RhOH (concentration = 1 × 10−5 M) with various metal ions (100 equiv).

or the emergence of new absorption bands. Upon the addition of a Hg2+ ion, RhOH was found to give electronic absorption and emission spectral changes similar to those of H+ (Figure 5 It is anticipated that the third optical output could be achievied for the formation of two ring-opened species, in which there is an extension of the π conjugation across the fused structure of five rings. An attempt has been made to open two spirolactone rings of RhOH on both the hydroxyl and amino sides, and a solution of a saturated fluoresein-like opened form was first prepared by a OH− ion (or Et3N). However, the subsequent addition of a Hg2+ ion would ring-close the fluoresein-like opened form and eventually only the rhodaminelike opened form could be obtained (Figure 6). In the reversed sequence of addition, i.e., a Hg2+ ion first and then a OH− ion, a

Figure 5. Absorption (left) and emission (right) spectral changes of cis-RhOH (concentration = 3 × 10−5 M) upon the addition of Hg2+ (concentration = 4 × 10−2 M) in MeOH. Insets: Plots of the absorbance at 495 nm or emission intensity at 575 nm as a function of the concentration of Hg2+ with a theoretical fit.

for cis-RhOH; Figure S12 for trans-RhOH). As in the acid titration study, 530 nm emission was observed before the addition of any Hg2+ ion because of the presence of a small amount of fluorescein ring-opened species. According to the spectroscopic response in an acidic environment, the spirolactone ring-opening process on the amino side, leading to the formation of extended π conjugation over a xanthene moiety with an ammounium group, is responsible for such spectral changes with a Hg2+ ion. Although there are two spirolactone rings in RhOH, it is worth noting that only the spirolactone ring near the amino group would undergo a ring-

Figure 6. Electronic absorption spectral changes of cis-RhOH (concentration = 3 × 10−5 M) in MeOH upon the sequence of the addition of OH− and then a Hg2+ ion. 208

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Inorganic Chemistry similar open−close−open process was observed to give the fluoresein-like opened form at the end. The clear solution was retained without observation of the HgO precipitate in the aforementioned experiments. The ring-opening processes for the formation of fluorescein-like and rhodamine-like species are mutally exclusive, presumably because of the opposite charge required for π conjugation of resonance structures. Although the species with two opened rings could not be obtained, we designed and synthesized a derivative of control, in order to verify the spectroscopic behaviors of the fluoresein-like and rhodamine-like opened forms as well as the two ring-opened species. A mimic to the molecular structure of fluorescein-like ring-opened species, control consists of an xanthene moiety with a ketone group and only one spirolactone ring on the amino side. The electronic absorption and emission spectra of control and its corresponding opened form, control + Hg2+ ion, are depicted in Figure 7. The absorption and emission

Figure 8. Energy-level diagrams of molecular orbitals for four compounds at B3LYP/6-31G(d).

Caenorhabditis elegans was incubated with cis-RhOH to investigate whether RhOH can be taken up by nematodes, and fluorescent signals are shown in the body. C. elegans is a well-known biological model organism because it is transparent and therefore ideal for fluorescent studies. Since RhOH was found to be soluble and C. elegans was alive for 6 h of incubation, 5% ethanol in double-distilled water was used as the buffer system for RhOH studies. Control experiments were also performed by incubating the worms in buffer only or supplemented with a Hg2+ ion for 6 and 4 h, respectively. No observable fluorescent signals (within 580−653 nm) were detected by using spinning-disk confocal microscopy (Figure 9, panels A and B; Figure 10), indicating that buffer and Hg2+ treatment were not able to increase the background signal in the worm body. On the other hand, weak fluorescent patches were observed in the body of worms after treatment with cisRhOH for 6 h (Figure 9, panel C, Figure 10). To further investigate the cis-RhOH-labeled structures, 60× objective lenses were used for high-resolution fluorescent imaging and differential interference contrast (DIC) imaging. As shown in Figure 9, panel G, the cis-RhOH-labeled structures were superimposed with the intestine granules. Strikingly, the intensity of these RhOH-labeled intestine granules was greatly increased after exposure of C. elegans to 50 μM Hg2+ ion for 4 h (Figure 9, panels D and H). According to quantitative analysis, the integrated intensity of cis-RhOH in C. elegans treated with a Hg2+ ion was found to increase about 9-fold (Figure 10). In addition, the tissue in the buccal cavity was clearly stained by cis-RhOH in Hg2+ treatment (Figure 9, panel I). This suggested that RhOH was taken up by worms through the buccal cavity, pharynx, then entered the gut, and eventually accumulated in the intestine granules. In view of the fact that cis-RhOH is biofriendly and it can be used to detect the accumulation of Hg2+ ions in the body of C. elegans, this probe is anticipated to be used for biological studies in a more advanced level. Fluorescence recovery after photobleaching (FRAP) is a powerful optical technique to characterize the kinetics of diffusion of cellular molecules within living cells. Typically, the nonprotein cellular molecules are made fluorescent by labeling with fluorescent dyes, while proteins are fused with fluorescent proteins.11 In FRAP, the fluorophore is irreversibly photobleached by a high-power laser illumination in the region of interest (ROI). After that, the

Figure 7. Electronic absorption and emission spectra of control and its corresponding opened form, control + Hg2+ ion, in MeOH.

bands of control are at energies similar to those of the fluoresein-like opened form of RhOH. The spirolactone ring could be opened by the addition of a Hg2+ ion or an acid to give the opened form of control. This opened control species possesses an extended π conjugation across the fused structure of two xanthene groups, similar to that of the two ring-opened species of RhOH. The absorption and emission were found to shift to the red, and the energies are lower than those of the fluorescein-like and rhodamine-like opened forms of RhOH. This could validate and confirm that only one spirolactone ring was opened upon the addition of a Hg2+ ion, although one could expect that both spirolactone rings could probably be opened. On the basis of the X-ray crystallographic data of cis-RhOH, the full geometry optimizations of four compounds (twoclosed, −OH-opened, −NEt2-opened, and two-opened) were calculated with the B3LYP hybrid functional of DFT (Figure S16). From the energy-level diagrams of their molecular orbitals (Figure 8), the band-gap values for π−π* transition were found to decrease from the −OH opened form to the −NEt2 opened form and would be further reduced in the twoopened form. Time-dependent DFT (TD-DFT)//B3LYP/631G(d) was also employed to obtain the nature and excitation energies of the four compounds. The wavelength, oscillator strength, and major contributions of the calculated transition were tabulated in Table 3. The excitation energies from this TD-DFT result give the same energy trend as the band-gap values. These calculation data are in agreement with the experimental findings in the spectroscopic properties of the fluoresein-like opened form, rhodamine-like opened form, and opened form of control. Biological Imaging. In order to test the feasibility of RhOH to act as a mercury sensor in living organisms, adult 209

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Inorganic Chemistry Table 3. Electronic Transition Data Obtained by TD-DFT for Four Compounds electron transition

main transition configuration (CI coefficient)

excitation energy (eV)

calculated wavelength (nm)

oscillator strength f

cis two-closed

S0−S15

4.70

264.06

0.1704

cis −OH-opened

S0−S3

2.92

425.07

0.3017

cis NR2-opened

S0−S3

2.81

440.61

0.4978

cis two-opened

S0−S4

HOMO−2 → LUMO+3 (0.47) HOMO → LUMO or LUMO+5 (0.22) HOMO → LUMO (0.49) HOMO−4 → LUMO (0.14) HOMO−2 → LUMO (0.66) HOMO−6 → LUMO (0.16) HOMO−2 → LUMO (0.65) HOMO−3 → LUMO (0.21)

2.72

456.42

0.3862

compound

Figure 11. Kinetics of cis-RhOH fluorescent signal after photobleaching. Worms were treated with cis-RhOH and Hg2+, and images were collected by a 63× objective lens. The ROIs (blue box) were irradiated by a high-power laser to photobleach the cis-RhOH-labeled intestine granule (left) and tissue in the buccal cavity (right).

Figure 9. Fluorescence confocal images (signal) and DIC images of C. elegans treated with cis-RhOH and a Hg2+ ion. Worms were treated with cis-RhOH (150 μM) and a Hg2+ ion (50 μM) as indicated. Images were collected at 580−653 nm by using a 10× objective lens (A−D) and a 63× objective lens (E−I).

intensity in the ROI was found to drop by about 30% after a short pulse of a high-power laser and then recover to around 80% of the original level within 15 s (Figure 11, right, insets). This suggested that some of the unbleached RhOH molecules outside the ROI were able to recover the fluorescent intensity by diffusion.

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CONCLUSIONS In conclusion, the present work represents the first report on the synthesis and isolation of a novel dual-output fluorescent sensor/probe, RhOH, based on a structural combination of fluorescein and rhodamine with two spirolactone rings. Various stimuli, such as a H+, a OH−, or a Hg2+ ion, could selectively open a particular spirolactone ring, leading to the fluoresceinlike or rhodamine-like optical output. RhOH was found to exhibit selective Hg2+-sensing behaviors. Confocal microscopic images of C. elegans incubated with RhOH showed enhanced fluorescent intensity with a Hg2+ ion, demonstrating the potential application of RhOH in biological imaging. The pKa values of 5.7 and 2.3, arising from the independent ringopening processes, provide wider working pH ranges, which will be beneficial for the development of an intracelluar pH indicator. The successful isolation of this class of complexes should open up new avenues for the design and development of molecular logic gates and fluorescent probes for biological studies. Modification of the spirolactone rings into other functionalities in order to alter the selectivity and sensitivity is in progress.

Figure 10. Integrated intensity of C. elegans treated with cis-RhOH. Worms were treated with cis-RhOH and Hg2+, and images were collected by a 10× objective lens. The average number of pixels with intensity over 3000 in a worm body was counted; n = number of worms investigated.

intensity of fluorescence recovery over time within the ROI is recorded, and then the dynamic behavior of the fluorescent reporter is calculated by mathematical analysis. In order to investigate the ability of RhOH for FRAP, we have applied a high-energy laser to the RhOH-labeled intestine granules and tissue in the buccal cavity. The diffusion behavior of RhOH was also examined by measuring the fluorescent recovery over time after photobleaching within a region of the structure (Figure 11). As shown in Figure 11, left, the fluorescence intensity of RhOH-labeled intestine granules was completely lost after photobleaching, and the whole granules did not show any fluorescent recovery over time. This indicated that RhOH will be photobleached irreversibly. Because the whole structure of intestine granules was bleached, free molecules of RhOH were not available to recover the intensity by diffusion. The fluorescent signal of a portion of the RhOH-labeled tissue in the buccal cavity, as shown in Figure 11, right, insets, was erased followed by time-resolved image recording. The RhOH

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EXPERIMENTAL SECTION

Materials and Reagents. Resorcinol (99%) and benzaldehyde (98%) were purchased from Energy Chemical, and fluorescein (99%, Shanghai Chemical Reagent Co., Ltd.), 3-(diethylamino)phenol (97%, Sigma-Aldrich Chemical Co.), phthalic anhydride (Tianjin Fuchen Chemial Reagents Factory), 2-methylresorcinol (98%, J&K Scientific Ltd.), and other materials for the synthesis were used without further 210

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Inorganic Chemistry

153.47, 151.98, 151.86, 150.91, 150.32, 136.28, 135.86, 130.77, 130.62, 130.12, 129.26, 129.23, 127.90, 127.12, 125.51, 124.93, 124.83, 123.98, 113.97, 113.73, 113.69, 109.83, 109.61, 106.74, 105.22, 102.74, 97.76, 83.26, 78.48, 45.04, 12.73. HRMS (ESI). Calcd for C38H28NO7 ([M + H]+): m/z 609.1788. Found: m/z 610.1871. Characterization Data of cis-RhOH. 1H NMR (400 MHz, CD3OD:CDCl3 = 1:1): δ 8.31−8.26 (m, 1H), 8.11−8.07 (m, 1H), 7.86−7.77 (m, 4H), 7.33−7.27 (m, 1H), 7.26−7.21 (m, 1H), 7.12 (d, J = 8.9 Hz, 1H), 6.92 (d, J = 8.9 Hz, 1H), 6.69 (d, J = 9.6 Hz, 1H), 6.55 (dd, J = 4.0 and 2.0 Hz, 4H), 6.01−5.97 (m, 1H), 3.49 (q, J = 7.0 Hz, 4H), 1.30−1.26 (t, J = 7.0 Hz, 6H). 13C NMR (100 MHz, CD3OD:CDCl3 = 1:1): δ 172.46, 170.72, 155.68, 153.80, 152.18, 151.85, 151.18, 150.30, 136.10, 135.82, 130.81, 130.57, 130.14, 129.27, 129.18, 127.92, 127.53, 125.58, 125.08, 124.80, 123.78, 114.19, 113.70, 109.82, 106.86, 105.25, 102.70, 97.76, 83.24, 78.62, 78.30, 77.97, 66.53, 45.05, 12.73. HRMS (ESI). Calcd for C38H28NO7 ([M + H]+): m/z 609.1788. Found: m/z 610.1872. Synthesis of control. A mixture of benzaldehyde (1.06 g, 10 mmol) and resorcinol (2.48 g, 20 mmol) in MeSO3H (50 mL) was heated at 70 °C for 24 h. After that, a dark solution was produced. The reaction mixture was cooled to room temperature and poured into 400 mL of a 3 M NaOAc solution. The resulting dark-red solid was collected by filtration to provide the crude product. The product was purified by column chromatography on silica gel with dichloromethane/MeOH (40:1, v/v) to afford the intermediate 6-hydroxy-9-phenyl-3H-xanthen3-one (0.89 g, 31%). To a mixture of S1 (0.25 g, 0.8 mmol) and 6-hydroxy-9-phenyl-3Hxanthen-3-one (0.23 g, 0.8 mmol) was added concentrated sulfuric acid (6 mL) dropwise at 0 °C. The resulting suspension was heated at 100 °C for 3 h. After the mixture was cooled to room temperature and poured into ice water (20 mL) with vigorous stirring, the pH of the mixture was adjusted to ∼7. The mixture was extracted with dichloromethane (20 mL) three times. The organic layers were dried over anhydrous magnesium sulfate and evaporated to give the crude product. Purification and separation of the product were achieved by silica column chromatography, eluting with dichloromethane and MeOH (100:1) to give the pure form of control (0.11 g, 24%). Characterization Data of control. 1H NMR (400 MHz, CDCl3): δ 7.87 (d, J = 7.6 Hz, 1H), 7.66 (td, J = 7.5 and 1.2 Hz, 1H), 7.61−7.37 (m, 1H), 7.48−7.37 (m, 2H), 7.30 (s, 1H), 7.27−7.20 (m, 2H), 7.17 (d, J = 7.6 Hz, 1H), 7.12 (d, J = 9.8 Hz, 1H), 6.93 (d, J = 7.7 Hz, 1H), 6.64−6.52 (m, 4H), 6.47 (d, J = 1.9 Hz, 1H), 6.41 (dd, J = 8.9 and 2.6 Hz, 1H), 3.40 (q, J = 7.1 Hz, 4H), 1.21 (t, J = 7.0 Hz, 6H). HRMS (ESI). Calcd for C37H28NO5 ([M + H]+): m/z 565.1889. Found: m/z 566.1969. X-ray Crystallography. Single-crystal X-ray diffraction analysis of cis-RhOH was performed on a Bruker APEX-II CCD diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) at room temperature. All absorption corrections were performed using a multiscan. The structure was solved by direct methods and refined by full-matrix least squares on F2 with the SHELXTL-97 program package.13 The intensity data of trans-RhOH were collected on a Rigaku Saturn944+ CCD diffractometer with graphite-monochromated Cu Kα radiation (λ = 1.54178 Å) at 113 K. All absorption corrections were performed using a multiscan. The structure was solved by direct methods and refined by full-matrix least squares on F2 with the SHELXTL-2014 program package.13,14 CCDC 1407618 (cisRhOH) and 1407619 (trans-RhOH) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www. ccdc.cam.ac.uk/data_request/cif. Buffers in Different pH Values. Different buffer solutions were prepared by using 50 mM potassium hydrogen phthalate (for the pH 1−5 buffer), 25 mM potassium dihydrogen phosphate (for the pH 6− 8 buffer), 10 mM sodium tetraborate (for the pH 9−10 buffer), and 50 mM sodium bicarbonate (for the pH 11−14 buffer). The pH was adjusted by adding 0.1 M NaOH or 0.1 M HCl solutions.

purification. All of the chemicals used for the synthesis were of analytical grade. Methanol (MeOH) for analysis was of spectroscopy grade. Copper(II) perchlorate, sodium(I) perchlorate, lead(II) perchlorate trihydrate, cadmium(II) perchlorate hexahydrate, lithium(I) perchlorate, magnesium(II) perchlorate, cobalt(II) perchlorate, and iron(II) perchlorate hydrate were purchased from Alfa Aesar with RG grade, and zinc(II) perchlorate hexahydrate (RG, Aladdin Chemical Co., Ltd.), barium(II) perchlorate (RG, Sigma-Aldrich Chemical Co.), silver(I) perchlorate (AR, Energy Chemical), nickel(II) perchlorate hexahydrate, calcium(II) perchlorate tetrahydrate, and mercury(II) perchlorate trihydrate were purchased from Strem Chemicals, Inc., with over 99.0% purity. Safety Precaution! Mercury(II) salt is hazard to health. Perchlorate salts of metal ion are potentially explosive. Both of them should be handled with care. Instruments. The UV−vis absorption spectra were taken on Cary 60 UV−vis spectrophotometer. Steady-state emission spectra at room temperature were recorded on an Edinburgh Instruments FLS980 fluorescence spectrometer. Quartz cuvettes (path length = 1 cm) were used in all spectrophotometric and fluorometric measurements. NMR spectra were recorded on a Bruker AVANCE 400 (1H NMR for 400 MHz and 13C NMR for 100 MHz) Fourier transform NMR spectrometer with chemical shifts reported relative to tetramethylsilane, (CH3)4Si. High-resolution MS spectra were performed on an Orbitrap Fusion Tribrid mass spectrometer. The UV−vis absorption spectra were obtained by utilizing a Cary 60 UV−vis spectrophotometer. Ion-Binding Studies. Binding constants for 1:1 complexation were determined by nonlinear least-squares fits to eq 1, in which the derivations were described previously.12

X = X0 +

Xlim − X 0 {[M]T + [Hg 2 +] + 1/K s 2[M]T

− [([M]T + [Hg 2 +] + 1/K s)2 − 4[M]T [Hg 2 +]]1/2 }

(1)

where X0 and X are the absorbance (or luminescence intensity) of RhOH at a selected wavelength in the absence and presence of the Hg2+ ion, respectively, [M]T is the total concentration of RhOH, [Hg2+] is the concentration of the Hg2+ ion, Xlim is the limiting value of the absorbance (or luminescence intensity) in the presence of excess HgII ion, and Ks is the stability constant. Synthesis. Synthesis of cis-RhOH and trans-RhOH. To a solution of (3-diethylamino)phenol (2.0 g, 12 mmol) in toluene (60 mL) was added phthalic anhydride (1.8 g, 12 mmol). The suspension was heated to reflux for 6 h. The resulting residue was filtered and washed with MeOH to provide the intermediate S1 (2.1 g, 55%) for the next step without further purification. To a mixture of S1 (0.313 g, 1.0 mmol) and fluorescein (0.332 g, 1.0 mmol) was added concentrated sulfuric acid (5 mL) dropwise at 0 °C. The resulting suspension was heated at 100 °C for 3 h. After the mixture was cooled to room temperature and poured into ice water (30 mL) with vigorous stirring, the pH of the mixture was adjusted to ∼7. The mixture was extracted with dichloromethane (30 mL) three times. The organic layers were dried over anhydrous magnesium sulfate and evaporated to give the crude product of a mixture of cisRhOH and trans-RhOH (0.37 g, 61%). Purification and separation of the stereoisomer were achieved by silica column chromatography, eluting with dichloromethane and MeOH (100:1) to give the pure form of cis-RhOH (0.10 g, 27%) and trans-RhOH (0.11 g, 29.7%) as a white solid in a ratio of about 1:1. Subsequent recrystallization of the respective compounds by diffusion of diethyl ether vapor into a solution of the product in dichloromethane afforded cis-RhOH and trans-RhOH as single crystals, respectively. Characterization Data of trans-RhOH. 1H NMR (400 MHz, CD3OD:CDCl3 = 1:1): δ 8.22−8.13 (m, 1H), 8.00 (dt, J = 7.6 and 1.0 Hz, 1H), 7.76 (td, J = 7.5 and 1.2 Hz, 1H), 7.72−7.65 (m, 3H), 7.25− 7.18 (m, 2H), 7.01 (d, J = 8.9 Hz, 1H), 6.84 (d, J = 8.9 Hz, 1H), 6.61 (d, J = 9.6 Hz, 1H), 6.54−6.42 (m, 4H), 6.10 (d, J = 2.0 Hz, 1H), 3.39 (q, J = 7.0 Hz, 4H), 1.19 (t, J = 7.0 Hz, 6H). 13C NMR (100 MHz, CD3OD:CDCl3 = 1:1): δ 172.54, 170.81, 160.23, 155.58, 153.80, 211

DOI: 10.1021/acs.inorgchem.5b02147 Inorg. Chem. 2016, 55, 205−213

Article

Inorganic Chemistry

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METHODOLOGY FOR BIOLOGICAL IMAGING C. elegans Strain. C. elegans N2 strains were maintained on nematode growth medium (NGM) plates using standard procedures. This strain is provided by the Caenorhabditis Genetics Center, which is funded by the National Center for Research Resources, National Institutes of Health. Worm Treatment, Imaging, and FRAP. N2 worms were transferred from the NGM plate to a 150 μL solution (buffer, 5% ethanol in double-distilled water; Hg2+ solution, 50 μM Hg2+ in buffer; cis-RhOH solution, 150 μM cis-RhOH in a buffer) in a 1.5 mL centrifuge tube and incubated at 22 °C. Worms were incubated for 4 h in a Hg2+ solution and 6 h for a buffer and a cis-RhOH solution. After that, worms were mounted on 3% agarose pads and sealed with Vaseline. For confocal imaging, worms were imaged with Nikon Eclipse Ti (Nikon, Japan) with 10× and 60× objective lenses, equipped with a Yokogawa CSU-X1 spinning-disk unit (Nikon, Japan), and illuminated with 50 mW 488 nm lasers (Andor, U.K.). Images were captured on an iXon Ultra camera (Andor, Belfast, U.K.) controlled by MetaMorph (Molecular Devices, Sunnyvale, CA). For spectrum scanning, worms were imaged with Leica TCS SP8 (Leica, Germany) with 63× objective lens every 5 nm from 500 to 705 nm. Image processing was performed with ImageJ. For FRAP experiments, worms were treated and imaged in the same conditions as the confocal imaging experiments. The pulses of a high-energy laser were generated by a Micropoint unit (Andor, U.K.) attached to the Nikon Eclipse Ti (Nikon, Japan) system.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS K.M.-C.W. acknowledges receipt of the “Young Thousand Talents Program” award and the start-up fund administrated by South University of Science and Technology of China. This project was also supported by the National Scientific Foundation of China (NSFC; Grant 21471074). Y.C.T. and J.W. thank the support National Scientific Foundation of China (grant no. 31301114) and the Shenzhen Key Laboratory of Cell Microenvironment (grant no. ZDSYS20140509142721429). We thank X-ray service open platform of Peking University Shenzhen Graduate School for structural determination of trans-RhOH.

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Calculations were performed with DFT and TD-DFT using the Gaussian 09 program.15 Full geometry optimizations of systems in the gas phase were calculated at hybrid DFT levels by the B3LYP function16 with a 6-31G(d) basis set.17 The simulation of the frequency based on the fully optimized geometries implied that optimized geometry was located at the lowest point of the potential energy surface. Meanwhile, charge distribution of the reactant fluorescein was performed using NBO analysis. The electronic properties, such as the HOMO−LUMO band gap, absorption wavelengths, and oscillator strengths, as well as excitation energies, are calculated using the TD-DFT method (B3LYP function) with a 631G(d) basis set. In fact, the TD-DFT method has been proven to be appropriate for investigation of the vertical transition energy of organic materials in some papers.18,19 S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.5b02147. 1 H and 13C NMR spectra, titration spectra, and the results of theoretical calculation (PDF) X-ray crystallographic data of cis-RhOH in CIF format (CIF) X-ray crystallographic data of trans-RhOH in CIF format (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ‡

L.L. and C.W. contributed equally to this work. 212

DOI: 10.1021/acs.inorgchem.5b02147 Inorg. Chem. 2016, 55, 205−213

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DOI: 10.1021/acs.inorgchem.5b02147 Inorg. Chem. 2016, 55, 205−213