A New Rhodamine-based Chemosensor Exhibiting Selective

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1 Supporting information for A New Rhodamine-based Chemosensor Exhibiting Selective Fe III -amplified Fluorescence Yu Xiang and Aijun Tong* Department of Chemistry, Tsinghua University, Beijing , P.R. China tongaj@mail.tsinghua.edu.cn Materials and Methods Absolute ethanol and deionized water were used as solvents. All the materials for synthesis were purchased from commercial suppliers and used without further purification. The solutions of metal ions were prepared from their nitrate salts, except for FeCl 2, CrCl 3 and MnCl 2. The use of Fe(NO 3 ) 3 and FeCl 3 yielded nearly the same results. Tris-HCl buffer solutions (ph = 7.15) were prepared using 0.01 M Tris, 0.1 M KNO 3 and proper amount of HCl (about 0.01 M). Absorption spectra were determined on a JASCO V-550 UV-VIS spectrophotometer. Fluorescence spectra measurements were performed on a JASCO FP-6500 spectrofluorimeter equipped with a xenon discharge lamp using 1 cm quartz cells. All ph measurements were made with a Model phs-3c ph meter (Shanghai, China). NMR spectra were recorded using a JOEL JNM-ECA300 spectrometer operated at 300MHz (TMS as internal standard). MS spectra were obtained on a HP 1100 LC-MS spectrometer. Element analysis was carried out on a FLASH EA1112 elemental analyzer. Synthesis of rhodamine-based compound 3: Rhodamine B acid chloride was synthesized from rhodamine B (1) and phosphorus oxychloride according the reported method 1. Then the crude acid chloride (2.3 mmol) was dissolved in 60 ml acetonitrile and added dropwise to a stirred solution of 5 ml diethylenetriamine (2) in 20 ml acetonitrile over 0.5~1 h in an ice bath. After the addition was completed, the reaction mixture was stirred at room temperature overnight. Solvent was removed under reduced pressure, and the residue was washed with water (20 ml 4). Then the resulting purple solid was dried and purified by column

2 chromatography (CH 2 Cl 2 : CH 3 OH = 10: 1, v/v). The first pink band was collected and dried to afford 198 mg 3 as light orange solid (yield: 18%). 1 H-NMR (CDCl 3 ) δ (ppm): 7.83 (2H), 7.40 (4H), 7.05 (2H), 6.39 (d, J = 8.4 Hz, 4H), 6.33 (4H), 6.24 (d, J = 8.4Hz, 4H), 3.31 (m, J = 6.9 Hz, 16H), 3.06 (4H), 2.19 (4H), 1.13(t, J = 6.9 Hz, 24H); 13 C-NMR (CDCl 3 ) δ (ppm): 167.3, 153.8, 153.3, 148.8, 132.4, 131.6, 128.8, 127.9, 123.8, 122.8, 108.2, 105.9, 97.8, 65.0, 47.6, 44.4, 40.3, 12.7; MS (ESI): found: m/z = (M+H) +, calcd for C 60 H 70 N 7 O 4 = Element analysis (%): found: C 74.90, H 7.21, N 9.82, calcd: C 75.68, H 7.30, N 10.03, O S-Scheme 1. Synthesis of 3.

3 S-Figure 1. Evaluation of the fluorescence intensity (at 575 nm) of 3 (10 um) in aqueous buffers of different ph. The buffers were prepared using 0.01 M Tris with different amount of HCl. The same result was also obtained using phosphate buffers. Excitation was at 510 nm. S-Figure 2. Upper photograph: 10 um 3 in ethanol in the presence of different metal ions (20 equiv); lower photograph: 10 um 3 in Tris-HCl buffer (ph = 7.15) in the presence of different metal ions (50 equiv). Since the excitation was performed by a 250nm/365nm UV light source, a mild background of purple color existed. However, only the solution of 3 with Fe III showed an obvious orange fluorescence.

4 S-Figure 3. Absorption spectra of 3 (10 um) upon the addition of different metal ions (50 equiv) in Tris-HCl buffer. S-Figure 4a. Fluorescence titration of Fe III (0 ~ 20 equiv) to 3 (30 um) in ethanol. Inset: fluorescence intensity at 573 nm as a function of the amount of Fe III added. Excitation was at 510 nm. S-Figure 4b. Fluorescence titration of Fe III (0 ~ 80 equiv) to 3 (30 um) in Tris-HCl buffer (ph = 7.15). Inset: fluorescence intensity at 575 nm as a function of the amount of Fe III added. Excitation was at 510 nm.

5 S-Figure 4c. Fluorescence titration of Cr III (bottom to top: 0, 20, 50, 100, 200 equiv) to 3 (30 um) in ethanol. Excitation was at 510 nm. S-Figure 4d. Fluorescence titration of Fe II (bottom to top: 0, 20, 50, 100, 200 equiv) to 3 (30 um) in ethanol. Excitation was at 510 nm.

6 S-Figure 4e. Fluorescence titration of Cu II (bottom to top: 0, 20, 50, 100, 200 equiv) to 3 (30 um) in ethanol. Excitation was at 510 nm. S-Figure 5. Changes in the fluorescence intensity of 3 at 573 nm in ethanol upon the titration of Fe III (purple), Fe II (blue), Cr III (red) and Cu II (black). Excitation was at 510 nm.

7 S-Figure 6a. Fluorescence excitation and emission spectra of 3 (30 um) with Fe III in ethanol. From bottom to top: 0, 1, 3, 5, 8, 10 equiv Fe III. (blue: excitation spectra, emission at 573 nm; red: emission spectra, excited at 557 nm). S-Figure 6b. Fluorescence excitation and emission spectra of 3 (30 um) with Fe III in Tris-HCl buffer (ph = 7.15). From bottom to top: 0, 20, 40, 50, 60 equiv Fe III. (blue: excitation spectra, emission at 576 nm; red: emission spectra, excited at 561 nm).

8 References (1) Kwon, J. Y.; Jang, Y. J.; Lee, Y. J.; Kim, K. M.; Seo, M. S.; Nam, W.; Yoon, J. J. Am. Chem. Soc. 2005, 127,

9 Review Only Following Pages: track changes version of the revised manuscript

10 A New Rhodamine-based Chemosensor Exhibiting Selective Fe III -amplified Fluorescence Yu Xiang and Aijun Tong* Department of Chemistry, Tsinghua University, Beijing , P. R. China Received Date (will be automatically inserted after manuscript is accepted) ABSTRACT A new fluorescent probe 3 was synthesized and it exhibited high selectivity for Fe III over other commonly coexistent metal ions in both ethanol and water. Upon the addition of Fe III, the spirocyclic ring of 3 was opened and a significant enhancement of visible color and fluorescence in the range of 500~600 nm was observed. During the recent two decades, there is a great emergence of interest in the development of fluorescent probes for various cations and anions. 1 Due to their importance in many biological and environmental processes, transition metal ions have received increasing 1 For reviews, see: (a) de Silva, A. P.; Gunaratne, H. Q. N; Gunnlaugsson, T; Huxley, A. J. M; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, (b) Callan, J. F.; de Silva, A. P.; Magri, D. C. Tetrahedron 2005, 61, (c) Pina, E.; Bernardo, M. A.; García- España, E. Eur. J. Inorg. Chem. 2000, (d) Valeur, B; Leray, I Acc. Chem. Res. 2000, 205, 3. (e) Rurack, K.; Resch-Genger, U. Chem. Soc. Rev. 2002, 31, 116. concern. Numerous excellent works focus on the selective and sensitive detection of transition metal ions, e.g., detection of Cu II, Pb II, Zn II and Hg II have been reported. 2 Surprisingly, the examples of Fe III -selective 2 For copper-responsive fluorescent sensors, see: (a) Wu, Q; Anslyn, E. V. J. Am. Chem. Soc. 2004, 126, (b) Gunnlaugsson, T.; Leonard, J. P.; Murray, N. S. Org. Lett. 2004, 6, (c) Royzen, M.; Dai, Z.; Canary, J. W. J. Am. Chem. Soc. 2005, 127, For lead-specified probes, see: (d) Hayashita, T.; Qing, D.; Minagawa, M.; Lee, J. C.; Ku, C. H.; Teramae, N. Chem.Commun. 2003, (e) Chen, C. T.; Huang, W. P. J. Am. Chem. Soc. 2002, 124, For znic-selective indicators, see: (f) Komatsu, K.; Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2005, 127, (g) Ajayaghosh, A.; Carol, P.; Sreejith, S.

11 fluorescent probes are still scarce 3 despite the indispensable role of Fe III in many biochemical processes at cellular level 4. In addition, the ferric ion is well known as a fluorescence quencher due to its paramagnetic nature, and most of the reported Fe III receptors, such as analogues of ferrichromes or siderophores, undergo a fluorescence quenching when bound with Fe III, 5 though it is generally believed that probes with a fluorescence enhancement signal when interacting with analytes are much more efficient. Therefore, the development of new fluorescent Fe III indicators, especially those exhibit selective Fe III -amplified emission, is still a challenge. On the other hand, rhodamine-based fluorescent chemosensors have received increasing interests in recent years by virtue of their long-wavelength emission and availability. 6 Moreover, it is well known that many derivatives of rhodamine undergo equilibrium between spirocyclic and ring-open forms, and the two forms always behave with completely different fluorescent properties. In fact, this is an ideal model for the design of light off-on switch sensors, because the commonly existent spirocyclic forms of these dyes are generally nonfluorescent ( off signal), and the formation of strongly fluorescent open-ring states will occur ( on signal) when guests, e.g., protons and metal ions, are bound to the host probes. 6b Herein we report a new rhodamine-based chemosensor 3 (Figure 1), which displayed highly selective Fe III - amplified fluorescence in both ethanol and buffered water. Compound 3 was facilely synthesized from rhodamine B (1) and diethylenetriamine (2) and obtained as light orange crystals. Its molecular structure was confirmed by MS, NMR and element analysis. 7 Although 3 is a derivative of rhodamine B, it forms a nearly colorless solution in either Tris-HCl aqueous buffer (ph = 7.15) or absolute ethanol, indicating that the spirocyclic form exists predominantly. The characteristic peak near 65.0 ppm (9-carbon) in the 13 C-NMR spectrum of 3 also supports this consideration. 8 Besides, neither the color nor the fluorescence (excited at 510 nm) characteristics of rhodamine could be observed for 3 between ph 5.0~9.0 in water, suggesting that the spirocyclic form was still preferred in this range. As the solution became even more acidic (ph < 4.5), however, an obvious enhancement (S-Figure 1) of color and fluorescence appeared due to the formation of the open-ring state (Figure 1). Interestingly, the addition of Fe III into the colorless solutions (in both neutral buffer and ethanol) of 3 also generated a purple color and orange fluorescence rapidly, while other ions, such as Co II, Ni II, Zn II, Cd II, Ag I, Pb II, Ba II, Mg II, Ca II, K I and Na I, gave no visible change except for Cr III, Fe II and Cu II, which caused a very mild effect compared to Fe III in ethanol but not in water. This intersting feature reveals that 3 can serve as a selective naked-eye chemosensor for Fe III (S-Figure 2). Figure 1. Structrues of ligand 1, 2 and 3 J. Am. Chem. Soc. 2005, 127, For mercury chemosensors, see: (h) Matsushita, M.; Meijler, M. M.; Wirsching, P.; Lerner, R. A.; Janda, K. D. Org. Lett. 2005, 7, (i) Guo, X.; Qian, X.; Jia, L. J. Am. Chem. Soc. 2004, 126, For iron-responsive probes, see: (a) Bricks, J. L.; Kovalchuk, A.; Trieflinger, C.; Nofz, M.; Büschel, M.; Tolmachev, A. I.; Daub, J.; Rurack, K. J. Am. Chem. Soc. 2005, 127, (b) Tumambac, G. E.; Rosencrance, C. M.; Wolf, C. Tetrahedron. 2004, 60, Aisen, P.; Wessling-Resnick, M.; Leibold, E. A. Curr. Opin. Chem. Biol. 1999, 3, (a) Ma, L.; Luo, W.; Quinn, P. J.; Liu, Z.; Hider, R. C. J. Med. Chem. 2004, 47, (b) Nudelman, R.; Ardon, O.; Hadar, Y.; Chen, Y.; Libman, J.; Shanzer, A. J. Med. Chem. 1998, 41, (c) Weizman, H.; Ardon, O.; Mester, B.; Libman, J.; Dwir, O.; Hadar, Y.; Chen, Y.; Shanzer, A. J. Am. Chem. Soc. 1996, 118, For recent rhodamine-based chemosensors for metal ions, see: (a) Yang, Y. K.; Yook, K. J.; Tae, J. J. Am. Chem. Soc. 2005, 127, (b) Kwon, J. Y.; Jang, Y. J.; Lee, Y. J.; Kim, K. M.; Seo, M. S.; Nam, W.; Yoon, J. J. Am. Chem. Soc. 2005, 127, (c) Dujols, V.; Ford, F.; Czarnik, A. W. J. Am. Chem. Soc. 1997, 119, Figure 2. Changes in the absorption spectra of 3 (100 um) in the presence of different metal ions in absolute ethanol. Inset: plots according to the method for continuous variations, indicating the 1:1 stoichiometry for 3-Fe III (the total concentration of 3 and Fe III is 100 um). 7 For more details, see Supporting Information Section. 8 Anthoni, U.; Christophersen, C.; Nielsen, P.; Puschl, A.; Schaumburg, K. Structural Chem. 1995, 3, 161.

12 Figure 2 shows the absorption spectra of 3 in the presence of various metal ions and different amounts of Fe III in absolute ethanol. When no metal ion was added to the solution of 3 (100 um), almost no absorption above 500 nm could be observed, whereas a significant enhancement of the characteristic absorption of rhodamine B emerged soon after Fe III was injected into the solution. There was a large enhancement factor (154- fold) of absorbance at λ max = 557 nm upon the addition of 1 equiv Fe III (100 um). A mild increase of absorbance at 557 nm was also detected when the same amount (100uM) of Cr III (causing 20-fold absorption enhancement), Fe II (24-fold) or Cu II (13-fold) was added due to their low binding affinity to 3. Other cations of our interest gave no response (Figure 2). Similar changes in absorption spectra was also observed when 3 was examined in Tris-HCl aqueous buffer of ph 7.15 (S- Figure 3). nm 9 (Figure 3). In the absence of metal ions, 3 exhibited a very weak fluorescence peak near 550 nm, which was probably the emission of trace open-ring molecules of 3. When Fe III was introduced to a 10 um solution of 3 in either ethanol or buffered water, obvious red shift (~25 nm) and enhancement of fluorescence spectra were observed, whereas other ions of interest displayed much weaker response. In absolute ethanol, 3 (10 um) exhibited a 114-fold enhancement of fluorescence intensity at peak wavelength λ max = 573 nm in the presence of 20 equiv Fe III. Very mild fluorescence enhancement factors (FEF) were also detected for Cr III (20-fold), Fe II (23-fold) and Cu II (6-fold), and Co II, Ni II, Zn II, Cd II, Ag I, Pb II, Ba II, Mg II, Ca II, K I or Na I showed nearly no response (Figure 3a). In Tris-HCl aqueous buffer (ph = 7.15), a lower FEF of 48-fold at λ max = 575 nm was obtained upon the addition of 50 equiv Fe III. Nevertheless, the selectivity was much higher than that in ethanol, since there was even no fluorescence response of Cr III, Fe II and Cu II (Figure 3b). The competition experiment was also carried out by adding Fe III to the aqueous solutions of 3 in the presence of other metal ions and showed in Figure 4. The results indicate that the sensing of Fe III by 3 is hardly affected by these commonly coexistent ions. Figure 3. Fluorescence spectra of 3 (10 um) in the absence and presence of different metal ions in (a) ethanol (20 equiv ions) and (b) Tris-HCl buffer (50 equiv ions, ph = 7.15). For all the tests, excitation and emission was performed at 510 and 575 nm respectively. The fluorescence enhancement effects of various metal ions on 3 were investigated under excitation at λ ex = Excitation was performed at 510 nm rather than 557 nm (the peak of the absorption spectra) to obtain a full view of the fluorescence spectra from 520 to 650 nm.

13 Figure 4. (a) Fluorescence enhancement factors (FEF) of 3 (10 um) upon the addtion of different metal ions in ethanol (black, 20 equiv ions) and Tris-HCl buffer (white, 50 equiv ions). X represents either Co II, Ni II, Zn II, Cd II, Ag I, Pb II, Ba II, Mg II, Ca II, K I or Na I. (b) Fluorescence enhancement response of 3 (10 um in Tris-HCl buffer containing 500 um Fe III ) to 500 um ifferent metal ions. X is a mixture of Ba II, Mg II, Ca II, K I and Na I. Excitation and emission was at 510 and 575 nm respectively. probably due to the space effect of large rhodamine units in 3. The fluorescence titration experiments were performed by means of mixing various amounts of metal ions with 30 um 3 (S-Figure 4a-e and 5). It was found that, to generate more than 90% of the total fluorescence enhancement, 15 and 50 equiv Fe III were necessary in ethanol and Tris-HCl buffer respectively. The inset of Figure 2 indicates that a 1: 1 stoichiometry is most possible for the binding mode of Fe III and 3 in ethanol. 10,11 The stability constant (K) of 3 with different metal ions was calculated according to the 1: 1 model (Table 1). 12 The moderate stability constant of the 3-Fe III complex in aqueous buffer is mainly because the need of Fe III for six-coordination is not satisfied, and, moreover, the strong hydration ability of iron in water. However, the detection of Fe III at 10 5 M level 3b with high selectivity is still available using 3 as an indicator. Figure 5. Proposed mechanism for the fluorescence enhancement of 3 upon the addtion of Fe III. The solvent oxygens and counter anions were omitted for clarity. Table 1. Stability constant (K) of 3 with different metal ions metal ions log K in ethanol log K in buffer a Fe III Fe II c Cr III c Cu II c other ions b - c - c a Tris-HCl aqeous buffer (ph = 7.15). b Including Co II, Ni II, Zn II, Cd II, Ag I, Pb II, Ba II, Mg II, Ca II, K I or Na I. c Too low to detect. As with many reported rodamine-based spirolactam chemosensors, the Fe III induced fluorescence enhancement of chemosensor 3 is most likely the result of the spiro ring-opening mechanism. That is, the chelation of Fe III with the xoygen atoms of the amide groups of 3 results in the formation of the open-ring form. 6 Furthermore, since the color and fluorescence of 3-Fe III disappeared immediately when excess EDTA or diethylenetriamine was added, the sensing process was considered to be reversible rather than an ion catalyzed reaction. The proposed binding mechanism of Fe III with 3 was shown in Figure 5. It should be noted that other coordination sties of the three-coordinate iron may be occupied by solvent oxygens and the counter anions of Fe III. The absence of the 1: 2 (Fe III : 3) binding mode is 10 Vosburgh, W. C.; Cooper, G. R. J. Am. Chem. Soc. 1941, 63, The repeatability of the Job s plot analysis in Tris-HCl buffer was poor due to the weak binding capacity of 3 and Fe III in aqueous media and the trend of 3 to precipate in netrual water at high concentration (200 um). 12 Connors, K. A. Binding Constants-The Measurement of Molecular Complex Stability; John Wiley & Sons: New York, 1987; Chapter 4. In conclusion, we synthesized a new fluorescent probe for Fe III using rhodamine as a fluorophore. This spirolactam compound showed highly selective Fe III - amplified fluorescence emission in both ethanol and aqueous Tris-HCl buffer (ph = 7.15). Commonly coexistent metal ions, e.g., Cr III, Fe II, Cu II, Co II, Ni II, Zn II, Cd II, Ag I, Pb II, Ba II, Mg II, Ca II, K I and Na I displayed little interference, especially when the experiments were operated in buffered water. The enhancement of color and fluorescence in the presence of Fe III was in the range of 500~650 nm, thus the compound was able to serve as a naked-eye chemosenor for ferric ion. The main limitation of this probe is probably its moderate binding capacity to Fe III in aqueous media, which hinders its usefulness in biochemical applications. However, its selectivity is excellent, and the detection of Fe III at 10 5 M level is still available. The modification of 3 to develop new fluorescent probes for Fe III with stronger binding ability (e.g., the proper connection of 3 with other receptor molecules to yield a six-coordinate probe) is now under our investigation. Acknowledgment. The authors are grateful for the financial support from the National Natural Science Foundation of China No Supporting Information Available: Experimental procedures, characterization data for the compounds described and selected spectroscopic data of 3. This material is available free of charge via the Internet at