Protein Detection and Quantitation by Tetraphenylethene-Based

J. Phys. Chem. B 2007, 111, 11817-11823

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Protein Detection and Quantitation by Tetraphenylethene-Based Fluorescent Probes with Aggregation-Induced Emission Characteristics Hui Tong,† Yuning Hong,† Yongqiang Dong,†,‡ Matthias Ha1 ussler,† Zhen Li,† Jacky W. Y. Lam,† Yuping Dong,† Herman H.-Y. Sung,† Ian D. Williams,† and Ben Zhong Tang*,†,‡ Department of Chemistry, The Hong Kong UniVersity of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China, and Department of Polymer Science and Engineering, Zhejiang UniVersity, Hangzhou 310027, China ReceiVed: April 24, 2007; In Final Form: July 15, 2007

Three functionalized derivatives of tetraphenylethylene (TPE), namely, 1,2-bis(4-methoxyphenyl)-1,2diphenylethene (1), 1,2-bis(4-hydroxyphenyl)-1,2-diphenylethene (2), and 1,2-bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-diphenylethene sodium salt (3), were synthesized and their fluorescence properties were investigated. All the TPE molecules are nonluminescent in the solution state but are induced to emit efficiently by aggregate formation. This novel process of aggregation-induced emission (AIE) is rationalized to be caused by the restriction of intramolecular rotations of the dye molecules in the aggregate state. The possibility of utilizing the AIE effect for protein detection and quantification is explored using bovine serum albumin (BSA) as a model protein, with salt 3 being found to perform as a stable, sensitive, and selective bioprobe.

Introduction Protein analysis is of fundamental importance to proteome research that aims to decipher biological processes at the protein level. A number of molecular probes for protein assays have been developed by utilizing the changes in their photophysical properties caused by their chemical reactions or physical interactions with proteins.1 Although these probes have worked for certain protein systems, there is still much room for improvement. The absorption-based protein assays, for example, are inherently limited in sensitivity and effective range (e.g., Bradford and Lowry assays).2 The fluorometric methods have proven useful for the assay of proteins in solutions and helped researchers to probe the concentrations, distributions, and even structures of proteins by means of fluorescence (FL) microscopy, flow cytometry, and FL spectroscopy. Several FL reagents for protein detections and quantifications based on emission enhancement have been developed, such as NanoOrange, fluorescamine, o-phthaldialdehyde, cyanine dyes, and SYPRO Ruby.3 The FL techniques offer high sensitivity, low background signals, and wide dynamic ranges. However, some FL bioprobes require lengthy procedures with carefully timed steps, some show small Stokes shifts and nonlinear calibration curves, and some are even not environmentally stable (e.g., Nile Red and fluorescamine).1,3 There thus is a high demand for the development of simple and stable FL bioprobes. A thorny problem encountered by almost all conventional FL probes is chromophore aggregation.4 Organic dye molecules tend to aggregate when dispersed in aqueous media or bound to proteins in large quantities. The aggregation usually quenches FL, which limits the effective ranges of the probes.3 This makes it particularly difficult to assay low-abundance proteins such * Corresponding author. Phone: +852-2358-7375. Fax: +852-23581594. E-mail: [email protected]. † The Hong Kong University of Science & Technology. ‡ Zhejiang University.

as transcription factors and kinases. The FL signals are too weak to record at the “normal” dye concentrations, while increasing the dye concentrations worsens, instead of improving, the situation. We have discovered a novel aggregation-induced emission (AIE) phenomenon: nonluminescent silole molecules are induced to emit efficiently by aggregate formation.5 Our study proves that the AIE effect is caused by the restrictions of intramolecular rotations of the chromophoric molecules in the aggregate state.6 A large number of AIE-active dyes have since been developed by various research groups, examples of which include 1-cyano-trans-1,2-bis(4-methylbiphenyl)ethylene, bis{4-[N-(1-naphthyl)phenylamino]phenyl}fumaronitrile, 1,4-bis(trans-2-phenyl-1-propenyl)benzene, N,N-bis(salicylidene)-pphenylenediamine, triphenylbenzenes, triarylethenes, tetraphenylbutadiene, diphenyldistyrylbenzenes, benzofurannaphthyridines, azobenzenes, and metalloles.6,7 The AIE molecules emit lights of various colors (blue, green, yellow, and red) in high quantum yields (ΦF up to 85%) in the solid state. The applications of the AIE luminophores have been mainly concentrated on their utilizations for the construction of organic light-emitting diodes.5-7 Their potential uses as FL probes for biological assays have, however, been virtually unexplored.8 We envision that binding of the AIE dyes to protein molecules may turn their light emissions on, thus offering a FL bioprobe system based on a new working mechanism. We have recently developed two AIE-active cationic tetraphenylethylene (TPE) derivatives containing quaternary ammonium groups, the emissions of which were switched on by their bindings with DNA and proteins.8 Although the work has proved the feasibility of using AIE dyes as bioprobes, their sensitivities need to be improved.8 The goal of our present work is thus to design and synthesize sensitive AIE bioprobes. In this study, we prepared a dimethoxylated TPE derivative 1,9 which was further derivatized to give dihydroxylated TPE 2 and then disulfonated TPE 3 (Scheme 1). Dye 2 can be

10.1021/jp073147m CCC: $37.00 © 2007 American Chemical Society Published on Web 09/18/2007

11818 J. Phys. Chem. B, Vol. 111, No. 40, 2007

Tong et al.

SCHEME 1: Syntheses of Functionalized TPE Derivatives 2 and 3 from 1

were fully characterized spectroscopically (see Experimental Section for details), with that of 1 verified crystallographically (cf., Figure 1 and Supporting Information). TPE dyes 1 and 2 are soluble in common organic solvents such as acetonitrile (AN) but are insoluble in water. On the other hand, salts 2-Na+2 and 3 are soluble in polar solvents including water, methanol, DMF, and DMSO. The FL spectrum of 1 in AN is barely discernible even after 5-times magnification, while its suspensions in water/AN mixtures with high water fractions is highly emissive (Figure 2). The ΦF value for the emission at 394 nm of 1 in AN is negligibly small (0.11%). The emission intensity starts to increase when the dyes begin to cluster together in the water/ AN mixtures with ∼70% water. It reaches 15.3% in the 99% water/AN mixture, which is ∼150-fold higher than that of its dilute AN solution (Table 1). The photographs given in Figure 2B clearly manifest the nonemissive and emissive nature of the molecular and aggregated species, respectively. Water is a nonsolvent of 1. In the aqueous mixtures with high water contents, the molecules of 1 should aggregate. This is confirmed by the leveled-off tails in the visible region of the absorption spectra of the dye in the aqueous mixtures with high water contents due to the light-scattering effects of the dye nanoparticles (e.g., Figure 3). At 99% volume fraction of water, the excitation maximum for the FL at 474 nm is located at 332 nm, coinciding with the absorption maximum (330 nm) in this aggregate state. Clearly, the emission of 1 is induced by the aggregate formation, or, in other words, 1 is AIE active. The real ΦF values of the aggregates could be even higher as the ΦF calculation does not consider the distorted strong absorption due to light-scattering effect of the nanoparticles.

transformed to a water-soluble salt 2-Na+2 by its reaction with ethanolic sodium, while 3 is already a water-soluble salt. In this paper, we prove that all the three TPE derivatives (1-3) are AIE active and demonstrate that 3 works as an excellent protein probe with high sensitivity and selectivity (Figure 1). Results and Discussion TPE derivative 1 was prepared in 91% yield by simply refluxing p-methoxybenzophenone in THF in the presence of TiCl3/AlCl3-Zn.9 Demethylation of 1 by BBr3 followed by treatment with water gave dihydroxylated TPE derivative 2 (cf., Scheme 1). Nearly quantitative transformation from 1 to 2 was confirmed by the absence of resonance peaks of the methoxyl protons in the 1H NMR spectrum of the reaction product. The reaction of a sodium salt of 2 with 1,3-propanesultone in ethanol resulted in the formation of 1,2-bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-diphenylethene (3). The molecular structures of 1-3

Figure 1. Left: crystal structure of 1 (see the Supporting Information for details). Right: photographs of an aqueous solution of 3, its aggregate suspension in a mixture of acetonitrile (AN) and water with 99 vol % of AN, and its complex with BSA in an aqueous phosphate buffer with pH 7.0.

Figure 2. (A) FL spectra of 1 in water/AN mixtures and (B) dependence of the fluorescence quantum yield of 1 on the solvent composition of the water/AN mixture. Concentration of 1, 10 µM; excitation wavelength, 350 nm.

Protein Detection by AIE-Active Fluorescent Probes

J. Phys. Chem. B, Vol. 111, No. 40, 2007 11819

TABLE 1: Photophysical Properties of TPE Derivatives 1-3 in Solution (soln),a Aggregate (aggr),b and Binding (bindg)c States λab, nmd

λem, nm (ΦF, %)e

TPE

soln

aggr

soln

aggr

bindg

1 2 3

311 312 312

330 316 320

394 (0.11) 393 (0.57) 398 (0.37)

477 (15.30) 439 (8.90) 442 (17.47)

467 (35.7) 472 (58.2)

a In AN for 1 and 2 (10 µM) and in water for 3 (5 µM). b In water/ AN mixture (with 99 vol % water) for 1 and 2 and in AN/water mixture (with 99 vol % AN) for 3. c In BSA solution of 2-Na+2 or 3 (5 µM) in an aqueous phosphate buffer with pH 7.0. d Absorption maximum. e Emission maximum (quantum yield given in the parentheses); excitation wavelength, 350 nm. Relative ΦF value was determined using 10 µM of quinine sulfate in 0.1 N H2SO4 solutions as standard.

Figure 5. (A) Change in FL spectrum of 2-Na+2 (5 µM) with addition of BSA in an aqueous phosphate buffer (pH 7.0). (B) Plot of FL intensity at 476 nm versus BSA concentration. (C) Linear region of the binding isotherm of 2 to BSA.

Figure 3. Absorption (ab), emission (em), and excitation (ex) spectra of 1 (10 µM) in pure AN (dotted line) and a water/AN mixture with 99 vol % of water (solid line).

Figure 6. Excitation (ex) and emission (em) spectra of (A) 2-Na+2 and (B) 3 in the phosphate buffer (pH 7) containing (A) 300 and (B) 500 µg/mL of BSA.

Figure 4. Absorption (ab) and emission (em) spectra of (A) 2 (10 µM) in AN and water/AN mixture (with 99 vol % water) and (B) 3 (5 µM) in water and AN/water mixture (with 99 vol % AN).

soluble but can be made miscible with aqueous media by transforming it to a negatively charged sodium salt (2-Na+2). A stock solution (0.5 mM) of the salt in a phosphate buffer was thus prepared and its behaviors of protein binding were investigated using BSA as a model protein. The aqueous solution of 2-Na+2 in the phosphate buffer is almost nonfluorescent (ΦF ∼ 0.57%; Figure 5A). When a small amount of BSA is added, its photoluminescence is immediately activated, showing an FL spectrum centered at ∼467 nm with a large Stokes shift (147 nm). Although the net increase in the FL intensity (I/I0 - 1) is only ∼2.5 at 1 µg/mL of BSA, it can be increased by up to ∼26-fold upon further addition of BSA. For our previous positively charged TPE derivative, the I/I0 1 values at 1 and 300 µg/mL of BSA were ∼0.3 and ∼20, respectively.8 Evidently, salt 2-Na+2 is a more sensitive protein probe than the cationic dye we previously developed. The ΦF value of the salt reaches ∼36% at a BSA concentration of 300 µg/mL (Table 1). At this BSA concentration, the excitation maximum for the emission at 467 nm is located at 331 nm (Figure 6A), very close to that of the dye aggregates (332 nm). This reveals a close relationship between the protein-bindinginduced emission of the salt and its AIE activity. The plot of the FL intensity of 2-Na+2 at 467 nm as a function of BSA concentration is shown in Figure 5B. In the BSA concentration range of 0-10 µg/mL, the plot displays a good linear relationship (R2 > 0.997, Figure 5C). It is obvious that 2 can be used as a “light-up” or “turn-on” FL bioprobe for protein detection and quantification. However, 2 is a water-insoluble

Similarly, dyes 2 and 3 are nonemissive when molecularly dissolved in good solvents but highly luminescent when aggregated in poor solvents (Figure 4). Thus, the nonemissive TPE derivatives can all be induced to emit by aggregate formation, proving that the form of the dye (neutral or negatively or positively8 charged) does affect its AIE activity. All the three TPE derivatives are strong blue emitters in the solid state. To investigate complexation or conjugation process of an AIE dye with a protein in an aqueous buffer solution, the dye must be soluble in water, and its emission and excitation spectra must be measurable in the buffer. Dye 1 thus cannot be used for such study, because of its insolubility in water. Dye 2 is not water-

11820 J. Phys. Chem. B, Vol. 111, No. 40, 2007

Figure 7. (A) Change in FL spectrum of 3 with addition of BSA in an aqueous phosphate buffer (pH 7.0). (B) Plot of FL intensity at 472 nm versus BSA concentration. (C) Linear region of the [BSA] vs (I/I0 - 1) plot in panel B.

neutral dye and must be converted to negatively charged sodium salt in a basic solution before use, which is not so convenient for practical applications. In the meantime, the phenol anion salt is not so stable because of its transformation from phenol structure to quinone form, as suggested by the experimental observation that the color of its stock solution changes from yellow to green after the solution has been stored under ambient conditions for about 2 weeks. In comparison to 2, the water-soluble salt 3 is much more stable and is therefore expected to be more suitable for protein detection and quantification as an FL bioprobe. A stock solution of 3 (0.5 mM) was prepared by directly dissolving it in a pH 7.0 phosphate buffer. The dye solution in the absence of BSA is almost nonluminescent (Figure 7A), although it shows a high molar absorptivity of 16 400 M-1 cm-1 at 312 nm. Its emission is again switched on instantly by the addition of BSA. Its intensity increase (up to ∼240; Figure 7B) and linear range (0100 µg/mL; Figure 7C) are ∼9- and 10-fold larger and wider than those of 2-Na+2, respectively. In the presence of 500 µg/mL of BSA, the ΦF value of 3 in the phosphate buffer is ∼58% (Table 1), which is >1.6-fold higher than that of 2-Na+2 under the same conditions. The Stokes shift is ∼152 nm, which is also larger than that of 2-Na+2. The excitation maximum for the emission at 472 nm is located at ∼332 nm, which is almost identical to that of 2-Na+2, suggesting the same origin for the enhanced emission in the two cases. However, no change in the emission spectrum of 3 is observed after its stock solution has been put on the shelf without any precautionary protections from light and air for more than 2 weeks. Clearly, salt 3 is a much more stable and readily applicable FL bioprobe for protein detection and quantification. As discussed in the Introduction, traditional molecular probes suffer from the self-quenching problem at high dye concentrations. The FL spectrum of salt 3 is, however, intensified with an increase in its solution concentration (Figure 8), thanks to its AIE characteristics. Its FL is further intensified by the addition of BSA (Figure 9A), suggestive of specific dye-protein interactions in the probing process. Salt 3 cannot react with amines, so it is impossible for it to form covalent adducts or conjugates with BSA as o-phthaldialdehyde does.3 The interactions between 3 and BSA should be mainly hydrophobic in nature. Adding sodium dodecyl sulfate (SDS) into a BSA solution of 3 dramatically weakens its FL (Figure 9B). The probe is thus folding-structure-sensitive, because SDS can effectively unfold BSA.10

Tong et al.

Figure 8. FL spectra of solutions of 3 with different concentrations in a phosphate buffer (pH 7.0).

Figure 9. (A) Effect of dye concentration on the FL intensity of buffer solution of 2-Na+2 at 467 nm or 3 at 472 nm in the absence or presence of BSA (10 µg/mL). (b) Effect of BSA (100 µg/mL) and/or SDS (1 mg/mL) on the FL spectrum of a buffer solution of 3 (5 µM).

What is the mechanistic cause of the AIE effect of 3 and how does it associate with the bioprobing process? In the solution state, torsional and rotational motions have been reported for TPE derivatives upon excitation,11 which can serve as nonradiative channels for the excited species to decay, whereas the formation of dye aggregates can obstruct these intramolecular motions.6,7 The aggregation-imposed conformational rigidification is thus the likely mechanistic origin of the AIE effect. Theoretically, high viscosity and low temperature should hamper the intramolecular rotations. Indeed, the FL intensity of a dilute solution of 3 in a viscous glycerol/water mixture (with 90 vol % of glycerol) at 25 °C is much higher than that in pure water (Figure 10A). When the solution temperature is slightly decreased, the FL intensity is dramatically increased. Note here that dye 3 becomes highly luminescent in the viscous media and at the low temperatures as isolated molecules in the dilute solution but not as supramolecular aggregates in the nanoparticle suspension. The absorption maximum of 3 is red-shifted to 321 nm in the glycerol/water mixture, which is very close to that of the aggregates (320 nm). Most importantly, the excitation maximum of 3 here is located at 330 nm (Figure 10B), which is almost identical to that of its aggregates or BSA complexes (332 nm). These experimental results support that the restriction of intramolecular rotations indeed plays a very important role in inducing the dye to emit in all the situations discussed above. To further understand the AIE processes, the geometric structure of 1 is optimized at the RHF/6-31G* level using

Protein Detection by AIE-Active Fluorescent Probes

Figure 10. (A) Emission spectra of 3 (5 µM) in a glycerol/water mixture with 99% of glycerol at -5, 0, and 25 °C. Data for 3 in water (5 µM) at 25 °C is shown for comparison. (B) Excitation (ex) and emission (em) spectra of 3 in the glycerol/water mixture at 25 °C.

J. Phys. Chem. B, Vol. 111, No. 40, 2007 11821 potentially adopt a large number of conformations that can interconvert rapidly through single-bond rotation.15 The absorption maximum of 1 in AN at room temperature is located at 312 nm, indicating that the conformations of the dye molecules in the solution are more twisted, possibly due to the thermally activated torsional motions allowed in this solvent. However, when the temperature is decreased or highly viscous solvents are used, the less twisted, most stable conformation cannot be easily altered due to the involved temperature or viscosity effect. The intramolecular rotations become more difficult, so the absorption is red-shifted, accompanying an intensified, red-shifted emission. In the aggregate state or in the solutions containing BSA, the bond rotation becomes even more difficult, so the intense AIE from the optimized geometry is easily observed. The native folding structure of BSA contains hydrophobic binding sites such as hydrophobic pockets.2-3 The dye molecules may bind to the hydrophobic regions of BSA chains and enter into the hydrophobic pockets of their folding structures, where the rotations of the dye molecules are frozen, hence inducing them to emit as aggregation does. It is thus the AIE nature that makes the dye useful as a protein probe. The BSA-bindinginduced red-shift in λem (74 nm) is much larger than that in λab (18 nm), which thus widens the Stokes shift. When the surfactant molecules of SDS destroys the native quaternary folding structure of BSA chains by binding to the hydrophobic regions of the protein,10 dye 3 ceases to function as a protein bioprobe (cf., Figure 9B). This phenomenon attests the hydrophobic nature of the 3-BSA interaction. Conclusion

Figure 11. Plots of potential energies of S0 and S1 of 1 and its transition energies from S0 to S1 as a function of rotation angle θ.

Gaussian 98.12 Its congeners 2 and 3 are assumed to take similar structures. Its molecular structure obtained from the single crystal analysis (cf., Figure 1 and Supporting Information) serves as a starting point for the computer simulation and structural optimization. The optimized geometry of dye 1 is very close to a C2 symmetry, with a rotation angle (θ) of ∼51°. On the basis of this structure, its electronic transitions are obtained by using the ZINDO/S method in HyperChem 7.5.13 Its electronic transition energy (Et ) 330 nm) from the ground state (S0) to the first singlet excited state (S1) is nearly identical to its excitation (332 nm) and absorption (330 nm) maxima in the aggregate state, indicating that its AIE is associated with the excitation of this optimized ground state. As the C-C single bond (or σ bond) is easy to rotate, the geometric structures and their corresponding transition energies at different θ angels are simulated in a similar way. The potential energies of S1 are then given by adding the Et values to the potential energies of S0. On the basis of the potential energy curve of S1,14 the energy minimum is found at θ ∼ 30° with a transition energy of 355 nm, which is very close to the excitation maximum (353 nm) of 1 in AN. It is possible that all other excited states relax through bond rotation to this conformation, which acts as a weakly radiative channel. According to the potential energy curve of S0 given in Figure 11, the energy surface around the calculated minimum is very flat. Thus, at ambient temperature and in low viscosity solvents, 1 can

In this work, we have developed an efficient protein assay system based on an AIE-active fluorophore. Dye 3 is stable: it does not decompose after storage for a long period of time under ambient conditions without taking any precautionary measures. The probe is selective: it works for native BSA but not its denatured form. The probe is also sensitive, giving clear FL signals at [BSA] ∼ 500 ng/mL at [3] ) 5 µM. It is envisioned that even lower [BSA] should be detectable at higher [3] without suffering from the self-quenching problem. Its calibration curve has a very wide linear range (0-100 µg/mL). Most importantly, it is a supramolecular bioprobe working by a new AIE mechanism, in comparison to the traditional molecular probes. This mechanistic feature endows the bioprobe with a large Stokes shift, because the aggregation greatly shifts its λem. The AIE nature allows the use of large fluorophore/protein ratios, enabling the detection of trace amounts of low-abundance proteins. FL intensity of an AIE dye is increased with increasing aggregation extent. This offers an intriguing possibility of “seeing” the hydrophobic pockets of proteins, active sites of enzymes, etc. by imaging shapes of the AIE aggregates therein by such techniques as confocal microscope. Tetraarylethenes with larger aryl rings emitting green, yellow, orange, and red lights are readily accessible via simple McMurry reactions. The studies of their further applications in the biological science and technology are in progress in our laboratories in collaboration with our biochemistry colleagues. Experimental Section General Information. p-Methoxybenzophenone, boron tribromide, titanium tetrachloride, sodium ethoxide, 1,3-propanesultone, titanium(III) chloride-aluminum(III) chloride, zinc dust, and SDS were purchased from Aldrich and used as received. BSA was purchased from Sigma and stored in a cold,

11822 J. Phys. Chem. B, Vol. 111, No. 40, 2007 dark place before use. All the solvents were purchased from Aldrich, some of which were purified by standard distillation procedures. Phosphate buffer solution with pH 7.0 was purchased from Merck. 1H and 13C NMR spectra were measured on a Bruker ARX 300 or Varian 300 spectrometer using deuterated chloroform as solvent. Tetramethylsilane (TMS) was used as internal reference for the NMR analyses. Mass spectra were recorded on a triple quadrupole mass spectrometer (Finnigan TSQ7000). UV-vis absorption spectra were recorded on a Milton Roy Spectronic 3000 Array spectrophotometer. FL spectra were measured on a Perkin-Elmer LS 50B spectrofluorometer with a Xenon discharge lamp excitation. X-ray diffraction intensity data of crystals were collected at 100 K on a Bruker-Nonius Smart Apex CCD diffractometer with graphite-monochromated Mo KR radiation (λ ) 0.7107 Å). A single crystal of 1 was grown from a THF/ methanol mixture. Lattice determination and data collection were carried out using SMART (version 5.625). Data reduction was performed using SAINT (version 6.26a). The data were corrected for absorption using SADABS (version 2.03), and the structure solution and refinement were carried out by SHELXTL (version 6.10). Stock solutions of 1 and 2 with a concentration of 1.0 mM were prepared by dissolving appropriate amounts of the dyes in AN. Stock solutions of 2-Na+2 and 3 with a concentration of 0.5 mM were prepared by dissolving appropriate amounts of the salts into a sodium hydroxide solution and an aqueous phosphate buffer, respectively. Stock solutions of BSA with concentrations of 0.01, 0.1, and 1 mg/mL were prepared by dissolving appropriate amounts of the protein in an aqueous phosphate buffer. Sample solutions for measuring absorption and emission spectra were prepared by adding 1 mL of a stock solution into 99 mL of AN or water under vigorous stirring at room temperature. FL titration was carried out by adding aliquots of a BSA solution in the aqueous phosphate buffer to 0.1 mL of a stock solution of 2-Na+2 or 3, followed by adding a proper amount of the pH 7.0 buffer to acquire a 10.00 mL solution. The mixture was stirred for 30 min prior to taking its spectrum. The ΦF value was determined by the standard method using 10 µM of quinine sulfate in 0.1 N H2SO4 as standard. Refractive indices of the solvents were taken into account in the quantum yield calculation. Geometrical structures of isolated molecules of dye 1 in the ground state were optimized on the basis of RHF/6-31G* calculations using Gaussian 98. On the basis of the optimized geometrical structures, their electronic spectra were calculated, using the semiempirical ZINDO/S method in HyperChem 7.5. Preparation of TPE Derivative 1. A suspension of pmethoxybenzophenone (1.06 g, 5.0 mmol), 1.34 equiv of TiCl3/ AlCl3 (5.81 g, 6.7 mmol), and 25 equiv of Zn dust (8.01 g, 122.0 mmol) in 100 mL of dry THF was refluxed for 20 h. The reaction mixture was cooled to room temperature and then filtered. The filtrates were evaporated, and the crude product was purified by a silica gel column using hexane as eluent. Product 1 was isolated in 91% yield (0.89 g). 1H NMR (CDCl3, 300 MHz), δ (TMS, ppm): 7.10-7.06 (m, 10H), 6.93 (t, 4H), 6.64 (t, 4H), 3.74 (s, 6H). 13C NMR (CDCl3, 75 MHz), δ (TMS, ppm): 158.0, 144.4, 139.7, 136.5, 132.6, 131.5, 127.8, 126.3, 113.2, 55.2. MS (TOF) m/e: 392.1 (M+, calcd 392.2). Singlecrystal file no.: CCDC 622769 (see Supporting Information for details). Synthesis of TPE Derivative 2. Into a 100 mL flask were added 1.40 g (3.56 mmol) of 1 and 20 mL of dichloromethane

Tong et al. (DCM). The flask was placed in an acetone-dry ice bath at -78 °C. A solution of 3.59 g (14.3 mmol) of boron tribromide in 10 mL of DCM was added carefully to the mixture under stirring. The resultant mixture was allowed to warm to room temperature and stirred overnight. The reaction product was hydrolyzed by careful shaking with 20 mL of water. The organic phase was separated and concentrated by a rotary evaporator. The crude product was purified by recrystallization from THF/ methanol to afford a white solid (1.26 g, 97% yield). 1H NMR (CDCl3, 300 MHz), δ (TMS, ppm): 7.11-7.02 (m, 10H), 6.88 (t, 4H), 6.56 (d, 4H). 13C NMR (CDCl3, 75 MHz), δ (TMS, ppm): 154.1, 144.2, 139.7, 135.5, 132.8, 131.5, 127.8, 126.3, 114.7. MS (TOF) m/e: 363.1 [(M - H)+, calcd 363.1]. Synthesis of TPE Salt 3. Into a 100 mL round-bottom flask were added 0.5 g (1.37 mmol) of 2 and 20 mL of anhydrous ethanol under nitrogen. The mixture was stirred until all solids disappeared. A mixture of NaOEt (0.20 g, 3.0 mmol) in 20 mL of ethanol was added dropwise and stirred for 1 h, causing the colorless solution to turn orange-red. Into the solution was added 0.35 g of 1,3-propanesultone (2.88 mmol) in 20 mL of ethanol. The mixture was vigorously stirred for 12 h, during which time a white product was precipitated out from the solution. The product was collected by filtration and washed with ethanol and acetone twice to give a white solid (0.55 g, 61% yield). 1H NMR (DMSO-d6, 300 MHz), δ (TMS, ppm): 7.25-7.13 (m, 6H), 7.08-7.02 (m, 4H), 6.95-6.90 (m, 4H), 6.81-6.73 (m, 4H), 4.09-4.02 (m, 4H), 2.66-2.58 (m, 4H), 2.08-2.02 (m, 4H). 13C NMR (DMSO-d , 75 MHz), δ (TMS, ppm): 157.0, 143.9, 6 139.2, 135.5, 131.9, 130.8, 127.8, 126.2, 113.8, 66.4, 47.9, 25.3. MS (TOF) m/e: 631.1 [(M + 2H)+ - Na, calcd 631.1], 609.2 [(M + 3H)+ - 2Na, calcd 609.1]. Acknowledgment. This project was partially supported by the Research Grants Council of Hong Kong (602706, HKU2/ 05C, 603505, and 603304), the National Natural Science Foundation of China (20634020), and the Ministry of Science and Technology of China (2002CB613401). B.Z.T. thanks the support from the Cao Guangbiao Foundation of Zhejiang University. Supporting Information Available: Single-crystal data of dye 1. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) (a) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals; Molecular Probes: Leiden, 2002. (b) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538-544. (c) Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science 2006, 312, 217-224. (2) (a) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-275. (b) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. (c) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 76-85. (d) Methods in Proteome and Protein Analysis; Kamp, R. M., Calvete, J. J., CholiPapadopoulou, T., Eds.; Springer-Verlag: New York, 2004. (3) (a) Fluorescent and Luminescent Probes for Biological ActiVity; Mason, W. T., Ed.; Academic Press: London, 1999. (b) Berggren, K.; Steinberg, T. H.; Lauber, W. M.; Carroll, J. A.; Lopez, M. F.; Chernokalskaya, E.; Zieske, L.; Diwu, Z.; Haugland, R. P.; Patton, W. F. Anal. Biochem. 1999, 276, 129-143. (c) Yarmoluk, S. M.; Kryvorotenko, D. V.; Balanda, A. O.; Losytskyy, M. Y.; Kovalska, V. B. Dyes Pigm. 2001, 51, 41-49. (d) Jones, L. J.; Haugland, R. P.; Singer, V. L. BioTechniques 2003, 34, 850-861. (e) Suzuki, Y.; Yokoyama, K. J. Am. Chem. Soc. 2005, 127, 17799-17802. (f) Granzhan, A.; Ihmels, H. Org. Lett. 2005, 7, 51195122. (g) Hoefelschweiger, B. K.; Duerkop, A.; Wolfbeis, O. S. Anal. Biochem. 2005, 344, 122-129. (h) Royer, C. A. Chem. ReV. 2006, 106, 1769-1784.

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