Synthesis of a New Water-Soluble Rhodamine Derivative and

Bioconjugate Chem. 2006, 17, 1219−1225

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Synthesis of a New Water-Soluble Rhodamine Derivative and Application to Protein Labeling and Intracellular Imaging Rakeshwar Bandichhor,† Anca D. Petrescu,‡ Aude Vespa,‡ Ann B. Kier,‡ Friedhelm Schroeder,‡ and Kevin Burgess*,† Department of Chemistry, Texas A & M University, Box 30012, College Station, Texas 77842-3012, and Departments of Physiology, Pharmacology, and Pathobiology, Texas A & M University, TVMC, College Station, Texas 77881. Received May 30, 2006; Revised Manuscript Received July 27, 2006

Synthesis of a new fluorescent rhodamine derivative, dye 1, is reported. This probe is different from other rhodamines insofar as it has several (four) carboxylic acid functionalities to promote water solubility and facilitate conjugation to proteins. It also has an aryl bromide functionality that could, in principle, be used to further functionalize the system for specialized applications. Dye 1 was conjugated to a model protein called ACBP (acyl-CoA binding protein). The properties of this conjugate were tested to establish that the label does not significantly perturb the binding function of the protein to its natural ligand in vitro and to confirm that its secondary structure was not significantly perturbed (circular dichroism). Experiments were performed to test if the labeled protein could be imported into liVing COS-7 cells (using the Chariot-peptide delivery system) and, if so, to observe, via fluorescence microscopy, which of the labeled protein was able to migrate to the nucleus, as expected for ACBP in cells. In the event, all these postulates were confirmed.

INTRODUCTION Intracellular imaging (1) requires a selection of water-soluble, fluorescent dyes that absorb and emit at wavelengths longer than those commonly associated with autofluorescence in cells (2), i.e., above around 550 nm. Some rhodamine dyes with these characteristics are commercially available, but specialized situations call for “tailor-made” probes that can be modified with other functionalities via organic transformations. Preparation and manipulation of water-soluble fluorescent dyes is challenging, because separation of gram amounts of material tends to be more difficult for polar, water-soluble materials relative to others that are soluble, and chromatographically separable, using organic solvents. Furthermore, even recrystallization of highly colored, fluorescent dyes is experimentally difficult, simply because it is difficult to see when crystals are forming. Our collaborative project on intracellular imaging of proteins involved in nuclear localization called for a special type of rhodamine dye. Specifically, the probe should (i) be water soluble; (ii) absorb and fluoresce above 550 nm; (iii) have functional groups that allow attachment to proteins; (iv) have another functional group that would allow attachment of other organic fragments to the dye via organometallic reactions involving conditions that would not perturb other functional groups in the dye; and (v) be synthetically accessible. Rhodamine derivative 1 was conceived on the basis of these considerations (Figure 1; “rosamine” is informal nomenclature introduced by Molecular Probes to denote rhodamine without a carboxylic acid on the peripheral benzene ring). This probe has four carboxylic acid groups to promote water solubility (at least above pH levels of approximately 4.5) and to provide points of attachment to proteins. Use of carboxylic acids means that most of the synthetic steps could * [email protected]. Ph: (979)845-4345. Fax: (979)845-8839. † Department of Chemistry. ‡ Departments of Physiology, Pharmacology, and Pathobiology.

Figure 1. The target probe 1.

be performed on tetraesters, and these compounds tend to be hydrophobic and more easily chromatographed on a gram scale. Finally, the aryl bromide functionality was included in the design to allow fusion of other entities to the fluorescent dye via palladium-catalyzed reactions (e.g., Suzuki (3) and Sonogashira (4) couplings). Our particular need was to attach other dye fragments to this site, to form “through-bond energy transfer cassettes” (5, 6). That particular goal is beyond the scope of this paper, but what is important here is simply the concept that this functional group could be used to attach other groups to create dual-purpose probes. We propose that the second site could be modified to include useful entities such as EPR spinlabels, chelating groups to facilitate MRI imaging, or radioactive reporter functionalities.

EXPERIMENTAL PROCEDURES Chemicals and Reagents. Unless otherwise noted, all nonaqueous reactions were carried out under an atmosphere of dry N2 in dried glassware. When necessary, solvents and

10.1021/bc0601424 CCC: $33.50 © 2006 American Chemical Society Published on Web 08/23/2006

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reagents were dried prior to use. Diethyl ether and dichloromethane were deoxygenated by purging with N2 and then dried by passing through activated alumina. Tetrahydrofuran and toluene were distilled from sodium and hexanes from calcium hydride. DMF was distilled from CaH2 under reduced pressure. K2CO3 was used as received from EMD ACS grade. 3-Aminophenol and acrylic acid from Across Organics were used as received, and 4-bromobenzaldehyde, N-hydroxysuccinimide, and DIC (diisopropylcarbodiimide) were purchased from Aldrich. Buffer ingredients and solutions for liquid chromatography, gel electrophoresis, and cell culture were purchased from Gibco BRL (Invitrogen, Carlsbad, CA) and Sigma-Aldrich (St. Louis, MO). Bio-Gel P-4 for gel filtration chromatography was from Bio-Rad (Hercules, CA). Instruments. Analytical thin layer chromatography (TLC) was performed on EM Reagents 0.25 mm silica gel 60-F plates. Visualization was accomplished with UV light. Chromatography on silica gel was performed using a forced flow of the indicated solvent system on EM Reagents Silica Gel 60 (230400 mesh). 1H NMR spectra were recorded (VXR-300 and Inova 500 MHz spectrometer) at room temperature, and chemical shifts are reported in parts per million (ppm) from the solvent resonance (CDCl3 7.26 ppm, CD2Cl2 5.30 ppm, DMSO-d6 2.49 ppm, D2O 4.80 ppm). Data are reported as follows: chemical shift, multiplicity (s ) singlet, d ) doublet, t ) triplet, q ) quartet, br ) broad, m ) multiplet), coupling constants, and number of protons. Proton decoupled 13C NMR spectra were also recorded (VXR and Inova spectrometers) at room temperature. Chemical shifts are reported in parts per million from tetramethylsilane resonance (CDCl3 77.36 ppm). MS were measured (Thermofinnigan LC-Q Deca spectrometer) under ESI, MALDI, or APCI conditions. IR spectra were recorded on a Bruker (Tensor 27), UV on an HP (Diode Array Spectrophotometer), and fluorescence spectra were recorded on a Fluorolog spectrometer. Preparation of the Diester 2. A solution of 3-aminophenol (1.00 mol, 109 g) in acrylic acid (3.00 mol, 185 mL) and water (93 mL) was heated to 70 °C. After stirring for 3 h, the reaction mixture was cooled, ethanol (180 mL) was added and the solution kept at 5° C. After 12 h, precipitate appeared and was filtered, washed with ethanol (50 mL), and dried under vacuum to afford the intermediate diacid as a solid (200 g, 80%). Mp ) 153-154 °C (lit (7) 149-150 °C); 1H NMR (300 MHz, acetone-d6) δ 7.02-6.98 (m, 1H), 6.28-6.24 (m, 2H), 6.196.17 (m, 1H), 3.65 (t, 4H, J ) 4.5 Hz), 2.60 (t, 4H, J ) 4.5 Hz); 13C NMR (75 MHz, acetone-d6) δ 172.6, 158.6, 148.6, 130.0, 104.0, 104.2, 99.6, 46.8, 31.7; MS (ESI) calcd for C12H15NO5 253.0950, found 253.0400. A solution of the diacid prepared above (39.5 mmol, 10.0 g) in methanol (500 mL) and HCl (10 M, 1 mL) was refluxed for 12 h, then cooled to room temperature, and MeOH evaporated under reduced pressure. The residue was dissolved in EtOAc (100 mL), and the organic layer was washed with water (5 × 20 mL). The organic layer was evaporated under reduced pressure to yield 2 (8) as a yellow semisolid (7.0 g, 63%). Rf ) 0.7 (50% EtOAc/hexane). 1H NMR (300 MHz, CD3OD) δ 7.05-6.95 (m, 1H), 6.25-6.15 (m, 3H), 3.65 (s, 6H), 3.60 (t, 4H, J ) 7.2 Hz), 2.58 (t, 4H, J ) 7.2 Hz); 13C NMR (75 MHz, CD3OD) δ 173.3, 158.36, 148.5, 130.1, 104.6, 104.2, 99.9, 51.1, 46.9, 32.1; MS (ESI) calcd for C14H19NO5 281.1263, found 281.3614. Bromorosamine Tetraacid 1. 4-Bromobenzaldehyde (3.6 mmol, 0.66 g) was added to a solution of dimethyl ester 2 (7.2 mmol, 2.0 g) in ether (5 mL). After stirring for 15 min, the ether was evaporated. The neat mixture was subjected to microwave irradiation (300 W, 150-155 °C, 20 min). Chloranil (4.1 mmol, 1.0 g) in 1:1 (CH2Cl2/MeOH) (10 mL)

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was then added, and the reaction was stirred for 1 h. The solvents were removed, and residue was chromatographed (SiO2; 20% MeOH/CH2Cl2) to afford 3 (0.5 g) in 20% yield (8085% purity) as a sticky solid. Rf ) 0.30 (10% MeOH/CH2Cl2); 1H NMR (500 MHz, CD OD/CDCl ) δ 7.89 (d, 2H, J ) 8.5 3 3 Hz), 7.49 (d, 2H, J ) 9.5 Hz), 7.46 (d, 2H, J ) 8.5 Hz), 7.21 (dd, 2H, J ) 2.5 Hz, 2.5 Hz), 7.13 (d, 2H, J ) 2.5 Hz), 4.03 (t, 8H, J ) 7.0 Hz), 3.72 (s, 12H), 2.81 (t, 8H, J ) 7.5 Hz); MS (ESI) calcd for C35H38BrN2O9+ 709.1755, found 709.1904, 711.1924. Solid K2CO3 (1.4 mmol, 193 mg) was added to a solution of compound 3 (0.07 mmol, 50 mg) in 1:1:1 (H2O/MeOH/THF) (10 mL). After stirring for 12 h at 40 °C, the MeOH and THF were removed, and the aqueous layer was extracted with EtOAc (3 × 50 mL). The aqueous layer was further acidified with conc HCl to attain pH ) 2-3, then extracted with 1:2 (iPrOH/CH2Cl2) and concentrated to afford crude compound 1 (30 mg). Crude compound 1 (30 mg) was dissolved in 1:1 (CH3CN/H2O) (30 mL) and subjected to reversephase preparative HPLC (C18, 5:95 (CH3CN/H2O), tR ) 12.0 min) to afford pure compound 1 (10 mg) in 22% yield as a solid: Rf ) 0.10 (10% MeOH/CH2Cl2); 1H NMR (500 MHz, CD3OD/CDCl3) δ 7.84 (d, 2H, J ) 10 Hz), 7.48 (d, 2H, J ) 10 Hz), 7.35 (d, 2H, J ) 10 Hz), 7.12 (dd, 2H, J ) 2.5 Hz, 2.0 Hz), 7.02 (d, 2H, J ) 5.0 Hz), 4.00 (t, 8H, J ) 5.0 Hz), 2.75 (t, 8H, J ) 5.0 Hz); 13C NMR (125 MHz, CD3OD/CDCl3) δ 173.1, 158.4, 158.0, 156.3, 132.6, 132.3, 131.2, 130.6, 125.5, 115.2, 114.1, 97.5, 77.8, 32.1; IR (neat) ν (cm-1) 1644; MS (ESI) calcd for C31H30BrN2O9+ 653.1129, found 653.1062, 655.1052. Activation of Bromorosamine 4. N-Hydroxysuccinimide (0.003 mmol, 0.36 mg) and DIC (0.004 mmol, 0.47 mg) were added to a solution of 1 (0.003 mmol, 2.0 mg) in dry DMF (0.5 mL) under N2. After stirring for 24 h at 25 °C, the solvent was removed, and the residue was dried under high vacuum. This sample, without purification, was used to label the protein. Labeling of Recombinant Mouse Acyl-CoA Binding Protein (ACBP). A recombinant mouse ACBP as described before (9) was tagged with five histidine (His) residues at its Cterminus, expressed in E. coli, and purified by affinity chromatography on Ni-CAM-Sepharose. Newly synthesized watersoluble rhodamine-based dye 1 was coupled to ACBP by using multiple dye-to-protein molar ratios, and the optimal ratios were 5:1 and 10:1, so that in the end a molecule of protein would be labeled with 0.5-5 dye molecules. Typically, 50 nmoles of ACBP were incubated with 250-500 nmoles of rhodamine derivative 1 in PBS, pH 8.5. Incubation of protein with dye was allowed at room temperature for 1 h, in the dark with gentle stirring. ACBP/dye reaction mixtures were then centrifuged at 13 000 rpm for 20 s in an Eppendorf centrifuge 5415 D to remove possible large particles and aggregates resulting from the coupling reaction. The supernatant containing soluble dyelabeled protein and unbound dye was collected and loaded onto a 20 mL Bio-Gel P-4 (fine, 45-90 µm, wet gel particles) column which had been equilibrated in PBS, pH 7.4, in order to separate the colored protein from unbound dye. The column separated two bands: the first one contained labeled ACBP, while the slower band consisted of unbound dye; this separation was confirmed by reading protein absorbance at 280 nm in all collected fractions. The dye-labeled ACBP was characterized in terms of protein and dye concentrations, as well as dye-toprotein molar ratio. Protein concentration was determined by Bio-Rad protein assay as recommended by the manufacturer. The molecular size of ACBP protein after labeling with 1 was checked by SDS-PAGE in 14% polyacrylamide electrophoresis gel following a Bio-Rad procedure based on Laemmli’s method (10).

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Water-Soluble Rhodamine in Intracellular Imaging

Absorbance and Fluorescence Spectroscopy of RhodamineDye-labeled ACBP. The absorbance properties of rhodamine dye after coupling to ACBP protein were measured by using a Carry 100 Scan UV-visible spectrophotometer from Varian (website at www.varianinc.com), while its fluorescence emission spectra were recorded with a PC1 photon-counting fluorometer from ISS (ISS, Inc., Urbana, IL). Fluorescence Binding Assays. cis-Parinaroyl-CoA, a long acyl-CoA with an 18-carbon chain and 4 conjugated double bonds, that emits with a maximum at 410 nm when it is excited at 320 nm, was used for titration of 0.02-0.2 µM unlabeled and dye-labeled ACBP, as described before (9, 11). Increasing concentrations of cis-parinaroyl-CoA from 2.5 to 500 nM were added to a constant amount of ACBP, and fluorescence emission spectra were recorded at each titration point from 350 to 500 nm, with excitation of cis-parinaroyl-CoA at 320 nm. Controls for unbound ligand (identical concentrations of cis-parinaroyl-CoA but in the absence of protein) and for protein-interfering emission were measured and subtracted. The maximum fluorescence intensities from recorded spectra were then plotted against total concentration of cis-parinaroylCoA at each titration point. Further analysis of these binding curves by nonlinear regression and fitting to ligand binding to one site equations was performed with Sigma Plot 8.0 software, and the values of Kd, i.e., dissociation constant, and Bmax, i.e., maximum binding fluorescence intensity, were then obtained. Circular Dichroism (CD) Analysis of ACBP Before and After Labeling with Dye 1. Far-UV CD spectra of unlabeled and 1-ACBP at a concentration of 4 µM in 20 mM potassium phosphate buffer, pH 7.4, were recorded by the use of a J-710 spectropolarimeter (Jasco, Baltimore, MD). Measurements were done in a 1 mm cuvette, and spectra were recorded from 260 to 190 nm at 50 nm/min with a time constant of 1 s and a bandwidth of 2 nm. For each CD profile, an average of 10 scans was obtained. Percentages of secondary structures in ACBP when unlabeled or 1-labeled were calculated from CD spectra by using the CD-PRO software (12). Cell Culture and Delivery of Dye-1 Labeled ACBP into Cells for Intracellular Imaging. COS-7 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and grown in DMEM with 10% fetal bovine serum (FBS), in 2-well chamber glasses (LabTek from VWR, Suwanee, GA) at 37 °C and 5% CO2. A sample of 1-ACBP was delivered to cells grown to about 80% confluence, according to a modified procedure recommended by Active Motif (Carlsbad, CA) for the use of Chariot transfection reagent. Briefly, dyelabeled ACBP from 500 ng to 2 µg were preincubated with Chariot (from Active Motif, Carlsbad, CA) in protein-to-peptide ratio of 1:20 (w/w) for 30 min at room temperature and then added to COS-7 cells in the presence of serum-free DMEM. After 30 min incubation, the protein-Chariot mixes were washed off cells with PBS, and cells were incubated with 10% FBS/DMEM at 37 °C, 5% CO2 for 2 h before confocal microscopy. Cell nuclei were counterstained by adding 1 µg/ mL Hoechst 33342 nuclear marker solution (from Molecular Probes, Eugene, OR) about 30 min before confocal microscopy observation. Intracellular Imaging of Labeled ACBP By Laser Scanning Confocal Microscopy (LSCM). An MRC1024 confocal imaging system (from Bio-Rad) consisting of an Axiovert 135 microscope (Zeiss, New York) equipped with three independent low-noise photomultiplier tube channels was used for LSCM. The excitation light (λ ) 488 and 568 nm) from a 15 mW krypton-argon laser was delivered to sample through a 63× Zeiss Plan-Fluor oil immersion objective, numerical aperture 1.45. Emission filters 540/30 (allowing

Scheme 1. Synthesis of Dye 1

emission light of 520-530 nm wavelengths) and HQ598/40 (allowing emission light of 580-660 nm wavelengths) were employed to detect fluorescence images in the green (PMT2) and red (PMT1) channels, respectively. Hoechst 33342 nuclear marker was excited at 408 nm by using a solid-state laser (from Power Technology, Little Rock, AR) and detected in the green channel with a 540/30 emission filter in PMT2. Colocalization analysis was achieved by using the LaserSharp v 3.0 software from Bio-Rad, and colocalization coefficients were calculated as described (13). Additional processing of images was performed by using MetaMorph Image Analysis from Advanced Scientific Imaging (Meraux, LA). Electronic Spectra. Figure 3 shows the UV and fluorescence spectra of dye 1 in PBS at pH 7.4. For this solution, the molar extinction coefficient, , was measured to be 91 000 and λmax abs ) 556 nm. It fluoresced at 579 nm with a quantum yield of 0.76. Fluorescence quantum yield measurements were measured on a photon counting spectrofluorometer PC1 SSI instrument equipped with an R928P photomultiplier tube which is sensitive up to ∼850-900 nm. The slit width was 0.5 nm for both excitation and emission. Relative quantum efficiencies of fluorescence of dye 1 were obtained by comparing the areas under the corrected emission spectrum of the test sample in PBS buffer (pH ) 7.4) with that of a solution of rhodamine B, which has a quantum efficiency of 0.97 according to the literature (14). DI water for the PBS buffer and a 10 mm quartz cuvette were used. Dilute solutions (0.01 < A < 0.05) were used to minimize reabsorption effects. The excitation wavelength was 556 nm for both compound 1 and the reference. Quantum yields were determined using eq 1 (15)

ΦX ) Φst(IX/Ist)(Ast/AX)(ηX2/ηst2)

(1)

where Φst is the reported quantum yield of the standard, I is the integrated emission spectra, A is the absorbance at the excitation wavelength, and η is the refractive index of the solvent used (η ) 1 if same solvent). The X subscript denotes unknown, and “st” denotes standard.

RESULTS Synthesis. Scheme 1 summarizes the synthesis of compound 1. Double Michael addition of acrylic acid to 3-aminophenol


via a literature procedure (7) gave the diacid as a precipitate. This material, without purification, was esterified to give the diester 2 which could be isolated on a multigram scale via extraction with ethyl acetate. The next step in the synthesis was to condense the phenol 2 with an aldehyde to give the rosamine framework. Such syntheses usually feature protic acid media (16); however, we found that the reaction proceeds without solvent or added catalysts. We were unable to identify reaction conditions for this condensation that featured heating under conventional conditions. However, in a microwave reactor specifically designed for holding reactions at a set temperature (Discover, CEM Corporation), the reaction was found to proceed with good conversion in only 20 min. Chloranil was added to oxidize the initial condensation product to the rosamine tetraester 3, which was then isolated by column chromatography. This tetraester was then hydrolyzed to the desired product 1. Crude samples of the rosamine 1 with quite good purity could be isolated via an acid-base extraction procedure. Pure material was isolated via preparative RP-HPLC to give a 22% yield of the product. Selected data for compounds 1 and 3 are shown in Figure 2. The dye is highly soluble in aqueous buffer at pH 7.4. Electronic spectra of compound 1 showed several desirable characteristics. The dye absorbs strongly at 556 nm and emits with a high quantum yield at 579 nm in aqueous buffer. Variation of the concentration of the dye in the buffer showed that the emission maxima wavelength did not shift; this indicates that the dye is not aggregated in this media (Figure 3). Monoactivation of tetraacid 1 to give the reactive ester 4 was performed by employing DIC and N-hydroxysuccinimide in DMF (Scheme 2). Compound 4 was used, without purification, to tag with an illustrative protein, as described below. The unimolecular binding of dye 1 to protein is confirmed by MALDI-TOF (Figure 3). Spectral Properties of Dye 1 After Conjugation to the Illustrative Protein: Acyl-CoA Binding Protein (ACBP). ACBP was chosen to test the possibility of using the newly synthesized water-soluble rhodamine derivative for intracellular imaging by laser scanning confocal microscopy. A recombinant mouse ACBP was purified and conjugated to dye 1 as described under Experimental Procedures. Tests to ensure that the basic physical and functional properties of fluorescent dyes were maintained after conjugation to the protein included absorbance and fluorescence spectra on the labeled protein and compared with dye 1. Figure 4 shows the absorbance spectra of 1-ACBP and the dye only, in PBS pH 7.4. The wavelength corresponding to maximum absorbance for the conjugate was slightly shifted from 553 nm for the dye only, to 560 nm for 1-ACBP conjugate. The fluorescence emission spectra of 1 either free in PBS or conjugated to ACBP protein demonstrated that the maximum fluorescence wavelength exhibited a small red shift from 574 nm for free dye 1 to 583 nm for ACBP-coupled dye 1 when excitation was at 568 nm. Physical and Functional Properties of Acyl-CoA Binding Protein (ACBP) After Conjugation to Dye 1. The structural and functional properties of the protein were also tested after conjugation to dye 1. Thus, SDS-polyacrylamide gel electrophoresis, fluorescence binding assays, and circular dichroism were performed on the 1-ACBP product and compared to unlabeled ACBP. An SDS-PAGE test demonstrated that the molecular size of 1-ACBP was very similar to that of ACBP (not shown). The small increase in molecular weight of ACBP after labeling was due to the additional weight of dye molecules that had been covalently attached to the protein by conjugation. Most importantly, the SDS-PAGE assay demonstrated that no intermolecular cross-linking occurred as a negative side effect during protein-

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Figure 2. Data for 1 and 3. (a) Electrospray MS showing molecular ion peak for 3; (b) analytical RP-HPLC (C18, 5% CH3CN in H2O and 0.1% TFA, tR ) 3.21 min) for 1; (c) electrospray MS showing molecular ion peak for 1. Scheme 2. Monoactivation of Dye 1

dye reaction. This result was possible by purposely activating dye 1 once to contain a single reactive group available for conjugation to amino groups on the surface of ACBP protein. One of the most important functions of ACBP is to bind medium- and long-chain fatty acyl-CoA thioesters with high affinity (i.e., low-range nanomolar dissociation constant, Kd). To check if this property was maintained after labeling, the recombinant ACBP was assayed by titration with a fluorescent ligand, namely, cis-parinaroyl-CoA up to saturation of binding, as described under Experimental Procedures. The binding curves as shown in Figure 5a,b were obtained by plotting fluorescence intensities versus total concentration of ligand (cis-parinaroylCoA) at each titration point, followed by nonlinear regression analysis and calculation of Kd and Bmax (maximum fluorescence intensity at binding saturation). The labeled 1-ACBP exhibited cis-parinaroyl-CoA binding to saturation, just as the unlabeled ACBP did, with a Kd of 6.3 ( 2.7 nM; this was a slightly higher affinity for cis-parinaroyl-CoA than unlabeled ACBP (Kd of 11.3 ( 2.8 nM). Circular dichroism (CD) spectra in the far UV were recorded to get more information about the structural and conformational



Figure 3. Mass spectra of free and labeled His-ACBP-1 and electronic spectra of 1 in PBS buffer (pH ) 7.4). (a) (1) MALDI-TOF of free His-ACBP [M+ + X.K+/Na+(10 928.20)]; (2) MALDI-TOF for His-ACBP-1 [MH+ + X.K+/Na+ (11 565.77) (before conjugate purification)]; (b) normalized UV spectrum at concentration 10-4 M, 91 000, λmax abs ) 556 nm; (c) fluorescence spectrum at 10-5 M, λmax emis ) 579 nm, using 556 nm excitation wavelength; (d) the fluorescence emission wavelength of compound 1 does not vary with concentration.

Figure 4. Absorbance and fluorescence spectra of free and 1-ACBP-bound in PBS at pH 7.4. (a) UV-vis spectra of free dye is shown in red line and that of ACBP-bound dye in black line; (b) emission spectra of 1-ACBP.

changes caused by dye 1 on ACBP. Figure 5c shows the CD spectra of ACBP and 1-ACBP. The two spectra are similar, suggesting that only small changes in the secondary structure of ACBP were introduced by coupling it to dye 1. Intracellular Imaging of 1-ACBP. COS-7 cells were incubated with 1-ACBP that had been labeled with dye 1 in

the presence of the Chariot system (17) to test whether the labeled protein could be imported into cells in this way. If free 1-ACBP is internalized in the cells, and functions in the same way as the unlabeled protein, then some of it should localize in the nucleus (18). The cell nuclei were counterstained with Hoechst 33342 dye to test for this. Figure 6a shows fluorescence


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Figure 5. (a) Binding curve for ACBP; (b) binding curve for 1-ACBP; (c) circular dichroic (CD) spectra of unlabeled ACBP (black circles) and 1-ACBP (white triangles).

Figure 6. Intracellular imaging of 1-ACBP labeled COS-7 cells by confocal microscopy. (A) 1-ACBP with excitation at 568 nm, and emission filter HQ 598/40; (B) overlay of 1-ACBP image (red) with nuclei image (green, given by the nuclear marker Hoechst 33342 that emits at 530 nm with excitation at 408 nm); (C) colocalized pixels (yellow) from 1-ACBP and the nuclear marker; and (D) fluorograph indicating the extent of colocalization.

images of 1-ACBP in cells (detected in the red channel, with an emission filter at 598 nm, excitation at 568 nm). In Figure 6b, fluorescence signals from 1-ACBP (in red) and Hoechststained nuclei (in green) are shown to demonstrate the distribution of ACBP molecules inside cells relative to nuclei. Most of the fluorescently labeled ACBP molecules appeared in the cytoplasm, around nuclei, but a fraction of the 1-ACBP molecules were observed inside nuclei. This demonstrates that the Chariot-mediated import of 1-ACBP was successful and that some of the labeled protein is gravitating to the region of the cell (the nucleus) that is expected from its postulated function (18). Yellow pixels in Figure 6c represent the colocalization of 1-ACBP with the nuclear marker. The fluorogram in Figure 6d shows a more quantitative colocalization analysis for the relative distribution and fluorescence intensities of the two signals (green and red).

water solubility and to facilitate coupling to proteins. It can be selectively coupled to just one protein molecule without significant cross-linking to a second one. In the particular case of the protein ACBP, the data presented here show that conjugation of this model probe does not significantly perturb the secondary structure or the natural binding function of the protein in vitro. Moreover, 1-ACBP can be imported into liVing cells using the novel Chariot-peptide carrier system wherein it accumulates, at least partially, in the nucleus as expected. Though the work presented here does not actually demonstrate this, it is clear that organometallic coupling reactions could be used to add different molecular entities to the aryl bromide functionality of compound 1. If this were done, a variety of “bifunctional” labels might be prepared. They too might be imported into living cells via the Chariot system, then studied in vivo.

DISCUSSION

ACKNOWLEDGMENT

This paper reports the synthesis of a new water-soluble dye, 1. This compound has four carboxylic acid groups to increase

We thank Mr. Jiney Jose for developing a procedure to prepare compound 2, CEM for support with the Discover unit



used in this research, and the TAMU/LBMS-Applications Laboratory directed by Dr. Shane Tichy for assistance with mass spectrometry. Support for this work was provided by The NIH (GM72041) and by The Robert A. Welch Foundation A-1121.

LITERATURE CITED (1) Weissleder, R., and Ntziachristos, V. (2003) Shedding light onto live molecular targets. Nat. Med. 9, 123-8. (2) Benson, R. C., Meyer, R. A., Zaruba, M. E., and McKhann, G. M. (1979) Cellular autofluorescence - is it due to flavins? J. Histochem. Cytochem. 27, 44-8. (3) Miyaura, N., and Suzuki, A. (1995) Palladium-catalyzed crosscoupling reactions of organoboron compounds. Chem. ReV. 95, 2457-83. (4) Sonogashira, K., Tohda, Y., and Hagihara, N. (1975) A convenient synthesis of acetylenes: catalytic substitutions of acetylenic hydrogen with bromoalkenes, iodoarenes, and bromopyridines. Tetrahedron Lett. 16, 4467-70. (5) Burghart, A., Thoresen, L. H., Chen, J., Burgess, K., Bergstro¨m, F., and Johansson, L. B.-A. (2000) Energy transfer cassettes based on BODIPY dyes. Chem. Commun. 2203-4. (6) Jiao, G.-S., Thoresen, L. H., and Burgess, K. (2003) Fluorescent, through-bond energy transfer cassettes for labeling multiple biological molecules in one experiment. J. Am. Chem. Soc. 125, 14668-9. (7) Mitskyavichyus, V. Y., and Bilinskaite, I. (1996) Alkyl derivatives of 1-aryldihydro-4(1H,3H)-pyrimidinone-2-thiones and their structure. Khim. Geterotsikl. Soedin. 1255-60. (8) Jose, J., and Burgess, K. (2006) Syntheses and Properties of Watersoluble nile red derivatives. J. Org. Chem. in press. (9) Chao, H., Martin, G., Russell, W. K., Waghela, S. D., Russell, D. H., Schroeder, F., and Kier, A. B. (2002) Membrane charge and curvature determine interaction with acyl CoA binding protein (ACBP) and fatty acyl CoA targeting. Biochemistry 41, 10540-53.

(10) Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. (11) Frolov, A. A., and Schroeder, F. (1998) Acyl coenzyme A binding protein: conformational sensitivity to long chain fatty acyl-CoA. J. Biol. Chem. 273, 11049-55. (12) Sreerama, N., and Woody, R. W. (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal. Biochem. 287, 252-60. (13) Manders, E. M. M., Verbeek, F. J., and Aten, J. A. (1993) Measurement of colocalization of objects in dual-colour confocal images. J. Microsc. 169, 375-82. (14) Vincett, P. S., Voigt, E. M., and Rieckhoff, K. E. (1971) Phosphorescence and fluorescence of phthalocyanines. J. Chem. Phys. 55, 4131-40. (15) Williams, A. T. R., Winfield, S. A., and Miller, J. N. (1983) Relative fluorescence quantum yields using a computer-controlled luminescence spectrometer. Analyst (Cambridge, U.K.) 108, 106771. (16) Jiao, G.-S., Castro, J. C., Thoresen, L. H., and Burgess, K. (2003) Microwave-assisted syntheses of regioisomerically pure bromorhodamin derivatives. Org. Lett. 5, 3675-7. (17) Morris, M. C., Depollier, J., Mery, J., Heitz, F., and Divita, G. (2001) A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19, 1173-6. (18) Petrescu, A. D., Payne, H. R., Boedeker, A. L., Chao, H., Hertz, R., Bar-Tana, J., Schroeder, F., and Kier, A. B. (2003) Physical and functional interaction of acyl CoA binding protein (ACBP) with hepatocyte nuclear factor-4R (HNF4R). J. Biol. Chem. 278, 51813. BC0601424

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