Bioconjugate Chem. 2007, 18, 1004−1009
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Testing Oligothiophene Fluorophores under Physiological Conditions. Preparation and Optical Characterization of the Conjugates of Bovine Serum Albumin with Oligothiophene N-Hydroxysuccinimidyl Esters Massimo Zambianchi,†,* Andrea Barbieri,† Alfredo Ventola,‡ Laura Favaretto,† Cristian Bettini,‡ Matteo Galeotti,‡ and Giovanna Barbarella†,* Istituto per la Sintesi Organica e la Fotoreattivita` (ISOF), Consiglio Nazionale delle Ricerche (CNR) and Mediteknology srl, Via Piero Gobetti 101, 40129 Bologna, Italy. Received October 24, 2006; Revised Manuscript Received January 22, 2007
Bovine serum albumin (BSA) was reacted with linear and newly synthesized branched oligothiophene N-hydroxysuccinimidyl ester fluorophores (TSEs) in moderately basic carbonate buffer solution. Optically stable BSA-TSE conjugates were obtained with a degree of labeling depending on experimental conditions. Conjugates with high fluorophore to BSA ratios (F/BSA ) 8) displayed fluorescence quantum yields in the range of 1030% in water at pH ) 7.2, comparable to the quantum yield (25%) of the BSA-FITC conjugate prepared under the same conditions and with the same degree of labeling.
INTRODUCTION Currently, there is a wide interest in polythiophenes as optical reporters of events of biological interest (1-4). Indeed, besides being important semiconductor materials, thiophene oligomers and polymers may also be strongly fluorescent compounds (5), and their optical properties are very sensitive to conformational changes induced by the environment (6). Charged polythiophenes have been used for polynucleotide detection, DNA hybridization, and sequence characterization, showing high selectivity and sensitivity useful for applications in medical diagnostics (1-4). The fluorescence properties of charged polythiophenes have been used for the specific detection of nucleic acids in sub-micromolar concentrations (7). Furthermore, it has been demonstrated that the optical phenomena observed in solution upon interaction of polynucleotides with charged polythiophenes can also be induced on surfaces, indicating that there is room for the development of inexpensive polythiophenebased gene chips for DNA detection (3). Our interest in the field of biodiagnostics has brought us to develop new classes of thiophene-based fluorophores emitting light in the visible range and characterized by remarkable optical stability and fluorescence intensity (8, 9). In particular, we have developed a rapid and high yield synthetic pattern for the preparation of oligothiophene N-hydroxysuccinimidyl esters and demonstrated that these fluorophores can be covalently linked to biological molecules and can be used to image specific antibody-antigen interactions in tumor cells by fluorescence microscopy or monitor polynucleotides hybridization by fluorescence spectroscopy methods (9). Development of innovative fluorescent probes is highly required by the increasing number of applications of fluorescence techniques in biotechnologies (10, 11). Fluorescence is a widely used readout modality employed in medical diagnostics, cellular imaging, microarrays, etc. and there is a recognized shortage of efficient fluorophores for biological applications. * Authors to whom the correspondence should be adressed. E-mail:
[email protected]. † Istituto per la Sintesi Organica e la Fotoreattivita` (ISOF). ‡ Mediteknology srl, Consiglio Nazionale Ricerche, Via Piero Gobetti 101, 40129 Bologna, Italy.
Developing new markers as probes for protein binding, DNA hybridization, immunoistochemistry, or other applications requires detailed knowledge on the spectroscopic and chemical properties of fluorophore-biopolymer conjugates to provide a basis for such applications. Newly developed markers should be easy to handle and to couple to biopolymers, be highly fluorescent under aqueous conditions, be resistant to photobleaching, and have large Stokes shifts between emission and absorption to facilitate fluorescence detection. The fluorophores should also be available in a great number of shades for multilabeling experiments and should be easy to couple to biopolymers with the same standard chemical methodology in all colors. Moreover, it is greatly desirable that all colors be excitable by means of the same light source. However, developing new fluorophores with all these characteristics at the same time is challenging, mainly because the optical response to excitation of a given molecular structure is not easy to predict. In this respect, thiophene oligomers are intriguing compounds, since the balance between radiative and nonradiative deexcitation pathways is greatly affected by the nature and the position of substituents, oligomers size, environment, etc. (6, 12, 13). We report here a study on the optical properties of a set of newly synthesized oligothiophene N-hydroxysuccinimidyl esters (TSEs), together with the preparation and the optical characterization of the conjugates of linear and branched TSEs with bovine serum albumin (BSA). BSA, a major component in plasma protein, is one of the cheapest commercially available proteins and has been used as a model in a variety of studies (14-16). In the context of the present investigation, BSA is used to test the amino-reactive TSEs in the formation of bioconjugates in aqueous solvents and assess their fluorescence properties in physiological conditions.
MATERIALS AND METHODS General Details. Bovine serum albumin (BSA), 2-bromothiophene (3a), succinic anhydride, aluminum chloride (AlCl3), N-hydroxysuccinimide (HOSu), N,N′-dicyclohexylcarbodiimide (DCC), N-bromosuccinimide (NBS), tris(dibenzylideneacetone)dipalladium(0)-chloroform adduct, Tween-20, and triphenylarsine are commercially available compounds.
10.1021/bc060332e CCC: $37.00 © 2007 American Chemical Society Published on Web 03/23/2007
Technical Notes Scheme 1
a
a
Reagents and conditions: (i) (I) Succinic anhydride, AlCl3, (II) aq. HCl 4N; (ii) HOSu, DCC; (iii) Pd(AsPh3)4.
Scheme 2
a
Bioconjugate Chem., Vol. 18, No. 3, 2007 1005
a
Reagents and conditions: (i) Pd(AsPh3)4; (ii) NBS.
Synthesis of Fluorophores. The synthesis of compounds 1 and 5 has already been reported (9). The synthesis of compound 3 was performed as shown in Scheme 1. The synthesis of compounds 2, 4, 6, 7 was performed as shown in Scheme 2. 4-(3′-Methylsulfanyl-[2,2′]bithiophenyl-5-yl)-4-oxo-butyric Acid 2,5-Dioxo-pyrrolidin-1-yl Ester (3). Yield: 55%. Pale yellow solid, mp 153-155 °C. EI-MS m/z 409 (M+). 1H NMR (CDCl3, TMS/ppm) δ 7.69 (d, 3J ) 4.0 Hz, 1H), 7.35 (d, 3J ) 4.0 Hz, 1H), 7.31 (d, 3J ) 4.8 Hz, 1H), 7.07 (d, 3J ) 4.8 Hz, 1H), 3.36 (t, 3J ) 6.8 Hz, 2H), 3.10 (t, 3J ) 6.8 Hz, 2H), 2.84 (s, 4H), 2.51 (s, 3H); 13C NMR (CDCl3, TMS/ppm) δ 189.1, 168.9, 168.2, 144.2, 141.4, 132.8, 132.3, 131.8, 130.4, 126.2, 125.5, 33.3, 25.6, 25.4, 18.6. Anal. Calcd for C17H15NO5S3 (409.50): C, 49.86; H, 3.69. Found: C, 49.95; H, 3.81. 3′-Methylsulfanyl-[2,2′]bithiophenyl-5-carboxylic Acid 2,5dioxo-pyrrolidin-1-yl Ester (2). Yield: 71%. Microcrystalline bright yellow solid, mp 131 °C; EI-MS m/z 353 (M+); 1H NMR (CDCl3, TMS/ppm) δ 7.94 (d, 3J ) 4.4 Hz, 1H), 7.38 (d, 3J ) 4.4 Hz, 1H), 7.34 (d, 3J ) 5.2 Hz, 1H), 7.09 (d, 3J ) 5.2 Hz, 1H), 2.90 (s, 4H), 2.51 (s, 3H); 13C NMR (CDCl3, TMS/ppm) δ 169.1, 157.4, 145.9, 136.5, 133.2, 131.7, 130.7, 126.1, 125.8, 125.1, 25.6, 18.7. Anal. Calcd for C14H11NO4S3 (352.99): C, 47.58; H, 3.14. Found: C, 47.66; H, 3.23. 3′’-Methylsulfanyl-[2,2′;5′,2′′]terthiophene-5-carboxylic Acid 2,5-Dioxo-pyrrolidin-1-yl Ester (4). Yield: 80%. Microcrystalline bright orange solid, mp 156 °C; EI-MS m/z 435 (M+); 1H NMR (CDCl , TMS/ppm) δ 7.92 (d, 3J ) 4.0 Hz, 1H), 7.29 3 (d, 3J ) 4.0 Hz, 1H), 7.28 (d, 3J ) 4.0 Hz, 1H), 7.25 (d, 3J ) 5.2 Hz, 1H), 7.24 (d, 3J ) 4.0 Hz, 1H), 7.06 (d, 3J ) 5.2 Hz, 1H), 2.90 (s, 4H), 2.50 (s, 3H); 13C NMR (CDCl3, TMS/ppm) δ 169.1, 157.1, 147.6, 137.6, 137.4, 135.2, 132.9, 130.7, 130.6, 126.8, 126.1, 124.3, 124.1, 124.0, 25.6, 18.8. Anal. Calcd for C18H13NO4S4 (434.97): C, 49.64; H, 3.01. Found: C, 49.73; H, 3.07. 5′-Bromo-3′-methylsulfanyl-[2,2′]bithiophenyl-5-carboxylic Acid 2,5-Dioxo-pyrrolidin-1-yl Ester (2b). Under exclusion of light N-bromosuccinimide (0.33 g, 1.87 mmol) was added stepwise to a solution of 2 (0.60 g, 1.70 mmol) in acetic acid/methylene chloride 1:1 (30 mL). The mixture was left to stir overnight and then quenched with ice. After separation of layers, the aqueous phase was extracted with dichloromethane. The resulting organic layers were washed with potassium hydroxide (2 × 50 mL, 10% aqueous) and then dried over anhydrous sodium sulfate. Evaporation of the solvent under reduced pressure gave
the crude product, which was isolated by flash chromatography on silica with increasing amounts of ethyl acetate in petroleum ether as eluent. Yield: 0.47 g (64%). Polycrystalline yellow solid; EI-MS m/z 432 (M+); 1H NMR (CDCl3, TMS/ppm) δ 7.13 (d, 3J ) 4.0 Hz, 1H), 7.27 (d, 3J ) 4.0 Hz, 1H), 7.04 (s, 1H), 2.90 (s, 4H), 2.50 (s, 3H). 5′′-Methylsulfanyl-3′-methylsulfanyl-[2,2′;5′,2′′]terthiophene5-carboxylic Acid 2,5-Dioxo-pyrrolidin-1-yl Ester (6). To a 5 mL dry toluene solution containing 0.0025 mmol of tetrakis(triphenylarsine)palladium(0) prepared in situ was added compound 2b (22 mg, 0.05 mmol) in toluene (2 mL). The mixture was heated to 80 °C, and 2-tributylstannyl-5-(methylthio)thiophene (21 mg, 0.05 mmol) dissolved in 2 mL of toluene was added dropwise. The reaction mixture was stirred for 4 h at this temperature, and then the solvent was removed under reduced pressure. The crude mixture was purified by flash chromatography (silica gel, petroleum ether/ ethyl acetate 1:1) to provide 16 mg (66% yield) of the title product as microcrystalline yellow ocher solid, mp 106-108 °C; EI-MS m/z 481 (M+); 1H NMR (CDCl3, TMS/ppm) δ 7.93 (d, 3J ) 4.0 Hz, 1H), 7.34 (d, 3J ) 4.0 Hz, 1H), 7.06 (d, 3J ) 4.0 Hz, 1H), 7.05 (s, 1H), 6.98 (d, 3J ) 4.0 Hz, 1H), 2.90 (s, 4H), 2.54 (s, 3H), 2.53 (s, 3H); 13C NMR (CDCl3, TMS/ppm) δ 169.2, 157.4, 145.5, 138.5, 137.2, 136.9, 136.5, 134.2, 131.3, 130.1, 126.3, 125.8, 125.0, 124.9, 25.8, 21.8, 18.7. Anal. Calcd for C19H15NO4S5 (481.65): C, 47.38; H, 3.14. Found: C, 47.61; H, 3.22. 3′′′-Methylsulfanyl-[2,2′;5′,2′′;5′’,2′′′]quaterthiophene-5-carboxylic Acid 2,5-Dioxo-pyrrolidin-1-yl Ester (7). Yield: 72%. Microcrystalline red-orange solid, mp 195 °C; EI-MS m/z 517 (M+); 1H NMR (CDCl3, TMS/ppm) δ 7.92 (d, 3J ) 4.0 Hz, 1H), 7.27 (d, 3J ) 4.0 Hz, 1H), 7.26 (d, 3J ) 4.0 Hz, 1H), 7.21 (d, 3J ) 5.2 Hz, 1H), 7.20 (d, 3J ) 4.0 Hz, 1H), 7.16 (d, 3J ) 4.0 Hz, 1H), 7.15 (d, 3J ) 4.0 Hz, 1H), 7.04 (d, 3J ) 5.2 Hz, 1H), 2.89 (s, 4H), 2.50 (s, 3H); 13C NMR (CDCl3/CS2, TMS/ ppm) δ 168.6, 156.8, 147.2, 139.1, 137.3, 136.2, 135.6, 134.0, 133.4, 130.6, 130.0, 127.0, 126.6, 124.5, 124.3, 124.1, 124.0, 123.8, 25.6, 18.8. Anal. Calcd for C22H15NO4S5 (516.96): C, 51.04; H, 2.92. Found: C, 51.27; H, 3.05. General Procedure for the Preparation of BSA-TSE Conjugates. In a 2 mL microtube with screw cap was dissolved the appropriate amount of BSA (calculated on the basis of a 1:15 BSA/fluorophore molar ratio) in a 0.05 M buffer carbonate/ bicarbonate solution (1 mL, pH 9.3) at room temperature. Tween-20 (0.5%, w/v) was added to the buffered solution to
1006 Bioconjugate Chem., Vol. 18, No. 3, 2007
promote the solubility of the fluorophore in the aqueous medium. Then 100 µL of a dry DMF solution containing the fluorophore dissolved in concentration 2.5 mg/mL was added slowly to the BSA solution with gentle swirling, care being taken to avoid foaming. The microtube was then put on a rotating plate mixer (25 rpm), and conjugation was allowed to proceed at room temperature (21-24 °C) with continuous mixing for 30 min. The unreacted fluorophore was removed by passing the conjugate mixture through a Sephadex column (G-25, medium grade), previously equilibrated using phosphate-buffered saline (PBS, 80 mL, pH 7.4). Using this buffer as eluent, 500 µL fractions were collected at room temperature. All fractions were analyzed using a Perkin-Elmer spectrophotometer Lamda 20 for reading the absorbances. The conjugate was found in fractions 6-8 that were pooled in a 2 mL micro tube. Since the absorbance of these fractions was sometimes above 2, a 500 µL aliquot of the pooled solution was diluited 1:10 with PBS to give measurable readings. Before storing the solutions of the conjugates at 4 °C, sodium azide (0.1%, w/v) was added to inhibit bacterial growth. The protein content and the degree of labeling were determined by spectroscopic measurements as described (17). Absorption and Photoluminescence Measurements. The absorption spectra of dilute solutions of TSEs alone and of their conjugates with BSA were obtained with a Perkin-Elmer Lambda 45 UV/vis spectrophotometer. The luminescence spectra were measured at room temperature using a Spex Fluorolog II spectrofluorimeter, equipped with a Hamamatsu R928 phototube. Air-equilibrated sample solutions were excited at the indicated wavelength, and the concentration was adjusted to obtain absorption values e0.15 at the excitation wavelengths. While uncorrected luminescence band maxima were used throughout the text, corrected spectra were employed for the determination of the luminescence quantum yields (φ). The correction procedure was based on the use of a software taking care of the wavelength dependent response of the phototube. Luminescence quantum efficiencies (φem) were evaluated by comparing wavelength integrated intensities (I) with reference to quinine sulfate as the standard (φr ) 0.546 in air-equilibrated 1 N H2SO4) and by using the following equation:
φem )
Zambianchi et al. Table 1. Effect of BSA to Fluorophore Molar Ratio, Reaction Time, pH, and Fluorophore Concentration on the Yields of Formation of the BSA-1 Conjugate in Phosphate-Buffered Solution BSA-fluorophore molar ratio (60 min, pH 9.3, fluorophore 10 mg/mL)
F/BSA ratioa
1:20 1:15 1:10 1:5
4.9 4.4 4.8 2.6
pH (30 min, 1:15, molar ratio fluorophore 10 mg/mL)
F/BSA ratioa
8.1 8.5 9.0 9.3
1.1 1.8 2.9 4.0
a
reaction time (1:15 molar ratio, pH 9.3, fluorophore 10 mg/ mL)
F/BSA ratioa
120 min 60 min 45 min 30 min 15 min
5.4 4.4 4.0 3.7 3.5
fluorophore concentration (30 min, pH 9.3, 1:15 molar ratio)
F/BSA ratioa
20 mg/mL 10 mg/mL 5 mg/mL 2.5 mg/mL 1.25 mg/mL
3.2 3.3 5.9 8.1 7.2
Expressed in moles of fluorophore per mole BSA.
Table 2. Molecular Structure, Maximum Absorption (λmax, nm) and Emission (λPL, nm) Wavelengths, and Molar Absorption Coefficients (E, cm-1 M-1) of Oligothiophene Fluorophores 1-7a and λmax, λPL Values of the Corresponding Conjugates with BSAb
In2 ODr ‚ ‚φ OD I n 2 r r r
where OD and n are absorbance values at the employed excitation wavelength, and refractive index of the solvent, respectively. Band maxima and relative luminescence quantum yields were obtained with uncertainty of 2 nm and 20%, respectively. The luminescence lifetimes (τ) were obtained with an IBH single photon counting equipment by using pulsed diode NanoLED sources with excitation at 278 and 331 nm. Analysis of the luminescence decay profiles against time was accomplished by using software provided by the manufacturers, with an estimated error on the lifetime results of 10%.
RESULTS The molecular structures of all oligothiophene N-hydroxysuccinimidyl esters employed in this study are reported in Table 2 (see below). The tuning of the emission color from blue to orange was obtained by changing the oligomer size from dimer to tetramer and the number and the position of methylsulfanyl (SCH3) substituents. All fluorophores were soluble in DMF, while solubility in water was achieved by addition of a small amount (90/% efficiency of FITC in alkaline aqueous solutions (22). However, the flexibility of conjugation conditions of TSEs illustrated in Table 1 should allow the optimization, case by case, of the labeling degree of proteins with oligothiophene fluorophores to minimize self-quenching phenomena. Work is currently under way in this direction to establish the dependence of the fluorescence quantum yield of TSEs on the degree of labeling. It is worth noting that what is important in the present context is that the molar absorption coefficients of TSEs fluorophores and their fluorescence quantum yields are sufficiently high to allow the easy detections of protein-fluorophore conjugates in micromolar and even lower concentrations. It is also to note that the fluorophores are characterized by linewidths on the order of 100 nm and by very large Stokes shifts between absorption and emission signals. These large Stokes shifts are even increased in the conjugates with proteins with respect to the free fluorophores. Moreover, as already observed in the case of oligothiophene isothiocyanates (8), the fluorophores show high resistence to photobleaching.
CONCLUSION We have shown that oligothiophene N-succinimidyl esters, built using a few repeating and simple structural elements, i.e., thienyl and R or β SCH3 substituents and characterized by efficient emission in the blue-orange interval, can easily be bound to BSA achieving high fuorophore to protein ratios. The fluorophore to protein ratio can be controlled by an accurate choice of the experimental conditions. BSA-TSE conjugates in water in physiological conditions display fluorescence quantum yields that are comparable to that of BSA-FITC conjugates in similar conditions. Considering the synthetic versatility of thiophene (5), it is easy to predict that thiophenebased fluorophores even more efficient than those described here will rapidly be developed.
ACKNOWLEDGMENT This work was partially supported by the project “Synthesis of novel organic materials and supramolecular architectures for high efficiency optoelectronic and photonic systems” (SYNERGY, FIRB RBNE03S7XZ_005) and by CNR Projects PM.P04.010 and “Functional organic materials for Hi-Tech applications” (MAFO-HT). Thanks are also due to Mediteknology srl for providing some of the compounds used in this study.
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