Synthesis and spectroscopic characterisation of

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Materiais Orgânicos, Av. Bento Gonçalves, 9500. CP 15003, CEP ..... 30 A. U. Acu ˜na, F. Amat-Guerri, A. Costela, A. Douhal, J. M. Figueira,. F. Florido and R.
FULL PAPER

PPS

Fabiano Severo Rodembusch,a Fernando Paulus Leusin,a Luis Fernando da Costa Medina,b Adriano Brandellib and Valter Stefani*a a Universidade Federal do Rio Grande do Sul - Instituto de Qu´ımica, Laborat´orio de Novos Materiais Orgˆanicos, Av. Bento Gonc¸alves, 9500. CP 15003, CEP 91501-970, Porto Alegre-RS, Brazil. E-mail: [email protected]; Fax: +55 (51) 33 16 73 04; Tel: +55 (51) 33 16 62 85; Web: www.iq.ufrgs.br/lnmo b Universidade Federal do Rio Grande do Sul, Departamento de Ciˆencia dos Alimentos, Laborat´orio de Bioqu´ımica e Microbiologia Aplicada, Av. Bento Gonc¸alves, 9500. CEP 91501-970, Porto Alegre-RS, Brazil

www.rsc.org/pps

Synthesis and spectroscopic characterisation of new ESIPT fluorescent protein probes

Received 17th June 2004, Accepted 5th January 2005 First published as an Advance Article on the web 20th January 2005

Three new benzazole isothiocyanate fluorescent dyes, 2-(4 -isothiocyanate-2 -hydroxyphenyl)benzoxazole, 2-(4 -isothiocyanate-2 -hydroxyphenyl)benzothiazole and 2-(4 -isothiocyanate-2 -hydroxyphenyl)benzimidazole were synthesised, purified until optical purity grade and characterised by spectroscopic techniques. UV/VIS and steady-state fluorescence were also applied to characterise the photophysical behaviour of the dyes. These dyes exhibit an intense fluorescence emission with a large Stokes shift, inherent to the class of benzazoles which relax by the excited state intramolecular proton transfer (ESIPT) mechanism. The dyes were studied for labeling bovine serum albumin (BSA), resulting conjugates BSA-dye with a remarkable photostability under UV/VIS radiation in relation to classical protein labels. The resulting conjugates presented fluorescence in the blue–green region. Direct fluorescence detection of protein-labeled with those dyes after polyacrylamide gel electrophoresis indicates their potential use as fluorescent probes for proteins.

1 Introduction Although some biomolecules present an intrinsic fluorescence, which allows some investigations by fluorescence measurements,1–3 many biological systems are not fluorescent or present a very weak fluorescence. Organic fluorescent dyes, as coumarin derivatives, fluorescein isothiocyanate and others have been synthesised and conjugated to biological system to act as fluorescent probes.4–9 Some dyes present very good results regarding the fluorescence sensitivity at very low concentration.9–11 They are useful tools in immunofluorescence and immunofluorometric assays,10–13 detection of compounds in HPLC10,14 and capillary electrophoresis.10,15,16 Benzazole isothiocyanates were also used successfully as protein probes.17 The high intensity of fluorescence emission and the large Stokes shift due to an intramolecular proton transfer phenomena18–24 (Fig. 1) allows these dyes to have many interesting applications.25–34

In this paper we present the synthesis and characterization of three new fluorescent dyes and their use to label bovine serum albumin (BSA) in a similar approach as presented in our previous paper.17 The new benzazoles isothiocyanates present fluorescence emission in a different region than those presented in the previous work, are more soluble and the methodology to prepare the conjugates was different. A detailed photophysical study of the dyes and conjugates was also performed.

2 2.1

DOI: 10.1039/b409233c 254

Photochem. Photobiol. Sci., 2005, 4, 254–259

Biological and chemical materials

Reagent grade thiophosgene (Aldrich) was used as received. 2(4 -amino-2 -hydroxyphenyl)benzazoles were prepared according to a methodology previously described.35 Silicagel 60 (Merck) was used for chromatographic column separations. Fluorescein isothiocyanate were from Aldrich. Spectroscopic grade solvents (Merck) were used for fluorescence and UV/VIS measurements. All other reagents and solvents were from Aldrich or Merck and were used as received. BSA fraction V was purchased from Sigma Chemical Co. SephadexR G-50 was from Pharmacia Biotech AB. 2.2

Fig. 1 ESIPT mechanism for the benzazole dyes.

Experimental

Methods and instruments

Melting points were measured with a Thermolyne and are uncorrected. Infrared spectra were recorded on a Mattson Galaxy Series FT-IR3000 model 3020 in Nujol mulls. NMR spectra were performed on a Varian spectrometer, model INOVA300, using tetramethylsilane (TMS) as the internal standard and dimethylsulfoxide-d 6 as the solvent at room temperature. UV/VIS absorption for the fluorescence quantum yield was taken on a Shimadzu UV-1601PC spectrophotometer. The absorption spectra of the dyes were measured on a Varian Cary 50 spectrophotometer. Fluorescence spectra were measured with This journal is

© The Royal Society of Chemistry and Owner Societies 2005

Fig. 2 Synthetic process for the synthesis of the isothiocyanate derivatives 4a–c.

a Hitachi spectrophotometer, model F-4500. Elemental analysis was performed by Perkin-Elmer model 240. 2.3 General procedure for synthesis of isothiocyanates derivatives The synthesis of 2-(4 -amino-2 -hydroxyphenyl)benzazoles 3a– c is presented in Fig. 2 and consists in the condensation of the 4-aminosalicilic acid (1) with o-substituted anilines (2) in polyphosphoric acid to yields the corresponding aminobenzazoles 3a–c.35 The isothiocyanate derivatives were obtained as previously described.17 A solution of 3a (or 3b–c) in dry acetone was added dropwise into a solution of thiophosgene in dry acetone (1 : 5), at 0 ◦ C. The reaction mixture was stirred for two hours at room temperature producing the isothiocyanate derivatives 4a–c (Fig. 2). The precipitate was filtered, washed with cold acetone and dried at room temperature. The purification was made by column chromatography eluted with dichloromethane. 2-(4-isothiocyanate–2-hydroxyphenyl)benzoxazole (4a). Yield 85%. Mp 145–147 ◦ C. Calc. for C14 H8 N2 O2 S: C, 62.67; H, 3.01; N, 10.44%. Found: C, 62.78; H, 3.24; N, 9.97%. IR (cm−1 ): 2063 (mN=C=S), 1634 and 1498 (marom C=C). 1 H NMR (d) = 11.48 (br s, 1 H, OH); 8.06 (d, 1 H, H6 , J o = 8.4 Hz); 7.80–7.90 (m, 2 H, H4 and H7 ); 7.54–7.42 (m, 2 H, H5 and H6 ); 7.16 (d, 1 H, H3 , J m = 1.8 Hz); 7.12 (dd, 1 H, H5 , J m = 1.8 Hz and J o = 8.14 Hz). 2-(4-isothiocyanate-2-hydroxyphenyl)benzothiazole (4b). Yield 58%. Mp 178–180 ◦ C. Calc. for C14 H8 N2 OS2 : C, 59.13; H, 2.84; N, 9.85%. Found: C, 59.88; H, 2.94; N, 9.28%. IR (cm−1 ): 2125 (mN=C=S), 1624 and 1572 (marom C=C). 1 H NMR (d) = 11.74 (br s, 1 H, OH); 8.30 (d, 1 H, H6 , Jo = 8.1 Hz); 8.16 (d, 1 H, H4 or H7 , J o = 7.2 Hz); 8.06 (d, 1 H, H7 or H4 , J o = 7.2 Hz); 7.56 (t, 1 H, H5 or H6 , J m = 1.2 Hz and J o = 7.5 Hz); 7.46 (t, 1 H, H6 or H5 , J m = 1.2 Hz and J o = 7.5 Hz); 7.06 (dd, 1 H, H5 , J m = 2.1 Hz and J o = 8.4 Hz); 7.04 (d, 1 H, H3 , J m = 2.4 Hz). (4c). 2-(4 -isothiocyanate–2 -hydroxyphenyl)benzimidazole Yield 99%. Mp 215–217 ◦ C (decomp.). Calc. for C14 H9 N3 OS: C, 62.91; H, 3.39; N, 15.72%. Found: C, 63.41; H, 3.72; N, 15.99%. IR (cm−1 ): 3361 (mNH), 2026 (mN=C=S), 1603 and 1460 (marom C=C). 1 H NMR (d) = 11.80 (br s, 1 H, OH); 8.04 (dd, 1 H, H4 or H7 , J m = 1 Hz and J o = 7.6 Hz); 7.90 (dd, 1 H, H7 or H4 , J m = 1 Hz and J o = 7.6 Hz); 7.64 (d, 1 H, H6 , J o = 8.4 Hz); 7.48 (t, 1 H, H5 or H6 , J m = 1 Hz and J o = 7.6 Hz); 7.34 (t, 1 H, H6 or H5 , J m = 1 Hz and J o = 7.6 Hz); 6.28 (dd, 1 H, H5 , J m = 2.2 Hz and J o = 8.4 Hz); 6.18 (d, 1 H, H3 , J m = 2.2 Hz); 5.96 (br s, 1 H, NH). 2.4 General procedure for protein labeling with dyes 4a–c Bovine serum albumin (BSA fraction V) was labeled with different concentrations of dyes 4a–c. The same procedure was previously described for other fluorescent probes.17,36 The isothiocyanates were dissolved in DMSO to a final concentration of 1 mg ml−1 . Small aliquots of this solution were added, slowly and with gentle stirring, to 1 ml of protein solution (10 mg ml−1 in sodium carbonate 0.1 M, pH 9). Doses of 20, 50 and 100 lg dyes

per ml protein solution were used. The protein-dye mixture was kept overnight at 4 ◦ C. Labelled protein was separated from free dyes by gel filtration chromatography on SephadexR G-50. The column was equilibrated and eluted with phosphate buffered saline (PBS, 10 mM phosphate, 150 mM NaCl, pH 7.4). 2.5

Polyacrylamide gel electrophoresis

Gel electrophoresis was performed by SDS-PAGE (4% stacking gel, pH 6.8, 10% separating gel, pH 8.8). After labeling with 4a–c, the protein suspension was diluted in an equal volume of sample buffer (125 mM Tris, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol). The suspension was boiled for 5 min and loaded (10 lg lane−1 ) onto gels. After SDS-PAGE, gels were observed under UV-light and photographed.

3 3.1

Results and discussion UV/VIS and fluorescence emission characterisation

The UV/VIS and the fluorescence emission spectra of the isothiocyanates derivatives were made in a solution concentration of 10−6 M using dichloromethane, ethyl acetate and ethanol as solvents. All experiments were performed at room temperature. Figs. 3, 4 and 5 present the curves for the dyes 4a–c, respectively. The relevant data are summarised in the Table 1. In Fig. 6 are presented the fluorescences of the dyes 4a–c in the solid state. As it can be seen in UV/VIS absorption spectra of the isothiocyanates 4a–c (Figs. 3–5), the maximum absorption wavelength (kabs max ) was not affected by the solvent polarity. Values of 323–350 nm could be observed. In the fluorescence emission spectra, the maximum wavelength (kem max ) presented small shifts, depending on the solvent polarities. The dyes 4a and 4c present fluorescence in the blue–purple region (450–478 nm) and the dye 4b presented fluorescence in the green region (503–509 nm). Quantum yields of fluorescence (φ fl ) values were obtained using quinine sulfate (Riedel) in 0.5 M H2 SO4 as quantum yield standards (φ fl = 0.55). The measurements were made at 25 ◦ C with a solution absorbance < 0.05.37,38 Table 1 shows the quantum yield of the studied dyes in three different solvents. The dye 4a, presents a quantum yield of fluorescence around 0.38, with the higher one in a non-protic solvent (0.478 in ethyl acetate). The dyes 4b and 4c presented values around 0.14 and 0.23, respectively. To these dyes, the maximum values of φ fl were also obtained in non-protic solvents (4b: 0.223 in ethyl acetate and 4c: 0.369 in dichloromethane). Since the ESIPT mechanism is quite dependent on the solvent polarity, this variation on the quantum yield of fluorescence was expected.39–42 Similar values than those observed in solution could be seen in the solid-state fluorescence (Fig. 6). The dyes 4a and 4c presented a maximum emission of fluorescence located in the blue–purple region, 481 and 463 nm, respectively. The dye 4b presented fluorescence in the green region with a maximum emission of fluorescence located at 507 nm. 3.2

Protein labeling with isothiocyanate derivatives

The dyes 4a–c are insoluble in water. However, using a methodology already described,17 these dyes can be used as protein Photochem. Photobiol. Sci., 2005, 4, 254–259

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Fig. 3 Normalised UV/VIS absorbance and fluorescence emission spectra of 4a.

Fig. 4 Normalised UV/VIS absorbance and fluorescence emission spectra of 4b.

probes in aqueous solutions. The dyes should be dissolved in DMSO and added slowly, in small aliquots, to the protein solution (sodium carbonate 0.1 M, pH 9) in order to warrant an efficacious label. The unbound dyes were efficiently removed by gel filtration chromatography on SephadexR G-50. It is worth to mention that BSA molecule have 60 lysine residues (NCBI database, accession AF542068.1). Labeling is done in excess of fluorochrome to attempt maximum yield. Although all these residues are potential sites for labeling, it is reasonable to consider that labeling will not occur in all residues that are placed very closely (e.g. 557–559), or also neighbor Lys residues (e.g. 155–156, 547–548). In addition, with protein folding, some additional residues may be also unavailable due to steric hindrance.

Fig. 5 Normalised UV/VIS absorbance and fluorescence emission spectra of 4c.

Fig. 7 presents the fluorescence emission spectra of BSA labeled with the dyes 4a–c (BSA–4), performed in PBS as a solvent. The fluorescence emission (kem max ) of the conjugates were at 464, 444 and 449 nm, respectively. Any comparison with the dyes 4a–c fluorescence emission can be done, since these values were performed in organic solvents. The degree of conjugation of BSA–4 was studied through abs the ratios of absorption (kabs max )dye /(kmax )BSA of various dye/protein ratios as presented in Fig. 8. The maximum absorptions of dyes 4a–c were 320, 350 and 540 nm, respectively; for the BSA, where used, 280 nm. An increase of the dye bounded in the protein can be seen as the dye/protein (wt/wt) increased from up to 0.1. As already observed, considering the increased at higher

Table 1 UV/VIS and fluorescence emission data of the dyes 4a–c Dye

Solvent

kabs max /nm

emax × 10−4 /l mol−1 cm−1

kem max /nm

Dk/nm

φfl

4a

Dichloromethane Ethyl acetate Ethanol Dichloromethane Ethyl acetate Ethanol Dichloromethane Ethyl acetate Ethanol

323 323 323 350 347 347 339 338 338

4.44 8.66 3.41 5.50 6.13 5.22 6.47 0.70 4.66

475 478 474 503 509 503 463 461 450

152 157 151 153 162 156 124 123 112

0.302 0.478 0.367 0.105 0.223 0.095 0.369 0.267 0.061

4b 4c

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Fig. 6 Normalised solid-state fluorescence emission spectra of isothiocyanate derivatives.

Fig. 7

Fig. 9

Stability of BSA–4 vs. daylight exposition.

The intensities presented a small decrease in the first two weeks and remained practically constant for the next three weeks. Fluorescein isothiocyanate, which is widely used as a fluorescent protein probe,43 loses the fluorescence quickly when exposed to light. The photobleaching occurs when a fluorophore permanently loses the ability to fluoresce due to photon-induced chemical damage and covalent modification. Other protein probes like 7-hydroxy-4-methylcoumarin and Rhodamine retain full fluorescence more than fluorescein isothiocyanate when exposed to light.4,44,45 To show the remarkable differences in photostability, the dyes 4a–c were analyzed and compared with fluorescein isothiocyanate (FITC) (percent decrease of the fluorescence intensity after illumination with UV radiation at the wavelength of maximum absorption). The values of the percent decrease of fluorescence were taken under different exposition times as presented in Fig. 10. The BSA–4 conjugates, described in this work, showed higher photostability than FITC and do not need to be stored in amber glass containers or even in the dark.

Normalised fluorescence emission spectra of BSA–4.

Fig. 10 Percent decrease of fluorescence vs. UV-light time exposition. Fig. 8 Conjugation of 4a–c with BSA obtained as a function of dye/protein ratio.

dye concentrations the recovery of protein decreased sharply, probably due to protein precipitation.17 The stability vs. photodecomposition of BSA–4 is depicted in Fig. 9. The same samples used in the fluorescence emission experiments were stored in clear glass containers (ordinary labware) in a lighted daylight room at room temperature. Aliquots of the labeled protein were taken at different times. For each BSAdye conjugate, the intensity value of the fluorescence emission at time 0 was used to normalise the follow measurements.

3.3

Polyacrylamide gel electrophoresis

The labeled bovine serum albumin (BSA–4) was submitted to polyacrylamide gel electrophoresis. Under visible light the gel is completely transparent and any vestige of BSA and/or the conjugates can be seen. However, under UV/VIS light, an intense fluorescence can be observed (Fig. 11). In Fig. 11 is presented the electrophoresis pattern for Coomassie-labeled BSA and for the BSA labeled with fluorescent isothiocyanates 4a–c. Although the labeling process converts a positively charged ammonium ion on a lysine to Photochem. Photobiol. Sci., 2005, 4, 254–259

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Fig. 11 SDS-PAGE of BSA–4 submitted to polyacrylamide gels electrophoresis. In a black box: (left) under visible light and (right) under UV-light. Since BSA is not fluorescent under UV-light, the BSA–4 gel was coloured with Coomassie.

an uncharged thiourea, the mobility of the protein in the electrophoresis of BSA–4 does appear to be different from the mobility of BSA, particularly considering that broad bands were observed. Mobility during electrophoresis in polyacrylamide gels containing the anionic detergent SDS is presumed to be only due to the molecular mass. In the presence of SDS, the protein– SDS complexes formed would have an excessive negative charge, to ensure polypeptide migration only according their molecular masses.

4 Conclusions Three new benzazole derivatives were synthesised, purified until optical purity grade and characterised by elemental analysis, 1 H NMR, IR, UV/VIS and fluorescence emission spectroscopy. These compounds were highly fluorescent when irradiated with UV-light. They presented kem max at 450–509 nm, with a Stokes shift of the order of 112–162 nm. They have a potential use as fluorescent probes for protein labeling, since the dyes could be conjugated to bovine serum albumin (BSA) with very good and stable fluorescence. When stored in clear glass containers at room temperature for five weeks, the BSA–4 still presented a very stable and intense fluorescence emission. A simple and highly sensitive assay for detection of these proteins was reported here. The method was based on the direct fluorescence detection of protein-labeled with isothiocyanate derivatives after polyacrylamide gel electrophoresis.

Acknowledgements We are grateful for financial support and scholarships from the Brazilian agencies CNPq, FAPERGS and CAPES.

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