Texas Red-X and Rhodamine Red-X, New Derivatives of

Sulforhodamine 101 and Lissamine Rhodamine B with Improved. Labeling and Fluorescence Properties. Charles Lefevre, Hee Chol Kang, Rosaria P. Haugland,...
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Bioconjugate Chem. 1996, 7, 482−489

482

Texas Red-X and Rhodamine Red-X, New Derivatives of Sulforhodamine 101 and Lissamine Rhodamine B with Improved Labeling and Fluorescence Properties Charles Lefevre, Hee Chol Kang, Rosaria P. Haugland,* Nabi Malekzadeh, Seksiri Arttamangkul, and Richard P. Haugland Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, Oregon 97402. Received February 2, 1996X

Texas Red sulfonyl chloride (TR-SC) and Lissamine rhodamine B sulfonyl chloride (LRB-SC) are popular dyes often used to prepare red fluorescent conjugates that are useful second labels in combination with fluorescein. Unfortunately, being sulfonyl chloride derivatives, both are unstable to moisture during storage and prone to hydrolysis in the conjugation reaction. Their instability causes the percentage of reactive dye to vary from lot to lot and requires use of low temperatures and a relatively high pH to optimize conjugation efficiency. Succinimidyl esters of the aminohexanoic acid sulfonamides of both dyes have been prepared, which are designated Texas Red-X succinimidyl ester (TR-X-SE) and Rhodamine Red-X succinimidyl ester, respectively. Their spectral properties are similar to those of their sulfonyl chloride analogs; moreover, incorporation of the succinimidyl ester at the end of the aliphatic chain spacer facilitates conjugation, decreases precipitation of proteins during conjugation and storage, and usually increases the fluorescence yield of the conjugate. Comparison of the rate of hydrolysis of TR-SC with that of TR-X-SE shows that, while the former was completely hydrolyzed within 5 min by exposure to water, TR-X-SE retains most of its reactivity for more than an hour. The reactivity of both new derivatives is high between pH 7.5 and 8.5, allowing conjugation of proteins that do not tolerate the high pH required for reaction with sulfonyl chlorides. In addition, Texas Red maleimides and haloacetamides containing spacer groups were prepared for labeling sulfhydryl groups. A Texas Red-X derivative of phalloidin has also been prepared, and its use for labeling F-actin has been characterized.

INTRODUCTION

Texas Red sulfonyl chloride (TR-SC) and Lissamine rhodamine B sulfonyl chloride (LRB-SC) are derivatives of sulforhodamine 101 and sulforhodamine B, respectively. Both dyes have strong red fluorescence and are frequently used in combination with fluorescein for dual or multiparameter fluorescence analysis (Titus et al., 1982; Wessendorf et al., 1990; Staines et al., 1988). The absorption and emission spectra of their conjugates are sufficiently shifted to the red so that they encounter little interference from the fluorescence of fluorescein conjugates when used for multicolor labeling experiments or the intrinsic fluorescence in biological samples. Texas Red conjugates absorb further in the red than Lissamine rhodamine B conjugates, and Texas Red is generally considered the more useful of the two dyes. However, being sulfonyl chlorides, both reactive dyes hydrolyze rapidly in aqueous environments and their conjugation to biomolecules usually requires a pH of 9-9.5, a pH value at which some proteins denature. In addition, sulfonyl chlorides form conjugates with tyrosine, serine, and histidine residues that may be unstable (Wong, 1991). Conjugations with TR-SC and LRB-SC must be performed at a low temperature to reduce the rate of dye hydrolysis. The low temperature and basic pH serve to maximize the reaction efficiency, though it often remains low. Furthermore, sensitivity of the reactive dyes to traces of moisture during storage affects the reactive dye’s purity and, consequently, the percentage of reactive sulfonyl chlorides fluctuates from batch to batch. Un* Author to whom correspondence should be addressed [telephone (541) 465-8300; fax (541) 344-6504; e-mail [email protected]]. X Abstract published in Advance ACS Abstracts, June 1, 1996.

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certainty regarding dye purity, combined with variations in the time that elapses between dissolving the dye and initiating the reaction, results in difficulty in reproducing conjugation reactions. In an effort to facilitate and improve conjugations, we have added a 6-aminohexanoic acid (“X”) spacer between the fluorophore and the reactive group. This allows us to prepare succinimidyl ester derivatives, the most amine-selective and stable reactive group for labeling amines (Drexage, 1990). We have named these derivatives Texas Red-X succinimidyl ester (TR-X-SE) and Rhodamine Red-X succinimidyl ester (RhR-X-SE). Their spectral properties and extinction coefficients are similar to those of their sulfonyl chloride analogs. However, they can be conjugated to proteins at a lower pH, at room temperature, and with greater reproducibility. In many cases, less precipitation of proteins occurs during conjugation and storage, and the products have increased fluorescence yields. Also, the greater stability of the reactive dyes permits use of less dye when using the succinimidyl esters to achieve the same degree of dye conjugation. In addition, we have prepared iodoacetamide, bromoacetamide, and maleimide derivatives of the Texas Red chromophore with similar aliphatic spacers, thus obtaining previously unavailable red fluorescenceemitting reagents that are useful for the labeling of thiol groups. Use of these reagents allows preparation of conjugates that are otherwise difficult to make. MATERIALS AND METHODS

TR-SC, Texas Red cadaverine, and LRB-SC were obtained from Molecular Probes, Inc., Eugene, OR. Methyl 6-aminohexanoate, O-(N-succinimidyl)-N,N,N′,N′tetramethyluronium tetrafluoroborate, iodoacetic anhy© 1996 American Chemical Society

New Improved Texas Red and Lissamine Rhodamine B

dride, and bromoacetyl bromide were purchased from Fluka Chemical Corp., Ronkonkoma, NY. All other common reagents were purchased from Aldrich Chemical Co., Milwaukee, WI. Thin-layer chromatography was performed on silica gel 60 F-254 sheets and column chromatography on silica gel 60 (230-400 mesh, E. Merck, No. 9385, Gibbstown, NJ). 1H NMR spectra were recorded on a Nicolet QE-400 MHz (General Electric) spectrometer. Absorption spectra were obtained using an IBM 9429 UV-visible spectrophotometer. Emission spectra were obtained using a Perkin-Elmer Model 65040 fluorescence spectrophotometer. Mass spectra were recorded on a Kratos MS-50 mass spectrometer (Manchester, England). 9-[2(or 4)-[[[5-[N-(Succinimidyl)oxy]carbonylpentyl]amino]sulfonyl]-4(or 2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-1H,5H,11H,15H-xantheno[2,3,4ij:5,6,7-i′j′]diquinolizin-18-ium, Inner Salt (3). To a solution of methyl 6-aminohexanoate hydrochloride (0.40 g, 2.20 mmol) and 0.6 mL of triethylamine in 40 mL of chloroform was added TR-SC (1) (1.00 g, 1.60 mmol) in small portions over a period of 10 min while the reaction mixture was stirred at 0 °C. After the reaction mixture was stirred at room temperature for 15 h, it was washed with three 50 mL portions of water. The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a dark purple solid. This crude methyl ester derivative was purified by silica gel column chromatography with 2.5% methanol in chloroform as eluant to give 0.80 g (68%) of the methyl ester derivative. To a suspension of the above methyl ester (0.80 g, 1.09 mmol) in 15 mL of dioxane was added 25 mL of 6 M HCl dropwise over a period of 5 min. After the reaction mixture was stirred at room temperature for 18 h, it was poured into 200 mL of water. The resulting solid was collected by filtration and purified by column chromatography on silica gel with 15% methanol in chloroform as eluant to give 0.68 g (87%) of 2 (TR-X) as a dark purple solid. Compound 2 had an absorption maximum at 591 nm with a molar absorptivity of 85 400 cm-1 M-1 and an emission maximum of 610 nm in pH 7.5 phosphate buffer solution. To a solution of compound 2 (300 mg, 0.42 mmol) in 3 mL of DMF was added O-(Nsuccinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (150 mg, 0.50 mmol). After the reaction mixture was stirred at room temperature for 1 h, it was diluted with 50 mL of chloroform, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to a volume of 10 mL. The resulting solution was added dropwise into 100 mL of ether while being stirred vigorously at room temperature. The resulting precipitate was collected by filtration and dried under vacuum to give 295 mg (87%) of 3 as a dark purple solid; one spot on TLC (Rf ) 0.20, 10% methanol in chloroform); 1H NMR (DMSO-d6) δ 8.39 (d, 1H, ArH), 7.92-7.84 (m, 1H, ArH), 7.48-7.33 (m, 1H, ArH), 6.53 (d, 2H, 2 × ArH), 3.54-3.40 (m, 8H, 4 × CH2), 3.132.57 (m, 8H, 4 × CH2), 2.80 (s, 4H, 2 × CH2), 2.08-1.74 (m, 8H, 4 × CH2), 1.65-1.12 (m, 6H, 3 × CH2); absorption maximum, 583 nm ( 116 300 cm-1 M-1), emission maximum, 603 nm in methanol; high-resolution FABMS, m/e (M + H) calcd 817.2580, found 817.2580. Anal. Calcd for C41H44N4O10S2‚2.5H2O: C, 57.13; H, 5.73; N, 6.50. Found: C, 57.38; H, 5.87; N, 7.14. 3,6-Bis(diethylamino)-9-[2(or 4)-[[[5-[N-(succinimidyl)oxy]carbonylpentyl]amino]sulfonyl]-4(or 2)sulfophenyl]xanthylium, Inner Salt (6). Compound 6 was prepared in a similar manner as described for the synthesis of 3 starting with LRB-SC instead of TR-SC. Compound 5 (RhR-X) was obtained as a dark red solid:

Bioconjugate Chem., Vol. 7, No. 4, 1996 483

yield, 62%; one spot on TLC (Rf ) 0.37, 20% methanol in chloroform); 1H NMR (DMSO-d6) δ 8.40 (s, 1H, ArH), 7.96-7.85 (m, 2H, 2 × ArH), 7.47 (d, 1H, ArH), 7.087.02 (m, 2H, 2 × ArH), 6.98 (d, 1H, ArH), 6.93 (s, 2H, 2 × ArH), 3.73-3.60 (q, 8H, 4 × CH2), 2.90-2.83 (m, 2H, CH2), 2.19-2.11 (m, 2H, CH2), 1.50-1.40 (m, 4H, 2 × CH2), 1.30-1.26 (m, 2H, CH2), 1.25 (t, 12H, 4 × CH3); absorption maximum, 570 nm ( 114 850 cm-1 M-1), emission maximum, 590 nm in pH 7.5 phosphate buffer; high-resolution FAB-MS, m/e (M + H) calcd 672.2416, found 672.2417. Compound 6 was obtained as a dark red solid: yield, 83%; one spot on TLC (Rf ) 0.36, 15% methanol in chloroform); 1H NMR (DMSO-d6) δ 8.41 (s, 1H, ArH), 7.96-7.88 (m, 2H, 2 × ArH), 7.48 (d, 1H, ArH), 7.07-7.01 (m, 2H, 2 × ArH), 6.99 (d, 1H, ArH), 6.96 (s, 2H, 2 × ArH), 3.70-3.57 (q, 8H, 4 × CH2), 2.89-2.81 (m, 2H, CH2), 2.80 (s, 4H, 2 × CH2), 2.69-2.63 (m, 2H, CH2), 1.70-1.49 (m, 2H, CH2), 1.52-1.35 (m, 4H, 2 × CH2), 1.20 (t, 12H, 4 × CH3); high-resolution FAB-MS, m/e (M + H) calcd 769.2580, found 769.2579. Anal. Calcd for C37H44N4O10S2‚0.5H2O: C, 57.14; H, 5.83; N, 7.21. Found: C, 57.08; H, 5.80; N, 7.20. 9-[2(or 4)-[[[5-(N-Iodoacetyl)aminopentyl]amino]sulfonyl]-4(or 2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-1H,5H,11H,15H-xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, Inner Salt (8). To a suspension of Texas Red cadaverine (7) (50 mg, 0.07 mmol) in 10 mL of chloroform was added N,N-diisopropylethylamine (15 µL, 0.09 mmol), followed by addition of iodoacetic anhydride (25 mg, 0.07 mmol), and the mixture was stirred at room temperature in the dark for 5 h. The reaction mixture was then diluted with 50 mL of chloroform, washed with water (3 × 50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product. The product was purified by column chromatography on silica gel with 5% methanol in chloroform as eluant to give 14 mg (88%) of 8 as a dark purple solid; one spot on TLC (Rf ) 0.21, 10% methanol in chloroform); 1H NMR (DMSO-d6) δ 8.38 (d, 1H, ArH), 7.90-7.80 (m, 1H, ArH), 7.38-7.35 (m, 1H, ArH), 6.50 (d, 2H, 2 × ArH), 3.60 (s, 2H, CH2I), 3.583.40 (m, 8H, 4 × CH2), 3.05 (m, 6H, 3 × CH2), 2.68-2.54 (m, 6H, 3 × CH2), 2.08-1.98 (m, 4H, 2 × CH2), 1.901.78 (m, 4H, 2 × CH2), 1.45-1.12 (m, 6H, 3 × CH2); absorption maximum, 591 nm, emission maximum, 613 nm in pH 7.5 phosphate buffer; high-resolution FAB-MS, m/e (M + H) calcd 859.1699; found 859.1700. 9-[2(or 4)-[[[5-(N-Bromoacetyl)aminopentyl]amino]sulfonyl]-4(or 2)-sulfophenyl]-2,3,6,7,12,13,16,17octahydro-1H,5H,11H,15H-xantheno[2,3,4-ij:5,6,7i′j′]diquinolizin-18-ium, Inner Salt (9). To a suspension of Texas Red cadaverine (50 mg, 0.07 mmol) in 10 mL of chloroform was added N,N-diisopropylethylamine (15 µL, 0.09 mmol), followed by addition of bromoacetyl bromide (7 µL, 0.08 mmol), and the reaction mixture was stirred at room temperature in the dark for 5 h. It was then diluted with 50 mL of chloroform, washed with water (3 × 50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product. It was purified by column chromatography on silica gel with 5% methanol in chloroform as eluant to give 41 mg (73%) of 9 as a dark purple solid; one spot on TLC (Rf ) 0.20, 10% methanol in chloroform); 1H NMR (DMSO-d6) δ 8.38 (d, 1H, ArH), 7.91-7.80 (m, 1H, ArH), 7.40-7.35 (m, 1H, ArH), 6.50 (d, 2H, 2 × ArH), 3.80 (s, 2H, CH2Br), 3.60-3.40 (m, 8H, 4 × CH2), 3.06-2.83 (m, 6H, 3 × CH2), 2.67-2.53 (m, 6H, 3 × CH2), 2.09-1.97 (m, 4H, 2 × CH2), 1.90-1.78 (m, 4H, 2 × CH2), 1.44-1.12 (m, 6H, 3 × CH2); absorption maximum, 583 nm ( 112 700 cm-1 M-1), emission

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maximum, 603 nm in methanol; high-resolution FABMS, m/e (M + H) calcd 811.1837, found 811.1840. 9-[2(or 4)-[[[(5-Maleimidylpentyl)-N-acetyl]amino]sulfonyl]-4(or 2)-sulfophenyl]-2,3,6,7,12,13,16,17octahydro-1H,5H,11H,15H-xantheno[2,3,4-ij:5,6,7i′j′]diquinolizin-18-ium, Inner Salt (10). To a solution of Texas Red cadaverine (50 mg, 0.07 mmol) and N,Ndiisopropylethylamine (25 µL, 0.14 mmol) in 5 mL of chloroform was added maleic anhydride (10 mg, 0.10 mmol). After the reaction mixture was stirred at room temperature for 5 h, it was concentrated to a volume of 1 mL under reduced pressure. The residual solution was poured into 10 mL of ether. The resulting precipitate was collected by filtration to give 43 mg of the maleamic acid intermediate as a dark purple solid. A suspension of this intermediate and 10 mg of sodium acetate in 2 mL of acetic anhydride was heated at 80 °C. After 0.5 h, the reaction mixture was concentrated under reduced pressure. The residue was diluted with 50 mL of chloroform, washed with water (3 × 50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product. It was purified by chromatography on silica gel with 5% methanol in chloroform as eluant to give 14 mg (25%) of 10 as a dark purple solid; one spot on TLC (Rf ) 0.13, 10% methanol in chloroform); 1H NMR (DMSO-d6) δ 8.41 (s, 1H, ArH), 8.08 (d, 1H, ArH), 7.44 (d, 1H, ArH), 7.01 (d, 2H, -CHdCH-), 6.44 (s, 2H, 2 × ArH), 3.58-1.19 (m, 34H, 17 × CH2), 1.97 (s, 3H, CH3); absorption maximum, 601 nm, emission maximum, 619 nm, in pH 7.5 phosphate buffer; high-resolution FAB-MS, m/e (M + 1) calcd 813.2631, found 813.2633. 9-[2(or 4)-[[(2-Maleimidylethyl)amino]sulfonyl]4(or 2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro1H,5H,11H,15H-xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, Inner Salt (11). To a solution of N-(2aminoethyl)maleimide trifluoroacetic acid salt (100 mg, 0.39 mmol) and N,N-diisopropylethylamine (135 µL, 0.78 mmol) in 30 mL of dichloromethane was added TR-SC (245 mg, 0.39 mmol) in small portions over a period of 5 min. After the reaction mixture was stirred at room temperature overnight, it was washed with water (2 × 30 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product. It was purified by column chromatography on silica gel with 5% methanol in chloroform to give 185 mg (65%) of 11 as a dark purple solid; one spot on TLC (Rf ) 0.45, 15% methanol in chloroform); 1H NMR (CDCl3) δ 8.51 (s, 1H, ArH), 7.79 (d, 1H, ArH), 7.13 (d, 1H, ArH), 6.70 (s, 2H, -CHdCH-), 6.68 (s, 2H, 2 × ArH), 3.60 (t, 2H, CH2), 3.50-3.34 (m, 8H, 4 × CH2), 3.20 (t, 2H, CH2), 3.042.90 (m, 4H, 2 × CH2), 2.72-2.52 (m, 4H, 2 × CH2), 2.101.90 (m, 8H, 4 × CH2); absorption maximum, 582 nm ( 108 100 cm-1 M-1), emission maximum, 600 nm in methanol; high-resolution FAB-MS, m/e (M + 1) calcd 729.2055, found 729.2050. Spectral Properties of Texas Red-X and Rhodamine Red-X Derivatives. Figures 4 and 5 show the similarity of the normalized absorption and fluorescence emission spectra of TR-X and RhR-X with those of sulforhodamine 101 and sulforhodamine B, dissolved in phosphate-buffered saline (PBS). The “X” derivatives exhibit a very small shift (∼5 nm) toward the red portion of the spectrum. RhR-X conjugates can be efficiently excited by the 568 nm line of the krypton ion laser. Both dyes are well suited for multiple labeling of biopolymers, as their fluorescence emission spectrum is further separated from fluorescein than that of tetramethylrhodamine. Conjugations of TR-X and RhR-X Succinimidyl

Lefevre et al. Table 1. Labeling Efficiency of Texas Red Sulfonyl Chloride versus Texas Red-X Succinimidyl Ester, and Lissamine Rhodamine B Sulfonyl Chloride versus Rhodamine Red-X Succinimidyl Ester As Determined by Preparing Conjugates of Selected Proteins with Different Dye-to-Protein Molar Ratiosa protein BSA GAM GAM GAM streptavidin streptavidin streptavidin streptavidin streptavidin streptavidin streptavidin streptavidin streptavidin streptavidin

fluorophores/protein (mol/mol) dye:protein ratio TR-SC LRB-SC TR-X-SE RhR-X-SE 15.0 2.5 5.5 8.0 1.0 2.5 5.5 8.0 10.0 3 5 7 10 15

3.2 0.6 1.0 1.3 0.1 1.2 2.4 3.0 3.9

4.5 1.3 2.5 3.3 0.8 1.6 4.4 5.6 6.0 0.8 1.2 1.4 1.8 2.2

2.4 3.5 4.9 5.7 5.8

a Conjugations with TR-SC and LRB-SC were performed at 4 °C and pH 9. TR-X-SE and RhR-X-SE conjugates were prepared at room temperature and pH 8.3. The results demonstrate that TR-X-SE and RhR-X-SE consistently yield conjugates with more dyes per protein.

Esters to Proteins. To compare the reactivity of TRX-SE and RhR-X-SE with that of the respective sulfonyl chloride analogs, the dyes were reacted with different proteins at the molar ratios of dye-to-protein of 1:1 to 1:15, as reported in Table 1. Proteins were either dissolved at 10 mg/mL in 0.1 M NaHCO3, pH 8.3, or, when already solubilized in PBS, diluted with 1/10 volume of 1 M NaHCO3, pH 8.3. The dye was then weighed and dissolved at 10 mg/mL in dimethylformamide (DMF). Aliquots of the dye solution were immediately added to the protein at the desired molar ratios. Reactions were run for 1 h at room temperature, at which point 1/10 volume of 1.5 M hydroxylamine (adjusted to pH 8) was added and the reaction was left for an additional 30 min. Hydroxylamine serves to stop the reaction and to dissociate the dye from unstable conjugates formed with alcohols (Brinkley, 1992). Any visible precipitate was removed by microcentrifugation, and the conjugate that remained in solution was then purified on Bio-Gel P-30, fine grade, spin columns (Bio-Rad, Hercules, CA). Conjugation of TR-SC and LRB-SC to Proteins. These conjugations were performed in parallel with the conjugations of TR-X-SE and RhR-X-SE (see above), except that the buffer pH was adjusted to pH 9.0 rather than pH 8.3. Conjugations with TR-SC were performed in an ice bath. Low temperatures and pH 9.0 are the optimal conditions for conjugations with TR-SC, as reported by Titus et al. (1982). The dyes were dissolved in DMF at 10 mg/mL with brief sonication, and aliquots were immediately added to the protein at the calculated molar ratios. The reactions were allowed to proceed for 1 h and then stopped by the addition of 1/10 volume of 1.5 M hydroxylamine, pH 8.0. Any visible precipitate was spun down, and the conjugates were purified on Bio-Gel P-30 spin columns. The absence of free dye in the conjugate solution was assayed by thin-layer chromatography in chloroform/methanol (70:30) and, when necessary, the column purification repeated until no free dye was detected. Dye/Protein Degree of Labeling. Molarities of dye and protein in the conjugates were calculated from the absorbances of the conjugates at 280 nm and at the absorbance peaks of the different dyes. Since both fluorophores also absorb at 280 nm, their contribution

New Improved Texas Red and Lissamine Rhodamine B

to the absorbance at that wavelength was subtracted to determine the absorbance due to protein. The absorbance contributions of conjugates of TR-X-SE and TRSC at 280 nm are both about 20% that of their peak absorbance at 595 nm, based on the spectra of the acid forms of the dyes, while absorbance at 280 nm of both LRB-SC and RhR-X-SE is about 17% that of the dyes at their absorbance peaks. The degree of labeling was calculated as the molarity of dye divided by the molarity of protein, as described by Haugland (1995). Cross-Binding of Anti-Texas Red and Anti-Tetramethylrhodamine Antibodies to TR-X and RhRX. Similarity between TR-X or RhR-X and sulforhodamine 101 or sulforhodamine B was investigated by measuring the fluorescence quenching capacity of rabbit polyclonal antibodies to these fluorophores. These antibodies were raised against KLH labeled with TR-SC or with tetramethylrhodamine isothiocyanate, respectively (Molecular Probes). Identical aliquots of antibody solutions at the concentration of 1 mg/mL were added to 2 mL of 5 × 10-8 M TR-X (2) or RhR-X (5). The fluorescence output was measured after each addition until no further quenching of the fluorescence was obtained (saturation). Time Course of the Hydrolysis of TR-SC and TRX-SE. The ability of TR-X-SE and TR-SC to form conjugates after exposure to water was investigated by dissolving the reactive dyes at 10 mg/mL in DMF and diluting 4-fold in water to bring the final composition of the solution to 25% DMF. Following incubation at room temperature for 1, 5, 10, 20, 30, and 60 min after dilution with water, aliquots of the dye solutions were used to label 2 mg of goat IgG dissolved in 0.2 mL of buffer that had been adjusted with a solution of sodium bicarbonate to pH 8.3 for TR-X-SE and to pH 9.0 for TR-SC. The labeling reactions were left for 1 h at room temperature, and then the conjugates were purified on spin columns as previously described. Phalloidin Labeling with TR-X-SE. To a solution of 3 mg of aminophalloidin p-toluenesulfonate (Wieland et al., 1983) and 4 mg of TR-X-SE in 300 µL of DMF was added 5 µL of triethylamine, and the mixture was stirred at room temperature for 1 h. To the reaction mixture was added 7 mL of ether, and the resulting precipitate was collected by centrifugation. The crude product was purified by chromatography over lipophilic Sephadex LH20 using water for elution. The desired fractions were combined and lyophilized to give 3 mg of a dark purple solid of the TR-X phalloidin conjugate; one spot on TLC (Rf ) 0.53, 70 chloroform/27 methanol/3 water). F-Actin Staining by TR-X Phalloidin. F-Actin in 2-day-old bovine aorta endothelial cell (BAEC) cultures was stained with TR-X phalloidin according to the standard method described by Haugland et al. (1994). Labeling of an Oligonucleotide Primer with TRX-SE. To a solution of 500 µg of a 5′-amine-modified, 24-base M13 primer sequence in 220 µL of sodium bicarbonate, pH 8.5, was added a solution of 1 mg of TRSC or TR-X-SE, each dissolved in 35 µL of DMF. After 16 h at room temperature, the conjugates were precipitated by the addition of salt and cold ethanol. The labeled oligonucleotides were purified by HPLC on a C8 reversed phase column (Rainin Instrument Co., Woburn, MA) with a gradient of acetonitrile in 0.1 M triethylamine, pH 7.0. Detection was accomplished using a Waters 490 detector, monitoring at 254 and 590 nm. Cell Staining and Photobleaching Studies Using Streptavidin Conjugates of TR-SC and TR-X-SE. Cell staining was performed on commercially prepared HEp-2 ANA slides (INOVA Diagnostics Inc., San Diego,

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Figure 1. Reaction scheme for the synthesis of TR-X-SE (3) from TR-SC (1).

CA). Human anti-nuclear autoantibody (INOVA Diagnostics Inc.) was applied to the positive wells, while negative control wells were incubated in PBS. All of the wells were then treated with mouse anti-human IgG (Zymed Laboratories, S. San Francisco, CA) followed by biotinylated protein A. Conjugates of streptavidin with either TR-SC or TR-X-SE were added at 1, 5, and 10 µg/ mL in 1% BSA/PBS. The slides were then viewed through a longpass filter optimized for Texas Red conjugates (Omega Optical Inc., Brattleboro, VT). The above HEp-2 cells, labeled with the Texas Redstreptavidin conjugates, were illuminated with a 100 W mercury arc lamp using a 40 × 0.65 NA E PLAN lens (Nikon, Inc., Melville, NY). Images were acquired using a Star-1 CCD camera (Photometrics, Tucson, AZ) at 5 s intervals over a period of continuous illumination totaling 100 s. The fluorescence intensity at each time point was integrated over the frame using an Image 1 processor (Universal Imaging, West Chester, PA) with the signal threshold set above background. Photobleaching in solution was conducted by illuminating streptavidin conjugates that had been adjusted to the same optical density at their excitation maxima while their emission intensity over time was measured. Fluorescence spectra were measured using an SPF-500C spectrofluorometer (SLM Instruments Inc., Urbana, IL). Conjugation of the Thiol-Reactive Texas Red Derivatives to β-Galactosidase. As an example of the possibility of obtaining thiol derivatives with the spectral properties of TR, β-galactosidase was conjugated with Texas Red iodoacetamide, bromoacetamide, or maleim-

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Figure 2. Structures of LRB-SC (4), RhR-X (5), and RhR-XSE (6).

ide. The protein was dissolved at 10 mg/mL in 0.1 M sodium phosphate, 0.1 M NaCl, and 2 mM EDTA, pH 7.5, and the dyes were dissolved at 10 mg/mL in DMF using a molar ratio of dye-to-protein of 15. The reactions were performed under argon for 1 h at room temperature. Because iodoacetamide derivatives are light sensitive, exposure to light was strictly limited throughout the conjugation procedure. The conjugates were purified by size exclusion chromatography on Cellufine GH-25 (Amicon, Beverly, MA) columns equilibrated in the same pH 7.5 buffer. RESULTS AND DISCUSSION

Synthesis of TR-X and RhR-X Reactive Forms. The synthesis of TR-X-SE, (3), which involved three

Lefevre et al.

steps, is outlined in Figure 1. The aminohexanoic acid derivative, TR-X (2), was prepared using modifications of a published procedure (Abuelyaman et al., 1994). Use of acidic hydrolysis went more smoothly than the published method. In a similar manner, RhR-X-SE (6) was prepared in three steps starting from LRB-SC (4) (Figure 2). Thiol-reactive Texas Red haloacetamide and maleimide derivatives were prepared as shown in Figure 3. To make a Texas Red maleimide derivative, Texas Red cadaverine (7) was converted to an intermediate maleamic acid, which was then cyclized under dehydrative conditions using acetic anhydride/sodium acetate (Mehta et al., 1960). Cyclization of the maleamic acid derivative produced compound 10, which contains an N-acetyl group in addition to the maleimide, as a major product. Its structure was confirmed by high-resolution mass spectrometry and 1H NMR analysis. Spectral Properties of TR-X-SE and RhR-X-SE. As illustrated in Figures 4 and 5, the spectral properties of TR-X-SE and RhR-X-SE are very similar to those of their parent molecules. The absorption and fluorescence emission spectra of both dyes shift only a few nanometers. Cross-Binding of Anti-Texas Red and Anti-Tetramethylrhodamine Antibodies. Similarity of TR-X and RhR-X to sulforhodamine 101 and sulforhodamine B was confirmed by comparing the fluorescence quenching ability of anti-Texas Red and anti-tetramethylrhodamine antibodies when added to the respective solutions of the above dyes. We determined that, by coincidence, the same amount of antibody (90 µg) was needed to maximally quench either form of dye, when either dye was dissolved at a concentration of 5 × 10-8 M. TR-X and RhR-X Conjugates. The fluorescence yields of protein conjugates with TR-X-SE and RhR-X-

Figure 3. Reaction scheme for the synthesis of haloacetamide and maleimide derivatives of Texas Red from Texas Red cadaverine (7).

New Improved Texas Red and Lissamine Rhodamine B

Bioconjugate Chem., Vol. 7, No. 4, 1996 487

Figure 4. Normalized absorption of sulforhodamine 101 (B) and TR-X (C) (panel a) and normalized emission spectra (panel b) of fluorescein (A), sulforhodamine 101 (B), and TR-X (C) in PBS. The patterns of the absorption and emission of both Texas Red dyes are very similar and well distinguished from that of fluorescein.

Figure 5. Normalized absorption of sulforhodamine B (B) and RhR-X (C) (panel a) and overlap of the normalized emission spectrum of fluorescein (A) with the normalized emission spectra of sulforhodamine B (B) and RhR-X (C) (panel b). The arrow at 568 nm of the absorption spectrum shows that this probe is suitable for excitation by the krypton ion laser.

SE are shown in Figures 6 and 7, respectively. Both new dyes produced conjugates of streptavidin and goat antimouse (GAM) that were more fluorescent than the conjugates prepared from their sulfonyl chloride analogs. We also observed less precipitation of protein conjugates prepared from TR-X-SE and RhR-X-SE than of conjugates prepared from the sulfonyl chloride derivatives. TRX-SE produced brighter conjugates with goat IgG, goat anti-rabbit IgG, streptavidin, wheat germ agglutinin, and concanavalin A than did TR-SC (part of the data not shown). All of the conjugates prepared from TR-X-SE that we have tried have higher fluorescence than conjugates of TR-SC, with the exception of bovine serum albumin, DNase I, human transferrin, and dextrans. However, TR-X-SE and RhR-X-SE clearly react with greater efficiency with biopolymers than TR-SC and LRBSC. Table 1 shows that much lower ratios of TR-X-SE and RhR-X-SE are required to achieve the same given degree of labeling. Banks and Paquette (1995) reported that, when compared to the isothiocyanate and dichlorotriazine derivatives, the succinimidyl ester derivatives of fluorophores are more reactive and yield more stable conjugates. Figure 6 shows that the total fluorescence of TR-X-SE GAM conjugate, determined as described in the legend, is about 65% higher than the fluorescence of TR-SC conjugate, while the total fluorescence of TR-X-SEstreptavidin is about double the fluorescence of TR-SCstreptavidin. The curves have a broad bell shape, because the fluorescence of the conjugates is being

quenched after the optimal number of fluorophores per protein is reached. The difference in the number of dyes that can be conjugated to the protein before quenching occurs is more similar for GAM (2-3) than for streptavidin. The latter can be labeled with 4 TR-X-SE/protein before the fluorescence is quenched, while the fluorescence of the TR-SC conjugate is already quenched by 2.5-3 TR-SC/streptavidin. Similar results are shown in Figure 7 for a comparison of conjugates of GAM or streptavidin with LRB-SC and RhR-X-SE. Thus, it appears that, especially for streptavidin, not only are the conjugates with TR-X-SE or RhR-X-SE more fluorescent, but they can be labeled with a larger number of fluorophores before their fluorescence is quenched. Stability of TR-X-SE. The resistance of TR-X-SE to hydrolysis is striking when compared to the hydrolysis of sulfonyl chloride, as can be seen in Table 2. To compare rates of hydrolysis of TR-SC and TR-X-SE, the dyes were reacted with GAM IgG following incubation in a mostly aqueous environment (75% buffer, 25% DMF) for various periods of time. The degrees of labeling of the resulting conjugates were used to evaluate the relative extent of hydrolysis that occurred before the beginning of the conjugation reaction. After 5 min in aqueous solution, TR-SC failed to produce any detectable conjugate, whereas TR-X-SE continued to produce satisfactory yields of conjugates, even after an hour. Qualitative evaluation of HEp-2 cells stained with streptavidin conjugates of TR-X-SE and/or TR-SC indicated that the streptavidin conjugate prepared with TR-

488 Bioconjugate Chem., Vol. 7, No. 4, 1996

Lefevre et al. Table 2. Stability of Texas Red Sulfonyl Chloride and Texas Red-X Succinimidyl Ester in Aqueous Solutiona degree of labeling (dyes:protein) time (min)

TR-SC

TR-X-SE

1 5 10 20 30 60

0.30