Bioconjugate Chem. 1997, 8, 495−497
495
Practical Method for the Multigram Separation of the 5- and 6-Isomers of Carboxyfluorescein Francis M. Rossi† and Joseph P. Y. Kao* Medical Biotechnology Center and Department of Physiology, School of Medicine, University of Maryland, Baltimore, Maryland 21201. Received April 9, 1997X
An efficient preparative method for separating 5- and 6-carboxyfluorescein is presented. 6-Carboxyfluorescein dipivalate is isolated as its diisopropylamine salt, which can be converted to the free acid or used directly in coupling reactions. The 5-isomer is isolated from the acidified mother liquor. Isomerically pure carboxyfluoresceins are prepared by hydrolysis of the corresponding dipivalates.
Fluorescently labeled biologically active molecules have many important analytical and biochemical applications (1-5). Carboxyfluorescein is a useful reagent for the preparation of hydrolytically stable fluorescent conjugates and is a useful starting material for the synthesis of other fluorescein-derived reagents (1, 4-7). It is prepared as a mixture of roughly equal amounts of the 5- and 6-isomers by the reaction of benzene tricarboxylic acid anhydride with resorcinol and zinc chloride (8, 9). Although these isomers have nearly identical properties, when conjugated to other molecules, differences have been observed in conjugate polarity, internal fluorescence quenching, and, for the case of a related rhodamine labeling reagent, labeling specificity (10, 11). Moreover, multistep synthetic preparations of fluorescein derivatives require isomerically pure starting material so that reaction products can be easily purified and identified. Although isomerically pure carboxyfluorescein is commercially available in milligram quantities (12), we are aware of no published method for the separation of these isomers that is simple and does not require extensive chromatography. With this in mind, we set out to develop a practical method for separating 5- and 6-isomers of carboxyfluorescein as shown in Scheme 1.1 The poor solubility of fluorescein derivatives and their existence as either lactone or free acid tautomers complicate isomeric separation. Both of the problems were circumvented by heating carboxyfluorescein in pivalic anhydride to give a crude mixture of dipivalates. When this mixture was dissolved in ethanol and treated with various amines, an insoluble salt selectively formed between the 6-isomer and hindered amines such as diisopropylamine and diisopropylethylamine. When other solvents were ex* Address correspondence to this author at the Medical Biotechnology Center and Department of Physiology, School of Medicine, University of Maryland, 725 W. Lombard St., Baltimore, MD 21201 [telephone (410) 706-4167; fax (410) 706-8184; e-mail
[email protected]]. † Present address: Department of Chemistry, Santa Clara University, Santa Clara, CA 95053 (e-mail
[email protected]). X Abstract published in Advance ACS Abstracts, June 15, 1997. 1 Two numbering schemes for fluorescein coexist in the literature. The numbering convention adopted here is based on the lactone form of fluorescein (see Scheme 1), with the spiro carbon being position 1. An alternate numbering convention is based on the ring-opened free acid form of fluorescein, with the point of attachment of the phenyl ring to the xanthene chromophore being position 1. In the latter convention, 4- and 5-carboxyfluorescein, respectively, correspond to 5- and 6-carboxyfluorescein in the lactone convention used here.
S1043-1802(97)00078-5 CCC: $14.00
Scheme 1
amined, e.g. methanol, isopropyl alcohol, acetonitrile, and acetone, no precipitate formed. NMR analysis of the diisopropylamine salt (1) indicated that the isomeric purity was >95%. The 5-isomer of carboxyfluorescein dipivalate (2) was crystallized from a nitromethane solution of the acidified mother liquor left from the preparation of 1. NMR analysis showed that it also had an isomeric purity >95%. Attempts to apply this method to separate the isomers of carboxyfluorescein diacetate failed to give an isomerically pure precipitate in all examined combinations of amines and solvents. From the separated dipivalates, the corresponding carboxyfluoresceins (3 and 4) were prepared as shown © 1997 American Chemical Society
496 Bioconjugate Chem., Vol. 8, No. 4, 1997
Rossi and Kao Scheme 2
Figure 1. HPLC analysis of 5- and 6-carboxyfluoresceins as shown by elution profiles of 5- and 6-carboxyfluoresceins, prepared according to the method of Scheme 1, and the parent isomeric mixture, prepared as previously described (8, 9). Mobile phase for isocratic elution was an 8:2 mixture of 10 mM sodium phosphate (pH 7.05) and methanol. Approximately 25-50 nmol of sample was injected for each run. Samples were prepared as described under Experimental Procedures.
in Scheme 1. To assess the effectiveness of the separation procedure more quantitatively, we analyzed the resulting 5- and 6-carboxyfluoresceins by HPLC. The results are shown in Figure 1. Peak integration revealed that the 5-carboxyfluorescein sample contained 1.4% 6-carboxyfluorescein, whereas the 6-carboxyfluorescein sample contained 4.6% 5-carboxyfluorescein. The percentages given are based on total carboxyfluorescein in each sample and are not relative percentages. Thus, HPLC analysis confirmed that the procedure we have described is quite efficient for separating the two positional isomers of carboxyfluorescein. Carboxyfluorescein dipivalates are useful synthetic intermediates in themselves (1), in addition to being precursors to carboxyfluoresceins 3 and 4. For example, the diisopropylamine salt (1) was converted to the carboxylic acid (5) by treatment with HCl or was used directly in reactions that normally require an equivalent of base, as shown in Scheme 2. Thus, when salt 1 was treated with bis(2-oxo-3-oxazolidinyl)phospinic chloride (BOP-Cl) and glycine methyl ester hydrochloride, amide conjugate 6 was isolated. EXPERIMENTAL PROCEDURES
General. Reagents and solvents used were of ACS or HPLC grade and used as received from Aldrich or Fisher. All oxygen and water-sensitive reactions were performed under a dry argon atmosphere. For water-sensitive reactions, glassware was dried at 130 °C for at least 3 h and cooled under a stream of argon gas or in a desiccator prior to use. Silica gel 60 (230-400 mesh, E. Merck) was used for compounds purified by chromatography. Melting points were recorded on a Melt-temp II (Laboratory Devices) apparatus coupled to an Omega (Omega Engineering) HH23 digital thermometer and are uncorrected. The structures of all purified products were established by NMR spectral analysis. Spectra were recorded on a
General Electric QE-300 (300 MHz) NMR spectrometer. Samples were dissolved in CDCl3 (0.03% TMS), unless otherwise stated, and were referenced to tetramethylsilane (TMS). Samples dissolved in solvents other than CDCl3 were referenced to the residual solvent peak. High-resolution mass spectral analysis was performed at the University of Maryland, College Park, on a Model VG707E spectrometer (VG Analytical). HPLC Analyses. HPLC analyses were performed on a Waters chromatograph (Waters Corp.) consisting of a Model 600E multisolvent delivery system coupled to a Model 996 photodiode array detector. An Inertsil ODS-2 column (250 × 4.6 mm, 5 µm particle size; MetaChem Technologies, Inc.) was used for analytical separations. The mobile phase for isocratic elution was an 8:2 mixture of 10 mM sodium phosphate (pH 7.05) and methanol. All elutions were performed at a flow rate of 1 mL‚min-1, at room temperature. Millenium 2010 software (Waters Corp.) was used for data acquisition and analysis. All samples were made by mixing 1 mg of carboxyfluorescein with 2 equiv of 1 M NaOH and diluting the mixture with 10 mM sodium phosphate buffer (pH 7.05) to yield 10 mM carboxyfluorescein. Aliquots of 2.5-5 µL were injected for analysis. Preparation of 6-Carboxyfluorescein Dipivalate Diisopropylamine Salt (1). 5(6)-Carboxyfluorescein (5.00 g, 13 mmol) was refluxed in trimethylacetic anhydride (10 mL, 49 mmol) for 2 h. The reaction mixture was cooled to room temperature and diluted with tetrahydrofuran (THF; 10 mL) and water (10 mL). After 2 h of vigorous stirring, ether (50 mL) was added and the aqueous layer was removed. The organic layer was washed with phosphate buffer (3 × 25 mL, 1.4 M, pH 7.0), aqueous HCl (50 mL, 1 M), and saturated NaCl and dried with MgSO4. The solvent was removed by evaporation, and the resulting yellow syrup was taken up in absolute ethanol (50 mL). Diisopropylamine (5.0 mL, 36 mmol) was added, and the solution was cooled to -20 °C. The resulting solid was removed by filtration and washed with cold ethanol and acetone to give 2.39 g (28%) of 1, which had an isomeric purity >95% as determined by proton NMR analysis: 1H NMR δ 1.23 (d, J ) 6.35 Hz, 12 H), 1.35 (s, 18 H), 3.21 (a quintet, J ) 6.35 Hz, 2 H), 6.75 (dd, J ) 1.95, 8.79 Hz, 2 H), 6.84 (d, J ) 8.79 Hz, 2 H), 7.03 (d, J ) 1.95 Hz, 2 H), 7.69 (s, 1 H), 7.99 (d, J ) 7.81 Hz, 1 H), 8.26 (d, J ) 7.81 Hz, 1 H); 13C NMR δ 19.0, 27.0, 39.1, 46.3, 110.1, 116.3, 117.6, 124.3, 124.5, 126.7, 129.0, 131.1, 145.5, 151.4, 152.4, 152.9, 169.2, 169.8, 176.5; FABMS (M + H+) 646; HRMS(EI) calcd for C31H29O9 (carboxylate + 2H+) 545.1812, observed 545.1835.
Bioconjugate Chem., Vol. 8, No. 4, 1997 497
Large-Scale Carboxyfluorescein Isomer Separation
Preparation of 5-Carboxyfluorescein Dipivalate (2). The solvent was evaporated from the filtrate obtained in the preparation of 1. The resulting syrup was diluted with ether (50 mL) and extracted with water (4 × 25 mL) and aqueous HCl (1 M, 25 mL). The organic layer was dried with MgSO4, and the solvent was evaporated. The resulting foam was dissolved in nitromethane (10 mL) and cooled to -20 °C. A solid formed and was isolated by filtration and washed with a small amount of cold nitromethane to give 1.39 g (19%) of 2 that was isomerically pure by proton NMR analysis: mp 220-222 °C; 1H NMR δ 1.36, (s, 18 H), 6.82, (s, 4 H), 7.08 (s, 2 H), 7.30 (d, J ) 8.3 Hz, 1 H), 8.41 (d, J ) 8.8 Hz, 1 H), 8.78 (s, 1H); 13C NMR δ 27.0, 39.1, 110.5, 115.3, 117.9, 124.4, 126.6, 127.6, 128.7, 131.7, 136.7, 151.4, 152.8, 157.5, 168.0, 169.7, 176.5; HRMS(CI) calcd for C31H29O9 (M + H+) 545.1812, obsd 545.1804. Preparation of 6-Carboxyfluorescein (3). Sodium hydroxide (1 M, 1.5 mL, 1.5 mmol) was added to a solution of pivalate 1 (0.161 g, 0.25 mmol) in methanol (3 mL). The colorless solution became orange on addition of the base. After 30 min of stirring, the solvent was evaporated, and the residue was dissolved in water and acidified with HCl (1 M, 1.5 mL, 1.5 mmol). The resulting solid was isolated by filtration and washed with water and ether to give 0.071 g (75%) of 3: purity 95.4% by HPLC; mp 352-356 °C; 1H NMR (NaOD/D2O) δ 6.50 (m, 4 H), 7.18 (d, J ) 9.8 Hz, 2 H), 7.83 (d, J ) 1.5 Hz, 1 H), 7.86 (d, J ) 8.3 Hz, 1 H), 8.09 (dd, J ) 1.5, 7.8 Hz, 1 H); 13C NMR δ 106.1, 114.4, 125.2, 130.4, 132.1, 132.9, 133.3, 133.4, 133.7, 139.6, 144.4, 159.9, 160.6, 176.8, 177.4; HRMS(CI) calcd for C21H13O7 (M + H+) 377.0661, obsd 377.0648. Preparation of 5-Carboxyfluorescein (4). Sodium hydroxide (1 M, 3 mL, 3 mmol) was added to a solution of 2 (0.272 g, 0.50 mmol) in THF (3 mL). After 15 min, the solvent was evaporated and the resulting foamy residue was dissolved in water. The solution was acidified with concentrated HCl, giving a precipitate that was isolated by filtration, washed with water, and air-dried to give 0.185 g (98%) of 4 as an orange solid: purity 98.6% by HPLC; mp 368-372 °C; 1H NMR (NaOD/D2O) δ 6.46 (d, J ) 1.96 Hz, 2 H), 6.52 (dd, J ) 1.96, 9.27 Hz, 2 H), 7.05 (d, J ) 9.28 Hz, 2 H), 7.11 (d, J ) 8.30 Hz, 1 H), 7.93 (d, J ) 9.28 Hz, 1 H), 8.16 (s, 1 H); 13C NMR δ 106.3, 114.3, 125.4, 130.9, 131.8, 132.5, 133.6, 136.3, 139.8, 142.2, 160.38, 160.39, 176.9, 177.2, 183.0; HRMS(EI) calcd for C21H12O7 (M+) 376.0583, obsd 376.0590. Preparation of 6-Carboxyfluorescein Dipivalate (5). Carboxyfluorescein salt 1 (1.50 g, 2.32 mmol) was dissolved in dichloromethane (25 mL) and extracted with aqueous HCl (1 M, 2 × 25 mL). The organic layer was dried with MgSO4, and the solvent was evaporated to give 1.20 g (95%) of 5: mp 198-200 °C; 1H NMR δ 1.36 (s, 18 H), 6.79 (s, 4 H), 7.08 (s, 2 H), 7.86 (s, 1 H), 8.13 (d, J ) 7.81 Hz, 1 H), 8.35 (d, J ) 7.81 Hz, 1 H); 13C NMR δ 27.0, 39.1, 110.4, 115.3, 117.8, 125.4, 125.8, 128.6, 130.1, 131.7, 135.8, 151.5, 152.7, 153.1, 168.1, 169.2, 176.4; HRMS(CI) calcd for C31H29O9 (M + H+) 545.1812, obsd 545.1784. Preparation of N-(Dipivaloylfluorescein-6-ylcarbonyl)glycine Methyl Ester (6). BOP-Cl (0.280 g, 1.10 mmol) was added to a suspension of fluorescein salt 1
(0.323 g, 0.50 mmol) in dichloromethane (3 mL). After 10 min, glycine methyl ester hydrochloride (0.138 g, 1.10 mmol) was added followed by triethylamine (0.17 mL, 1.10 mmol). The reaction mixture was stirred overnight, taken up in ethyl acetate (25 mL), and extracted with water and 1 M HCl. The organic layer was dried with MgSO4, and the solvent was evaporated. The resulting solid was chromatographed with hexane/ethyl acetate (1: 1) to give 0.169 g (55%) of 6: 1H NMR δ 1.36 (s, 18 H), 3.71 (s, 3 H), 4.19 (d, J ) 4.9 Hz, 2 H), 6.68 (br s, 1 H), 6.80 (s, 4 H), 7.07 (s, 2 H), 7.52 (s, 1 H), 8.10 (s, 2 H); 13C NMR δ 27.0, 39.1, 41.7, 52.4, 110.4, 115.4, 117.8, 122.5, 125.6, 128.4, 128.8, 129.5, 140.3, 151.4, 152.7, 153.4, 165.6, 168.2, 169.9, 176.5; HRMS(CI) calcd for C34H34NO10 (M + H+) 616.2183, obsd 616.2164. ACKNOWLEDGMENT
This work was supported by NIH Grant GM46956 to J.P.Y.K. Supporting Information Available: 1H-NMR spectra of compounds 1-6 (8 pages). Ordering information is given on any current masthead page. LITERATURE CITED (1) Theisen, P., McCollum, C., Upadhya, K., Jacobson, K., Vu, H., and Andrus, A. (1992) Fluorescent dye phosporamidite labelling of oligonucleotides. Tetrahedron Lett. 33, 50335036. (2) Goodchild, J. (1990) Conjugates of oligonucleotides and modified oligonucleotides: a review of their synthesis and properties. Bioconjugate Chem. 1, 165-187. (3) Brinkley, M. (1992) A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents. Bioconjugate Chem. 3, 2-13. (4) Mattingly, P. (1992) Preparation of 5- and 6-(aminomethyl)fluorescein. Bioconjugate Chem. 3, 430, and ref 2 of this paper. (5) Souto, A. A., Acun˜a, U. A., Andreu, J. M., Barasoain, I., Abal, M., and Amat-Guerri, F. (1995) New fluorescent water-soluble taxol derivatives. Angew. Chem., Intl. Ed. Engl. 34, 27102712. (6) Fiechtner, M., Wong, M., Bieniarz, C., and Shipchandler, M. T. (1989) Hydrophilic fluorescent derivatives: useful reagents for liposome immunolytic assays. Anal. Biochem. 180, 140-146. (7) Bertrand, R., Derancourt, J., and Kassab, R. (1995) Production and properties of skeletal myosin subfragment 1 selectively labeled with fluorescein at lysine-553 proximal to the strong actin binding site. Biochemistry 34, 9500-9507. (8) Chen, C.-S., and Poenie, M. (1993) New fluorescent probes for protein kinase C. J. Biol. Chem. 268, 15812-15822. (9) Orndorff, W. R., and Hemmer, A. J. (1927) Fluorescein and some of its derivatives. J. Am. Chem. Soc. 49, 1272-1280. (10) Ajtai, K., Ringler, D., Toft, D., Hellen, E. H., Ilich, P. J., and Burghardt, T. P. (1992) Stereospecific reaction of muscle fiber proteins with the 5′ or 6′ iodoacetamido derivative of tetramethylrhodamine: Only the 6′ isomer is mobile on the surface of S1. Biophys. J. 61, A287, Abstract 1647. (11) Buolamwini, J. K., Craik, J. D., Wiley, J. S., Robins, M. J., Gati, W. P., Cass, C. E., and Paterson, A. R. P. (1994) Conjugates of fluorescein and SAENTA (5′-S-(2-aminoethyl)N6-(4-nitrobenzyl)-5′-thioadenosine): flow cytometry probes for the ES nucleoside transport elements of the plasma membrane. Nucleosides Nucleotides 13, 737-751. (12) Molecular Probes, Eugene, OR.
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