Bioconjugate Chem. 1997, 8, 253−255
253
Preparation of Succinimidyl and Pentafluorophenyl Active Esters of 5- and 6- Carboxyfluorescein Maciej Adamczyk,* Jeffrey R. Fishpaugh, and Kevin J. Heuser Diagnostic Division, Division Organic Chemistry (D-9NM), Building AP-20, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-3500. Received November 8, 1996X
A mixture of 5- and 6-carboxyfluorescein was activated with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride in the presence of either N-hydroxysuccinimide or pentafluorophenol to give the corresponding succinimidyl and pentafluorophenyl esters. The regioisomeric mixtures were separated to give the 5- and 6- succinimidyl and pentafluorophenyl active esters in >98% purity.
INTRODUCTION
Fluorescein derivatives have been widely used as fluorescent markers in bioanalytical chemistry (1-5) and have found exceptional utility as the fluorophores of choice in fluorescent polarization immunoassays (FPIA) used for monitoring the level of drugs and hormones in human fluids (6, 7). Active esters of 5- and 6-carboxyfluorescein1 are used for production of labels and conjugates (1, 4, 8-10). The 5- and 6-carboxyfluorescein mixture (61:39 isomer ratio) is commercially available (11). The pure isomers are also available, but at a high cost, while the pure succinimidyl active esters can be purchased at a cost of $7600/g (12). From the literature (13, 14) only limited information is available for the preparation, purification, and analysis of the fluorescein succinimidyl active esters, while the corresponding pentafluorophenyl active esters, which usually possess higher hydrolytic stability (15), have not been previously reported. Thus, there is a need for an efficient and cost effective method for the preparation of these active esters. We now report a method for the preparation of 5- and 6-carboxyfluorescein succinimidyl (2a and 2b, respectively) and new pentafluorophenyl (2c and 2d) active esters from the inexpensive, commercially available mixture of 5(6)-carboxyfluorescein (1) as shown in Scheme 1. MATERIALS AND METHODS
All reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI) except for 5(6)-carboxyfluorescein (1), which was purchased from Kodak (Rochester, NY), and these chemicals were used without further purification. Solvents were of HPLC grade and used without further purification. Silica gel 60 (230-400 mesh) was purchased from EM Science (Gibbstown, NJ). Biotage FLASH 75 purification system and KP-SIL silica cartridges (32-63 µm, 60A, 7.5 × 30 cm) were purchased from Biotage, Inc. (Charlottesville, VA). 1H NMR spectra were recorded at 300 MHz, on a Varian Gemini 300 spectrometer. Electrospray ionization mass spectra were recorded on a Perkin-Elmer Sciex API 100 instrument. Elemental analyses were performed by Robertson Microlit Laboratories, Inc. (Madison, NJ). Analytical HPLC * Author to whom correspondence should be addressed [telephone (847) 937-0225; fax (847) 938-8927; e-mail adamczykm@ apmac.abbott.com]. X Abstract published in Advance ACS Abstracts, February 1, 1997. 15(6)-Carboxyfluorescein is also known as 4(5)-carboxyfluorescein.
S1043-1802(96)00087-0 CCC: $14.00
was performed using a Waters (Milford, MA) 8 × 100 mm µBondapak radial compression C18 column with a flow rate of 2 mL/min and UV detection at 225 nm. Preparative HPLC was performed using a Waters 40 × 100 mm µBondapak radial compression C18 column with a flow rate of 45 mL/min and UV detection at 225 nm. 5- and 6- Carboxyfluorescein Succinimidyl Active Esters (2a, 2b). To a solution of 5(6)-carboxyfluorescein (5.0 g, 13.3 mmol) in anhydrous N,N-dimethylformamide (DMF; 50 mL) was added 1-[3-(dimethylamino)propyl]3-ethylcarbodiimide hydrochloride (EDAC; 3.1 g, 16.0 mmol) followed by N-hydroxysuccinimide (HOSu; 1.9 g, 16.0 mmol). The reaction was covered with foil, stirred under nitrogen, and monitored by analytical HPLC (22% CH3CN/22% MeOH/56% aqueous 0.1% formic acid). After 4.5 h, additional EDAC (510 mg, 0.2 equiv) was added, and the reaction was stirred for an additional 18.5 h. The reaction mixture was rinsed into a separatory funnel with DMF (10 mL) and diluted with acetone (200 mL). Buffer (0.05 M, pH 6 phosphate buffer, 250 mL) was added, and the mixture was extracted with diethyl ether (Et2O)/ethyl acetate (EtOAc) (2:1, 300 mL). The organic layer was separated, and the aqueous layer was extracted two times with Et2O/EtOAc (2:1, 250 mL). The combined organic extracts were washed with water (3 × 200 mL) and brine (1 × 250 mL), dried over Na2SO4, and filtered, and the solvents were removed in vacuo to afford 4.7 g of crude 5(6)-carboxyfluorescein succinimidyl active esters. Separation of the isomers on the Biotage system was performed as follows: (a) the Biotage column was equilibrated with 2 L of eluent (15% acetone/85% toluene containing 0.05% acetic acid); (b) crude active ester mixture, adsorbed onto silica gel (50 g), was loaded into the SIM (16); and (c) the isomers were eluted at a main system pressure of 100 psi and solvent pressure of 5560 psi, to afford 1.7 g of 2a (98%) pentafluorophenyl active esters. These procedures allow for the rapid and cost effective production of these carboxyfluorescein active
esters, and the Biotage FLASH 75 system offers the advantages of safety, reproducibility, and speed over other techniques for the separation of larger amounts of material. LITERATURE CITED (1) Haughland, R. P. (1991) Biosensors with Fiberoptics (D. L. Wise and D. Wingard, Eds.) p 85-109, Humana Press, Clifton, NJ. (2) Bright, F. V. (1988) Bioanalytical Applications of Fluorescence Spectroscopy. Anal. Chem. 60, 1031A-1039A. (3) Owens, M. A., and Loken, M. R. (1995) Flow Cytometry: Principles for Clinical Laboratory Practice, pp 28-29, WileyLiss, New York. (4) Haughland, R. P. (1992) Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene, OR. (5) Miki, M. (1987) The Recovery of the Polymerizability of Lys61-Labelled Actin by the Addition of Phalloidin. Eur. J. Biochem. 164, 229-235. (6) Lu-Steffes, M., et al. (1982) Fluorescence Polarization Immunoassay IV. Determination of Phenytoin and Phenobarbital in Human Serum and Plasma. Clin. Chem. 28, 22782282. (7) Adamczyk, M., Harrington, C., and Johnson, D. (1994) Reagents and Methods for the Quantification of Imipramine and Desipramine in Biological Fluids. U.S. Pat. 5,340,750. (8) Bodanszky, M. (1979) The Peptides, Vol. 1, pp 106-175, Academic Press, New York. (9) Bodanszky, M. (1984) Principles of Peptide Synthesis, pp 2849, Springer-Verlag, New York. (10) Reference 4, p 26. (11) The mixture is available from Fluka (Ronkonkoma, NY) and Kodak. Analytical HPLC revealed a 61:39 ratio using the Waters system described under Materials and Methods and eluting with 20% CH3CN/20% MeOH/60% aqueous 0.1% formic acid. (12) Molecular Probes, Eugene, OR. 1996 price $380 per 50 mg, 20 × $380/50 mg ) $7600/g. (13) Yoshida, R. A. (1980) Support Ligand Analog Fluorescer Conjugate and Serum Assay Method Involving Such Conjugate. Eur. Pat. Appl. 0015695. (14) Vanderbilt, A. S., Osikowicz, E. W., Fino, J. R., and Shipchandler, M. T. (1986) Total Estriol Fluorescence Polarization Immunoassay. Eur. Pat. Appl. 0200960. (15) Imming, P., and Jung, M-H. (1995) Pentafluorophenyl Esters of Dicarboxylic Acids. Arch. Pharm. 328, 87-91. (16) Stainless steel sample injection module (SIM) which delivers sample onto the cartridge. (17) Eluted with 15% acetone/85% toluene containing 0.05% acetic acid. (18) Eluted with 70% ethyl acetate/30% toluene (6000 mL), then 90% ethyl acetate/10% toluene (4000 mL), then 100% acetone containing 0.1% acetic acid (2000 mL). (19) Biotage FLASH 75 system user’s manual, version 2.0 (1995).
BC9600877
