Bioconjugate Chem. 1992, 3, 430-431
430
TECHNICAL NOTES Preparation of 5- and 6-(Aminomethy1)fluorescein Phillip G. Mattingly Abbott Laboratories, Diagnostics Division, One Abbott Park Road, Abbott Park, Illinois 60064. Received May 11,1992
5(6)-Carboxyfluorescein is protected as the diacetate then reduced to 5(6)-(hydroxymethyl)fluorescein diacetate. The separated isomers are subjected to a Mitsunobu reaction with dibenzyl imidodicarbonate, yielding diprotected 5-and 6-(aminomethyl)fluoresceindiacetate. Methanolysis of the acetates followed by deprotection with HBr/acetic acid gives 5- and 6-(aminomethyl)fluorescein hydrobromide.
INTRODUCTION
Many fluoresceinderivatives have functional groups that are suitable for reaction with other molecules, and can therefore serve as labels in a variety of analytical applications ranging from probing cell functions (1) to monitoring the level of drugs in human fluids via immunoassays (2). The choice of the fluorescein derivative can be crucial to the optimal performance of the conjugate formed and cannot usually be made a priori. This is especially true when the conjugate is to be used in an immunoassay. In such cases a series of conjugates are prepared from a variety of fluorescein derivatives. Commonly used derivatives include 5- and 6-carboxyfluorescein, 5- and 6-aminofluorescein, 5- and 6-fluorescein isothiocyanate, and 4'(aminomethy1)fluorescein(4'-AMF) (3). Of these, the 5and 6-aminofluorescein compounds are the most difficult to work with since the amino group is not very nucleophilic. This is an expected consequence of the amino group being directly bound to the deactivating aromatic ring. 4'(Aminomethy1)fluoresceinwas an earlier attempt to solve this problem by adding a methylene group between the amino group and the aromatic ring. While successful in restoring normal amino group reactivity, 4'-AMF derivatives do have some drawbacks, including a tedious purification in the preparation. In light of this, a fluorescein derivative substituted with a reactive amino group at the 5 or 6 position was desired. While 5-(aminomethyl)fluorescein has recently become commercially available, as of yet though there is not a reported preparation of the compound. The present work details the preparation of 5- and 6-(aminomethy1)fluorescein hydrobromide from the readily available mixture of 5(6)-carboxyfluorescein,thereby giving useful alternatives to currently used fluorescein labels. EXPERIMENTAL PROCEDURES
General Comments. All reagents were purchased from Aldrich Chemical Company, Inc., Milwaukee, WI, and were used without further purification, except where noted. Solvents employed were of reagent or HPLC grade and were used as received. lH NMR spectra were recorded at 200 MHz on a Chemagnetics A-200 spectrometer in CDC13 with TMS as a standard. High-resolution mass spectra were recorded on a Kratos MS-50 double-focusing mass spectrometer. 5(6)-(Hydroxymethy1)fluorescein Diacetate (3a,b). 5(6)-Carboxyfluoresceindiacetate was prepared from 5(6)-
carboxyfluorescein (Eastman Chemicals) following the procedure for unsubstituted fluorescein (4). 5(6)-Carboxyfluorescein diacetate (2.5 g, 5.5 mmol) and triethylamine (845pL, 6.0 mmol) are dissolved in THF (25 mL, distilled from benzophenone potassium ketyl) under a nitrogen atmosphere and cooled to 0 "C. Ethyl chloroformate (845 pL, 6.0 mmol) was added at once and the mixture stirred for 1h. Afterward, the mixture was fiitered and added to sodium phosphate buffer (15 mL, 1 M, pH 6.0) which had been cooled to 0 "C. NaBH4 (500 mg, 13.2 mmol) in water (5mL) was then added portionwise with efficient stirring over 5 min. The reaction was then immediately poured into cold water (100 mL) and extracted with ethyl acetate (3 X 50 mL). The extract was washed with saturated sodium chloride (2 X 25 mL), dried over anhydrous NazSO4, and evaporated to give the mixed 5(6)-(hydroxymethyl)fluorescein diacetate (1.73 g, 3.74 mmol, 68%). The mixture was separated by filtration through silica gel (5), eluting with methylene chloride/ diethyl ether (9:l). The first eluting compound was 3a. 'H NMR (6): 2.24 (6 H, s), 4.73 (2 H, d, J = 4.9 Hz), 6.86 (4 H, m), 7.1 (3 H, m), 7.55 (1 H, d, J = 8.2 Hz), 7.96 (1 H, d, J = 8.2 Hz). Calculated mass for C25H1908 447.1080, found 447.1075. The second eluting compound was 3b. lH NMR (6): 2.29 (6 H, s), 4.82 (2 H, d, J = 4.2 Hz), 6.78 (4 H, m), 7.1 (3 H, m), 7.63 (1H, d, J = 8.2 Hz), 8.0 (1H, 8 ) . Calculated mass for C25H1908447.1080, found 447.1075. 6 4[NP-Bis(carbobenzyloxy)amino]methyl]fluorescein Diacetate (4a). Compound 3a (1g, 2.2 mmol), triphenylphosphine (690 mg, 2.6 mmol), and dibenzyl imidodicarbonate (640 mg, 2.2 mmol) are stirred in THF (50 mL, distilled from benzophenone potassium ketyl) under a nitrogen atmosphere. To this solution is added diethyl azodicarboxylate (415 pL, 2.6 mmol) in THF (25 mL) dropwise over 30 min. After stirring for an additional hour, the solution is evaporated and the residue purified by filtration through silica gel by eluting with methylene chloride/diethyl ether (9:l) to give 4a (923 mg, 1.3mmol, 59%). 'H NMR (6): 2.32 (6 H, s), 4.9 (2 H, s), 5.14 (2 H, s), 5.16 (2 H, s), 6.73 (4 H, m), 6.94-7.35 (13 H, m), 7.47 (1H, d, J = 8.2 Hz), 7.88 (1H, d, J = 8.2 Hz). Calculated mass for C41H32NOll 714.1975, found 714.1977. 5- [[N,N-Bis(carbobenzyloxy )aminolmet hyl]fluorescein diacetate (4b)was prepared from 3b in the same manner as 4a. Compound 4b (1g, 1.4 mmol, 61%). lH NMR (6): 2.31 (6 H, s), 5.03 (2 H, s), 5.19 (2 H, s), 5.27 (2 H, s), 6.73 (4 H, m), 6.93-7.35 (13 H, m), 7.51 (1 H, d, 0 1992 American Chemical Society
Biocon/ugete Chem., Vol. 3, No. 5, 1992 431
Technical Notes
Scheme I' 0
OR
a
b-c
I
CWH 1
3a R=CH.CO- X=OH 6 isomer 3b R=CH;CO-, X=OH. 5 isomer 4a R=CH,CO, X=-N(& 6 isomer 4b R=CH,CO X=-N(& 5 isomer
2 R-CHJCO
5a X=-NLZb 6 isomer 5b X=-N(& 5 isomer
6a X=-NH3*Bi,6 isomer 6b X=-NH3'Bi, 5 isomer
carboxyfluorescein diacetate (2) (6)derivative was soluble in organic solvents commonly used for the reduction of carboxylic acid derivatives. The carboxylic acid was reduced to the alcohol by treatment with borane-tetrahydrofuran complex, or borane-dimethyl sulfide complex (7)over the course of 1-2 days. Loss of the acetyl groups was a concomitant side reaction which lessened the yield of the alcohol 3a,b. Alternatively, the carboxylic acid was converted into the mixed carbonic anhydride with ethyl chloroformate and reduced with NaBH4 (8) in pH 6 buffered, aqueous THF (5 min, 0 "C) to the alcohol 3a,b. The mixture of alcohols thus formed were conveniently separated at this stage by chromatography on silica gel. The alcohol 3a or 3b was converted into the protected amine 4a, 4b under Mitsunobu conditions (9) using dibenzyl imidodicarbonate (IO). Subsequent removal of the acetyl groups with methanol containing catalytic 4-(dimethylamino)pyridine,followed by treatment with HBr/acetic acid, gave (aminomethy1)fluorescein hydrobromide 6a or 6b.
(a) acetic anhydride, sodium acetate; (b) (i) ethyl chloroformate, (ii) sodium borohyride, pH 6; (c) diethyl azodicarboxylate, triphenylphosphme, dibenzyl imidodicarbonate; (d) methanol, DMAP; (e) HBr/acetic acid.
ACKNOWLEDGMENT
J = 8.2 Hz), 7.92 (1 H, s, J = 8.2 Hz). Calculated mass for C41H32NOll 714.1975, found 714.1977. 6-(Aminomethy1)fluorescein Hydrobromide (6a). Compound 4a (675 mg, 0.95 mmol) was stirred at reflux in methanol (50 mL) containing 44dimethylamino)pyridine (10 mg) for 24 h. The solvent was evaporated to give 5a. lH NMR (6): 4.92 (2 H, s), 5.13 (2 H, s), 5.20 (2 H, s), 6.61 (4 H, m), 6.94-7.35 (13 H, m), 7.49 (1H, d, J = 8.2 Hz), 8.0 (1H, d, J = 8.2 Hz). Calculated mass for C37H28N09 630.1764, found 630.1763. Without further purification 5a was dissolved in methylene chloride (50 mL, distilled from PzOd and treated with HBr/HOAc (315% , 5 mL). After stirring for 4 h the reaction mixture was filtered and the precipitate dried in vacuo for 12 h to give 6a (410 mg, 0.93 mmol, 98%). lH NMR (&-DMSO) (6): 4.12 (2 H, m), 6.53 (4 H, m), 7.06 (1H, s), 7.35 (2 H, d, J = 8.2 Hz), 7.76 (1H, d), 8.2 (1H, d, J = 8.2 Hz). Calculated mass for C21H~N05362.1028, found 362.1027. Compound 6a was 97% pure by HPLC 13.6 mm X 25 cm pBondaPak, C-18,25:5025 MeOH/H20/ 10% aqueous HOAc, 2 mL/min, t R = 4.97 minl when monitored by UV (254 nm) or fluorescence (480ed520em) detection. 5-(Aminomethy1)fluoresceinhydrobromide (6b)was prepared from 4b (1 g, 1.4 mmol) by the procedure used for 6a. Compound 5b. 'H NMR (6): 5.03 (2 H, s), 5.20 (2 H, s), 5.27 (2 H, s), 6.78 (4 H, m), 6.94-7.35 (13 H, m), 7.54 (1H, d, J = 8.2 Hz), 7.94 (1H, 8). Calculated mass for C37HzeN09 630.1764, found 630.1763. Compound 6b (600 mg, 1.36mmol,98%1. 'H NMR (d6-DMSO) (6): 4.24 (2 H, br s), 6.53 (4 H, m), 6.7 (2 H, m), 7.35 (1H, d, J = 8.2 Hz), 7.84 (1H, d, J = 8.2 Hz), 8.1 (1H, 8). Calculated mass for C21H16N05362.1028,found 362.1027. Compound 6b was 94% pure by HPLC [3.6 mm X 25 cm pBondapak, C-18, 25:50:25 MeOH/HzO/10% aqueous HOAc, 2 mL/ min, t~ = 6.14 minl when monitored by UV (254 nm) and 99 7% pure when monitored by fluorescence (480ed520,m) detection.
LITERATURE CITED
Mass spectra were recorded by D418, PPD Structural Chemistry.
RESULTS AND DISCUSSION
(1) See, for example: (a) Dive, C., Cox, H., Watson, J. V., and Workman, P. (1988)Polar fluorescein derivatives as improved substrate probes for flow cytoenzymologicalassay of cellular esterases. Mol. Cell.Probes 2,131. (b) Graber, M. L., DiLillo, D. C., Friedman, B. L., and Pastoriza-Munoz, E. (1986) Characteristics of fluoroprobes for measuring intracellular pH. Anal. Biochem. 156, 202. (2) See, for example: (a) Brynes, P. J., Martinus, J. A., Smith, C. M., Molina, C. M., and Vaughn, K. S. (1988) Fluorescence polarization immunoassay for amphetamine/metamphetamine. Eur. Pat. Appl. EP279213 (Chem.Abstr. 110,205680). (b) Wang, N. Y., Keegan, C. L., Hieman, D. F., Flentge, C. A., and Wang, P. P. (1988)New substituted 5-aryl-benzodiazepin2-one compounds useful as immunogens and tracers for fluorescence polarization immunoassay of benzodiazepin derivatives. Eur. Pat. Appl. EP264797 (Chem.Abstr. 110,91693). (3) Fino, J. R., Shipchandler, M. T., Klein, L. D., and Kirkemo, C. L. (1987) 4'-[AminomethylJfluorescein and its N-alkyl derivatives: useful reagents in immunodiagnostic techniques. Anal. Biochem. 162, 89. (4) Orndorff, W. R., and Hemmer, A. J. (1927) Fluorescein and some of it's derivatives. J. Am. Chem. Soc. 49, 1272. ( 5 ) Yau, E. K., and Coward, J. K. (1988) Filtering-column chromatography-a fast, convenient chromatographic method. Aldrichimica Acta 21, 106. (6) Bruning, J. W., Kardol, M. J., and Arnetzen, F. (1980) Carboxyfluorescein fluorochromasia assays. I. Non-radioactively labeled cell mediated lympholysis. J. Zmmunol. Methods 33, 33. (7) (a) Brown, H. C . (1972) Boranes in Organic Chemistry, Cornel1 University Press. (b) Lane, C. F. (1976) Reductions of organic compounds with diborane. Chem. Rev. 76, 773. (8) Ramasamy, K., Olsen, R. K., and Emery, T. (1982)Synthesis of N-t-Boc-L-a-aminoadipicacid 1-t-butyl-6-ethyl ester from L-aspartic acid: a new route to L-a-aminoadipic acid. Synthesis 42. (9) Mitsunobu, 0. (1981) The use of diethyl azodicarboxylate and triphenylphosphine in synthesis and transformation of natural products. Synthesis 1. (10) Grehn, L., Lurdes, M., Almeida, S., and Ragnarsson, U. (1988) Convenient preparation of alkyl benzyl imidodicarbonates, useful reagents for the direct synthesis of protected amines. Synthesis 992.
5(6)-Carboxyfluorescein (1,roughly 40:60 mixture) was modified to increase its solubility in organic solvents and to differentiate the two carboxyl groups, by treatment with acetic anhydride (Scheme I). Once so treated the 5(6)-
Registry No. 1 (isomer A), 76823-03-5; 1 (isomer B), 330179-9;2 (isomerA), 3348-03-6;2 (isomerB), 79955-27-4;3a, 14310655-2; 3b, 143106-56-3; 4a, 143106-57-4; 4b, 143142-35-2; Sa, 143106-58-5;5b, 143106-59-6;6a, 143106-60-9;6b, 143106-61-0.
