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Bioconjugate Chem. 2001, 12, 76−83
A New Approach to the Synthesis of APTRA Indicators Pieter A. Otten, Robert E. London, and Louis A. Levy* National Institute of Environmental Health Sciences, Laboratory of Structural Biology, P.O. Box 12233, MR-01, Research Triangle Park, North Carolina 27709. Received June 13, 2000; Revised Manuscript Received November 5, 2000
We have devised a general synthesis of Mg2+ indicators which is based on the aminophenol triacetic acid (APTRA) structure. The key step is a palladium-catalyzed coupling reaction of a precursor of the APTRA ligand with a fluorescent group. This strategy resulted in new ratioable fluorescent APTRA indicators and the finding that the fluorescence response of these indicators is different for Mg2+ and Ca2+ in some cases. We believe that this represents a generally useful approach for combining fluorophore and chelator functionalities.
INTRODUCTION
Fluorescent probes have proven to be valuable tools for the determination of the concentration of free metal ions in the cell cytosol (1) and have provided important insight into the function of these ions. As an analytical methodology, fluorescence spectroscopy is an attractive technique as it is very sensitive and only minute intracellular amounts of the indicator are necessary to obtain good signal-to-noise ratios. Intracellular loading of a fluorescent probe leaves the cells intact (in contrast to atomic absorption spectroscopy), thereby allowing one to study the functioning cell. Fluorescent probes can be used to study whole cell populations, single cells, or even subcellular domains. Time dependent changes in intracellular ion distribution can be studied with high spatial and temporal resolution. Fluorescent indicators can also be used in conjunction with flow cytometry to sort cells on the basis of their free metal content or other properties. Magnesium, the most abundant divalent metal ion in tissues, participates in a broad range of biochemical and regulatory processes. Magnesium ions are involved in many important functions in the cell such as enzyme activity, the conformation of DNA, ion transport through the cell membrane, the maintenance of cell shape, and in signaling pathways (2). Magnesium ions are essential cofactors for most of the ATP-dependent phosphorylation reactions which take place in the cell. Furthermore, the distribution of cytosolic magnesium and subcellular magnesium appears to be important in cell cycle control and regulation of cell differentation (3). Mag-fura-2 (1) [also known as FURAPTRA (4)], Magfura-5 (1), and Mag-indo (1) are three, widely used, commercially available, fluorescent probes for magnesium. A literature search revealed that in the period 1997-1998, over 40 papers have described the use of these indicators. In some recent literature examples, fluorescent indicators have been used to study the mechanism of Mg2+ transport across the cell membrane (5-8); to determine the influence of hormones on Mg2+ regulation in the cell (9-12); and in the investigation of this ion’s role in the fructose-induced activation of * To whom correspondence should be addressed. Phone: (919) 967-5736. E-mail:
[email protected].
glycogen phosphorylase (13). The use of Mag-fura 2 has revealed the high levels of cytosolic free Mg2+ in platelets of patients with essential hypertension (14) and vasospastic angina (15). An important and desirable feature of Mag-furas and Mag-indo is that they are ratioable indicators. Upon binding Mg2+, the excitation maximum (of all three indicators) and the emission maximum (Mag-indo) of the indicator-cation complex display a significant wavelength shift relative to the free indicator (1, 4, 16-18). This allows the ion concentration to be measured as a function of the ratio of the fluorescence at two different wavelengths, preferably the fluorescence maxima of both species (16). By using a ratioable fluorophore, the measurements of Mg2+ are not influenced by variations in the efficiency of the instrument and the dye concentration in the cell (18). Other commercially available fluorescent probes (1), Magnesium Green, Magnesium Orange, and Mag-Fura Red, are not ratioable but have excitation wavelengths in the visible region thereby eliminating the interference from the fluorescence of intracellular chromophores (18). The chelating moiety of the magnesium probes is based on the aminophenol triacetic acid (APTRA) structure, which was developed for this purpose by Levy et al. (19). At physiological pH, the deprotonated carboxylate groups, the anilino nitrogen atom, and the ether oxygen are all available for complexation (19). The dissociation constants (KMg D ), determined in vitro, of APTRA indicators fall in the low millimolar range [1.0-3.9 mM at physiological pH (1)]. These values correspond well with the low and submillimolar levels of cytosolic free Mg2+ (2), and thus, one can make use of the optimum sensitivity of the fluorophores. Care must be taken in using in vitro determined KMg D values as recent publications on the in vivo calibration of APTRA-type indicators, reported higher KMg D values [5.2 mM for Mag-indo (20) and 5.4 mM for Mag-fura-2 (21)]. It should also be noted that APTRA chelators have a higher affinity for Ca2+ than for Mg2+; the KCa D values were found to be around 30 µM. However, these values are 2 orders of magnitude higher than basal, free Ca2+ levels (17), and calcium, found at levels of about 100 nM, does not interfere with magnesium measurements under normal conditions. However, APTRA indicators also have been used as low affinity
10.1021/bc000069w Not subject to U.S. Copyright. Published 2001 by American Chemical Society Published on Web 12/21/2000
Synthesis of APTRA Indicators Scheme 1. Indicator
Retrosynthetic Approach to an APTRA
Ca2+ indicators (22-24). In addition, APTRA-type fluorophores can be applied to measure Zn2+ and Cd2+ (1). Recently, Prodi et al. has reported a nonratioable fluorescent l,10-diaza-18-crown-6 with a higher affinity for Mg2+ over Ca2+ (25). For the chelator to have fluorescent properties, the APTRA unit is connected to a reporter group, preferably a polyaromatic group, or the APTRA chelator may be incorporated as part of a polyaromatic structure. The development of new fluorescent magnesium indicators has been limited because of the lack of an efficient and general synthesis of these compounds. The present approach of a unique synthesis for each candidate is unattractive because the fluorescent response of an indicator upon metal complexation is unpredictable. From a retrosynthetic perspective, a general approach to the synthesis of new APTRA fluorophores would employ in one step, the joining of an APTRA chelator, or some precursor thereof, with a fluorescent group. The method of choice for the formation of such a carboncarbon bond between two aryl groups is a palladium catalyzed coupling reaction (26). A coupling reaction, catalyzed by an appropriate palladium catalyst, between triflate 1 (X ) OTf) and an organometallic reaction partner 2, carrying the fluorescent group, would give the basic structure of a new fluorophore (Scheme 1). Further elaboration of the obtained protected nitrophenol through established chemistry would afford the desired APTRA indicator. Using the strategy described above, we have synthesized three new fluorescent APTRA chelators carrying a naphth-1-yl, a naphth-2-yl, and a benzo[b]fur-2-yl substituent. The fluorescent response of these three fluorophores was found to depend strongly on the nature of the polyaromatic group and on whether the chelated ion is Mg2+ or Ca2+. EXPERIMENTAL PROCEDURES
General Remarks. Tetrahydrofuran (THF) was dried and distilled (Na/benzophenone) prior to use. Dimethylformamide (DMF) was dried over freshly activated (300 °C) molecular sieves (4 Å) and acetonitrile was distilled from P2O5. NaI was dried at 100 °C in vacuo. Ethyl acetate, hexane, diethyl ether, and all other commercially available chemicals were used as received. After extractive workup, the combined organic phases were dried over MgSO4. Water used in the UV and fluorescence experiments was filtered through a Hydro Picopure2 filter. Thin-layer chromatography was carried out on SigmaAldrich TLC Plates. Chromatographic purifications were done by flash chromatography using Merck Silica Gel, grade 9385, 230-400 mesh, 60 Å. Melting points were measured on a Fisher-Johns melting point apparatus and are uncorrected. 1H NMR spectra and 13C NMR spectra were recorded on a Nicolet NT 360 MHz NMR Spectrometer and a Varian UNITY plus 400 MHz NMR Spectrometer. Chemical shifts are reported in parts per million (δ), using TMS as an internal standard, and signals are
Bioconjugate Chem., Vol. 12, No. 1, 2001 77
expressed as an s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br (broad). UV spectra were recorded with a Beckman DU 640 Spectrophotometer using Fisherbrand semimicro quartz cells (Fisher Scientific). Fluorescence measurements were recorded with an Aminco-Bowman Series 2 Spectrometer using quartz fluorimeter cuvettes from Sigma. 2-Benzyloxy-4-fluoro-nitrobenzene (4). To a suspension of K2CO3 (5.18 g, 38 mmol) in 15 mL of DMF was added 2-nitro-5-fluorophenol (3) (3.93 g, 28 mmol) in one portion at room temperature. After gas evolution ceased, the temperature of the bright orange reaction mixture was raised to 70 °C and a solution of benzyl bromide (3.25 mL, 28 mmol) in 10 mL of DMF was added rapidly. After 4 h, TLC (10% EtOAc in hexane) indicated complete conversion of the starting material. The reaction mixture was poured into water and extracted three times with Et2O. The combined organic layers were washed twice with water. Evaporation of the solvent in vacuo gave a viscous yellow oil (5.96 g, 95%) which slowly solidified at room temperature, mp 58-60 °C. 1H NMR (CDCl3) δ 5.22 (s, 2H), 6.72 (ddd, 1H, J ) 2.6 Hz, J ) 7.0 Hz, J ) 9.1 Hz), 6.82 (dd, 1H, J ) 2.6 Hz, J ) 10.2 Hz), 7.32-7.48 (m, 5H), 7.95 (dd, 1H, J ) 5.9 Hz, J ) 10.2 Hz). 13C NMR (CDCl3) δ 72.52, 103.88 (d, J ) 26 Hz), 108.70 (d, J ) 24 Hz), 128.02, 129.10, 129.22, 129.52, 129.88, 135.87. 3-Benzyloxy-4-nitrophenol (5). To a solution of 4 (4.36 g, 17 mmol) in 50 mL of DMSO was added 11 mL of a 20% NaOH solution which resulted in a mildly exothermic reaction. The orange reaction mixture was stirred overnight and TLC (10% EtOAc in hexane) indicated complete consumption of the starting material. The reaction mixture was poured into 250 mL of water containing 5 mL of concentrated HCl and extracted three times with Et2O. The combined organic layers were washed two times with water. Evaporation of the solvent gave 5 as a yellow solid (4.04 g, 95%), mp 134-6 °C. 1H NMR (CDCl3) δ 5.21 (s, 2H), 6.44 (dd, 1H, J ) 2.6 Hz, J ) 9.0 Hz), 6.55 (d, 1H, J ) 2.2 Hz), 7.22-7.40 (m, 5H), 7.95 (d, 1H, J ) 9.2 Hz). 13C NMR (CDCl3) δ 72.00, 102.34, 107.66, 126.94, 128.23, 128.42, 128.71, 135.49, 154.84, 160.97. One quaternary carbon not observed. 2-Benzyloxy-4-(trifluoromethyl)sulfonyl-nitrobenzene (1). A solution of 5 (3.92 g, 16 mmol) in a mixture of 25 mL CH2Cl2, distilled from CaH2, and 10 mL of pyridine was cooled with an ice-bath. Over a period of 1 h, a solution of trifluoromethanesulfonic anhydride (5 g, 18 mmol) in 15 mL of CH2Cl2 was added. The reaction mixture was allowed to warm to room temperature and stirring was continued overnight. Subsequently, 25 mL of water and 100 mL of CH2Cl2 was added. The organic layer was separated and washed twice with 10% HCl. The organic layer was then washed twice with water. Evaporation of the solvent resulted in a yellow oil which was further purified by flash chromatography (30% EtOAc in hexane) to give a pale yellow oil which slowly solidified upon standing at room temperature (4.87 g, 80%), mp 56-8 °C. 1H NMR (CDCl3) δ 5.25 (s, 2H), 6.96 (dd, 1H, J ) 2.6 Hz, J ) 8.9 Hz), 7.03 (d, 1H J ) 2.4 Hz), 7.30-7.50 (m, 5H), 7.96 (d, 1H, J ) 9.1 Hz). 13C NMR (CDCl3) δ 72.18, 109.00, 113.26, 118.66 (q, J ) 320 Hz), 127.14, 127.19, 128.66, 128.84, 134.30, 139.64, 151.97, 153.21. 2-[3-Benzyloxy-4-nitrophenyl]benzo[b]furane (7a). A 50 mL three-necked, round-bottomed flask, well-purged with argon, was charged with 1 (1.31 g, 3.70 mmol), benzo[b]furan-2-boronic acid (6a) (470 mg, 3.0 mmol), Pd[P(C6H5)3]4 (20 mg, 0.017 mmol), 1 M Na2CO3 (5 mL) and
78 Bioconjugate Chem., Vol. 12, No. 1, 2001
10 mL of DME. The reaction mixture was heated for 4.5 h at 80 °C, cooled, poured into water and extracted three times with CH2Cl2. Evaporation of the solvent gave an orange solid which was crystallized twice from benzene to give 7a as bright yellow crystals (782 mg, 75%), mp 137-9 °C. 1H NMR (CDCl3) δ 5.37 (s, 2H), 7.20-7.70 (m, 12H), 7.99 (d, 1H, J ) 8.5 Hz), 13C NMR (CDCl3) δ 71.77, 104.72, 111.42, 117.03, 121.53, 123.51, 125.65, 126.39, 126.46, 127.18, 128.31, 128.72, 135.52, 135.99, 152.73, 153.32, 155.39. One quaternary carbon not observed. MS: (m/z) 345.0994, required for C21H15NO4 345.1001. 1-[3-Benzyloxy-4-nitrophenyl]naphthalene (7b). In a manner similar to the preparation of 7a, compound 7b was prepared and isolated pure (91%) as a white powder by flash chromatography (10% EtOAc in hexane), mp 115-8 °C. 1H NMR (CDCl3) δ 5.28 (s, 2H), 7.18 (dd, 1H, J ) 1.7 Hz, J ) 8.1 Hz), 7.30-7.50 (m, 6H), 7.507.60 (m, 2H), 7.69 (d, 1H, J ) 8.8 Hz), 7.93 (d, 1H, J ) 8.1 Hz), 7.94 (d, 1H, J ) 8.1 Hz), 8.02 (d, 1H, J ) 8.1 Hz). 13C NMR (CDCl3) δ 71.42, 117.23, 122.33, 125.13, 125.19, 125.62, 126.11, 126.67, 126.76, 127.05, 128.17, 128.46, 128.67, 128.81, 130.97, 133.83, 135.53, 137.95, 147.12, 151.66. MS: (m/z) 355.1233; required for C23H17NO3, 355.1208. 2-[3-Benzyloxy-4-nitrophenyl]naphthalene (7c). In a manner similar to the preparation of 7a, compound 7c was prepared and isolated pure (92%) as an off-white powder by flash chromatography (10% EtOAc in hexane), mp 114-6 °C. 1H NMR (CDCl3) δ 5.36 (s, 2H), 7.30-7.46 (m, 5H), 7.50-7.58 (m, 4H), 7.64 (dd, J ) 9 Hz, J ) 1.5 Hz), 7.84-8.08 (m, 5H). 13C NMR (CDCl3) δ 71.71, 114.54, 119.69, 124.83, 126.27, 126.59, 126.76, 126.79, 127.15, 127.70, 128.26, 128.34, 128.71, 128.87, 133.28, 133.48, 135.67, 136.38, 139.26, 147.39, 152.44. MS: (m/z) 355.1196; required for C23H17NO3, 355.1208. 2-(3-Hydroxy-4-aminophenyl)benzo[b]furan-N,N,Otriacetic acid trimethylester (9a). Compound 7a (345 mg, 1.00 mmol) and 100 mg of 5% Pd on C were suspended in 25 mL of ethanol. This reaction mixture was hydrogenolyzed over 16 h in a Parr apparatus at an initial H2 pressure of 30 psi. The reaction mixture was filtered and the solvent evaporated in vacuo. TLC (50% EtOAc in hexane) indicated complete consumption of the starting material and 1H NMR of the crude product confirmed complete removal of the benzyl group. The crude aminophenol 8a was dissolved in 10 mL of acetonitrile and transferred to a 100 mL oven-dried, threenecked, round-bottomed flask, equipped with a stirring bar and oven-dried condenser and charged with NaI (1.0 g, 6.7 mmol), Proton Sponge (1.7 g, 7.9 mmol), and methyl bromoacetate (1.0 mL, 10 mmol). The reaction mixture was refluxed for 16 h under an argon atmosphere, allowed to cool to room temperature, and poured into 200 mL of Et2O. The resulting white suspension was filtered and the filtrate was washed with 50 mL portions of buffer (pH 2, Hydrion), until the aqueous layer remained acidic. The ether layer was dried, filtered and 5 g of silica gel was added. After evaporation of the solvent, the silica gel was loaded on a pre-packed column (33% EtOAc in hexane). Triester 9a was obtained as a colorless oil (210 mg, 47% overall). 1H NMR (CDCl3) δ 3.75 (s, 6H), 3.82 (s, 3H), 4.26 (s, 4H), 4.76 (s, 2H), 6.87 (s, 1H), 6.92 (d, 1H, J ) 8.5 Hz), 7.20-7.28 (m, 2H), 7.30 (d, 1H, J ) 1.5 Hz), 7.42 (dd, 1H), J ) 1.8 Hz, J ) 8.2 Hz), 7.49 (d, 1H, J ) 8.2 Hz), 7.54 (d, 1H, J ) 8.7 Hz). 13C NMR (CDCl3) δ 51.70, 52.07, 53.82, 66.56, 100.29, 110.96, 111.88, 119.80, 119.85, 120.59, 122.84, 123.87, 124.84, 129.40, 140.07, 149.67, 154.75, 155.60, 168.98, 171.35. MS: (m/ z) 441.1400; required for C23H23NO3, 441.1424.
Otten et al.
1-(3-Hydroxy-4-aminophenyl)naphthalene-N,N,Otriacetic Acid Trimethylester (9b). In a manner similar to the preparation of 9a, compound 9b was obtained as a colorless oil (64% overall yield) by flash chromatography (25% EtOAc in hexane). 1H NMR (CDCl3) δ 3.76 (s, 3H), 3.77 (s, 6H), 4.30 (s, 4H), 4.68 (s, 2H), 6.93 (d, 1H, J ) 2.0 Hz), 6.98 (d, 1H, J ) 8.4 Hz), 7.07 (dd, 1H, J ) 2.0 Hz, J ) 8.4 Hz), 7.37 (dd, 1H, J ) 0.8 Hz, J ) 7.0 Hz), 7.41 (ddd, 1H, J ) 1.6 Hz, J ) 6.8 Hz, J ) 8.4 Hz), 7.45-7.50 (m, 2H), 7.82 (d, 1H, J ) 8.0 Hz), 7.88 (d, 1H, J ) 8.8 Hz), 7.91 (d, 1H, J ) 8.8 Hz). 13C NMR (CDCl3) δ 52.89, 53.16, 54.69, 67.13, 117.47, 120.36, 125.27, 126.40, 126.81, 127.02, 127.07, 127.83, 128.54, 129.30, 132.67, 134.86, 135.87, 139.72, 140.61, 150.12, 170.22, 172.89. MS: (m/z) 451.1621; required for C25H23NO7, 451.1631. 2-(3-Hydroxy-4-aminophenyl)naphthalene-N,N,Otriacetic Acid Trimethylester (9c). In a manner similar to the preparation of 9a, compound 9c was obtained as a colorless oil (51% overall yield) by flash chromatography (25% EtOAc in hexane). 1H NMR (CDCl3) δ 3.75 (s, 6H), 3.81 (s, 3H), 4.27 (s, 4H), 4.77 (s, 2H), 7.00 (d, 1H, J ) 8.0 Hz), 7.18 (d, 1H, J ) 2.0 Hz), 7.31 (dd, 1H, J ) 2.0 Hz, J ) 8.0 Hz), 7.43-7.51 (m, 2H), 7.65 (dd, 1H, J ) 2.0 Hz, J ) 8.4 Hz), 7.85 (dd, 1H, J ) 9.6 Hz, J ) 9.6 Hz), 7.87 (d, 1H, J ) 8.8 Hz), 7.93 (s, 1H). 13C NMR (CDCl ) δ 52.90, 53.24, 54.68, 67.50, 115.34, 3 121.08, 122.79, 126.19, 126.34, 126.85, 127.36, 128.68, 129.13, 129.45, 133.50, 134.71, 136.55, 138.85, 140.06, 150.89, 170.50, 172.73. MS: (m/z) 451.1618, required for C25H23NO7 451.1631. 2-(3-Hydroxy-4-aminophenyl)benzo[b]furan-N,N,Otriacetic Acid (10a). In a 25 mL round-bottomed flask, equipped with a condenser, triester 9a (60 mg, 0.17 mmol) was suspended in a mixture of 2 mL of methanol and 2 mL of 1 M NaOH. The reaction mixture was refluxed for 4 h and allowed to cool to room temperature. The solvent was evaporated in vacuo and the remaining solid material was dissolved in 2 mL of water to give a clear yellow solution. The aqueous solution was cooled with a ice-bath and acidified with 1 M HCl to pH 3. Stirring was continued for 15 min. The precipitate was collected by filtration and dried in vacuo over P2O5 to give 50 mg (89%) of an off-white powder which decomposes above 240 °C. 1H NMR (DMSO-d6) δ 3.53 (s, 4 H), 4.17 (s, 3 H), 6.73 (d, 1H, J ) 8.4 Hz), 6.76 (s, 1H), 6.90 (d, 1H, J ) 1.7 Hz), 7.02-7.10 (m, 3H), 7.28 (d, 1H, J ) 8.0 Hz), 7.37 (d, 1H, J ) 7.0 Hz). 13C NMR [D2O (1.0 mL and 50 µL of 40% NaOD)] δ 57.78, 68.67, 100.85, 110.44, 111.94, 118.81, 119.31, 121.86, 123.36, 124.15, 125.03, 130.38, 150.39, 155.27, 156.91, 178.12, 180.42. 1-(3-Hydroxy-4-aminophenyl)naphthalene-N,N,Otriacetic acid (10b). In a manner similar to the preparation of 10a, triacid 10b was obtained as an off-white powder (57%), which decomposes above 230 °C. 1H NMR [D2O (1.0 mL, and 50 µL of 40% NaOD)] δ 3.68 (s, 4H), 4.33 (s, 2H), 6.83 (d, 1H, J ) 8.3 Hz), 6.95 (s, 1H), 7.08 (d, 1H, J ) 7.7 Hz), 7.30-7.48 (m, 2H), 7.55 (d, 1H, J ) 8.0 Hz), 7.60-7.80 (m, 3H), 7.83 (s, 1H). 13C NMR [D2O (1.0 mL and 50 µL of 40% NaOD)] δ 58.13, 68.54, 112.50, 119.65, 121.01, 125.42, 126.00, 127.09, 127.62, 128.59, 129.05, 129.47, 133.07, 134.74, 138.34, 140.55, 150.93, 171.97, 180.33. 2-(3-Hydroxy-4-aminophenyl)naphthalene-N,N,Otriacetic acid (10c). In a manner similar to the preparation of 10a, triacid 10c was obtained as a slightly pink powder (49%), which decomposes above 250 °C. 1H NMR [D2O (1.0 mL, and 50 µL of 40% NaOD)] δ 3.86 (s, 4H), 4.44 (s, 2H), 6.87 (s, 1H), 6.92 (d, 1H, J ) 8.42 Hz), 6.99
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Bioconjugate Chem., Vol. 12, No. 1, 2001 79
Table 1. Spectroscopic Data and KD Values of Indicators 10a-c Mg2+
Ca2+
compd
abs. max (nm)
λemiss (nm)b
λexcit (nm)b,c
KD (mM)
λemiss (nm)
λexcit (nm)b,c
KD (µM)
10a 10b 10c
330 310 316
416 f 407 499, 358 497
340 f 328 (410) 325 f 295 (499) 328 f 305 (498)
2.3 1.8 2.1
414 f 411 500, 362 498
340 f 328 325 f 499 328 f 498
70 17 28
a Absorption maximum of the free indicator. b An arrow denotes a shift of the fluorescence maximum upon ion complexation. c The emission wavelength used is given in brackets.
(d, 1H, J ) 7.7 Hz), 7.40-7.58 (m, 4H), 7.87 (d, J ) 8.05, 1H), 7.90-8.02 (m, 2H). 13C NMR [D2O (1.0 mL and 50 µL of 40% NaOD)] δ 57.91, 68.51, 115.75, 118.88, 123.81, 126.77, 126.84, 127.22, 127.49, 127.92, 128.44, 129.37, 132.04, 134.06, 134.62, 140.22, 140.53, 150.33, 178.05, 180.61. Fluorescence Measurements. The buffered solutions used in all experiments contained 120 mM KCl, 20 mM NaCl, and 15 mM Tris and were brought to pH 7.20 with concentrated HCl. Using sonication, a stock solution of indicator 10b was prepared by dissolving 3.2 mg (7.8 µmol, 1.6 mM) in 5 mL of buffer. To record a UV spectrum, the stock solution of 10b was diluted 40 times (25 µL in 975 µL of buffer). For the fluorescence titration experiments, 25 µL of stock solution was diluted 100 times (to 16 µM) with 2475 µL of buffer. A fluorescence emission and a fluorescence excitation spectrum were recorded (for excitation and emission wavelengths, see Table 1). To 2475 µL of 500 mM MgCl2 solution was added 25 µL of stock solution of the indicator. To the solution of the free indicator, increasing amounts of the MgCl2/indicator solution (5, 10, 20, 40, 80, and 160 µL) were added and after each titration step, a fluorescence emission and a fluorescence excitation spectrum were recorded. In the case of a fluorescence titration with CaCl2, a 1 mM solution was used and portions of 5, 10, 20, 40, 80, 160, 320, and 320 µL of the CaCl2/indicator solution were added. Using this procedure, fluorescence titrations of indicators 10a, and 10c were performed with stock solutions of these indicators of 0.81 and 0.54 mM, respectively. and The corresponding dissociation constants (KMg D Ca KD ) were determined from the fluorescence/titration experiments as follows. In the case of Mg2+, the free Mg2+ concentration, [Mg2+], was approximated by the total Mg2+ added, since the correction for bound Mg2+ is minimal. The data were fit to a straight line according to
[
[Mg2+] ) KMg D
(F - F0)
(Fsat - F)
]
(1)
[
(R - R0)
(Rsat - R)
]( ) F0,λ2
Fsat,λ2
Scheme 3. Suzuki Coupling of 1
Scheme 4. Completion of the APTRA Synthesis
Since for Ca2+ binding, the Ca2+ dissociation constant, is of the same order of magnitude as the indicator concentration, the data were analyzed using a two parameter fit using Mathematica to fit a nonlinear function according to
KCa D ,
where F is the fluorescence at a given wavelength (preferably the fluorescence maximum) and at a given [Mg2+], and F0 and Fsat are the fluorescence values for zero and saturating Mg2+ . For a ratioable indicator, with fluorescence maxima at λ1 and λ2 for the free and metalbound indicator, respectively, the data were fit to a straight line applying the following equation:
[Mg2+] ) KMg D
Scheme 2. Synthesis of Triflate 1
(2)
where R is the ratio of the fluorescence values at λ1 and λ2 at a given [Mg2+], R0 and Rsat are the ratios at zero and saturating Mg2+, and F0,λ2 and Fsat,λ2 represent the fluorescence values of the free and saturated indicator at λ2.
Fobs ) FF(1 - pB) + FBpB
(3)
where Fobs is the observed intensity of the fluorescence at the measured wavelength, FF and FB are the indicator fluorescence values of the uncomplexed and Ca2+-complexed forms, and pB the fraction of indicator bound to Ca2+. The latter were calculated from the equilibrium equation assuming 1:1 binding stoichiometry:
pB ) leading to
Ca - I I0
(4)
80 Bioconjugate Chem., Vol. 12, No. 1, 2001
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Figure 1. Fluorescent responses of compound 10b to Mg2+ and Ca2+. (A) Emission spectra as a function of [Mg2+] (in mM), excitation at 310 nm; (B) emission spectra as a function of [Ca2+] (in µM), excitation at 310 nm; (C) Excitation spectra as a function of [Mg2+] (in mM), emission measured at 499 nm; (D) Excitation spectra as a function of [Ca2+] (in µM), emission measured at 499 nm. Spectra obtained at 20 °C. SYNTHESIS
2+ pB ) (I0 + KCa D + [Ca ] 2+ 2 2+ x(I0 + KCa D + [Ca ]) - 4I0[Ca ])/2
(5)
where I0 is the total indicator concentration, the two parameters to be fit are the fluorescence of the Ca2+indicator complex, FB, and the Ca2+-indicator dissociation constant, KCa D . The value of FF is obtained directly from the spectrum of the sample in the absence of added calcium.
Triflate 1 was obtained in three steps (Scheme 2) starting from commercially available 2-nitro-5-fluorophenol 3. The starting phenol 3 was protected as its benzyl ether 4 by a reaction with benzyl bromide in DMF/ K2CO3. The crude benzyl ether proved to be sufficiently pure for further manipulation. The key step in the synthesis of 1 stems from the realization that the electron-poor aromatic ring of compound 4 readily un-
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Bioconjugate Chem., Vol. 12, No. 1, 2001 81
Figure 2. Fluorescent excitation spectra for compound 10c. (A) as a function of [Mg2+] (in mM), emission measured at 498 nm; (B) as a function of [Ca2+] (in µM), emission measured at 498 nm. Spectra obtained at 20 °C.
dergoes a nucleophilic aromatic substitution at the carbon bound to fluorine. Thus, when 4 was subjected to NaOH in DMSO/H2O at room temperature, the fluorine substituent was replaced by a hydroxyl group to give phenol 5 in quantitative yield. Phenol 5 is then converted to the triflate 1 in good yield. Protection of the phenol 3 as its benzyl ether serves two purposes. First, it prevents deprotonation of the phenol which would prevent the substitution reaction from taking place. Second, it allows a regioselective preparation of triflate 1 via the newly created hydroxyl group para to the nitro substituent of 4. Standard conditions for the Suzuki-coupling reaction (Scheme 3) of boronic acids 6a-c with triflate 1 were employed (27). It was found that using the weaker base NaHCO3 instead of Na2CO3 did not result in conversion of 1. Consumption of 1 was complete after several hours at 80 °C in all cases studied. The coupling products were obtained pure and in excellent yields after extractive workup, followed by flash chromatography. The highly crystalline nature of benzo[b]fur-2-yl derivative 7a allowed its purification by crystallization from benzene. Reduction of the nitro group and deprotection of the adjacent benzyl ether of compounds 7a-c, was accomplished in one step by palladium-catalyzed hydrogenation and debenzylation in ethanol. The 1H NMR spectrum of the crude products showed that complete reduction/deprotection was achieved after 16 h. The use of ethyl acetate, although a better solvent, led to incomplete removal of the benzyl ether. The crude aminophenol derivatives 8a-d were found to be sufficiently pure for further manipulation. Alkylation of aminophenols 8a-d with methyl bromoacetate was achieved in refluxing acetonitrile in the presence of Proton Sponge and sodium iodide. After extractive workup and flash chromatography the corresponding triesters 9a-d were obtained in good overall yields (Scheme 4). It was found that the use of carefully dried acetonitrile and the use of NaI (to form, in situ, the more reactive methyl iodoacetate) were necessary for success of this reaction.
The triesters were readily saponified in refluxing aqueous MeOH/NaOH. After acidic workup, tri-acids 10a-c, R2dH were isolated in a pure state by filtration. FLUORESCENCE
The emission and excitation fluorescence spectra of 10a-c were recorded using 5.4-16 µM solutions of the chelator, buffered at pH 7.2. The solutions also contained 120 mM KCl and 20 mM NaCl to mimic the ionic strength of the intracellular milieu. These solutions were titrated with stock solutions of MgCl2 or CaCl2 containing an equimolar concentration of the APTRA chelator to cancel out dilution effects during the titration. After each titration step, both the emission and excitation fluorescence spectra were recorded. The dissociation constants of indicators 10a-c for Mg2+ were determined as originally described by Grynkiewicz et al. (18). It should be noted that the micromolar range concentrations of the APTRA indicators used here are of the same order of magnitude as the expected KDs for Ca2+. We found that when using APTRA concentrations in the nanomolar range for the fluorescence titrations with Ca2+, the likely presence of trace metal impurities in the materials used caused nonreproducible fluorescent responses. We therefore chose to apply higher indicator concentrations to minimize the influence of trace metal impurities on the total fluorescence. That the problem was with the adventitious trace metals was confirmed by the fact that addition of EGTAsto capture trace metal impuritiessto micromolar solutions of the free indicator did not change the fluorescence intensities. The KD values for Ca2+ were determined by applying a nonlinear leastsquares module of Mathematica (see Experimental Procedures). Table 1 shows the results of the spectral analysis of indicators 10a-c. Because of the buffering effect of the indicator on the Ca2+ concentration, it is not possible to compare changes in the fluorescence intensities to those observed with Mg2+.
82 Bioconjugate Chem., Vol. 12, No. 1, 2001 RESULTS
Indicator 10a proved to be a nonratioable fluorescent indicator for Mg2+ and Ca2+. The change in fluorescent response to increasing Mg2+ concentrations of APTRA chelator 10a, carrying a benzo[b]fur-2-yl group, is small. Upon saturation of the chelator with Mg2+, the fluorescence emission intensity increases 25%. In addition, a small shift of the emission maximum from 407 to 416 nm is observed. The change in fluorescence response to increasing Ca2+ concentrations is similar. When saturated with Mg2+, the fluorescence excitation maximum of 10a at 340 nm displays an overall decrease of its intensity. In addition, a shift of the excitation fluorescence maximum to 328 nm from 340 nm is observed. An identical shift of the excitation fluorescence maximum of 10a upon an increase of the Ca2+ concentration was found. Figures 1 and 2 show the titration/fluorescence spectra of compounds 10b and 10c with Mg2+ and Ca2+. The emission fluorescence spectrum of naphth-1-yl-substituted APTRA ligand 10b shows two maxima at 358 and 499 nm (Figure 1A). Saturation of the chelator with Mg2+ results in an increase of the fluorescence intensities at both wavelengths; a 7-fold increase at 358 nm and a 1.8fold increase at 499 nm are observed. No shifts of either emission maxima were found. Titration of 10b with Ca2+ resulted in a different response in its fluorescence spectrum (Figure 1B). The fluorescence intensity at 362 nm increased, as was observed with Mg2+, but the fluoresence intensity at 500 nm was found to decrease upon increasing Ca2+ concentration. An isosbestic point was found at 400 nm. Thus, APTRA chelator 10b is a ratioable indicator for Ca2+. Titration of 10b with Mg2+ and measurement of its fluorescence excitation spectra shows that this APTRA derivative can be used as a ratioble indicator (Figure 1C). The excitation fluoresence at 325 nm (emission observed at 499 nm), corresponding to the free chelator, decreases upon complexation whereas a peak at 295 nm of the Mg2+ complex appears and increases in intensity upon further titration. An isoexcitation point was observed at 318 nm. In contrast, upon titration with Ca2+, the fluorescence excitation spectrum displayed a different behavior. Again, the intensity of the excitation fluorescence at 325 nm decreases with increasing Ca2+ concentration (emission observed at 499 nm), while there is a very small increase in intensity of the excitation fluorescence at lower wavelengths (Figure 1D), in contrast with the case of a titration with Mg2+ (Figure 1C). This difference between the responses of the fluorescence excitation of 10b could allow one to make a distinction between changes in Mg2+ or Ca2+ levels in the cell. In the latter case, a change in fluorescence excitation at 325 nm will not be accompanied by a change in fluorescence at 295 nm. In contrast to the naphth-1-yl-substituted APTRA chelator, the naphth-2-yl-substituted derivative 10c shows only one maximum in its emission spectrum at 498 nm. Upon saturation of the chelator with Mg2+, the fluorescence intensity does not change significantly; a shift of fluorescence maximum to 482 nm being observed. Saturation of 10c with Ca2+ results in a reduction of the fluorescence intensity at 498 nm. Titration of 10c with Mg2+ and recording the excitation fluorescence spectra shows that this chelator possesses the spectral characteristics required for a ratioable indicator (Figure 2A). The fluorescence excitation at 328 nm (emission observed at 498 nm) decreases upon increasing Mg2+ concentration, whereas the fluorescence at 305 nm increases. An isoexcitation point is found at 313 nm. Titration of 10c
Otten et al.
with Ca2+ only results in a decrease of the fluorescence at 328 nm (emission observed at 498 nm), but no response of the fluorescence was observed at lower wavelengths (Figure 2B). Thus, chelator 10c could also be useful to distinguish changes of intracellular Mg2+ levels from “calcium spikes” in the cell. CONCLUSIONS
The palladium catalyzed coupling reaction has proven to be a useful tool for the synthesis of new fluorescent APTRA indicators. Triflate 1 could be readily coupled with polyaromatic organoboron species and the resulting coupling products could then be easily carried on to the desired APTRA derivatives in good overall yields. The fluorescent response of the indicators on complexation of Mg2+ and Ca2+ depends greatly on the nature of the polyaromatic group coupled to the chelating APTRA moiety. Benzo[b]fur-2-yl-substituted ligand 10a was found to be a nonratioable indicator for both Mg2+ and Ca2+. However, the APTRA-ligand substituted with a naphth1-yl or a naphth-2-yl group did result in the discovery of two new ratioable fluorescent indicators for Mg2+. Indicators 10b and 10c showed an appreciable spectral shift of 30 and 23 nm, respectively in their fluorescent excitation spectra upon complexation of Mg2+ and with useful dissociation constants. Thus, these new ratioable indicators may be expected to find use in studying intracellular Mg2+ levels by fluorescence microscopy/imaging techniques. We also found that the nature of the fluorescent response of indicators 10b and 10c depends strongly on the metal ion. This is most notable for indicators 10b and 10c. When titrated with Mg2+, both indicators displayed the desired ratioable characteristics in their excitation fluorescence spectra. On the other hand, the excitation fluorescence spectra showed only a decrease of the fluorescence intensity upon increasing Ca2+ levels. This observation is important because corrections made for (high intracellular levels of) Ca2+ when measuring intracellular Mg2+ are often based on the assumption that the fluorescent response of an indicator to both metal ions is identical (13). The dissociation constants for Mg2+ found for all three indicators 10a-c, ranging from 1.8 mM to 2.3 mM, are similar to the values reported for Mag-indo and Magfura (1). Analysis of the Ca2+ fluorescence/titration data via a two parameter nonlinear fit resulted in KCa D values for indicators 10a-c (70, 17, and 28 µM) which correspond very well with the values found for the commercially available APTRA indicators. The lower Ca2+ affinity found for benzo-[b]-fur-2-yl substituted indicator 10a may be explained by the stronger electron-withdrawing character of this hetero-aromatic system compared to a naphthyl group. ACKNOWLEDGMENT
Mass spectrometry was provided by the Washington University Mass Spectrometry Resource with support from the NIH National Center for Research Resources (Grant P41RR0954). LITERATURE CITED (1) Haugland, R. P. (1996) Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes Inc., Eugene, OR. (2) Romani, A., and Scarpa, A. (1992) Regulation of Cell Magnesium. Arch. Biochem. Biophys. 298, 1-12. (3) Di Francesco, A., Desnoyer, R. W., Covacci, V., Wolf, F. I., Romani, A., Cittadini, A., and Bond, M. (1998) Changes in
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