Anal. Chem. 2003, 75, 549-555
A Fluorescent Chemosensor for Sodium Based on Photoinduced Electron Transfer Huarui He,*,† Mark A. Mortellaro,† Marc J. P. Leiner,‡ Susanne T. Young,† Robert J. Fraatz,† and James K. Tusa†
Roche Diagnostic Corporation, 235 Hembree Park Drive, Roswell, Georgia 30076, and Roche Diagnostic GmbH, 1 Hans-List-Platz, A-8020 Graz, Austria
A new optical sensor suitable for practical measurement of sodium in serum and whole blood samples is described. The optical sensor is based on a novel PET (photoinduced eletron transfer) fluoroionophore immobilized in a hydrophilic polymer layer. The design concept of the fluoroionophore follows the receptor-spacer-fluorophore approach to sensor design using intramolecular PET-based signal transduction. Key to the development of this sensor is the identification of a nitrogen-containing, sodium-binding ionophore, coupled with a fluorophore having the correct spectral and electron-accepting properties. The slope of the sensor is ∼0.5%/mM in the typical clinically significant range of 120-160 mM. This sensor has been implemented into a disposable cartridge, used for a commercially available critical care analyzer (Roche OPTI CCA) with precision better than (1 mM (1 SD). The sensor displays excellent stability against hydrolysis and oxidation, leading to slope changes 50 nm), and
low sensitivity to pH because of the absence of ionizable functional groups within the physiological pH range. We chose N-(omethoxyphenyl)aza-15-crown-519 as a suitable ionophore because of its sodium binding in the desired measuring range, minimal interference from pH, and the presence of a tertiary amine that acts as a PET fluorescence quencher. The synthesis described below starts with the bifunctional commercially available starting material, 4-chloro-1,8-naphthalic anhydride; its two reactive functional groups allow convenient synthesis of an “ionophorespacer-fluorophore” type construct.15 EXPERIMENTAL SECTION Reagents. Solvents and reagents used in the synthesis of fluoroionophore 11 were purchased form Aldrich (Milwaukee, WI) and used without further purification. Analytical grade buffer and inorganic salts were purchased from either Fluka AG (Buchs, Switzerland) or Sigma Co. (St. Louis, MO). The hydrophilic hydrogel D4 was purchased from Tyndale Plains-Hunter Ltd. (Ringoes, NJ); the polyester sheets (MELINEX), from Polymer Supplier (Atlanta, GA). Absorbance and Fluorescence Titrations. Absorption measurements were performed with a Shimadzu UV2101PC spectrophotometer. Titration of ionophore 3 was carried out in the following manner: A methanolic solution of ionophore was diluted with deionized water in a volumetric flask, the required amount of solid sodium chloride was added, and the solution’s absorption spectrum was measured. Fluorescence measurements were performed with an ISS PC1 photon-counting spectrofluorometer with a xenon lamp as excitation source. A sensor disk (25 mm, see below) was placed into a custom-made flow-through cell fitted into the fluorometer. N-(2-Hydroxyethyl)piperazine-N′-(ethanesulfonic acid) (HEPES) buffer solutions of the appropriate pH and ion concentrations were then pumped through the cell prior to collection of emission and excitation spectra. Buffer pH values were measured at room temperature (∼22 °C) and 37 °C with a Corning pH meter 125 and AVL 987 analyzer, respectively. All NMR data were collected at room temperature by QE 300 (Nicolet/GE), 300 MHz, with 0.1% TMS as standard. Elemental analyses were performed by Galbraith Laboratories, Inc., Knoxville, TN. Immobilization of Sodium Fluoroionophore (11) onto Aminocellulose. Aminocellulose (5 g)20 was suspended in 50 mL of 2.5% aqueous sodium carbonate for 30 min, filtered, resuspended in 50 mL DMF for 30 min, filtered, and then washed twice more with DMF in order to replace trapped water. The washed cellulose was then transferred into a flask containing 11 (0.21 g, 0.3 mmol), N,N-dicyclohexyl-1,3-carbodiimide (DCC, 0.62 g, 3 mmol), and N-hydroxysuccinimide (NHS, 0.35 g, 3 mmol) in anhydrous DMF (20 mL), and the suspension was stirred at room temperature for 20 h. The yellow cellulose fiber was filtered, washed with DMF (5 × 50 mL) until the filtrate became colorless, and then washed with water (50 mL), 0.2 N HCl (2 × 50 mL), water (50 mL), 0.2 N NaOH (2 × 50 mL), water (10 × 50 mL), acetone (2 × 50 mL), and ether (2 × 50 mL), after which it was then dried at room temperature for 16 h. The resulting cellulose powder was yellow-colored, and the exact amount of the immobilized dye was not measured. Preparation of Sensor Disk. Sieved (25-µm) indicatorimmobilized amino-cellulose fiber (0.5 g) was stirred 16 h into a
D4 hydrogel dispersion (9.5 g) containing 10% solids in 90% w/w ethanol/water. The resulting dispersion was knife-coated onto a 125-µm polyester sheet such that the indicator layer dried to a thickness of 10 µm. A second hydrogel layer consisting of 3% w/w carbon black (Degussa Corporation) in the same hydrogel dispersion was then knife-coated and allowed to dry overnight. A sensor disk 25 mm in diameter was then punched out and soaked in buffer for at least 16 h prior to use. Syntheses. The synthesis of the sodium fluoroionophore 11 from commercially available compounds is illustrated in Figure 1. o-Anisidine (1) was dialkylated with 2-chloroethanol then reacted with bis[(2-chloro-ethoxy)]ethane. The resultant phenylazacrown ether (3) was formylated, and the aldehyde (4) was condensed with nitromethane to afford β-nitrostyrene (5). Reduction of the nitrostyrene with lithium aluminum hydride gave the phenethylamine derivative (6), which was coupled with 4-chloronaphthalimide (9) (prepared from 4-chloronaphthalic anhydride in two steps) to form the fluoroionophore (10). Hydrolysis of the tert-butyl ester afforded carboxylic acid derivative 11 suitable for immobilization onto a solid support. N,N-bis(2-Hydroxylethyl)-2-methoxyaniline (2). A solution of 452 g (4 mol) of 1 in 2-chloro-ethanol (1932 g, 24 mol) and 350 mL of water was heated to 80 °C for 15 min. K2CO3 (829 g, 6 mol) was slowly added such that the temperature of this exothermic reaction was kept below 110 °C. The mixture was heated at 95 °C for 22 h, cooled, and ∼800 mL of unreacted 2-chloroethanol was removed under vacuum. The residue was diluted with water (2 L) and extracted with CHCl3 (2 L). The CHCl3 solution was dried over K2CO3, and the solvent was evaporated to afford 802 g (98%) of brown oil. 1H NMR (300 MHz, CDCl3) δ ) 3.18 (t, 4H, N CH2), 3.50 (t, 4H, O CH2), 3.60 (br, 2H, O H), 3.82 (s, 3H, Ar O CH3), 6.90-7.19 (m, 4H, Ar H). Anal. Calcd for C11H17NO3: C, 62.54; H, 8.11; N, 6.63. Found: C, 61.33; H, 8.28; N, 6.43. 2-Methoxyphenylaza-15-crown-5 (3). Compound 2 (403 g, 1.91 mol) was dissolved in dioxane (2.21 L) and heated at 80 °C for 20 min. Powdered NaOH (168 g, 4.20 mol) was added slowly within about 3 h. The temperature was then increased to 95 °C, bis(2-chloroethanoxyethane) (300 mL, 1.93 mol) was added in one portion, and the mixture was kept at 95 °C for 30 h. The suspension was then filtered hot, the solvent was evaporated, and the residue was treated with a solution of NaClO4 (234 g, 1.91 mol) in methanol (640 mL). The mixture was stirred at 60 °C for 30 min and concentrated to ∼300 mL. Ethyl acetate (860 mL) was added and the mixture was stirred at room temperature for 20 min, then allowed to stand at room temperature for 2 h. The resulting precipitate was filtered, washed with ethyl acetate (2 × 200 mL), and dried at room temperature for 30 min to give 199 g of azacrown-sodium perchlorate complex as a soft white powder. This powder was dissolved in a mixture of CH2Cl2 (600 mL) and water (600 mL), the layers were separated, and the aqueous phase was extracted with CH2Cl2 (400 mL). The organic solutions were combined, washed with water (8 × 600 mL), dried over Na2SO4, then evaporated to afford 100.4 g (16%) of pale yellow oil. 1H NMR (300 MHz, CDCl3) δ ) 3.49 (t, 4H, N CH2), 3.68 (m, 16H, O CH2), 3.82 (s, 3H, Ar O CH3), 6.88-7.12 (m, 4H, Ar H). Anal. Calcd for Analytical Chemistry, Vol. 75, No. 3, February 1, 2003
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C17H27NO5: C, 62.70; H, 8.36; N, 4.30. Found: C, 61.63; H, 8.44; N, 4.26. 4-Formyl-2-methoxyphenylaza-15-crown-5 (4). Compound 3 (100 g, 308 mmol) was dissolved in DMF (145 mL, 1850 mmol) in a 500-mL three-neck flask and cooled to -5 °C. POCl3 (57.4 mL, 616 mmol) was added dropwise via an addition funnel such that the solution temperature did not exceed 5 °C. After stirring at room temperature for 16 h, the solution was heated to 60 °C for 1 h, cooled, and poured into 500 g of ice; the flask was rinsed with 70 mL of water; and the combined aqueous solutions were adjusted to pH 7 (by pH paper) with saturated K2CO3. The solution was extracted with CHCl3 (2 × 500 mL), the CHCl3 phase washed with water (2 × 500 mL) then dried over MgSO4 (100 g) for 1 h. Evaporation of the solvent afforded 85 g of light yellow oil that crystallized upon standing overnight. Recrystallization from ethyl acetate/hexane (1:4) afforded 56 g (51%) of light orange crystals. 1H NMR (300 MHz, CDCl ) δ ) 3.68 (t, 16H, O CH ), 3.78 (t, 4H, 3 2 N CH2), 3.82 (s, 3H, Ar O CH3), 7.05-7.28 (m, 3H, Ar H), 9.78 (s, 1H, -CHO). Anal. Calcd for C18H27NO6: C, 61.17; H, 7.70; N, 3.96. Found: C, 61.05; H, 8.01; N, 4.04. 4-Nitroethylenyl-2-methoxyphenylaza-15-crown-5 (5). Compound 4 (18.8 g, 50 mmol) and ammonium acetate (38.5 g, 500 mmol) were suspended in acetic acid (100 mL) and stirred at room temperature for 10 min. Nitromethane (59.4 mL, 1100 mmol) was added, and the mixture was heated at 60 °C for 5 h and then poured into ice water. The resulting crystals were collected by filtration, washed with water, then dried in a desiccator with P2O5 to afford 11.9 g (60%) dark red needles. 1H NMR (300 MHz, CDCl3) δ ) 3.68 (t, 16H, O CH2), 3.78 (t, 4H, N CH2), 3.82 (s, 3H, Ar O CH3), 6.88-7.08 (m, 3H, Ar H), 7.45 (d, 1H, CdC H), 7.85 (d, 1H, NO2CdC H). Anal. Calcd for C19H28N2O6: C, 57.56; H, 7.12; N, 7.07. Found: C, 56.91; H, 7.42; N, 7.12. 4-Aminoethyl-2-methoxyphenylaza-15-crown-5 (6). LiAlH4 (3.8 g, 100 mmol) was slowly added into THF (200 mL) in a 500mL three-neck flask. A solution of 5 (4.0 g, 10 mmol) in THF (50 mL) was then added dropwise within 2 h. The mixture was heated to reflux for 4 h, cooled in an ice bath, then quenched with 6 N KOH. The slurry was filtered, the solvent was evaporated, and the residue was dissolved in CHCl3 (150 mL). The CHCl3 was washed with water (2 × 150 mL), dried over K2CO3 (15 g), and evaporated to afford 3.7 g (101%) orange oil. TLC appeared about 90% purity, and the major spot turned bluish violet when heated with ninhydrin solution. This amine was used directly without further purification. 1H NMR (300 MHz, CDCl3) δ ) 2,62 (t, 2H, Ar CH2), 2.95 (t, 2H, H2N CH2), 3.68 (t, 16H, O CH2), 3.78 (t, 4H, N CH2), 3.82 (s, 3H, Ar O CH3), 6.68-6.98 (m, 3H, Ar H). Anal. Calcd for C19H32N2O5: C, 61.93; H, 8.75; N, 7.60. Found: C, 60.98; H, 8.97; N, 6.67. 4-Chloro-N-(4-carboxyphenylmethyl)-1,8-naphthalimide (8). 4-Chloro-1,8-naphthalic anhydride (7, 46.4 g, 200 mmol), 4-(aminomethyl)benzoic acid (30.2 g, 200 mmol), and K2CO3 (13.8 g, 100 mmol) were suspended in DMF (2 L), stirred at room temperature for 16 h, then at 60 °C for 6 h. The mixture was poured into water (4 L), the solution was adjusted to pH 4 with 6 N HCl, and the resulting precipitate was collected by filtration. Drying the solid at 60 °C for 18 h afforded 36 g (51%) of an off-white powder. Product showed ∼90% purity by thin-layer chromatography (TLC) and was used directly for the next step. 552
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NMR (300 MHz, DMSO-D6) δ ) 5.30 (s, 2H, Ar CH2), 7.458.60 (m, 9H, Ar H). t-Butyl 4-Chloro-1,8-naphthalimidylmethyl Benzoate (9). A suspension of 8 (29.2 g, 80 mmol) in DMF (320 mL) was stirred at 40 °C under a stream of nitrogen, and then 1,1′-carbonyldiimidazole (52.0 g, 320 mmol) was added slowly over 20 min. The suspension clarified and then became turbid again in 15 min. tertButyl alcohol (52 mL, 1600 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 48 mL, 320 mmol) were added, and the mixture was heated to 60 °C for 18 h. The mixture was then cooled and poured into ice-cold 1 N HCl (2 L) with vigorous stirring. The resulting precipitate was filtered, washed with 1 N HCl (2 × 300 mL), and dried in a desiccator with P2O5 for 18 h to afford 28.5 g of crude product. Purification by silica gel column chromatography (1:1 CHCl3/cyclohexane) afforded 12.0 g of white powder (36%). 1H NMR (300 MHz, CDCl3) δ ) 1.50 (s, 9H, t-Bu H), 5.30 (s, 2H, Ar CH2), 7.45-8.60 (m, 9H, Ar H). Anal. Calcd for C24H21ClNO4: C, 68.33; H, 4.78; N, 3.32. Found: C, 67.79; H, 5.01; N, 3.24. t-Butyl 4-{4′-[4′′-C-(aza-15-crown-5)-3′′-Methoxyphenylethylamino]-1′,8′-naphthalimidylmethyl} Benzoate (10). Compound 9 (2.11 g, 5 mmol), 6 (2.62 g, 10 mmol), and diisopropylethylamine (1.63 g, 12.5 mmol) were suspended in N-methylpyrrolidinone (12.5 mL) and heated at 100 °C for 15 h. The mixture was poured into water (238 mL), and the precipitate was collected by filtration, washed with water (2 × 50 mL), and dried in a desiccator with P2O5 for 18 h to afford 3.8 g brownish-yellow solid. This solid was purified on a silica gel column eluted with a CHCl3/ ethyl acetate gradient, affording 2.34 g (62%) of orange gum. 1H NMR (300 MHz, CDCl3) δ ) 1.55 (s, 9H, t-Bu H), 3.05 (t, 2H, Ar CH2), 3.45 (t, 2H, HN CH2), 3.50 (t, 4H, N CH2), 3.65 (t, 16H, O CH2), 3.78 (s, 3H, Ar O CH3), 5.25 (s, 2H, Ar CH2), 6.78 (m, 3H), 7.05-8.55 (m, 12H, Ar H). FABMS (70 eV, NBA/Li matrix): 754 (100%), (M + H); 313 (64%) (phenylazacrown + Li). Anal. Calcd for C43H51N3O9: C, 65.51; H, 6.82; N, 5.57. Found: C, 65.82; H, 7.00; N, 5.71. 4-[4′-[4′′-C-(aza-15-crown-5)-3′′-Methoxyphenylethylamino]-1′,8′-naphthalimidylmeth-yl] Benzoic Acid (11). To a solution of 10 (1.88 g, 2.5 mmol) in CH2Cl2 (40 mL) was added 10 mL trifluoroacetic acid. The orange solution was stirred at room temperature for 50 min. The solution was diluted with CHCl3 (50 mL) and evaporated to dryness. The residue was dissolved in CHCl3/CH3OH (9/1, v/v) and evaporated to dryness again. This process was repeated twice more to remove any remaining trifluoroacetic acid, and 1.70 g of yellow gum was isolated. This powder was dissolved in 50 mL of CHCl3/CH3OH (9/1, v/v) and washed with 50 mL of 5% TFA. The organic layer was dried over K2SO4. Solvent was evaporated to give 1.75 g (98%) of yellow foam. 1H NMR (300 MHz, CDCl ) δ ) 2.90 (t, 2H, Ar CH ), 3.25 (t, 4H, 3 2 HN CH2), 3.50 (t, 16H, O CH2), 3.60 (t, 4H, N CH2), 3.75 (s, 3H, Ar O CH3), 5.25 (s, 2H, Ar CH2), 6.78-8.75 (m, 12H, Ar H). FABMS (70 eV, NBA/Na matrix): 720 (60%), (M + Na); 607 (100%), (debenzylated + 2Na+ - H+). Anal. Calcd for C43H51N3O9 + CF3COOH + 3H2O: C, 56.87; H, 5.82; N, 4.85. Found: C, 56.87; H, 5.56; N, 4.59. 1H
RESULTS AND DISCUSSION Ionophore Selection. Selection of a suitable ionophore was driven by several design criteria. First, the ionophore must contain
Figure 2. Absorption spectra of sodium ionophore 3 in 95/5 (v/v) water/methanol.
a tertiary nitrogen that can act as an electron donor and will also interact with a bound sodium cation. Additionally, the ionophore’s binding properties should be insensitive to pH changes in the clinically important range of 6.6-7.8 so as to minimize undesirable pH interference to the measurement of sodium. Finally, the ionophore should preferentially bind sodium with an aqueous complex dissociation constant (Kd) near the desired measuring range of 100-180 mM while in the presence of typical blood concentrations of potassium (4.5 mM) and lithium (
