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Synthesis and luminescence properties of lanthanide(III) chelates with polyacid derivatives of thienyl-substituted terpyridine analogues
ARTICLE IN PRESS
Journal of Luminescence 106 (2004) 91–101
Synthesis and luminescence properties of lanthanide(III) chelates with polyacid derivatives of thienyl-substituted terpyridine analogues Jingli Yuan*, Mingqian Tan, Guilan Wang Department of Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116012, China Received 14 July 2003; accepted 15 August 2003
Abstract Two new polyacid derivative ligands of thienyl-substituted terpyridine analogues, N,N,N1,N1-[40 -(2000 -thienyl)2,20 :60 ,200 -terpyridine-6,600 -diyl]bis(methylenenitrilo) tetrakis(acetic acid) (TTTA) and N,N,N1,N1-[2,6-bis(30 -aminomethyl-10 -pyrazolyl)-4-(200 -thienyl)pyridine] tetrakis(acetic acid) (BTTA), were synthesized, and the luminescence properties of their Eu3+ and Tb3+ chelates were investigated. The Eu3+chelates of the two ligands are strongly luminescent having luminescence quantum yields of 0.150 (TTTA-Eu3+) and 0.114 (BTTA-Eu3+), and lifetimes of 1.284 ms (TTTA-Eu3+) and 1.352 ms (BTTA-Eu3+), whereas their Tb3+ chelates are weakly luminescent. The TTTAEu3+ chelate was used for streptavidin (SA) labeling, and the labeled SA was used for time-resolved fluoroimmunoassay of insulin in human sera. The method gives the detection limits of 33 pg ml1. r 2003 Elsevier B.V. All rights reserved. Keywords: Lanthanide(III); Terpyridine; Quantum yield; Insulin
1. Introduction In recent 20 years, lanthanide chelates have been successfully developed as the luminescence labels for highly sensitive diagnostics and bioassays based on the microsecond time-resolved luminescence measurement technique [1–10]. Because the luminescence of lanthanide chelates has the properties of very long lifetime, large Stokes shift, and sharp emission band, the microsecond timeresolved luminescence measurement using a lanthanide chelate as the label can effectively *Corresponding author. Tel./fax: +86-411-3693509. E-mail address: [email protected] (J. Yuan).
eliminate the short-lived background signals from the biological samples and the optical components, whereas this background problem cannot be removed by the conventional fluorescence method. Recently, many luminescent lanthanide chelates have been synthesized and their luminescence properties were investigated [11–25]. Some of them have been used as the luminescence labels for improving the sensitivities of time-resolved luminescence bioassays and simplifying the assay procedures [26–30]. The luminescent lanthanide chelates mainly include the Eu3+, Tb3+, Sm3+ and Dy3+ chelates with b-diketone ligands and aromatic amine derivative ligands, in which the chelates of b-diketones have the drawbacks of
0022-2313/$ - see front matter r 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2003.08.001
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lower solubility in water-based buffer and lower chelate stability (smaller association constant) when they are used as the labels for bioassays. In the present work, two new polyacid derivative ligands of thienyl-substituted terpyridine analogues, N,N,N1,N1-[40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine-6,600 -diyl]bis(methylenenitrilo) tetrakis(acetic acid) (TTTA) and N,N,N1,N1-[2,6-bis(30 aminomethyl-10 -pyrazolyl)-4-(200 -thienyl)pyridine] tetrakis(acetic acid) (BTTA), were synthesized, and the luminescence properties of their Eu3+ and Tb3+ chelates were studied. Although the luminescence properties of lanthanide(III) chelates with terpyridine derivatives have been extensively investigated [19], the synthesis and luminescence properties of the lanthanide(III) chelates with thienyl-substituted terpyridine derivatives have not been reported. It has been known that the thienyl substituent in a b-diketone ligand, such as thenoyltrifluoroacetone, can strongly enhance the luminescence of the b-diketonate-Eu3+ chelate due to its electron-donating property [31]. The same effect could be expected for the Eu3+ chelates with thienyl-substituted terpyridine derivatives. The luminescence measurement results showed that the thienyl substituent in the two new ligands enhances the luminescence of their Eu3+ chelates, but weakens the luminescence of their Tb3+ chelates, compared with the phenyl substituent. These results indicate that the terpyridine derivative having a thienyl substituent is also favorable for the luminescence enhancement of its Eu3+ chelate. To evaluate the usefulness of the new chelate as a label for time-resolved fluoroimmunoassay (TR-FIA), the TTTA-Eu3+-labeled streptavidin (SA) was prepared and used for TRFIA of insulin in human sera. The results show that the TTTA-Eu3+ chelate is suitable for use as a luminescence label for TR-FIA and the method is highly sensitive with good accuracy and precision.
2. Experimental 2.1. Synthesis of TTTA The ligand TTTA was synthesized following the eight-step reactions shown in Scheme 1. The
details of the procedure are described in the following. 2.1.1. Synthesis of 40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine ð1Þ The starting materials (E)-3-(200 -thienyl)-1-(pyrid-20 -yl)prop-2-enone [13] and N-[2-(pyrid-20 -yl)-2oxoethyl] pyridinium iodide [32] were prepared by the literature methods. A mixture of 23.10 g dry AcONH4 (300 mmol), 16.30 g N-[2-(pyrid-20 -yl)-2oxoethyl] pyridinium iodide (50.0 mmol), 10.76 g (E)-3-(2 00 -thienyl)-1-(pyrid-2 0 -yl)prop-2-enone (50.0 mmol), and 500 ml dry methanol was refluxed for 24 h. After the mixture was cooled at 15 C for 1 h, the precipitate was filtered and washed with cold methanol. After the precipitate was recrystallized from acetonitrile, compound 1 was obtained (6.91 g, 43.8% yield). 1H NMR (CDCl3) d 8.74 (d, J; 7.9 Hz, 2H), 8.69 (s, 2H), 8.64 (d, J; 7.9 Hz, 2H), 7.87 (t, J; 7.9 Hz, 2H), 7.78 (d, J; 3.6 Hz, 1H), 7.44 (d, J; 5.1 Hz, 1H), 7.38–7.32 (m, 2H), 7.19–7.15 (m, 1H). 2.1.2. Synthesis of 40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine-1,100 -dioxide ð2Þ To 500 ml of CH2Cl2 was added 12.60 g of compound 1 (40.0 mmol) and 40 g 3-chloroperoxybenzoic acid. The solution was stirred at room temperature for 20 h. After the solution was washed with 4 200 ml of 10% Na2CO3, dried with Na2SO4 and evaporated, the residue was dissolved in 300 ml of methanol. After filtering, the solvent was evaporated, and the product was washed with acetonitrile and dried. Compound 2 was obtained (8.53 g, 61.4% yield). 1H NMR (CDCl3) d 9.23 (s, 2H), 8.35 (d, J; 6.6 Hz, 2H), 8.23 (d, J; 7.9 Hz, 2H), 7.70 (d, J; 3.6 Hz, 1H), 7.45– 7.28 (m, 5H), 7.16–7.13 (m, 1H). 2.1.3. Synthesis of 40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine-6,600 -dicarbonitrile ð3Þ To 300 ml of CH2Cl2 was added 8.69 g of compound 2 (25.0 mmol) and 24.80 g of (CH3)3SiCN (250 mmol) with stirring. After stirring for 20 min, 14.05 g of benzoyl chloride (100 mmol) was added dropwise within 20 min, and the solution was stirred at room temperature for 20 h. The solution was evaporated to halve
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Scheme 1. Synthesis of TTTA.
volume, to the solution was added 600 ml of 10% K2CO3, and the mixture was further stirred at room temperature for 1 h. The precipitate was filtered and washed with water and cold CH2Cl2, and dried. Compound 3 was obtained (9.00 g, 98.5% yield). 1H NMR (DMSO-d6) d 8.95 (d, J; 7.9 Hz, 2H), 8.62 (s, 2H), 8.32-8.26 (m, 2H), 8.19 (d, J; 7.6 Hz, 2H), 8.07 (d, J; 3.6 Hz, 1H), 7.86 (d, J; 5.1 Hz, 1H), 7.28–7.31 (m, 1H). 2.1.4. Synthesis of 40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine-6,600 -dicarboxylate dimethyl ð4Þ After a mixture of 4.40 g of compound 3 (12.0 mmol), 45 ml of concentrated H2SO4, 45 ml
of acetic acid, and 12 ml of water was stirred at 75–80 C for 48 h, the solution was added to 300 ml of ice-water. The precipitate was filtered and washed with water and ethanol, and dried. To 400 ml of cooled methanol (ice-water bath) was added dropwise 8 g of thionyl chloride. After stirring 15 min at room temperature, the above precipitate was added, and the mixture was refluxed for 8 h. After evaporation, the residue was dissolved in 300 ml of chloroform. The chloroform solution was washed with 5% of NaHCO3 and dried with Na2SO4. After the solvent was evaporated, the residue was purified by silica gel column chromatography using
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CH2Cl2–CH3OH (99:1, w/w) as eluent and then was recrystallized from toluene. Compound 4 was obtained (2.50 g, 48.1% yield). Anal. Calcd for C23H17N3O4S: C, 64.03; H, 3.97; N, 9.74. Found: C, 63.76; H, 3.83; N, 9.52. 1H NMR (CDCl3) d 8.82 (d, J; 7.8 Hz, 2H), 8.78 (s, 2H), 8.20 (d, J; 7.6 Hz, 2H), 8.04 (t, J; 7.8 Hz, 2H), 7.82 (d, J; 3.4 Hz, 1H), 7.49 (d, J; 4.9 Hz, 1H), 7.22–7.19 (m, 1H), 4.08 (s, 6 H).
2.1.5. Synthesis of 40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine-6,600 -dimethanol ð5Þ A mixture of 200 ml dry ethanol, 2.89 g compound 4 (6.7 mmol) and 1.05 g NaBH4 was stirred at room temperature for 3 h, and further refluxed for 1 h. After the solvent was evaporated, 100 ml of saturated NaHCO3 was added, and the solution was heated to boiling. The cold mixture was filtered and washed with water. After drying, the product was dissolved in 200 ml of THF, and THF solution was filtered. After the solvent was evaporated, the product was washed with acetonitrile and dried. Compound 5 was obtained (1.82 g, 72.2% yield). 1H NMR (DMSO-d6) d 8.63 (s, 2H), 8.50 (d, J; 7.3 Hz, 2H), 8.03 (t, J; 7.3 Hz, 2H), 7.93 (d, J; 3.6 Hz, 1H), 7.82 (d, J; 5.1 Hz, 1H), 7.61 (d, J; 7.1 Hz, 2H), 7.28–7.31 (m, 1H), 4.74 (s, 4H).
2.1.6. Synthesis of 40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine-6,600 -dibromomethyl ð6Þ To a mixture of 200 ml dry THF, 30 ml dry DMF, and 2.17 g of compound 5 (5.8 mmol) was added 4.75 g of PBr3 with stirring. After the solution was refluxed for 5 h, the solvent was evaporated. The residue was dissolved in 300 ml chloroform, and the chloroform solution was washed with 4 100 ml of 5% Na2CO3 and dried with Na2SO4. After evaporation, the product was washed with hexane and dried. Compound 6 was obtained (2.02 g, 69.7% yield). 1H NMR (CDCl3) d 8.71 (s, 2H), 8.54 (d, J; 7.8 Hz, 2H), 7.87 (t, J; 7.8 Hz, 2H), 7.78 (d, J; 3.6 Hz, 1H), 7.52 (d, J; 7.8 Hz, 2H), 7.48 (d, J; 5.1 Hz, 1H), 7.21–7.19 (m, 1H), 4.70 (s, 4H).
2.1.7. Synthesis of tetraethyl N,N,N1,N1-[40 -(2000 thienyl)-2,20 :60 ,200 -terpyridine-6,600 -diyl]bis (methylenenitrilo) tetrakis(acetate) ð7Þ After a mixture of 200 ml dry acetonitrile, 50 ml dry THF, 2.04 g compound 6 (4.0 mmol), 1.53 g diethyl iminodiacetate (8.1 mmol), and 5.52 g K2CO3 was refluxed for 24 h with stirring, the mixture was filtered. After evaporation, the residue was dissolved in 200 ml of chloroform, and the solution was washed with 4 100 ml of saturated Na2SO4, dried with Na2SO4, and then the solvent was evaporated. The oily residue was washed with petroleum ether, and purified by silica gel column chromatography using ethyl acetate-methanolTHF (10:3:2, w/w/w) as eluent. Compound 7 was obtained (1.95 g, 67.9% yield). 1H NMR (CDCl3) d 8.67 (s, 2H), 8.51 (d, J; 7.8 Hz, 2H), 7.86 (t, J; 7.8 Hz, 2H), 7.78 (d, J; 3.6 Hz, 1H), 7.63 (d, J; 7.8 Hz, 2H), 7.45 (d, J; 5.1 Hz, 1H), 7.20–7.17 (m, 1H), 4.20 (s, 4H), 4.18 (q, J; 7.3 Hz, 8H), 3.72 (s, 8H), 1.26 (t, J; 7.3 Hz, 12H). 2.1.8. Synthesis of N,N,N1,N1-[4’-(2000 -thienyl)2,20 :60 ,200 -terpyridine-6,600 diyl]bis(methylenenitrilo) tetrakis(acetic acid) (TTTA) A mixture of 120 ml ethanol, 4 g KOH, 10 ml H2O, and 1.94 g compound 7 (2.7 mmol) was refluxed with stirring for 3 h. After the solvent was evaporated, the residue was dissolved in 150 ml water, and the solution was filtered. To the solution was added dropwise 1:1 CF3COOHH2O to adjust the pH to B1, and the solution was stirred for 3 h at room temperature. The precipitate was collected by filtration and washed with 1% CF3COOH. After drying, the product was added to 200 ml of acetonitrile, and the mixture was refluxed 1 h with stirring. The precipitate was filtered and dried. TTTA was obtained (0.89 g, 51.4% yield). Anal. Calcd for C29H31N5O9S (TTTA 2H2O): C, 54.29; H, 4.87; N, 10.91. Found: C, 54.09; H, 4.57; N, 10.41. 1H NMR (DMSO-d6) d 8.75 (s, 2H), 8.59 (d, J; 7.8 Hz, 2H), 8.18 (t, J; 7.8 Hz, 2H), 8.10 (d, J; 3.6 Hz, 1H), 7.87 (d, J; 5.1 Hz, 1H), 7.75 (d, J; 7.8 Hz, 2H), 7.34–7.31 (m, 1H), 4.72 (s, 4H), 4.28 (s, 8H).
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2.2. Synthesis of BTTA The ligand BTTA was synthesized following the six-step reactions shown in Scheme 2. The details of the procedure are described in the following. 2.2.1. Synthesis of 2,6-dibromo-4-(20 thienyl)pyridine ð8Þ The starting compound 4-amino-2,6-dibromopyridine was synthesized by using a previous method [25]. To 250 ml acetic acid containing 3.25 g of 4-amino-2,6-dibromopyriding (12.9 mmol) and 12.90 g of thiophene (146 mmol) was added dropwise the solution of 13 ml acetic acid containing1.93 g of iso-amyl nitrite (16.0 mmol) with stirring. After stirring at room temperature for 24 h, the solution was further stirred at 45 C for 3 h. The solvent was evaporated, and the residue was neutralized with 40 ml
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of 5% K2CO3. The aqueous phase was extracted with 4 60 ml chloroform, and organic phase was dried with Na2SO4. After filtration and evaporation, the residue was purified by silica gel column chromatography using CH2Cl2–CH3OH (99:1, w/ w) as eluent, and then was recrystallized from methanol. Compound 8 was obtained (1.97 g, 47.9% yield). Anal. Calcd for C9H5NBr2S: C, 33.88; H, 1.58; N, 4.39. Found: C, 33.46; H, 1.46; N, 4.23. 1H NMR (CDCl3) d 7.60 (s, 2H), 7.50– 7.48 (m, 2H), 7.16–7.13 (m, 1H). 2.2.2. Synthesis of 2,6-bis(30 -methoxycarbonyl-10 pyrazolyl)-4-(200 -thienyl)pyridine ð9Þ Potassium (3.12 g, 80.0 mmol) was added in small portions to a solution of 10.10 g of 3methoxycarbonylpyrazole (80.0 mmol) in 150 ml of dry THF at 60–70 C with stirring. After the metal was dissolved, 6.38 g of compound 8
Scheme 2. Synthesis of BTTA.
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(20.0 mmol) was added, and the mixture was refluxed for 1 week. The solvent was evaporated, and the residue was extracted with 6 150 ml of chloroform. After evaporation, the residue was purified by silica gel column chromatography using CH2Cl2–CH3OH (99:1, w/w) as eluent, and then was recrystallized from benzene. Compound 9 was obtained (3.00 g, 36.7% yield). Anal. Calcd for C19H15N5O4S: C, 55.74; H, 3.69; N, 17.10. Found: C, 55.47; H, 3.62; N, 16.82. 1H NMR (CDCl3) d 8.60 (d, J; 2.7 Hz, 2H), 8.22 (s, 2H), 7.79 (d, J; 3.6 Hz, 1H), 7.53 (d, J; 5.0 Hz, 1H), 7.20–7.18 (m, 1H), 7.03 (d, J; 2.7 Hz, 2H), 4.01 (s, 6H).
2.2.3. Synthesis of 2,6-bis(30 -hydroxymethyl-10 pyrazolyl)-4-(200 -thienyl)pyridine ð10Þ To 300 ml of dry THF containing 1.30 g LiAlH4 was added 2.72 g (6.6 mmol) of compound 9. After stirring for 4 h at room temperature, 1.1 ml of water, 1.1 ml of 15% NaOH and 4.5 ml of water were added dropwise to the solution. The solution was filtered to remove precipitate, and the solvent was evaporated. The product was washed with acetonitrile and dried. Compound 10 was obtained (1.60 g, 68.2% yield). 1H NMR (DMSO-d6) d 8.88 (d, J; 2.6 Hz, 2H), 7.96 (d, J; 3.6 Hz, 1H), 7.90 (s, 2H), 7.85 (d, J; 5.1 Hz, 1H), 7.29–7.26 (m, 1H), 6.60 (d, J; 2.5 Hz, 2H), 4.58 (s, 4H).
2.2.4. Synthesis of 2,6-bis(30 -bromomethyl-10 pyrazolyl)-4-(200 -thienyl)pyridine ð11Þ To 200 ml of dry THF containing 1.59 g compound 10 (4.5 mmol) was added 3.65 g of PBr3 with stirring. After the solution was refluxed 4 h, the solvent was evaporated. The residue was dissolved in 100 ml CHCl3, and the solution was washed with 3 50 ml 5% NaHCO3. After drying with Na2SO4, the solvent was evaporated. The product was washed with hexane and dried. Compound 11 was obtained (2.03 g, 94.1% yield). 1 H NMR (CDCl3) d 8.50 (d, J; 2.7 Hz, 2H), 8.02 (s, 2H), 7.72 (d, J; 3.6 Hz, 1H), 7.50 (d, J; 5.1 Hz, 1H), 7.19–7.16 (m, 1H), 6.56 (d, J; 2.7 Hz, 2H), 4.59 (s, 4H).
2.2.5. Synthesis of tetraethyl N,N,N1,N1-[2,6bis(30 -aminomethyl-10 -pyrazolyl)-4-(200 thienyl)pyridine] tetrakis(acetate) ð12Þ To a solution of 100 ml dry CH3CN, 30 ml dry THF, and 1.20 g of compound 11 (2.5 mmol) was added 984 mg of diethyl iminodiacetate (5.2 mmol) dissolved in 30 ml of CH3CN, and 3.45 g of dry K2CO3 (25 mmol). The mixture was refluxed for 24 h with stirring. After filtering, the solvent was evaporated. The residue was dissolved in 200 ml of CHCl3, washed with 2 100 ml of water, dried with Na2SO4, and then the solvent was evaporated. The oily residue was purified by silica gel column chromatography using ethyl acetateCH2Cl2 (10:1,w/w) as eluent. After evaporation and drying, compound 12 was obtained (0.87 g, 50.0% yield). 1H NMR (CDCl3) d 8.51 (d, J; 2.7 Hz, 2H), 8.00 (s, 2H), 7.72 (d, J; 3.6 Hz, 1H), 7.49 (d, J; 5.1 Hz, 1H), 7.19–7.16 (m, 1H), 6.56 (d, J; 2.7 Hz, 2H), 4.20 (q, J; 7.2 Hz, 8H), 4.09 (s, 4H), 3.66 (s, 8H), 1.28 (t, J; 7.2 Hz, 12H). 2.2.6. Synthesis of N,N,N1,N1-[2,6-bis(30 aminomethyl-10 -pyrazolyl)-4-(200 -thienyl)pyridine] tetrakis(acetic acid) To a solution of 1.80 g KOH dissolved in 60 ml of ethanol and 7 ml of water was added 850 mg of compound 12 (1.2 mmol). After the solution was refluxed for 3 h with stirring, the solvent was evaporated. The residue was dissolved in 50 ml of water, and the solution was filtered. To the solution was added dropwise 3 M HCl to adjust the pH to B1, and the solution was stirred for 3 h at room temperature. The precipitate of BTTA was collected by filtration and washed with 1% HCl. After drying, 660 mg of BTTA was obtained (92.6% yield). Anal. Calcd for C25H27N7O9S (BTTA H2O): C, 49.92; H, 4.52; N, 16.29. Found: C, 50.08; H, 4.38; N, 16.09. 1H NMR (DMSO-d6) d 8.90 (d, J; 2.5 Hz, 2H), 7.98 (d, J; 3.6 Hz, 1H), 7.90 (s, 2H), 7.86 (d, J; 5.1 Hz, 1H), 7.30–7.27 (m, 1H), 6.59(d, J; 2.5 Hz, 2H), 4.00 (s, 4H), 3.52 (s, 8H). 2.3. Labeling of SA with TTTA-Eu3 The labeling of SA with TTTA-Eu3+ was carried out basically by using a previous method
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[25]. Before use in immunoassay, the TTTA-Eu3+labeled SA solution was diluted 500-fold with a 0.05 M Tris-HCl buffer of pH 7.8, containing 0.2% BSA, 0.9% NaCl, and 0.1% NaN3. 2.4. Application of TTTA-Eu3+-labeled SA to TRFIA
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The wells were washed with buffer 1 and buffer 2, and 45 ml of TTTA-Eu3+-labeled SA was added to each well. After the plate was incubated at 37 C for 1 h, the wells were washed four times with buffer 1, and then subjected to solid-phase timeresolved fluorometric measurement. 2.5. Instrumentation and measurement conditions
2.4.1. Preparation of biotinylated anti-human insulin monoclonal antibody After twice dialyses of 1.0 ml anti-human monoclonal antibody solution (0.3 mg ml1, International Reagents Co., Japan) for 24 h at 4 C against 3 l of saline water, 8.4 mg NaHCO3 and 3 mg sulfosuccinimidyl-6-(biotin-amido)hexanoate (NHS-LC-biotin, Pierce Chemical Co.) were added with stirring. After stirring for 1 h at room temperature, the solution was further incubated for 24 h at 4 C. The solution was twice dialyzed each for 24 h at 4 C against 3 l of 0.1 M NaHCO3 containing 0.25 g NaN3, then 5 mg BSA and 5 mg NaN3 were added. The solution was stored at 20 C before use. When the biotinylated antibody solution was used for immunoassay, it was diluted 200-fold with a 0.05 M Tris-HCl buffer of pH 7.8, containing 0.2% BSA, 0.9% NaCl, and 0.1% NaN3. 2.4.2. TR-FIA of human insulin using TTTA-Eu3+labeled SA The standard solutions of human insulin were prepared by diluting human insulin (Sigma) with a 0.05 M Tris-HCl buffer of pH 7.8 containing 5% BSA, 0.9% NaCl and 0.1% NaN3. After anti-human insulin monoclonal antibody (International Reagents Co., Japan, diluted to 10 mg ml1 with 0.1 M carbonate buffer of pH 9.6) was coated on the wells (50 ml per well) of 96-well microtiter plate (FluoroNunc plate) by the physical absorption method [33], 45 ml of human insulin standard solution or serum sample was added to each well. After incubation at 37 C for 1.5 h, the wells were washed twice with a 0.05 M Tris-HCl buffer of pH 7.8 containing 0.05% Tween 20 (buffer 1) and once with a 0.05 M Tris-HCl buffer of pH 7.8 (buffer 2). Then 45 ml of biotinylated anti-human insulin antibody was added to each well and the plate was incubated at 37 C for 1 h.
The 1H NMR spectra were recorded on a Bruker DRX 400 spectrometer. The luminescence excitation and emission spectra, and lifetime were measured on a Perkin-Elmer LS 50B luminescence spectrometer. The luminescence quantum yields (f1 ) of the Eu3+ and Tb3+ chelates were measured in a 0.05 M borate buffer of pH 9.1, and calculated by using the equation f1 ¼ I1 e2 C2 f2 =I2 e1 C1 with a standard quantum yield of f2 ¼ 0:160 for the Eu3+ chelate with N,N,N1,N1-[4’-phenyl2,20 :600 ,200 -terpyridine-6,600 -diyl]bis(methylenenitrilo) tetrakis(acetic acid) (PTTA-Eu3+, molar absorption coefficient e335 nm =14 300 cm1 M1) or f2 ¼ 0:100 for the Tb3+ chelate with N,N,N1,N1-[40 phenyl-2,20 :60 ,200 -terpyridine-6,600 -diyl]bis(methylenenitrilo) tetrakis(acetic acid) (PTTA-Tb3+, molar absorption coefficient e337 nm ¼14 000 cm1 M1) [19]. In the equation, I1 and I2 ; e1 and e2 ; and C1 and C2 are the luminescence intensities, molar absorption coefficients, and concentrations for the measured chelate and the standard chelate, respectively. The time-resolved luminescence measurement for TR-FIA was performed on a PerkinElmer Victor 1420 Multilabel Counter with the measurement conditions of excitation wavelength, 340 nm, emission wavelength, 615 nm, delay time, 0.2 ms, window time, 0.4 ms, and cycling time, 1.0 ms.
3. Results and discussion The excitation and emission spectra of the Eu3+ chelates with TTTA and BTTA are shown in Fig. 1. The luminescence properties of the Eu3+ and Tb3+ chelates are shown in Table 1. Two Eu3+ chelates emit strong luminescence when they are excited by ultraviolet light. The TTTA-Eu3+ and BTTA-Eu3+ show the excitation maximum
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Tb3+ chelates. When the substituent is phenyl, both the 2,20 :60 ,200 -terpyridine and 2,6-bis(pyrazol1-yl)pyridine derivatives form strongly luminescent chelates with Tb3+. After the substituent is changed to thienyl, the luminescence of their Tb3+ chelates becomes remarkably weak. It has been reported [19] that two polyacid derivative ligands of thienyl-substituted pyridine, N,N,N1,N1-[4-(20 thienyl)pyridine-2,6-diyl] bis(methylenenitrilo) tetrakis(acetic acid) and 4-(20 -thienyl)pyridine-2,6dicarboxylic acid, can form strongly luminescent chelates with Eu3+, but not Tb3+, since their triplet state levels are higher than the lowest excited resonance level of Eu3+ (5D0, ca. 17 250 cm1) but lower than that of Tb3+(5D4, ca. 20 500 cm1). In addition, when a thienyl substituent is introduced to pyridine-2,6-dicarboxylic acid, the triplet state level of the ligand is decreased from 27 050 to 19 800 cm1. According to these facts and referring to the triplet state levels of N,N,N1,N1-[2,20 :60 ,200 -terpyridine-6,600 -diyl] bis(methylenenitrilo) tetrakis(acetic acid) (22 400 cm1) and N,N,N1,N1-[2,6-bis(30 -aminomethyl-10 -pyrazolyl)pyridine] tetrakis(acetic acid) (25 150 cm1), it can be predicted that the triplet state levels of two thienyl-substituted ligands, TTTA and BTTA, are below the lowest excited resonance level 5D4 of Tb3+ ion, therefore their Tb3+ chelates are weakly luminescent. However, the change of phenyl to thienyl is favorable for the luminescence enhancement of the Eu3+ chelates since the molar absorption coefficients of the thienyl-substituted derivatives are larger remarkably than those of phenyl-substituted
wavelengths at 336 and 319 nm, and the emission maximum wavelengths at 615 and 620 nm, respectively. However, the luminescence of the Tb3+ chelates with TTTA and BTTA is very weak. Compared to the ligands of similar structures, PTTA and N,N,N1,N1-[2,6-bis(30 -aminomethyl10 -pyrazolyl)-4-phenylpyridine] tetrakis(acetic acid) (BPTA, eBPTA-Eu;325 nm ¼ 9290 cm1 M1, fBPTA-Eu ¼ 0:134; eBPTA-Tb;325 nm ¼ 9290 cm1M1, fBPTA-Tb ¼ 1:00) [25], although the structures of PTTA and TTTA (2,20 :60 ,200 -terpyridine derivatives) as well as BPTA and BTTA (2,6-bis(pyrazol1-yl)pyridine derivatives) are rather similar, the variation of the substituent in middle pyridine ring from phenyl to thienyl led to the remarkable change of the luminescence properties of their
Fig. 1. Excitation and emission spectra of TTTA-Eu3+ (1.0 106 M, solid line) and BTTA-Eu3+ (1.0 106 M, dashed line) in a 0.05 M borate buffer of pH 9.1. Ex: excitation spectrum. Em: emission spectrum.
Table 1 Luminescence properties of the Eu3+ and Tb3+ chelates of TTTA and BTTA in 0.05 M borate buffer of pH 9.1 Chelates
lex, 3+
TTTA-Eu TTTA-Tb3+ BTTA-Eu3+ BTTA-Tb3+ TTTA-Eu3+ (bound to SA)
336 336 319 319 336
max
(nm)
e (cm1 M1)
lex,
24 600 24 300 25 200 25 000
615 545 620 545 615
max
(nm)
fa
ef (cm1 M1)
t (ms)
0.150 p0.001 0.114 p0.002
3690 Intensity too low 2873 Intensity too low 1710
1.284b — 1.352 — 1.009b
The experimental uncertainty for quantum yield is o15%. In 0.05 M NaHCO3–H2O and 0.05 M NaHCO3–D2O buffers, the luminescence lifetimes of free TTTA-Eu3+ chelate are 1.206 and 1.523 ms, and those of TTTA-Eu3+ bound to SA are 0.910 and 1.248 ms, respectively. a
b
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derivatives, whereas the luminescence quantum yields of the Eu3+ chelates are not obviously changed with the change from phenyl to thienyl. For Eu3+ chelates, depending on the changes of the substituents in middle pyridine ring among proton, phenyl and thienyl, the f; e and t are changed with 0.176, 0.160, 0.150 (f), 12 100, 14 300, 24 600 cm1 M1 (e), and 1.310, 1.210, 1.284 ms (t), for terpyridine-type chelates, and 0.030, 0.134, 0.114 (f), 8100, 9290, 25 200 cm1 M1 (e), and 1.300, 1.353, 1.352 ms (t), for 2,6-bis(pyrazol-1-yl)pyridine-type chelates, respectively. These results obviously show that the e is affected strongly, whereas f and t are affected slightly [except the chelate of N,N,N1,N1-[2,6bis(30 -aminomethyl-10 -pyrazolyl)pyridine] tetrakis(acetic acid)] by the substituent change for the Eu3+ chelates. The change tendencies of the luminescence intensities (ef) for the phenyl and thienyl-substituted chelates are in the following order: Eu3+ chelates, TTTA>BTTA>PTTA >BPTA; Tb3+ chelates, BPTA>PTTA>BTTA ETTTA. The luminescence of TTTA-Eu3+ and BPTA-Tb3+ is the strongest for Eu3+ and Tb3+ chelates, respectively. After TTTA-Eu3+ was bound to SA, its luminescence intensity and lifetime decreased to about 46% and 78%, respectively. These results can be explained by the fact that since one of the chelating carboxylic groups of TTTA is used for coupling to SA through amide bond, the number of water molecules in the first coordination sphere of Eu3+ ion and the quenching effect of O–H vibrations are increased. Using the luminescence lifetimes of TTTA–Eu3+ (free and bound to SA) in D2O and H2O buffers, the average number (q) of water molecules in the first coordination sphere of Eu3+ ion is calculated from the equation of q ¼ 1:05 (1/tH2O1/tD2O) [19] to be 0.18 (free chelate) and 0.31 (bound to SA), respectively. The effects of pH on the luminescence intensity and the lifetime were measured by using a solution of 1.0 mM TTTA-Eu3+ in 0.05 M Tris-HCl buffer with several pHs (from 5.4 to 10.0). As shown in Fig. 2, both luminescence intensity and lifetime are changed to the largest at pHB8.0. However, the effects of pH on luminescence intensity and lifetime are not large. The changes of luminescence
99
Fig. 2. Influences of pH on the luminescence intensity (solid line) and lifetime (dashed line) of TTTA-Eu3+ chelate (1.0 106 M in 0.05 M Tris-HCl buffer).
Fig. 3. Log(luminescence count) vs. log(Eu3+ chelate concentration) in a 0.05 M Tris-HCl buffer of pH 8.0. (A) TTTAEu3+, (B) BTTA-Eu3+. The concentration of the Eu3+ chelate is in molar and the luminescence count is in arbitrary units.
intensity and lifetime in the range of pH 6–9 are less than 10%. The diluting curves of TTTA-Eu3+ and BTTA-Eu3+ in a 0.05 M Tris-HCl buffer of pH 8.0 were determined by time-resolved luminescence measurement. The results are shown in Fig. 3. The detection limits, calculated as the
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100
85–110%. These results show that the TR-FIA using TTTA-Eu3+-labeled SA has good accuracy and precision for clinic insulin assay.
4. Conclusion
Fig. 4. Calibration curve of TR-FIA for human insulin. Background: bg. Table 2 Analytical precision and recovery of insulin added to human serum samples Added (ng ml1)
Found (ng ml1)
CV (%, n ¼ 6)
Recovery (%)
0.00 0.50 2.50 5.00 0.00 0.50 2.50 5.00
0.19 0.67 2.61 4.49 0.17 0.70 2.70 4.75
1.85 7.08 4.00 2.77 7.82 9.12 7.54 6.22
— 96.0 96.8 86.0 — 106.0 101.2 91.6
concentration corresponding to three standard deviations (SD) of the background intensity, are 1.1 1012 M (TTTA-Eu3+) and 1.4 1012 M (BTTA-Eu3+), respectively. The calibration curve of TR-FIA for human insulin using TTTA-Eu3+-labeled SA is shown in Fig. 4. The detection limit, calculated as the concentration corresponding to 3SD of the background signal, is 33 pg ml1. This detection limit is low enough for the assay of human sera [34]. The dynamic range is up to B5 ng ml1. The analytical precisions and recoveries of insulin in human sera are summarized in Table 2. The coefficient variations (CVs) of all assays are below 10%, and the analytical recoveries are in the range of
Two polyacid derivative ligands of thienylsubstituted terpyridine analogues, TTTA and BTTA, were synthesized, and the luminescence properties of their Eu3+ and Tb3+ chelates were studied in the present work. The results revealed that the thienyl substituent in a terpyridine derivative is favorable for the luminescence enhancement of the Eu3+ chelate, but unfavorable for that of the Tb3+ chelate. Another usefulness of this thienyl substituent in the ligand is expected that an amino-reactive group can be introduced to the thienyl group for biomolecule labeling. This approach was unsuccessful until now, and is going to continue on the way. However, the application of TTTA-Eu3+-labeled SA for TR-FIA of human insulin has shown that the chelate is useable as a luminescence label for highly sensitive immunoassay.
Acknowledgements The present work was financially supported by the National Natural Science Foundation of China (No. 20175027).
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[22] C. Galaup, J.M. Couchet, C. Picard, P. Tisn"es, Tetrahedron Lett. 42 (2001) 6275. [23] J. Yuan, K. Matsumoto, J. Pharm. Biomed. Anal. 15 (1997) 1397. [24] J. Yuan, K. Matsumoto, H. Kimura, Anal. Chem. 70 (1998) 596. [25] J. Yuan, G. Wang, K. Majima, K. Matsumoto, Anal. Chem. 73 (2001) 1869. [26] J. Yuan, G. Wang, H. Kimura, K. Matsumoto, Anal. Biochem. 254 (1997) 283. [27] G. Mathis, F. Socquet, M. Viguier, B. Darbouret, Anticancer Res. 17 (1997) 3011. [28] K. Blomberg, P. Hurskainen, I. Hemmil.a, Clin. Chem. 45 (1999) 855. [29] S. Sueda, J. Yuan, K. Matsumoto, Bioconjugate Chem. 11 (2000) 827. [30] G. Wang, J. Yuan, K. Matsumoto, Z. Hu, Talanta 55 (2001) 1119. [31] H.G. Huang, K. Hiraki, Y. Nishikawa, Nippon Kagaku Kaishi (1981) 66. . [32] F. Krohnke, Angew. Chem. Int. Ed. 2 (1963) 225. [33] K. Matsumoto, J. Yuan, G. Wang, H. Kimura, Anal. Biochem. 276 (1999) 81. [34] P.M. Clark, Ann. Clin. Biochem. 36 (1999) 541.
Journal of Luminescence 106 (2004) 91–101
Synthesis and luminescence properties of lanthanide(III) chelates with polyacid derivatives of thienyl-substituted terpyridine analogues Jingli Yuan*, Mingqian Tan, Guilan Wang Department of Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116012, China Received 14 July 2003; accepted 15 August 2003
Abstract Two new polyacid derivative ligands of thienyl-substituted terpyridine analogues, N,N,N1,N1-[40 -(2000 -thienyl)2,20 :60 ,200 -terpyridine-6,600 -diyl]bis(methylenenitrilo) tetrakis(acetic acid) (TTTA) and N,N,N1,N1-[2,6-bis(30 -aminomethyl-10 -pyrazolyl)-4-(200 -thienyl)pyridine] tetrakis(acetic acid) (BTTA), were synthesized, and the luminescence properties of their Eu3+ and Tb3+ chelates were investigated. The Eu3+chelates of the two ligands are strongly luminescent having luminescence quantum yields of 0.150 (TTTA-Eu3+) and 0.114 (BTTA-Eu3+), and lifetimes of 1.284 ms (TTTA-Eu3+) and 1.352 ms (BTTA-Eu3+), whereas their Tb3+ chelates are weakly luminescent. The TTTAEu3+ chelate was used for streptavidin (SA) labeling, and the labeled SA was used for time-resolved fluoroimmunoassay of insulin in human sera. The method gives the detection limits of 33 pg ml1. r 2003 Elsevier B.V. All rights reserved. Keywords: Lanthanide(III); Terpyridine; Quantum yield; Insulin
1. Introduction In recent 20 years, lanthanide chelates have been successfully developed as the luminescence labels for highly sensitive diagnostics and bioassays based on the microsecond time-resolved luminescence measurement technique [1–10]. Because the luminescence of lanthanide chelates has the properties of very long lifetime, large Stokes shift, and sharp emission band, the microsecond timeresolved luminescence measurement using a lanthanide chelate as the label can effectively *Corresponding author. Tel./fax: +86-411-3693509. E-mail address: [email protected] (J. Yuan).
eliminate the short-lived background signals from the biological samples and the optical components, whereas this background problem cannot be removed by the conventional fluorescence method. Recently, many luminescent lanthanide chelates have been synthesized and their luminescence properties were investigated [11–25]. Some of them have been used as the luminescence labels for improving the sensitivities of time-resolved luminescence bioassays and simplifying the assay procedures [26–30]. The luminescent lanthanide chelates mainly include the Eu3+, Tb3+, Sm3+ and Dy3+ chelates with b-diketone ligands and aromatic amine derivative ligands, in which the chelates of b-diketones have the drawbacks of
0022-2313/$ - see front matter r 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2003.08.001
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lower solubility in water-based buffer and lower chelate stability (smaller association constant) when they are used as the labels for bioassays. In the present work, two new polyacid derivative ligands of thienyl-substituted terpyridine analogues, N,N,N1,N1-[40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine-6,600 -diyl]bis(methylenenitrilo) tetrakis(acetic acid) (TTTA) and N,N,N1,N1-[2,6-bis(30 aminomethyl-10 -pyrazolyl)-4-(200 -thienyl)pyridine] tetrakis(acetic acid) (BTTA), were synthesized, and the luminescence properties of their Eu3+ and Tb3+ chelates were studied. Although the luminescence properties of lanthanide(III) chelates with terpyridine derivatives have been extensively investigated [19], the synthesis and luminescence properties of the lanthanide(III) chelates with thienyl-substituted terpyridine derivatives have not been reported. It has been known that the thienyl substituent in a b-diketone ligand, such as thenoyltrifluoroacetone, can strongly enhance the luminescence of the b-diketonate-Eu3+ chelate due to its electron-donating property [31]. The same effect could be expected for the Eu3+ chelates with thienyl-substituted terpyridine derivatives. The luminescence measurement results showed that the thienyl substituent in the two new ligands enhances the luminescence of their Eu3+ chelates, but weakens the luminescence of their Tb3+ chelates, compared with the phenyl substituent. These results indicate that the terpyridine derivative having a thienyl substituent is also favorable for the luminescence enhancement of its Eu3+ chelate. To evaluate the usefulness of the new chelate as a label for time-resolved fluoroimmunoassay (TR-FIA), the TTTA-Eu3+-labeled streptavidin (SA) was prepared and used for TRFIA of insulin in human sera. The results show that the TTTA-Eu3+ chelate is suitable for use as a luminescence label for TR-FIA and the method is highly sensitive with good accuracy and precision.
2. Experimental 2.1. Synthesis of TTTA The ligand TTTA was synthesized following the eight-step reactions shown in Scheme 1. The
details of the procedure are described in the following. 2.1.1. Synthesis of 40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine ð1Þ The starting materials (E)-3-(200 -thienyl)-1-(pyrid-20 -yl)prop-2-enone [13] and N-[2-(pyrid-20 -yl)-2oxoethyl] pyridinium iodide [32] were prepared by the literature methods. A mixture of 23.10 g dry AcONH4 (300 mmol), 16.30 g N-[2-(pyrid-20 -yl)-2oxoethyl] pyridinium iodide (50.0 mmol), 10.76 g (E)-3-(2 00 -thienyl)-1-(pyrid-2 0 -yl)prop-2-enone (50.0 mmol), and 500 ml dry methanol was refluxed for 24 h. After the mixture was cooled at 15 C for 1 h, the precipitate was filtered and washed with cold methanol. After the precipitate was recrystallized from acetonitrile, compound 1 was obtained (6.91 g, 43.8% yield). 1H NMR (CDCl3) d 8.74 (d, J; 7.9 Hz, 2H), 8.69 (s, 2H), 8.64 (d, J; 7.9 Hz, 2H), 7.87 (t, J; 7.9 Hz, 2H), 7.78 (d, J; 3.6 Hz, 1H), 7.44 (d, J; 5.1 Hz, 1H), 7.38–7.32 (m, 2H), 7.19–7.15 (m, 1H). 2.1.2. Synthesis of 40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine-1,100 -dioxide ð2Þ To 500 ml of CH2Cl2 was added 12.60 g of compound 1 (40.0 mmol) and 40 g 3-chloroperoxybenzoic acid. The solution was stirred at room temperature for 20 h. After the solution was washed with 4 200 ml of 10% Na2CO3, dried with Na2SO4 and evaporated, the residue was dissolved in 300 ml of methanol. After filtering, the solvent was evaporated, and the product was washed with acetonitrile and dried. Compound 2 was obtained (8.53 g, 61.4% yield). 1H NMR (CDCl3) d 9.23 (s, 2H), 8.35 (d, J; 6.6 Hz, 2H), 8.23 (d, J; 7.9 Hz, 2H), 7.70 (d, J; 3.6 Hz, 1H), 7.45– 7.28 (m, 5H), 7.16–7.13 (m, 1H). 2.1.3. Synthesis of 40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine-6,600 -dicarbonitrile ð3Þ To 300 ml of CH2Cl2 was added 8.69 g of compound 2 (25.0 mmol) and 24.80 g of (CH3)3SiCN (250 mmol) with stirring. After stirring for 20 min, 14.05 g of benzoyl chloride (100 mmol) was added dropwise within 20 min, and the solution was stirred at room temperature for 20 h. The solution was evaporated to halve
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Scheme 1. Synthesis of TTTA.
volume, to the solution was added 600 ml of 10% K2CO3, and the mixture was further stirred at room temperature for 1 h. The precipitate was filtered and washed with water and cold CH2Cl2, and dried. Compound 3 was obtained (9.00 g, 98.5% yield). 1H NMR (DMSO-d6) d 8.95 (d, J; 7.9 Hz, 2H), 8.62 (s, 2H), 8.32-8.26 (m, 2H), 8.19 (d, J; 7.6 Hz, 2H), 8.07 (d, J; 3.6 Hz, 1H), 7.86 (d, J; 5.1 Hz, 1H), 7.28–7.31 (m, 1H). 2.1.4. Synthesis of 40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine-6,600 -dicarboxylate dimethyl ð4Þ After a mixture of 4.40 g of compound 3 (12.0 mmol), 45 ml of concentrated H2SO4, 45 ml
of acetic acid, and 12 ml of water was stirred at 75–80 C for 48 h, the solution was added to 300 ml of ice-water. The precipitate was filtered and washed with water and ethanol, and dried. To 400 ml of cooled methanol (ice-water bath) was added dropwise 8 g of thionyl chloride. After stirring 15 min at room temperature, the above precipitate was added, and the mixture was refluxed for 8 h. After evaporation, the residue was dissolved in 300 ml of chloroform. The chloroform solution was washed with 5% of NaHCO3 and dried with Na2SO4. After the solvent was evaporated, the residue was purified by silica gel column chromatography using
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CH2Cl2–CH3OH (99:1, w/w) as eluent and then was recrystallized from toluene. Compound 4 was obtained (2.50 g, 48.1% yield). Anal. Calcd for C23H17N3O4S: C, 64.03; H, 3.97; N, 9.74. Found: C, 63.76; H, 3.83; N, 9.52. 1H NMR (CDCl3) d 8.82 (d, J; 7.8 Hz, 2H), 8.78 (s, 2H), 8.20 (d, J; 7.6 Hz, 2H), 8.04 (t, J; 7.8 Hz, 2H), 7.82 (d, J; 3.4 Hz, 1H), 7.49 (d, J; 4.9 Hz, 1H), 7.22–7.19 (m, 1H), 4.08 (s, 6 H).
2.1.5. Synthesis of 40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine-6,600 -dimethanol ð5Þ A mixture of 200 ml dry ethanol, 2.89 g compound 4 (6.7 mmol) and 1.05 g NaBH4 was stirred at room temperature for 3 h, and further refluxed for 1 h. After the solvent was evaporated, 100 ml of saturated NaHCO3 was added, and the solution was heated to boiling. The cold mixture was filtered and washed with water. After drying, the product was dissolved in 200 ml of THF, and THF solution was filtered. After the solvent was evaporated, the product was washed with acetonitrile and dried. Compound 5 was obtained (1.82 g, 72.2% yield). 1H NMR (DMSO-d6) d 8.63 (s, 2H), 8.50 (d, J; 7.3 Hz, 2H), 8.03 (t, J; 7.3 Hz, 2H), 7.93 (d, J; 3.6 Hz, 1H), 7.82 (d, J; 5.1 Hz, 1H), 7.61 (d, J; 7.1 Hz, 2H), 7.28–7.31 (m, 1H), 4.74 (s, 4H).
2.1.6. Synthesis of 40 -(2000 -thienyl)-2,20 :60 ,200 terpyridine-6,600 -dibromomethyl ð6Þ To a mixture of 200 ml dry THF, 30 ml dry DMF, and 2.17 g of compound 5 (5.8 mmol) was added 4.75 g of PBr3 with stirring. After the solution was refluxed for 5 h, the solvent was evaporated. The residue was dissolved in 300 ml chloroform, and the chloroform solution was washed with 4 100 ml of 5% Na2CO3 and dried with Na2SO4. After evaporation, the product was washed with hexane and dried. Compound 6 was obtained (2.02 g, 69.7% yield). 1H NMR (CDCl3) d 8.71 (s, 2H), 8.54 (d, J; 7.8 Hz, 2H), 7.87 (t, J; 7.8 Hz, 2H), 7.78 (d, J; 3.6 Hz, 1H), 7.52 (d, J; 7.8 Hz, 2H), 7.48 (d, J; 5.1 Hz, 1H), 7.21–7.19 (m, 1H), 4.70 (s, 4H).
2.1.7. Synthesis of tetraethyl N,N,N1,N1-[40 -(2000 thienyl)-2,20 :60 ,200 -terpyridine-6,600 -diyl]bis (methylenenitrilo) tetrakis(acetate) ð7Þ After a mixture of 200 ml dry acetonitrile, 50 ml dry THF, 2.04 g compound 6 (4.0 mmol), 1.53 g diethyl iminodiacetate (8.1 mmol), and 5.52 g K2CO3 was refluxed for 24 h with stirring, the mixture was filtered. After evaporation, the residue was dissolved in 200 ml of chloroform, and the solution was washed with 4 100 ml of saturated Na2SO4, dried with Na2SO4, and then the solvent was evaporated. The oily residue was washed with petroleum ether, and purified by silica gel column chromatography using ethyl acetate-methanolTHF (10:3:2, w/w/w) as eluent. Compound 7 was obtained (1.95 g, 67.9% yield). 1H NMR (CDCl3) d 8.67 (s, 2H), 8.51 (d, J; 7.8 Hz, 2H), 7.86 (t, J; 7.8 Hz, 2H), 7.78 (d, J; 3.6 Hz, 1H), 7.63 (d, J; 7.8 Hz, 2H), 7.45 (d, J; 5.1 Hz, 1H), 7.20–7.17 (m, 1H), 4.20 (s, 4H), 4.18 (q, J; 7.3 Hz, 8H), 3.72 (s, 8H), 1.26 (t, J; 7.3 Hz, 12H). 2.1.8. Synthesis of N,N,N1,N1-[4’-(2000 -thienyl)2,20 :60 ,200 -terpyridine-6,600 diyl]bis(methylenenitrilo) tetrakis(acetic acid) (TTTA) A mixture of 120 ml ethanol, 4 g KOH, 10 ml H2O, and 1.94 g compound 7 (2.7 mmol) was refluxed with stirring for 3 h. After the solvent was evaporated, the residue was dissolved in 150 ml water, and the solution was filtered. To the solution was added dropwise 1:1 CF3COOHH2O to adjust the pH to B1, and the solution was stirred for 3 h at room temperature. The precipitate was collected by filtration and washed with 1% CF3COOH. After drying, the product was added to 200 ml of acetonitrile, and the mixture was refluxed 1 h with stirring. The precipitate was filtered and dried. TTTA was obtained (0.89 g, 51.4% yield). Anal. Calcd for C29H31N5O9S (TTTA 2H2O): C, 54.29; H, 4.87; N, 10.91. Found: C, 54.09; H, 4.57; N, 10.41. 1H NMR (DMSO-d6) d 8.75 (s, 2H), 8.59 (d, J; 7.8 Hz, 2H), 8.18 (t, J; 7.8 Hz, 2H), 8.10 (d, J; 3.6 Hz, 1H), 7.87 (d, J; 5.1 Hz, 1H), 7.75 (d, J; 7.8 Hz, 2H), 7.34–7.31 (m, 1H), 4.72 (s, 4H), 4.28 (s, 8H).
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2.2. Synthesis of BTTA The ligand BTTA was synthesized following the six-step reactions shown in Scheme 2. The details of the procedure are described in the following. 2.2.1. Synthesis of 2,6-dibromo-4-(20 thienyl)pyridine ð8Þ The starting compound 4-amino-2,6-dibromopyridine was synthesized by using a previous method [25]. To 250 ml acetic acid containing 3.25 g of 4-amino-2,6-dibromopyriding (12.9 mmol) and 12.90 g of thiophene (146 mmol) was added dropwise the solution of 13 ml acetic acid containing1.93 g of iso-amyl nitrite (16.0 mmol) with stirring. After stirring at room temperature for 24 h, the solution was further stirred at 45 C for 3 h. The solvent was evaporated, and the residue was neutralized with 40 ml
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of 5% K2CO3. The aqueous phase was extracted with 4 60 ml chloroform, and organic phase was dried with Na2SO4. After filtration and evaporation, the residue was purified by silica gel column chromatography using CH2Cl2–CH3OH (99:1, w/ w) as eluent, and then was recrystallized from methanol. Compound 8 was obtained (1.97 g, 47.9% yield). Anal. Calcd for C9H5NBr2S: C, 33.88; H, 1.58; N, 4.39. Found: C, 33.46; H, 1.46; N, 4.23. 1H NMR (CDCl3) d 7.60 (s, 2H), 7.50– 7.48 (m, 2H), 7.16–7.13 (m, 1H). 2.2.2. Synthesis of 2,6-bis(30 -methoxycarbonyl-10 pyrazolyl)-4-(200 -thienyl)pyridine ð9Þ Potassium (3.12 g, 80.0 mmol) was added in small portions to a solution of 10.10 g of 3methoxycarbonylpyrazole (80.0 mmol) in 150 ml of dry THF at 60–70 C with stirring. After the metal was dissolved, 6.38 g of compound 8
Scheme 2. Synthesis of BTTA.
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(20.0 mmol) was added, and the mixture was refluxed for 1 week. The solvent was evaporated, and the residue was extracted with 6 150 ml of chloroform. After evaporation, the residue was purified by silica gel column chromatography using CH2Cl2–CH3OH (99:1, w/w) as eluent, and then was recrystallized from benzene. Compound 9 was obtained (3.00 g, 36.7% yield). Anal. Calcd for C19H15N5O4S: C, 55.74; H, 3.69; N, 17.10. Found: C, 55.47; H, 3.62; N, 16.82. 1H NMR (CDCl3) d 8.60 (d, J; 2.7 Hz, 2H), 8.22 (s, 2H), 7.79 (d, J; 3.6 Hz, 1H), 7.53 (d, J; 5.0 Hz, 1H), 7.20–7.18 (m, 1H), 7.03 (d, J; 2.7 Hz, 2H), 4.01 (s, 6H).
2.2.3. Synthesis of 2,6-bis(30 -hydroxymethyl-10 pyrazolyl)-4-(200 -thienyl)pyridine ð10Þ To 300 ml of dry THF containing 1.30 g LiAlH4 was added 2.72 g (6.6 mmol) of compound 9. After stirring for 4 h at room temperature, 1.1 ml of water, 1.1 ml of 15% NaOH and 4.5 ml of water were added dropwise to the solution. The solution was filtered to remove precipitate, and the solvent was evaporated. The product was washed with acetonitrile and dried. Compound 10 was obtained (1.60 g, 68.2% yield). 1H NMR (DMSO-d6) d 8.88 (d, J; 2.6 Hz, 2H), 7.96 (d, J; 3.6 Hz, 1H), 7.90 (s, 2H), 7.85 (d, J; 5.1 Hz, 1H), 7.29–7.26 (m, 1H), 6.60 (d, J; 2.5 Hz, 2H), 4.58 (s, 4H).
2.2.4. Synthesis of 2,6-bis(30 -bromomethyl-10 pyrazolyl)-4-(200 -thienyl)pyridine ð11Þ To 200 ml of dry THF containing 1.59 g compound 10 (4.5 mmol) was added 3.65 g of PBr3 with stirring. After the solution was refluxed 4 h, the solvent was evaporated. The residue was dissolved in 100 ml CHCl3, and the solution was washed with 3 50 ml 5% NaHCO3. After drying with Na2SO4, the solvent was evaporated. The product was washed with hexane and dried. Compound 11 was obtained (2.03 g, 94.1% yield). 1 H NMR (CDCl3) d 8.50 (d, J; 2.7 Hz, 2H), 8.02 (s, 2H), 7.72 (d, J; 3.6 Hz, 1H), 7.50 (d, J; 5.1 Hz, 1H), 7.19–7.16 (m, 1H), 6.56 (d, J; 2.7 Hz, 2H), 4.59 (s, 4H).
2.2.5. Synthesis of tetraethyl N,N,N1,N1-[2,6bis(30 -aminomethyl-10 -pyrazolyl)-4-(200 thienyl)pyridine] tetrakis(acetate) ð12Þ To a solution of 100 ml dry CH3CN, 30 ml dry THF, and 1.20 g of compound 11 (2.5 mmol) was added 984 mg of diethyl iminodiacetate (5.2 mmol) dissolved in 30 ml of CH3CN, and 3.45 g of dry K2CO3 (25 mmol). The mixture was refluxed for 24 h with stirring. After filtering, the solvent was evaporated. The residue was dissolved in 200 ml of CHCl3, washed with 2 100 ml of water, dried with Na2SO4, and then the solvent was evaporated. The oily residue was purified by silica gel column chromatography using ethyl acetateCH2Cl2 (10:1,w/w) as eluent. After evaporation and drying, compound 12 was obtained (0.87 g, 50.0% yield). 1H NMR (CDCl3) d 8.51 (d, J; 2.7 Hz, 2H), 8.00 (s, 2H), 7.72 (d, J; 3.6 Hz, 1H), 7.49 (d, J; 5.1 Hz, 1H), 7.19–7.16 (m, 1H), 6.56 (d, J; 2.7 Hz, 2H), 4.20 (q, J; 7.2 Hz, 8H), 4.09 (s, 4H), 3.66 (s, 8H), 1.28 (t, J; 7.2 Hz, 12H). 2.2.6. Synthesis of N,N,N1,N1-[2,6-bis(30 aminomethyl-10 -pyrazolyl)-4-(200 -thienyl)pyridine] tetrakis(acetic acid) To a solution of 1.80 g KOH dissolved in 60 ml of ethanol and 7 ml of water was added 850 mg of compound 12 (1.2 mmol). After the solution was refluxed for 3 h with stirring, the solvent was evaporated. The residue was dissolved in 50 ml of water, and the solution was filtered. To the solution was added dropwise 3 M HCl to adjust the pH to B1, and the solution was stirred for 3 h at room temperature. The precipitate of BTTA was collected by filtration and washed with 1% HCl. After drying, 660 mg of BTTA was obtained (92.6% yield). Anal. Calcd for C25H27N7O9S (BTTA H2O): C, 49.92; H, 4.52; N, 16.29. Found: C, 50.08; H, 4.38; N, 16.09. 1H NMR (DMSO-d6) d 8.90 (d, J; 2.5 Hz, 2H), 7.98 (d, J; 3.6 Hz, 1H), 7.90 (s, 2H), 7.86 (d, J; 5.1 Hz, 1H), 7.30–7.27 (m, 1H), 6.59(d, J; 2.5 Hz, 2H), 4.00 (s, 4H), 3.52 (s, 8H). 2.3. Labeling of SA with TTTA-Eu3 The labeling of SA with TTTA-Eu3+ was carried out basically by using a previous method
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[25]. Before use in immunoassay, the TTTA-Eu3+labeled SA solution was diluted 500-fold with a 0.05 M Tris-HCl buffer of pH 7.8, containing 0.2% BSA, 0.9% NaCl, and 0.1% NaN3. 2.4. Application of TTTA-Eu3+-labeled SA to TRFIA
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The wells were washed with buffer 1 and buffer 2, and 45 ml of TTTA-Eu3+-labeled SA was added to each well. After the plate was incubated at 37 C for 1 h, the wells were washed four times with buffer 1, and then subjected to solid-phase timeresolved fluorometric measurement. 2.5. Instrumentation and measurement conditions
2.4.1. Preparation of biotinylated anti-human insulin monoclonal antibody After twice dialyses of 1.0 ml anti-human monoclonal antibody solution (0.3 mg ml1, International Reagents Co., Japan) for 24 h at 4 C against 3 l of saline water, 8.4 mg NaHCO3 and 3 mg sulfosuccinimidyl-6-(biotin-amido)hexanoate (NHS-LC-biotin, Pierce Chemical Co.) were added with stirring. After stirring for 1 h at room temperature, the solution was further incubated for 24 h at 4 C. The solution was twice dialyzed each for 24 h at 4 C against 3 l of 0.1 M NaHCO3 containing 0.25 g NaN3, then 5 mg BSA and 5 mg NaN3 were added. The solution was stored at 20 C before use. When the biotinylated antibody solution was used for immunoassay, it was diluted 200-fold with a 0.05 M Tris-HCl buffer of pH 7.8, containing 0.2% BSA, 0.9% NaCl, and 0.1% NaN3. 2.4.2. TR-FIA of human insulin using TTTA-Eu3+labeled SA The standard solutions of human insulin were prepared by diluting human insulin (Sigma) with a 0.05 M Tris-HCl buffer of pH 7.8 containing 5% BSA, 0.9% NaCl and 0.1% NaN3. After anti-human insulin monoclonal antibody (International Reagents Co., Japan, diluted to 10 mg ml1 with 0.1 M carbonate buffer of pH 9.6) was coated on the wells (50 ml per well) of 96-well microtiter plate (FluoroNunc plate) by the physical absorption method [33], 45 ml of human insulin standard solution or serum sample was added to each well. After incubation at 37 C for 1.5 h, the wells were washed twice with a 0.05 M Tris-HCl buffer of pH 7.8 containing 0.05% Tween 20 (buffer 1) and once with a 0.05 M Tris-HCl buffer of pH 7.8 (buffer 2). Then 45 ml of biotinylated anti-human insulin antibody was added to each well and the plate was incubated at 37 C for 1 h.
The 1H NMR spectra were recorded on a Bruker DRX 400 spectrometer. The luminescence excitation and emission spectra, and lifetime were measured on a Perkin-Elmer LS 50B luminescence spectrometer. The luminescence quantum yields (f1 ) of the Eu3+ and Tb3+ chelates were measured in a 0.05 M borate buffer of pH 9.1, and calculated by using the equation f1 ¼ I1 e2 C2 f2 =I2 e1 C1 with a standard quantum yield of f2 ¼ 0:160 for the Eu3+ chelate with N,N,N1,N1-[4’-phenyl2,20 :600 ,200 -terpyridine-6,600 -diyl]bis(methylenenitrilo) tetrakis(acetic acid) (PTTA-Eu3+, molar absorption coefficient e335 nm =14 300 cm1 M1) or f2 ¼ 0:100 for the Tb3+ chelate with N,N,N1,N1-[40 phenyl-2,20 :60 ,200 -terpyridine-6,600 -diyl]bis(methylenenitrilo) tetrakis(acetic acid) (PTTA-Tb3+, molar absorption coefficient e337 nm ¼14 000 cm1 M1) [19]. In the equation, I1 and I2 ; e1 and e2 ; and C1 and C2 are the luminescence intensities, molar absorption coefficients, and concentrations for the measured chelate and the standard chelate, respectively. The time-resolved luminescence measurement for TR-FIA was performed on a PerkinElmer Victor 1420 Multilabel Counter with the measurement conditions of excitation wavelength, 340 nm, emission wavelength, 615 nm, delay time, 0.2 ms, window time, 0.4 ms, and cycling time, 1.0 ms.
3. Results and discussion The excitation and emission spectra of the Eu3+ chelates with TTTA and BTTA are shown in Fig. 1. The luminescence properties of the Eu3+ and Tb3+ chelates are shown in Table 1. Two Eu3+ chelates emit strong luminescence when they are excited by ultraviolet light. The TTTA-Eu3+ and BTTA-Eu3+ show the excitation maximum
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Tb3+ chelates. When the substituent is phenyl, both the 2,20 :60 ,200 -terpyridine and 2,6-bis(pyrazol1-yl)pyridine derivatives form strongly luminescent chelates with Tb3+. After the substituent is changed to thienyl, the luminescence of their Tb3+ chelates becomes remarkably weak. It has been reported [19] that two polyacid derivative ligands of thienyl-substituted pyridine, N,N,N1,N1-[4-(20 thienyl)pyridine-2,6-diyl] bis(methylenenitrilo) tetrakis(acetic acid) and 4-(20 -thienyl)pyridine-2,6dicarboxylic acid, can form strongly luminescent chelates with Eu3+, but not Tb3+, since their triplet state levels are higher than the lowest excited resonance level of Eu3+ (5D0, ca. 17 250 cm1) but lower than that of Tb3+(5D4, ca. 20 500 cm1). In addition, when a thienyl substituent is introduced to pyridine-2,6-dicarboxylic acid, the triplet state level of the ligand is decreased from 27 050 to 19 800 cm1. According to these facts and referring to the triplet state levels of N,N,N1,N1-[2,20 :60 ,200 -terpyridine-6,600 -diyl] bis(methylenenitrilo) tetrakis(acetic acid) (22 400 cm1) and N,N,N1,N1-[2,6-bis(30 -aminomethyl-10 -pyrazolyl)pyridine] tetrakis(acetic acid) (25 150 cm1), it can be predicted that the triplet state levels of two thienyl-substituted ligands, TTTA and BTTA, are below the lowest excited resonance level 5D4 of Tb3+ ion, therefore their Tb3+ chelates are weakly luminescent. However, the change of phenyl to thienyl is favorable for the luminescence enhancement of the Eu3+ chelates since the molar absorption coefficients of the thienyl-substituted derivatives are larger remarkably than those of phenyl-substituted
wavelengths at 336 and 319 nm, and the emission maximum wavelengths at 615 and 620 nm, respectively. However, the luminescence of the Tb3+ chelates with TTTA and BTTA is very weak. Compared to the ligands of similar structures, PTTA and N,N,N1,N1-[2,6-bis(30 -aminomethyl10 -pyrazolyl)-4-phenylpyridine] tetrakis(acetic acid) (BPTA, eBPTA-Eu;325 nm ¼ 9290 cm1 M1, fBPTA-Eu ¼ 0:134; eBPTA-Tb;325 nm ¼ 9290 cm1M1, fBPTA-Tb ¼ 1:00) [25], although the structures of PTTA and TTTA (2,20 :60 ,200 -terpyridine derivatives) as well as BPTA and BTTA (2,6-bis(pyrazol1-yl)pyridine derivatives) are rather similar, the variation of the substituent in middle pyridine ring from phenyl to thienyl led to the remarkable change of the luminescence properties of their
Fig. 1. Excitation and emission spectra of TTTA-Eu3+ (1.0 106 M, solid line) and BTTA-Eu3+ (1.0 106 M, dashed line) in a 0.05 M borate buffer of pH 9.1. Ex: excitation spectrum. Em: emission spectrum.
Table 1 Luminescence properties of the Eu3+ and Tb3+ chelates of TTTA and BTTA in 0.05 M borate buffer of pH 9.1 Chelates
lex, 3+
TTTA-Eu TTTA-Tb3+ BTTA-Eu3+ BTTA-Tb3+ TTTA-Eu3+ (bound to SA)
336 336 319 319 336
max
(nm)
e (cm1 M1)
lex,
24 600 24 300 25 200 25 000
615 545 620 545 615
max
(nm)
fa
ef (cm1 M1)
t (ms)
0.150 p0.001 0.114 p0.002
3690 Intensity too low 2873 Intensity too low 1710
1.284b — 1.352 — 1.009b
The experimental uncertainty for quantum yield is o15%. In 0.05 M NaHCO3–H2O and 0.05 M NaHCO3–D2O buffers, the luminescence lifetimes of free TTTA-Eu3+ chelate are 1.206 and 1.523 ms, and those of TTTA-Eu3+ bound to SA are 0.910 and 1.248 ms, respectively. a
b
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derivatives, whereas the luminescence quantum yields of the Eu3+ chelates are not obviously changed with the change from phenyl to thienyl. For Eu3+ chelates, depending on the changes of the substituents in middle pyridine ring among proton, phenyl and thienyl, the f; e and t are changed with 0.176, 0.160, 0.150 (f), 12 100, 14 300, 24 600 cm1 M1 (e), and 1.310, 1.210, 1.284 ms (t), for terpyridine-type chelates, and 0.030, 0.134, 0.114 (f), 8100, 9290, 25 200 cm1 M1 (e), and 1.300, 1.353, 1.352 ms (t), for 2,6-bis(pyrazol-1-yl)pyridine-type chelates, respectively. These results obviously show that the e is affected strongly, whereas f and t are affected slightly [except the chelate of N,N,N1,N1-[2,6bis(30 -aminomethyl-10 -pyrazolyl)pyridine] tetrakis(acetic acid)] by the substituent change for the Eu3+ chelates. The change tendencies of the luminescence intensities (ef) for the phenyl and thienyl-substituted chelates are in the following order: Eu3+ chelates, TTTA>BTTA>PTTA >BPTA; Tb3+ chelates, BPTA>PTTA>BTTA ETTTA. The luminescence of TTTA-Eu3+ and BPTA-Tb3+ is the strongest for Eu3+ and Tb3+ chelates, respectively. After TTTA-Eu3+ was bound to SA, its luminescence intensity and lifetime decreased to about 46% and 78%, respectively. These results can be explained by the fact that since one of the chelating carboxylic groups of TTTA is used for coupling to SA through amide bond, the number of water molecules in the first coordination sphere of Eu3+ ion and the quenching effect of O–H vibrations are increased. Using the luminescence lifetimes of TTTA–Eu3+ (free and bound to SA) in D2O and H2O buffers, the average number (q) of water molecules in the first coordination sphere of Eu3+ ion is calculated from the equation of q ¼ 1:05 (1/tH2O1/tD2O) [19] to be 0.18 (free chelate) and 0.31 (bound to SA), respectively. The effects of pH on the luminescence intensity and the lifetime were measured by using a solution of 1.0 mM TTTA-Eu3+ in 0.05 M Tris-HCl buffer with several pHs (from 5.4 to 10.0). As shown in Fig. 2, both luminescence intensity and lifetime are changed to the largest at pHB8.0. However, the effects of pH on luminescence intensity and lifetime are not large. The changes of luminescence
99
Fig. 2. Influences of pH on the luminescence intensity (solid line) and lifetime (dashed line) of TTTA-Eu3+ chelate (1.0 106 M in 0.05 M Tris-HCl buffer).
Fig. 3. Log(luminescence count) vs. log(Eu3+ chelate concentration) in a 0.05 M Tris-HCl buffer of pH 8.0. (A) TTTAEu3+, (B) BTTA-Eu3+. The concentration of the Eu3+ chelate is in molar and the luminescence count is in arbitrary units.
intensity and lifetime in the range of pH 6–9 are less than 10%. The diluting curves of TTTA-Eu3+ and BTTA-Eu3+ in a 0.05 M Tris-HCl buffer of pH 8.0 were determined by time-resolved luminescence measurement. The results are shown in Fig. 3. The detection limits, calculated as the
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85–110%. These results show that the TR-FIA using TTTA-Eu3+-labeled SA has good accuracy and precision for clinic insulin assay.
4. Conclusion
Fig. 4. Calibration curve of TR-FIA for human insulin. Background: bg. Table 2 Analytical precision and recovery of insulin added to human serum samples Added (ng ml1)
Found (ng ml1)
CV (%, n ¼ 6)
Recovery (%)
0.00 0.50 2.50 5.00 0.00 0.50 2.50 5.00
0.19 0.67 2.61 4.49 0.17 0.70 2.70 4.75
1.85 7.08 4.00 2.77 7.82 9.12 7.54 6.22
— 96.0 96.8 86.0 — 106.0 101.2 91.6
concentration corresponding to three standard deviations (SD) of the background intensity, are 1.1 1012 M (TTTA-Eu3+) and 1.4 1012 M (BTTA-Eu3+), respectively. The calibration curve of TR-FIA for human insulin using TTTA-Eu3+-labeled SA is shown in Fig. 4. The detection limit, calculated as the concentration corresponding to 3SD of the background signal, is 33 pg ml1. This detection limit is low enough for the assay of human sera [34]. The dynamic range is up to B5 ng ml1. The analytical precisions and recoveries of insulin in human sera are summarized in Table 2. The coefficient variations (CVs) of all assays are below 10%, and the analytical recoveries are in the range of
Two polyacid derivative ligands of thienylsubstituted terpyridine analogues, TTTA and BTTA, were synthesized, and the luminescence properties of their Eu3+ and Tb3+ chelates were studied in the present work. The results revealed that the thienyl substituent in a terpyridine derivative is favorable for the luminescence enhancement of the Eu3+ chelate, but unfavorable for that of the Tb3+ chelate. Another usefulness of this thienyl substituent in the ligand is expected that an amino-reactive group can be introduced to the thienyl group for biomolecule labeling. This approach was unsuccessful until now, and is going to continue on the way. However, the application of TTTA-Eu3+-labeled SA for TR-FIA of human insulin has shown that the chelate is useable as a luminescence label for highly sensitive immunoassay.
Acknowledgements The present work was financially supported by the National Natural Science Foundation of China (No. 20175027).
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