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Bioconjugate Chem. 1904, 5, 270-202
Synthesis of Europium(111) Chelates Suitable for Labeling of Bioactive Molecules Harri Takalo,*j+Veli-Matti Mukkala,’ Heikki Mikola,’ Paivi Liitti,+and Ilkka Hemmila Wallac Oy, P. 0. Box 10, FIN-20101 Turku, Finland, Department of Chemistry, University of Turku, FIN-20500 Turku, Finland, and Centre for Biotechnology, P. 0. Box 123, FIN-20521 Turku, Finland. Received March 30, 1993”
Two different kinds of europium(II1) chelates, luminescent and nonluminescent, were prepared. The chelates were coupled to bioanalytical reagents, such as antibodies, after activations of the amino group on the chelates with thiophosgene, 2,4,6-trichloro-1,3,5-triazine, or iodoacetic anhydride. The reactivities of the activated luminescent chelates in the labeling of antibodies as well as the effects of both the coupling ratio and the linkage group to the luminescence quantum yield of the antibody-bound chelate were studied in aqueous buffer solution.
INTRODUCTION Due to their unique luminescence properties lanthanide chelates have recently been developed and used as labels both in immunological and in DNA hybridization assays (1,2). The types of chelates synthesized include lanthanide cryptates (3),macrocyclic Schiff bases (41, and polyaminopolycarboxylates (2, 5-11 ). The commonly used commercial system, DELFIA (Wallac Oy, Turku, Finland), is based on the use of nonluminescent europium(II1) chelates (2,5-7) as the labels. After completion of the specific binding reaction the lanthanide ions are dissociated from the nonluminescent transporting chelates, and the luminescence is enhanced in a micellar chelating environment. In DELFIA-type immunoassays, the label is a lanthanide (Eu(III),Sm(III), or Tb(II1)) chelate of N-(isothiocyanatobenzy1)diethylenetriamine-N,N’,N”,N’’-tetrakis(aceticacid) (5)whereas in DNA hybridization assays the chelate is composed of 2,2’,2”,2’”- [ [4-[2-(4-isothiocyanatophenyl)ethyllpyridine2,6-diyllbis(methylenenitrilo)ltetrakis(acetic acid) (loa in Scheme 1)because that chelate better tolerates conditions used in hybridization (6, 7). The marker release prohibits the application of DELFIA labels in areas where the luminescence signals have to be localized, e.g., in situ immunostaining, in situ and Southern blot nucleic acid hybridization, DNA sequencing, or in cytofluorometry. The other way to utilize lanthanide chelates is to use a free luminogenic ligand as a label and saturate the ligand afterwards with the ion (12). There are, however, numerous reasons why a stable luminescent chelate would be preferred. The ligands which can be used as luminogenic labels are generally only three dentate, and before measurement the surface has to be dried to avoid aqueous quenching (13). In addition, the use of free ligand requires an additional step in the indirect staining process to avoid ligand contamination with endogenous ions originating from the sample and the final luminescence depends, amongst other things, on the chelate stoichiometry and humidity of the surface. A stable, multidentate, luminescent chelate would eliminate all these problems and make the staining simple and the response quantitative. +
Wallac Oy and Centre for Biotechnology.
* Wallac Oy and University of Turku.
Abstract published in Advance ACS Abstracts, April 15, 1994. @
1043-1802/94/2905-0270$04.50/0
A large number of ligands capable of forming stable and luminescent complexes with lanthanides have been prepared (14-18). As a part of these studies, the europium(111)chelate of 2,2’,2”,2”’- [ 14-[ (4-isothiocyanatopheny1)ethynyl]pyridine-2,6-diyllbis(methylenenitri1o)ltetrakis(acetic acid) (13a in Scheme 1) was used as a luminescent label in time-resolved fluorescencemicroscopy for localization of antigens, mRNAs and gene sequences on the cell and tissue level (9). As yet, though, a good preparation method of the europium(II1) chelate 10a (DELFIA DNA labeling chelate) has not been reported, and the only reported synthesis of ligand 8 contains many steps (19). The present work describes in detail the preparation of 13a and gives a simple method for the synthesis of 10a from the same key intermediate 6 (18). In the present work we have also used different activation methods including 2,4,6-trichloro1,3,5-triazine (DTA) and iodoacetic anhydride (I-Acet) in addition to thiophosgene (NCS) for coupling the chelates to biomolecules. The effect of both coupling ratio (chelates per biomolecule) and the chemical linkage between the chelate and a biomolecule to luminescence properties were studied by conjugating luminescent chelates 13a-c to an antibody, rabbit anti-mouse IgG. EXPERIMENTAL PROCEDURES General Comments. All reagents were purchased from Aldrich-Chemie GmbH & Co.KG, Steinheim, and used without further purification. The solvents employed were of reagent grade and were used as received. lH NMR spectra were recorded a t 400 MHz on a Jeol-GX-400 spectrometer with TMS as a standard. UV and IR spectra were recorded on Shimadzu-UV-2100 and Perkin-Elmer 1600 FTIR spectrophotometers, respectively. Elemental analyses were recorded on the Perkin-Elmer 2400 CHNS/O elemental analyzer. Tetra(tert-buty 1) 2,2’,2”,2”’-[[4- [2-(4-Aminopheny l )ethyl]pyridine-2,6-diyl]bis(methylenenitrilo)]tetrakis(acetate) (7). A mixture of tetra(tert-butyl) 2,2’,2”,2”’14- [(4-aminophenyl)ethynyllpyridine-2,6-diyllbis(methylenenitrilo)ltetrakis(acetate) (18)(6;3.45 g, 5.00 mmol), 10% Pd on carbon (0.5 g), and MeOH (40mL) was stirred in a hydrogen atmosphere (0.69 MPa) for 6 h. After filtration, the filtrate was evaporated and the residue purified by flash chromatography on silica gel by eluting with petroleum ether (40-60 “C)/ethyl acetate (53) to give 7 (3.10 g, 89%). IR (film): 1716, 1368, 1157 cm-1 v(C=O @ 1994 American Chemical Society
Bioconjugate Chem., Vol. 5, No. 3, 1994 270
Technlcal Notes
IR (KBr pellet): 2190 v(C=C), 2096 v(SCN), 1610, 1405 and CO). UV (EtOH): 294,271 (sh), 239 nm. ‘H NMR: cm-1 v(C=O and CO). UV (HzO): 334,321,300 (sh), 285 6 (DMSO-&) 1.42 (36 H, s), 2.66-2.72 (2 H, m), 2.74-2.80 (sh), 225 nm. Anal. Calcd for C Z ~ H ~ ~ N ~ S O ~ E U N ~ . ~ H Z O (2 H, m), 3.39 (8 H, s), 3.85 (4 H, s), 4.83 (2 H, s), 6.47 (2 C, 35.79; H, 3.75; N, 6.96. Found C, 36.19; H, 3.65; N, H, d, J = 8.6 Hz), 6.85 (2 H, d, J = 8.6 Hz), 7.24 ppm (2 6.79. H, s). Anal. Calcd for C39H~N40rHzO:C, 64.09; H, 8.55; N, 7.67. Found: C, 64.10; H, 8.48; N, 7.59. Synthesis of Compounds 10b and 13b. A solution of 2,4,6-trichloro-1,3,5-triazine(18 mg, 0.1 mmol), acetone 2,2’,2”,2”’-[[4 4 2-(4-Aminopheny1)ethyllpyridine(1.0 mL) and water (1.0 mL), was added to a solution of 2,b-diylIbis(methylenenit rilo)]tetrakis (acetic acid) amino chelate (9 or 12,O.l mmol) and 0.1 M NaOAc (1.5 (8). A solution of compound 7 (3.15 g, 4.4 mmol) in mL, pH 4.9). After being stirred for 30 min, the reaction trifluoroacetic acid (120 mL) was stirred for 1.5 h a t room mixture was treated with acetone, and the precipitate was temperature. After evaporation, the residue was triturated filtered and washed with acetone. with diethyl ether and filtered to give 8 (3.50 g, 96% 1. IR (KBr pellet): 1735, 1671, 1413,1197 v(C=O, CO), 1197 Europium(111) Chelate of 2,2’,2””’’’-[ [4-[2-[4-[ (4,6cm-l v(CF). UV (HzO): 268 (sh), 261 nm. lH N M R 6 Dichloro-l,3,5-triazin-2-yl)amino]phenyl]ethyl]pyri(DMSO-&) 2.84-2.92 (2 H, m), 2.97-3.05 (2 H, m), 3.57 dine-2,6-diyl]bis(methylenenitrilo)]tetrakis(acetic (8 H, s), 3.89 (4 H, s), 4.18 (2 H, s), 6.82 (2 H, d, J = 7.8 acid) (lob). Yield: 72%. IR (KBr pellet): 1601, 1400 Hz),7.07 (2 H,d, J = 7.8 Hz), 7.57 ppm (2 H, s). Anal. cm-l v(C=O and CO). UV (H20): 270 nm. Anal. Calcd Calcd for C Z ~ H Z ~ N ~ O ~ ~ C FC,~ 41.94; O O H H, : 3.76; N, for C Z ~ H Z ~ C ~ Z N ~ O ~ E UC, N 34.11; ~ . ~ HH, ZO 3.85; : N, 10.71. 6.75. Found: C, 41.64; H, 3.96; N, 6.28. Found C, 34.75; H, 3.68; N, 10.17. Synthesis of Europium(II1) Chelates 9 and 12. Europium(II1) Chelate of 2,2’,2”””’-[ [4-[[4-[(4,6Tetraacid (8 or 11 (18),6.7 mmol) was dissolved in water Dichloro-1,3,5-triazin-2-yl)amino]phenyl]et hynyllpyridine-2,6-diyl]bis(methylenenitrilo)]tetrakis(75 mL) and pH was adjusted to 6.5 with solid NaHC03. Europium(II1) chloride (2.7 g, 7.4 mmol) in water (30 mL) (acetic acid) (13b). Yield 78%. IR (KBr pellet): 2208 was added over 15 min and the pH was maintained in the v(C=C), 1601, 1405 cm-l v(C=O and CO). UV (H20): range 5-7. After the mixture was stirred for 1.5 h, the pH 325 nm. Anal. Calcd for C Z ~ H ~ ~ C ~ Z N ~ O S E U C, N~-~HZO: was raised to 8.5 with 1M NaOH and the precipitate was 34.26;H,3.43;N,10.76. Found: C,34.14;H,3.74;N,10.95. filtered off. Acetone was added, and the precipite was Europium(II1) Chelate of 2,2’,2””’-[[4-[[4-(Iodoacfiltered and washed with acetone. etamido)phenylethynyl]pyridine-2,6-diyl]bis( methylenenitrilo)]tetrakis(acetic acid) (13c). A solution Europium(II1) Chelate of 2,2”2”,2’”-[[4-[2-(4-Amiof iodoacetic anhydride (165 mg, 0.47 mmol) and CHCl3 nophenyl)ethyl]pyridine-2,6-diyl]bis(methyleneni(1.5 mL) was added to a solution of chelate 12 (51 mg, trilo)]tetrakis(acetic acid) (9). Yield: 82%. IR (KBr 0.078 mmol), N,N-diisopropylethylamine (81 pL, 0.47 pellet): 1602,1403 cm-l v(C=O and CO). UV (HzO): 263, mmol), 235 nm. Anal. Calcd for C Z ~ H Z ~ N ~ O ~ E U N ~ . ~ H Z O - ~ N ~and C ~water : (1.5 mL). After the mixture was stirred for 1 h, the phases were separated and the water phase C, 31.24; H, 4.10; N, 6.34. Found: C, 31.85; H,3.37; N, was washed with CHC13 (2 X 4 mL). The aqueous solution 6.36. was treated with acetone, and the precipitate was filtered Europium(II1) Chelate of 2,2”2’”2’”-[[4-[(4-Amiand washed with acetone. Yield: 65 mg (90%). IR (KBr nophenyl)ethynyl]pyridine-2,6-diyl]bis(methylenepellet): 2207 v(C=C), 1600, 1406 cm-l v(C=O and CO). nitrilo)]tetrakis(acetic acid) (12). Yield: 77 % . IR UV (HzO): 317 nm. Anal. Calcd for C26H21(KBr pellet): 2196 v(C=C), 1597,1406 cm-l v(C=O and IN409EuNa.6HzO: C, 32.24; H, 3.57; N, 6.02. Found: C, CO). UV (HzO): 341,261 nm. Anal. Calcd for Cz3HzoN432.06; H, 3.33; N, 5.65. OsEuNa.7Hz0*2NaC1: C, 30.75; H, 3.81; N, 6.24. Found: Conjugation of the Europium(II1) Chelates 13a-c C, 30.24; H, 3.32; N, 6.61. to an Antibody. The IgG fraction of rabbit anti-mouse Synthesis of Chelates 10a and 13a. An aqueous IgG (Dako, Copenhagen, Denmark) was coupled with solution (15 mL) of amino chelate (9 or 12,0.91 mmol) was increasing amounts of the chelate 13a by incubating the added over 15 min to a mixture of thiophosgene (115 pL, protein with the chelate a t different molar ratios in 3.66 mmol), NaHC03 (380 mg, 4.57 mmol), and CHC13. carbonate buffer, pH 9.3, a t room temperature for 16 h. After the mixture was stirred for 0.5-1 h, the phases were The protein-chelate conjugates were purified by gel separated and the water phase was washed with CHC13 (3 chromatography and the labeling ratio analyzed by X 15mL). The aqueous solution was extracted with phenol DELFIA system (20). The labeling efficiency test was (about 3 g), and the phenol phase was treated with water performed by using fixed concentrations of the chelates (3 mL) and diethyl ether (60 mL). The water phase was 13a-c (at a 35-fold molar excess) during the labeling. separated and washed with diethyl ether (15 mL). The Luminescent Properties of the Antibody ConjupH was adjusted to 7 with 1M acetic acid, and acetonitrile gates. Luminescent properties (excitation and emission was added (HzO/MeCN, 1:2). The mixture was filtered spectra, decay times, and emission intensities) of the Euthrough silica gel by elution with HZO/MeCN (1:2). The labeled antibodies were investigated using a Perkin-Elmer solution was evaporated to 1-2 mL and treated with Model LS 5 spectrofluorometer (Beaconsfield, UK). The acetone. The precipitate was filtered and washed with luminescent properties were analyzed in an aqueous buffer, acetone. Tris-HC1 buffer, pH 7.5. Europium(II1)Chelate of 2,2’,2”,2”’-[[4-[2-(4-Isothiocyanatophenyl)ethyl]pyridine-2,6-diyl]bis(methylRESULTS AND DISCUSSION enenitrilo)]tetrakis(acetic acid) (loa). Yield: 69%. IR (KBr pellet): 2115 v(SCN), 1618, 1401 cm-’ u(C=O Europium(II1) chelates 9 and 12were prepared according and CO). UV (HzO): 278 (sh), 268,223 nm. Anal. Calcd to the routes shown in Scheme 1. The synthesis of the for C Z ~ H Z Z N ~ S O ~ E U N ~C, . ~ 35.61; H Z O :H, 4.23; N, 6.92. common intermediate, tetra(tert-butyl)2,2’,2”,2”’- [ [4-(4Found: C, 35.51; H, 3.82; N, 7.06. aminophenyl)ethynyllpyridine-2,6-diyllbis(methy1enenitrilo)ltetrakis(acetate) (6) (181, started from 4-hydroxyEuropium(II1) Chelate of 2,2’,2”””’-[[4-[ (4-Isothiopyridine-2,6-dicarboxylic acid (11, which reacted with cyanatophenyl)ethynyl]pyridine-2,6-diyl]bis(methphosphorus pentabromide to yield 4-bromopyridine-2,6ylenenitrilo)]tetrakis(acetic acid) (13a). Yield: 73 %
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Technical Notes
dicarboxylic dibromide (21,22). Treatment with EtOH generated the corresponding ester 2. Reduction with sodium borohydride resulted in the bis(methano1) 3, and bromination with phosphorus tribromide gave the bis(bromomethyl) derivative 4. After ita reaction with di(tert-butyl) iminobis(acetate), the 4-bromo atom of compound 5 reacted with 4-aminophenylacetylene in the presence of a palladium catalyst and copper(1) iodide. The triple bond was easily reduced with hydrogen and palladium on carbon. The reduction of the triple bond of compound 6 can be seen from the disappearance of u( C d ) a t about 2210 cm-' in the IRspectra. This synthesis of compound 7 is much more convenient and shorter than the previously reported method (19). Moreover, we can prepare both the luminescent (12 and 13a-c) and nonluminescent chelate (9 and loa-b) from the same intermediate 6. The ester groups of compounds 6 and 7 were hydrolyzed with trifluoroacetic acid. Finally, europium(II1) chelates 9 and 12 as well as their activated products 10 and 13with thiophosgene, iodoacetic anhydride (51,and 2,4,6-trichloro1,3,5-triazine (11) were prepared in accordance with standard methods. According to TLC, the activated reactions were nearly quantitative. After the activation reaction with thiophosgene, the products were purified from inorganic salts by phenol extraction (23). Usually, activated chelates contain inorganic impurities and are difficult to purify. We found that simple phenol extraction can be used for desalting the chelates. The formation of an isothiocyanato group can be seen from the appearance of u(S=C=N) a t about 2100 cm-l in the IR spectra. After the activation of chelate 12,the products 13a-c gave strong red luminescence both in solution and on TLC. Phenol extraction cannot be done with chelates activated by 2,4,6trichloro-1,3,5-triazinebecause of the high reactivity of the products. The efficencies of the activated groups in forming covalent bonds with a protein were elucidated by labeling of a model antibody. The dichlorotxiazinyl group produced clearly the most efficient labeling reagent with a labeling level of 12 Eu/IgG, the isothiocyanato group yielded under same conditions 6.5 Eu/IgG, and the iodoacetamido group yielded 1.2 Eu/IgG. Another important factor relating to the choice of reaction is its effect on the affinity and nonspecific binding properties of the used antibodies, which has to be elucidated for each particular antibody to be labeled. The most strongly reactive intermediate, dichlorotriazinyl activated chelate, may also cause decreased affinities when used in high excess conditions. It is generally assumed that, because of the very long Stokes' shift of the chelates, there is no problem of innerfilter quenching. This was verified by labeling IgG with up to 25 chelates per IgG and studying the respective luminescence quantum yields. As shown in Figure 1, the increasing amount of chelates in a protein does not have any major effect on quantum yield. Accordingly, the total luminescence can be increased by more efficient labeling as long as immunoreactivity is retained. The effect of different activated groups and different linkages between the chelate and the protein on the luminescent properties of Eu-chelates is shown in Table 1. Only the isothiocyanato group has a slightly negative effect on luminescence quantum yield and decay time. ACKNOWLEDGMENT The financial support of the Academy of Finland is gratefully acknowledged.
I
1
h
0
v
2 $.h
10 20 Labeling level, EdIgG
Figure 1. Effect of labeling level (Eu3+/IgG) to total luminescenceintensity (A)(arbitrary units) and to luminescencequantum yield ( 0 ) .
Table 1. Effect of Linkage Group on the Luminescence of Eu Chelates (13) Coupled to an Antibody chelate active group &., nm 7,~s c@ @ 13 NCS 330 380 550 0.025 13b DTA 341 400 1100 0.05 130 I-Acet 330 395 1150 0.05 LITERATURE CITED (1) Soini, E., and Hemmila, I. (1979) Fluoroimmunoassay:
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(13) Evangelista, R. A., Pollak, A., Allore B., Templeton E. F., Morton, R. C., and Diamandis, E. P. (1988) A new europium chelate for protein labelling and time-resolved fluorometric applications. Clin. Biochem. 21, 173-178. (14) Kankare, J.,Latva, M., and Takalo, H. (1991)Fluorescence intensities of Eu(II1) complexes with substituted 4-phenylethynylpyridines as ligands. Eur. J. Solid State Inorg. Chem. 28, 183-186. (15) Mukkala, V.-M., and Kankare, J. (1992)New 2,2'-bipyridine derivativesand their luminescence properties with europium(111) and terbium(II1) ions. Helv. Chim. Acta 75, 1578-1592. (16) Mukkala, V.-M., Sund, C., Kwiatkowski, M., Pasanen, P., Hogberg, M., Kankare, J., and Takalo, H. (1992) New heteroaromatic complexing agents and luminescence of their europium(II1) and terbium(II1) chelates. Helv. Chim. Acta 75, 1621-1632. (17) Mukkala,V.-M., Kwiatkowski, M., Kankare, J.,andTakalo, H. (1993) Influence of chelating groups on the luminescence properties of europium(II1) and terbium(II1) chelates in the 2,2'-bipyridine series. Helv. Chim. Acta 76, 893-899.
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