MAY/JUNE 1995 Volume 6, Number 3 0Copyright 1995 by the American Chemical Society
Bioconjugate Chemistry
ARTICLES Syntheses and Properties of Luminescent Lanthanide Chelate Labels and Labeled Haptenic Antigens for Homogeneous Immunoassays Heikki Mikola,*,T,*Harri Takalo,+ and Ilkka Hemmilat Wallac Oy, P.O. Box 10, FIN-20101 Turku, Finland, and Department of Chemistry, University of Turku, FIN-20500 Turku, Finland. Received October 17, 1994@
Lanthanide chelate labels containing substituted 4-(arylethynyl)pyridine as the chromogenic moiety and iminobidacetic acid) groups as the chelating part were synthesized. N-Succinimidyl esters of the carboxy derivatives of thyroxine and progesterone were prepared and coupled to the aliphatic amino groups of the synthesized lanthanide chelates. The luminescence properties of the chelates and labeled haptenic antigens were measured in ethanol and in an aqueous buffer containing either albumin or detergents as luminescence-modulating compounds. The energy transfer enhanced ion luminescence of the derivatives containing a para-amino-substituted phenyl ring showed particularly strong dependence on environmental changes, which makes these derivatives well suited for homogeneous time-resolved fluoroimmunoassay based on the use of external luminescence modulators.
INTRODUCTION
Time-resolved fluorometry combined with the use of luminescent long decay time emitting lanthanide chelate labels provides an excellent way to develop highly sensitive bioaffinity assays ( I ) . The assay technology based on dissociative fluorescence enhancement ( 2 ) ,DELFIA (Wallac Oy), is already widely applied, particularly in the field of clinical immunodiagnostics (31, but it is also finding applications in DNA hybridization assays (4). The use of time-resolved fluorometry is desirable for homogeneous assays because conventional fluorometry suffers from interference by the sample constituents present during the fluorometric measurement. Timeresolved fluorometry can completely eliminate the interference originating, e.g., from sample autofluorescence. +
Wallac Oy.
t University of Turku. @
Abstract published in Advance ACS Abstracts, April 15,
1995.
However, the DELFIA technology is not suited to homogeneous assays because the enhancement step applied requires a heterogeneous approach. To be applicable in homogeneous assays, the chelate label has to be stable and luminescent in situ, and it must in addition enable direct monitoring of the immunoreaction without any physical separation. Two assay principles have been applied in homogeneous time-resolved fluorometric immunoassays. One of these utilizes an energy transfer between a europium cryptate and a phycobiliprotein in a concentrated fluoride solution (5). This assay principle is applicable to different types of assays but requires two separate labelings for each analyte. We have developed a straightforward system based on the use of environmentally sensitive chelate labels and the addition of luminescence-modulating compounds in the assay buffer (6). This assay principle is already employed with some steroid glucuronides (7, 8) and thyroxine (6). The weak affinity of aromatic structures to serum proteins (or detergents)-a
1043-1802/95/2906-0235$09.00/00 1995 American Chemical Society
Mikola et al.
236 Bioconjugate Chem., Vol. 6,No. 3, 1995 r C O O H
Scheme 1. Synthesis of Iodobenzamides 7 and 8 /-7
H:b---$N b O O H
Nr C O O H k O O H
1 R=OH
5
3 R=OH 4 R=NY
6
R=OH R=NH2
2 R=NH2
Figure 1. Structures of chelating ligands 1 and 2. property which generally causes problems in, e.g., polarization-based homogeneous fluoroimmunoassays (914s made use of in this system to create an alternativebinding direct homogeneous assay. The matrix effect related to sample variations is avoided by using a large excess of the luminescence-modulating compound and diluting the samples prior to assay. Earlier investigations have shown that ligands containing 4-(arylethynyl)pyridineas the chromophoric group strongly enhance the europium ion luminescence (up to 9.5 x 106-fold) (IO), and chelates incorporating this structure have been used as labels in time-resolved spectroscopy (10-13). The effects of ligand substituents on the luminescence of a chelated europium ion have been studied both in aqueous solution and in ethanol (10).An amino or a hydroxy group at the para-position of the phenyl ring renders the europium chelates of these ligands sensitive to environmental changes, which can be exploited in direct homogeneous assays. For further studies we chose ligands 1 and 2 (Figure l),in which the carboxyl group at the meta-position makes it possible to couple them to bioaffinity reagents (IO). In the present paper we describe the syntheses of a series of 44arylethyny1)pyridine derivatives bearing a short spacer arm at the meta-position. To study the effects of the coupling method and ratio on the ion luminescence, we have previously coupled luminescent lanthanide chelates to proteins (13, 141,to be used in heterogeneous assays. In the present article we describe the conjugation of luminescent lanthanide chelates to small haptenic molecules to be applied as tracers in homogeneous assays. The coupling of the europium and terbium chelates was accomplished with N-hydroxysuccinimide-activatedderivatives of thyroxine and progesterone. We employed here activated carboxyl derivatives to selectively use aliphatic amino groups of the synthesized chelates for couplings. The luminescence properties of the lanthanide chelates and labeled haptenic antigens are discussed, and their suitability as tracers in direct homogeneous time-resolved fluoroimmunoassays is elucidated. EXPERIMENTAL PROCEDURES
Materials. The reagents for syntheses were purchased from Aldrich-Chemie,E. Merck, and Fluka and used as received. The solvents from E. Merck and Fluka were of p.a. grade. TLC plates and silica for flash chromatography were obtained from E. Merck. 'H NMR spectra were recorded on a JEOL JNM-GX400 FT-NMR spectrometer using tetramethylsilane as internal standard. IR and UV spectra were recorded on a PerkinElmer 1600 FTIR and a Shimadzu UV-2100 spectrophotometer, respectively. Melting points were measured on a Gallenkamp capillary apparatus and are uncorrected. Ligands 1 and 2 (Figure 1)were prepared according to Takalo and co-workers (IO). The luminescence properties of the europium and terbium chelates and the labeled hapten derivatives were
7 R'=OCOCF3 8 R'=NHCOCF3
+&
Scheme 2. Synthesis of Ligands 14-16 R'
rCOOC(CH3)3 i
HC-C$N
~ O O C ( C H ~ ) B rCOOC(CH3)3 N LCOOC(CH3)~
7 R' = OCOCF3, R' = CONH(CH2)2NHCOCF3
10
8 R' = NHCOCF3, R" = CONH(CH2)2NHCOCF3
9 R' = H, R" = CH2NHz
.*--eN 11-13
-
KOH. CF3COOH
rCOOH
R"'
b O O H
rCOOH N LCOOH
14 R"' =OH, R
"
= CONH(CH2)2NH2
15 R"' = NH2, R"" = CONH(CH2)2NH2
16 R"' = H. R"' = CHzNH2
measured using a 1234 DELFIA time-resolved fluorometer (Wallac). The measurements were done in 10 nM chelate derivative concentrations both in ethanol and in Tris-HC1 buffer (50 mM, pH 7.75) containing 0.9 g/L NaC1, with or without added albumin (0.5%). The measurements were standardized using 1nM europium in the DELFIA Enhancement Solution (Wallac) as a standard, known to have a luminescence quantum yield of 0.69 and a relative fluorescence intensity ( E x CP.1 of 24 840 (3). The luminescence intensities of the studied conjugates are expressed either as counts (integrated counts within the measuring time of 1 s) or as a percentage compared to the standard. Preparation of Chelating Ligands. The syntheses of the chelating ligands are shown in Schemes 1 and 2. Preparation of Amides 5 and 6. A mixture of ester 3 or 4 (10 mmol) (15) and ethylenediamine (6.01 g, 100 mmol) was stirred at the desired temperature until the reaction was complete (0.5 h at 60 "C for 5; 0.5 h at 75
Luminescent Chelates and Hapten Derivatives
"C for 6). The reaction mixture was evaporated to dryness, and the product was purified by a suitable method. N-(2-Aminoethyl)-2-hydroxy-5-iodobenzamide, 5. The product was crystallized from acetonitrile. Yield: 89%. Mp: 163-164 "C. 'H NMR: 6 (de-DMSO)2.82 (2 H, t, J = 6.1 Hz), 3.38 (2 H, t, J = 6.1 Hz), 4.30 (4 H, broad s), 6.45 (1H, d, J = 8.8 Hz), 7.32 (1H, dd, J = 2.5 and 8.8 Hz), 7.98 ppm (1H, d, J = 2.5 Hz). IR (KBr pellet): 1620, 1575, 1540, 1450, 1295 cm-l v(C0NH and NH). 2-Amino-N-(2-aminoethyl)-5-iodobenzamide, 6. The product was purified by flash chromatography on silica gel using MeOWCHC13 (first 0/1, then 5/3) as an eluent and finally crystallized from CHzClz after decantation from insoluble material. Yield: 73%. Mp: 110-112 "C. 'H NMR: 6 (ds-DMSO) 2.70 (2 H, t, J = 6.4 Hz), 3.23 (2 H, tt, J = 6.4 and 6.4 Hz), 3.31 (4 H, broad SI, 6.55 (1H, d, J = 9.4 Hz), 7.37 (1H, dd, J = 2.4 and 9.4 Hz), 7.78 (1 H, d, J = 2.4 Hz), 8.34 ppm (1H, t, J = 6.4 Hz). IR (KBr pellet): 3440, 3360, 3320, 1630, 1570, 1535, 1480, 1305 cm-I v(CONH and NH). Preparation of Compounds 7 and 8. Benzamide 5 or 6 (3.0 mmol) was added in small portions to cold (below 5 "C) trifluoroacetic anhydride (4.6 g, 22 mmol) during 0.5 h. After being stirred for another 0.5 h below 5 "C, the reaction mixture was kept a t room temperature for 2 h. Ice-cold water was added to the cooled reaction mixture. The formed solid material was filtered and washed with cold water. N-[2-(Trifluoroacetamido)ethyl]-2-(trifluoroacetoxy)-5iodobenzamide, 7. Yield: 60%. Mp: 173-175 "C. 'H NMR: 6(CD3COCD3)3.57-3.67 (4 H, m), 6.75 (1H, d, J = 8.8 Hz), 7.71 (1H, dd, J = 2.4 and 8.8 Hz), 8.04 (1H, d, J = 2.4 Hz), 8.56 (1H, s), 8.80 ppm (1H, s). IR (KBr pellet): 3415 and 3315 v(NH), 1705, 1635, 1575, 1560, 1530 and 1245 v(CONH, C=O and C-01, 1185 cm-l v(CF). 2-(Trifluoroacetamido)-N-[2-(trifluoroacetamido)ethyl/5-iodobenzamide, 8. The product was purified by flash chromatography on silica gel using petroleum ether (bp 40-60 "C)/ethyl acetate (5/3) as an eluent. Yield: 59%. Mp: 201-202°C (subl). 'H NMR: G(CD3COCD3) 3.593.70 (4 H, m), 7.96 (1H, dd, J = 1.9 and 8.8 Hz), 8.18 (1 H, d, J = 1.9 Hz), 8.38 (1H, d, J = 8.8 Hz), 8.65 ppm (3 H, broad s). IR (KBr pellet): 3305 v(NH), 1735, 1705, 1635, 1585, 1515, 1160 cm-l v(CONH and CF). Coupling of Compounds 7, 8, and 3-Iodobenzylamine Hydrochloride (9) to 10. Bis(tripheny1phosphine)palladium(II) chloride (7 mg, 0.01 mmol) and copper(1) iodide (4 mg, 0.02 mmol) were added under nitrogen to a mixture of 7, 8, or 3-iodobenzylamine hydrochloride (9) (0.5 mmol), tetra(tert-butyl) 2,2',2",2"'[(4-ethynylpyridine-2,6-diyl)bis(methylenenitrilo)]tetrakis(acetate) (16)(10,0.34 g, 0.56 mmol), dry triethylamine (2mL), and tetrahydrofuran (2.5 mL). After being stirred for 2 h a t room temperature, the mixture was filtered and the filtrate was evaporated to dryness. The residue was dissolved in CHC13 (15 mL), washed with water (2 x 5 mL) and dried with sodium sulfate. The product was purified by flash chromatography on silica gel. Tetra(tert-butyl) 2,2',2",2"'- { { 4-{{ 3 '-[[N-(2-(trifluoroacetamido)ethyl)amino/carbonyl-4'-(trifZphenyl}ethynyl}pyridine-2,6-diyl} bis(methy1enenitriZo))tetrakis(acetate), 11. Eluent: petroleum ether (bp 4060 "C)/ethyl acetate (first 10/3, then 1/11. Yield: 53%. 'H NMR: 6 (ds-DMSO) 1.40 (36 H, s), 3.37-3.44 (4 H, m), 3.42 (8 H, s), 3.89 (4 H, s), 6.99 (1H, d, J = 8.6 Hz), 7.53 (2 H, s), 7.61 (1H, dd, J = 2.5 and 8.6 Hz), 8.12 (1 H, d, J = 2.5 Hz), 9.08 (1H, broad, s), 9.54 ppm (1 H, broad, s).
Bioconjugate Chem., Vol. 6,No. 3, 1995 237
Tetra(tert-butyl) 2,2',2",2'"-{ { 4-{{ 3'-[[N-(2-(trifluoroacetamido~ethyl~amino/carbonyl/-4'-(trifluoroacetamido)phenyl}ethynyl}pyridine-2,6-diyl}bis(methylenenitrilo)}tetrulzis(acetate), 12. Eluent: petroleum ether (bp 4060 "CYethyl acetate (513). Yield: 79%. 'H NMR: 6 (d6DMSO) 1.42 (36 H, SI, 3.22-3.41 (4 H, m), 3.44 (8 H, s), 3.91 (4 H, s), 7.58 (2 H, s), 7.85 (1H, dd, J = 2.4 and 8.1 Hz), 8.16 (1H, d, J = 2.4 Hz), 8.42 (1H, d, J = 8.1 Hz), 9.24 (1 H, broad s), 9.50 ppm (1 H, t, J = 5.9 Hz). IR (KBr pellet): 2220 v(C=C), 1735, 1650, 1590,1545,1225, 1155 cm-' v(CONH, C=O, C-0 and CF). Tetrdtert-butyl) 2,2',2",2- { {4-{[3'-(aminomethyl)phnyllethynyl}pyridine-2,6-diyl} bis(methylenenitrilo)}tetrukis(acetate), 13. Eluent: first petroleum ether (bp 40-60 "C)/ethyl acetate (5/3) and then MeOH. Yield: 75%. lH NMR: 6 (d6-DMSO) 1.41 (36 H, s), 3.43 (8 H, s), 3.73 (2 H, s), 3.90 (4 H, SI, 7.35-7.45 (3 H, m), 7.55 (2 H, SI, 7.58 (1H, SI. Preparation of Compounds 14-16. A mixture of compound 11 or 12 (0.47 mmol), 0.5 M KOH in ethanol (16 mL), and water (13 mL) was stirred for 35 min at room temperature. The mixture was extracted with CHC4 (2 x 50 mL). The combined organic phases were washed with saturated NaCl solution (15 mL), dried with sodium sulfate, and evaporated to dryness. The residue, or 13 (0.47 mmol), was dissolved in trifluoroacetic acid (20 mL), and the mixture was kept at room temperature for 1.5 h. Trifluoroacetic acid was evaporated without heating. The residue was triturated with diethyl ether (50 mL), and the product was filtered and washed with diethyl ether. The yields were 100% for all compounds. 2,2',2",2"'- { (4{ (3'-[[N-(2-Aminoethyl)amino]carbonyl]4'-hydroxyphenyl}ethynyl}pyridine-2,6-diyE} bidmethylenenitrilo)}tetrakis(acetic acid), 14. 'H NMR: 6 (d6DMSO) 3.00-3.06 (2 H, m), 3.52 (8 H, s), 3.54-3.58 (2 H, m), 3.98 (4 H, s), 7.02 (1H, d, J = 8.8 Hz), 7.57 (2 H, s), 7.68 (1H, dd, J = 2.2 and 8.8 Hz), 7.84 (3 H, broad s), 8.15 (1H, d, J = 2.2 Hz), 9.05 ppm (1H, broad s). IR (KBr pellet): 2210 v(C=C), 1730, 1680, 1635, 1200 cm-' v(CONH, C=O and C-0). 2,2',2",2-{ (4-{{4'-amino-3'-[[N-(2-aminoethyl)amino]carbonyl]phenyl}ethynyl}pyridine-2,6-diyl} bis(methyZenenitriZo)}tetrakis(acetic acid), 15. 'H NMR: 6 (d6DMSO) 3.01-3.03 (2 H, m), 3.40 (5 H, broad s), 3.47 (8 H, s), 3.54-3.57 (2 H, m), 3.94 (4 H, s), 7.55 (2 H, s), 7.88 (1 H, dd, J = 1.5 Hz and 8.9 Hz), 8.23 (1 H, d, J = 1.5 Hz), 8.39 ppm (1H, d, J = 8.9 Hz). IR (KBr pellet): 2210 v(C=C), 1720,1680,1640,1200 cml v(CONH, C=O and c-0). 2,2',2",2"'- { { 4-{[3'-(Aminomethyl)phenyllethynyl}pyridine-2,6-diyl}bis(methylenenitrib)}tetrakis(acetzk aczd), 16. 'H NMR: 6 (dtj-DMSO) 3.52 (8 H, s), 3.98 (4 H, s), 4.09 (2 H, s), 7.50-7.65 (3 H, m), 7.59 (2 H, SI, 7.78 (1H, s). IR (KBr pellet): 2215 (CEC), 1725, 1675, 1630, 1200 cm-l v(C=O and C-0). Preparation of Europium and Terbium Chelates. The tetraacid ligands (14-16)were dissolved in water, and the pH was adjusted to 6-7 using solid NaHC03. An equimolar amount of aqueous solution of europium or terbium chloride hexahydrate was added during 15 min, and the pH was maintained in the range of 6-7. After 1 h of stirring at room temperature, the pH was raised to 8.5 with 1M NaOH and the formed precipitate was removed by centrifugation. The aqueous solution was concentrated to 1-2 mL, and acetone was added to precipitate the chelates. Precipitated chelates were washed with acetone and used without additional purification. Preparation of Octanedioic Acid Bis(N-succinimidyl ester). Octanedioic acid (1.0 g, 5.7 mmol),
Mikola et al.
238 Bioconjugate Chem., Vol. 6,No. 3, 1995 Scheme 3. Synthesis of the Activated Thyroxine Derivative 17
Scheme 4. Synthesis of the Activated Progesterone Derivative 18 7H3
HooC-COOH
U
0
0
I
THYROXINE
,NH
HO-@-O-&CH2-~-COOH I
I
o=c
\
,(CH2)6 o=c\
N
0,
17
O
/
e
N-hydroxysuccinimide (NHS) (1.4 g, 11.7 mmol), and N,N'-dicyclohexylcarbodiimide (DCC) (2.4 g, 11.7 mmol) were dissolved in dry 1,4-dioxane (25 mL), and the reaction mixture was stirred at room temperature overnight (17). Precipitated dicyclohexylurea was removed by filtration, and the solvent was evaporated under reduced pressure. The crude product was first purified by flash chromatography using acetone/toluene (1/9) as eluent. Upon crystallization from acetone/diethyl ether a white solid (1.10 g, 52%) was obtained. Mp: 159 "C. lH NMR: 6 (d6-DMSO) 1.46-1.50 (4 H, m), 1.68-1.73 (4 H, m), 2.66 (4 H, t, J = 7.3 Hz), 2.86 (8 H, SI. IR (KBr pellet): 2919, 1818, 1787, 1734, 1212, 1062, 868 cm-l. Preparation of N-Hydroxysuccinimide-Activated L-ThyroxineDerivative (17) (Scheme 3). A solution of octanedioic acid bis(N-succinimidylester) (100 mg, 0.27 mmol) in dry N,N-dimethylformamide (1.0 mL) was added to a solution of L-thyroxine (100 mg, 0.13 mmol) in dry N,N-dimethylformamide (1.0 mL) and dry triethylamine (40 yL). The reaction mixture was stirred overnight at room temperature. After concentration under reduced pressure, the activated thyroxine derivative 17 was purified by preparative TLC using toluene/ethanol (10)to develop the plates. Rf 0.5. Yield: 64%. lH NMR: 6(d6-DMSO)1.19-1.23 (2 H, m), 1.32-1.36 (2 H, m), 1.41-1.45 (2 H, m), 1.58-1.61 (2 H, m), 2.59 (1 H, s), 2.62 (2 H, t, J = 7.3 Hz), 2.73-2.79 (1 H, m), 2.81 (4 H, s), 3.06 (1H, dd, J = 4.7 and 13.91, 4.22-4.25 (1H, m), 7.05 (2 H, s), 7.74 (2 H, 9). IR (KBr pellet): 2925, 1701, 1636, 1436, 1226 cm-I. Preparation of Progesterone S-(O-Carboxymethylloxime and its N-SuccinimidylEster (18)(Scheme 4). Progesterone (1.7 g, 5.4 mmol) was added to a mixture of pyrrolidine (1.2 mL, 14 mmol) and methanol (100 mL). After 15 min (amino0xy)acetic acid hemihydrochloride (1.05 g, 4.8 mmol) was added, and the mixture was stirred for 1.5 h at room temperature according to Janoski and co-workers (18).Methanol was evaporated, the residue was dissolved in water, and the slightly alkaline solution was extracted once with ethyl
/" O\ /CH2 o=c I
18
?
"YNYO u acetate to remove any unreacted steroid. The aqueous solution was acidified to pH 2 and extracted three times with ethyl acetate. The combined organic phases were evaporated to dryness to yield progesterone 3-(O-carboxymethy1)oxime (1.5 g, 72%), which was used without further purification. 'H NMR: G(CDCl3) 0.65 (3 H, s), 1.06 and 1.09 (3 H, s, s), 2.11 (3 H, SI, 4.09 (2 H, m), 5.78 and 6.43 (1H, s, s). IR (KJ3r pellet): 3300-2500 (broad), 2937,1740,1703,1628,1208,1101 cm-l. UV (CH3CH2OH): A,, 249 nm. Progesterone 3-(O-carboxymethyl)oxime (1.00 g, 2.50 "011, N-hydroxysuccinimide (NHS)(317 mg, 2.75 mmol), and N,W-dicyclohexylcarbodiimide(DCC) (568 mg, 2.75 mmol) were dissolved in dry 1,4-dioxane (3.0 mL), and the reaction mixture was stirred overnight at room temperature (19,20). The precipitated dicyclohexylurea was removed by filtration, and the solvent was evaporated under reduced pressure. The product, N-succinimidyl ester of progesterone 3-(O-carboxymethyl)oxime, was purified on a TLC plate using acetone/toluene (1/9) or chlorofodmethanol(9/1) for developing. Yield: 62%. 'H NMR: G(CDC13) 0.65 (3 H, s), 1.06 and 1.09 (3 H, s, s), 2.17 (3 H, s), 2.87 (4 H, s), 4.90 (2 H, m), 5.78 and 6.43 (1H, s, 9). Labeling of N-Succinimidyl Esters of Haptens with Chelates. The N-succinimidyl esters of hapten derivative 17 o r 18 were dissolved in 1,4-dioxane, and the europium or terbium chelate bearing an aliphatic
Luminescent Chelates and HaDten Derivatives
Bioconjugate Chem., Vol. 6,No. 3, 1995 239
triazinyl derivatives of europium chelates have been used to label amino groups on proteins or hapten derivatives (13, 14, 21). Carboxyl derivatives of steroids have also been labeled in the presence of water-soluble carbodiimide using an aromatic amino derivative of a chelate (2224). However, as ligand 15 incorporates both aliphatic and aromatic amino groups, these labeling methods cannot be applied because of the presence of two reactive amino groups. Therefore, we used N-succinimidyl esters of carboxylic acid derivatives of the haptens, which made it possible to selectively derivatize only the aliphatic amino group of the chelating ligand. Thyroxine is an amino acid, and for immunoassays the amino group of the molecule is usually used for derivatization. In this work we had to use a preactivated bifunctional reagent to obtain the N-succinimidyl ester derivative because of the carboxyl group of thyroxine. Octanedioic acid bis(N-succinimidyl ester) was synthesized (Scheme 3) in 1,Cdioxane using N-hydroxysuccinimide and carbodiimide (171,and the product was purified by crystallization after flash chromatography. In the lH NMR spectrum the characteristic singlet of eight protons of succinimidyl groups at 6 2.86 could be detected. In the IR spectrum the absence of carboxylic acid absorption (3300-2500 cm-l) and the presence of new carbonyl bands near 1800 cm-l, a C-N stretch band at 1212 cm-l, and the band of cyclic imide a t 868 cm-l were characteristic to the synthesized bifunctional reagent. Nsuccinimidyl derivative of thyroxine 17 was synthesized (Scheme 3) using a 2.1-fold molar excess of the bisactivated octanedioic acid in N,N-dimethylformamide, and the product was purified on TLC plates. In the lH NMR spectrum the characteristic chemical shift of the succinimidyl group at 6 2.81 could be detected, as could the new carbonyl band a t 1701 cm-l in the IR spectrum. Progesterone 3-(0-~arboxymethyl)oximewas synthesized using the method of Janoski and co-workers (18) (Scheme 4), in which the 3-oxo group of the steroid dione is activated using pyrrolidine before the reaction with (amino0xy)acetic acid, whereby selective formation of 3-(O-carboxymethyl)oximeis achieved. In the U V spectra a characteristic shift of the absorption maximum from 240 to 249 nm was detected. In this reaction, cis- and trans-isomers of progesterone oxime were produced. The isomers were not separated, but they were clearly distinguishable in the lH NMR spectrum. The chemical shifts of the olefinic proton at carbon C-4 were 6.43 and 5.78, and those of the protons of the methyl group at carbon C-19 were 1.09 and 1.06 for the cis- and transisomers, respectively. According to NMR data, the ratio (cis/trans) of these isomers was 2/3. In the IR spectrum of the 3-(O-carboxymethyl)oximederivative the characteristic carboxylic acid absorption bands, the absence of a %oxo band (1662 cm-l), and the presence of a 20-oxo band (1703 cm-l) were clearly detected. This carboxyl derivative of progesterone was then activated with Nhydroxysuccinimide in dioxane (Scheme 4) using carbodiimide as the condensing agent (19,20). In the IH NMR spectrum the characteristic four-proton singlet at 6 2.87 was indicative of the produced N-succinimidyl ester derivative (18). Labeling of N-succinimidyl ester derivatives with lanthanide chelates was carried out in 1,Cdioxane-water solution, and the produced compounds were purified only on TLC plates, which is not sufficient for immunoassays but adequate for studies of luminescence properties. On a TLC plate the unreacted chelate and hapten derivatives, as well as most of the byproducts of the labeling
1 WNLcm.
O=C
YH
/-coo.
L"3+
N/-COO'
R
b O O .
J
7H3
/
0 ,w2
o=c,
I1
NH
R & - d $ N
R = OH
R = NH2 R=H
1
Loo. /-COO' L"3+
A = ( C H ~ ) ~ N H C(Ligand O 14) A = (CH&.NHCO (Ligand 15) A=cH? (Ligand 16)
Figure 2. Structures of the lanthanide-labeled thyroxine (I) and progesterone (11) derivatives.
amino group was added as a water solution. The mixtures were stirred at room temperature for 2-3 h and concentrated under reduced pressure, acetone was added to precipitate the labeled hapten, and the solvents were removed after centrifugation. The products (Figure 2) were purified on TLC plates using acetonitrile/water (41 1)for developing. The labeled haptens obtained were used for luminescence measurements without any further purification. RESULTS AND DISCUSSION
Syntheses of Ligands and Lanthanide Chelates. In addition to ligands 1 and 2 (Figure 11, we prepared chelates having a substituent containing an aliphatic amino group at the meta-position. The ester group of compounds 3 and 4 (15) reacted readily with ethylenediamine to produce amides 5 and 6. Before coupling to compound 10 (161, the hydroxy and amino groups were protected to give trifluoroacetamides 7 and 8. 3-Iodobenzylamine (91, on the other hand, did not need any protection. The iodo group of compounds 7-9 reacted with the terminal acetylene of 10 in the presence of a catalytic amount of palladium catalyst and copper(1) iodide (16). The protecting trifluoroacetamido, trifluoroacetoxy, and tert-butyl ester groups of compounds 1113 were hydrolyzed using alkaline and acidic hydrolysis, respectively, to the tetraacetic acids 14-16. The lanthanide chelates were synthesized using the method of Takalo and co-workers (13) in a slightly modified form. Syntheses and Labeling of Hapten Derivatives. Most commonly isothiocyanato, haloacetyl, or 2,4,6-
Mikola et al.
240 Bioconjugate Chem., Vol. 6,No. 3, 1995
Table 1. Relative Luminescence Intensities of Europium and Terbium Chelates in Ethanol and in an Aqueous Buffer with or without Albumin substituents re1 luminescence intensity (%I ligand para meta lanthanide ethanol buffer buffer albumin Tb(II1) 1.53 2.0 1.67 16 H CHzNHz Eu(II1) 3.21 1.83 1.53 16 H CHzNHz Eu(II1) 5.40 1.44 2.86 1 OH COOH Eu(II1) 0.43 0.05 0.08 CONHCHzCHzNHz 14 OH Eu(II1) 9.34 0.13 0.38 2 NH2 COOH 1.70 0.07 0.09 CONHCHzCHzNHz Eu(II1) 15 NH2
+
Table 2. Relative Luminescence Intensities of Europium- or Terbium-LabeledProgesterone and Thyroxinea in Ethanol and in an Aqueous Buffer with or without Albumin relative luminescence intensity (%) buffer + ligand lanthanide haDten ethanol buffer albumin 0.55 0.55 0.20 16 Tb(II1) Progesterone 0.34 0.09 0.09 16 Tb(II1) Thyroxine 14 Eu(II1) Progesterone 5.0 0.04 0.20 0.16 4.5 0.02 14 Eu(I1Ij Thyroxine 3.8 0.11 0.53 15 Eu(II1) Progesterone 1.31 4.3 0.18 15 Eu(1IIj Thyroxine ~~
1000000
~
a
For structures, see Figure 2.
reaction, could be removed, whereafter the only luminescent compound was the labeled hapten derivative (Figure 2). Luminescence Properties of Chelates and Chelate-Labeled Haptenic Antigens. Table 1 shows the luminescence properties of the studied chelates in ethanol and in a Tris-HC1 buffer with or without added albumin. Because of its tendency to bind aromatic structures, albumin is used as a luminescence-modulating compound to enable the construction of homogeneous assays. A high concentration of albumin also levels the inherent variations in patient sera, avoiding some of the problems derived from sample-to-sample deviations. The paruamino derivatives (2 and 151,in particular, demonstrate high sensitivity to environmental changes, which may occur during immunoreaction. The solvent sensitivity and the chelate affinity to the added albumin is greatly dependent on the type of substituents at the metaposition, and hence the final suitability of a particular chelate derivative to a homogeneous assay can only be judged after coupling of the chelate to the antigen. In Table 2, the luminescence properties of thyroxine and progesterone derivatives labeled with three different types of chelates are compared. Chelates without parasubstitution in the phenyl ring (16) show very little sensitivity to environmental changes. As an exception, the luminescence of the thyroxine conjugate with a terbium chelate is enhanced by albumin, probably because of strong internal quenching. In line with the results obtained with FITC-conjugated thyroxine (251,the direct quenching is diminished upon binding of the conjugate to proteins. The effect of added albumin on the luminescence of some of the labeled antigens is demonstrated in Figure 3. The maximum luminescence enhancement was 50fold when using thyroxine labeled with the para-hydroxy derivative of the europium chelate (141, whereas when using progesterone labeled with the para-unsubstituted derivative of the terbium chelate (16)the albumin effect was negative. To obtain the maximum enchancement, relatively high concentrations of albumin were required; i.e., the binding of the chelate to albumin is relatively weak. On the other hand, the lability of albumin binding speeds up the replacement reaction by anti-hapten antibodies in cases of low antigen concentrations in a
100000
B 8-
C
8
v)
.10000
-I
1
1
loooj.-jk,,,n, 0
0,Ol
I
I
i
0,1
1
3
Albumin, %
Figure 3. Effect of the albumin concentration on the luminescence of europium- or terbium-labeled haptens (for structures, see Figure 2): thyroxine labeled with the europium chelate of ligand 15 (A) and ligand 14 (B) and thyroxine and progesterone labeled with the terbium chelate of ligand 16 (C and D, respectively).
Table 3. Effect of Albumin and Various Detergents on the Luminescence Intensities of Europium (ligand 14)and Terbium (ligand 16)-LabeledProgesteronea re1 luminescence intensity (%j EuIII(14) TbIII(16)
modulators in buffer none 0.5% albumin 0.1% Triton X-100 0.1% sodium dodecyl sulfate 0.1% cetyltrimethylammonium bromide a
0.04 0.20
0.20 0.16 0.05
0.55 0.55 2.74 0.36 0.10
For structures, see Figure 2.
competitive assay, and thus facilitates the development of rapid homogeneous assays. In addition t o albumin, different detergents can also be used as luminescence modulating compounds. The effects of detergents depend on their charge and can be either luminescence enhancing or quenching (Table 3). Detergents can function as efficient modulators in assays in which the analyte matrix does not inherently contain high concentrations of binding proteins, such as albumin. They can be used e.g., for homogeneous analysis of urine steroids. Combining two homogeneous assays with two different lanthanide labels, europium and terbium, would even make it possible to construct simultaneous doublelabel homogeneous assays. The basic structure of the chelating ligand [ 4-(phenylethynyl)pyridinederivatives]
Bioconjugate Chem., Vol. 6, No. 3, 1995 241
Luminescent Chelates and Hapten Derivatives
presented here and previously is, however, not suitable for terbium assays (26). ACKNOWLEDGMENT
The excellent technical assistance of Ms. Airi Toivonen and the language checking of Ms. Teija Ristela are gratefully acknowledged. This work was financially supported in part by the Academy of Finland. LITERATURE CITED (1) Soini, E., and Hemmila, I. (1979) Fluoroimmunoassay: Present status and key problems. Clin. Chem. 25, 353-361. (2) Hemmila, I., Dakubu, S., Mukkala, V.-M., Siitari, H., and Lovgren, T. (1984) Europium as a label in time-resolved immunofluorometric assays. Anal. Biochem. 137, 335-343. (3) Hemmila, I. (1991) Application of fluorescence in immunoassays, Wiley Interscience, New York. (4) Hurskainen, P., Dahlen, P., Mikoski, J., Kwiatkowski, M., Siitari, H., and Lovgren, T. (1991) Preparation of europiumlabelled DNA probes and their properties. Nucleic Acids Res. 19, 1057-1061. (5) Mathis, G. (1993) Rare earth cryptates and homogeneous fluoroimmunoassayswith human sera. Clin. Chem. 39,19531959. (6) Hemmila, I., Malminen, O., Mikola, H., and Lovgren, T. (1988) Homogeneous time-resolved fluoroimmunoassay of thyroxin in serum. Clin. Chem. 34, 2320-2322. (7) Barnard, G., Kohen, F., Mikola, H., and Lovgren, T. (1989) Measurement of estrone-3-glucuronide in urine by rapid, homogeneous time-resolved fluoroimmunoassay. Clin. Chem. 35,555-559. ( 8 ) Barnard, G., Kohen, F., Mikola, H., and Lovgren, T. (1989) The development of non-separation time-resolved fluoroimmunoassays for the measurement of urinary metabolites. J. Biolumin. Chemilumin. 4 , 177-184. (9) Dandliker, W. B., Hsu, M.-L., Levin, J.,and Rao, B. R. (1981) Equilibrium and kinetic inhibition assays based upon fluorescence polarization. Methods Enzymol. 74, 3-28. (10) Takalo, H., Hanninen, E., and Kankare, J. (1993) Luminescence of europium(II1) chelates with 4-(arylethynyl)pyridines as ligands. Helv. Chim. Acta 76, 877-883. (11) 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. (12) Seveus, L., Vaisala, M., Syrjanen, S., Sandberg, M., Kuusisto, A., Harju, R., Salo, J.,Hemmila, I., Kojola, H., and Soini, E. (1992) Time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization. Cytochemistry 13, 329-338.
(13) Takalo, H., Mukkala, V.-M., Mikola, H., Liitti, P., and Hemmila, I. (1994) Synthesis of europium(II1) chelates suitable for labeling of bioactive molecules. Bioconjugate Chem. 5, 278-282. (14) Mukkala, V.-M., Helenius, M., Hemmila, I., Kankare, J., and Takalo, H. (1993)Development of luminescent europium(111)chelates of 2,2’:6’,2”-terpyridinederivatives for protein labelling. Helv. Chim. Acta 76, 1361-1378. (15) Takalo, H., Kankare, J., and Hbninen, E. (1988)Synthesis of some substituted dimethyl and diethyl 4-(phenylethynyl)2,6-pyridinecarboxylates.Acta Chem. Scand. B42,448-454. (16) Hanninen, E., Takalo, H., and Kankare, J. (1988) Preparation of new complexing agents containing a highly conjugated ethynylated pyridine subunit. Acta Chem. Scand. B42,614619. (17) Pilch, P. F. (1979) Interaction of cross-linking agents with the insulin effector system of isolated fat cells. J. Biol.Chem. 254, 3375-3381. (18) Janoski, A. H., Shulman, F. C., and Wright, G. E. (1974) Selective 3-(0-~arboxymethyl)oximeformation in steroidal 3,20-diones for hapten immunospecificity. Steroids 23, 49-64. (19) Anderson, G. W., Zimmerman, J. E., and Callahan F. M. (1964) The use of esters of N-hydroxysuccinimide in peptide synthesis. J . Am. Chem. SOC.86, 1839-1842. (20) Hosoda, H., Sakai, Y., Yoshida, H., Miyairi, S., Ishii, K., and Nambara, T. (1979) The preparation of steroid Nhydroxysuccinimide esters and their reactivities with bovine serum albumin. Chem. Pharm. Bull. (Tokyo) 27, 742-746. (21) Mukkala, V.-M., Mikola, H., and Hemmila, I. (1989) The synthesis and use of activated N-benzyl derivatives of diethylenetriaminetetraacetic acids: alternative reagents for labeling of antibodies with metal ions. Anal. Biochem. 176, 319325. (22) Mikola, H., and Miettinen, P. (1991) Preparation of europium labeled derivatives of cortisol for time-resolved fluoroimmunoassays. Steroids 56, 17-21. (23) Mikola, H., Hoglund, A.-C., and Hanninen, E. (1993) Labeling of estradiol and testosterone alkyloxime derivatives with a europium chelate for time-resolved fluoroimmunoassays. Steroids 58, 330-334. (24) Mikola, H., and Hedlof, E. (1994) Syntheses of europiumlabeled digoxin derivatives and their use in time-resolved fluoroimmunoassay. Steroids 59, 472-478. (25) Smith, D. S. (1977) Enhancement fluoroimmunoassay of thyroxine. FEBS Lett. 77, 25-27. (26) Takalo, H., Hanninen, E., and Kankare, J. (1995) The influence of substituents on the luminescence properties of the Eu(II1) and Tb(II1)chelates of 4-(phenylethynyl)pyridine derivatives. J. Alloys Compd (in press). BC950018C
