Figure 1. Synthesis of BHHT and BHHCT.
to proteins. One such ligand, 5-chlorosulfonyl-2-thenoyltrifluoro-
acetone (CTTA), has been prepared, but its application in
immunoassay did not give any improvement of the sensitivity.13,14
The unexpectedly small improvement is due to the low stability
of the CTTA-Eu3+ complex and the decreased fluorescence
intensity caused by the protein-binding group attached to the
aromatic ring of the ligand.
pg/ mL (solution measurement after solid dissolution) for AFP
were obtained. The latter value is 4.3 orders of magnitude lower
than those of all of the previously reported immunoassays.
BHHCT was also used for labeling of BSA, and the detection limit
of labeled BSA was 6.5 × 10-15 M.
EXPERIMENTAL SECTION
Synthesis of BHHCT. The labeling reagent BHHCT was
In this report, synthesis of a new tetradentate â-diketone ligand,
4,4′-bis(1′′,1′′,1′′,2′′,2′′,3′′,3′′-heptafluoro-4′′,6′′-hexanedion-6′′-yl)-
chlorosulfo-o-terphenyl (BHHCT, Figure 1) is reported. The
ligand forms a very stable complex with Eu3+ (owing to the
tetradentate structure), emits very strong fluorescence when
complexed with Eu3+ (owing to the heptafluoropropyl group on
one side of the â-diketone groups),17 and can be covalently bound
to proteins. Remarkable superiority of BHHCT over conventional
organic fluorescent labels and other lanthanide labels has been
proved in the time-resolved fluorometric determination of bovine
serum albumin (BSA) and TR-FIA of R-fetoprotein (AFP). Human
AFP is one of the carcinoembryonic proteins and has been
measured with various immunoassays, i.e., radioimmunoassay
(RIA),18,19 enzyme immunoassay (EIA),19 luminescence EIA,20
chemiluminescence EIA (CL-EIA),21,22 latex particle immunoas-
say,23 electorochemiluminescence immunoassay,24 and TR-FIA.25-29
These methods provide detection limits of ∼10-12 M (1 × 10-1
ng/ mL) or higher for AFP. In the present study, detection limits
of 4.6 × 10-2 pg/ mL (solid-phase measurement) and 4.1 × 10-3
prepared according to the three steps in Figure 1, as described
in the following.
(i) P reparation of 4 ,4 ′-Diacetyl-o-terphenyl. To 200 mL
of CH2Cl2 containing 28 g (210 mmol) of anhydrous AlCl3 and
16.1 g (205 mmol) of CH3COCl was added dropwise 100 mL of
CH2Cl2 containing 23 g (100 mmol) of o-terphenyl at 0 °C with
stirring. The solution was further stirred at 0 °C for 30 min and
then at room temperature for 24 h. After 2 h refluxing, the
solution was poured into an ice-HCl solution with vigorous
stirring. The CH2Cl2 was removed by vacuum distillation, and
the residue was collected by filtration. After the precipitate was
washed with water, it was recrystallized from 250 mL of 2-bu-
tanone. Pale yellow prismatic crystals were obtained (24.6 g, 78%
yield). Anal. Calcd for C22H18O2: C, 84.05; H, 5.77. Found: C,
1
84.06; H, 5.87. The compound was also confirmed by H NMR.
(ii) P reparation of 4 ,4 ′-Bis(1 ′′,1 ′′,1 ′′,2 ′′,2 ′′,3 ′′,3 ′′-hep-
tafluoro-4 ′′,6 ′′-hexanedion-6 ′′-yl)-o-terphenyl (BHHT). A so-
lution of 70 mL of dry ether containing 3.0 g (55.5 mmol) of
NaOCH3, 4.84 g (20 mmol) of C3F7COOC2H5, and 3.14 g (10
mmol) of 4,4′-diacetyl-o-terphenyl was stirred at room temperature
in a sealed flask for 36 h. The ether solution was poured into
100 mL of 15% H2SO4, after 15 min of stirring at room temperature,
ether was removed by distillation. The sticky solid obtained was
washed with water and dissolved in 200 mL of boiling ethanol.
The solution was filtered while hot, and the filtrate was condensed
to ∼20 mL and added to 200 mL of petroleum ether (30-60 °C).
The solution was further stirred for 10 min at room temperature
and was then filtered. The oily â-diketone residue, obtained after
vacuum distillation of the filtrate, was vacuum-dried for several
days, and a yellow solid was obtained (5.1 g, 72% yield). Anal.
Calcd for C30H16F14O4: C, 51.00; H, 2.28. Found: C, 51.22; H,
(17) Yuan, J.; Matsumoto, K. Anal. Sci. 1 9 9 6 , 13, 31-36.
(18) Nomura, M.; Imai, M.; Takahashi, K.; Kumakura, T.; Tachibana, K. J.
Immunol. Methods 1 9 8 3 , 58, 293-300.
(19) Christensen, R. L.; Rea, M. R.; Kessler, G.; Crane, J. P.; Valdes, R. Clin.
Chem. 1 9 8 6 , 32, 1812-1817.
(20) John, R.; Henley, R.; Shankland, D. Clin. Chem. 1 9 8 6 , 32, 2066-2069.
(21) Nishizono, I.; Iida, S.; Suzuki, N.; Kawada, H.; Murakami, H.; Ashihara, Y.;
Okada, M. Clin. Chem. 1 9 9 1 , 37, 1639-1644.
(22) Arakawa, H.; Ikegami, T.; Maeda, M.; Tsuji, A. J. Biolumin. Chemilumin.
1 9 9 3 , 8, 135-139.
(23) Passelecq, B.; De Bo, M.; Huber, C.; Gennart, J. P.; Bernard, A.; Lauwerys,
R. J. Immunol. Methods 1 9 8 8 , 109, 69-74.
(24) Blackburn, G. F.; Shah, H. P.; Kenten, J. H.; Leland, J.; Kamin, R. A.; Link,
J.; Peterman, J.; Powell, M. J.; Shah, A.; Talley, D. B.; Tyagi, S. K.; Wilkins,
E.; Wu, T.-G.; Massey, R. J. Clin. Chem. 1 9 9 1 , 37, 1534-1539.
(25) Suonpa¨a¨, M.; Lavi, J.; Hemmila¨, I.; Lo¨ vgren, T. Clin. Chim. Acta 1 9 8 5 , 145,
341-348.
(26) Chan, M. A.; Bellem, A. C.; Diamandis, E. P. Clin. Chem. 1 9 8 7 , 2000-
2003.
(27) Diamandis, E. P.; Morton, R. C.; Reichstein, E.; Khosravi, M. J. Anal. Chem.
1
2.61. The compound was also confirmed by H NMR.
(iii) P reparation of 4 ,4 ′-Bis(1 ′′,1 ′′,1 ′′,2 ′′,2 ′′,3 ′′,3 ′′-hep-
tafluoro-4′′,6′′-hexanedion-6′′-yl)chlorosulfo-o-terphenyl (BH-
HCT). To 5 mL of stirred HSO3Cl was gradually added 1.41 g (2
mmol) of BHHT. After the solution was stirred for 7 h at room
temperature, it was added dropwise with stirring to 150 mL of
1 9 8 9 , 61, 48-53.
(28) Christopoulos, T. K.; Lianiolou, E. S.; Diamandis, E. P. Clin. Chem. 1 9 9 0 ,
36, 1497-1502.
(29) Christopoulos, T. K.; Diamandis, E. P. Anal. Chem. 1 9 9 2 , 64, 342-346.
Analytical Chemistry, Vol. 70, No. 3, February 1, 1998 597