Synthesis of fluorine-containing tetraketones and diketo esters and luminescence-spectral properties of their complexes with lanthanide ions

Article abstract of DOI:10.1007/s11172-006-0248-5

The Claisen condensation of diesters of fluorinated dicarboxylic acids with 2-acetyl-naphthalene affords the corresponding naphthyl-containing tetraketones and diketo esters. The luminescence-spectral properties and stability constants of complexes of these compounds with Eu³⁺ ions were estimated. The characteristics of the complexes are comparable with those of fluorinated β-diketones containing chromophoric substituents, which are widely used in luminescence analysis. The synthesized naphthyl-containing diketo esters are efficiently conjugated with proteins and can be used for detection of biospecific interactions.

Full text of DOI:10.1007/s11172-006-0248-5

Russian Chemical Bulletin, International Edition, Vol. 55, No. 2, pp. 276—280, February, 2006
276
Synthesis of fluorineꢀcontaining tetraketones and diketo esters
and luminescenceꢀspectral properties of their complexes
with lanthanide ions
a
b
b
b
D. V. Romanov,a N. V. Vasil´ev, A. I. Lyamin, N. P. Ivanovskaya, and N. S. Osin
ꢀ
aMilitary University of Radioactive, Chemical and Biological Defense,
13 Brigadirsky per., 105005 Moscow, Russian Federation.
Tel.: +7 (495) 265 9318
bState Scientific Center of the Russian Federation "State Research Institute of Biological Engineering,"
building 1, 75 Volokolamskoe shosse, 125424 Moscow, Russian Federation.
Tel.: +7 (495) 490 5704. Еꢀmail: immunosc@online.ru
The Claisen condensation of diesters of fluorinated dicarboxylic acids with 2ꢀacetylꢀ
naphthalene affords the corresponding naphthylꢀcontaining tetraketones and diketo esters.
The luminescenceꢀspectral properties and stability constants of complexes of these compounds
with Eu3+ ions were estimated. The characteristics of the complexes are comparable with those
of fluorinated βꢀdiketones containing chromophoric substituents, which are widely used in
luminescence analysis. The synthesized naphthylꢀcontaining diketo esters are efficiently conꢀ
jugated with proteins and can be used for detection of biospecific interactions.
Key words: βꢀdiketones, tetraketones, diketo esters, lanthanides, fluorineꢀcontaining comꢀ
pounds, luminescence analysis.
Metal complexes with fluorineꢀcontaining βꢀdiketones
However, the complexones recommended for use in nonꢀ
dissociation assay are not efficient enough because they
require a high excess of Eu3+ and are multiply inferior to
NTA in quantum efficiency.
Thus, the search for complexones with high binding
constants and quantum efficiency of luminescence reꢀ
mains topical. Therefore, it seemed of interest to prepare
models of bisꢀβꢀdiketonate complexones with different
lengths of the fluorineꢀcontaining chain and estimate their
luminescenceꢀspectral and complexing properties.
as ligands find wide use in various analytical applicaꢀ
tions.1—3 In particular, similar rareꢀearth metal (REM)
complexes are used in variants for luminescence detecꢀ
tion of biospecific interactions.3—8
As other lanthanide chelates they are characterized by
unique luminescenceꢀspectral properties (considerable
Stokes shift >200 nm, narrow luminescence emission band
~14 nm, long lifetime of the excited state τ up to 800 µs).
At the same time, these compounds have additional adꢀ
vantages: high quantum efficiency (εϕ) and longerꢀwaveꢀ
length position of the luminescence excitation maximum.
All these data are very significant for sensitivity increasing
and specificity of analysis and for possibility of its instruꢀ
mental realization.
At the same time, βꢀdiketonate complexes of REM
are not sufficiently stable for nonꢀseparable analysis or
analysis in situ. The binding constant of the europium
ion (Eu3+) by the most known complexone of this
type, namely, naphthoyltrifluoroacetone (NTA),3 is only
~107 L mol–1. Therefore, this complexone is used only in
high excess over Eu3+ ions in the variant of dissociaꢀ
tion enhanced lanthanide fluorescence immunoassay
(DELFIA). The introduction of additional βꢀdiketonate
residues (two5,6,8 or even four7) into a chelating molecule
for enhancing the dentate character of ligands was used in
recent attempts to increase the stability of complexes.
Results and Discussion
Naphthylꢀcontaining tetraketones 1a—d, whose βꢀdiꢀ
ketonate moieties are separated by a perfluoroalkylene
chain (CF2)n of different length (n = 1, 2, 4, 6) were
synthesized according to the Claisen method (Scheme 1).
The reactions of dimethyl perfluorodicarboxylates with
2ꢀacetylnaphthalene were carried out in the presence of
anhydrous sodium alkoxide in ether. The maximum conꢀ
version of the reactants was achieved on refluxing and
stirring of the reaction mixture for 3—4 h. In all cases,
diketo esters 2a—d are formed along with the expected
tetraketones. Unlike compounds 2a—d, tetraketones 1a—d
are virtually insoluble in ether and other solvents with low
polarity, and this property was used to isolate them. Diketo
esters 2a—d were additionally purified by fractionation
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 269—273, February, 2006.
1066ꢀ5285/06/5502ꢀ0276 © 2006 Springer Science+Business Media, Inc.

Fluorinated tetraketones
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 2, February, 2006
277
in vacuo. The 1H and 19F NMR spectra show that
tetraketones 1a—d and diketo esters 2a—d exist excluꢀ
sively in the enole form.
Table 1. Longꢀwavelength absorption band values (λmax) and
molar absorption coefficients (ε) for the Eu3+ complexes of the
studied compounds in aqueous mediaa
Compꢀ
lexꢀ
one
Ib
IIc
Scheme 1
λ
/nm
ε•10–4
λ
/nm
ε•10–4
max
max
/L mol–1 cm–1
/L mol–1 cm–1
NTA
1a
1b
1c
1d
2a
2b
2c
2d
—
346→348
340
340
342
335→340
—
—
2.55→2.62
1.03→1.10
2.40
2.80
1.30→1.25
—
334→336
346→350
340→344
340→342
340→342
338→338
334→340
336→340
338→340
2.00→2.10
3.00→3.05
2.85→3.10
3.80
3.90
2.00→2.20
2.00→1.92
1.78→1.92
2.45→2.52
340
342
1.15
1.70
a The arrow means a change in λ
and ε upon the addition of
max
ЕuCl3 (final concentration 2•10–4 mol L–1) to the complexone
(10–5 mol L–1).
b Distilled water.
c Solution of trioctylphosphine oxide (5•10–5 mol L–1) and
Triton Xꢀ100 (0.1%) in water.
agent favoring Eu3+ insulation in a complex from the
quenching effect of water molecules, and micelleꢀformꢀ
ing agent Triton Xꢀ100,3 the compounds of the studied
series in a wide interval of stoichiometric ratios exhibit
emission at 613—616 nm (characteristic of Eu3+) for
excitation of the complexone in the absorption reꢀ
gion (~340 nm) beginning from concentrations about
n = 1 (a), 2 (b), 4 (c), 6 (d)
It should be mentioned that the formation of diketo
esters of similar structure has not previously5,9 been reꢀ
ported in the synthesis of different tetraketones.
10–10 mol L–1
.
The luminescence intensities and lifetimes of the comꢀ
plexes formed at the stoichiometric ratio ligand : ion =
1 : 500 (10–8 mol L–1 : 5•10–6 mol L–1) are given in
Table 2.
The chelating properties of the synthesized compounds
toward lanthanide ions were estimated by luminescenceꢀ
spectral methods. The longꢀwavelength absorption band
values and molar absorption coefficients of the complexes
of tetraketones 1a—d and diketo esters 2a—d with Eu3+
are presented in Table 1. An ion : ligand ratio of 20 : 1, at
which the formation of the 1 : 1 complex is most probꢀ
able, was used in the estimation of the luminescenceꢀ
spectral characteristics. The formation of similar comꢀ
plexes is also most probable for ligand conjugation with a
biological object labeled by this ligand.
As follows from the data presented, the spectral charꢀ
acteristics of the Eu3+ complexes with compounds 1a—d
and 2a—d are close to those for the Eu3+ complex with
NTA: their longꢀwavelength absorption maximum lies at
340—350 nm. The molar absorption coefficients for diketo
esters 2a—d are close to the value for NTA. The ε value
for tetraketones 1a—d is 1.5—2 times higher, and a tenꢀ
dency for increasing the molar absorption coefficients
with an increase in the number of difluoromethylene
groups is observed.
As follows from the data obtained, no explicit depenꢀ
dence is observed for the lifetime and luminescence inꢀ
tensity of the Eu3+ complexes of the synthesized comꢀ
pounds on the difluoromethylene chain length and the
number of C=O groups in the βꢀdiketonate fragments.
The highest luminescence intensity (154 rel. units) is
achieved for ligand 1d (six CF2 groups and four C=O
groups), while the τ parameter for this ligand is one of the
lowest (666 µs). Ligand 2a (one CF2 group and two C=O
groups) forms a complex with the lowest luminescence
intensity (44 rel. units) but higher τ value (724 µs). The
highest τ value (856 µs) belongs to the complex based
on ligand 1b (two CF2 groups and four C=O groups);
however, its luminescence intensity is insignificant
(63 rel. units).
The binding constants for the Eu3+ ion and comꢀ
plexones (Table 3) were determined from the luminesꢀ
cence characteristics of the complexes. The data obtained
show that the increase in the dentate character of the
complexones, which is achieved in molecules of tetraꢀ
When europium salt are added to aqueous solutions of
trioctylphosphine oxide (TOPO), which is a synergetic

278
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 2, February, 2006
Romanov et al.
Table 2. Parameters of prolong luminescence of the Eu3+ comꢀ
plexes of the synthesized compounds (fluorescence relative to
NTA (Frel) and luminescence lifetime (τ))*
served for diketo esters 2a—d. Compound 2d (six CF2
groups) forms complexes that are threefold more stable
than those with NTA. The elongation of the difluoroꢀ
methylene chain sterically facilitates, most likely, a more
favorable arrangement of the groups (most probably, the
diketonate moieties) involved in complex formation.
Note that none of the synthesized compounds forms
luminescent complexes with the Tb3+ ion.
Complexone
n
z**
Frel
τ/µs
(%)
NTA
1a
1
1
2
4
6
1
2
4
6
2
4
4
4
4
2
2
2
2
100 56
(6)
827 37
[0.9999]
644 30
[0.9999]
81
9
Diketo ester 2d was examined as a potential marker of
biological structures. In its 20ꢀfold mole excess in a conꢀ
jugating medium relatively to the labeled molecule of boꢀ
vine serum albumin (BSA), conjugates containing about
five molecules of marker 2d per one protein molecule
were obtained. The conjugate yield based on protein
was 71%. Conjugation occurs, most likely, due to acylaꢀ
tion of the amino groups of the protein by the highly
reactive methoxycarbonyl groups of compound 2d.
The ability of the complexone to form luminescent
complexes with Eu3+ in the BSAꢀbonded state was estiꢀ
mated in 0.05 М solutions of Tris buffer (pH 7.4) and after
TOPO (5•10–5 mol L–1) and Triton Xꢀ100 (0.1%) were
added to this solution. The results are given in Table 4.
As follows from the data obtained, in the absence of
the additional insulating shell, which is optimally created
by TOPO with Triton Xꢀ100, the luminescence of the
complex is quenched with water. However, the presence
of BSA and, the more so, conjugation with it favor insulaꢀ
tion, which is especially pronounced at high concentraꢀ
tions of the complex (for the luminescence intensities in
parentheses of the second column, see Table 4).
(3)
63 35
(6)
79 14
(4)
154 18
(2)
44 36
(6)
70 25
(6)
56 31
(3)
1b
856 9
[0.9998]
615 22
[0.9990]
1c
1d
666 1
[0.9999]
724 16
[0.9998]
724 40
[0.9998]
2a
2b
2c
760
[0.9999]
685
[0.9999]
5
2d
70
4
2
(2)
* The number of independent entries performed on different
days is given in parentheses, and the pair correlation coefficient
calculated using the leastꢀsquares method by approximation of
the obtained data according to the monoexponential law of luꢀ
minescence intensity decay is presented in brackets.
** Here and in Table 3, the number of the C=O groups in the
βꢀdiketonate moieties.
In all cases, the addition of TOPO increases the lumiꢀ
nescence of Eu3+ in the complex with ligand 2d, being the
lowest in the conjugated state. Evidently, the sites of ligand
binding in the conjugated and BSAꢀadsorbed states differ,
which appears as an insufficient increase in the luminesꢀ
cence in the presence of TOPO.
ketones 1a—d, affects dramatically the binding constants:
they are increased by four and more orders of magnitude.
Some tendency for increasing the binding constant with
an elongation of the difluoromethylene chain is also obꢀ
Table 3. Binding constants of the synthesized luminescent
complexones with the Eu3+ ion
Table 4. Luminescence intensity (I) of the 2d•Eu3+ complex in
the free and BSAꢀbonded states in different media*
Complexone
n
z
Binding constant
/L mol–1
Complex
2d•Eu3+
I (rel. units)
NTA
1a
1b
1c
1d
2a
2b
2c
2d
—
1
2
4
6
1
2
4
6
2
4
4
4
4
2
2
2
2
(1.7—1.9)•107
>1011
TB**
TB +
TB + TOPO +
+ TOPO
+ Triton Xꢀ100
>1011
>1011
Free
In the presence
of BSA
7 (70) 1070 (10500)
7 (170) 820 (14900)
1350
1090
>1011
(0.5—3.0)•107
(0.5—2.0)•107
(2.0—3.3)•107
(3.6—10.0)•107
In the conjugate
with BSA
7 (365) 250 (6550)
80
* [2d] = 10–8 (3.2•10–7) mol L–1, [Eu3+] = 5•10–6 mol L–1
[BSA] = 2•10–9 (6.3•10–8) mol L–1 (the I values in parentheses
correspond to the concentrations given in parentheses); λ
,
Note. The constants were determined in aqueous media with
TOPO, Triton Xꢀ100, and Tris buffer (pH 7.2) from the depenꢀ
dence of the luminescence intensity of the system on the conꢀ
centration at the ratios ligand : ion = 1 : 1 (for 1a—d) and
ligand : ion = 1 : (100—1000) and 1 : 1 (for 2a—d).
=
exc
340 nm, λ = 614 nm, td = 0.05 (0.1) ms, tg = 1 ms (see
em
Experimental).
** Tris buffer.

Fluorinated tetraketones
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 2, February, 2006
279
The addition of Triton Xꢀ100 to the free complexone
results in the further increase in the luminescence intenꢀ
sity (in the presence of BSA, this is less pronounced).
However, an additional (almost threefold) quenching of
luminescence is observed in the conjugated state. In this
case, the Eu3+ ions with TOPO are redistributed, most
likely, from the conjugated complexone to Triton Xꢀ100
micelles containing no ligands, due to which the lumineꢀ
scence intensity decreases additionally.
The luminescence lifetimes of the complex of 2d with
Eu3+ in the free, adsorbed, and BSAꢀconjugated states in
the presence of TOPO are almost the same, being 390,
394, and 400 µs, respectively, which agrees with the
τ values obtained for the complexones of this series in the
absence of Triton Xꢀ100.
The estimations for the newly synthesized diketo
esters 2a—d show that they have comparable characterꢀ
istics with fluorinated βꢀdiketones containing the chroꢀ
mophore substituents, which are widely used for lumiꢀ
nescence analysis. At the same time, these compounds
possess a basically novel property: they are efficiently
conjugated with protein. The complexing properties
of tetraketones 1a—d undergo jumpwise changes due to
an increase in the dentate character of these molecules,
which can find use in the practice of fluorescence immuꢀ
noassay.
Table 5. Physicochemical characteristics, yields, elemental analysis data, and 1H, 19F NMR and IR spectra of the synthesized
compounds
Comꢀ
poꢀ
B.p.
/°C
M.p. Yield Found
Molecular
formula
NMR, δ (J/Hz)
IR,
(%)
/°C
Calculated
ν/cm–1
1H
19F
und (p/Torr)
(%)
12
С
H
1a
1b
1c
1d
2a
—
—
—
—
171
72.95 4.31 C27H18F2O4 6.9 (s, 1 H, =CH);
39.8 (s, 2 F, CF2)
1581
(C=О)
72.97 4.05
7.6 (m, 2 H, H arom.);
7.9 (m, 4 H, H arom.);
8.6 (s, 1 H, H arom.)
180—190 28
(decomp.)
67.75 3.27 C28H18F4O4 6.8 (s, 1 H, =CH);
68.02 3.64
42.0 (s, 4 F, 2 CF2)
1610
(C=О)
7.6 (m, 2 H, H arom.);
7.9 (m, 4 H, H arom.);
8.5 (s, 1 H, H arom.)
150—154 11
136—138 14
60.28 3.67 C30H18F8O4 6.8 (s, 1 H, =CH);
60.60 3.30
44.6, 45.9
1661
(C=О)
7.6 (m, 2 H, H arom.);
8.0 (m, 4 H, H arom.);
8.5 (s, 1 H, H arom.)
(both m, 4 F each,
2 CF2)
55.35 2.73 C32H18F12O4 6.75 (s, 1 H, =CH);
41.5 (t, 4 F, 2 CF2,
J = 11.0); 43.1, 43.7
(both m, 4 F each,
4 CF2)
1605
(C=О)
55.33 2.59
7.60 (m, 2 H, H arom.);
8.00 (m, 4 H, H arom.);
8.55 (s, 1 H, H arom.)
174
(1)
68
—
45
—
17
22
20
10
62.60 3.78 C16H12F2O4 3.9 (s, 3 H, Me);
62.75 3.95
37.4 (s, 2 F, CF2)
1770
(C=О);
1600
6.8 (s, 1 H, =CH);
7.6 (m, 2 H, H arom.);
7.9 (m, 4 H, H arom.);
8.5 (s, 1 H, H arom.)
(C=О)
2b
2c
2d
180
(1)
56.90 3.30 C17H12F4O4 4.0 (s, 3 H, Me);
57.30 3.37
41.6, 42.2
1776
(C=О);
1605
6.8 (s, 1 H, =CH);
(both t, 2 F each,
2 CF2, J = 11.0)
7.6 (m, 2 H, H arom.);
7.9 (m, 4 H, H arom.);
8.5 (s, 1 H, H arom.)
(C=О)
160
(1)
50.15 2.96 C19H12F8O4 4.0 (s, 3 H, Me);
50.00 2.63
40.1, 41.7
1785
6.8 (s, 1 H, =CH);
(both t, 2 F each, 2 CF2, (C=O);
7.6 (m, 2 H, H arom.);
7.9 (m, 4 H, H arom.);
8.5 (s, 1 H, H arom.)
J = 11.0); 43.7, 42.2
(both m, 2 F each,
2 CF2)
1611
(C=O)
195
(1)
45.51 2.23 C21H12O4F12 4.00 (s, 3 H, Me);
40.0, 41.5 (both t,
2 F each, 2 CF2,
J = 10.5);
43.2, 43.4, 43.7, 44.5
(all m, 2 F each, 4 CF2)
1785
(C=O);
1630
45.32 2.16
6.75 (s, 1 H, =CH);
7.60 (m, 2 H, H arom.);
7.95 (m, 4 H, H arom.);
8.55 (s, 1 H, H arom.)
(C=O)

280
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 2, February, 2006
Romanov et al.
sodium phosphate, 0.005 М solution of EDTA sodium salt,
pH 8.0). After continuous stirring in the dark for 2.5 h, a mixture
of products was separated by gel filtration on a column packed
with Sephadex G25 (column height 10 cm, diameter 1 cm,
elution rate 16 mL h–1, 0.05 М solution of Tris buffer as eluent,
pH 7.5), collecting the fractions containing protein and diketo
ester 2d.
The label to protein ratio in the conjugate was determined
from the spectrophotometric characteristics of the conjugate
assuming that the absorption of protein and diketo ester 2d in
the free and conjugated states remains unchanged. The followꢀ
ing ε values were used in the calculations: for BSA, 3.3•104 and
0 L mol–1 cm–1; for 2d, 7.2•103 and 1.36•104 L mol–1 cm–1 at
absorption wavelengths of 278 and 333 nm, respectively.
Experimental
1
H NMR spectra were recorded on a Bruker ACꢀ300 specꢀ
trometer (300.13 MHz). 19F NMR spectra were measured on a
Bruker WPꢀ200 SY spectrometer (188.31 MHz). Chemical
shifts (δ) are presented in ppm relative to external standards:
Me4Si (1H) and CF3COOH (19F). IR spectra of substances in
thin layer were obtained on a URꢀ20 spectrometer. The physiꢀ
cochemical characteristics, yields of substances, elemental analyꢀ
sis data, and 1H, 19F NMR and IR spectra are given in Table 5.
The luminescenceꢀspectral properties of compounds 1a—d
and 2a—d were estimated in 10–10—10–5 М solutions of the
complexones in an aqueous medium containing TOPO (5•10–5
mol L–1) and Triton Xꢀ100 (0.1%) in the presence of Eu3+
((1—5)•10–5—(1—5)•10–6 mol L–1) (the ligand to ion ratio at
which the 1 : 1 complex can be formed). Absorption spectra
were recorded on a Perkin—Elmer М 555 spectrophotometer.
Phosphorescence spectra with a time delay (td) of 0.05—0.10 ms
relatively to the exciting pulse and a gate time (tg) of 1.00 ms
were obtained on an LSꢀ5B spectrofluorimeter (Perkin—Elmer).
Commercial reagents and solvents, which were prepared acꢀ
cording to known recommendations,10 were used. Sodium
methoxide was dried at 130 °C and 1 Torr prior to use.
References
1. K. I. Pashkevich, V. I. Saloutin, and I. L. Postovskii, Usp.
Khim., 1981, 50, 325 [Russ. Chem. Rev., 1981, 50 (Engl.
Transl.)].
2. N. Ishikawa and Y. Kobayashi, Fluorine Compounds — Chemꢀ
istry and Application, Kodansha Scientific, Tokyo, 1979.
3. I. Hemmila, Anal. Biochem., 1984, 137, 335.
4. R. P. Ekins, International Application WO 86/01604, 1986;
Chem. Abstrs, 1986, 105, P57494e.
5. J. Yuan and K. Matsumoto, J. Pharm. Biomed. Anal., 1997,
15, 1397.
6. S. Sueda, J. Yuan, and K. Matsumoto, Bioconjug. Chem.,
2000, 11, 827.
7. A. V. Chudinov, Novosti nauki i tekhniki. Seriya Meditsina.
Allergiya, astma, klinicheskaya immunologiya [Scientific and
Technical News. Series Medicine. Allergy, Asthma, Clinical
Immunology], 2001, 1, 143 (in Russian).
8. F. B. Wu and C. Zhang, Anal. Biochem., 2002, 311, 57.
9. D. L. Chizhov, V. G. Ratner, M. I. Kodes, and K. I.
Pashkevich, Zh. Org. Khim., 1988, 34, 371 [J. Org. Chem.
USSR, 1988, 34 (Engl. Transl.)].
Reaction of 2ꢀacetylnaphthalene with dimethyl perfluoroꢀ
alkanedicarboxylates (general procedure). The corresponding diꢀ
ester of perfluoroalkanedicarboxylic acid (30 mmol) was added
with stirring for 15 min to a suspension of sodium methoxide
(30 mmol) and ether (30 mL). After stirring for 30 min,
2ꢀacetylnaphthalene (30 mmol) and ether (30 mL) were added.
The reaction mixture was refluxed for 3—4 h with stirring
and then cooled to 0—5 °C, and a solution of sulfuric acid
(100 mmoles in 30 mL of water) was added for 10 min. The preꢀ
cipitate that formed was filtered off, washed with ether (3×15 mL)
and water (3×15 mL), and dried. Tetraketones 1a—d were obꢀ
tained by recrystallization from heptane, which is difficult.
Therefore, pure samples of tetraketones 1a—d can be obtained
already after washing of the precipitate with hot heptane. After
tetraketones 1a—d were separated, the ethereal solution was
washed with water (3×10 mL) and dried with sodium sulfate.
Diketo esters 2a—d were obtained by fractionation in vacuo.
Conjugation of BSA with diketo ester 2d and estimation of
10. A. J. Gordon and R. A. Ford, The Chemist´s Companion,
WileyꢀIntersci. Publ., New York—London—Sydney—
Toronto, 1972.
labeling. A solution of diketo ester 2d (56.6 µL, 3•10–7 mol L–1
)
in ethanol was added to 0.5 mL of a solution containing BSA
(1.5•10–8 mol L–1) in a conjugating buffer (0.05 М solution of
Received June 14, 2005;
in revised form November 23, 2005