Novel rhenium(I) polypyridine biotin complexes that show luminescence enhancement and lifetime elongation upon binding to avidin

Article abstract of DOI:10.1021/ja026598n

While most biotin-fluorophore conjugates suffer from significant emission quenching upon binding to avidin due to resonance energy-transfer, three novel rhenium(I) polypyridine biotin complexes have been designed in view of their characteristic photophysical properties, in particular their large Stokes shifts. In contrast to most biotin-fluorophore conjugates, the ³MLCT emission intensities and lifetimes of these rhenium(I) complexes are increased upon binding to avidin, rendering them luminescent probes for avidin and biotinylated species. Copyright

Full text of DOI:10.1021/ja026598n

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Novel Rhenium(I) Polypyridine Biotin Complexes That Show Luminescence
Enhancement and Lifetime Elongation upon Binding to Avidin
Kenneth Kam-Wing Lo,* Wai-Ki Hui, and Dominic Chun-Ming Ng
Department of Biology and Chemistry, City UniVersity of Hong Kong, Tat Chee AVenue, Kowloon,
Hong Kong, P. R. China
Received April 19, 2002
Chart 1. Structures of Complexes 1-3
Since the noncovalent binding mode between avidin and biotin
is the strongest biological interaction between a protein and a ligand,
it plays an extremely important role in biochemistry, immunology,
and bioanalytical chemistry.1 Biotinylated biomolecules such as
DNA, peptides, and proteins can be recognized by avidin conjugated
with a marker such as a fluorescent label. Although a similar
recognition and detection procedure can be carried out using avidin
as the bridge and biotin-fluorophore conjugates as the reporter,
the use of fluorescent biotin reagents is much less common. The
major reason is that most conventional biotin-fluorophore conju-
gates, such as those of fluorescein, pyrene, tetramethylrhodamine,
and Cy dyes, lose their fluorescence when they are bound to avidin,
unless there are exceptionally long spacers between the biotin and
the fluorophore units.2 We anticipate that this problem can be solved
using luminescent biotin-transition metal complex conjugates in
view of their characteristic photophysical properties, in particular,
the large Stokes shifts. In addition, the tunability of the emission
energy and relatively long emission lifetimes of luminescent
transition metal complexes with a charge-transfer excited state are
also attractive properties for the purpose of a recognition assay.
While there have been reports on the conjugation of the biotin
moiety to different transition metal complexes,3 the use of biotin-
transition metal complex conjugates as luminescent probes has not
been explored.
We report here that the metal-to-ligand charge-transfer (MLCT)
emission of rhenium(I) polypyridines can be exploited to provide
a new generation of luminescent biotin derivatives with emission
properties remarkably different from those of traditional biotin-
fluorophores. The complexes [Re(N-N)(CO)3(py-CH2-NH-biotin)]-
(PF6) [N-N ) 1,10-phenanthroline (1); 3,4,7,8-tetramethyl-1,10-
phenanthroline (2); 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline
(3); py-CH2-NH-biotin ) N-[(4-pyridyl)methyl]biotinamide] were
prepared, in moderate yields, from the reactions of [Re(N-N)(CO)3-
(CH3CN)](CF3SO3) with py-CH2-NH-biotin in THF under refluxing
conditions, followed by metathesis with KPF6 and purification by
column chromatography (alumina).4 The structures of the complexes
are shown in Chart 1.
Table 1. Photophysical Data for Complexes 1-3 at 298 K
λem
/
τo/
µsa
I
I
I
a
complex medium nm
Φa
(τ/µs)b,c
(τ/µs)b,d
(τ/µs)b,e
1
2
3
CH3CN 552 1.37 0.079 1.00 (0.56) 1.42 (0.90) 0.98 (0.55)
CH2Cl2 536 2.69
CH3CN 518 7.32 0.072 1.00 (1.23) 2.25 (2.96) 1.04 (1.25)
CH2Cl2 510 7.81
CH3CN 550 6.78 0.079 1.00 (1.84) 2.98 (2.70) 0.96 (1.90)
CH2Cl2 540 7.22
a In degassed solvents. b Relative emission intensities in aerated 50 mM
potassium phosphate buffer pH 7.4. c [Re] ) 15.2 µM, [avidin] ) 0 µM,
[unmodified biotin] ) 0 µM. d [Re] ) 15.2 µM, [avidin] ) 3.8 µM,
[unmodified biotin] ) 0 µM. e [Re] ) 15.2 µM, [avidin] ) 3.8 µM,
[unmodified biotin] ) 380.0 µM.
cence titrations using the rhenium(I) complexes as titrants show
that complex 1 displays a higher emission intensity in the presence
of avidin. At [1]:[avidin] ) 4:1, the emission intensity is ca. 1.4
times that of the complex in the absence of avidin (Table 1). The
increase in emission intensity is ascribed to the binding of the
complex into the biotin-binding sites of avidin because no increase
in emission intensity is observed when excess unmodified biotin
is present (Table 1). These observations are of great interest because
most biotin-fluorophore conjugates suffer from severe emission
quenching upon binding to avidin due to resonance-energy transfer
(RET), unless exceptionally long spacers such as poly(ethylene
glycol) are present between the fluorophore and the biotin units.2
This provides sufficient separation between the fluorophores bound
to the same avidin molecule to eliminate this effect. It is also well-
known that biomolecules labeled with organic fluorophores do not
become more highly fluorescent with higher degrees of labeling.7
The absence of emission quenching in the present case is due to
the insignificant overlap between the absorption and emission
spectra of 1, which renders quenching by RET unfavorable. The
enhancement in emission intensity is in line with the observations
that when luminescent transition metal complexes are conjugated
to protein molecules, the emission is enhanced and oxygen-
quenching becomes much less effective.8 Interestingly, more
Complexes 1-3 exhibit intense and long-lived greenish-yellow
to yellow luminescence upon photoexcitation. The photophysical
data are summarized in Table 1. The emission energy, band shapes,
lifetimes, and luminescence quantum yields resemble those of
typical [Re(N-N)(CO)3(py)]+ systems, and the emission is assigned
3
to originate from an MLCT [dπ(Re) f π*(N-N)] excited state.5
The binding of the complexes to avidin has been investigated
using the standard HABA (4′-hydroxyazobenzene-2-carboxylic
acid) assay.6 The results reveal that 1-3 bind to the avidin molecule
with the same stoichiometry as unmodified biotin (4:1). Lumines-
* To whom correspondence should be addressed. E-mail: bhkenlo@cityu.edu.hk.
9
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J. AM. CHEM. SOC. 2002, 124, 9344-9345
10.1021/ja026598n CCC: $22.00 © 2002 American Chemical Society

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C O M M U N I C A T I O N S
maximum (560 nm) overlaps significantly with the emission
maximum of 3 (556 nm) in phosphate buffer. It appears that RET
plays an important role in this emission quenching since the lifetime
decreases from 6.02 to 4.90 µs and a similar decrease in the
emission lifetime is not observed when unmodified biotin (300.0
µM) is initially present (τ ) 5.94 µs). On the basis of the spectral
data of the donor and acceptor, a Fo¨rster distance of 44.5 Å is
estimated.
In conclusion, these novel luminescent rhenium(I) polypyridine
biotin complexes offer remarkable advantages over traditional
biotin-fluorophores as probes for avidin and can be utilized in
homogeneous assays for biotin and biotinylated biomolecules.
Related studies of other luminescent biotin-transition metal
complex conjugates are in progress.
Acknowledgment. This work was fully supported by a Direct
Allocation Grant from the City University of Hong Kong (Project
No. 7100212). W.K.H. acknowledges the receipt of a postgraduate
studentship and a Research Tuition Scholarship, both administered
by the City University of Hong Kong. We sincerely thank Professor
Vivian W. W. Yam of The University of Hong Kong for access to
the equipment for photophysical measurements and for her helpful
discussion.
Figure 1. Luminescence titration curves for the titrations of (i) 3.8 µM
avidin (b), (ii) 3.8 µM avidin and 380.0 µM unmodified biotin (2), and
(iii) a blank phosphate buffer solution (0) with complex 3.
significant luminescence enhancement has been observed for 2 and
3. At [Re]:[avidin] ) 4:1, the emission intensities of 2 and 3 are
increased by factors of ca. 2.3 and 3.0, respectively (Table 1).
Emission titration curves for 3 are shown in Figure 1.4 The emission
lifetimes have also been found to increase by ca. 1.5-2.4-fold when
1-3 are bound to avidin (Table 1). Since the lifetimes of the excited
complexes are sensitive to the hydrophobicity of the environment,
evidenced by the fact that the lifetimes of all three complexes
increase from CH3CN to CH2Cl2 (Table 1), it is conceivable that
the enhancement in emission intensities and lifetimes result from
the hydrophobicity associated with the binding pockets of avidin.
This is in agreement with the finding that 2 and 3, being more
hydrophobic than 1, exhibit more significant increases in emission
intensities and lifetimes after binding to avidin. We, however, notice
that at [Re]:[avidin] > 4:1, the titration curves are not exactly
parallel to (i) those of the solutions without avidin and (ii) those
with avidin in the presence of excess unmodified biotin. Therefore,
the possibility of nonspecific interactions between the free com-
plexes and bound complexes or rhenium-bound avidin cannot be
excluded.
Supporting Information Available: Synthetic procedures and
characterization data for the ligand py-CH2-NH-biotin and complexes
1-3, electronic absorption spectral data for 1-3, luminescence titration
curves for 1 and 2, details of competitive association and dissociation
assays for the interactions between complexes 1-3 and avidin, and
procedure for the conjugation of QSY-7 isothiocyanate to avidin (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Wilchek, M.; Bayer, E. A. Methods of Enzymology; Academic Press: San
Diego, 1990; Vol. 184.
(2) (a) Gruber, H. J.; Marek, M.; Schindler, H.; Kaiser, K. Bioconjugate Chem.
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1997, 8, 560. (c) Gruber, H. J.; Hahn, C. D.; Kada, G.; Riener, C. K.;
Harms, G. S.; Ahrer, W.; Dax, T. G.; Knaus, H.-G. Bioconjugate Chem.
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(3) See, for example, (a) Anne, A. Tetrahedron Lett. 1998, 39, 561. (b) Wilbur,
D. S.; Chyan, M. K.; Pathare, P. M.; Hamlin, D. K.; Frownfelter, M. B.;
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Organometallics 2000, 19, 1789.
(4) Details are included in the Supporting Information.
(5) Kalyanasundaram, K. Photochemistry of Polypyridine and Porphyrin
Complexes; Academic Press: San Diego, 1992.
The binding properties of 1-3 to avidin with respect to
unmodified biotin have been studied by competitive association
and dissociation assays.9 The assays show that ca. 12-23% avidin
molecules remain bound by the complexes in the presence of biotin.4
Dissociation constants (Kd) of ca. 3.2 × 10-10, 5.2 × 10-11, and
4.0 × 10-9 mol dm-3 have been determined for the avidin
complexes of 1-3, respectively, from luminescence titration
experiments.10
(6) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego,
1996; p 591.
(7) Hemmila¨, I. A. Applications of Fluorescence in Immunoassays; John Wiley
& Sons: New York, 1991; p 113.
In view of the long emission lifetimes of these rhenium(I)
polypyridine biotin complexes, we have investigated the possibility
of their utilization as probes for biotin and biotinylated species based
on the RET principle.11 As an example, a degassed buffer solution
of 3 (12.0 µM) exhibits emission quenching (I/Io ) 0.37) in the
presence of avidin (6.0 µM) that has been modified with the
nonfluorescent energy-acceptor dye QSY-7 isothiocyanate (Mo-
lecular Probes).4 The dye QSY-7 is chosen because its absorption
(8) (a) Lo, K. K. W.; Ng, D. C. M.; Chung, C. K. Organometallics 2001, 20,
4999. (b) Lo, K. K. W.; Hui, W. K.; Ng, D. C. M. Inorg. Chem. 2002,
41, 40 and references therein.
(9) Wilbur, D. S.; Pathare, P. M.; Hamlin, D. K.; Frownfelter, M. B.; Kegley,
B. B.; Leung, W. Y.; Gee, K. R. Bioconjugate Chem. 2000, 11, 584.
(10) Wang, Z. X.; Kumar, N. R.; Srivastava, D. K. Anal. Biochem. 1992, 206,
376.
(11) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer
Academic and Plenum Publishers: New York, 1999; p 367.
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