Radioactivity-synchronized fluorescence enhancement using a radionuclide fluorescence-quenched dye

Article abstract of DOI:10.1021/ja903685b

(Chemical Equation Presented) We demonstrate the first evidence of radioactivity-synchronized fluorescence quenching of a near-infrared light-emitting dye by a radionuclide, ⁶⁴Cu, and subsequent fluorescence enhancement upon ⁶⁴Cu decay to the daughter isotopes ⁶⁴Ni and ⁶⁴Zn. The dynamic switch from high radioactivity and low fluorescence to low radioactivity and high fluorescence is potentially useful for developing complementary multimodal imaging and detection platforms for chemical, environmental, and biomedical applications as well as for unraveling the mechanisms of metal-induced dynamic fluorescence changes.

Full text of DOI:10.1021/ja903685b

Published on Web 06/10/2009
Radioactivity-Synchronized Fluorescence Enhancement Using a Radionuclide
Fluorescence-Quenched Dye
†
†
‡
†
†
Mikhail Y. Berezin, Kevin Guo, Bao Teng, W. Barry Edwards, Carolyn J. Anderson,
‡
§
‡
,†,|
Olga Vasalatiy, Amir Gandjbakhche, Gary L. Griffiths, and Samuel Achilefu*
Department of Radiology, Washington UniVersity School of Medicine, 4525 Scott AVenue,
St. Louis, Missouri 63110, Imaging Probe DeVelopment Center, National Institutes of Health, NHLBI, 9000 Medical
Center DriVe, Bethesda, Maryland 20892, Physical Biology Program, National Institutes of Health, Eunice ShriVer
NICHD, 9000 Medical Center DriVe, Bethesda, Maryland 20892, and Department of Biochemistry and Molecular
Biophysics, Washington UniVersity School of Medicine, 660 South Euclid AVenue, St. Louis, Missouri 63110
Received May 6, 2009; E-mail: achilefus@mir.wustl.edu
Metal-sensitive fluorescent probes have been shown to accurately
and rapidly report metal complexation through a change in the
5-nitro-2,3,3-trimethylindolenine to provide indole analogue 2 in
60% yield. Reduction of the nitro group followed by N-alkylation
using N,N-bis[(tert-butyloxycarbonyl) methyl]-2-bromoethylamine
(1) afforded indole derivative 4 in an overall yield of 65%.
Treatment of 1,2,3,3-tetramethylindolinium iodide with glutacon-
dialdehyde dianil, acetyl chloride, and acetic anhydride produced
hemicyanine intermediate 5 in 87% yield. Reaction of 5 with 4
under reflux in anhydrous ethanol with anhydrous sodium acetate
afforded Boc-protected intermediate 6, which was isolated in good
yield (74.5%). Hydrolysis of 6 to afford LS479 was accomplished
with trifluoroacetic acid, and the desired product was purified by
preparative HPLC.
1-3
fluorophore’s spectral properties.
Previous studies have focused
on quasistatic metal-fluorophore complexes, where a single metal
of interest complexes with the fluorophore, altering its emission in
some discernible fashion. The resultant emission could be further
modified by changing the ligands attached to the metal or altering
the metal oxidation states. Neither experimental nor theoretical
studies have been conducted regarding dynamic metal-fluorophore
complexes in which the nature of the bound metal itself is variable.
Such changes, for example, may transform a nonfluorescent
complex into a fluorescent system if a quencher metal could convert
into a nonquencher metal. The difficulty in achieving this trans-
formation arises from the high stability of metals. However, a select
group of metals, namely, radionuclides, are ideally suited for this
application because spontaneous radioactive decay of an unstable
parent metal could transform it to a different, more stable metal.
Herein we propose the first molecular system that demonstrates
the feasibility of altering the fluorescence properties of an organic
dye as a function of radionuclide decay. Previous studies have
Scheme 1. Synthesis of LS479 and Structure of HITC
shown that 64Cu decays to Zn and Ni through two pathways, as
64
64
shown in the following equation:
ꢀ-
+ 3 Zn r Cu f Ni + ꢀ
64
0
64
29
64
28
+
3
9%
61%
2
+
We found a clear differentiation in fluorescence between Cu
2+
2+
and its decay products, with Cu acting as a strong quencher, Zn
as an enhancer, and Ni2 having no net effect on the dye
fluorescence. Correlation of the radionuclide decay with the dye
fluorescence enhancement validates the hypothesized metal effect
and suggests a realm of possible applications for complementing
fluorescence with radioactivity.
+
We used absorption studies to establish that metal complexation
6
4
2+
would not be lost upon radioactive decay of Cu and that the
parent and daughter nuclides remain bound to LS479. A solution
of HITC or LS479 in water was titrated with Cu , Ni , and Zn
To study the influence of intermetal conversion through radioac-
tive decay on the fluorescence properties of organic dyes, we
prepared LS479, a novel near-infrared fluorescent dye with a
covalently linked metal-chelating group (Scheme 1). LS479 is
structurally similar to the known dye 1,1′,3,3,3′,3′-hexamethylin-
dotricarbocyanine (HITC), which is widely used in analytical
chemistry and biological optical-imaging studies. The chelating
group is derived from diethylenetriaminepentaacetic acid (DTPA).
The synthesis of LS479 (Scheme 1) involved the methylation of
2
+
2+
2+
chlorides, and the absorption and emission spectra were recorded.
HITC, lacking the chelating group, did not show any isosbestic
points, and the shape of the absorption spectra upon metal titration
remained the same (Figures S1-S3 in the Supporting Information),
suggesting the absence of metal chelation to the dye molecule.
Similarly, we did not observe significant changes in the fluorescence
of HITC upon addition of metal salts (Figure S4). In contrast,
transformations in the absorption spectra of LS479 (Figures S5-S7)
showed that all of the metals remained bound to the dye. In all
4
†
Department of Radiology, Washington University School of Medicine.
‡
National Institutes of Health, NHLBI.
§
National Institutes of Health, Eunice Shriver NICHD.
Department of Biochemistry and Molecular Biophysics, Washington University
2+
|
cases, clear isosbestic points were observed, especially for Zn
and Ni .
2
+
School of Medicine.
9
198 9 J. AM. CHEM. SOC. 2009, 131, 9198–9200
10.1021/ja903685b CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. (left) Titration emission spectra of LS479 with (top) a [61%
nat
2+
nat
2+
64
2+
Ni + 39% Zn ] mixture and (bottom) decayed Cu (λex ) 675
64
nm). (right) Fluorescence intensity at 760 nm as a function of decayed Cu
addition, where the saturation point apparently corresponds to a 1:1 metal/
dye ratio. Addition of either solution to LS479 resulted in marked
fluorescence enhancement.
64Cu2+ to a solution of LS479 rapidly quenched the dye fluorescence
with a concomitant increase in fluorescence over time (Figure
3
).There were no changes in the absorption spectra of LS479, which
Figure 1. (left) Titration emission spectra of LS479 with (top to bottom)
CuCl , NiCl , and ZnCl in water (excitation at 675 nm). Arrows indicate
indicates that the observed fluorescence increases were not dimin-
ished or altered by radiolysis. The resultant fluorescence enhance-
2
2
2
2
+
the direction of increasing metal concentration. Cu was shown to quench
the fluorescence, Zn2 enhanced it, and Ni had no net effect. (right)
Fluorescence intensities at the LS479 emission maximum (760 nm) as a
function of metal/dye ratio.
+
2+
64
2+
64
2+
64
2+
ment reflected the transformation of Cu into Ni and Zn .
As Cu further decayed to Ni and 64Zn , the quenching effect
decreased and the fluorescence increased to 130% relative to the
emission of unquenched LS479 for a net enhancement of fluores-
6
4
64
2+
2+
Analysis of the fluorescence spectra showed that the three metals
displayed different effects on the emission from LS479 (Figure 1).
6
4
64
2+
cence. After 72 h, nearly all of the Cu decayed into stable Ni
and Zn (2% of initial activity remaining). Consequently, there
were no changes in fluorescence intensity thereafter.
6
4
2+
2+
Cu had a strong quenching ability, achieving maximum quenching
at a 1:1 molar ratio to the dye. The fluorescence intensity of the
2
+
2+
Fluorescence quenching by Cu is a general phenomenon with
Ni -LS479 complex remained the same as that of LS479 at all
6
2
2+
few exceptions, spanning the range from organic molecules to
quantum dots and fluorescent proteins. However, the mechanism
of fluorescence quenching still remains obscure. Quenching has
been attributed to a spheres-of-action static-quenching mechanism,
the paramagnetic nature of the metal, a heavy-atom effect, energy
transfer from the dye (donor) to Cu (acceptor), and charge
transfer from π* to d orbitals. We ruled out the first mechanism,
spheres-of-action static quenching, because it considers a certain
molar ratios examined. Furthermore, Zn significantly enhanced
7
8
2+
the fluorescence of LS479: at a 1:1 Zn /LS479 molar ratio, the
3
2+
emission of the Zn -LS479 complex was ∼5 times higher than
that of LS479. The metal effects on fluorescence and absorption
of LS479 were concentration-dependent. The metal effects on the
dye fluorescence were mostly linear until a metal/dye ratio of ∼1:1
was established. Obviously, at this point, all the dye molecules were
chelated to the metal, and no free dye existed in solution. Further
addition of metal resulted in minimal alteration of the spectra.
8
9
10
2
+
11
1
2
1
3
probability of collision quenching, which is not the case for the
2
+
2+
Cu -LS479 complex, where the probability reaches 100% with
maximum quenching occurring at 1:1 metal/dye ratio. Paramagnet-
ism is also an inadequate explanation because another paramagnetic
complex, octahedral Ni , did not exhibit any quenching. A heavy-
atom effect was discounted on the grounds that the heaviest ion,
Having demonstrated that Cu was a fluorescence quencher of
LS479, Ni2 did not have an effect, and Zn significantly enhanced
+
2+
the fluorescence, we hypothesized that a solution of completely
2+
14
decayed 64CuCl
would possess similar effects on the fluorescence
2
2
+
2+
spectra of LS479 as a result of the daughter nuclides Ni and Zn .
2
+
64
Zn , enhanced the fluorescence of LS479 instead of quenching it.
Since the half-life of Cu is 12.7 h, we used a completely decayed
solution of 64CuCl
in water (>12 half-lives), which ensured that
the solution contained 61% Ni and 39% Zn in addition to
any other incipient metals. The remaining amount of Cu was
virtually zero. As expected, the emission spectra were drastically
different from the “cold” Cu2 titration spectra (Figure 2). The
2
To delineate the contribution of metal chelation to the observed
fluorescence properties of LS479 metal chelates, we prepared and
compared the absorption spectra of Cu -, Ni -, and
Zn -DTPA complexes and the emission spectra of free LS479
(Figure 4). The result showed a large spectral overlap between the
64
2+
64
2+
5
64
2+
2+
2+
2
+
+
64
2+
2+
spectral changes induced by decayed Cu on LS479 were similar
to those caused by a model daughter-isotope mixture of Cu
dye and the Cu -DTPA complex and insignificant overlap for
the Ni and Zn complexes, thus supporting the conclusion that
6
4
2+
2+
2+
decay products (Ni/Zn ratio ≈ 1.5) (see Figures S8 and S9 for the
an energy-transfer mechanism is responsible for the strong quench-
2
+
2+
2+
absorption spectra).
ing by Cu and the neutrality by Ni and Zn . Similarly,
quenching could be explained by excited-state charge transfer,
where the excited electron travels from the π* orbital of the dye to
The above results using “cold” metal models suggest that “hot”
Cu would quench the fluorescence of LS479. Indeed, addition of
J. AM. CHEM. SOC. 9 VOL. 131, NO. 26, 2009 9199

C O M M U N I C A T I O N S
2
+
a d orbital of similar energy on Cu , where the absorbed energy
nonradiatively dissipates. The higher energy of the d orbitals in
supports our model of fluorescence enhancement synchronized with
radioactive decay. This new method of monitoring fluorescence
enhancement through the decay of radionuclides has a variety of
potential applications. For example, it may open new possibilities
for studying fundamental molecular transformations, such as
determining how quickly a molecule readjusts from one state to
another after radioactive decay. Furthermore, combining radionu-
clide decay with fluorescence enhancement could provide a unique
complementary signaling mechanism for radionuclear-optical
multimodal biological imaging. In fact, the new radiolabeled LS479
represents a paradigm-shifting strategy in multimodal molecular
2+
Ni compared with the π* orbitals precludes this transfer, allowing
the fluorescence of LS479 to occur naturally via a π* f π
transition. Indeed, the absorbance maximum in the visible spectra
2+
of Cu -DTPA was 740 nm, while that for Ni-DTPA was at a
shorter wavelength, 610 nm. A similar mechanism of quenching
might occur in porphyrin, which in its free form emits at ∼600 nm
but for which both Cu2 and Ni cause fluorescence quenching.
+
2+
15
6
4
2+
imaging because Cu and near-infrared fluorescent dyes such as
1
7
LS479 are routinely used in positron emission tomography and
1
8
optical imaging of living organisms, respectively. Moreover, the
strong correlation between fluorescence quenching and the presence
6
4
2+
of Cu chelation provides a dynamic method for studying the in
vivo stability of the radionuclide chelates, where fluorescence
enhancement reports demetalation of the radionuclide. Finally, the
new molecular construct and mechanism could be used to develop
visible radioactivity decay devices for environmental and nuclear
forensic applications. Future work will focus on these potential
applications.
Figure 3. Percent changes in 64Cu2+ concentration as a result of decay
calculated from exponential decay) and in relative fluorescence of Cu-
LS479 solution as a function of time. Unquenched LS479 (0.5 µM)
corresponded to a hypothetical zero-time point for fluorescence. Relative
fluorescence was measured as an area under the emission curve after addition
(
Acknowledgment. This study was support in part by the NIH
extramural grants R01 CA109754, R33 CA123537, R33 CA100972,
U54 CA136398, and U54 CA119342, the NIH Roadmap for
Medical Research as the funding source for the IPDC, and the
Intramural Research Program of Eunice Shriver NICHD.
64
of Cu solution divided by the area before the addition (λex ) 675 nm).
An overall increase in fluorescence intensity was seen as a function of time.
Supporting Information Available: Synthesis, experimental pro-
cedures, calculations, and optical spectra. This material is available
free of charge via the Internet at http://pubs.acs.org.
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Figure 4. Normalized emission of LS479 (λex ) 690 nm) and absorption
2+
2+
2+
spectra of Cu -DTPA, and Ni -DTPA and Zn -DTPA (the absorption
maximum for the Zn2 -DTPA complex was <450 nm). Substantial overlap
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plexation with Zn suggests that the lack of a vacant d orbital
precludes ligand-to-metal charge transfer and the absence of
absorption bands prevents energy transfer. A distinguishing char-
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typically act as highly efficient fluorescence quenchers via an
2
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6
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excited-state charge transfer (ESCT) mechanism. Indeed, the
quantum yield of LS479 (Φ ) 0.018) in unbuffered water was ∼6
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5
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The addition of 64CuCl
The net fluorescence enhancement upon Cu decay strongly
also resulted in fluorescence quenching.
2
64
2+
JA903685B
9
200 J. AM. CHEM. SOC. 9 VOL. 131, NO. 26, 2009