Polyvalent carbocyanine molecular beacons for molecular recognitions

Article abstract of DOI:10.1021/ja049441z

Polyvalent carboxylate-terminating near-infrared (NIR) carbocyanine molecular beacons were prepared by homologation of reactive carboxyl groups of the beacon with imino diacetic acid. Their conjugation with unprotected d-(+)-glucosamine gave dendritic arrays of the carbohydrate on an inner NIR chromophore core. In vivo evaluation of the dendritic glucosamine constructs shows enhanced uptake in proliferating tumor cells relative to surrounding normal tissue. The structural framework of these polyvalent beacons permits the amplification by synergistic effects of a variety of bioactive motifs or chemical sensors in molecular recognition interactions without dramatic change of their desirable NIR spectral properties. Copyright

Full text of DOI:10.1021/ja049441z

Published on Web 06/02/2004
Polyvalent Carbocyanine Molecular Beacons for Molecular Recognitions
Yunpeng Ye, Sharon Bloch, and Samuel Achilefu*
Department of Radiology, Washington UniVersity School of Medicine, St. Louis, Missouri 63110
Received January 30, 2004; E-mail: achilefus@wustl.edu
Scheme 1. Synthesis of Polycarboxylate-Containing
Synthetic dendrimers and other polyvalent compounds use
synergistic multivalent interactions to amplify desired chemical or
biological molecular recognitions.1,2 Modification of their peripheral
functional groups with fluorescent antennas provides a highly
sensitive approach to track, visualize, and quantify different
molecular interactions by optical methods. Whereas numerous
fluorescent molecular beacons are conventionally used for these
studies, background interference in heterogeneous media such as
cells and tissues can be minimized by using probes that absorb
light in the near-infrared (NIR) wavelengths between 700 and 900
nm.3,4 Unfortunately, conjugating such multiple hydrophobic bea-
cons at the outer sphere of polyvalent compounds may quench
fluorescence, induce aggregation, disrupt bioactive conformations
of adjacent small molecules, and destabilize the chromophore
systems because of their exposure to harsh and metabolically active
media. Therefore, an alternative strategy to overcome these limita-
tions is to construct polyvalency on a fluorescent inner core.
Currently, only nanoparticles and quantum dots provide inner
core NIR chromophore system with outer core functional groups.5-7
However, the biological applications of these particulates are limited
by their potential to elicit immunogenic responses in vivo. Ad-
ditionally, the constituents of quantum dots can be cytotoxic. Herein,
we report the first polyvalent NIR carbocyanine-based molecular
beacons, in which the carbocyanine moiety serves as not only the
chromophore but also an inner core for constructing novel dendritic
arrays of small molecules for molecular recognition studies.
On the basis of a recent report describing a convenient method
to synthesize dicarboxylic acid-containing carbocyanine dye (cypate,
1) from carboxyethyl benzoindole,8 we used 1 as the central core
for the polyvalent constructs. Thus, reaction of 1 with di-tert-butyl
imino diacetate in the presence of EDCI/HOBt, followed by TFA-
mediated deprotection, gave two second-generation polyvalent
beacons (tri (2) and tetra (3) carboxylate-containing derivatives).
Further reactions of 2 and 3 with di-tert-butyl imino diacetate in
the presence of EDCI/HOBt and subsequent TFA-mediated depro-
tection afforded two third-generation derivatives (hexa (4) and octa
(5) carboxylate-containing derivatives; Scheme 1). The compounds
were purified and obtained in moderate to good yields as follows:
2 (33%), 3 (60%), 4 (30%), and 5 (30%).
Carbocyanine Fluorescent Probesa
a
Reagents and conditions: (a) EDCI/HOBT/di-tert-butyl iminodiacetate/
rt/5h (b) TFA.
Chart 1. Structures of Representative Dendritic Arrays of
Glucosamine on an Inner NIR Carbocyanine Core
To explore the potential of using the polyvalent molecular beacon
for molecular recognition studies, we constructed a small library
of dendritic arrays of glucosamine because synergistic effects of
glycosidic clustering play important roles in carbohydrate-mediated
interactions.9 Accordingly, reaction of unprotected D-(+)-glucos-
amine with 1-5 in the presence of HBTU/HOBT/DIEA afforded a
series of compounds including 6-11 in moderate yields (Chart 1).
The absorption and emission spectra of all the compounds
evaluated are similar, with their maxima centered at about 780 and
810 nm, respectively. Expectedly, the spectral properties of the
dendritic arrays are also similar to the precursor polyvalent beacons
because conjugation occurred at distal positions to the inner
chromophore core, which is another advantage of the polyvalent
beacons. Figure 1 shows the normalized absorption and emission
spectra of 5. Interestingly, all the compounds have exceptionally
high molar absorptivity in 20% DMSO/H2O [ꢀmax mol-1 cm-1: 1
(224 060), 2 (190 408), 3 (215 447), 4 (201 181), 5 (205 111), 6
(210 653), 7 (214 629), 8 (209 389), 9 (199 027), 10 (219 160), and
9
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10.1021/ja049441z CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Figure 1. Spectral properties of a representative dendritic array of
glucosamine 5 in 20% aqueous DMSO.
Figure 2. Biodistribution of dendritic arrays of glucosamine in CA20948
pancreatic tumor-bearing mice at 24 h post-injection of the probes.
11 (207 156)]. The concentrations of solutions used for molar
absorptivity determinations ranged from 0.3 to 8 µM.
In conclusion, we have described the synthesis of novel poly-
valent molecular beacons that used a NIR carbocyanine as the inner
core. On the basis of the synthetic strategy described above, higher
generations of the polyvalent compounds are accessible. The
strategic “encapsulation” of the chromophore system in the inner
core prevents fluctuations in the spectral properties of the com-
pounds. This improves data reproducibility and enables using the
same excitation source and emission filter for the analyses of
samples by optical methods. Our preliminary in vivo results showed
enhanced uptake of the beacons in proliferating tumor cells, possibly
mediated by GLUTs. Finally, the polyvalent structural framework
could be used to amplify a variety of biological and chemical
molecular recognition interactions.
The glycolytic pathway for energy production by cells requires
delivery of glucose to the mitochondria, where it is phosphorylated
by an enzyme called hexokinase.10 Glucose transporters (GLUTs)
are responsible for channeling the carbohydrate into cells. Because
of their high energy needs, proliferating cells overexpress GLUTs
to enhance glycolysis relative to normal cells. Consequently,
radiolabeled glucose derivatives are used to detect cancers in
humans.11 Previous studies have shown that a fluorescent probe-
labeled glucosamine derivative, 2-(N-(7-nitrobenz-2-oxa-1,3- diazol-
4-yl)amino)-2-deoxyglucose (2-NBDG), is a viable nonradioactive
method to monitor glucose transport and uptake in living cells.12
Therefore, we evaluated the retention of representative polyvalent
molecular beacons in a proliferating tumor model (CA20948) in
nude mice.
Acknowledgment. Funding for this project was provided by
Each tumor-bearing mouse received 0.1 µmol of the beacons/
kg of body weight via tail vein injection. The in vivo and ex vivo
distribution of the molecular beacons were monitored by NIR
fluorescence imaging using two collimated solid 780-nm laser
sources for excitation and a CCD camera equipped with 830-nm
interference filter to capture the fluorescence emission.4 An
advantage of the optical method is that the blood clearance profile
and the tumor uptake of the beacons can be monitored continuously
and in real time. At 24 h post-injection, the nude mice were
sacrificed. Aliquots of blood and some major organs (tumor, kidney,
muscle, kidney, and liver) were harvested and washed with
phosphate-buffered saline. The mean fluorescence count in each
tissue was determined for each compound and normalized. Negative
control studies show that the precursor dye 1 was not retained in
the tumor but accumulated in the liver at about 1 h post-injection.
On the contrary, the dendritic arrays of glucosamine were retained
in the tumor up to 24 h post-injection (Figure 2). Uptake of 6 by
the liver was slightly higher than uptake in the tumor and the
kidneys. Evaluation of the retention rate and biodistribution suggests
that compound 8 rapidly accumulated in the tumor and had the
lowest overall uptake in nontarget tissues. These preliminary results
also show that each compound can be used for different applica-
tions. For example, compound 9, which is retained in blood for
>24 h, could serve as a blood pool agent for monitoring blood
flow and imaging blood vessels.
the NSF Bioengineering Grant (BES 0119489).
Supporting Information Available: Description of synthetic
procedures, spectral characterization, and fluorescence imaging method.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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