Libraries of composite polyfluors built from fluorescent deoxyribosides

Article abstract of DOI:10.1021/ja027197a

We report on a new class of water-soluble fluorescent molecules (polyfluors) that are composed of multiple individual fluorophores assembled on a DNA-like backbone. Four fluorophore deoxyribosides were synthesized, and these individual molecules were asse

Full text of DOI:10.1021/ja027197a

Published on Web 09/04/2002
Libraries of Composite Polyfluors Built from Fluorescent Deoxyribosides
Jianmin Gao,† Christoph Stra¨ssler,† Deborah Tahmassebi,‡ and Eric T. Kool*,†
Department of Chemistry, Stanford UniVersity, Stanford, California 94305-5080, and
Department of Chemistry, UniVersity of San Diego, San Diego, California 92110
Received June 6, 2002
Fluorescent labels are universally applied in biology, biotech-
nology, and medicine.1 In addition, fluorophores are becoming
increasingly useful in combinatorial chemistry and biology, both
as encoders of library members and as reporters of chemical
reactions.2 As a result of this greatly expanding use of fluorescence,
there is a corresponding increase in the need for new fluorophores
having a wider range of spectral characteristics.3 One way to
generate new fluorescence properties is to encourage two dyes to
interact photophysically. A prominent and useful example of this
is FRET dye pairs,4 which have been instrumental in decoding the
human genome. Electronically interacting fluorophores can display
new properties that individual dyes do not have, including increased
Figure 1. Structures of “fluoroside” monomers in this study (a) and of a
selected tetrafluor oligomer (b) having the sequence 5′-DEYD. R ) H or
dimethoxytrityl.
Stokes shifts, specifically tuned excitations, or increased brightness.
Here we report on a new class of fluorescent molecules that are
composed of multiple individual fluorophores. To encourage various
forms of energy transfer, including the possibilities of FRET,
excimer, exciplex, and charge transfer, we encourage close interac-
tion by mimicking the structure of natural DNA. The individual
fluorophores are constructed as deoxyribosides, with the flat
aromatic fluorophores replacing a DNA base (Figure 1).5 These
individually fluorescent molecules are assembled into oligofluor
strings that resemble single-stranded DNA. This strategy not only
allows for ready preparation on a synthesizer, but it also encourages
the closest possible interactions by allowing them to stack, as do
nucleobases in DNA. Finally, the DNA backbone negative charges
maintain water solubility in what would otherwise be insoluble dyes.
To test whether this potentially stacked oligofluor design would
result in useful fluorophore interactions, we prepared four fluores-
cent deoxyribosides (“fluorosides”) as monomeric components of
a combinatorial set. We chose pyrene (Y, a violet fluorophore),
oxoperylene (E, green), dimethylaminostilbene (D, blue), and
quinacridone (Q, yellow) as a simple set of test dyes (Figure 1).
Their synthesis is described in the Supporting Information. We
prepared them as 5′-dimethoxytrityl-3′-phosphoramidite derivatives
for automated incorporation into DNA-like strings on a commercial
synthesizer. We also prepared a set of 12 binary encoding
compounds,6 so that individual polyfluorophores could later be
decoded after selection.
Figure 2. Fluorescence microscope image from a 256-member tetrafluor
library composed of all possible tetrameric combinations of monomers D,
E, Y, Q. Excitation was at 340-380 nm. Beads are PEG-polystyrene and
are dry during image acquisition. At right is a representative spectrum of
bead colors and intensities from the library.
We then prepared an initial small (16-member) test library
comprised of these four monomers two units in length, on 130 µm
PEG-polystyrene beads, using standard split-and-mix methods, and
on a 1 µmol scale. Trityl cation monitoring demonstrated coupling
yields that were acceptably high (at >93.6%) for each of the four
monomers. We imaged the bead-based library by epifluorescence
microscope, with excitation at 340-380 nm. Approximately 8-10
variations of intensity and hue were distinguishable by eye. This
suggested that neither strong radiationless quenching mechanisms,
nor overwhelming of apparent library color by a single bright dye,
were dominant outcomes for this molecular design.
Encouraged by this, we prepared a larger 256-member binary-
encoded tetrafluor library composed of all combinations of fluo-
rosides Y, E, D, and Q. On the basis of the scale of the library,
each member was represented with an ca. 40-fold redundancy. This
new set of tetrafluors exhibited at least 50 different hues and
intensities distinguishable by eye (Figure 2), ranging from violet
to yellow-orange in color, and from relatively bright to nearly
completely dark. As an initial sample, we picked and sequenced a
set of tetrafluors based on bright beads of various hues as well as
a few especially dark examples. Approximately 90% of 50 selected
We characterized the fluorescence properties of the individual
monomer fluorosides Y, E, D, and Q (see Supporting Information).
They display absorbance maxima ranging from 342 to 509 nm and
emission maxima from 375 to 541 nm. Molar absorptivities vary
from 8.8 × 103 to 4.7 × 104 L mol-1 cm-1. Stokes shifts range
from 17 to 93 nm, and quantum yields (in air-saturated methanol)
vary from 0.055 to 0.81. The properties are close to those of the
parent fluorophores.7
* To whom correspondence should be addressed. E-mail: kool@stanford.edu.
† Stanford University.
‡ University of San Diego.
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C O M M U N I C A T I O N S
prepared earlier. This is because a greater number of different
fluorophores can be introduced into a smaller molecule and because
we are not limited by commercially available phosphoramidite-
derivatized dyes. Third, the present tetrafluors are much smaller,
simpler, and less expensive to prepare (previous triple-fluorophore
molecules are a total of 13 nt in length10). The relatively small
size of the current molecules makes them good candidates for
conjugation to proteins and DNA.
In summary, we have described a new strategy for the construc-
tion and screening of a wide variety of discrete, water-soluble
fluorescent species from a small set of monomer “fluoroside” dyes.
Future work will be aimed at characterizing properties of individual
tetrafluors from this library, at constructing a wider variety of
monomer dyes and larger libraries that contain them, and at
developing methods to conjugate them to other molecules of
interest. We expect that oligofluorosides may find use as labels
having new characteristics for applications in the biomedical
sciences and in combinatorial encoding and discovery.
Figure 3. Fluorescence of selected library members. (a) Aqueous solutions
of three tetrafluors having sequence 5′-YDDD (left), 5′-YEEY (center), and
5′-QYYY (right), under UV excitation. (b) Normalized emission spectra
of the same three tetrafluors (excitation 345 nm).
beads yielded a decodable sequence; a full analysis of properties
of some of these tetrafluors is underway.
Screening of molecules on solid supports does not guarantee that
they will retain the same properties in solution. In the present case,
it is possible that effects arising from the solid-supported methods
might in some cases affect the outcome. As a test, we synthesized
and purified three tetrafluors, identified under the microscope as
bright blue (5′-YDDD), green (5′-YEEY), and yellow dyes (5′-
QYYY) on the bead support. These were conveniently purified by
gel electrophoresis, as if they were oligonucleotides, and were
characterized by MS and spectroscopically. Results showed that
the new tetrafluors exhibited the same apparent color in solution
as on the solid support (Figure 3). This does not, of course,
guarantee that other members will not exhibit such methodology-
related effects (we have observed a few cases that do8). However,
the findings establish that a major fraction of the selected beads in
such a library will yield comparable results in solution.
Acknowledgment. This work was supported by a grant from
the U.S. Army Research Office and by the NSF. J.G. acknowledges
support from a Stanford Graduate Fellowship. We thank Nicholas
Salzameda for assistance in synthesis.
Supporting Information Available: Experimental details of mono-
mer synthesis, library preparation, and fluorescence measurements
(PDF). This material is available free of charge via the Internet at http://
pubs.acs.org.
References
Importantly, these three selected tetrafluors display properties
markedly different than any of the monomeric fluorophores that
make them up. Their colors differ in nontrivial ways from the
monomers that compose them; for example, QYYY is yellow-
orange despite being composed of three violet dyes and only one
yellow dye. Stokes shifts are often much larger for these tetrafluors
(40-221 nm) than for the monomers, and the three display their
varied emission colors with a single excitation wavelength.
Ordinarily the observation of three differently colored commercial
monomeric dyes under the microscope requires three different
excitations with three different filter sets, and they could only be
observed simultaneously by artificially overlaying three digital
images. Thus, the preliminary results quite successfully demonstrate
new and useful fluorescence properties.
(1) (a) Mayer, A.; Neuenhofer, S. Angew. Chem., Int. Ed. Engl. 1994, 33,
1044-1072. (b) Zimmer, M. Chem. ReV. 2002, 102, 759-781.
(2) (a) Hoveyda, A. H. Chem. Biol. 1999, 6, R305-R308. (b) Copeland, G.
T.; Miller, S. J. J. Am. Chem. Soc. 2001, 123, 6496-6502. (c) Battersby,
B. J.; Bryant, D.; Meutermans, W.; Matthews, D.; Smythe, M. L.; Trau,
M. J. Am. Chem. Soc. 2000, 122, 2138-2139. (d) Nanthakumar, A.; Pon,
R. T.; Mazumder, A.; Yu, S. Y.; Watson, A. Bioconjugate Chem. 2000,
11, 282-288. (e) Gauglitz, G. Curr. Opin. Chem. Biol. 2000, 4, 351-
355. (f) Reetz, M. T. Angew. Chem., Int. Ed. 2001, 40, 284-310. (g)
Stambuli, J. P.; Stauffer, S. R.; Shaughnessy, K. H.; Hartwig, J. F. J. Am.
Chem. Soc. 2001, 123, 2677-2678. (h) Moreira, R.; Havranek, M.; Sames,
D. J. Am. Chem. Soc. 2001, 123, 3927-3931. (i) Battersby, B. J.; Lawrie,
G. A.; Trau, M. Drug DiscoVery Today 2001, 6, S19-S26. (j) Farrer, R.
A.; Copeland, G. T.; Previte, M. J. R.; Okamoto, M. M.; Miller, S. J.;
Fourkas, J. T. J. Am. Chem. Soc. 2002, 124, 1994-2003.
(3) (a) Isacsson, J.; Westman, G. Tetrahedron Lett. 2001, 42, 3207-3210.
(b) Schiedel, M. S.; Briehn, C. A.; Bauerle, P. Angew. Chem., Int. Ed.
2001, 40, 4677-4680.
(4) Glazer, A. N.; Mathies, R. A. Curr. Opin. Biotechnol. 1997, 8, 94-102.
(5) Kool, E. T. Acc. Chem. Res. 2002, 35, in press.
Future work will be needed to study in detail the photophysical
properties of a broader set of tetrafluors. We expect that such
compounds might exhibit properties that individual commercially
available fluorophores cannot. One example is the aforementioned
large effective Stokes shifts of greater than 200 nm in some cases.
In addition, the high total molar absorptivity of four chromophores
implies the possibility of light-harvesting effects,9 allowing for
brighter emission than a single dye might offer on a per-molecule
basis. Finally, the closeness of the monomers in the library, and
their potentially stacked arrangement, offer the possibility of forms
of energy transfer beyond simple FRET, to include excimer,
exciplex, and charge-transfer mechanisms. Indeed, we have ob-
served multiple examples of non-FRET forms of energy transfer
in this library (J. Gao, work in progress).
A few recent studies have applied up to three commercial dyes
in DNA-based FRET libraries, generating up to eight different
molecules spaced by 5-10 nucleotides.10 The present approach
offers a number of potentially useful differences from previous
strategies. First, it allows for a broader array of energy transfer
mechanisms, resulting in greater diversity in photophysical out-
comes. Second, the present library is considerably larger than those
(6) Ohlmeyer, M. H. J.; Swanson, M.; Dillard, L. W.; Reader, J. C.; Asouline,
G.; Kobayashi, R.; Wigler, M.; Still, W. C. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 10922-10926.
(7) (a) Letard, J. F.; Lapouyade, R.; Rettig, W. J. Am. Chem. Soc. 1993, 115,
2441-2447. (b) Zaharia, C. N.; Tarabasanu-Mihaila, C. ReV. Roum. Phys.
1975, 20, 769-780. (c) Nijegorodov, N. I.; Downey, W. S. J. Phys. Chem.
1994, 98, 5639-5643.
(8) Experiments revealed that the quinacridone fluoroside is somewhat labile
to the acidic detritylation conditions; thus, if the Q fluorophore appears
toward the 3′ end of a sequence, it is lost to some degree.
(9) (a) Adronov, A.; Frechet, J. M. J. Chem. Commun. 2000, 1701-1710.
(b) Burrell, A. K.; Officer, D. L.; Plieger, P. G.; Reid, D. C. W. Chem.
ReV. 2001, 101, 2751-2796. (c) Weil, T.; Wiesler, U. M.; Herrmann, A.;
Bauer, R.; Hofkens, J.; De Schryver, F. C.; Mullen, K. J. Am. Chem. Soc.
2001, 123, 8101-8108. (d) Peng, K. Y.; Chen, S. A.; Fann, W. S. J. Am.
Chem. Soc. 2001, 123, 11388-11397. (e) Sugou, K.; Sasaki, K.; Kitajima,
K.; Iwaki, T.; Kuroda, Y. J. Am. Chem. Soc. 2002, 124, 1182-1183. (f)
Kodis, G.; Liddell, P. A.; de la Garza, L.; Clausen, P. C.; Lindsey, J. S.;
Moore, A. L.; Moore, T. A.; Gust, D. J. Phys. Chem. A 2002, 106, 2036-
2048. (g) Loewe, R. S.; Lammi, R. K.; Diers, J. R.; Kirmaier, C.; Bocian,
D. F.; Holten, D.; Lindsey, J. S. J. Mater. Chem. 2002, 12, 1530-1552.
(10) (a) Kawahara, S.; Uchimaru, T.; Murata, S. Chem. Commun. 1999, 563-
564. (b) Ohya, Y.; Yabuki, K.; Komatsu, M.; Ouchi, T. Polym. AdV.
Technol. 2000, 11, 845-855. (c) Tong, A. K.; Li, Z. M.; Jones, G. S.;
Russo, J. J.; Ju, J. Y. Nat. Biotechnol. 2001, 19, 756-759. (d) Tong, A.
K.; Jockusch, S.; Li, Z.; Zhu, H.; Akins, D. L.; Turro, N. J.; Ju, J. J. Am.
Chem. Soc. 2001, 123, 12923-12924. (e) Tong, A. K.; Ju, J. Y. Nucleic
Acids Res. 2002, 30, e19.
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