Sensing metal ions with DNA building blocks: Fluorescent pyridobenzimidazole nucleosides

Article abstract of DOI:10.1021/ja0581806

We describe novel fluorescent N-deoxyribosides (1 and 2) having 2-pyrido-2-benzimidazole and 2-quino-2-benzimidazole as aglycones. The compounds were prepared from the previously unknown heterocyclic precursors and Hoffer's chlorosugar, yielding alpha ano

Full text of DOI:10.1021/ja0581806

Published on Web 04/12/2006
Sensing Metal Ions with DNA Building Blocks: Fluorescent
Pyridobenzimidazole Nucleosides
Su Jeong Kim and Eric T. Kool*
Contribution from the Department of Chemistry, Stanford UniVersity,
Stanford, California 94305-5080
Received December 1, 2005; E-mail: kool@stanford.edu
Abstract: We describe novel fluorescent N-deoxyribosides (1 and 2) having 2-pyrido-2-benzimidazole and
2-quino-2-benzimidazole as aglycones. The compounds were prepared from the previously unknown
heterocyclic precursors and Hoffer’s chlorosugar, yielding alpha anomers as the chief products. X-ray crystal
structures confirmed the geometry and showed that the pyridine and benzimidazole ring systems deviated
from coplanarity in the solid state by 154° and 140°, respectively. In methanol compounds 1 and 2 had
absorption maxima at 360 and 370 nm, respectively, and emission maxima at 494 and 539 nm. Experiments
revealed varied fluorescence responses of the nucleosides to a panel of 17 monovalent, divalent, and
trivalent metal ions in methanol. One or both of the nucleosides showed significant changes with 10 of the
metal ions. The most pronounced spectral changes for ligand-nucleoside 1 included red shifts in
fluorescence (Au+, Au3+), strong quenching (Cu2+, Ni2+, Pt2+), and substantial enhancements in emission
intensity coupled with red shifts (Ag+, Cd2+, Zn2+). The greatest spectral changes for ligand-nucleoside 2
included a red shift in fluorescence (Ag+), a blue shift (Cd2+), strong quenching (Pd2+, Pt2+), and substantial
enhancements in emission intensity coupled with a blue shift (Zn2+). The compounds could be readily
incorporated into oligodeoxynucleotides, where an initial study revealed that they retained sensitivity to
metal ions in aqueous solution and demonstrated possible cooperative sensing behavior with several ions.
The two free nucleosides alone can act as differential sensors for multiple metal ions, and they are potentially
useful monomers for contributing metal ion sensing capability to DNAs.
species, such as peptides,4 proteins,5a,b and small molecules,6a-c
and sensing physical changes, such as pH7 and light.8 Few
Introduction
Although in nature DNA acts primarily as a storehouse of
genetic information, recent studies have been aimed at endowing
new properties on this genetic molecule. Among these properties
is the ability to act as a sensor of physical conditions or
molecular species. Because DNA has the innate ability to
recognize other nucleic acids, many studies have reported on
the use of modified DNAs as sensors of RNA and DNA.1a-n
However, DNA (especially in the single-stranded form) can fold
into diverse structures that allow for recognition of varied types
of molecules,2a-e and many changes can be made to add
functional groups of DNA to increase chemical diversity
further.3a-d As a result, an increasing number of studies have
focused on the use of modified DNA in detection of other
experiments have been aimed at sensing metal ions with DNAs
as well.9a-d The properties of DNA as a sensor offer a number
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3793. (c) Ortiz, E.; Estrada, G.; Lizardi, P. M. Mol. Cell Probes 1998, 12,
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2004, 69, 3271-3275. (f) Saito, Y.; Miyauchi, Y.; Okamoto, A.; Saito, I.
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10.1021/ja0581806 CCC: $33.50 © 2006 American Chemical Society

Fluorescent Nucleoside Metal Ligands
A R T I C L E S
Chart 1. Structures 1 and 2
because they have a rotatable bond that alters conjugation, and
because they were known to be fluorescent. We added a
dimethylamino group to the benzimidazole portion, placing this
donor in conjugation with the pyridine ring nitrogen. Thus, the
act of chelating a metal ion might be expected to alter
conjugation by changing the dihedral angle of the single bond,
which would alter the donation by the amino group. In addition,
varied metals in the act of binding the ligand could also further
alter the electronic states of the conjugated systems. Thus, we
anticipated the possibility that these somewhat generic nitrogen
ligands might yield diverse signals for different metal ions as a
result of these variable electronic and structural factors.
We prepared 1 and 2 starting with commercial 5-chloro-2-
nitroaniline (Scheme 1). The chloride was displaced by N,N-
dimethylamine. Reduction of the nitro group to the amine
followed by subsequent oxidative condensation with pyridine
2-carboxaldehyde in nitrobenzene yielded the heterocycle 3 and
with quinoline 2-carboxaldehyde13 yielded 4. These were reacted
with Hoffer’s R-chlorosugar14 in the presence of sodium hydride
in N,N-dimethylacetamide. The chief products were assigned
as R-anomers by proton NOE measurements. This stereochem-
ical outcome is atypical for this reaction; we hypothesize that
the steric bulk of the 2-aryl-benzimidazoles was better accom-
modated from the R face of the bis-toluoyl ester intermediate,
yielding the R isomer in an SN1 mechanism. Although there
are two possible benzimidazole isomers for each nucleoside,
we only observed the isomers in which the dimethylamino group
is closer to the sugar; the origin of this preference is not yet
clear but may be related to electron donation from this amino
group into the imidazole ring. The ester protecting groups were
subsequently removed under basic conditions. We were able to
obtain single crystals of the free nucleosides 1 and 2 by
crystallization from chloroform/methanol/hexane (Figure 1). The
X-ray structures confirmed the R-anomeric geometry and
showed that the pyridyl(quinolinyl) and benzimidazolyl rings
deviated from syn coplanarity in the solid state by 154° and
140°, respectively. This apparent near-anti conformational
preference may arise from avoidance of lone pair repulsions
from the two nitrogens and/or possible steric repulsion of the
pyridine protons away from the deoxyribose substituent.
of advantages in application over other organic scaffolds; among
these are water solubility, ease of synthesis (including automated
methods), and predictable assembly and recognition properties.
As part of a larger program aimed at development of water-
soluble, diverse fluorescent species, we adopted a strategy of
replacing DNA bases with varied fluorescent groups.10a-d We
considered the possibility that adding metal ligand functionality
to fluorescent systems might render such species able to
recognize and report on metal ions in solution. In this regard,
a number of laboratories have recently described deoxyribosides
with metal ligands as replacements for nucleobases,11a-h but
these were designed for cross-linking nucleic acid strands, and
use in ion detection was not reported.
Here we report on two new nucleosides in which the
nucleobase is replaced by pyridobenzimidazoles that are de-
signed to act as metal ligands. Their preparation, structure, and
optical properties are described. The nucleosides are fluorescent
and were found to be sensitive to the presence of 12 different
metal ions, displaying significant changes in their emission
spectra, both as free nucleosides and in the context of DNA.
The compounds are expected to be useful in lending sensing
properties to DNA.
Results
Nucleoside Design and Synthesis. Compounds 1 and 2 are
bifunctional and possess both the structure of a DNA building
block (i.e., deoxyribose) and structures designed to bind metals
with conjugated systems (Chart 1). We chose these pyrido- (and
related quino-) benzimidazole heterocycles (3 and 4), both of
which were previously unknown, because simple pyridobenz-
imidazoles were previously known as ligands for metals,12
Nucleoside Optical Properties. We examined the optical
properties of the free nucleosides in solution. They were poorly
soluble in water and dissolved in methanol and ethyl acetate
for the measurements. In MeOH, both compounds showed small
absorption maxima at 260 nm; 1 also had a major longer-
wavelength absorption band at 360 nm, while 2 had an
absorption at 370 nm (Table 1 and Supporting Information).
Emission spectra were measured with excitation at the long-
wavelength bands; they revealed fluorescence in the visible
ranges for both compounds (Figure 2) with emission maxima
at 494 and 539 nm for 1 and 2, respectively. Quantum yields
for these emission bands were 0.052 and 0.0085, respectively.
The emission maxima in methanol were red shifted relative to
the corresponding maxima in lower-polarity ethyl acetate
solution (464 and 520 nm, respectively), consistent with
(10) (a) Ren, R. X.-F.; Chaudhuri, N. C.; Paris, P. A.; Rumney, S., IV; Kool,
E. T. J. Am. Chem. Soc. 1996, 118, 7671-7678. (b) Gao, J.; Stra¨ssler, C.;
Tahmassebi, D.; Kool, E. T. J. Am. Chem. Soc. 2002, 124, 11590-11591.
(c) Cuppoletti, A.; Cho, Y.; Park, J. S.; Stra¨ssler, C.; Kool, E. T.
Bioconjugate Chem. 2005, 16, 528-534. (d) Liu, H.; Gao, J.; Lynch, S.;
Maynard, L.; Saito, D.; Kool, E. T. Science 2003, 302, 868-871.
(11) (a) Tanaka, K.; Shionoya, M. J. Org. Chem. 1999, 64, 5002. (b) Meggers,
E.; Holland, P. L.; Tolman, W. B.; Romesberg, F. E.; Schultz, P. G. J.
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Am. Chem. Soc. 2001, 123, 3375-3376. (d) Zimmermann, N.; Meggers,
E.; Schultz, P. G. J. Am. Chem. Soc. 2002, 124, 13684-13685. (e) Brotschi,
C.; Leumann, C. J. Nucleosides, Nucleotides Nucletic Acids 2003, 22,
1195-1197. (f) Morales, H.; Kool, E. T. Org. Lett. 2002, 12, 4377-4380.
(g) Tanaka, K.; Tengeiji, A.; Kato, T.; Toyama, N.; Shionoya, M. Science
2003, 299, 1212-1213. (h) Zhang, L.; Meggers, E. J. Am. Chem. Soc.
2005, 127, 74-75.
(12) (a) Gumus, F.; Pamuk, I.; Ozden, T.; Yildiz, S.; Diril, N.; Oksuzoglu, E.;
Gur, S.; Ozkul, A. J. Inorg. Biochem. 2003, 94, 255-262. (b) Chanda, N.;
Sarkar, B.; Kar, S.; Fiedler, J.; Kaim, W.; Lahiri, G. K. Inorg. Chem. 2004,
43, 5128-5133. (c) Sokolov, M. N.; Fedorova, N. E.; Peresypkina, E. V.;
Paetow, R.; Fedorov, V. E.; Fenske, D. Inorg. Chim. Acta 2005, 358, 3914-
3918. (d) Chanda, N.; Paul, D.; Kar, S.; Mobin, S. M.; Datta, A.; Puranik,
V. G.; Rao, K. K.; Lahiri, G. K. Inorg. Chem. 2005, 44, 3499-3511. (e)
Liu, Q.-D.; Jia, W.-L.; Wang, S. Inorg. Chem. 2005, 44, 1332-1343.
(13) (a) Amendola, V.; Fabbrizzi, L.; Gianelli, L.; Maggi, C.; Mangano, C.;
Pallavicini, P.; Zema, M. Inorg. Chem. 2001, 40, 3579-3587. (b) Spencer,
D. J. E.; Johnson, B. J.; Johnson, B. J.; Tolman, W. B. Org. Lett. 2002, 4,
1391-1393. (c) Scolaro, L. M.; Mazzaglia, A.; Romeo, A.; Plutino, M.
R.; Castriciano, M.; Romeo, R. Inorg. Chim. Acta 2002, 330, 189-196.
(14) Hoffer, M. Chem. Ber. 1960, 93, 2777-2781.
9
J. AM. CHEM. SOC. VOL. 128, NO. 18, 2006 6165

A R T I C L E S
Kim and Kool
Scheme 1. Synthesis of Deoxyribosides 1 and 2
Figure 1. Structures of deoxynucleosides 1 (A) and 2 (B) in the solid state, determined by X-ray crystallography and shown in ORTEP form. Details are
given in Supporting Information.
(K2+, Mg2+, Ca2+, Mn2+, La2+, Eu2+) had little or no effect on
Table 1. Optical Data for Nucleosides 1 and 2
the emission from the pyridobenzimidazole. Co2+ and Pd2+
absorbance
maxa (nm)
emission
max (nm)
1
compd
solvent
ꢀ
(M-1 cm-
)
Φfl
nd
0.050
nd
τ
(ns)b
showed weak quenching of this fluorophore, while Pt2+, Cu2+
,
and Ni2+ showed apparently strong quenching. In contrast, both
Au+ and Au3+ gave marked red shifts in emission (shifts of 55
1
1
2
2
EtOAc
MeOH
EtOAc
MeOH
360
360
370
384
nd
460
494
521
543
nd
0.45
nd
1.37 × 104
nd
and 37 nm) with little effect on intensity. Finally, Ag+, Cd2+
,
1.36 × 104
0.0085 0.36
and Zn2+ yielded both red shifts and marked increases in
emission intensity; red shifts increased in the order Ag+ (26
nm shift with 3-fold increase in intensity) < Cd2+ (59 nm with
8-fold increase) < Zn2+ (79 nm with 3-fold increase). The
effects are summarized in Table 2.
a Long-wavelength band is listed; shorter wavelength bands at 260 nm
were also present. b Conditions: 10 µM nucleoside in methanol, 25 °C,
with excitation at 370 nm. Compound. 1 was measured at 450 nm with a
430 nm UV filter; compound 2 was measured at 550 nm with a 515 nm
UV filter.
Metal ion effects on quinobenzimidazole 2 were also mea-
sured (Figure 4, Table 2, and Supporting Information) and
revealed both similarities and differences with 1. With 2, eight
metal ions (K+, Mg2+, Ca2+, Hg2+, Mn2+, La2+, Eu2+) had little
or no effect on the emission from ligand 1. Co2+, Au3+, and
Ni2+ showed slight quenching of this fluorophore, while Cu2+
showed moderate quenching in addition to a 5 nm red shift.
Addition of Pd2+ and Pt2+ gave apparently strong quenching
coupled with red shifts (shifts of 38 and 20 nm, respectively).
In contrast, Ag+ gave a 36 nm red shift with little effect on
intensity. Finally, unlike ligand 1, ligand 2 yielded blue shifts
of the emission band in two cases. Cd2+ yielded a shift from
the original 543 nm emission to dual emission at 460 and 510
nm with little change in intensity, while Zn2+ yielded a shift to
476 nm and a substantial (2-fold) increase in emission intensity.
A summary of the results for both fluorescent nucleoside
ligands is outlined in Table 2 and points out the similarities
formation of a strong excited-state dipole due to resonance
overlap of the dimethylamino group with the pyridine ring
nitrogen.
Nucleoside Response to Metal Ions. We then investigated
the effects of metal ions on the fluorescence of nucleosides 1
and 2 in methanolic solution. Nucleosides and metals were
studied at 50 and 100 µM concentrations, respectively, and we
screened the effects of 17 metal species on the two nucleosides
by monitoring emission spectra. Note that the ligands could in
principle bind with varied stoichiometry with a given metal;
some may bind as 1:1 ligand:metal complexes, while other
combinations may form complexes with other stoichiometry.
Metals (as their chloride salts) were added from concentrated
stock solutions in DMSO, giving a final concentration of 5%
DMSO in methanol. Results with compound 1 showed (Figure
3, Table 2, and Supporting Information) that a few metal ions
9
6166 J. AM. CHEM. SOC. VOL. 128, NO. 18, 2006

Fluorescent Nucleoside Metal Ligands
A R T I C L E S
Figure 2. Excitation and emission spectra of 1 (A) and 2 (B) (2 µM) in
methanol (25 °C). Excitation spectra are shown with dashed lines and were
monitored at the emission maxima. Emission spectra are shown with solid
lines and were measured with excitation at the long-wavelength absorption
maxima.
and differences. As free nucleosides in methanol, neither
compound showed significant changes with seven ions (K2+
,
Mg2+, Ca2+, Hg2+, Mn2+, La2+, Eu2+). Both compounds showed
similar changes for Co2+ (slight quenching) and Pt2+ (strong
quenching with red shift). Interestingly, although they are similar
ligands, the compounds showed different responses for the other
eight metal ions. The most dramatic differences occurred with
Ag2+ (>3-fold enhancement of fluorescence with 1 versus a
red shift with 2) and especially Zn2+ and Cd2+, which showed
marked red shifts and fluorescence enhancements with 1 but
strong blue shifts with 2.
Responses of Ligand Pairs in DNA. We then investigated
the incorporation of fluorescent nucleosides 1 and 2 into DNA
oligonucleotides. Both compounds were converted to the 5′-
dimethoxytrityl-3′-phosphoramidite derivatives using standard
methods (Supporting Information). They were then incorporated
into two 13mer complementary oligonucleotides using auto-
mated synthesizer coupling programs. The presence of the intact
ligands in the DNAs was confirmed by MALDI-TOF mass
spectrometry. We also prepared complementary DNAs in which
A, C, G, or T was paired opposite the ligands; these showed
little or no differential effect on fluorescence emission (data
not shown).
Figure 3. Fluorescence response of nucleoside 1 to 17 metal salts in
methanolic solution (25 °C). (A) Photograph showing varied responses of
1 to the metals using 365 nm light from a UV box as the excitation source.
(B) Spectra of 1 (2 µM) in the presence of metal ions that change the
emission intensity or wavelength of the nucleoside. (C) Spectra of 1 with
metal ions that yield little or no change in the emission properties. Metals
are present at 100 µM as chloride salts except AgNO3.
which nucleosides 1 and 2 were paired opposite themselves or
each other (Figure 5). Four pairing geometries were tested: 1-1,
2-2, and the similar (but not identical) 2-1 and 1-2 cases. In
the DNA context, nucleoside 1 (as a 1-1 pair) showed only a
small change (a 5 nm red shift) compared to its behavior in
methanol as a free nucleoside. Nucleoside 2, on the other hand,
showed a marked change with its major emission band appearing
in the blue (440 nm) in DNA as a 2-2 pair, whereas alone in
methanol 2 emits in the yellow range (543 nm). For the mixed
1-2 and 2-1 pairings, the emission behavior in the absence of
To investigate whether the fluorescence and recognition
properties of these ligands were retained in water, we performed
a screen of fluorescence responses of 13mer DNA duplexes in
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J. AM. CHEM. SOC. VOL. 128, NO. 18, 2006 6167

A R T I C L E S
Kim and Kool
Table 2. Fluorescence Responses of Nucleosides 1 and 2 to
Metal Salts in Methanolic Solution
emission
max (nm)
intensity
(rel)
emission
max (nm)
intensity
(rel)
+
Mn
+
Mn
compd
compd
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
none
K+
494
494
494
494
494
494
496
486
573
503
520
553
508
549
531
498
496
498
1.0
1.1
1.2
1.1
1.0
0.6
0.1
0.1
2.9
0.7
3.4
8.3
0.3
1.1
0.9
1.0
1.0
0.9
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
none
K+
543
544
543
541
540
539
542
548
476
581
579
510
563
577
552
552
548
540
1.0
1.1
1.1
1.0
1.0
0.9
0.9
0.6
2.0
0.2
1.0
1.2
0.3
0.8
0.8
1.0
1.0
0.8
Mg2+
Ca2+
Mn2+
Co2+
Ni2+
Cu2+
Zn2+
Pd2+
Ag+
Mg2+
Ca2+
Mn2+
Co2+
Ni2+
Cu2+
Zn2+
Pd2+
Ag+
Cd2+
Pt2+
Cd2+
Pt2+
Au+
Au+
Au3+
La3+
Eu3+
Hg2+
Au3+
La3+
Eu3+
Hg2+
a Conditions: 50 µM nucleoside, 100 µM metal ion, 95% methanol, 5%
DMSO, 25 °C. All counterions were chloride except AgNO3.
metals resembled that of the 2-2 pairing more than the 1-1
pairing (Supporting Information), suggesting that 2 may quench
the emission of 1. However, in the presence of metals, the
responses were more similar to those of the 1-1 pairing (see
below).
We then screened these four different ligand “base pairs” for
their responses to the addition of metal ions (Figure 5). In some
cases we found roughly parallel responses to those seen for the
free nucleosides in methanol; for example, the 1-1 pairing
showed strong quenching by Pd2+ and Pt2+ and displayed
enhancements of fluorescence and red shifts with Zn2+ and
Cd2+, similar to what was observed for 1 alone in methanol. In
another example Cd2+ gave essentially the same results whether
with 2 alone or with the 2-2 pairing in DNA. For the mixed
1-2 and 2-1 pairings, the emission behavior in the presence
of metals resembled the results with the 1-1 pairing more than
the 2-2 pairing.
However, there were some marked differences between the
fluorescence emission behavior in the DNA setting and that of
the ligand nucleosides alone. For ligand 1, Cd2+, Ag+, and Zn2+
yielded considerably greater enhancements of fluorescence with
the 1-1 pairing in DNA, giving 20-, 15-, and 11-fold overall
enhancements, respectively, in emission intensity. Au3+ yielded
a red shift in methanol, whereas with 1-1 in DNA it quenched
fluorescence. In another example Pd2+ did not quench 1 alone,
whereas it strongly quenched 1-1 in DNA. For ligand 2, Zn2+
yielded a new band in the red for the 2-2 pairing in DNA,
whereas alone in methanol it gave enhancement plus a blue shift.
The Ag+ ion yielded dual bands at 440 and 552 nm with 2-2
in DNA, whereas with 2 in methanol it yielded only a 36 nm
red shift.
In a few other cases the ligand-nucleosides showed ad-
ditional striking evidence of apparently cooperative behavior
in a “paired” configuration (see Figure S2 in Supporting
Information). For example, the 1-1 pairing yielded enhanced
signals in the presence of Eu3+, whereas 1 alone in methanol
showed little or no effect with this lanthanide. In a second
example La3+ yielded a strong change for the 2-2 pairing with
a new red band appearing at 541 nm, whereas 2 alone in
Figure 4. Fluorescence response of nucleoside 2 to 17 metal salts in
methanolic solution (25 °C). (A) Photograph showing varied responses of
2 to the metals using 365 nm light from a UV box as the excitation source.
(B) Spectra of 2 (2 µM) in the presence of metal ions that change the
emission intensity or wavelength of the nucleoside. (C) Spectra of 2 with
metal ions that yield little or no change in the emission properties. Metals
are present at 100 µM as chloride salts except AgNO3.
methanol yielded no change with this ion. In a third example
the 2-1 pairing showed a nonquenched red shift with Ni2+
,
whereas this metal strongly quenched 1 alone in methanol and
had no effect on 2. Finally, Hg2+, another ion to which neither
1 nor 2 responded alone, yielded strong quenching in DNA
containing the 2-2 pairing.
Discussion
These early experiments show that combinations of one or
both of these nucleoside ligands, either as free nucleosides or
in DNA, could sense 12 of the 17 metal ions in this initial panel
9
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Fluorescent Nucleoside Metal Ligands
A R T I C L E S
2-2 pairing in DNA show that this combination of two ligands
can apparently act cooperatively to recognize and give a
response signal for Hg2+. Beyond the current studies, we do
expect that the current nucleosides may be able to respond to
the presence of other cations not included in this initial panel,
and future studies will address this. One limitation should be
noted for these nucleosides, namely, that they are only weakly
water soluble and thus limited in their aqueous applications
unless organic cosolvent can be added. Future studies will
address whether more polar derivatives can be prepared. Of
course, in the DNA context (see below) water solubility is no
longer problematic.
As a whole, the fluorescence responses of nucleosides 1 and
2 are remarkably diverse. Nucleoside 1 in methanol gives
emission maxima over an 87 nm wavelength range and a >80-
fold intensity range depending on the metal ion. Nucleoside 2
gives a similar 85 nm emission wavelength range, yielding a
red-shifted emission with Ag+ and a blue-shifted emission with
Cd2+. Overall, ligand 1 gives at least five qualitatively different
responses (strong quenching, weak quenching, strong emission
enhancement, red shift, multiple emission bands) for different
ions, and ligand 2, differing from 1 only by benzo substitution,
also gives at least five qualitatively different responses, in some
cases markedly different than 1. Taken together, the data suggest
that monomer 1 can act as a useful “turn on” sensor of Ag+,
Cd2+, Zn2+, and Au+ ions, and the responses to these are
different enough to allow separate identification of these metals
in an unknown sample. Because the responses to Au+ and Au3+
are similar, 1 would not be useful in discriminating between
the latter two. In the quenching mode both Ni2+ and Cu2+ give
very strong quenching of this nucleoside, allowing one to
discriminate between this pair and the above fluorescence-
enhancing ions. Monomer 2, like 1, can also be used as
enhanced-signal reporters of Zn2+ and Cd2+, and it also gives
a marked positive response for Ag+ and Au+, although it is
difficult to distinguish between the last two. Unlike 1, 2 can
distinguish between Au+ and Au3+, by a 25 nm difference in
wavelength. In quenching applications ligand 2 is not as strongly
quenched by the metals in this panel as 1 and would not be as
useful as the latter ligand.
Figure 5. Fluorescence responses of 1 and 2 to metal ions in aqueous
solution, measured in the context of 13mer duplex DNAs having ligands 1
and/or 2 placed opposite each other, as pseudo base pairs. (A) Emission
spectra of DNA with nucleoside 1 paired opposite itself in the presence of
varied metal ions. (B) Emission spectra of DNA with ligand 2 paired
opposite itself. See Figure S2 (Supporting Information) for cases with ligand
2 paired with 1. Solutions contained 3 µM DNA duplex and 100 µM metal
ion at 25 °C. Buffers also contained 100 mM NaCl and 10 mM Na‚PIPES
(pH 7.0). Excitation was at 370 nm.
In addition to fluorescence responses involving an increase
or a decrease in intensity, these ligand nucleosides in a number
of cases change their emission wavelengths markedly in the
presence of some metal ions. This is especially useful for two
reasons: first, it allows for ratiometric measurements of
concentration, as the ratio of two wavelengths can easily be
measured. Second, it allows for distinguishing between back-
ground signal and positive signal in applications where separat-
ing the reporter from the analyte is undesirable or impossible,
such as in real-time solution-phase assays and intracellular
assays. Nucleoside 1 shows its most marked red shifts in
wavelength for Cd2+ (59 nm), Zn2+ (79 nm), and Au+ (54 nm)
and thus might in principle be used ratiometrically for these.
Nucleoside 2 shows the greatest shifts for Zn2+ (67 nm) and
Cd2+ (33 nm) (these are blue shifts rather than red shifts).
of salts and allow one to distinguish between all 12 (except
Pd2+ and Pt2+, which give similar quenching effects with both
ligands). We expect that the diverse responses arise from the
combination of the rotatable bond, the electron-donating amino
group in conjugation with the pyridine nitrogen, and variable
electronic interactions of the ligands with the individual metals.
Further experiments will be needed to understand the details of
these mechanisms for the individual ligand-ion combinations.
With the current ligand design, which employs bipyridyl-
like structure, it is not surprising that the fluorescence responses
are weak or absent for the more oxophilic metals in this panel
of analytes, such as K+, Mg2+, Ca2+, Hg2+, and Mn2+. These
metals are expected to have weak affinity for the current nitrogen
ligands; future nucleoside designs for sensing these species may
require different liganding groups. However, our data for the
A comparison of the fluorescence responses of 1 and 2 with
other recently reported sensors reveals some similarities and
differences. There exist a substantial number of reports of
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A R T I C L E S
Kim and Kool
sensors for Zn2+ 15
,
including some that respond with a single
between multiple ligands as our preliminary studies suggest.
Finally, the DNA portion of such molecules could be useful
for self-assembly of sensors on surfaces and in arrays.
wavelength and a few that give a change in wavelength,15b,d,g
which is useful for ratiometric measurements. Compounds 1
and 2 fall into the latter category; it is not yet clear whether
they would be useful in detecting Zn2+ in cellular media as their
cell permeability and affinity for low biological concentrations
of this ion are not yet known. Compounds 1 and 2 also detect
Cd2+, Au+, and Ag+ with enhancements of emission and
wavelength shifts; although Zn2+ sensors often respond to Cd2+
as well, compound 1 is unusual in that it can readily distinguish
between these two closely related ions by wavelength. Finally,
we have shown that base pairs involving these nucleosides can
also respond to lanthanides with wavelength shifts. Thus,
although metal ion sensing (especially of Zn2+) is by now well
established in the literature, the diversity of responses of these
two nucleoside-ligands with a range of metal ions is rare.
Our data show that these nucleosides can be readily used as
monomers in the synthesis of DNA and that they retain metal
ion recognition properties in aqueous buffers and in the context
of the DNA double helix. We have shown that pairing of two
nucleoside ligands opposite one another can in some cases yield
new recognition and sensing properties that the monomers alone
do not have. A number of laboratories have described the
strategy of assembling base pairs in which a metal ion cross
links two paired ligands as DNA base replacements.11a-h The
current studies build on that concept by showing a fluorescence
reporting response as a result of that apparent interaction.
Examples of responses that may be specially useful in applica-
tion are those of the 1-1 pairing, which gives strong fluores-
Experimental Section
Nucleoside Synthesis. 5-N,N-Dimethylamino-2-(2-pyridinyl)-1H-
benzimidazole (3).
Nitrobenzene (200 mL) was added to 4-N,N-(dimethylamino)-
phenylenediamine (19.2 mmol) to make a solution in a round-bottom
flask. 2-Pyridinecarboxaldehyde (1.83 mL, 19.2 mmol) was added, and
then the solution was slowly heated to 100 °C. After 1 h stirring, the
reaction mixture was further heated to 150 °C and stirred for an
additional 2 h. This solution was cooled to room temperature and
directly loaded on a silica gel column for chromatographic purification
(eluent CH2Cl2/hexane (1:1) followed by CH2Cl2/EtOAc (2:3)). The
product was obtained as a dark yellow powder (3.57 g, 15.0 mmol,
1
78% yield over two steps). H NMR (400 MHz, DMSO-d6): δ 8.64
(d, J ) 5.2 Hz, 1H), 8.20 (d, J ) 8.0 Hz, 1H), 7.92 (t, J ) 8.0 Hz,
1H), 7.46-7.40 (m, 2H), 6.82-6.78 (m, 2H), 2.88 (s, 6H). 13C NMR
(100.7 MHz, DMSO-d6): δ 149.9, 149.8, 149.3, 148.8, 138.1, 124.8,
121.5, 112.1, 96.9, 41.9. HRMS (DEI): [M]+ calcd for C14H14N4,
238.1218; found, 238.1226.
5-N,N-Dimethylamino-2-(2-quinolinyl)-1H-benzimidazole (4). Ni-
trobenzene (250 mL) was added to 4-N,N-(dimethylamino)phenylene-
diamine (27.1 mmol) to make a solution in a round-bottom flask.
2-Quinolinecarboxaldehyde (4.14 g, 26.3 mmol) was added, and then
the solution was slowly heated to 80 °C. After 1 h stirring, the reaction
mixture was further heated to 150 °C and stirred for an additional 24
h. This solution was cooled to room temperature and directly loaded
onto a silica gel column for chromatographic purification (eluent CH2-
Cl2/hexane (1:1) followed by CH2Cl2/EtOAc (1:1)). The product was
obtained as an orange yellow powder (4.86 g, 16.9 mmol, 62% yield
cence enhancements and red shifts with Cd2+, Ag+, and Zn2+
;
1
it is even possible to distinguish between the three by wave-
length. The 2-2 pairing may also be especially useful in sensing
of Cd2+ and Zn2+, although these cannot be distinguished from
one another. More useful may be the application of 2-2 in
sensing of Ag+ and La3+, where the original blue short-
wavelength band is strongly quenched and new long-wavelength
yellow bands appear. These findings, coupled with those of the
nucleosides alone, suggest that DNAs containing 1 and 2 might
have useful ion sensing applications in both single-stranded and
double-stranded forms. Future studies will investigate this in
detail.
In summary, the present compounds show promise not only
as sensors alone but also as components for introducing metal
ion sensing capability into DNA. There is now a broad number
of literature reports describing fluorescence sensing of metal
ions by ligands in solution,15,16 but few if any of these are
directly adaptable to DNA.16h We anticipate that the use of DNA
as a scaffold for this fluorescence sensing will be advantageous
by adding aqueous solubility to these heterocyclic species, thus
allowing possible applications in biological systems and aqueous
waste streams. In addition, the use of DNA double-helical or
folded structure may allow for useful cooperative sensing
over two steps). H NMR (400 MHz, CDCl3) δ 8.56 (d, J ) 8.8 Hz,
1H), 8.25 (d, J ) 8.4 Hz, 1H), 8.09 (d, J ) 8.4 Hz, 1H), 7.83 (d, J )
8.0 Hz, 1H), 7.71-7.65 (m, 2H), 7.51 (t, J1 ) 7.3 Hz, 1H), 6.81 (dd,
J1 ) 9.2, J2 ) 2.4 Hz, 1H), 6.28 (s, 1H), 2.84 (s, 6H). 13C NMR (100.7
MHz, CDCl3): δ 149.4, 149.2, 149.1, 147.8, 137.4, 137.3, 136.0, 129.1,
128.5, 128.1, 127.1, 120.6, 119.4, 111.6, 93.5, 41.5. HRMS (DEI):
[M]+ calcd for C18H16N4, 288.1375; found, 288.1375.
1-(5-N,N-Dimethylamino-2-(2-pyridinyl)benzimidazole)-1′-R-deox-
yriboside (1). 5-N,N-Dimethylamino-2-(2-pyridinyl)-1H-benzimidazole
(1.1 g, 4.54 mmol) and NaH (0.20 g, 4.99 mmol) were charged into a
100 mL round-bottom flask. A 15 mL amount of N,N-dimethylaceta-
mide was added into the flask via syringe under an atmosphere of argon.
The reaction mixture was stirred at room temperature for 30 min, then
Hoffer’s chlorosugar (2.12 g, 5.39 mmol) was added in one portion,
and the reaction mixture was stirred for 2 h at room temperature. The
solvent was evaporated under vacuum. The crude mixture was extracted
with water and CH2Cl2. The organic layer was dried with Mg2SO4 and
evaporated under reduced pressure. The crude product was dissolved
in 30 mL of CH2Cl2/MeOH (1:1), and then 4.5 mL of NaOMe (0.5 N
in MeOH) was added into the flask via syringe under an atmosphere
of argon. The reaction mixture was stirred at room temperature for 1
h. The solvent was evaporated under reduced pressure. The crude
(16) (a) Dujols, V.; Ford, F.; Czarnik, A. W. J. Am. Chem. Soc. 1997, 119,
7386-7387. (b) Zeng, H. H.; Thompson, R. B.; Maliwal, B. P.; Fones, G.
R.; Moffett, J. W.; Fierke, C. A. Anal. Chem. 2003, 75, 6807-6812. (c)
Obare, S. O.; Murphy, C. J. Inorg. Chem. 2001, 40, 6080-6082. (d) Nolan,
E. M.; Lippard, S. J. J. Am. Chem. Soc. 2003, 125, 14270-14271. (e) Kwon,
J. Y.; Jang, Y. J.; Lee, Y. J.; Kim, K. M.; Seo, M. S.; Nam, W.; Yoon, J.
J. Am. Chem. Soc. 2005, 127, 10107-10111. (f) Bricks, J. L.; Kovalchuk,
A.; Trieflinger, C.; Nofz, M.; Buschel, M.; Tolmachev, A. I.; Daub, J.;
Rurack, K. J. Am. Chem. Soc. 2005, 127, 13522-13529. (g) Matsushita,
M.; Meijler, M. M.; Wirsching, P.; Lerner, R. A.; Janda, K. D. Org. Lett.
2005, 7, 4943-4946. (h) Mokhir, A.; Kiel, A.; Herten, D. P.; Kraemer, R.
Inorg. Chem. 2005, 44, 5661-5666. (i) Yang, L.; McRae, R.; Henary, M.
M.; Patel, R.; Lai, B.; Vogt, S.; Fahrni, C. J. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 11179-11184.
(15) (a) Zalewski, P. D.; Forbes, I. J.; Betts, W. H. Biochem. J. 1993, 296,
403-408. (b) Maruyama, S.; Kikuchi, K.; Hirano, T.; Urano, Y.; Nagano,
T. J. Am. Chem. Soc. 2002, 124, 10650-10651. (c) Shults, M. D.; Pearce,
D. A.; Imperiali, B. J. Am. Chem. Soc. 2003, 125, 10591-10597. (d) Chang,
C. J.; Jaworski, J.; Nolan, E. M.; Sheng, M.; Lippard, S. J. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 1129-1134. (e) Yuasa, H.; Miyagawa, N.;
Nakatani, M.; Izumi, M.; Hashimoto, H. Org. Biomol. Chem. 2004, 2,
3548-3556. (f) Wu, Y.; Peng, X.; Guo, B.; Fan, J.; Zhang, Z.; Wang, J.;
Cui, A.; Gao, Y. Org. Biomol. Chem. 2005, 3, 1387-1392. (g) Woodroofe,
C. C.; Won, A. C.; Lippard, S. J. Inorg. Chem. 2005, 44, 3112-3120. (h)
Komatsu, K.; Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am. Chem.
Soc. 2005, 127, 10197-10204.
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Fluorescent Nucleoside Metal Ligands
A R T I C L E S
products were purified by silica gel chromatography (eluting with
hexane/ethyl acetate 1:1 to 1:3) to give the desired product (562 mg,
1.59 mmol, 35% yield). 1H NMR (400 MHz, CDCl3/ CD3OD (1:1)) δ
8.65 (d, J ) 4.8 Hz, 1H), 8.03 (d, J ) 8.0 Hz, 1H), 7.90 (td, J1 ) 8.0,
J2 ) 2.0 Hz, 1H), 7.56 (d, J ) 8.4 Hz, 1H), 7.41 (dd, J1 ) 7.6, J2 ) 5.2
Hz, 1H), 7.34 (d, J ) 2.4 Hz, 1H), 7.12 (t, J ) 8.0 Hz, 1H), 6.94 (dd,
J1 ) 9.2, J2 ) 2.4 Hz, 1H), 4.55-4.50 (m, 1H), 4.31-4.27 (m, 1H),
3.75 (dd, J1 ) 12.0, J2 ) 3.6 Hz, 1H), 3.65 (dd, J1 ) 12.0, J2 ) 4.8 Hz,
1H), 3.02 (s, 6H), 2.96-2.88 (m, 1H), 2.72-2.65 (m, 1H). 13C NMR
(100.7 MHz, CDCl3/CD3OD (1:1)): δ 150.0, 149.1, 148.9, 148.8, 137.5,
135.2, 134.6, 125.1, 124.1, 119.5, 112.4, 96.9, 87.2, 86.8, 71.0, 62.2,
41.4, 39.2. MS (EI): m/z ) 355.4 [M + H]+. HRMS (DCI): [M +
H]+ calcd for C19H23N4O3, 355.1770; found, 355.1780.
deprotection of the 5′-terminal DMT. After 30 min at ambient
temperature, the AcOH was evaporated under reduced pressure. The
residue was diluted with water (2 mL), and the solution was purified
by HPLC again. Modified oligonucleotides in the study were character-
ized by matrix-assisted laser-desorption-ionization time-of-flight (MALDI-
TOF) mass spectrometry (see Supporting Information).
Fluorescence Measurements. Fluorescence measurements were
carried out on a Spex Fluorolog 3 fluorescence spectrometer equipped
with a Lauda Brinkmann RM 6 temperature controller. Fluorescence
quantum efficiency (φsamp) was obtained by comparing the integrated
fluorescence spectra of a sample (Isamp) with that of a reference sample
(Iref) having a known fluorescence quantum efficiency (φref). We used
9,10-diphenylanthracene (10 µM) dissolved in EtOH (φref ) 0.81,
excitation at 366 nm) as a standard. Compounds 1 and 2 were prepared
as 10 µM solutions in methanol. Quantum yields were calculated by
the following equation
1-(5-N,N-Dimethylamino-2-(2-quinolinyl)benzimidazole)-1′-R-deoxy-
riboside (2). 5-N,N-Dimethylamino-2-(2-quinolinyl)-1H-benzimidazole
(1.13 g, 3.9 mmol) and NaH (264 mg, 6.6 mmol) were charged into a
100 mL round-bottom flask. A 20 mL amount of N,N-dimethylaceta-
mide was added into the flask via syringe under an atmosphere of argon.
The reaction mixture was stirred at room temperature for 30 min; then
solvent was evaporated under vacuum. The crude mixture was extracted
with water and CH2Cl2. The organic layer was dried with Mg2SO4.
The crude products were purified by silica column chromatography
(eluting with hexane/ethyl acetate/dichloromethane 2:1:2 to 1:1:1) to
give the desired product (971.4 mg, 1.52 mmol, 39% yield). This bis-
toluoyl-protected deoxyriboside (971.4 mg 1.52 mmol) was dissolved
in 50 mL of CH2Cl2/MeOH (1:1), and then 10 mL of NaOMe solution
(0.5 N in MeOH) was added into the flask via syringe under an
atmosphere of argon. The reaction mixture was stirred at room
temperature for 1 h. The solvent was then evaporated under reduced
pressure, and the crude products were purified by silica column
chromatography (eluting with hexane/ethyl acetate 1:1 to 1:3) to give
φsamp ) φref × (Aref/Asamp)(ηsamp/ηref)2(Isamp/Iref)
Absorption spectra were measured with a Cary I UV-vis spectrometer,
and excitation and emission spectra were observed by a Spex Fluorolog
3 fluorescence spectrometer. Fluorescence lifetimes were measured on
a Photon Technologies, Inc. Easylife LS instrument; a 465 nm pulsed
LED was utilized for excitation, and a 535 nm long-pass filter selected
the desired emission wavelengths.
Screening of Metal Ions. Seventeen metal salts were tested for
fluorescence responses with 1 and 2: KCl, MgCl2, AgNO3, NiCl2,
PdCl2, PtCl2, CuCl2, ZnCl2, CoCl2, AuCl, LaCl3, EuCl3, AuCl3, CdCl2,
HgCl2, MnCl2, CaCl2. All metal salt solutions were prepared as stock
solutions (5.0 mM) in DMSO. Note that AuCl had poor solubility and
left some precipitate. Nucleosides 1 and 2 were separately prepared as
50 µM solutions in methanol, and each metal salt stock solution was
added to give 2-fold excess metal concentration (100 µM). Photographs
were taken over a UV light box (354 nm), and fluorescence spectra
were measured as described above.
Crystal Structure Determination. Single crystals of free ligand-
nucleosides 1 and 2 were grown by a bilayer method. The compounds
were dissolved in a 1:1 mixture of dry CHCl3 and MeOH. These
solutions (ca. 1 mL) were divided to NMR tubes (5 × 200 mm), and
hexane (ca. 2 mL) was added very slowly, making a second layer. At
the junction between the sample layer (lower) and the hexane (upper)
layer we obtained crystals for X-ray diffraction. The X-ray data for 1
and 2 were collected on a Siemens SMART-APEX CCD single-crystal
X-ray diffractometer. The structures were solved and refined by full-
matrix least-squares techniques on F2 using SHELX-97. Additional
details are in the Supporting Information.
1
the desired product (541 mg, 1.34 mmol, 88% yield). H NMR (400
MHz, DMSO-d6) δ 8.45 (d, J ) 8.8 Hz, 1H), 8.30 (d, J ) 8.8 Hz,
1H), 8.06 (d, J ) 8.4 Hz, 1H), 7.99 (d, J ) 7.6 Hz, 1H), 7.80 (t, J )
8.0 Hz, 1H), 7.66-7.55 (m, 3H), 7.33 (s, 1H), 6.88 (dd, J1 ) 8.8, J2 )
2.4 Hz, 1H), 5.63 (br, 1H), 4.95 (t, J ) 5.2 Hz, 1H), 4.45 (br, 1H),
3.58-3.47 (m, 2H), 3.04-2.93 (m, 7H), 2.49-2.43 (m, 1H). 13C NMR
(100.7 MHz, DMSO-d6): δ 150.7, 148.7, 148.3, 147.1, 137.6, 135.9,
135.7, 131.0, 129.8, 128.6, 128.1, 127.7, 122.3, 120.6, 112.0, 96.8,
88.2, 87.3, 71.4, 62.5, 41.6, 39.2. MS (EI): m/z ) 405.2 [M + H]+.
HRMS (DCI): [M + H]+ calcd for C23H25N4O3, 405.1927; found,
405.1924.
Oligonucleotide Synthesis. We prepared the 5′-O-DMTr-protected
3′-2-cyanoethyl phosphoramidite building blocks of 1 and 2 following
standard methods (see Supporting Information). We directly applied
them using solid-phase oligodeoxynucleotide (ODN) synthesis protocols
on an automated DNA synthesizer (Applied Biosystems 394 DNA/
RNA synthesizer). All oligodeoxynucleotides were synthesized on 1.0
µmol scale using standard â-cyanoethyl phosphoramidite chemistry but
with extended (200 s) coupling times, double coupling, and high
concentration (0.13 mM) for modified nucleosides. Stepwise coupling
yields for nonnatural nucleosides were all greater than 95% as
determined by trityl monitoring. The synthesized oligonucleotides were
cleaved from the solid support by treatment with 30% aqueous NH4OH
(1.0 mL) for 10 h at 55 °C. The crude products from the automated
ODN synthesis were lyophilized and diluted with distilled water (2
mL). The ODNs were purified by HPLC (Zorbax ODS column, 9.4 ×
250 mm). The fractions containing the purified ODN were pooled and
lyophilized. Aqueous AcOH (80%) was added to the ODN for
Acknowledgment. This work was supported by the Army
Research Office and the U.S. National Institutes of Health
(GM067201) and by a fellowship to S.J.K. from the KOSEF
Foundation. We thank Dr. J. Wilson for assistance with
fluorescence lifetime measurements.
Supporting Information Available: Details of the synthesis
and characterization of phosphoramidite derivatives of 1 and
2; characterization of modified oligonucleotides; fluorescence
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
JA0581806
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