FRET enabled real time detection of RNA-small molecule binding

Article abstract of DOI:10.1021/ja905767g

A robust analysis and discovery platform for antibiotics targeting the bacterial rRNA A-site has been developed by incorporating a new emissive U surrogate into the RNA and labeling the aminoglycosides with an appropriate fluorescence acceptor. Specifical

Full text of DOI:10.1021/ja905767g

Published on Web 11/12/2009
FRET Enabled Real Time Detection of RNA-Small Molecule
Binding
Yun Xie, Andrew V. Dix, and Yitzhak Tor*
Department of Chemistry and Biochemistry, UniVersity of California, San Diego,
La Jolla, California 92093-0358
Received July 12, 2009; E-mail: ytor@ucsd.edu
Abstract: A robust analysis and discovery platform for antibiotics targeting the bacterial rRNA A-site has
been developed by incorporating a new emissive U surrogate into the RNA and labeling the aminoglycosides
with an appropriate fluorescence acceptor. Specifically, a 5-methoxyquinazoline-2,4(1H,3H)-dione-based
emissive uracil analogue was identified to be an ideal donor for 7-diethylaminocoumarin-3-carboxylic acid.
This donor/acceptor pair displays a critical Fo¨rster radius (R0) of 27 Å, a value suitable for an A-site-
aminoglycoside assembly. Titrating the coumarin labeled aminoglycosides into the emissive A-site construct,
labeled at position U1406, shows a decrease in donor emission (at 395 nm) and concurrent increase of
the acceptor emission (at 473 nm). Titration curves, obtained by fitting the donor’s emission quenching or
the augmentation of the acceptor’s sensitized emission, faithfully generate EC50 values. Titration of unlabeled
ligands into the preformed FRET complex showed a continuous increase of the donor emission, with a
concurrent decrease of the acceptor emission, yielding valuable data regarding competitive displacement
of aminoglycosides by A-site binders. Detection of antibiotic binding is therefore not dependent on changes
in the environment of a single fluorophore, but rather on the responsive interaction between two
chromophores acting as a FRET pair, facilitating the determination of direct binding and competitive
displacement events with FRET accuracy.
Introduction
The ribosomal decoding site, also known as the A-site,
ensures high fidelity in protein synthesis by appraising
codon-anticodon matching.1-3 Numerous naturally occurring
potent antibiotics, particularly the aminoglycosides family, have
evolved to meddle with this precise monitoring and corrupt
bacterial protein production.4-8 Specifically, the aminoglyco-
sides bind a small loop within the 16S rRNA and interfere with
the conformational flexibility of A1492 and A1493, two key
adenine residues (Figure 1a).9-12 The aminoglycosides stabilize
(1) Green, R.; Noller, H. F. Annu. ReV. Biochem. 1997, 66, 679–716.
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D.; Lynch, S. R.; Recht, M. I.; Yoshizawa, S. Aminoglycosides
Antibiotics and Decoding. In The Ribosome: Structure, Function,
Antibiotics, and Cellular Interactions; Garrett, R. A., Douthwaite, S. R.,
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Press: Washington, DC, 2000; pp 419-429.
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124–130.
Figure 1. (a) Binding of aminoglycosides to the bacterial A-site impacts
the placement and dynamics of the unpaired A1492 and 1493 residues. (b)
By replacing one of the nucleosides in the A-site with an isosteric emissive
nucleoside analogue as a donor (D) and tagging the antibiotics with an
appropriate acceptor (A), binding and displacement events can be accurately
monitored using FRET.
(4) Gale, E. F.; Cundliffe, E.; Renolds, P. E.; Richmond, M. H.; Waring,
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an RNA conformation similar to the one induced by the cognate
acyl-tRNA-mRNA complex, causing the ribosome to lose its
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Xie et al.
ability to distinguish between the correct and incorrect
anticodon-codon hybrids.13-18
The A-site, the Achilles heel of the bacterial ribosome, has
remained one of the most attractive targets for the discovery and
development of new antibiotics.19-25 A number of tools have been
developed to assess ligand binding to this unique RNA site.26,27
Fluorescent A-site constructs, which contain emissive and respon-
sive nucleoside analogues, such as 2-aminopurine at positions 1492
or 1493, have shown great promise.28-31 Their fluorescence
response is, however, antibiotic-dependent.28,32 To overcome
this drawback and devise a robust analysis and discovery
platform for A-site binders, we have envisioned an approach
where detection of antibiotic binding is not dependent on
changes in the environment of a single fluorophore, but rather
on the interaction between two chromophores acting as a Fo¨rster
Resonance Energy Transfer (FRET) pair (Figure 1b). In this
fashion, we hypothesized that one could follow direct binding
of appropriately labeled antibiotics and their displacement by
competing binders with “FRET accuracy” without relying on a
fluorescent nucleobase as the sole sensing moiety (Figure 1b).
Figure 2. Structure of the donor 1 and acceptor 2, as well as 3 (the parent
donor heterocycle) and 4 (an isomeric methoxy substituted heterocycle).40
as a fluorescent donor, is incorporated into the A-site and
coumarin-labeled aminoglycosides act as FRET acceptors.
Results
Selection of the Donor and Acceptor. While diverse FRET
donors and acceptors exist, small chromophores capable of
serving as nonperturbing nucleobase surrogates are exceedingly
rare.34 In pursuing such unique probes, we have synthesized,
examined, and implemented new emissive nucleosides with
absorption maxima between 290 and 350 nm and emission bands
between 350 and 440 nm.35-39 We have identified 5-methoxy-
quinazoline-2,4-(1H,3H)-dione 1, an emissive uracil analogue,
as an ideal donor for 7-diethylaminocoumarin-3-carboxylic acid
2 (Figure 2). The extinction coefficient of 2 at 320 nm, the
absorption maximum of 1, is minimal, while the emission of 1,
centered around 395 nm (ΦF ) 0.16), overlaps perfectly with
the absorption band of 2, which emits at 473 nm (ΦF ) 0.83),
suggesting excellent FRET pairing (Figure 3).40,41 Indeed, the
critical Fo¨rster radius (R0) for the 1/2 pair was experimentally
determined to be 27 Å, a suitable value for the proposed A-site
assembly.42
To realize such a system, we have relied on two key features:
(a) one of the native nucleobases in the A-site, proximal to the
binding site, but not part of it, had to be replaced with an
emissive isomorphic nucleobase analogue acting as a FRET
donor, and (b) aminoglycosides, the cognate binders of the
A-site, had to be labeled with an appropriate FRET acceptor in
positions that are not essential for RNA binding (Figure 1b).33
By monitoring the interactions of ligands and their RNA targets
based on distance and location, convoluting factors such as
modes of binding can be eliminated when studying binding and
displacement. Here we describe the design, assembly, and utility
of such a FRET-friendly and minimally perturbed RNA
construct, where a new fluorescent uridine analogue, serving
The presence and position of the methoxy group on the
quinazoline-2,4-(1H,3H)-dione core impact the photophysical
properties and are critical for the compatibility of the FRET
partners, as evident by a comparison of the parent and to two
isomeric methoxy substituted heterocycles. The emission profile
of quinazoline-2,4-(1H,3H)-dione 3, the parent unsubstituted
heterocycle, is hypsochromically shifted compared to the
emission of 1, with a maximum at 370 nm (Figure 4). The
emission of the isomeric 3-methoxyquinazoline-2,4-(1H,3H)-
dione 4 is even further shifted to the blue with the emission
maximum at 356 nm (Figure 4). Importantly, glycosylation of
these heterocycles to yield the corresponding nucleosides
negligibly impacts the photophysical properties.40
(13) Purohit, P.; Stern, S. Nature 1994, 370, 659–662.
(14) Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D. Science
1996, 274, 1367–1371.
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(24) Hermann, T. Biochimie 2006, 88, 1021–1026.
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Liu, M.; Vakulenko, S. B.; Chow, C. S.; Mobashery, S. J. Am. Chem.
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(34) See, however: (a) Mart´ı, A. A.; Jockusch, S.; Li, Z.; Ju, J.; Turro,
N. J. Nucleic Acids Res. 2006, 34, e50. (b) Borjesson, K.; Preus, S.;
El-Sagheer, A. H.; Brown, T.; Albinsson, B.; Wilhelmsson, L. M.
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(28) Kaul, M.; Barbieri, C. M.; Pilch, D. S. J. Am. Chem. Soc. 2004, 126,
3447–3453.
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(33) Aminoglycosides have been fluorescently labeled before, but typically
by modifying the amines that are also essential for specific RNA
binding. See: (a) Wang, Y.; Hamasaki, K.; Rando, R. R. Biochemistry
1997, 36, 768–779. (b) Hamasaki, K.; Rando, R. R. Biochemistry 1997,
36, 12323–12328. (c) Hamasaki, K.; Ueno, A. Bioorg. Med. Chem.
Lett. 2001, 11, 591–594.
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1334–1338.
(40) See Supporting Information for additional details.
(41) Note the red-shifted absorption of 1 compared to the native nucleobases
(λmax 250-270 nm) and the intense blue emission of the acceptor.
(42) Based on crystal structures (PDB 2ET4 and 1LC4), the estimated
distance between U1406 and the fluorophore on 11 or 12 would be
less than 15 Å.
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Real Time Detection of RNA-Small Molecule Binding
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Scheme 1. Synthesis of the Modified Nucleoside 5 and Its
Phosphoramidite 8a
Figure 3. Normalized absorption (---) and emission (s) spectra of 1 (black)
and 2 (red) in water.40
a Reagents: (a) NaOCN, NaOH, conc. HCl, water, 90%. (b) (i) N,O-
bis(trimethylsilyl)acetamide, CF3SO3Si(CH3)3, ꢀ-D-ribofuranose 1-acetate
2,3,5-tribenzoate, CH3CN; (ii) conc. NH4OH, 81%. (c) DMTrCl, Et3N,
pyridine, 85%. (d) iPr2NEt, nBu2SnCl2, (iPr3SiO)CH2Cl, ClCH2CH2Cl, 30%.
(e) iPr2NEt, (iPr2N)P(Cl)O-CH2CH2CN, ClCH2CH2Cl, 60%.
Figure 4. Normalized absorption (---) and emission (s) spectra of 4 (black),
3 (red), and 1 (blue) in water.
Synthesis and Photophysical Properties of Modified RNA.
To modify the A-site RNA oligonucleotide, nucleoside 5 and
its phosphoramidite 8 were prepared as shown in Scheme 1.40
The commercially available 2-methoxy-5-aminobenzoic acid
was cyclized with sodium cyanate to yield the emissive
heterocycle 1, which was glycosylated to provide the modified
nucleoside 5 after saponification of all esters. Protection of the
5′-hydroxyl as the 4,4′-dimethoxytrityl (DMT) derivative and
the 2′-hydroxyl as the (trisisopropylsiloxy)methyl (TOM)
derivative, followed by phosphitylation of the 3′-hydroxyl,
provided phosphoramidite 8 (Scheme 1).40
Standard solid-phase oligonucleotide synthesis was utilized
to prepare the 27-mer bacterial A-site model construct 10,43
where the fluorescent U analogue 5 replaces U1406 (Figure 5).44
The oligonucleotide was purified by PAGE, and MALDI-TOF
mass spectrometry confirmed its full length and the presence
of the intact emissive nucleoside 5.40 Thermal denaturation
Figure 5. Unmodified 9 and modified 10 A-site constructs.
studies showed that the incorporated modified nucleobase had
minimal impact on the stability of the folded RNA. The emissive
RNA construct 10 displayed a Tm of 71 °C, while the unmodified
control A-site construct 9 had a Tm of 72 °C (cacodylate buffer
pH 7.0, NaCl 1.0 × 10-1 M).40 The emission profile of the
emissive A-site construct 10, excited at 320 nm, resembled that
of the parent nucleoside in water, with a maximum emission at
395 nm, albeit with a lower emission quantum yield (ΦF )
0.03).45
(43) The minimal A-site construct has been demonstrated to be an
autonomous RNA domain capable of mimicking the function and
antibiotics recognition features of the 16S rRNA. See ref 13 and
24.
(44) Modification of U1406 with isosteric nucleosides has been demon-
strated to have a minimal detrimental effect on the A-site construct’s
folding and antibiotic recognition properties. See ref 36.
(45) Such a decrease in the quantum yield is common among fluorescent
nucleosides upon incoporation into an RNA or DNA duplex. See ref
34-39 and (a) Hawkins, M. E.; Pfleiderer, W.; Balis, F. M.; Porter,
D.; Knutson, J. R. Anal. Biochem. 1997, 244, 86–95. (b) Hawkins,
M. E. Cell Biochem. Biophys. 2001, 34, 257–281.
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Scheme 2. Structure of the Aminoglycosides Used for Binding and Displacement Studies (11 and 12) and the Synthesis of
Coumarin-Labeled Neomycin 11a
a Reagents: (a) 7-(Et2N)coumarin-3-carboxylic acid, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, 4-dimethylaminopyridine, iPr2EtN, Cl2CH2, 84%.
(b) Trifluoroacetic acid, triisopropylsilane, CH2Cl2 82%.
Synthesis and Photophysical Properties of Modified Aminogly-
cosides. To complete the FRET pair, neomycin B and tobramy-
cin, two distinct 2-deoxystreptamine-based aminoglycosides,
were conjugated to 7-diethylaminocoumarin-3-carboxylic acid
to afford 11 and 12, respectively, by modifying their primary
hydroxymethyl groups, previously shown not to be critical for
RNA binding (Scheme 2).7,17,40,46 Using previously reported
procedures, the Boc-protected aminoglycosides were activated
at the primary hydroxymethyl group on the ribose by 2,4,6-
triisopropylbenzenesulfonyl chloride (TIBS-Cl).47 Treatment
with ammonia in MeOH provided the aminomethyl substituted
product (e.g., 13, scheme 2). The newly installed amine was
coupled to the coumarin carboxylic acid, using standard peptide
coupling conditions. The resulting Boc-protected, coumarin-
labeled aminoglycosides were treated with trifluoroacetic acid
to remove the Boc groups, yielding 11 and 12 as their TFA
salts. The emission profile of 11 and 12 resembled that of the
parent heterocycle 2, displaying an emission maximum at 473
nm upon excitation at 400 nm, maintaining a quantum yield of
80%.
Binding of Coumarin-Labeled Aminoglycosides to the
Fluorescently Labeled A-Site. Titration of the coumarin-labeled
neomycin B derivative 11 into the emissive A-site construct,
excited at 320 nm, showed a continuous decrease of the donor
Figure 6. (a) Example of binding study. 11 is titrated into 10. (b) Example
emission at 395 nm, with a concomitant increase of the acceptor
emission at 473 nm (Figure 6a).40 Similarly, titrating the
coumarin-labeled tobramycin derivative 12 into the emissive
A-site construct, excited at 320 nm, showed a decrease of the
donor emission at 395 nm and a concurrent increase of the
of displacement study. Tobramycin is titrated into 10 saturated with 12.
Conditions: 10 (1 × 10-6 M), cacodylate buffer pH 7.0 (2.0 × 10-2 M),
NaCl (1.0 × 10-1 M).
acceptor emission at 473 nm. Figure 7 graphically illustrates
the titration curves generated by plotting the fractional fluores-
cence saturation of the donor and acceptor. Identical EC50 values
of 0.84 ((0.03) × 10-6 M are obtained from curve fitting the
quenching of the donor’s emission or the augmentation of the
acceptor’s sensitized emission (Figure 7a). Analogous behavior
(46) Wang, H.; Tor, Y. J. Am. Chem. Soc. 1997, 119, 8734–8735.
(47) (a) Wang, H.; Tor, Y. Angew. Chem., Int. Ed. 1998, 37, 109–111. (b)
Michael, K.; Wang, H.; Tor, Y. Bioorg. Med. Chem. 1999, 7, 1361–
1371. (c) Boer, J.; Blount, K. F.; Luedtke, N. W.; Elson-Schwab, L.;
Tor, Y. Angew. Chem., Int. Ed. 2005, 44, 927–932.
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Figure 7. Fractional fluorescence saturation of the donor (9) in the labeled A-site and the emissive acceptor (O) in the labeled aminoglycosides in the
following experiments: (a) titration of 10 with 11; (b) titration of 10 with 12; (c) displacement of the A-site bound 11 with neomycin; (d) displacement of
the A-site bound 12 with neomycin; (e) displacement of the A-site bound 11 with tobramycin; (f) displacement of the A-site bound 12 with tobramycin.
Conditions: 10 (1.0 × 10-6 M), cacodylate buffer pH 7.0 (2.0 × 10-2 M), NaCl (1.0 × 10-1 M).
is observed for the labeled tobramycin derivative 12 (Figure
7b), yielding a higher EC50 value of 2.3 ((0.2) × 10-6 M.
place. It is the only validated drug target.10,11,23,25,48-50 This
conformational switch is the cognate binding site of aminogly-
coside antibiotics, a diverse family of natural products evolved
to interfere with the decoding process in bacteria.2 These highly
effective bactericidal agents alter the conformational flexibility
of two key residues, A1492 and A1493, inducing a conformation
similar to the one found in the cognate acyl-tRNA-mRNA
complex (Figure 1).51,52 This impairs the fidelity of ribosomal
protein synthesis, ultimately leading to bacterial death.
The utility of aminoglycosides has declined over the years
due to their diminished potency in resistant bacteria, their
adverse side effects, and, consequently, the availability of newer,
potent, and safer drugs.23,25,27,53 The rapid emergence of resistant
pathogens and the time-consuming and minimally productive
development of new broad-spectrum antibiotics have created,
however, alarming circumstances, where new antibiotics are
needed to replace existing compromised drugs.54,55 These
developments have generated a renewed interest in the A-site
and triggered the development of new tools to assess ligand
binding to this bacterial RNA site. In particular, fluorescent
Displacement of A-site Bound Coumarin-Labeled Aminogly-
cosides with Unlabeled RNA Binders. Preformation of the FRET
complex by saturating the modified A-site construct with the
labeled neomycin or tobramycin derivatives facilitates the
evaluation of the relative A-site affinity of other antibiotics by
competition experiments, where the resonance energy transfer
is disrupted by displacing the bound coumarin-labeled antibi-
otic.40 Titration of the unlabeled aminoglycosides into the
preformed FRET complex, excited at 320 nm, showed continu-
ous increase of the donor emission at 395 nm, with a concurrent
decrease of the acceptor emission at 473 nm (Figure 6b).
Titration curves were generated by plotting the fractional
fluorescence saturation of the donor and acceptor against the
concentration of the unlabeled aminoglycoside. Figure 7 (panels
c-f) provides selected examples (see also Figures S4,S5).
Competing off a bound neomycin, one of the strongest A-site
binders, with other aminoglycosides (paromomycin, apramycin,
hygromycin, kanamycin A, Figure 8), requires relatively high
competitor concentrations (Table 1). In contrast, the same
competitor antibiotics more easily displace the lower affinity
tobramycin. Additionally, two synthetic amino-aminoglycosides
(6′′-amino-6′′-deoxy-kanamycin A and 6′′-amino-6′′-deoxy-
tobramycin prepared in our lab47a) are found to be rather potent
A-site binders (Table 1). Importantly, spermidine, serving as a
negative control polyamine, is unable to displace any of the
bound aminoglycosides (up to 5 × 10-3 M).
(48) Schluenzen, F.; Tocilj, A.; Zarivach, R.; Harms, J.; Gluehmann, M.;
Janell, D.; Bashan, A.; Bartels, H.; Agmon, I.; Franceschi, F.; Yonath,
A. Cell 2000, 102, 615–623.
(49) Yoshizawa, S.; Fourmy, D.; Puglisi, J. D. Science 1999, 285, 1722–
1725.
(50) O’Connor, M.; Brunelli, C. A.; Firpo, M. A.; Gregory, S. T.;
Lieberman, K. R.; Stephen Lodmell, J.; Moine, H.; Ryk, D. I. V.;
Dahlberg, A. E. Biochem. Cell Biol. 1995, 73, 859–868.
(51) Vicens, Q.; Westhof, E. Structure 2001, 9, 647–658.
(52) Ogle, J. M.; Brodersen, D. E.; Clemons, W. M., Jr.; Tarry, M. J.;
Carter, A. P.; Ramakrishnan, V. Science 2001, 292, 897–902.
(53) Vakulenko, S. B.; Mobashery, S. Clin. Microbiol. ReV. 2003, 16, 430–
450.
(54) Smolinski, M. S.; Hamburg, M. A.; Lederberg, J. Microbial Threats
to Health: Emergence, Detection, and Response; National Academy
of Sciences: Washington DC, 2003.
(55) Wax, R. G.; Lewis, K.; Salyers, A.; Taber, H. Bacterial Resistance to
Antimicrobials, 2nd ed.; CRC Press: Boca Raton, FL, 2007.
Discussion
Among the RNA targets explored over the past two decades,
the bacterial ribosomal decoding site (or A-site) holds a unique
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Figure 8. Aminoglycosides used for displacement studies.
Table 1. IC50 Values of Aminoglycosides Competing off 11 and
In designing such chromophoric RNA-small molecule as-
semblies, two major issues need to be addressed. The first
involves the position and type of modification of the RNA target
with a donor, and the second, which is of related significance
and is partially coupled, is the selection and placement of an
appropriate chromophoric partner. The identification of the
donor is the most challenging, as it must fulfill strict functional
criteria: it needs to be small and structurally nonperturbing, while
at the same time displaying useful photophysical characteristics
that can be matched to a suitable and small acceptor. Due to its
imposed small molecular footprint and high similarity to the
native nucleobases, such a nucleobase analogue is likely to emit
below or close to the visible range with a moderate emission
quantum yield at best. This, in turn, impacts the selection of
the acceptor to be placed on the aminoglycoside. In addition to
being structurally and functionally nonperturbing (i.e., minimally
impacting the recognition properties of the antibiotic), the
acceptor needs to have a high degree of spectral overlap with
the donor, while displaying intense emission in the visible range.
As a FRET pair, the selected donor and acceptor need to have
a critical Fo¨rster radius that matches the recognition phenom-
enon and the anticipated distances between the RNA and the
bound antibiotic.
To confer useful emissive properties upon nucleic acids, while
minimally perturbing their folded structure, we have been
pursuing the development of isosteric/isomorphic nucleobase
analogues.35-39 These heterocyclic surrogates can replace a
native nucleobase without significantly altering the folding and
recognition features of the native target, but with the added
benefit of being fluorescent.60 Specifically, we have previously
demonstrated that replacing the U residue at position 1406 with
emissive nucleoside analogues retains the folding and antibiotic
recognition properties of the A-site.36,61 Its proximity to the
aminoglycoside binding site ensures adequate photophysical
12a
Displacement
of 11b
Displacement
of 12b
Aminoglycosides
Neomycin
Tobramycin
Paromomycin
Apramycin
Hygromycin
0.03 ( 0.01
0.50 ( 0.02
1.14 ( 0.08
3.00 ( 0.09
1.46 ( 0.06
1.73 ( 0.07
3.30 ( 0.09
0.18 ( 0.03
0.02 ( 0.01
0.02 ( 0.01
0.16 ( 0.03
0.06 ( 0.03
1.00 ( 0.05
1.00 ( 0.05
0.56 ( 0.04
1.61 ( 0.06
0.05 ( 0.02
0.01 ( 0.01
Amikacin
Kanamycin A
Amino-Kanamycin A
Amino Tobramycin
a Conditions: 10 (1 × 10-6 M), cacodylate buffer pH 7.0 (2.0 × 10-2
M), NaCl (1.0 × 10-1 M). b Aminoglycoside concentration is given in
10-3 M.
A-site constructs, which contain responsive nucleoside analogues
such as 2-aminopurine, have proven useful,56-59 although their
response is antibiotic-dependent, which is most likely due to
the presence of distinct binding modes, which impact the
emission readout. This could affect the ability of fluorescent
nucleosides to accurately respond to ligand binding. To create
a robust analysis and discovery platform for A-site binders, we
have developed a FRET-enabled assembly, where the A-site
serves as the donor and the aminoglycoside as the acceptor.
The detection of antibiotic binding and competitive displacement
is highly sensitive and is no longer dependent on arbitrary
changes in the environment of a single fluorophore, but rather
on the interaction between two matching chromophores acting
as a FRET pair (Figure 1b).
(56) Ward, D. C.; Reich, E.; Stryer, L. J. Biol. Chem. 1969, 244, 1228–
1237.
(57) Kawai, M.; Lee, M. J.; Evans, K. O.; Nordlund, T. M. J. Fluorescence
2001, 11, 23–32.
(58) Jean, J. M.; Hall, K. B. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 37–
41.
(59) Rachofsky, E. L.; Osman, R.; Ross, J. B. A. Biochemistry 2001, 40,
(60) Tor, Y. Tetrahedron 2007, 63, 3425–3426.
946–956.
(61) Srivatsan, S. G.; Tor, Y. Nat. Protocols 2007, 2, 1547–1555.
9
17610 J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009

Real Time Detection of RNA-Small Molecule Binding
A R T I C L E S
place of native U1406 in the bacterial A-site for affinity assays
to recognize RNA-small molecule interactions.36,61 These
modifications have been found to be minimally perturbing and
effective at monitoring binding events. Thus, we chose to replace
U1406 with the fluorescent U analogue 5 for our labeled A-site
model. According to thermal denaturation studies, the substitu-
tion of U1406 by 5 appeared to be insignificantly destabilizing,
as the modified construct 10 and unmodified control construct
9 had essentially the same melting temperatures within error.
The photophysical properties of 5 remained adequate in the
A-site construct. The emission maximum of 10 was the same
as that of fluorescent ribonucleoside 5 at 395 nm; however, the
emission quantum yield dropped from 0.16 to 0.03. Such a
diminished quantum yield upon incorporation into a RNA or
DNA duplexes is not uncommon among fluorescent nucleo-
sides.45 Nevertheless, with an almost perfect spectral overlap
between 1 and 2, the “modified” Fo¨rster radius remains suitable
for this assembly (R0 ) 20 Å). As for the acceptors in our FRET
pair, conjugating 7-diethylaminocoumarin 2 to the aminogly-
cosides had little effect on its photophysics; 11 and 12 resembled
2 in absorption and emission profiles and maintained a high
emission quantum yield.
Figure 9. A model of neomycin bound to the A-site (PDB 2ET4). The
distance from the center of U1406 (green) to the primary 5′ hydroxymethyl
group on the ribose (orange) is less than 10 Å.
interaction with suitably labeled aminoglycoside, as shown in
the modeled structure (Figure 9). Out of the possible motifs,
we focused on a size-expanded U analogue with a methoxy
group in the 5 position, which is a superior isostere to similar
benzo[g]quinazoline nucleosides that have been previously
utilized.62-64
Titrating the labeled aminoglycosides into the donor-containing
A-site construct yielded the expected spectral behavior, with
continuous quenching of the donor’s emission and concomitant
enhancement of the acceptor’s sensitized emission. As stated above,
the ideal spectral overlap between 1 and 2 provides efficient energy
transfer even with the reduced quantum yield of the donor’s
emission within the RNA construct. Based on the efficiency of
energy transfer at saturation, the calculated FRET distance between
the bound coumarin-labeled neomycin 11 and tobramycin 12 to
the modified RNA construct 10 are 14 ( 1 Å and 12 ( 2 Å,
respectively. This is in good agreement with the distances predicted
byexaminingthesolid-statestructuresoftheseRNA-aminoglycoside
complexes.40,42 Consequently, binding and competition experiments
faithfully reproduce the expected trends in affinity of the diverse
ligands tested and suggest that this FRET assembly is indeed a
useful tool for the analysis of A-site binders. Furthermore, the
discovery of two high RNA affinity amino-aminoglycoside
derivatives, previously untested for A-site binding, demonstrates
the potential utility of this tool for the discovery of new A-site
binders.
The emission profile of the fluorescent nucleobase 1 comple-
ments the absorbance of 7-diethylaminocoumarin (Figure 3), a
commonly used fluorophore that can be conjugated to ami-
noglycosides at positions that do not significantly impact their
binding to the A-site (Figure 9).65 Coumarin-based fluorophores
are among the smallest organic chromophores displaying
emission in the visible range with high quantum yields (Φf >
0.5).66,67 Specifically, 7-diethylaminocoumarin lacks any sig-
nificant molar absorptivity at 320 nm, which facilitates selective
excitation of donor 1. It is worth noting that placing the electron-
donating methoxy group on the quinazoline-2,4-(1H,3H)-dione
skeleton is essential for maximizing the spectral overlap between
the donor and acceptor. The absence of the methoxy group, as
in the unsubstituted parent heterocycle 3, or its placement in
an electronically related position, such as in the isomeric 4,
causes a significant blue shift in the donors’ emission, diminishes
the spectral overlap, and lowers FRET efficiency between the
quinazoline and coumarin chromophores (Figure 4). We pos-
tulate that excited state stabilization (likely due to charge transfer
from the methoxy to the conjugated carbonyl generating
opposing dipoles in 1 only) leads to lower energy emission and
optimal spectral overlap between 1 and 2.
The results of FRET-monitored binding and displacement
experiments reveal distinct advantages over traditional methods
that rely on singly labeled RNA constructs. While the latter
can monitor the binding of small molecules to RNA targets,
multiple ligand binding modes might limit their accuracy and
dependability. In fact, the popular 2-AP-modified A-site
construct,56-59 while responding well to most aminoglycoside
antibiotics, does not reliably detect the binding of neomycin,
the strongest naturally occurring A-site binder.36 In addition,
singly labeled RNA constructs are unable to consistently perform
in evaluating competitive binding, as displacing a bound ligand
with an equipotent one is unlikely to be “visible” to a probe
only reporting the “bound” or “unbound” status of the RNA
construct. Fo¨rster Resonance Energy Transfer between strategi-
cally placed donors and acceptors, on the other hand, can clearly
overcome such limitations, as unequivocally demonstrated by
our results. As FRET is distance dependent, when the ligand is
bound, the donor and acceptor would be brought together,
leading to the increased emission of the acceptor and the
quenching of the donor. If another small molecule was added
to the mix and competed off the original ligand, the donor and
In labeling the A-site, it was important to place 5 close to
the recognition site without distorting the binding properties of
the model RNA construct. Functional, isosteric nucleosides, such
as furan-modified ribonucleosides, have been incorporated in
(62) Liu, H.; Gao, J.; Kool, E. T. J. Org. Chem. 2005, 70, 639–647.
(63) Lee, A. H. F.; Kool, E. T. J. Am. Chem. Soc. 2006, 128, 9219–9230.
(64) Godde, F.; Toulm, J.-J.; Moreau, S. Biochemistry 1998, 37, 13765–
13775.
(65) According to crystal structures PDB 2ET4 and 1LC4, the positions
of modification on the aminoglycosides are not involved in binding
to the RNA.
(66) Gold, H. Fluorescent brightening agents. In The Chemistry of Synthetic
Dyes; Venkataraman, K., Ed.; Academic Press: New York, 1971; Vol.
V, pp 535-542.
(67) Baindur, N.; Triggle, D. J. Med. Res. ReV. 1994, 14, 591–664.
9
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A R T I C L E S
Xie et al.
utilizing either an LCQDECA (Finnigan) ESI with a quadrapole
ion trap or an MAT900XL (ThermoFinnigan) FAB double focusing
mass spectrometer. UV-vis spectra were recorded on an Agilent
8453 Diode Array Spectrometer. MALDI-TOF spectra were col-
lected on a PE Biosystems Voyager-DE STR MALDI-TOF
spectrometer in positive-ion, delayed-extraction mode. Reversed-
phase HPLC (Vydac C18 column) purification and analysis were
carried out using a Hewlett-Packard 1050 Series instrument. Steady-
state fluorescence experiments were carried out in a microfluores-
cence cell with a path length of 1.0 cm (Hellma GmH & Co KG,
Mullenheim, Germany) on a Jobin Yvon Horiba FluoroMax-3
luminescence spectrometer. A background spectrum (buffer) was
subtracted from each sample. Modified oligonucleotides were
synthesized on a Biosearch Cyclone Plus DNA synthesizer using
a 0.2 µmol scale 500 Å CPG column. All hybridization and UV
melting experiments were done with a Beckman-Coulter DU 640
spectrometer with a high performance temperature controller and
micro auto six-cell holder.
acceptor would be parted, decreasing the emission of the
acceptor and simultaneously increasing the fluorescence of the
donor. A FRET based system, therefore, unambiguously pro-
vides both association and competitive dissociation information
that is independent of specific binding modes. Most importantly,
while singly labeled RNA constructs might generate false
positive signals due to remote binding at a nonfunctional state
(which could alter the environment of a responsive probe), a
FRET based technique, as described here, requires specific
binding to the A-site pocket. A nonspecific RNA binder, for
example, would not be able to displace an antibiotic from its
cognate recognition site, nor would it generate an intense FRET
signal in monitoring direct binding experiments.
Summary and Implications
We have constructed a useful and effective FRET pair system
for the evaluation of antibiotics binding to the bacterial ribosomal
decoding site. It relies on the incorporation of a new emissive
uridine analogue into an A-site construct, which serves as a donor
for a highly emissive coumarin acceptor that is attached to
aminoglycosides. The strong dependence of FRET on the
donor-acceptor distance is unambiguously used to detect the onset
and offset of binding more effectively than a single chromophore
system (which is also incapable of monitoring displacement and
competition events). As a proof of concept, we have demonstrated
binding of neomycin B and tobramycin, two A-site binding
antibiotics displaying distinct affinity to their native rRNA target,
and their competitive displacement by a variety of aminoglycoside
antibiotics. It is worth noting here that antibacterial activity does
not directly correlate with affinity to the A-site, which functions
as a conformational switch.18 This further justifies the use of
multiple probes that facilitate screening of potential new A-site
binders with diverse affinity. We note that our design is not limited
to the A-site and its binders; related FRET assemblies can be
developed for other RNA-ligand recognition events. Additionally,
further development of ideal FRET pairs, composed of isomorphic
nucleosides and corresponding minimally perturbing FRET ac-
ceptors, will facilitate the analysis of other RNA targets with
therapeutic potential.
Synthesis. 5-Methoxyquinazoline-2,4-(1H,3H)-dione (1). Water
(36 mL) and glacial acetic acid (0.7 mL) were added to 2-methoxy-
5-aminobenzoic acid (1.00 g, 5.98 mmol). The slurry was stirred
at 35 °C for 15 min. Sodium cyanate (0.97 g, 14.92 mmol) was
dissolved in water (4 mL) and added slowly to the slurry. The
reaction was stirred at 35 °C for 30 min. Sodium hydroxide (10.68
g, 267 mmol) was added slowly to the reaction. The reaction was
cooled to room temperature. The pH was adjusted to 4 with
concentrated hydrochloric acid. The white precipitate was collected
and washed with water (200 mL). Product: white solid (1.03 g,
1
5.38 mmol, 90% yield). H NMR (400 MHz, DMSO-d6): δ 10.97
(s, NH, 1H), 10.87 (s, NH, 1H), 7.47 (t, J ) 2.0 Hz, 1H), 6.68 (d,
J ) 14 Hz, 1H), 6.67 (d, J ) 7.2 Hz, 1H), 3.79 (s, OCH3, 3H); 13
C
NMR (100 MHz, DMSO-d6): δ 161.54. 161.14, 150.81, 143.79,
136.05, 107.96, 105.57, 104.81, 56.53; ESI-MS calculated for
C9H9N2O3 [M + H]+ 193.1, found 193.1.
5-Methoxyquinazoline-2,4-(1H,3H)-dione Ribonucleoside (5). To
a suspension of 1 (0.10 g, 0.52 mmol) in anhydrous acetonitrile (5
mL) N,O-bis(trimethylsilyl)acetamide (0.64 mL, 2.6 mmol) was
added dropwise under argon. The reaction was stirred at 25 °C for
2 h. The reaction temperature was raised to 50 °C. TMSOTf (0.14
mL, 0.77 mmol) and ꢀ-D-ribofuranose-1-acetate-2,3,5-tribenzoate
(0.26 g, 0.52 mmol) were added at the same time under argon.
The reaction was stirred at 50 °C for 24 h. The reaction was cooled
to room temperature and diluted with dichloromethane (10 mL).
The solution was washed with saturated sodium bicarbonate and
brine. The organic layer was dried over sodium sulfate. The solvent
was removed under reduced pressure, and the crude product was
dissolved in dioxane (5 mL) and transferred to a 200 mL pressure
tube. Ammonium hydroxide (28%, 80 mL) was added. The reaction
was stirred at 80 °C for 24 h. The solvent was removed was
removed under reduced pressure, and the product was isolated by
flash chromatography (88/12 dicholromethane/methanol). Product:
white solid (0.14 g, 0.42 mmol, 81% yield over two steps). 1H NMR
(400 MHz, DMSO-d6): δ 11.25 (s, NH, 1H), 7.55 (t, J ) 8.4 Hz,
1H), 7.26 (d, J ) 8.4 Hz, 1H), 6.88 (d, J ) 8.8 Hz, 1H), 6.02 (d,
J ) 5.2 Hz, 1′-H, 1H), 5.22 (d, 2′-OH, 1H), 4.99 (b, 3′-OH, 1H),
4.94 (b, 5′-OH, 1H), 4.47 (t, J ) 5.6 Hz, H-2′, 1H), 4.08 (t, J )
5.6 Hz, H-3′, 1H), 3.82 (s, OCH3, 3H), 3.66 (m, H-4′, 1H),
3.46-3.57 (m, H-5′, 2H); 13C NMR (100 MHz, DMSO-d6): δ
161.33, 160.08, 150.81, 142.99, 135.74, 109.25, 107.27, 106.47,
91.48, 85.02, 73.97, 69.47, 61.78, 56.74; ESI-MS calculated for
C14H17N2O7 [M + H]+ 325.1, found 325.1.
Experimental Section
Materials. Unless otherwise specified, materials obtained from
commercial suppliers were used without further purification.
2-Methoxy-5-aminobenzoic acid and spermidine were purchased
from VWR. Neomycin, tobramycin, paromomycin, amikacin,
apramycin, and hygromycin were purchased as their sulfate salts
from Sigma-Aldrich and were converted into the corresponding
neutral form by passing through DOWEX MONOSPHERE 550 Å
(OH) anion exchange resin. The identities of the aminoglycosides
1
were confirmed by mass measurements, H NMR, and 13C NMR.
7-(Diethylamino)coumarin-3-carboxylic acid was purchased from
Sigma-Aldrich. Anhydrous pyridine, dichloroethane, and acetonitrile
were obtained from Fluka. Anhydrous N,N-diisopropylethylamine
and triethylamine were obtained from Acros. NMR solvents were
purchased from Cambridge Isotope Laboratories (Andover, MA).
The unmodified oligonucleotide was purchased from Thermo
Scientific. Standard phosphoramidites and solutions necessary for
solid phase RNA synthesis were purchased from Glen Research.
Oligonucleotides were purified by gel electrophoresis and desalted
on a Sep-Pak (Waters Corporation). Chemicals for preparing buffer
solutions were purchased from Fisher Biotech (enzyme grade).
Autoclaved water was used in all fluorescence titrations.
5′-Dimethoxytrityl-5-methoxyquinazoline-2,4-(1H,3H)-dione Ri-
bonucleoside (6). Anhydrous pyridine (3 mL), anhydrous triethy-
lamine (51 µL, 0.37 mmol), and 4,4′-dimethoxytrityl chloride (0.12
g, 37 mmol) were added to 3 (0.10 g, 0.31 mmol) over argon. The
reaction was stirred at room temperature for 8 h and quenched with
methanol (0.5 mL). The solvent was removed under reduced
pressure, and the product was isolated by flash chromatography
(1% triethylamine, 2% methanol, 97% dichloromethane). Product:
Instrumentation. NMR spectra were recorded on a Varian
Mercury 400 MHz spectrometer. Mass spectra were recorded at
the UCSD Chemistry and Biochemistry Mass Spectrometry Facility,
9
17612 J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009

Real Time Detection of RNA-Small Molecule Binding
A R T I C L E S
1
white solid (0.16 g, 0.26 mmol, 85% yield). H NMR (400 MHz,
0.026 mmol) were added. The reaction was stirred for 18 h. The
solvent was removed under reduced pressure, and the resulting solid
was dissolved in ethyl acetate and washed with water and brine.
The organic layer was dried over sodium sulfate, and the solvent
was removed under reduced pressure. The product was isolated by
flash chromatography (3% methanol in dichloromethane). Product:
yellow powder (26.8 mg, 0.0184 mmol, 84% yield). 1H NMR (400
MHz, CD3OD): δ 8.66 (s, 1H), 7.45 (d, J ) 10.5 Hz, 1H), 6.81 (d,
J ) 9 Hz, 1H), 6.56 (s, 1H), 5.34 (s, 1H), 5.12 (s, 1H), 5.02 (s,
1H), 4.28 (s, 3H), 4.09-4.06 (m, 1H), 4.01-3.98 (m, 1H),
3.90-3.87 (m, 4H), 3.82-3.79 (m, 2H), 3.76-3.71 (m, 4H),
3.61-3.44 (m, 24H), 3.34-3.15 (m, 2H), 2.63-2.57 (m, 1H),
CDCl3): δ 7.46-7.16 (m, 12H), 6.97 (t, J ) 8.8 Hz, 1H), 6.75 (d,
J ) 8.8 Hz, 2H), 6.60 (d, J ) 8.4 Hz, 1H), 6.32 (d, J ) 5.6 Hz,
1H), 4.81 (t, J ) 6.4 Hz, 1H), 4.57 (t, J ) 6.4 Hz, 1H), 4.05 (br,
1H), 3.88 (s, 3H), 3.73 (s, 6H), 3.47-3.55 (m, 2H); 13C NMR (100
MHz, CDCl3): δ 161.48, 160.50, 158.66, 150.60, 144.99, 142.68,
136.04, 135.97, 135.81, 130.48, 128.56, 128.02, 127.01, 113.30,
109.53, 106.56, 90.79, 86.48, 83.56, 69.96, 69.39, 63.42, 63.29,
56.58, 55.45, 53.13; ESI-MS calculated for C35H34N2NaO9 [M +
Na]+ 649.2, found 649.2.
2′-(Trisisopropylsiloxy)methyl-5′-dimethoxytrityl-5-methoxyqui-
nazoline-2,4-(1H,3H)-dione Ribonucleoside (7). Anydrous dichlo-
roethane (3 mL) and N,N-diisopropylethylamine (0.17 mL, 1.0 mmol)
were added to 4 (0.20 g, 0.32 mmol). Dibutyltin dichloride (0.10 g,
0.33 mmol) was added to the reaction under argon and stirred at room
temperature for 1 h. The reaction was placed into a 80 °C water bath
and stirred for 10 min. (Triisopropylsiloxy)methyl chloride (87 µL,
38 mmol) was added, and the reaction was stirred at 80 °C for 15
min. The reaction was diluted with dichloromethane (10 mL) and
poured into saturated sodium bicarbonate (15 mL). The mixture was
stirred vigorously for 15 min. The organic layer was extracted, and
the aqueous layer was washed with dichloromethane (5 mL). The
organic layers were pooled and dried over sodium sulfate. The solvent
was removed under reduced pressure, and the product was isolated
by flash chromatography (1% triethylamine, 35% ethyl acetate, 64%
hexanes). Product: white foam (0.078 g, 0.01 mmol, 30% yield). 1H
NMR (400 MHz, CDCl3): δ 7.43-7.47 (m, 3H), 7.26-7.34 (m, 4H),
7.18-7.26 (m, 4H), 7.06 (t, J ) 8.4 Hz, 1H), 6.78 (d, J ) 8.8 Hz,
3H), 6.67 (d, J ) 8.4 Hz, 1H), 6.35 (d, J ) 5.2 Hz, 1H), 5.07 (d, J )
4.8 Hz, 1H), 4.90-4.95 (m, 2H), 4.61 (t, J ) 6.4 Hz, 1H), 4.07 (dd,
J1 ) 4.0 Hz, J2 ) 3.2 Hz, 1H), 3.95 (s, 3H), 3.77 (s, 6H), 3.51 (dd, J1
) 8.0 Hz, J2 ) 2.4 Hz, 1H), 3.39 (dd, J1 ) 6.4 Hz, J2 ) 4.0 Hz, 1H),
3.28 (q, J ) 7.2 Hz, 1H), 2.70 (q, J ) 6.8 Hz, 1H), 1.07-1.00 (m,
21H); 13C NMR (100 MHz, CDCl3): δ 161.71, 160.02, 158.72, 149.92,
144.96, 143.91, 136.00, 135.94, 130.45, 128.53, 128.05, 127.08,
113.33, 109.71, 106.75, 106.13, 91.08, 88.99, 86.63, 83.70, 69.70,
63.44, 59.85, 56.71, 55.45, 29.95, 17.95, 12.02; ESI-MS calculated
for C45H56N2NaO10Si [M + Na]+ 835.4, found 835.4.
3′-2-Cyanoethyldiisopropylphosphoramidite-2′-(trisisopropylsi-
loxy)methyl-5′-dimethoxytrityl-5-methoxyquinazoline-2,4-(1H,3H)-
dione Ribonucleoside (8). Anhydrous dichloromethane (0.6 mL)
and N,N-diisopropylethylamine (0.13 mL, 0.75 mmol) were added
to 5 (0.05 g, 0.062 mmol). The reaction was cooled on ice, and
2-cyanoethyl N,N-diisopropylchlorophosphoramidite (28 µL, 0.13
mmol) was added. The reaction was stirred at room temperature
for 18 h. The solvent was removed under reduced pressure, and
the product was isolated by flash chromatography (1% triethy-
lamine, 15-30% ethyl acetate in hexanes). Product: white foam
(0.038 g, 0.037 mmol, 60% yield). 1H NMR (300 MHz, CDCl3): δ
7.44-7.46 (m, 3H), 7.32-7.34 (m, 4H), 7.22-7.26 (m, 4H), 7.07
(t, J ) 8.4 Hz, 1H), 6.78 (d, J ) 8.1 Hz, 3H), 6.68 (d, J ) 8.4 Hz,
1H), 6.35 (d, J ) 5.4 Hz, 1H), 5.07 (d, J ) 4.2 Hz, 1H), 4.91-4.94
(m, 2H), 4.62 (t, J ) 6.4 Hz, 1H), 4.07 (b, 1H), 3.96 (s, 3H), 3.78
(s, 6H), 3.51 (m, 1H), 3.40 (m, 1H), 2.81 (q, J ) 6.9 Hz, 1H), 2.01
(b, 1H), 1.20-1.15 (m, 8H), 1.01 (d, J ) 6.8 Hz, 4H), 0.92-0.89
(m, 21H); 13C NMR (100 MHz, CDCl3): δ 171.37, 161.62, 160.19,
158.67, 158.65, 144.88, 136.10, 136.03, 135.88, 135.50, 130.47,
130.44, 128.67, 128.56, 127.99, 127.94, 127.05, 126.99, 117.94,
117.53, 113.23, 106.61, 106.23, 86.50, 86.46, 64.56, 60.60, 59.02,
56.66, 55.40, 55.35, 43.58, 43.45, 43.31, 43.19, 30.84, 29.90, 24.82,
24.75, 24.69, 21.24, 21.21, 19.23, 17.87, 17.81, 14.40, 13.92, 12.06,
12.01, 11.98; ESI-MS calculated for C54H73N4NaO11PSi[M + Na]+
1035.5 and [M + K]+ 1051.4, found 1035.4 and 1051.4.
1.99-1.89 (2H), 1.46-1.38 (m, 54H), 1.37 (t, J ) 5 Hz, 6H); 13
C
NMR (125 MHz, CD3CN): δ 164.66, 163.53, 158.72, 158.21,
157.86, 157.62, 157.29, 156.55, 154.02, 149.22, 132.40, 111.34,
110.52, 109.87, 109.06, 101.22, 99.69, 97.06, 80.24, 79.90, 79, 81,
79.64, 79.56, 74.99, 73.82, 70.88, 68.43, 56.72, 53.13, 51.65, 45.70,
45.28, 42.54, 41.44, 28.69, 12.67, 12.21; ESI-MS calculated for
C67H108N8O27 [M + Na]+ 1479.72, found 1479.71.
Coumarin-Labeled Neomycin (11). Anhydrous dichloromethane
(2 mL) and triisopropylsilane (200 µL) were added to 14 (26.8
mg, 0.0184 mmol). To this trifluoroacetic acid was added (2 mL),
and the reaction was stirred at RT for 15 min. The reaction was
diluted with toluene (5 mL), and the solvent was removed under
reduced pressure. The resulting solid was dissolved in water and
washed with dichloromethane. The aqueous layer was dried and
concentrated under reduced pressure and further purified by
reversed-phase HPLC using a gradient of 10-30% acetonitrile
(0.1% TFA) in water (0.1% TFA) over 30 min, eluting at 14.78
min. Product: yellow powder (21.7 mg, 0.0151 mmol, 82% yield).
1H NMR (400 MHz, D2O): δ 8.65 (s, 1H), 7.62 (d, J ) 9.2 Hz,
1H), 6.92 (d, J ) 9.2 Hz, 1H), 6.67 (s, 1H), 5.96 (s, 1H), 5.36, (s,
1H), 5.29 (s, 1H), 4.49 (t, J ) 5.5 Hz, 1H), 4.39 - 4.35 (m, 2H),
4.28 (t, J ) 5 Hz, 1H), 4.21 (t, J ) 3.5 Hz, 1H), 3.99-3.95 (m,
2H), 3.89-3.83 (m, 2H), 3.79 (s, 2H), 3.64 (t, J ) 9.5 Hz, 1H),
3.59 (s, 1H), 3.56-3.51 (q, J1 ) 6.5 Hz, J2 ) 7.0 Hz, 4H), 3.33
(d, J ) 4 Hz, 4H), 3.18-3.12 (m, 1H), 2.42-2.33 (m, 1H),
1.83-1.71 (m, 1H), 1.21 (t, J ) 7 Hz, 6H); 13C NMR (125 MHz,
D2O): δ 166.19, 164.44, 163.17, 162.99 (J1 ) 27.8 Hz, J2 ) 58.4
Hz), 157.78, 154.03, 149.05, 131.83, 116.25 (J ) 231 Hz, J2 )
465 Hz), 115.08, 111.53, 110.43, 108.14, 106.39, 95.97, 95.44,
94.46, 79.69, 76.50, 73.24, 69.96, 67.52, 67.27, 50.7416, 48.49,
44.99, 40.32, 39.83, 11.44; ESI-MS calculated for C37H60N8O15 [M
+ 2H]2+ 429.21, [M + H]+ 857.43, and [M + Na]+ 879.41, found
429.35, 857.43, and 879.59.
Aminoglycoside Titrations. All titrations were performed with
working solutions of 1.0 × 10-6 M 10 in 20 × 10-6 M calcodylate
buffer (pH 7.0, 1.0 × 10-1 M NaCl, 5.0 × 10-4 M EDTA). The
solutions were heated to 75 °C for 5 min, cooled to room temperature
over 2 h, and placed on ice for 30 min prior to titrations. For binding
studies, 10 was excited at 320 nm, and changes in emission upon
titration with 11 or 12 were monitored at 395 and 473 nm. The
concentrations of 11 and 12 were determined by UV absorbance at
400 nm (ꢀ ) 20 000 M-1 cm-1). For competition studies, 11 or 12
was titrated into 10 until saturation. 10 was excited at 320 nm, and
changes in emission upon displacement of 11 or 12 by aminoglycosides
were monitored at 395 and 473 nm. EC50 and IC50 values were
calculated using OriginPro 8 software by fitting a dose response curve
(eq 1) to the fractional fluorescence saturation (Fs) plotted against the
log of aminoglycoside (AG) concentration.
Fs ) F0 + (F∞[AG]n)/([EC50]n + [AG]n)
(1)
Fi is the fluorescence intensity at each titration point. F0 and F∞
are the fluorescence intensity in the absence of aminoglycoside or
at saturation, respectively, and n is the Hill coefficient or degree
of cooperativity associated with the binding.
Boc6-Protected Coumarin-Labeled Neomycin (14). Anhydrous
dichloromethane (300 µL) and 7-diethylaminocoumarin-3-carboxy-
lic acid (6.8 mg, 0.0263 mmol) were added to 13 (26.58 mg, 0.0219
mmol). To this, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (5.03 mg, 0.0262 mmol), N,N-diisopropylethylamine
(8.62 µL, 0.048 mmol), and 4-(dimethylamino)pyridine (5.8 mg,
Acknowledgment. We thank the National Institutes of Health
for their generous support (GM 069773), Mary Noe´ for her
9
J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009 17613

A R T I C L E S
Xie et al.
assistance with MALDI experiments, and the National Science
Foundation (Instrumentation Grants CHE-9709183 and CHE-
0741968).
Supporting Information Available: Additional synthetic
details, thermal denaturation measurements, titration spectra,
photophysical data, and MALDI-TOF MS spectrum. This
information is available free of charge via the Internet at http://
pubs.acs.org.
Note Added after ASAP Publication. The fluorescence acceptor
emission was incorrect in the abstract published ASAP November 12,
2009. The corrected version was published November 13, 2009.
JA905767G
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17614 J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009