A fluorescein-containing, small-molecule, water-soluble receptor for cytosine free bases

Article abstract of DOI:10.1016/j.bmc.2010.08.013

In this study, we synthesized small-molecule, water-soluble, fluorescein-containing ureido compounds 6 and 8 as target receptors for cytosine free bases and then investigated the binding of cytosine free bases with the receptors using ¹⁵N NMR spectroscopy and partially labeled cytosine-2,4-¹³C-1,3,4-¹⁵N-cytosine. Binding with the receptor 6a (the disodium form of 6) caused the chemical shift of the nitrogen atom of the amino group of cytosine to move downfield; binding of the receptor 8a (the disodium form of 8), which is possessing no corresponding aryl nitrogen atom, had no effect on this signal. Fluorescence spectroscopy revealed that binding of cytosine and its derivatives led to quenching of the fluorescence of receptor 6a; in contrast, the quenching of receptor 8a was only slightly affected by cytosine. Because the fluorescence of 6a was not quenched by either deoxycytidine or uracil, it appears that this receptor is a specific for cytosine among the DNA bases. We used the fluorescence of 6a to measure the apparent binding constants for various cytosine derivatives, including the anticancer prodrug 5-fluorocytosine. Receptor 6a is the first small-molecule, water-soluble fluorescent receptor for the specific binding of cytosine free bases in aqueous solution.

Full text of DOI:10.1016/j.bmc.2010.08.013

Bioorganic & Medicinal Chemistry 18 (2010) 7034–7042
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A fluorescein-containing, small-molecule, water-soluble receptor
for cytosine free bases
⇑
Yu Lin Jiang , Puneet Patel, Suzane M. Klein
Department of Chemistry, College of Arts and Sciences, East Tennessee State University, Johnson City, TN 37614, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we synthesized small-molecule, water-soluble, fluorescein-containing ureido compounds 6
and 8 as target receptors for cytosine free bases and then investigated the binding of cytosine free bases
with the receptors using 15N NMR spectroscopy and partially labeled cytosine-2,4-13C-1,3,4-15N-cytosine.
Binding with the receptor 6a (the disodium form of 6) caused the chemical shift of the nitrogen atom of
the amino group of cytosine to move downfield; binding of the receptor 8a (the disodium form of 8),
which is possessing no corresponding aryl nitrogen atom, had no effect on this signal. Fluorescence spec-
troscopy revealed that binding of cytosine and its derivatives led to quenching of the fluorescence of
receptor 6a; in contrast, the quenching of receptor 8a was only slightly affected by cytosine. Because
the fluorescence of 6a was not quenched by either deoxycytidine or uracil, it appears that this receptor
is a specific for cytosine among the DNA bases. We used the fluorescence of 6a to measure the apparent
binding constants for various cytosine derivatives, including the anticancer prodrug 5-fluorocytosine.
Receptor 6a is the first small-molecule, water-soluble fluorescent receptor for the specific binding of
cytosine free bases in aqueous solution.
Received 29 June 2010
Revised 2 August 2010
Accepted 4 August 2010
Available online 9 August 2010
Keywords:
Fluorescein
Receptor
Cytosine
5-Fluorocytosine
Binding
15N NMR spectroscopy
Fluorescence quenching
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
with less selectivity compared with that of UDG.2,9 This cleavage
is pH-dependent; CDG is most reactive at pH 6.2.1 Notably, many
Human cytosine-DNA glycosylase (CDG) is a mutant of uracil-
DNA glycosylase (UDG) in which residue Asn123 has been replaced
by an Asp123 unit.1,2 Recently, thymine-DNA glycosylase (TDG),
involving a residue Glu42, was found to also have the ability to re-
move cytosine and derivatives from DNA.3 The common feature for
CDG and TDG is that both of them possess acidic residues for bind-
ing and catalysis of the glycosylation. This common feature also ex-
ists in other cytosine-related enzymes, including activation-
induced cytosine deaminase (AID) and yeast cytosine deaminase
(YCD).4–6 These two cytosine deaminases use their acidic residues
Glu58 and Glu64, respectively, for binding and catalysis. Therefore,
it appears that cytosine prefers to interact with these units when
protonated. Indeed, this situation exists in the Watson–Crick base
pairing between cytosine and guanine,7 where cytosine donates
one NH hydrogen atom for hydrogen bonding with guanine but re-
ceives two NH hydrogen atoms in return. Overall, cytosine obtains
one net NH hydrogen atom donated from guanine and becomes
protonated.8 Although the binding of cytosine with guanine is
common in DNA, the binding of cytosine’s free base with a syn-
thetic receptor is rarely studied.
human diseases are exacerbated at low pH; for example, acidic
extracellular pH promotes vascular endothelial growth factor
(VEGF) in human glioblastoma cells, tumor microenvironments in-
duce metastasis of human hepatocellular carcinoma cells, and acid
mediates tumor invasion.10–12 Therefore, cancers are more likely to
form under acidic conditions. A lower pH will more likely result in
higher levels of the cytosine free bases, not only because cancer is
more likely to deteriorate under acidic conditions but also because
cytosine is more likely to be cleaved.1 As a result, systems that rec-
ognize cytosine free bases might aid in monitoring of such
diseases.
At present, the analysis of cytosine from DNA relies mainly on
measuring the radioactivity of cytosine that has been pre-labeled
with tritium.2 The operation of the experiments is tedious.
Although a macrobicyclic compound has been reported to bind
cytosine,13 it exhibits low selectivity. In addition to cytosine, it
binds the nucleobases adenine and guanine, that is, as long as the
guest compounds have pyridine nitrogen atoms. Furthermore, this
molecule (molecular weight: 1812 g/mol) is much larger than the
molecular weight cut-off (500 g/mol) for small-molecule drugs.14
Recently, small-molecule ureidoquinoline and diaminonaphthyri-
dine derivatives have been developed as intercalators for DNA-con-
taining cytosine.15,16 When binding with DNA, these compounds
pair with cytosine units by forming three hydrogen bonds while
In addition to cleaving mismatched and unwanted cytosine
bases from DNA, CDG also removes healthy cytosine bases, but
⇑
Corresponding author. Tel.: +1 423 439 6917; fax: +1 423 439 5835.
E-mail address: jiangy@etsu.edu (Y.L. Jiang).
simultaneously taking part in
p–p stacking interactions with
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.013

Y. L. Jiang et al. / Bioorg. Med. Chem. 18 (2010) 7034–7042
7035
nucleobases in duplex DNA. Several receptors—based on pyridyl
ureas, imidazolyl ureas, and guanine/pyrene conjugates—have
been developed for the recognition of cytosine free bases and their
derivatives in organic solvents.17–19 Furthermore, a perylene-
containing DNA receptor has been developed to specifically bind
to cytosine in DNA.20 To the best of our knowledge, small-molecule
water-soluble receptors for cytosine free base have not been
reported previously.
to give the 6-carboazidofluorescein 3 in 71% yield (Scheme 2).28
Compound 3 was then decomposed to compound 4, which was re-
acted directly with 2-aminoimidazole to give compound 5 in 63%
yield.18 Compound 5 was hydrolyzed to give receptor 6 in 65%
yield.29 Likewise, we synthesized receptor 8, which features a phe-
nyl unit in place of the imidazole ring of 6, from compound 3
(Scheme 3). Both 6 and 8 were converted to their disodium forms,
6a and 8a, respectively, using 1 N NaOH.
In order to find a convenient method to analyze cytosine, we
employed an imidazolyl urea motif, which mimics the binding mo-
tifs in guanine, to develop a small-molecule (424 g/mol), water-
soluble receptor 6 for cytosine that features 2-aminoimidazole
and fluorescein units, both of which are water-soluble. The salient
feature of the imidazolyl urea moiety is that it forms an intramo-
lecular hydrogen bond between its imidazolyl NH with urea oxy-
gen atom.18 We also designed compound 6 so that it would be
amiable to future in vivo studies because, when hydrolyzed, it
forms 2-aminoimidazole and 6-aminofluorescein; the former oc-
curs naturally in marine sponges and it is not toxic,21–23 whereas
the latter is used widely in the diagnosis of human diseases (e.g.,
through fluorescein injection).24–27 This paper describes the syn-
thesis of the cytosine receptor 6 using a general binding mode
(Scheme 1) and a molecular recognition study of the hydrogen
bonding of receptor 6a (the disodium form of 6) with cytosine
using 15N nuclear magnetic resonance spectroscopy [using par-
tially labeled cytosine-2,4-13C-1,3,4-15N-cytosine (C1315)]. For
comparison, we have also synthesized the receptor 8. By taking
advantage of quenching of fluorescence of 6a by cytosine and its
derivatives, including the anticancer drug 5-fluorocytosine
(Chart 1), the apparent binding constants for them have been mea-
sured conveniently.
Compound 6a exhibits UV absorption maxima at 239, 276, 319,
and 490 nm in aqueous phosphate buffer (50 mM) at pH 7.4. Its
maximum fluorescence emission is located at 518 nm. Compound
8a features UV absorption maxima at 239, 270, 321, and 491 nm
in the same buffer, with its maximum fluorescence emission at
517 nm.
Our syntheses of compounds 6 and 8 were rather straightfor-
ward, each involving two one-pot transformations. The dipivaloyl
6-carboazidofluorescein 3 is a new building block that functions
as a fluorescent probe. We suspect that it might find use in the syn-
thesis of other ureido derivatives from such amino group-contain-
ing biomolecules as peptides, proteins, DNA, RNA, and peptide
nucleic acids, thereby allowing their highly fluorescent probing.
Although a few ureidopyridine and ureidoquinoline derivatives
have been reported to bind specifically to cytosine in DNA,15,16
compound 6a is the first water-soluble, fluorescent probe-labeled,
small-molecule receptor that exhibits long-wavelength in UV
absorption and fluorescence emission.
2.2. Monitoring the dimerization of receptor 6b in aqueous
solution using 1H NMR spectroscopy30
Receptor 6 was converted into its disodium form 6b using NaOD
solution (Scheme 4). The resulting solution was diluted with phos-
phate (50 mM) buffer (pD 7.4) in D2O to prepare a stock solution;
the pD was adjusted to 8.75 using NaOD and DCl solutions. At this
pD, the receptor 6 should also exist as a form of 6b because the
fluorescein group has values of pKa of 6.3 and 6.8.31
2. Results and discussion
2.1. Syntheses of 6 and 8
Next, we performed a 1H NMR spectroscopic dilution study of
6b, changing the concentration from 10.0 to 0.16 mM at 298 K,
using a phosphate (50 mM) buffer (pD 8.75) in D2O, to investigate
its dimerization patterns and its monomer structure (Fig. 1). In
D2O, all of the active hydrogen atoms (NH, OH) were replaced by
D atoms; therefore, they did not appear in the 1H NMR spectra.
As revealed in Figure 1, dilution caused most of the signals
of the aromatic protons of 6b, except that for H14, to move down-
field in the NMR spectra, suggesting that 6b dimerized at high
concentration. Interestingly, among all of the signals, that for
In one-pot, the 6-carboxyfluorescein 1 was converted through
sequential reactions with N-hydroxysuccinimide (NHS) and NaN3
H10 underwent the largest downfield movement (Dd = 0.34 ppm)
upon decreasing the concentration of 6b from 10 to 0.16 mM.
This behavior can be explained by considering that, at low
Scheme 1. Possible mode of binding between 6a (two sodium ions are omitted for
clearance) and cytosine.
Chart 1. Cytosines.

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Y. L. Jiang et al. / Bioorg. Med. Chem. 18 (2010) 7034–7042
Scheme 2. Synthesis of 6.
can pack through van der Waals interactions. Furthermore, in 6b,
the hydrogen atom H10 is located at the para position of the xan-
thenyl ring with respect to the sp2-hybridized carbon atom C22;
the
p electrons of the xanthenyl ring become conjugated with
those of the phenyl ring containing H10, as indicated in Scheme 5.
Because the quinonyl ring, just like a C@O group, is electron-with-
drawing, it causes the C10 and H10 atoms to be slightly electron-
deficient. Furthermore, H10 becomes even more electron-deficient
in the form of its monomer 6b because of possible contact with the
C@O oxygen atom at position 7.34,35 Therefore, the chemical shift of
H10 appears relatively downfield. In contrast, the carbon atom C22
is sp3-hybridized in 6c. Thus, the chemical shift of H10 is relatively
upfield.
More interestingly, only the signal of proton H14 moved upfield
(Dd = 0.30 ppm) when we decreased the concentration of 6b from
10 to 0.16 mM. This behavior also can be explained by considering
the tautomerization of 6b to 6c at high concentrations (Scheme 5).
In 6b, the hydrogen atom H14 is located above the six-membered
aromatic ring containing the carbon atom C22; its signal in the 1H
NMR spectra was, therefore, shielded by this xanthenyl group. In
6c, H14 sits above the nonaromatic six-membered ring containing
carbon atom C22; its signal is not shielded. Notably, dilution re-
solved the signals of all of the CH units of receptor 6b.
Scheme 3. Synthesis of 8.
Using nonlinear fitting and Grafit 6 software, we estimated the
dimerization constant of 6b to be 604 68 Mꢀ1 (Supplementary
Fig. S1).30,34 This relatively large value is consistent with the large
changes in the chemical shifts of protons H10 and H14 (Scheme 5).
Scheme 6 presents a possible structure for the dimer of 6c in D2O,
involving four possible [N–Dꢁ ꢁ ꢁN] hydrogen bonds.18
Scheme 4. Preparation of 6b.
2.3. Monitoring the binding of receptor 6a with cytosine in
aqueous solution using 15N NMR spectroscopy30
concentrations, the monomeric form of 6b should exist as depicted
in Scheme 4, whereas, when the concentration of 6b is high, its
tautomer 6c might form (Scheme 5).32,33 In its dimeric form, tauto-
mer 6c features hydrophilic and hydrophobic groups at its exterior
and interior, respectively; the hydrophilic groups are presented to
interact with the solvent (water) molecules through hydrogen
bonding and electrostatic interactions, while its hydrophobic units
For this study using 15N NMR spectroscopy, we used solutions
comprising 90% H2O and 10% D2O. First, receptor 6 was converted
into 6a using NaOH solution. The resulting solution was diluted
with phosphate (50 mM) buffer (pH 7.4) to form the stock solution
of 6a. Likewise, we prepared a stock solution of 8a. In addition, we

Y. L. Jiang et al. / Bioorg. Med. Chem. 18 (2010) 7034–7042
7037
Figure 1. Partial 1H NMR spectra of 6b recorded at various concentrations in 50 mM phosphate buffer in D2O (pD 8.75) at 293 K.
for C1315 (10.0 mM) alone and for its mixtures with the receptors
6a and 8a, individually (each concentration: 10.0 mM). Figure 2
displays three corresponding partial NMR spectra; Figure S2 pro-
vides calculated chemical shift changes for the nitrogen atoms at
the N1 and N4 positions of C1315 upon binding. In the presence
of receptor 6a, these signals for C1315 underwent downfield shifts
of +0.008 and +0.053 ppm, respectively. In the Watson–Crick base
pair between cytosine and guanine, guanine transfers a proton to
cytosine. In the binding between cytosine and receptor 6a, we
would also expect receptor 6 to donate a proton to cytosine.8 As re-
ported, protonation of a cytosine base in an oligonucleotide will re-
sult in downfield shifts of the signals of N1 and N4 in 15N NMR
spectra.38 The formation of a DNA base pair involving cytosine also
causes a downfield shift of the signal for N4 in 15N NMR spectra.39
Therefore, our observation of downfield chemical shifts of the sig-
nals of the nitrogen atoms N1 and N4 in cytosine (C1315) in the
presence of 6a is consistent with a protonated cytosine unit that
is bound to the receptor. Therefore, these spectra reveal that Wat-
son–Crick-type hydrogen bonding exists between the receptor 6a
and the cytosine free base under these conditions.
Scheme 5. Plausible tautomerization of 6b and 6c in pD (8.75) buffer. The low-
energy, three-dimensional structures 6b and 6c were calculated using Spartan 06 at
RM1 level.36
Figure S2B reveals that the shifts in the signals of the nitrogen
atoms N1 and N4 in the NMR spectra of C1315 were ꢀ0.008 and
0.000 ppm, respectively, in the presence of 8a, suggesting that little
or no hydrogen bonding occurred to the receptor. This behavior
can be explained by considering that receptor 8a lacks the aryl
nitrogen atom that receptor 6a possesses for additional hydrogen
bonding with cytosine. Any possible interaction between receptor
8a and cytosine will also evoke steric repulsion between the phe-
nyl group of 8a and the amino group of cytosine (Scheme 7). Thus,
receptor 8a is a suitable control that suggests positive binding oc-
curs between receptor 6a and cytosine.
Scheme 6. Plausible structure for the dimer of 6c, stabilized through hydrogen
bonding interactions.
also prepared a stock solution of the partially labeled cytosine-
2,4-13C-1,3,4-15N-cytosine (C1315) in phosphate buffer (pH 8.75).
We used these stock solutions to prepare the samples for NMR
spectroscopic analysis, adjusting their values of pH to 8.75 using
NaOH and HCl solutions.
15N NMR spectroscopy is used widely to study the hydrogen
bond-mediated base pairing of cytosine units in DNA and RNA.
These cytosine bases are usually also involved in
p–p stacking
interactions. In this study, we found that 15N NMR spectroscopy
is also useful for investigating the binding between a cytosine free
base and a synthetic receptor, mediated through hydrogen bonding
in aqueous solutions.
Next, we performed an 15N NMR spectroscopic study of the
binding between the receptors 6a and 8a and C1315 in 50 mM
phosphate buffer (pH 8.75) at 293 K.37 We obtained NMR spectra

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Y. L. Jiang et al. / Bioorg. Med. Chem. 18 (2010) 7034–7042
Figure 2. Partial 15N NMR spectra of (top) cytosine (C1315, 10.0 mM) in the presence of receptor 8a (10.0 mM), (middle) cytosine (C1315, 10.0 mM) only, and (bottom)
cytosine (C1315, 10.0 mM) in the presence of receptor 6a (10.0 mM); spectra were recorded from solutions in 50 mM phosphate buffer (pH 8.75) in 10% D2O at 293 K.
we performed this study of the binding between receptor 6a and
cytosine using the weaker excitation signal at 300 nm at 298 K.
Here, we monitored the change in fluorescence intensity of 6a
during the titration of cytosine into the mixture (Fig. 3), observing
a decrease in the fluorescence intensity at 518 nm to almost com-
plete quenching (80%). From the fluorescence intensities of cyto-
sine and nonlinear square curve fitting, we estimated the
Scheme 7. Plausible structure of the sterically encumbered complex formed
between 8a and cytosine.
apparent binding constant for the complex formed between recep-
tor 6 and cytosine to be 60 2 Mꢀ1 (Fig. 4).30 Using the control,
receptor 8a exhibited less-dramatic fluorescence quenching in
the presence of cytosine, with an estimated apparent binding con-
stant of 6.9 0.2 Mꢀ1. Our finding of the apparent binding constant
for 6a being about 8.7-fold greater than that for 8a reveals the
importance of the imidazole unit’s nitrogen atom in the formation
of the complex. This result is consistent with the chemical shift of
the amino nitrogen atom in the 15N NMR spectra of cytosine C1315
undergoing a significant downfield shift upon binding with 6a,
whereas it underwent no change in the presence of 8a.
2.4. Binding of 6a with cytosine quenches its
fluorescence30,34,35,40
For this study, we used a dilute solution of 6a (50 lM) in phos-
phate buffer (50 mM, pH 7.4) in water. At this pH, the receptor 6
would exist in the form of 6a because the values of pKa of the fluo-
rescein group are ca. 6.3 and 6.8.31 Although receptor 6a is a fluo-
rescent molecule exhibiting a fluorescence maximum at 518 nm,
The binding constant for the complex formed between receptor
6a and cytosine would be higher if there were no energetic penalty
Figure 3. Incremental quenching of the fluorescence of 6a (50 lM) at 518 nm upon
the addition of cytosine at concentrations of 0.0, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, 16.0,
Figure 4. Relative fluorescence of 6a plotted as a function of the concentration of
24.0, 28.0, and 32.0 mM in phosphate buffer (50 mM, pH 7.4) at 298 K.
cytosine in phosphate buffer (50 mM, pH 7.4) in H2O at 298 K.

Y. L. Jiang et al. / Bioorg. Med. Chem. 18 (2010) 7034–7042
7039
for breaking the dimers of 6a (dimerization constant:
604 68 Mꢀ1). Nevertheless, the receptor 6a is an effective binding
partner for cytosine, as revealed in both the 15N NMR and fluores-
cence spectroscopy studies. The apparent binding constant of
60 2 Mꢀ1 for the complexation of receptor 6a with cytosine is
similar to that (110 18 Mꢀ1) reported for a pyrene-containing
receptor with uracil;30 notably, both of these complexes are stabi-
lized by three hydrogen bonds in aqueous solution.
The binding of cytosine free base with receptor 6a in water
must also overcome the very high polarity of the solvent, which
forms hydrogen bonds with all of the active hydrogen atoms and
nitrogen and oxygen atoms in both cytosine and 6a. Our observa-
tion of downfield chemical shifts for the signals of the nitrogen
atoms in the 15N NMR spectra suggests that there is binding be-
tween receptor 6a and cytosine, presumably because three hydro-
gen bonds are involved in this complex. Although a couple of
water-soluble, fluorescent receptors for cytosine have been re-
ported, they were applied to studies of the binding of cytosine unit
within DNA.15,16 Neither the direct contact nor hydrogen bonding
between the receptors and cytosine was investigated in those stud-
ies. Here, we demonstrate that direct contact and hydrogen bond-
ing between receptors and cytosine can be revealed using both
NMR and fluorescence spectroscopy.
To explore the specificity of the receptor 6a in the binding of
cytosine free base, we examined its behavior toward the water-sol-
uble nucleobase uracil and the nucleoside deoxycytidine. We
found that neither compound quenched the fluorescence of recep-
tor 6a, indicating that little or no binding occurs between 6a and
either uracil or deoxycytidine. The absence of binding between
6a and uracil suggests that this receptor is specific for the nucleo-
base cytosine; the absence of binding between 6a and deoxycyti-
dine suggests that the sugar ring in deoxycytidine impedes the
binding through a steric effect (Scheme 1). Therefore, 6a is a spe-
cific receptor for cytosine free base, and not for cytosine-containing
nucleosides.
squares fitting, we estimated the apparent binding constant to be
560 40 Mꢀ1 (Table 1)—approximately 9.3-fold better than that
for cytosine. We used the same methodology to determine the
binding constants for the complexes formed between 6a and
other halogenated cytosines: 5-chlorocytosine (1690 122 Mꢀ1),
5-bromocytosine (1806 143 Mꢀ1), and 5-iodocytosine (4366
345 Mꢀ1). Interestingly, the larger the halogen atom, the greater
the apparent binding constant to receptor 6a; this trend might
be related to the acidity of the amino hydrogen atoms of the halo-
genated cytosines.30
The large difference between the apparent binding constants of
6a for cytosine and the anticancer drug 5-fluorocytosine shows
that the receptor 6a can distinguish cytosine from 5-fluorocytosine
using fluorescence quenching experiments. The significance of the
experiments is that the receptor 6a may be used in medical appli-
cation such as therapeutic-drug-monitoring of 5-fluorocytosine.
The advantage of the method is convenience, compared to tradi-
tional methodology based on the radioactivity of cytosine that
has been pre-labeled with tritium.2
Many 5-substituted cytosines, such as 5-methylcytosine,
5-hydroxymethylcytosine, and 5-carboxycytosine, are related to
epigenetics and DNA damage.47,48 Therefore, we also measured,
monitoring the quenching of the fluorescence of 6a (Table 1), the
apparent binding constants for the cytosines 5-methylcytosine
(334 10 Mꢀ1), 5-hydroxymethylcytosine (151 4 Mꢀ1), and 5-
carboxycytosine (913 88 Mꢀ1). The apparent binding constant
of 6a to 5-methylcytosine is 5.6-fold greater than that of 6a to
cytosine. The apparent binding constant of 6a to 5-hydroxymethyl-
cytosine is also greater (by ca. 2.5-fold) than that of 6a to cytosine.
Therefore, receptor 6a can bind cytosine, 5-methylcytosine, and 5-
hydroxymethylcytosine with different strengths. Receptor 6a is the
first water-soluble, fluorescent receptor to tell differences between
these three cytosine free bases using binding constants. The appar-
ent binding constant for 5-carboxycytosine is about 2.7- and 6.0-
fold of those for 5-methylcytosine and 5-hydroxymethylcytosine,
respectively, suggesting that the receptor 6a can bind 5-carboxycy-
tosine more tightly than cytosine, 5-methylcytosine, and 5-
hydroxymethylcytosine.
Furthermore, we also measured the apparent binding constant
for the complex formed between 5-cyanocytosine and 6a
(1100 79 Mꢀ1; Table 1). This apparent binding constant is similar
to that for 5-carboxycytosine—not unexpected because both car-
bon atoms of the substituents at the 5-position have the same oxi-
dation level and both are strongly electron-withdrawing.49 The
binding constant for isocytosine was 55 5 Mꢀ1, similar to that
for cytosine (Table 1). Again, this situation is predictable because
both isocytosine and cytosine are unsubstituted and have similar
structures. Thus, 6a is a versatile fluorescent indicator for various
cytosine derivatives.
As in other fluorescein-containing compounds, we expect the
xanthenyl unit and the phenyl ring of 6a to be aligned perpendic-
ular to each other.41,42 Therefore, in the complex formed between
6a and cytosine, it is unlikely that cytosine will undergo efficient
p–p stacking with the xanthenyl ring. The quenching of the fluo-
rescence of receptor 6a by cytosine is probably due to photoin-
duced electron transfer from cytosine, which is located in close
proximity to the fluorophore of 6a after binding.43–46 Our observa-
tion of little or no fluorescence quenching of 6a by uracil is consis-
tent with their poor association. The relatively dramatic quenching
of the fluorescence of 6a by cytosine suggests that these molecules
exist in close proximity in solution; in other words, cytosine and 6a
form a complex.
We also used 5-fluorocytosine, an anticancer prodrug of 5-fluo-
rouracil, as a component for a study of its molecular recognition
with 6a.5,6 Using fluorescence spectroscopy and nonlinear least-
Table 1
Apparent binding constants (Kb) of cytosine and its derivatives to
receptor 6a in phosphate buffer (50 mM, pH 7.4) at 298 K
Substrate
Kb (Mꢀ1
)
Cytosine
60
2
5-Fluorocytosine
5-Chlorocytosine
5-Bromocytosine
5-Iodocytosine
5-Methylcytosine
5-Hydroxymethylcytosine
5-Carboxycytosine
5-Cyanocytosine
Isocytosine
560 40
1690 122
1806 143
4366 345
334 10
151
4
913 88
1100 79
Figure 5. Calculated structure of the complex formed between the receptor 6a and
cytosine. Dotted lines: Hydrogen bonds. The two Na+ ions have been omitted for
clarity.
55
5

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Y. L. Jiang et al. / Bioorg. Med. Chem. 18 (2010) 7034–7042
Table 2
ical shifts of carbon nuclei are reported in ppm relative to solvent
signals used as internal standards. Fluorescence spectroscopy mea-
surements were performed using a FluoroMax-3 spectrometer
(HORIBA JOBIN YNON). Melting points were determined using a
MEL-TEMP apparatus. IR spectra were recorded using a Genesis II
FTIR spectrometer. UV–vis absorption spectra were recorded using
a Shimadzu UV-1700 UV–vis spectrophotometer. Buffer pH was
adjusted using an Accumet 925 pH meter (Fisher Scientific). The
structure of the complex formed between cytosine and receptor
6a was calculated using Spartan’06.36
Hydrogen bonding distances and angles in the complex formed between cytosine and
receptor 6a, calculated using the Spartan’06 software package at the RM1 level36
Hydrogen bond
Distance (Å)
Bond angle (°)
Oꢁ ꢁ ꢁH–N
Nꢁ ꢁ ꢁH–N
N–Hꢁ ꢁ ꢁN
1.942
1.933
1.936
164.5
161.3
167.4
2.5. Binding of receptor 6a with cytosine revealed through
structural calculations30,34,35
4.2. Synthesis of 30,60-di(trimethylacetyloxy)-3-oxospiro
[isobenzofuran-1(3H),90-[9H]xanthene]-6-carbonyl azide (3)
We used Spartan’06 software at the RM1 level to calculate the
structure of the complex formed between 6a with cytosine
(Fig. 5).36 Table 2 lists the distances and bond angles for the hydro-
gen bonds. The two ureido NH hydrogen atoms of receptor 6a
formed two strong hydrogen bonds with the oxygen and pyridine
nitrogen atoms of cytosine, with distances (bond angles) of 1.942
(164.5) and 1.933 Å (161.3°), respectively. The pyridine nitrogen
atom of the receptor 6a formed one hydrogen bond with the amino
group NH unit of cytosine, with a distance of 1.936 Å and a bond
angle of 167.4° (Fig. 5). All the bond lengths and angles satisfy
the criteria for strong hydrogen bonding, suggesting that cytosine
binds tightly to receptor 6a.50 Furthermore, the xanthenyl ring of
receptor 6a is positioned near the hydrogen atom H1 of cytosine
with a distance of 3.215 Å; this close proximity between the xan-
thenyl unit and cytosine would permit quenching of the fluores-
cence of receptor 6a through photoinduced electron transfer
(Scheme 1).43–46
NHS (0.53 g, 4.6 mmol) and N,N0-dicyclohexylcarbodiimide
(DCC, 0.96 g, 4.8 mmol) were added sequentially to a solution of
6-carboxyfluorescein (1, 1.68 g, 3.1 mmol) in 1,4-dioxane
(20 mL). The mixture was stirred under N2 for 25 min. The white
precipitate, N,N0-dicyclohexylurea, was removed through vacuum
filtration. The filtrate was concentrated in vacuo to afford com-
pound 2, which was used directly for next step without
purification.
The crude product obtained above was diluted with acetone
(50 mL) and then a solution of NaN3 (0.78 g, 12 mmol) in water
(20 mL) was added. The resulting mixture was then stirred at room
temperature for 3.5 h. After evaporation of the solvent in vacuo, the
residue was purified through column chromatography (eluent: 0–
20% EtOAc/hexane) to afford compound 3 as a white foam (1.25 g,
71%). Rf = 0.44 [EtOAc/hexane, 1:4 (v/v)]. Mp 158–160 °C. IR (neat,
cmꢀ1) 2978, 2145, 1760, 1695, 1611, 1495, 1481, 1423, 1274, 1249,
1205, 1154, 1111, 1078, 995, 908, 730. 1H NMR (400 MHz, CDCl3,
ppm) d 1.35 (s, 18H), 6.78 (s, 4H), 7.07 (s, 2H), 7.79 (s, 1H), 8.12
(d, J = 8.0 Hz, 1H), 8.30 (dd, J = 8.0, 1.5 Hz, 1H). 13C NMR
(100 MHz, CDCl3, ppm) d 27.16, 39.31, 82.21, 110.6, 115.3, 118.0,
125.2, 125.8, 128.8, 130.4, 131.1, 137.0, 151.6, 152.9, 153.5,
168.0, 171.1, 176.6. HRMS: calcd for C31H27N3O8Na+ (M+Na)
592.169, found 592.169.
3. Conclusions
We have synthesized the water-soluble, small-molecule, fluo-
rescein-containing ureido compounds 6 and 8, each through two
one-pot transformations, using 6-carboxyfluorescein as a starting
material. 1H NMR spectroscopic dilution experiments revealed evi-
dence for strong
p–p stacking in the dimer formed from 6a in
phosphate buffer (pD 8.75) in D2O. We then used 15N NMR spec-
troscopy to investigate the binding of compounds 6a and 8a with
cytosine at pH 8.75. In the presence of compound 6a, but not 8a,
the signals of nitrogen atoms N1 and N4 in cytosine underwent
downfield shifts, suggesting that 6a binds cytosine, but 8a does
not. We also observed that the binding of 6a to cytosine resulted
in quenching of the fluorescence of 6a at pH 7.4, allowing us to
determine the apparent binding constant using nonlinear least-
squares fitting. Uracil and deoxycytidine did not quench the fluo-
rescence of 6a in related binding studies, indicating that receptor
6a is a specific receptor for cytosine free base. Measurements of
the apparent binding constants of 6a to other cytosine derivatives,
such as 5-fluorocytosine, indicated that 6a is a versatile fluorescent
indicator for various cytosine free bases. Overall, receptor 6a is the
first water-soluble, small-molecule fluorescent receptor that binds
specifically to cytosine free bases in aqueous solution.
4.3. Synthesis of N-(30,60-di(trimethylacetyloxy)-3-oxospiro
[isobenzofuran-1(3H),90-[9H]xanthen]-5-yl)-N0-(2-
imidazoyl)urea (5)
A solution of compound 3 (0.121 g, 0.20 mmol) in toluene
(1 mL) was heated at 100 °C for 1 h. The solvent was then evapo-
rated under a N2 stream to afford compound 4, which was used di-
rectly in the next step without purification.
The residue obtained above was dissolved in 1,4-dioxane (2 mL)
and then 2-aminoimidazole (0.017 g, 0.21 mmol) was added. The
mixture was then stirred at 80 °C for 1.5 h. After evaporation of
the solvent, the residue was purified through column chromatog-
raphy (eluent: 0–4% MeOH/CH2Cl2) to afford 5 as a white solid in
65% yield. Rf = 0.26 [MeOH/CH2Cl2, 1:20 (v/v)]. Mp >217 °C (dec).
IR (neat, cmꢀ1) 3362, 2934, 2847, 1756, 1608, 1557, 1495, 1423,
1216, 1158, 1114, 759. 1H NMR (400 MHz, CDCl3/DMSO-d6, ppm)
d 1.31 (s, 18H), 6.65 (s, 2H), 6.92–6.99 (m, 4H), 7.24 (s, 2H), 7.55
(d, J = 8.4 Hz, 1H), 7.79 (s, 1H), 7.89 (d, J = 8.4 Hz, 1H), 9.67 (s,
2H). 13C NMR (100 MHz, CDCl3/DMSO-d6, ppm) d 27.26, 39.10,
80.39, 110.8, 117.1, 117.4, 119.1, 120.4, 126.2, 129.7, 148.5,
4. Materials and methods
4.1. Materials and instruments
þ
151.2, 152.7, 155.4, 168.9, 176.6. HRMS: calcd for C34H33N4O8
Chemicals were used as purchased without further purification.
Flash chromatography was performed over silica gel (70–230
mesh) and monitored through thin layer chromatography (TLC)
on silica gel plates. 1H and 13C NMR spectra were recorded using
a Varian 400 (400 MHz) instrument with CDCl3, D2O, DMSO-d6,
and CD3OD as solvents. Chemical shifts of protons are given in
ppm relative to the signal of TMS as the internal standard; chem-
(M+H) 625.229, found 625.230.
4.4. Synthesis of N-(30,60-dihydroxy-3-oxospiro[isobenzofuran-
1(3H),90-[9H]xanthen]-5-yl)-N0-(2-imidazoyl)urea (6)
1 N NaOH (0.47 mL) was added to a solution of compound 5
(0.050 g, 0.080 mmol) in MeOH (1 mL) and THF (1 mL). The

Y. L. Jiang et al. / Bioorg. Med. Chem. 18 (2010) 7034–7042
7041
resulting solution was stirred at room temperature for 1 h. After
evaporation of the solvents, the residue as dissolved in water
(2 mL). The solution was filtered and acidified to pH 2–3 using
6 N HCl. The resulting solid was filtered off, washed with water,
and dried to yield as a deep-red solid (23 mg, 65%). Mp >216 °C
(dec). UV–vis (disodium salt in 50 mM phosphate buffer, pH 7.4,
nm) kmax 239, 276, 319, 490. IR (neat, cmꢀ1) 3094, 2942, 1717,
1586, 1459, 1386, 1209, 1111, 846, 737, 690. 1H NMR (400 MHz,
D2O, ppm) d 6.21–6.24 (m, 2H), 6.39 (s, 4H), 6.84 (d, J = 9.2 Hz,
3H), 7.59 (d, J = 8.4 Hz, 1H), 7.95 (s, 1H). 13C NMR (100 MHz,
CD3OD/DMSO-d6, ppm) d 82.39, 102.5, 110.4, 111.4, 113.0, 118.9,
120.0, 125.8, 129.6, 147.2, 152.2, 155.6, 159.7, 169.1. HRMS: calcd
with AcOH (2 mL), dried in air, and then recrystallized from water
(15 mL) to afford 5-cholorcytosine (0.46 g, 62%). Mp >288 °C (dec).
1H NMR (400 MHz, DMSO-d6, ppm) d 10.84 (s, 1H), 7.70 (s, 1H),
7.07 (s, 2H).
4.8. Determination of dimerization constants of 6b through 1H
NMR spectroscopic dilution experiments
During the preparation of the buffer for the NMR spectroscopy
experiments, pre-weighed Na2HPO4 was exchanged with D2O
(99.5%) three times under a lyophilizer. The resulting Na2DPO4
was then dissolved in D2O to provide a total phosphate concentra-
tion of 50 mM. Buffers having values of pD of 7.4 and 8.75 were
prepared by adjusting the pD of the Na2DPO4 solution using NaOD
and DCl solutions and a pH meter.
A stock solution of 6b was prepared by dissolving 6 into 0.33 N
NaOD (2 equiv) and adjusting the volume to the required concen-
trations (usually 40 mM) using phosphate buffer (pD 7.4). The
solution of 6b (10 mM) for the NMR spectroscopic dilution exper-
iment was then prepared by diluting the stock solution with the
phosphate buffer (pD 7.4) and adjusting the pD to 8.75. In other
dilution experiments, the phosphate buffer was applied at a pD
of 8.75.
The dimerization constant for 6b was determined by diluting a
sample from a high concentration (10.0 mM) to the minimum con-
centration (0.16 mM) required for detection of a signal by 1H NMR
spectroscopy at 293 K. The chemical shift of proton H14 at
7.956 ppm at high concentration was followed; it decreased grad-
ually to 7.656 ppm upon decreasing the concentration of 6b. The
data were then fitted to a nonlinear regression curve on a PC using
the following Eq. 1:
þ
for C24H17N4O6 (M+H) 457.114, found 457.114.
4.5. Synthesis of N-(30,60-di(trimethylacetyloxy)-3-
oxospiro[isobenzofuran-1(3H),90-[9H]xanthen]-5-yl)-N0-
phenylurea (7)
A solution of compound 3 (0.107 g, 0.18 mmol) in toluene
(1 mL) was heated at 100 °C for 1 h. The solvent was then evapo-
rated under a N2 stream to afford compound 5, which was used di-
rectly in the next step without purification.
The residue obtained above was dissolved in 1,4-dioxane (2 mL)
and then aniline (0.017 g, 0.017 mL, 0.19 mmol) was added. The
mixture was stirred at 80 °C for 2 h. After evaporation of the sol-
vent, the residue was purified through column chromatography
(eluent: 0–60% EtOAc/hexane) to afford 7 as a white solid in 90%
yield. Rf = 0.46 [EtOAc/hexane, 1:1 (v/v)]. Mp 182–184 °C (dec). IR
(neat, cmꢀ1) 3369, 2978, 2927, 1756, 1709, 1597, 1539, 1495,
1423, 1289, 1220, 1154, 1111, 995, 893, 752, 690. 1H NMR
(400 MHz, CDCl3/DMSO-d6, ppm) d 1.31 (s, 18H), 6.93–6.99 (m,
5H), 7.21–7.25 (m, 4H), 7.40 (d, J = 8.0 Hz, 2H), 7.52 (dd, J = 8.4,
1.4 Hz, 1H), 7.60 (d, J = 1.2 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 8.8 (s,
1H), 9.4 (s, 1H). 13C NMR (100 MHz, CDCl3/DMSO-d6/CD3OD
ppm) d 27.11, 39.20, 80.62, 110.8, 111.3, 117.0, 118.3, 119.0,
119.1, 119.2, 120.4, 120.5, 122.9, 126.5, 129.2, 129.5, 139.3,
147.3, 147.4, 151.3, 152.4, 152.5, 152.8, 155.2, 168.7, 176.5. HRMS:
calcd for C37H34N2O8Na+ (M+Na) 657.221, found 657.222.
1=2
d ¼ dm ꢀ ½ðdm ꢀ ddÞ=cꢂ½c þ 0:25=Kd ꢀ ð0:5c=Kd þ 0:0625=KdÞ
ꢂ
ð1Þ
where d, dm, dd, and c are the observed chemical shift, the chemical
shift of the monomer, the chemical shift of the dimer, and the total
concentration of the receptor; Kd is the dimerization constant.
4.6. Synthesis of N-(30,60-dihydroxy-3-oxospiro[isobenzofuran-
1(3H),90-[9H]xanthen]-5-yl)-N0-phenylurea (8)
4.8.1. 15N NMR spectroscopic study of the binding between
receptor 6a and cytosine
In this experiment, partially labeled cytosine-2,4-13C-1,3,4-15N-
cytosine (C1315, Sigma) was used. The stock solution (usually
40 mM) of C1315 was prepared by dissolving C1315 into phos-
phate buffer in water at pH 7.4. Stock solutions of 6a and 8a were
prepared using a similar method as described above, except that
NaOH was used in place of NaOD.
1 N NaOH (0.40 mL) was added to a solution of compound 7
(0.042 g, 0.065 mmol) in MeOH (1 mL) and THF (1 mL). The result-
ing solution was stirred at room temperature for 1 h. After evapo-
ration of the solvents, the residue as dissolved in water (2 mL) and
the solution filtered and acidified to pH 2–3 using 6 N HCl. The
resulting solid was filtered off, washed with water, and dried to
yield 8 as deep-red solid (29 mg, 95%). Mp >216 °C (dec). UV–vis
(disodium salt in 50 mM phosphate buffer, pH 7.4, nm) kmax 239,
270, 321, 491. IR (neat, cmꢀ1) 3290, 3072, 1688, 1601, 1546,
1495, 1445, 1386, 1267, 1209, 1180, 1111, 850, 748, 690. 1H
NMR (400 MHz, CD3OD, ppm) d 6.54–6.68 (m, 6H), 7.00 (t,
J = 7.2 Hz, 1H), 7.24 (t, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.44
(s, 1H), 7.63 (dd, J = 8.4, 1.8 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 8.4 (s,
1H), 8.9 (s, 1H). 13C NMR (100 MHz, CD3OD, ppm) d 102.1, 110.3,
112.1, 112.4, 119.2, 119.7, 120.0, 123.0, 125.5, 128.6, 129.0,
Three samples for NMR spectroscopy (0.6 mL) were prepared
having a D2O content of 10% each. The first sample, which con-
tained C1315 (10 mM) only, was prepared by diluting the stock
solution of C1315 with phosphate buffer (pH 8.75) and adding
D2O. The second sample, which contained C1315 (10 mM) and 8a
(10.0 mM), was prepared by mixing the stock solutions of C1315
and 8a, phosphate buffer (pH 8.75), and D2O. The third sample,
which contained C1315 (10 mM) and 6a (10.0 mM), was prepared
in a manner similar to for the second sample, except that the stock
solution of 6a was applied. The final pH of each sample was ad-
justed to 8.75 using NaOH and HCl solutions.
138.5, 146.6, 152.8, 153.1, 160.1, 170.2. HRMS: calcd for
þ
C
27H19N2O6 (M+H) 467.124, found 467.123.
15N NMR spectra were recorded using a Varian 400 spectrome-
ter; the chemical shifts of the nitrogen atoms are reported in ppm.
The samples were scanned 3000–6000 times for 4–8 h.
4.7. Synthesis of 5-chlorocytosine51
N-Chlorosuccinimide (NCS, 0.801 g, 6.00 mmol) was added to a
solution of cytosine (0.55 g, 5.0 mmol) in a mixture of water (2 mL)
and AcOH (20 mL) and then the resulting solution was stirred for
72 h at room temperature. The precipitate was filtered off, washed
4.9. Fluorescence titration
Stock solutions of 6a and 8a were prepared as described in Sec-
tion 4.8. Stock solutions of cytosine (0.200 M) and 5-fluorocytosine

7042
Y. L. Jiang et al. / Bioorg. Med. Chem. 18 (2010) 7034–7042
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Acknowledgments
34. Lamale, B.; Henry, W. P.; Daniels, L. M.; Zhang, C.; Klein, S. M.; Jiang, Y. L.
Tetrahedron 2009, 65, 62.
35. Jiang, Y. L. Bioorg. Med. Chem. 2008, 16, 6406.
This study was supported by the Office of Research and Spon-
sored Programs (RD09010, E82016, RS0026) and SFRA Grants (for
P.P. and Y.L.J.) from the Honors College at East Tennessee State
University.
36. Shao, Y.; Fusti-Molnar, L.; Jung, Y.; Kussmann, J.; Ochsenfeld, C.; Brown, S. T.;
Gilbert, A. T. B.; Slipchenko, L. V.; Levchenko, S. V.; O’Neill, D. P.; Distasio, R. A.,
Jr.; Lochan, R. C.; Wang, T.; Beran, G. J. O.; Besley, N. A.; Herbert, J. M.; Lin, C. Y.;
Van Voorhis, T.; Chien, S. H.; Sodt, A.; Steele, R. P.; Rassolov, V. A.; Maslen, P. E.;
Korambath, P. P.; Adamson, R. D.; Austin, B.; Baker, J.; Byrd, E. F. C.; Daschel, H.;
Doerksen, R. J.; Dreuw, A.; Dunietz, B. D.; Dutoi, A. D.; Furlani, T. R.; Gwaltney,
S. R.; Heyden, A.; Hirata, S.; Hsu, C.-P.; Kedziora, G.; Khalliulin, R. Z.; Klunzinger,
P.; Lee, A. M.; Lee, M. S.; Liang, W.; Lotan, I.; Nair, N.; Peters, B.; Proynov, E. I.;
Pieniazek, P. A.; Rhee, Y. M.; Ritchie, J.; Rosta, E.; Sherrill, C. D.; Simmonett, A.
C.; Subotnik, J. E.; Woodcock, H. L., III; Zhang, W.; Bell, A. T.; Chakraborty, A. K.;
Chipman, D. M.; Keil, F. J.; Warshel, A.; Hehre, W. J.; Schaefer, H. F., III; Kong, J.;
Krylov, A. I.; Gill, P. M. W.; Head-Gordon, M. Phys. Chem. Chem. Phys. 2006, 8,
3172.
Supplementary data
Supplementary data (1H and 13C NMR spectra of compounds 3
and 5–8) associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2010.08.013.
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