Binding Expedient of 2-carbamido-1,3-indandione to Nucleic Acids: Potential Fluorescent Probe

Article abstract of DOI:10.1111/php.13378

Fluorescent and computational methods were used to elucidate the binding expedient of 2-carbamido-1,3-indandione (CAID) tautomers to nucleotides. The dependence of the fluorescence emission of CAID loaded nucleic acids sequences to compound concentration, temperature and time variation was investigated. It was found that the subject compound binds to nucleic acids but does not intercalate. According to our quantum-chemical calculations on the conjugation between CAID and nucleotides, the binding in the formed complexes may be through hydrogen bonds. Two possible types of complexes were considered—CAID to the phosphate group and CAID to the nucleobase. To estimate the binding affinity, the interaction energies of the formed complexes were calculated. Tautomer 2-carboamide-1-hydroxy-3-oxo-indane is preferred in the formation of complexes, and the phosphate group complexes were more stable. Generally, the guanosine and deoxyguanosine monophosphate complexes were the most preferred regardless of the complex type. Because of the lack of cytotoxic effect on untransformed cell lines of mouse embryo fibroblasts Balb/c 3T3 according to our previous report (Markova et al, (2017) Bulg Chem Commun, 49D, 221–226) and the affinity to nucleic acids, we can suggest that the subject compound could be suitable to be used as a novel type of fluorescent biomarker.

Full text of DOI:10.1111/php.13378

Photochemistry and Photobiology, 20**, **: *–*
Research Article
Binding Expedient of 2-carbamido-1,3-indandione to Nucleic Acids:
Potential Fluorescent Probe
Nina Stoyanova1, Nadezhda Markova2* , Ivan Angelov2, Irena Philipova2 and Venelin Enchev1
1Institute of General and Inorganic Chemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria
2Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria
Received 17 November 2020, accepted 1 January 2021, DOI: 10.1111/php.13378
ABSTRACT
of new compounds suitable for biomarkers and therapeutics is an
important challenge for the scientific community worldwide.
In the most cases, fluorescent labels are distributed randomly
and nonhomogenously in the cells and tissues. Therefore, the
intensity of the observed fluorescence signal depends not only on
the local concentration, but also on the intensity of lighting,
medium, optical path length, bleaching and other processes. That
is why fluorescence probes with significant spectral shift after
binding to the observed object are preferred. It should also be
noted that a good fluorescent marker should form hydrogen
bonds with biomolecules, and the formed complex is stabilized
in solution. There are many biomarkers that bind spontaneously
to DNA and exhibit higher emission (2). Some of the most used
biomarkers Hoechst 33258, Hoechst 33342 and Hoechst 34580
have NH group and bind to DNA via bifurcated hydrogen bonds
and/or electrostatic interactions (3). Unlike TAMRA and DAPI,
which damage the cell membranes and the DNA structure, these
markers are suitable for application on live-cell cultures because
they do not damage them and have lower cytotoxicity.
2-Substituted-1,3-indandiones are biologically active com-
pounds which are used in the medicine and biology (4). Studies
on their biological activity indicate the presence of anticoagulant
(5), anti-inflammatory (6), antibacterial (7), protein binding (4),
antineoplastic (8,9) and neuroprotective action (10). 2-Car-
bamido-1,3-indanedione (CAID), also known as 1,3-dioxo-2-
indane-carboxamide, belongs to the group of 2-substituted
indan-1,3-dione. The compound was synthesized by Horton and
Murdock (11) in 1960. In solid state, it exists as yellow-orange
needle-like crystals with melting point in the range of
180–220°C. The initially proposed structure (11) was later cor-
rected by Enchev et al. (12) It has been found that in solid state
and in solution, CAID coexists in two tautomeric forms: 2-(hy-
droxylaminomethylidene)-indan-1,3-dione, (CAIDA), and 2-car-
boamide-1-hydroxy-3-oxo-indane, (CAIDB), shown in Fig. 1. In
2007, CAID derivatives were synthesized (13), and it was found
that some of them crystallize in two tautomeric forms with dif-
ferent colors of the crystals.
Fluorescent and computational methods were used to elucidate
the binding expedient of 2-carbamido-1,3-indandione (CAID)
tautomers to nucleotides. The dependence of the fluorescence
emission of CAID loaded nucleic acids sequences to com-
pound concentration, temperature and time variation was
investigated. It was found that the subject compound binds
to nucleic acids but does not intercalate. According to our
quantum-chemical calculations on the conjugation between
CAID and nucleotides, the binding in the formed complexes
may be through hydrogen bonds. Two possible types of com-
plexes were considered—CAID to the phosphate group and
CAID to the nucleobase. To estimate the binding affinity, the
interaction energies of the formed complexes were calculated.
Tautomer 2-carboamide-1-hydroxy-3-oxo-indane is preferred
in the formation of complexes, and the phosphate group com-
plexes were more stable. Generally, the guanosine and deoxy-
guanosine monophosphate complexes were the most
preferred regardless of the complex type. Because of the lack
of cytotoxic effect on untransformed cell lines of mouse
embryo fibroblasts Balb/c 3T3 according to our previous
report (Markova et al, (2017) Bulg Chem Commun, 49D,
221–226) and the affinity to nucleic acids, we can suggest that
the subject compound could be suitable to be used as a novel
type of fluorescent biomarker.
INTRODUCTION
Malignant and infectious diseases (including COVID-19) account
for a large proportion of morbidity and mortality in the world.
The early detection, intervention and expedient treatment is
mandatory for a favorable prognosis. Modern imaging methods
are powerful tool in medicine. As part of optical molecular imag-
ing techniques, the fluorescence spectroscopy is promising tech-
nique for provision of more precise molecular and physiological
information. The diagnosis of infectious and other diseases is
most often performed by biomarker detection in laboratories
using conventional tests (1). There are many shortcomings in the
diagnosis of viral infections, including COVID-19, due to the
low sensitivity and specificity of the diagnostic tests, as well as
the long time to obtain the result. The search and development
The photophysical properties of CAID have recently been
investigated as well as its possible use as a sunscreen and bio-
marker (14). It exhibits very good photostability and absorbs in
the visible area (400–450 nm), UVA (320–400 nm), UVB
(290–320 nm) and UVC (200–290 nm) spectrum. CAID is also
suitable to be used as a biomarker for biologically relevant mole-
cules because it absorbs and fluoresces in very wide spectral
*Corresponding author email: nadya@orgchm.bas.bg (Nadezhda Markova)
© 2021 American Society for Photobiology
1

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Nina Stoyanova et al.
Figure 1. Two tautomeric forms of 2-carbamido-1,3-indanedione.
B3LYP/6-31 + G(d,p) computational level without symmetry constraints.
There are two possible ways of association between CAID and the
nucleotides: (i) to the nitrogen base denoted as XMP-CAID and (ii) to
the phosphate group denoted as CAID-XMP (see Figs. 3 and 4). The
local minima were verified by establishing that the Hessians had zero
eigen values. The values of Gibbs free energies (ΔG) were calculated for
a temperature of 298.15 K.
range and has high quantum yield. A hypsochromic shift by
25–30 nm of the fluorescence band of CAID-tagged DNA
sequences was observed after addition of the compound to the
DNA sequences. This suggests that CAID could bind to DNA
via hydrogen bonds. Fluorescence and microscopic analysis of
CAID in Balb/c3T3 cell cultures (15) shows that 2-carbamido-
1,3-indanedione has ability to penetrate through cell membranes.
The microscopic analyses indicate that the compound is not toxic
for the cell lines used. 2-Carbamido-1,3-indanedione also inter-
acts with serum albumin (4); thus, it could be a fluorescence
marker not only for DNA and RNA but also for other biomole-
cules. For live-cell probing penetration and cytotoxicity, as well
as side effects on cellular function are an important aspect that
should be taken into account when developing novel fluorescent
probes. Such a study related to the interaction of the title com-
pound with nucleic acids and the resulting biological applications
has not yet been performed. Therefore, it would be useful to
examine the binding affinity of the two stable tautomers of 2-car-
bamido-1,3-indanedione to nucleotides. Such study would
explain important aspects of the binding mode of the compound
in relation to its biomarker properties.
Spectral measurements. The solvent used for the spectrophotometric
and spectrofluorimetric investigations and analyses were purchased form
Sigma-Aldrich, Germany as commercial products of analytical grade, and
were used without further purification. 2 mL TRIS buffer (tris
hydroxymethyl aminomethane) at pH = 7.2 was used for preparing of
the solutions. The Ethidium bromide (Sigma-Aldrich, Germany) in
concentration 20 μM was used to prepare the solutions required for the
study. Double stranded DNA (dsDNA) and RNA sequences, purchased
from Metabion, Germany, in concentrations 40 and 200 μg mL−1
,
respectively, were used in the experiments. The concentration of the title
compound varies from 10 to 200 μM in different experiments. A stock
solution of CAID in dimethyl sulfoxide (DMSO) (10 mM) was prepared
and stored in the dark and diluted prior the measurements. The dye-
solution in microliter volume was used to obtain the desired ratio of the
DNA/CAID or RNA/CAID complexes. The final DMSO concentration in
the experimental volume did not exceed 0.5%. At this concentration,
DMSO does not affect the observed spectra and the tested biological
objects. The preparations of the complexes for investigation were carried
out in
a thermostated (37°C) quartz cell at constant stirring. The
fluorescence spectra of DNA/EthBr/CAID and DNA/CAID were recorded
after excitation at 500 and 330 nm, respectively, while those of RNA/
EthBr and RNA/CAID—after excitation at 500 and 350 nm, respectively.
The influence of temperature on fluorescence spectra of dsDNA/CAID
(λex = 330 nm) recorded at room temperature (initially and after 24 h of
the experiment), after heating (25–90°C for 1.5 h) and after cooling for
4 h was studied.
Fluorescence measurements were performed using 10 mm path-length
quartz cuvettes on a Perkin Elmer LS55 spectrofluorimeter at room tem-
perature or after heating the cuvette holder to specific, digitally controlled
temperature. The spectra were recorded, and the data were analyzed and
graphically represented by means of computer program Origin 8.0
(Microcal Software, Inc., Northampton, MA).
MATERIALS AND METHODS
Synthesis of 1,3-dioxo-2-indane-carboxamide. 1,3-Dioxo-2-indane-
carboxamide was synthesized via a two-step procedure (11). Thus, the
condensation of diethyl phthalate with acetonitrile in the presence of
sodium methoxide afforded 1,3-dioxo-2-indancarbonitril in 91% yield
after flash column chromatography. The desired amide was obtained by
subsequent hydrolysis of the corresponding nitrile with concentrated
sulfuric acid. Recrystallization from methanol gave 1,3-dioxo-2-indane-
carboxamide in 64 % yield. The structure of the obtained compound was
proved by 1H and 13C NMR spectra in DMSO-d6. NMR spectra were
recorded on
a Bruker Avance II + 600 spectrometer, operating at
600.13 MHz for 1H and 150.92 MHz for 13C, using a 5 mm dual probe
head. The measurements were carried out at ambient temperature
(293 K). Sample concentration of 0.01 M for 1H and saturated solution
for 13C NMR spectra were used. The chemical shifts are identical with
these presented in paper (12): 1H NMR: (DMSO-d6, 600 MHz): δ 9.08
(br, 1H, NH), 8.32 (br, 1H, NH), 7.65–7.63 (m, 2H, arom.), 7.607.50 (m,
2H, arom.) ppm. 13C NMR: (DMSO-d6, 150.9 MHz): δ 191.13 (C-1,
C-3), 167.50 (C-10), 138.21 (C-8, C-9), 133.22 (C-5, C-6), 120.93 (C-4,
C-7), 92.27 (C-2) ppm. The 1H and 13C NMR spectra of CAID in
DMSO-d6 are presented in Figures S1 and S2 in the Supporting
Information.
Computational details. The quantum-chemical calculations were
performed using Gaussian09 software package (16). Geometric
optimization of the tautomers CAIDA and CAIDB; the nucleotides AMP,
GMP, UMP and CMP; the deoxynucleotides dAMP, dGMP, TMP and
dCMP; and the complexes of the tautomers with nucleotides/
deoxynucleotides (XMP-CAID or CAID-XMP) were carried out at
RESULTS AND DISCUSSION
Fluorescent probes (fluorophores) as molecules that absorb light
of a specific wavelength and emit light of a different, typically
longer wavelength are administered to investigate biological sam-
ples. The role of these probes is to attach to target molecule and
act as a marker for analysis with fluorescence microscopy. The
fluorophores are distributed randomly and unevenly in cells and
tissues, and this way the intensity of the observed fluorescence
signal depends on different factors as local concentration, illumi-
nation intensity, surroundings and other processes. In order to
avoid the influence of these factors, fluorescent probes that exhi-
bit a significant spectral shift upon reaction or binding to the
investigated object are preferred for use. Furthermore, a required

Photochemistry and Photobiology
3
feature for fluorescent probes is the absence of photobleaching,
as well as to show two or more well-separated emission bands in
organic solvents. Such characteristics of fluorescence signals are
often manifested by compounds with native tautomerism.
maximum is lower ca. 4% compared with the peak of the pure
EthBr in dsDNA at the conditions described above. Insert in
Fig. 2 shows dependence of the F/F0 to CAID concentration (F0
and F are the measured fluorescence intensities of DNA/EthBr
without and with CAID, respectively). We can conclude that
CAID does not displace the intercalating agent and interacts with
dsDNA in a different manner, probably.
The fluorescence spectra of dsDNA/CAID (λex = 330 nm)
were recorded initially and after 24 h of the experiment at room
temperature. The solution was heated gradually from 25 to 90°C
Excited-state intramolecular proton transfer (ESIPT) is very
effective for designing dual-band fluorescent signal probes.
ESIPT leads to the formation of two tautomeric forms in the
excited state of the probe (17), which have well-separated emis-
sion maxima. According to our previous investigations (12,14),
2-carbamido-1,3-indanedione is exactly such compound. CAID
can exist in gas phase and solution in two tautomeric forms,
CAIDA and CAIDB, close by energy (Fig. 1). Two tautomeric
forms exist both in the ground and in the first excited state. Due
to its molecular structure, CAID could act as a proton donor in
hydrogen bonding interactions. Moreover, this compound demon-
strates the appropriate spectral ranges of absorption and fluores-
cence, and the high fluorescence quantum yield (0.78 in saline
buffer) which are important advantages for probing biomolecules
of high importance for biology and medicine (14).
To elucidate the possibility of CAID binding to nucleic acids
and its use as a fluorescent probe in biological investigations, flu-
orescence spectra of DNA and RNA sequences loaded with
CAID (DNA/CAID or RNA/CAID) were recorded. The depen-
dence of the CAID fluorescence to the temperature, the time and
the compound concentration was studied. Several different fluo-
rescent measurements were performed to investigate the possibil-
ity of conjugation between CAID and DNA sequences. The
fluorescence spectra of DNA, DNA loaded with 20 μM ethidium
bromide (DNA/EthBr) and DNA/EthBr + CAID (50, 100 or,
200 μM CAID) were recorded over time and are presented in
Fig. 2.
Double stranded DNA (dsDNA) was diluted in 2 mL TRIS
buffer at pH = 7.2 to achieve concentration 40 µg mL−1. The
DNA/EthBr (EthBr as intercalating agent) was used as a target
for verification of the title compound capability for DNA label-
ing as well as to clarify the possibility and manner of binding of
CAID to DNA in various concentrations of CAID (50–200 μM).
As can be seen from Fig. 2, even at ten-fold higher concentra-
tions of the test substance, the fluorescence spectrum of the
ethidium bromide does not change. The value of the peak
Figure 3. Fluorescence spectra of DNA/CAID after heating and cooling
(a), dependence of the fluorescent intensities ratio, F518/F440, to the tem-
perature (b) as well as fluorescence spectra of DNA/CAID at 0 h and
after 24 h of the measurements (c).
Figure 2. Fluorescence spectra of DNA, DNA/EthBr and DNA/EthBr +
CAID at different compound concentrations.

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Nina Stoyanova et al.
Figure 4. Fluorescence spectra of RNA/CAID over time (a) and at different temperatures (b). Fluorescence spectra of RNA/EthBr over time and at dif-
ferent temperatures (c).

Photochemistry and Photobiology
5
for 1.5 h and was cooled for 4 h. The spectra are presented in
Fig. 3. Two maxima at 440 and 518 nm were observed.
the peak at 518 nm is more defined. This fluorescent behavior is
probably due to the existence of the two CAID tautomers in
ground state and their fluorescence after excitation at 330 nm
(14).
At the starting point of the experiment, the two peaks seem
approximately the same (Fig. 3a–c). The intensity of the maxima
increases with incubation time and that one at 440 nm becomes
more intense in the end of measurement. The intensity of the
maxima changes in different manner during the experiments. The
fluorescent intensities ratio, F518/F440, increases during the heat-
ing while at cooling it decreases on the same pattern up to 55°C
(Fig. 3b). In the interval 55–25°C, the intensity of the maximum
at 518 nm does not change substantially unlike the one at
440 nm (Fig. 3a). After the sample was cooled to room tempera-
ture, it was left for up to 24 h and then a fluorescence spectrum
was recorded. As a result, the maximum at 440 nm gets more
intensive than the one at 518 nm (Fig. 3c). It should be noted
that there is no shifting in both of the fluorescent maxima, and
To investigate the binding expedient of CAID to RNA, several
fluorescent experiments were performed. The fluorescence spectra
of RNA/CAID in TRIS buffer at pH = 7.2 were scanned. We
have measured the emission of the solution at different tempera-
tures and over time (Fig. 4a,b). The dependence of the CAID flu-
orescence intensity on the incubation time is presented in Fig. 4a.
The intensity increases, and the slight red shift of the maximum
were observed. This is due to the fact that time is needed for
RNA to undergo conformational changes and to bind to CAID
through hydrogen bonds, probably. It is seen that there is similar-
ity of the fluorescent spectra of DNA/CAID and RNA/CAID
Figure 5. Complexes of the two tautomers, CAIDA and CAIDB, of 2-carbamido-1,3-indanedione with guanosine monophosphate (GMP) and deoxy-
guanosine monophosphate (dGMP), optimized at B3LYP/6-31+G(d,p) level.

6
Nina Stoyanova et al.
shown in Figs. 3c and 4a, respectively, due to the disruption of
the double-helix DNA structure after heating.
To clarify the affinity of CAID conjugation to nucleic acids,
quantum-chemical calculations at B3LYP/6-31+G(d,p) level
were performed. We started by modeling hydrogen bonded com-
plexes between the two tautomers of CAID and all possible
RNA and DNA nucleotides with purine and pyrimidine nitrogen
containing nucleobases: uridine monophosphate (UMP), thymi-
dine monophosphate (TMP), guanosine and deoxyguanosine
monophosphates (GMP and dGMP), cytidine and deoxycytidine
monophosphates (CMP and dCMP) as well as adenosine and
deoxyadenosine monophosphates (AMP and dAMP). There are
two possible sites of association of CAID to nucleotide by
hydrogen bonding—to the nucleobases (XMP-CAID) or to the
phosphate group (CAID-XMP). 2-carbamido-1,3-indanedione
takes part in hydrogen bonding by -NH2 and =O (-OH) groups.
The complexes formed between guanosine, deoxyguanosine,
cytidine and deoxycytidine monophosphates and CAID tautomers
are shown in Figs. 5 and 6. The structures of all other complexes
are presented in Figures S3 and S5 in the Supporting Informa-
tion.
Fluorescence spectra at different temperatures were recorded
(Fig. 4b). The fluorescence emission decreases when the temper-
ature increases. Such behavior could be explained by the rupture
of hydrogen bonds between the test molecule and RNA by heat-
ing.
Fluorescence spectra of RNA/EthBr over time and at different
temperatures were scanned. The experiment was performed to
study the behavior and binding possibility of CAID to RNA
compared with EthBr. As can be seen from Fig. 4c, changes in
the fluorescence emission were not observed. Therefore, the
ethidium bromide as an intercalating agent interacts with RNA in
a different manner from that one of CAID under changing med-
ium conditions. Thus, it can be concluded that CAID is suitable
for RNA containing biological objects detection. The fluorescent
properties of conjugation between CAID and nucleic acid
sequences were studied for evaluation of the potential for apply-
ing of CAID as a fluorescent probe in biological investigations.
Figure 6. Complexes of the two tautomers, CAIDA and CAIDB, of 2-carbamido-1,3-indanedione with cytidine monophosphate (CMP) and deoxycy-
tidine monophosphate (dCMP), optimized at B3LYP/6-31+G(d,p) level.

Photochemistry and Photobiology
7
All species were optimized at the B3LYP/6-31+G(d,p) level
of theory. The relative total energies (ΔEt) as well as the relative
Gibbs free energies (ΔG298) of all CAIDA and CAIDB dimers
in the gas phase are presented in Table 1.
ΔEint of all dimers are presented in Table 1. When CAID tau-
tomers are attached to the phosphate group of RNA nucleotides,
the most stable complexes are those formed between CAIDB
and pyrimidine base nucleotides: CAIDB-UMP and CAIDB-
According to the computed energies and energy differences,
the nucleotides form more stable complexes with tautomer
CAIDB when the hydrogen bonding is realized to their phos-
phate group. The free energy differences between dimers vary
from 0.62 to 5.52 kcal mol−1, and in all cases CAIDB com-
plexes are more stable than CAIDA ones. When CAID is hydro-
gen bounded to the nitrogen base of the nucleotides, CAIDB
complexes with GMP, dGMP, UMP and TMP are again pre-
ferred structures. According to our results, the structures of the
CAID dimers of the other nucleotides are isoenergetic, with a
slight predominance of CAIDA complexes.
CMP. The interaction energies are 17.67 and 17.51 kcal mol−1
,
respectively. The picture is slightly different in DNA complexes:
the CAIDB complexes are again more stable than those of
CAIDA, but the complexes CAIDB-dGMP and CAIDB-dCMP
have the highest interaction energy. When the hydrogen bonding
is realized with the nitrogen base of the nucleotides, the most
stable structures are dimers of CAIDB with GMP and dGMP:
ΔEint of the complexes formed are 17.10 and 14.99 kcal mol−1
respectively.
,
As a summary, the 2-carbamido-1,3-indanedione could form
stable complexes with nucleotides, and tautomer B is the pre-
ferred structure. Generally, the strongest complexes with CAIDB
form the G and dG nucleotides, and the hydrogen bonds are situ-
ated preferably in the phosphate group region. The TMP dimers
have the lowest interaction energy, and therefore, form the weak-
est complexes with CAID.
In order to evaluate the possibility of intermolecular hydrogen
bonds formation between CAID and nucleotides, the energies of
interaction (Eint) were calculated by eq. (1).
Eint ¼ ECAID þEXMP ꢀEcomplexð1Þ
EXMP, ECAID and Ecomplex are the Et energies, calculated at
B3LYP/6-31+G(d,p) level, for each nucleotide molecule, CAID
tautomer and its nucleotide complex, respectively. The results for
CONCLUSION
Quantum-chemical and fluorescent analyses were performed to
investigate the binding expedient of 2-carbamido-1,3-indandione
tautomers (CAIDA and CAIDB) to nucleotides. Such a study on
the binding ability of the title compound to DNA and RNA and
the resulting biological applications has not yet been performed.
According to our fluorescence experiments, CAID does not act as
an intercalating agent. The fluorescent behavior of the compound
shows that there is association between CAID and DNA and
RNA, and the binding is realized by hydrogen bonds, probably.
To elucidate the possibility of conjugation of CAID tautomers to
NA, two types of complexes were considered—when the com-
pound is attached to the nitrogen base and to the phosphate group
of the monophosphate. The calculated interaction energies of the
formed complexes indicate that tautomer CAIDB formed more
stable complexes than tautomer CAIDA. The preferred nucleotide
moiety for hydrogen bonding is the phosphate group. In general,
guanosine and deoxyguanosine monophosphate form the most
stable complexes depending on the complex type. As a summary,
it can be concluded that CAID has binding affinity to nucleic
acids and does not show a cytotoxic effect on untransformed cell
lines Balb/c 3T3. Thus, we could suggest the subject compound,
2-carbamido-1,3-indandione as a new reliable fluorescent biomar-
ker with future wide application in medicine and biology as well.
Table 1. Interaction energies, ΔEint, energy differences, ΔEt and ΔG298
(in kcal mol−1), for the complexes of the tautomers CAIDA and CAIDB
with nucleotides and deoxynucleosides calculated at B3LYP/6-31+G(d,p)
level.
Complex
type
Complex
type
ΔEint ΔEt ΔG298
ΔEint ΔEt ΔG298
To the phosphate group
To the phosphate group
4.57 5.52 CAIDA- 13.33 3.12 2.47
dGMP
CAIDA-
GMP
9.59
CAIDB-
GMP
CAIDA-
AMP
CAIDB-
AMP
CAIDA-
UMP
CAIDB-
UMP
CAIDA-
CMP
CAIDB-
CMP
15.65 0.00 0.00 CAIDB-
17.91 0.00 0.00
12.06 2.92 1.77
16.43 0.00 0.00
10.46 3.99 3.16
15.91 0.00 0.00
12.62 3.39 2.31
17.47 0.00 0.00
dGMP
10.42 3.60 2.46 CAIDA-
dAMP
15.48 0.00 0.00 CAIDB-
dAMP
12.4
3.78 2.49 CAIDA-
TMP
17.67 0.00 0.00 CAIDB-
TMP
12.62 3.42 0.62 CAIDA-
dCMP
17.51 0.00 0.00 CAIDB-
dCMP
To the nitrogen base
To the nitrogen base
GMP-
CAIDA
GMP-
CAIDB
AMP-
CAIDA
AMP-
CAIDB
UMP-
CAIDA
UMP-
CAIDB
CMP-
CAIDA
CMP-
CAIDB
9.82
17.1
8.81
5.78 3.76 dGMP-
CAIDA
0.00 0.00 dGMP-
CAIDB
0.58 0.00 dAMP-
CAIDA
11.78 1.75 1.20
14.99 0.00 0.00
9.16 0.46 0.00
11.08 0.00 0.04
6.63 1.54 0.67
9.63 0.00 0.00
10.76 1.40 0.00
13.62 0.00 0.67
Acknowledgements—The calculations were performed on the
AVITOHOL supercomputer at the Institute of Information and
Communication Technologies at the Bulgarian Academy of Sciences.
10.88 0.00 0.28 dAMP-
CAIDB
SUPPORTING INFORMATION
9.58
1.24 0.26 TMP -
CAIDA
12.25 0.00 0.00 TMP -
CAIDB
Additional supporting information may be found online in the
Supporting Information section at the end of the article:
1
Figure S1. H NMR spectra of CAID in DMSO-d6.
Figure S2. 13C NMR spectra of CAID in DMSO-d6.
Figure S3. Complexes of the two tautomers of 2-carbamido-
1,3-indanedione with cytidine monophosphate and deoxycytidine
monophosphate, optimized at B3LYP/6-31+G(d,p) level.
10.41 1.63 0.04 dCMP-
CAIDA
13.5
0.00 0.00 dCMP-
CAIDB

8
Nina Stoyanova et al.
9. Jung, J. K., J. Ryu, S. I. Yang, J. Cho and H. Lee (2004) Synthesis
and in vitro cytotoxicity of 1,3-dioxoindan-2-carboxylic acid ary-
lamides. Arch. Pharmacal Res. 27, 997–1000.
Figure S4. Complexes of the two tautomers of 2-carbamido-
1,3-indanedione with adenosine monophosphate and deoxyadeno-
sine monophosphate, optimized at B3LYP/6-31+G(d,p) level.
Figure S5. Complexes of the two tautomers of 2-carbamido-
1,3-indanedione with uridine monophosphate and thymidine
monophosphate, optimized at B3LYP/6-31+G(d,p) level.
Table S1. Total energies, Et and interaction energies, ΔEint
and ΔHint (in kcal mol-1), for nucleotides and deoxynucleosides
as well as their complexes with the tautomers CAIDA and
CAIDB calculated at B3LYP/6-31+G(d,p) level.
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