Tropolone-Conjugated DNA: Fluorescence Enhancement in the Duplex

Article abstract of DOI:10.1002/cbic.201800822

Tropolone (2-hydroxycyclohepta-2,4,6-triene-1-one and tautomer) is a non-benzenoid bioactive natural chromophore with pH-dependent fluorescence character and extraordinary metal binding affinities, especially with transition-metal ions Cu²⁺/Zn<

Full text of DOI:10.1002/cbic.201800822

Accepted Article
Title: Tropolone-conjugated DNA: Fluorescence Enhancement in the
Duplex
Authors: Nagendra K Sharma and Amarnath Bollu
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ChemBioChem
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Tropolone-conjugated DNA: Fluorescence Enhancement in the
Duplex
[
a]
[a]
Amarnath Bollu and Nagendra K. Sharma*
(
Dedicated to 65th birthday of Prof. Krishna. N. Ganesh, IISER-Tirupati)
Abstract: Tropolone is non-benzenoid bioactive natural chromophore
comprising pH dependent fluorescent character and extraordinarily
metal binding affinity especially with transition metal ions
tropolone exhibits interesting photophysical properties
(absorptions & emission) and considered as potential structural
moiety for designing the small molecule derived chromophores.
Absorption spectra of tropolone exhibits two peaks at wavelength
ranges 200-260nm and 260-380nm due to two types of electronic
2+
2+
2+
Cu /Zn /Ni . This report describes the syntheses and biophysical
studies of new tropolonyl thymidine (tr-T) nucleoside and tropolone
conjugated DNA oligonucleotides which form B-form DNA duplex
structure with complementary DNA strand. Though their duplex
structures are less stable as compare to the control. Further the
stability of those DNA duplex structures are lowered by increasing
number of tr-T residue or decreasing pH of environments. Most
importantly, these duplex structures become fluorescent because of
the conjugated tropolone moiety at thymidine residue. The
fluorescence behavior of those duplex structures exhibit pH
dependent such as more at low pH and less at high pH environments.
Importantly, the fluorescence characters of tr-DNA oligonucleotides
are significantly enhanced, nearly three folds, after duplex structure
formation with complementary control DNA oligonucleotide. Further
fluorescence behavior of tr-DNA duplex structures are also
dependents on pH conditions. Hence tropolonyl conjugated DNA is
new fluorescent analogues which could be employed for sensing the
DNA duplex formation and provide opportunities to further improve
fluorescence properties.
[9]
transitions: two π-π* and n-π* transitions. . Further, the pK
a
of
tropolone is reported as pK 6.5 and is reported as weak
a
fluorescent molecule.[10] Its fluorescence properties are described
in terms of the existence of the (n-π*) singlet state. Martin and co-
workers show that the fluorescent characters of tropolone, at
excitation wavelength (λ) 315nm, are dependent upon the solvent
polarity and pH conditions such as quantum yield of tropolone is
measured as 0.005 in cyclohexane (neutral), 0.003 in acidic water
(pH3.0) and 0.005*10-2 in basic conditions (pH12.0).[11] These
variation are due to the different form of tropolone structures as
neutral or anion forms due to hydroxyl protonation and
deprotonation process under different pH conditions (Figure 1a).
Thus we realized that tropolone could be interesting scaffold to
incorporate into peptides and nucleic acids for tuning their
physical and structural properties. Recently we have reported the
tropolonyl bearing amino acid/peptides and established the role
of tropolonyl carbonyl in conformational changes of peptides.[
However, we have found the role of tropolone in an unusual
12]
cleavage of adjacent amide bond under mild pH conditions and
reversible amidation in mild basic conditions.[
12-13]
To explore the
Introduction
photophysical properties of tropolone related compounds, we
have also explored the tropolone derived BODIPY analogues and
demonstrated their photophysical properties with fluorescent
Tropolone is α-hydroxy derivative of Tropone molecule which is
non-benzenoid seven-membered aromatic compound with a
carbonyl functional group.[1] Tropolone derivatives, tropolonoids,
are occurred in nature mostly in plants and Fungi.[2] Tropolone
and related derivatives are possessing enormous biological
activities, such as antibacterial,[3] antifungal,[4] antitumor,[5] and
antiviral[6] activities. Recent highlights show that tropolones could
be potent and selective inhibitors for enzymes with Zinc-
cofactor.[ Tropolone is also reported as distinctive metal
[14]
quantum yield upto ~16%. The incorporation of Tropolone into
nucleic acid is yet to bring in light as new conjugated DNA/RNA
analogues. However, the various chromophore conjugated
nucleosides and their DNA are reported and some of them have
shown distinctive photophysical properties as the enhancement
in fluorescence after formation of duplex with native
complimentary DNA oligonucleotide. Recent examples include 2-
aminopurine containing DNA as first potential fluorescent adenine
7]
2
+
2+
2+
chelating scaffold especially for Cu /Zn /Ni , because of α-
hydroxy carbonyl functional group.[8] The aromatic structure of
[15]
[16]
surrogates, Tor’s isomorphic RNA alphabet, Wilhelmsson’s
internucleobase FRET pair,[17] Sasaki’s fluorescent sensors for
nucleobase damage,[18] Purse’s tricyclic cytidine analogue,[19]
Luedtke’s DMAT nucleoside,[20] and
a number of modified
[
a]
A. N. Bollu and, Dr. N. K. Sharma* (Corresponding Author(s)
School of Chemical Science, Institution National Institute of Science
Education and Research -(NISER) Bhubaneswar,Jatani-752050,
Odisha, India & HBNI-Mumbai, Mumbai-India
Phone no. +91-674-249-4141
nucleobases oriented in major groove of DNA duplex which
interact with protein.[21] Their outcomes suggest that the photo
physical behavior of chromophores are affected with alteration in
microenvironment of DNA duplex such as polarity, hydrophobicity,
and pH conditions. Importantly, the photophysical behavior as
fluorescent character of tropolone decreases with increasing the
polarity and pH conditions of environment.[11] In repertoire of small
chromophore conjugated DNA analogues, we hypothesized to
conjugate tropolone moiety into thymidine (at C-5) via rigid and
conjugated alkyne linker which might be beneficial to utilize the
fluorescent character of tropolone moiety in the sensing of DNA
E-mail: nagendra@niser.ac.in
Supporting information for this article is given via a link at the end of
the document. Supplementary Information (ESI) available: [NMR
(1H/13C/31P), ESI-HRMS, of all new compounds of this reports are
provided as pdf file. HPLC of oligonucleotides are provided. Single
crystal X-ray data of compound (4) as Cif-file also provided
separately]. See DOI: 10.1039/x0xx00000x.
1
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ChemBioChem
10.1002/cbic.201800822
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duplex formation. Herein, this report describes the synthesis and
photo physical studies of new tropolonylated thymidine
nucleoside and its incorporated DNA oligonucleotides. This report
also demonstrates the formation of tr-DNA duplex structure as B-
form helix and enhancement in fluorescence’s intensity with
formation of duplex structure. Importantly this fluorescence event
is pH dependent.
thymidine phsophoramidite (6) in good yield. The characterization
data ( H-/ C-NMR/ P-NMR/ESI-HRMS) of compound 6 are
provided in Supporting Information.
1
13
31
Figure 1. (a) Tropolone; (b) tropolonylated nucleoside and DNA
Results
We began the synthesis of the rationally designed tropolonylated
thymidine nucleoside and its phophoramidites from commercially
available 5-Iodo-2’-deoxyuridine (1) and Tropolone (Scheme 1).
Scheme 1. Synthesis of Tropolonylated thymidine phosphoramidites and
ORTEP diagram of crystal compound (4).
5-Iodo-2’-deoxyuridine (1) was converted into 5-Ethynyl-2’-
deoxyuridine (2) in two steps by following the literature
procedure.[ The alkyne group of nucleoside (2) was coupled
with benzoyl protected 5-Iodotropolone (3) under Sonogashira
coupling reaction conditions using Pd-catalyst which produced 5-
tropolonylated thymidine nucleoside analogue (4) in a good yield.
The 5-iodotropolone derivative (3), however, was prepared from
commercially available Tropolone by following the reported
procedures.[ This tropolonylatedthymidine nucleoside (4) was
Further we attempted to incorporate new tropolonylated
phosphoramidite (6) into DNA oligonucleotides at different
positions with DNA oligo synthesizer. Herein we synthesized
duplex forming DNA from known oligonucleotides (ON1 & ON2)
with commercially available DNA phosphoramidites for control
studies (Table 1, Entry 1-2).Tropolonylated phosphoramidite (6)
was incorporated into DNA oligonucleotide ON2 and prepared
tropolonylated DNA (tr-DNA) oligonucleotides such as mono-
tropolonylated DNA oligonucleotide (ON3), di-tropolonylated DNA
22]
23]
1
13
characterized by
H-/ C-NMR/ESI-HRMS analyses (See
Supporting Information). Pleasantly we obtained the single crystal
of tropolonylated thymidine derivative (4) from 2% MeOH in DCM
solvent. The X-ray analysis of that single crystal confirms the
structure of new tropolonylated thymidine nucleoside (4). The
single crystal X-ray data are deposited to Cambridge
Crystallographic Data Centre with CCDC 1830067. The ORTEP
diagram of modified thymidine derivative (4) is also depicted in
Scheme 1, while its X-ray parameters are provided in the
Supporting Information. For synthesis of phosphoramidities, 5’-
OH of tropolonylatednucleoside (4) was protected with DMTr as
compound 5, and employed for phosphorylation, at 3’-OH, with
phosphorylating agent (diisopropylchlorophosporamidites) under
basic conditions which produced the desired tropolonylated
oligonucleotide
(ON4)
and
tri-tropolonylated
DNA
oligonucleotides (ON5) (Entry 3-5, Table 1). All these
oligonucleotides (ON1-ON5) were purified by Sephadex column
followed by HPLC and then characterized by ESI-MS which
confirmed the syntheses of oligonucleotides (Column 3-4, Table
1), their ESI-Mass spectra are provided in the Supporting
Information.
2
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Table 1. Synthesized DNA oligonucleotides and their mass
Entry
Name and Sequence of
synthesized DNA Oligo-
nucleotides (5’-3’) [a]
Calculated
Mass (M)
Observed
Deconvoluted
Mass
1
2
3
4
5
ON1: GCGTACACATGCG
ON2: CGCATGTGTACGC
ON3: CGCATGT*GTACGC
ON4: CGCATGT*GT*ACGC
ON5: CGCAT*GT*GT*ACGC
3959.639
3950.626
4080.639
4210.639
4340.639
3956.521 (M-3)-
3947.636 (M-3)-
4076.615 (M-4)-
4205.630 (M-5)-
4334.503 (M-6)-
[
a] T* is tropolonylated thymidine residue.
After successful syntheses of tr-DNA oligonucleotides we
attempted to explore their biophysical and photophysical
properties. We determined extinction coefficient (ɛ) of
debenzolylated tropolonylated nucleoside as 4-OH under
aqueous solution in presence of little NH
solubility in neutral pH, and found as 29123ꢀM cm wavelength
λ) 260nm (see in supporting information).
4
OH (pH10) due to poor
−
1
-1
(
Figure 2. (a) CD spectra of DNA duplexes (2.0M) in sodium phosphate buffer
CD Studies. Further we examined the formation of tropolonylated
DNA hybrid duplex from tr-DNA (ON3-ON5) and unmodified
complementary DNA oligonucleotide (ON1) near physiological
conditions by Circular Dichroism (CD) studies. To avoid the
photodecomposition of tropolone under acidic pH conditions
(100 mM) at pH7.0 conditions (A); (b) CD ellipticity vs number of tropolonyl
nucleoside unit at different pH conditions (B)
UV-Thermal melting: Further the stability of tropolonylated DNA
duplex structures were examined by UV-thermal melting or
thermal denaturation experiments. We prepared tropolonylated
DNA hybrid duplex structure as ON1:ON3, ON1-ON4, and
ON1:ON5 by annealing of equimolar DNA strands under different
pH conditions. We then performed UV-Melting experiments at
wavelength (λ) 260nm. For comparative studies, the similar
experiment was performed with unmodified DNA strands
(ON1:ON2) near physiological conditions (pH7.0) and considered
as control experiments. Their melting profiles (Absorbance vs
Temperature plots) at pH7.0 are depicted in Figure 3, while other
melting profiles at different pH conditions are provided in
Supporting Information. The typical melting profile of DNA duplex
structure is characteristic positive sigmoidal curve.[25] At pH7.0,
the UV melting profiles of tr-DNA duplex (ON1:ON3, ON1:ON4,
&ON1:ON5) exhibit positive sigmoidal curves as like control DNA
duplex structure and their first derivative curves are given as inset
(Figure 3A). The melting profile of same tr-DNA duplex at different
pH conditions are also exhibit as positive sigmoidal curves
(
pH3.0), we recorded the CD spectra of tropolonylated DNA
o
complexes (ON1:ON3, ON1-ON4, ON1:ON5) at 10 C under
different pH 6.0/7.0/8.0 conditions. For comparative studies we
recorded the CD spectra of control DNA complex (ON1:ON2) in
pH7.0. Their CD spectra at pH7.0 are depicted in Figure 2, CD
spectra at other pH conditions are provided in Supporting
Information. In Figure 2A, the CD spectra of tr-DNA duplexes
(
ON1:ON3/ON1:ON4/ON1:ON5) exhibit as minima (λ252nm,) and
maxima (λ284nm), which are characteristic of DNA B-form helices.
In literature, DNA B-form helices have similar CD spectra with
characteristic signals as minima (~λ250nm) and maxima
(
~λ280nm).[24] Though, the CD spectra of tri-tropolonylated tr-
DNA duplex (ON1:ON5) shows the destabilization in B-form helix
under similar pH conditions. For comparative studies, we plotted
CD ellipticity vs number of tropolonyl nucleoside unit at different
pH conditions (Figure 2B). This plot shows the depletion in the CD
signals of tr-DNA duplex with increasing tr-T nucleoside in DNA
strand. At pH 6.0, The CD spectra of tr-DNA duplexes
(
ON1:ON3/ON1:ON4/ON1:ON5) exhibit almost similar signals as
m
(Supporting Information). The T values of tr-DNA duplexes are
B-form DNA helix (Figure 2B). At pH8.0, The CD spectra of mono-
tropolonylated DNA duplex (ON1:ON3) also exhibit almost similar
as B-form DNA helix.
extracted from the first derivative curve of their respective melting
profiles which are described in Table 2.
Herein we noticed that mono-tropolonylated tr-DNA duplex
(
ON1:ON3) was less stable as compared to the control DNA
3
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duplex (ON1:ON2) near physiological conditions (pH 7.0) by
pH dependent UV/Fluorescence studies of 4-OH. The UV and
emission spectra of nucleoside tropolonyl thymidine (4-OH) at
different pH conditions are depicted in (Figure 4A), while spectra
of free Tropolone are provided in the Supporting Information. The
o
9
.5 C. Further the stability of di-/tri-tropolonylated DNA duplex
o
o
(
ON1:ON4, &ON1:ON5) were decreased by 15 C and 22.6 C
respectively at same pH conditions. In pH dependent comparative
studies, we found that these tr-DNA duplex (ON1:ON3, ON1:ON4, UV spectra of nucleoside 4-OH exhibit red shift and hyperchromic
&
ON1:ON5) were marginally stabilized by increasing pH
shift with increasing the pH from 3.7 to 10.8, whereas the
emission spectra at λex 395nm exhibit the blue shift with
remarkable depletion in the fluorescence intensity by increasing
pH (3.7 to 10.8) conditions. The similar effects were noticed with
control molecule (Tropolone). Here we extracted pH dependent
UV-absorbance (λ = 398nm) and fluorescence intensity (λ =
conditions to pH 8.0, while destabilized by lowering pH condition
to pH 6.0. Thus stability of tr-duplex is dependent upon pH
condition and number of incorporated tropolonyl residue.
4
(
90nm) values of nucleoside 4-OH, then plotted pH profiles
Figure 4B) for determination of pK . The mid-points of sigmoidal
pH profile is ~5.6 which is pKa of nucleoside 4-OH. Similarly, we
determined the pK of Tropolone as ~6.9 for comparative studies
Supporting Information). Thus pK of nucleoside 4-OH is lower
than free Tropolone molecule. This pK change in nucleoside (4-
a
a
(
a
a
OH) is attributed because of thymine residue which is known as
acceptor for charge transfer process in fluorophore conjugated
nucleosides.
Figure 3. UV thermal melting profiles of tr-DNA duplexes (2 µM) at pH7.0 in
sodium phosphate buffer. Inset: First derivative curves of UV-thermal melting
profiles of tr-DNA duplexes (2 µM) at pH7.0 in sodium phosphate buffer.
Table 2. Synthesized DNA oligonucleotides and their mass
o
Entry
Duplex
Tm ( C) of DNA duplex different pH conditions
pH6.0
pH7.0(ΔTm*
)
pH8.0
1
2
3
4
ON1:ON2
ON1:ON3
ON1:ON4
ON1:ON5
-
63.2
-
50.8
40.9
37.5
53.7 (-9.5)
54.3
49.9
47.5
47.4 (-15.8)
40.6 (-22.6)
m
*ΔTm: Difference in T of control DNA duplex (pH7.0) and Tr-DNA duplex
UV/Fluorescence studies: Since Tropolone is chromophore
comprising fluorescence properties, though its quantum yield is
low. Importantly, the fluorescence properties of Tropolone are
influenced by polarity and pH conditions of solvents.[11] Quantum
efficiency of tropolone is 0.005 (in cyclohexane/neutral pH),
-
2
0
.0025 (low pH conditions), and negligible (0.005*10 ) in alkaline
conditions (pH12).[11] The UV-absorption peaks (~λ250nm and
400nm) of Tropolone molecule are mainly due to due to the
Figure 4. (A) pH dependent UV absorbance spectra (Left) and emission spectra
at λex. 400nm (Right) ; (B) pH dependent UV absorbance value at λ398nm (Red, Left)
and emission intensity value at λ490nm (Blue, Right). Sample concentration:
tropolonyl thymidine, 4-OH, (22 µM) in 100 mM buffer (pH3.7-5.6, sodium
acetate buffer; pH6.0-8.0 phosphate buffer; pH9.2-10.8 sodium carbonate
buffer).
~
λ
electronic transitions (π-π* & n-π*) and intramolecular proton
transfer, though the fluorescence properties owing to the n-π* and
intramolecular proton transfer.[11] Herein we also performed
UV/Fluorescence studies of tr-nucleoside (4 & 4-OH) and tr-DNA
(
ON3-ON5) near physiological pH conditions. For control studies
We recorded the UV spectra of single strand tr-DNA
we performed the similar studies with free Tropolone.
(ON3/ON4/ON4)
and
their
duplex
(
ON1:ON3/ON1:ON4/ON1:ON5) with unmodified DNA (ON1)
4
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under different pH range (6.0-8.0). The UV spectra of tr-DNA
duplex, at pH7.0, are provided in Figure 4, while the UV spectra
of tr-DNA duplex at different pH are provided in Supporting
Information. The UV-spectra of tr-DNA and its duplex exhibit two
absorption peaks at λ250nm and λ400nm which are almost similar to
the tropolone molecule near physiological pH range (6.0-8.0). The
similar observations are noticed in UV spectra of other tr-DNA and
their duplexes under different pH conditions (pH6.0/8.0). Most
importantly, the UV spectra of tr-DNA duplex exhibits a little blue
shift, ~λ250nm and ~λ400nm, with increasing the tr-T nucleoside
residue into tr-DNA duplex. This blue shift indicates some sort of
π-π interactions with neighboring nucleobases.
ON1:ON5) are enhanced at pH6.0 conditions. These variations
are more pronounced in tr-DNA duplexes as compare to single
strand tr-DNA oligonucleotides. Importantly we also notice that
fluorescence efficiency of tr-DNA oligonucleotides and their
duplexes are marginally enhanced by increasing the tr-T residue
in DNA.
Figure 5. UV spectra of tr-DNA duplex (ON1:ON3, ON1:ON4, ON1:ON5) and
control under pH 6.0/7.0/8.0 conditions (sodium phosphate buffer) with 2.0 µM.
Further we determined the quantum yield of mono-tropolonylated
DNA (ON3)1.2% at excitation wavelength 400nm (λex.400nm
under aqueous pH7.0 condition, using quinine sulphate as
standard reference (0.1M H SO ). The quantum yield (1.2%) of
)
2
4
tr-DNA (ON3) is significantly enhanced almost five folds as
compare to tropolone molecule (~0.25%, at acidic pH3.0). We
recorded the fluorescence spectra of tr-DNA oligonucleotides
Figure 6. (a) Fluorescence spectra of single strand tr-DNA (ON3/ON4/ON5) and
tr-DNA (ON1:ON3/ON1:ON4/ON1:ON5) under pH6.0 (sodium phosphate
buffer) conditions (A); (b) Comparative bar diagram of Fluorescence intensity vs
tr-DNAs under different pH conditions.
(
ON3/ON4/ON5) and their tr-DNA duplex (ON1:ON3, ON1:ON4,
ON1:ON5) near physiological pH range (6.0-8.0). The
&
fluorescence spectra of tr-DNA (ON3) and its duplex ON1:ON3 at
different pH6.0/7.0/8.0 are depicted in Figure 5, while
fluorescence spectra of other tr-DNA (ON4/ON5) and their
duplexes at different pH6.0/7.0/8.0 are provided in the Supporting
Information. In Figure 6A, the fluorescence intensity of hybrid tr-
Discussion
Since tropolone experiences intramolecular hydrogen bonding at
acidic pH and neutral pH conditions.[11] and to explore the role of
tropolonyl residue in the stability of DNA duplex structure, we,
therefore, have synthesized tropolone conjugated thymidine
nucleoside (4-OH) and its oligonucleotides (ON3-ON5). Since
tropolone is excellent chromophore comprising weak
fluorescence properties. So we also studied the photophysical
properties of tr-T nucleoside (4-OH), tr-oligonucleotides (ON3-
ON5) and tr-DNA duplex.
DNA duplexes (ON1:ON3, ON1:ON4,
& ON1:ON5) are
remarkably enhanced as compare to single strand tr-DNA
oligonucleotides (ON3/ON4/ON5). Though the fluorescence
intensity of tr-DNA oligonucleotides (ON3/ON4/ON5) under same
acidic pH(6.0) conditions are almost equal with little variations,
especially with tri-tropolonylated DNA oligonucleotide (ON5).
These results suggest that fluorescence efficiency of tr-DNA
oligonucleotides is increased after formation of duplex. For
comparative studies, we extracted fluorescence intensity value of
tr-DNA oligonucleotides and their duplex under different pH
conditions, and generated a bar-diagram as florescence intensity
vs. tr-DNA (Figure 6b). This bar diagram shows that the
First, the CD spectral analyses suggest the formation of B-form
type of tr-DNA duplex structures near physiological pH conditions
as like control. Importantly, the perturbation of their CD signal are
noticed with increasing tr-T nucleoside unit into DNA which reveal
the destabilization of B-form type of DNA duplex structures. These
fluorescence
efficiency
of
tr-DNA
oligonucleotides
(
ON3/ON4/ON5) and their duplexes (ON1:ON3, ON1:ON4, &
5
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CD studies suggest the conformational changes of tropolone in
DNA duplex in different pH conditions.
The UV spectra of nucleoside (4-OH) exhibit red shift with
increasing the pH value (pH3.7-10.7), while its fluorescence
spectra shows slight blue shift with remarkable depletion in
fluorescence intensity with increasing the pH value (pH3.7-10.7).
Further the UV-melting results of tr-DNA duplex structures
indicate that tropolone residue of tr-DNA duplex has critical role
in destabilization of their duplex structures. Herein the stability of
tr-DNA duplex increases with increasing the pH value i.e. more
stable under pH8.0 as compare to pH6.0. The stability of tr-DNA
duplex, however, decreases with increasing the number of tr-T
nucleoside. In literature, nucleoside conjugated chromophores of
DNA are involved in π-π interactions with neighboring nucleobase
residues.[26] Moreover, the destabilization of DNA B-form duplex
structures are reportedly known because of the π-π interactions
The pK
fluorescence profile, which is same as from pH dependent UV
absorbance profile (pK = 5.8±0.2). Since Tropolone occurs in
a
of nucleoside 4-OH is also extracted from pH dependent
a
different structural form, as neutral form near neutral pH and
anionic form near alkaline pH conditions. The photophysical
properties of such molecules are severely affected with pH and
polarity of environments. To support the fluorescence character
of nucleoside tr-T (4-OH), we theoratically optimized the structure
of nucleoside (4-OH) under neutral/anionic form of tropolone
residue by DFT (B3YLP-6, 31-6G*) calculation, and then
pleasently extracted their frontier molecular orbitals (HOMO-
LUMO) and their energy lavel (Figure 7). In neutral form of
nucleoside (4-OH), both HOMO and LUMO are localized on
tropolone and extended up to thymine residue with an energy
difference (ΔE) of 3.31 eV. In contrast, in anionic form of 4-OH,
the HOMO is localized on tropolone residue, while LUMO is
localized on thymine residue but their energy difference (ΔE =
2.352 eV) is lower than its neutral form (ΔE = 3.31 eV). Generally
delocalization of HOMO-LUMO decreases ΔE and enhances
fluorescence character. It is also well known that whwn HOMO-
LUMO are localized and decreases energy gap, then the decay
of excited state to ground state dominates through radiationless
pathways which results in lowering of fluorescence.[20, 27] These
calculations support the pH dependent UV/fluorescence
behaviour of nucleoside 4-OH under different pH conditions due
to different sets of HOMO-LUMO under different pH conditions.
between
nucleobases.[
conjugated
chromophore
and
neighboring
19a]
Tropolone residue of tr-DNA duplex, therefore,
may have similar types of π-π interactions with neighboring G-
nucleobase. The increment in tr-T nucleosides is probably
enhanced the perturbation in base stacking structure of DNA
duplex which are responsible for destabilization of tr-DNA duplex
structure.
The fluorescence character of tropolone is appeared because of
the intramolecular charge transfer (n-π* transition) which is
sensitive to the pH and polarity of environments.[11] Firstly, we
determined the pK
We noticed the pK
a
of nucleoside (4-OH) and control Tropolone.
of tropolone residue of tr-T nucleoside (4-OH)
a
is marginally lower (0.9) than free Tropolone, possibly due the
presence of conjugated thymine nucleobase which is known as
acceptor in charge transfer molecular system. Thus charge
transfer ability is enhanced in thymine conjugated tropolone
nucleoside 4-OH. Hence fluorescence property of tr-T (4-OH) is
relatively higher than free Tropolone.
1
HOMO
LUMO
LUMO (E = 0.523 eV)
0
E = 2.352 eV
-
-
-
-
-
-
1
2
3
4
5
6
LUMO (E = -2.724 eV)
ANIONIC
FORM
HOMO (E = -1.828 eV)
ANIONIC FORM

E = 3.31 eV
HOMO (E = -6.033 eV)
NEUTRAL
FORM
NEUTRAL Form
Figure 7. HOMO-LUMO and their calculated energies in neutral form and anionic form of tropolonylated thymidine nucleoside (4-OH). Calculated using DFT-
B3LYP-6 from their optimized structures, neutral form (left) anionic form (right).
6
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ChemBioChem
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The fluorescent characters of intramolecular charge transfer
fluorophores are influenced with polarity of solvent as enhanced
in non-polar solvent.[28] The intramolecular charge transfer
carrying fluorophores also exhibit pH dependent fluorescence
character and explained by that mechanism.[20] Further, the
fluorescence character of fluorophore due to intramolecular
charge transfer, especially because of push-pull systems, is
significantly higher in non-polar solvents as compare to polar
solvents. Importantly the fluorescence quantum yield of
Tropolone is higher in hexane (non-polar solvent) as compare to
aqueous (pH7.0). Thus by changing the microenvironment of
nucleoside (4-OH) could affect its fluorescence properties. Thus
the quantum yield of tr-DNA oligoucleotide (ON3) is enhanced in
aqueous medium near physiological pH conditions as compare to
free Tropolone molecule, and further enhanced even by slight
lowering the pH conditions possibly because of push-pull
mechanism (Table S1). At pH 6.0, the intramolecular hydrogen
bonded tropolone (neutral form) are relatively populated in tr-DNA
oligonucleotide and experience non-polar micro-environment
The strength of W-C hydrogen bonding is relatively maximum in
control DNA duplex is maximum as compared to tr-T incorporated
tr-DNA duplex.
Conclusions
We have successfully synthesized the tropolone conjugated
thymidine nucleoside and DNA oligonucleotides. Since tropolone
is excellent non-benzenoid chromophores comprising pH
dependent weak fluorescent properties. We studies
UV/fluorescence properties of tr-T and determined the pKa 5.9 of
nucleoside which is lower than control tropolone (pKa 6.8). We
have accomplished pH dependent UV/fluorescence properties of
tropolone conjugated DNA nucleoside/oligonucleotides/DNA
duplexes. Our results support the enhancement of quantum yield
of oligonucleotides after the formation of DNA duplex structure (B-
form helix) and that is more pronounced at low pH (6.0) as
compare to pH 7.0/8.0. However the stability of tr-DNA duplex
structures is relatively higher at pH8.0 as compare to pH6.0. The
increasing the number of tr-T incorporation in DNA
oligonucleotides destabilize their DNA duplex B-form helical
structure under all pH conditions. We demonstrate that Tr-T
nucleoside and its oligonucleotides comprise pH dependent UV-
Fluorescence properties. Which are further supported by DFT
calculation Hence tr-T DNA nucleoside and its oligonucleotides
are new fluorescent DNA analogues which are pH sensitive and
enabling the sensing of duplex formation.
The DNA duplex (B-form) has non-polar microenvironment due to
the presence of grooves (major & minor) in aqueous medium. CD
results of tr-DNA duplex strongly support the formation of B-form
helical structure. Thus the fluorescence character of tr-DNA
duplex may affect in different pH conditions due to the presence
of fluorophores nucleoside tr-T in one strand. Presumably,
tropolone ring of tr-DNA duplex (ON1:ON3) is occurred in non-
polar microenvironment and experience π-π interactions with
neighboring nucleobases, guanosine. Such interactions affect
Watson-Crick (W-C) hydrogen bonding between complementary
nucleobases and play critical role in stability of DNA duplex
Experimental Section
structure.[
19a]
Since π-π attractive interactions between neutral
aromatic ring and nucleobase ring system suppress the W-C
hydrogen bonding ability at pyrimidine nucleobases (T/U/C), while
π-π repulsive interactions between anionic aromatic ring and
nucleobase ring system enhance the W-C hydrogen bonding
ability at pyrimidine nucleobases (T/U/C).[29] More W-C hydrogen
All required materials were obtained from commercial suppliers and used
without any further purification. Pyridine, methylene chloride, were distilled
over calcium hydride. Tetrahydrofuran was distilled over sodium and
benzophenone. Reactions were monitored by thin layer chromatography,
visualized by UV and Ninhydrin. Column chromatography was performed
in 230-400 mesh silica. Mass spectra and HRMS were obtained from
Bruker micrOTOF-Q II Spectrometer. 1H NMR, 13C NMR, and 31P NMR
spectra were recorded on Bruker AV-400 or 700 MHz at 298 K. H and 13C
NMR chemical shifts were recorded in ppm downfield from
tetramethylsilane or residual solvent peak. Splitting patterns are
abbreviated as: s, Singlet; d, doublet; dd, doublet of doublet; t, triplet; q,
quartet; dq, doublet of quartet; m, multiplet.
bonding quenches fluorescence properties of ICT-fluophore
conjugated DNA at pyrimidine nucleobases. At pH6.0, the
population of neutral form of tropolone structure in tr-DNA duplex
is relatively higher. In contrast, at pH 8.0, the population of anionic
form of tropolone is high. Thus π-π attractive interactions between
neutral tropolone ring and neighboring nucleobases are relatively
higher at low pH 6.0, and consequently decrease the W-C
hydrogen bonding in tr-DNA duplex. As resultant, fluorescence
character as quantum yield is enhanced at low pH but reduced
the stability of DNA structures. In contrast, the π-π repulsive
interactions between anionic tropolone ring and neighboring
nucleobases are relatively higher at high pH8.0, and
consequently increase the W-C hydrogen bonding in tr-DNA
duplex. As resultant, the fluorescence character as quantum yield
is significantly reduced in tr-DNA at pH8.0 and enhanced the
stability of DNA structures. Further the increasing tr-T nucleoside
residue in tr-DNA duplex, the B-form helical structures
significantly destabilize at pH6.0 but their fluorescence properties
exhibit almost same. However at pH8.0 fluorescence properties
of di-/tri-incorporated tr-T nucleoside are remarkably reduced.
The stability of three tr-T incorporated tr-DNA duplex structure is
relative stable at pH 8.0 as compare to pH6.0. Moreover, tr-DNA
duplex structures destabilize with increasing the tr-T nucleoside
and the similar trend is consistent under all three pH conditions.
1
5-Ethynyl 2’- deoxy uridine (2): 5-Iodo 2’-deoxy Uridine (1.0 g, 2.82 mmol)
dissolved in dry DMF (10 ml), trimethyl silyl acetylene (1.6 ml, 11.29 mmol)
Tetrakis (triphenylphosphine) palladium (0.326 g, 0.028 mmol), copper (I)
iodide (0.107 g, 0.564 mmol) and triethylamine (0.8 ml, 5.64 mmol) were
added and stirred under argon atmosphere over night at room temperature.
The solvents were removed under reduced pressure and the reaction
mixture passed through silica column (using methanol and chloroform) to
obtain a beige solid. The solid dissolved in dry methanol (10 ml) potassium
carbonate (0.781 g, 5.66 mmol) was added and stirred overnight under
argon atmosphere. The reaction mixture concentrated and purified by
column chromatography on silica gel using methanol and chloroform as
solvents to obtain 0.533 g of 5-Ethynyl 2’- deoxy uridine as white solid in
7
6
4
3
2
5% yield 1H NMR (700 MHz, DMSO-D
) δ 11.62 (s, 1H), 8.29 (s, 1H),
6
.10 (t, J = 6.6 Hz, 1H), 5.24 (d, J = 4.3 Hz, 1H), 5.13 (t, J = 4.9 Hz, 1H),
.23 (dt, J = 8.6, 4.4 Hz, 1H), 4.10 (s, 1H), 3.90 (s, 1H), 3.79 (dd, J = 6.5,
.3 Hz, 1H), 3.67 – 3.59 (m, 1H), 3.59 – 3.53 (m, 1H), 2.19 – 2.08 (m,
H) .(13C NMR , 175 MHz, DMSO –D
6
) δ ppm 40.33, 60.85, 70.18,76.38,
83.58, 84.95,87.79, 97.68, 144.67,149.53, 161.81. (ESI-TOF) m/z: [M +
Na],.for C11 Na 275.0638, Found 275.0646.
12 2 5
H N O
7
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ChemBioChem
10.1002/cbic.201800822
FULL PAPER
5-Iodo-2-bezoyloxy tropone (3): 5-Iodo tropolone synthesized starting from
temperature. After stirred for 3.5 h subjected to column chromatography
tropolone by following reported procedure.1 5-Iodo tropolone (1.0 g 4.0
mmol) dissolved in dry dichloromethane(10 ml) triethyl amine (1.12 ml
3
(1% Et N /60% EtOAc/ n-Hexanes) to yield 0.150 gm (61%) as pale yellow
1
3
foam. H NMR (400 MHz, CDCl ) δ 8.61 (d, J = 13.0 Hz, 1H), 8.14 (d, J =
8
.09 mmol) was added and stirred at room temperature under nitrogen
atmosphere for 10 minutes. Benzoyl chloride (0.702 ml, 6.0 mmol) was
added and stirred the reaction at room temperature for hour.
7.3 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H), 7.48 (t, J = 8.0 Hz, 4H), 7.40 – 7.27
(m, 5H), 7.24 – 7.13 (m, 1H), 6.81 (dt, J = 8.8, 3.6 Hz, 6H), 6.42 – 6.29 (m,
3H), 4.81 – 4.47 (m, 1H), 4.31 – 4.08 (m, 1H), 3.92 – 3.44 (m, 11H), 3.18
(dd, J = 8.3, 3.2 Hz, 1H), 2.61 (dd, J = 11.8, 5.5 Hz, 2H), 2.53 – 2.32 (m,
2H), 1.15 (dd, J = 9.0, 3.8 Hz, 8H), 1.02 (d, J = 6.8 Hz, 3H), 0.92 – 0.81
1
Dichloromethane (50 ml) added to the reaction and extracted with water.
The organic layer washed with sodium bicarbonate and brine solutions
followed by drying on anhydrous sodium sulphate and concentrated to
obtain 1.35 g of 5-Iodo 2 benzoyloxytropone as white solid in 95%
(m, 3H). 31P NMR (162 MHz, CDCl
3
) δ 149.67 (s), 149.25 (s). HRMS (ESI-
55 4
TOF) m/z: [M + Na] Calcd for C55H N O11PNa 1001.3497; Found
1
1
yield.( HNMR , 700 MHz, CDCl
3
) δ ppm 6.86 ( H, d J= 14 Hz),7.18(1H, d
1001.3522.
J= 14 Hz),7.47-7.51 (2H, m),7.60-7.65(1H, m), 7.68 (1H, d J= 14 Hz), 8.09-
8
1
1
.10 (1H, d J= 7 Hz),8.15-8.17 (2H, m) (13C NMR , 175 MHz, CDCl
03.64, 128.60, 128.63, 128.73, 129.46, 130.3,
33.82,134.07,139.03,141.1, 145.99,163.8,171.27. (ESI-TOF) m/z: [M +
INa 374.9489, found 374.9487
3
δ) ppm
130.65,
Acknowledgements
9 3
Na], for C14H O
We thank to Tiwari Ranjay Kumar and Dr. C. S. Purohit for helping
in X-ray studies of compound 4. We also thank Dr. Himansu
Biswal (NISER) for helping in theoratical calculations.
5
- (2-benzoyloxy tropone) ethynylthymidine (4):
5-Ethynyl 2’-deoxy
uridine 2 (0.2 g, 0.793 mmol) , 5-Iodo 2-benzoyloxytropone (0.335 g, 0.952
mmol) , Tetrakis (triphenylphosphine) palladium (0.0916 g, 0.079 mmol ),
copper (I) iodide (0.03 g, 0.158 mmol) and triethylamine ( 0.221 ml, 1.58
mmol ) were dissolved in dry DMF (5 ml) and stirred overnight under argon
atmosphere. The solvents were evaporated under reduced pressure and
the reaction mixture purified on silicagel chromatography by chloroform
Keywords: Tropolone 1 • Modified nucleoside 2 • DNA 3 •
a
Fluorophore 4 • pK of nucleoside 5
1
and methanol to obtain 0.245 g of yellow solid in 65 % yield. H NMR (700
Reference
6
MHz, DMSO-D ) δ 11.84 (s, 1H), 8.53 (s, 1H), 8.08 (d, J = 7.1 Hz, 2H),
7
6
=
2
8
1
.77 (t, J = 7.5 Hz, 1H), 7.61 (t, J = 7.8 Hz, 2H), 7.42 (d, J = 9.9 Hz, 4H),
.12 (t, J = 6.4 Hz, 1H), 5.28 (s, 1H), 5.21 (s, 1H), 4.27 (s, 1H), 3.83 (q, J
[
1]
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3.4 Hz, 1H), 3.68 (d, J = 11.8 Hz, 1H), 3.61 (d, J = 11.8 Hz, 1H), 2.24 –
.10 (m, 2H). (13C NMR, 175 MHz, DMSO –D
6
) δ ppm 40.33, 60.7, 69.72,
9305; (c) D. Burnham, M. Cook, Tetrahedron Lett. 1968, 9, 3771-3774.
5.17, 87.67, 87.86, 93.55, 97.16, 128.29, 128.61, 129.08, 129.29, 129.98,
32.89, 134.34, 145.46, 149.37, 161.22, 163.31, (ESI-TOF) m/z: [M + Na],
[
[
2]
3]
R. J. Cox, A. Al-Fahad, Curr. Opin. Chem. Biol. 2013, 17, 532-536.
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20 2 8
for C25H N O Na 499.1112 found 499.1111.
[
4]
R. F. Angawi, D. C. Swenson, J. B. Gloer, D. T. Wicklow, Tetrahedron
Lett. 2003, 44, 7593-7596.
Tropolonylated thymidine (4-OH). Compound 4 (15 mg, 0.031 mmol) was
treated with 1,4- dioxane and ammonium hydroxide (1:1), 0.6 ml and
stirred at room temperature for 1.5 h. The solvents were evaporated and
resulting solid is washed with ether and EtOAc each time and dissolved in
water and adjusted to pH6-7 with 1N HCl and dried under high vacuum to
[5]
[6]
[7]
P. S. Yang, M. J. Wang, T. Jayakumar, D. S. Chou, C. Y. Ko, M. J. Hsu,
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1
get tittle compound. H-NMR (400 MHz, DMSO) δ 12.19 – 10.88 (b, 1H),
8
2
2
1
.42 (s, 1H), 7.10 (s,b, 2H), 6.10 (d, J = 2.9 Hz, 1H), 5.32 (d, J = 30.0 Hz,
H), 4.26 (s, 2H), 3.81 (d, J = 2.8 Hz, 1H), 3.58 (dd, J = 18.9, 7.7 Hz, 2H),
.14 (t, 2H). 13C-NMR (176 MHz, DMSO) δ 172.59, 161.33, 149.40, 144.11,
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39.01, 124.14, 97.99, 87.66, 84.94, 69.89, 60.74, 40.26. HRMS (ESI-
-
TOF) m/z: [M - H] Calcd for C18
15 2 7
H N O , 371.0871; Found 371.1204
[
9]
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5
1
’-DMTr-(O-benzoyl-tropolonylated) thymidine (5). Compound 4 (0.937 gm,
.96 mmol) was co evaporated with anhydrous Pyridine for 3 times and
Cl (20 ml). To the resultant solution anhydrous
N (1.36 ml, 9.84 mmol) followed by DMTr-Cl (1.0 g, 2.95 mmol) was
[10] W. v. E. Doering, L. H. Knox, J. Am. Chem. Soc. 1951, 73, 828-838.
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Et
2
2
3
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added and stirred at room temperature for 7h. The crude reaction mixture
evaporated to dryness and then subjected to column chromatography
(
0
0.5% Et
.38 (2.5% MeOH/ CH
δ 8.55 (s, 1H), 8.13 (d, J = 7.5 Hz, 2H), 7.61 (t, J = 7.3 Hz, 1H), 7.51 – 7.43
m, 4H), 7.38 – 7.26 (m, 6H), 7.18 (d, J = 7.2 Hz, 1H), 6.81 (dd, J = 8.8,
.5 Hz, 6H), 6.42 (t, J = 8.5 Hz, 3H), 4.57 (s, 1H), 4.10 (s, 1H), 3.81 – 3.50
m, 8H), 3.19 (d, J = 8.7 Hz, 1H), 2.54 (dd, J = 8.1, 2.9 Hz, 1H), 2.45 – 2.31
m, 1H).13C NMR (101 MHz, CDCl
) δ 163.83, 161.01, 158.75, 158.74
49.04, 144.61, 143.94, 135.78, 135.34, 133.84, 130.48, 129.94, 129.83,
3
N/ 2.5% MeOH/CH
2
Cl
2
) to yield 0.9 gm (59%) as yellow solid. Rf
1
2
Cl
2
); H NMR (400 MHz, CDCl & 1-2 drops Et N )
3
3
(
2
[16] (a) D. Shin, R. W. Sinkeldam, Y. Tor, J. Am. Chem. Soc. 2011, 133,
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(
(
1
1
9
3
[17] Y. Shi, A. Dierckx, P. H. Wanrooij, S. Wanrooij, N.-G. Larsson, L. M.
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28.69, 128.57, 128.47, 128.19, 127.85, 127.25, 113.46, 113.43, 99.53,
4.90, 87.18, 86.94, 86.01, 84.94, 71.91, 63.2, 55.30, 42.04. HRMS (ESI-
38 2
TOF) m/z: [M + Na], Calcd for C46H N O10Na, 801.2419; Found 801.2394.
[18] Y. Koga, Y. Fuchi, O. Nakagawa, S. Sasaki, Tetrahedron 2011, 67, 6746-
6752.
[
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Tropolonylated-thymidine phosphoramidites (6): To a stirred solution of
compound 5 (0.2 g, 0.25 mmol) in anhydrous THF (1.5 ml) and N,N-
Diisopropyl ethylamine (0.27 ml, 15.4 mmol) was treated with 2-cyanoethyl
diisopropylchlorophosphoramidite (0.114 ml, 5.13 mmol) at room
8
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ChemBioChem
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FULL PAPER
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[
[
6
3
.
9
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Text for Table of Contents
Amarnath Bollu [a] and Nagendra K.
Sharma*[a(s)*
Page No. – Page No.
Tropolone conjugated DNA: pH
Induced Fluorescence Enhancement
of tropolone in DNA Duplex
(
(
))
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Amarnath Bollu[a] and Nagendra K.
Sharma*[a](s)*
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(
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Tropolone conjugated DNA: pH
Induced Fluorescence Enhancement
of tropolone in DNA Duplex
Text for Table of Contents
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