Molecular encapsulation of berberine by a modified β-cyclodextrin and binding of host: guest complex to G-quadruplex DNA

Article abstract of DOI:10.1080/15257770.2019.1618469

The capacity to control quadruplex formation, especially in cancer cells, is captivating and entails a reasonable comprehension of the ligand-G-quadruplex binding. Herein, we report an iminopyrenyl-β-cyclodextrin conjugate interacting with duplex and G-quadrulex DNAs. In addition, the host: guest association of the established G-quadruplex binder, berberine, with the β-cyclodextrin derivative is studied employing 2-D ROESY. NMR, UV-visible, and fluorescence spectroscopic techniques are utilized to explore the β-cyclodextrin conjugate's interaction with the quadruplexes. The Binding constants are accounted for the association of the ligands to each of the DNAs viz., calf thymus DNA (duplex), kit22, telo24, and myc22 (quadruplexes). The modulation of the iminopyrenyl-β-cyclodextrin binding to the DNAs are observed when berberine is loaded in the host molecule. A vivid distinction between the interactions of the ligands with duplex and quadruplex structures is inferred. Berberine-loaded iminopyrenyl-β-cyclodextrin shows a higher affinity for binding to kit22.

Full text of DOI:10.1080/15257770.2019.1618469

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: https://www.tandfonline.com/loi/lncn20
Molecular encapsulation of berberine by a
modified β-cyclodextrin and binding of host: guest
complex to G-quadruplex DNA
Soundrapandian Suganthi, Ramasamy Sivaraj & Israel V. M. V. Enoch
To cite this article: Soundrapandian Suganthi, Ramasamy Sivaraj & Israel V. M. V. Enoch
(
2019): Molecular encapsulation of berberine by a modified β-cyclodextrin and binding of
host: guest complex to G-quadruplex DNA, Nucleosides, Nucleotides and Nucleic Acids, DOI:
0.1080/15257770.2019.1618469
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2019.1618469
Molecular encapsulation of berberine by a modified
b-cyclodextrin and binding of host: guest
complex to G-quadruplex DNA
a
b
a,b
Soundrapandian Suganthi , Ramasamy Sivaraj , and Israel V. M. V. Enoch
a
Department of Chemistry, Karunya Institute of Technology and Sciences (Deemed-to-be
b
University), Coimbatore, Tamil Nadu, India; Nanotoxicology Research Lab, Department of
Nanoscience, Karunya Institute of Technology and Sciences (Deemed-to-be University),
Coimbatore, Tamil Nadu, India
ABSTRACT
ARTICLE HISTORY
The capacity to control quadruplex formation, especially in
cancer cells, is captivating and entails a reasonable compre-
hension of the ligand-G-quadruplex binding. Herein, we report
an iminopyrenyl-b-cyclodextrin conjugate interacting with
duplex and G-quadrulex DNAs. In addition, the host: guest
association of the established G-quadruplex binder, berberine,
with the b-cyclodextrin derivative is studied employing 2-D
ROESY. NMR, UV-visible, and fluorescence spectroscopic tech-
niques are utilized to explore the b-cyclodextrin conjugate’s
interaction with the quadruplexes. The Binding constants are
accounted for the association of the ligands to each of the
DNAs viz., calf thymus DNA (duplex), kit22, telo24, and myc22
Received 2 January 2019
Accepted 9 May 2019
KEYWORDS
b-cyclodextrin; berberine;
G-quadruplex; fluorescence
spectroscopy; ROESY;
binding constant
(
quadruplexes). The modulation of the iminopyrenyl-b-cyclo-
dextrin binding to the DNAs are observed when berberine is
loaded in the host molecule. A vivid distinction between the
interactions of the ligands with duplex and quadruplex struc-
tures is inferred. Berberine-loaded iminopyrenyl-b-cyclodextrin
shows a higher affinity for binding to kit22.
1
. Introduction
The standard double helix of DNA remain a subject for investigating lig-
[
1,2]
and-nucleic acid interactions by chemists, biologists, and theoreticians.
However, the double helical structure is not the only target for biophysical
studies. It has been recognized currently that the DNA strands adopt alter-
[
3]
native conformations specific to functions during cell life. Therefore, sev-
eral researchers are intrigued by the non-canonical DNA structures in the
regulation of gene expression and proliferation of tumor cells. Such struc-
[
2,4]
tures have been identified as potential therapeutic targets.
CONTACT Israel V. M. V. Enoch
drisraelenoch@gmail.com
Department of Chemistry and Department of
Nanoscience, Karunya Institute of Technology and Sciences (Deemed-to-be University), Coimbatore 641114,
Tamil Nadu, India.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.
ß 2019 Taylor & Francis Group, LLC

2
S. SUGANTHI ET AL.
Guanine (G) is one among the DNA nucleobases. It self-assembles and
forms G-quartets (or tetrads) with the structure being constructed by
hydrogen bonding. The stability is extended by the so-called Hoogstein
[
5–8]
hydrogen bonds.
fascinating structural motif prevalent in the genome of human beings.
More than 40% of human beings carry G-quadruplex forming sequences
Such guanine-rich oligonucleotide G-quartets are a
[
9,10]
[
11]
which are located within the gene start site.
G-Quadruplex-small mol-
ecule interaction can affect its formation selectively. This is a basis for the
[
12]
development of new anticancer therapeutics.
The promoter regions of
oncogenes including c-kit, c-myc, and telo are enriched with G-quadruplex
[
13]
forming sequences.
of telomere DNA contributes about 80–85% of tumor cells.
necessitates the induction or stabilization of G-quadruplexes through small
The functional telomerase involved in the elongation
[
14,15]
This
[
16]
molecule binding which could inhibit telomerase activity.
Most of the compounds studied as G-quadruplex binders possess aromatic
rings that are planar. They undergo p–p stacking and stabilize the G-quar-
[
17]
tet.
Bindings to minor/major grooves of the double-helix and/or intercal-
ation remain the frequently observed modes of interaction between ligands
[
18]
and duplex DNAs.
Pyrene is one such planar molecule that interacts with
[
19,20]
dsDNA through intercalation.
Benzimidazole derivatives have been syn-
thesized as DNA intercalators. Contrarily, the class of compounds containing
bis-benzimidazole group (for instance, Hoechst 33342 and Hoechst 33258)
[21]
reveal minor groove binding of duplex DNA.
Fused aromatic systems,
coronenes, naphthalene diimide derivatives, carbazoles, quinolones, and ber-
berines are the lead molecules identified as effective G-quadruplex bind-
[
22]
ers.
Selective drug-like chromones have been synthesized as G-quadruplex
[
23]
binders that do telomere damage and cancer cell death.
Cavity-containing molecules which can contain organic molecules of
appropriate size as guest species through non-covalent interaction can serve as
[
24]
carriers of drugs.
They can also influence the association of the encapsu-
[25–29]
lated guest molecule with DNA.
One of such host structures is b-cyclo-
dextrin (b-CD) which is a bucket-shaped oligosachcharide containing seven
a-D-glucopyranose rings. The inner cavity of b-CD is lined with ethereal oxy-
gens and the hydroxyls are projected outward at the exterior rim. These make
the molecule soluble in aqueous medium and able to accommodate organic
molecules through non-covalent forces. In recent years, native b-CD has been
studied as modulator of the binding of small molecules to DNA. However,
exploration of modified b-CD carrying DNA-binding moieties are scarcely
found in the literature. An encapsulation of DNA binding molecules by a
derivatized b-CD having another (covalently attached) DNA binding unit rep-
resents an intriguing and robust approach of targeting DNA, especially G-
quadruplexes. In this paper, we report the host: guest complexation of a

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
known G-quartet binder, i.e., berberine (Ber), with an iminopyrenyl modified
b-CD and their binding with duplex and quaduplex DNAs.
2
. Materials and methods
2
.1. Materials
1-Pyrenecarboxaldehyde was procured from Merck (India). b-CD and
ethylene diamine were purchased from Hi Media (India). Calf thymus
DNA (CT-DNA) was puchased from Genei (India). HPLC-purified
0
0
oligonucleotides viz., 5 -TTAGGGTTAGGGTTAGGGTTAGGG-3 (telo24),
0
0
0
5
-GAGGGTGGGGAGGGTGGGGAAG-3 (myc22), and 5 -AGGGAGGG
CGCTGGGAGGAGGG-3 (kit22) were the custom-synthesized products of
0
Shrimpex Biotech Services (India). The solvents viz., dimethyl sulfoxide
(
DMSO) and ethanol, and the DNA binder Ber were procured from Merck
and Alfa Aesar (India) respectively.
2
.2. Instruments
UV-visible absorption spectra were measured on a Jasco V630 spectropho-
tometer using quartz cuvettes of path length 1 cm. Fluorescence spectra
were recorded on a Jasco FP8300 spectrofluorimeter equipped with a xenon
source lamp. Proton NMR spectra were recorded on a Bruker FT-NMR
spectrometer operating at the frequency of 500 MHz. Tetramethylsilane
(
TMS) was used as the internal standard. NMR samples are prepared by
dissolving the samples in the deuteriated solvent DMSO. The bound com-
plexes were prepared by adding equimolar amounts of the G-quadruplexes
and IPCyD, stirring gently for about 15 minutes. Chemical shift values were
collected downfield from TMS in parts per million. Mass spectrum was
recorded on a Waters Q ToF–micro mass spectrometer.
2
.3. Solutions
The purity of CT-DNA was checked by an optical measurement (absorb-
ance ration A260/A280 > 1.80). CT-DNA was dissolved in 50 mM of NaCl
prior to use to achieve the desired concentration. The samples of oligonu-
ꢀ
cleotides were heated to 90 C for 5 minutes and gradually cooled overnight
to room temperature. Under non-denaturing conditions, polyacrylamide gel
electrophoresis (PAGE) was used to assess the formation of G-quadru-
ꢀ
plexes. DNA samples were warmed to 95 C for 5-6 minutes in buffer
(
50 mM Tris/HCl, pH 7.4, and 50 mM NaCl). It was then gradually cooled
for over 3 hours. When room temperature was reached, the gels were
visualized by UV shadowing to observe separated intensities.

4
S. SUGANTHI ET AL.
The concentration was derived following molar extinction coefficient at
60 nm. The stock solution of IPCyD was prepared in DMSO and test solu-
2
tions were prepared by appropriate dilutions of the stock solution in phos-
phate buffer. Thrice-distilled water was used throughout the experiments.
The test solutions contained a net amount of 1% DMSO.
2
.4. Syntheses of 6-deoxy-6-monotosyl-b-cyclodextrin (Ts-CD) and 6-deoxy-6-
aminoethylamino-b-cyclodextrin (CD-en)
[
30]
Ts-CD was prepared following a report in the literature.
In the typical
synthesis procedure, a suspension of b-CD and p-toluenesulfonic anhydride
in water was stirred for 2 hours at room temperature. Sodium hydroxide
(
10%) was added and stirred. Ammonium chloride (0.1 M) solution was
then added to bring the pH to 8.0 and precipitate the product of Ts-CD.
[
31]
For the preparation of CD-en, the procedure reported by Izawa et al.
was followed. An equimolar mixture of Ts-CD and ethylene diamine was
ꢀ
stirred at 60 C for 12 hours. The mixture was poured into ethanol after
cooling down to room temperature. The precipitate was filtered, washed
with ethanol, and dried.
2
.5. Synthesis of iminopyrenyl derivative of CD-en (IPCyD)
The iminopyrenyl derivative was synthesized by mixing equimolar amounts of
-pyrene-2-carboxaldehyde with CD-en, both dissolved in ethanol. The mix-
1
ture was heated for 6 hours. The solvent was then evaporated, the solid residue
was crystallized from anhydrous ethanol and dried under reduced pressure
ꢀ
1
after filtration (m. p. 300 C). H NMR (DMSO-D6), d ppm: 8.77–8.15 (m,
9
4
H), aromatic protons of the pyrenyl ring; 5.78 (t, 7 H), H-1 protons of b-CD;
.91–4.85 (t, 6 H), -CH - protons of aminoethylamino moiety; 4.53 (s, 1 H),
2
NH proton; 3.7–3.4 (14 H), H-3 and H-5 protons of b-CD; 3.2–3.3 (7 H), H-4
of b-CD.
3
. Results and discussion
.1. Synthesis of IPCyD and its binding to DNA duplex
-Deoxy-6-aminoethylamino-b-cyclodextrin (CD-en) was synthesized by
3
6
a two-step reaction starting from b-CD as discussed in the experimental
section. An imino coupling reaction between CD-en and 1-pyrene-2-carboxal-
1
dehyde yielded IPCyD. The H NMR spectrum of IPCyD is shown in Fig. 1
(the Supporting Information, SI 1, carries the mass spectrum of IPCyD).
Binding titrations of IPCyD vs. CT-DNA were carried out employing
absorption and fluorescence spectroscopic techniques. The absorption and

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Figure 1. Proton NMR spectrum of IPCyD.
fluorescence spectra are shown in Fig. 2 (a) and (b) respectively. The con-
ꢂ5
ꢂ3
centration of IPCyD was kept constant at 1 ꢁ 10 mol dm and CT-DNA
was added in aliquots up to a concentration slightly above the equimolar
concentration of the quadruplexes to equilibrate the binding. The final ratio
þ
of the concentrations was 1: 1. An optimal concentration of Na ions cor-
responds to less entropy loss upon binding of the ion and therefore enhan-
[
32]
ces ion-binding.
Therefore, we carried out the CT-DNA binding
experiment in 50 mM NaCl. The absorption band observed at 260 nm is
typical of CT-DNA. The broad band centered at the longer wavelength
(
45 nm) is attributed to the presence of conjugated pyrene moiety of
IPCyD. Addition of CT-DNA shows a hyperchromic shife of the 415 nm
ꢃ
(
corresponding to the n–p transition. The enhancement of the spectral
band shows the IPCyD molecule binds with the CT-DNA. There is no
apparent isosbestic point in the absorption spectra. Nevertheless, the
absorbance at 340 nm is similar for all the spectra and, on that account, is
chosen is the excitation wavelength for recording fluorescence spectra.
There are two bands observed. Both the bands show quenching of fluores-
cence at the addition of CT-DNA in increasing concentration. The quench-
ing data were furnished in a linear fashion following the typical Stern-
[
33]
Volmer equation:

6
S. SUGANTHI ET AL.
Figure 2. (a) UV-vis and (b) fluorescence spectral titrations of IPCyD–CT-DNA interaction. The
-
5
-3
concentration of IPCyD was kept constant at 1 ꢁ 10 mol dm . Concentration of CT-DNA in
6
-3
increasing order: 0, 2, 3, 4, 5, 6, 7, 8, 9, 10 (ꢁ 10 ) mol dm . The direction of spectral response
at the addition of DNA in incremental concentrations are marked with arrows. (c) Stern-Volmer
plot for the IPCyD–CT-DNA interaction.
F0
¼
1 þ KSV ½IPCyDꢄ
(1)
F
where the fluorescence intensities of IPCyD without and with added CT-
0
DNA are represented by F and F respectively. K is the Stern-Volmer
SV
5
ꢂ1
ꢂ3
constant, determined in the present case as 2.02 ꢁ 10 mol dm from the
plot shown in Fig. 2 (c). An intercalative binding interaction between the
[
34]
ligand and DNA typically results in a hyperchromic shift of absorption.
In addition, the radiationless deactivation of the excited state, accompanied
by an enhancement of fluorescence
[
35]
supports the inference of intercala-
tive mode of binding. However, the results observed in the present case of
IPCyD–CT-DNA binding is vividly different from the above-mentioned
spectral response. The hyperchromic effect of absorbance and quenching of
fluorescence suggest the existence of the electrostatic groove binding–possi-
bly because of the predominant interaction of the aminoethylamino moiety

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
of IPCyD with CT-DNA. b-CD does not appreciably bind to CT-DNA as
[
36]
influencing as planar aromatics or other small molecules.
3
.2. G-Quadruplex binding of IPCyD
The oligomers viz., kit 22, myc 22, and telo 24, which form G-quadruplexes,
were chosen for the present study. The nucleotide sequences of these are
shown in the experimental section. The absorption spectrum (Fig. 3 (a))
shows a vivid enhancement of absorbance (i.e., hyperchromism). The strik-
ing inference represents the substantial contribution due to non-covalent
[
2]
and non-intercalative binding. In the fluorescence spectrum (Fig. 3 (b)),
the two spectral bands get modulated in ways opposite to each other; the
shorter wavelength band presents an enhancement of fluorescence at each
addition of kit 22. Contrary to this, the longer wavelength band gets
quenched when kit 22 is added in increasing concentrations. This result
suggests there is more than one mode of interaction of IPCyD with kit 22,
unlike with duplex DNA discussed in the preceding paragraph. Apart from
Figure 3. (a) UV-vis and (b) fluorescence spectra of the binding of IPCyD to kit22. (c) Stern-
ꢂ5
ꢂ3
Volmer plot of the IPCyD–kit22 binding. Concentration of IPCyD: 1ꢁ 10 mol dm . Increasing
ꢂ6
ꢂ3
order of concentration of kit22: 0, 5, 6, 7, 8, 9, 10, 11, 12, 13 (ꢁ 10 ) mol dm
.

8
S. SUGANTHI ET AL.
Figure 4. (a) UV-vis and (b) fluorescence spectra of IPCyD–telo24 binding titrations. (c) Benesi-
ꢂ6
ꢂ3
Hildebrand plot made using fluorescence data. Concentration of IPCyD: 1 ꢁ 10 mol dm
.
ꢂ6
ꢂ3
Increasing order of concentration of telo24: 0, 5, 6, 7, 8, 9, 10, 11, 12 (ꢁ 10 ) mol dm
.
electrostatic interaction of the aminoethylamino group, the planar aromatic
pyrenyl moiety also gets bound to the quadruplex of kit 22. The latter
observation is furnished support by changes of the shorter wavelength
fluorescence band, which is largely contributed by the conjugated pyrenyl
moiety. The Stern-Volmer plot for the quenching of fluorescence is dis-
6
ꢂ1
3
played in Fig. 3 (c). The KSV value is 6.23 ꢁ 10 mol dm , larger than
that observed for the binding of IPCyD to CT-DNA.
The interaction of IPCyD with telo 24 was followed using UV-vis and
fluorescence spectra (Fig. 4 (a) and (b) respectively). The absorption spectra
report an enhancement (hyperchromism) of the shorter wavelength
(
280 nm) band and a parallel opposite hypochromism of the 375 nm band.
There is a clear isosbestic point at ꢅ295 nm suggesting there exists an equi-
librium namely, IPCyD þ telo 24ꢀIPCyD ꢆ telo 24: The fluorescence spec-
trum of IPCyD shows enhancements at the incremental addition of telo 24
as shown in Fig. 4 (b). The enhancement of fluorescence provides another
evidence for the predominant electrostatic mode of binding as discussed in
the case of CT-DNA. The binding constant value, determined from the

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
Figure 5. (a) UV-vis and (b) fluorescence spectra of IPCyD–myc22 binding titrations. (c) Benesi-
Hildebrand plot made using fluorescence data. Increasing order of concentration of myc22: 0, 5,
ꢂ6
ꢂ3
6
, 7, 8, 9, 10, 11, 12 (ꢁ 10 ) mol dm
.
[
37]
double reciprocal Benesi-Hildebrand plot
(Fig. 4 (c)) of the IPCyD–telo
5
ꢂ1
3
24 binding is 1.31 ꢁ 10 mol dm .
Figs. 5 (a) and (b) show the absorption and fluorescence spectra correspond-
ing to the binding titrations of myc 22 against IPCyD. A hyperchromic shift of
the shorter wavelength absorption band and a hypochromic effect on the
370nm band are seen. These results provide evidence for the existence of both
electrostatic and intercalative modes of binding of IPCyD with myc 22. The
fluorescence spectral band at ꢅ410 nm is enhanced when myc 22 is added.
Therfore, there is a predominant intercalation-like mode of binding. The bind-
ing constant determined employing the fluorescence spectral data is
5
ꢂ1
3
5
.22 ꢁ 10 mol dm . The Benesi-Hildebrand plot is displayed in Fig. 5 (c).
3
.3. NMR spectral study of IPCyD–quadruplex interaction
To assess the involvement of aromatic protons of the pyrenyl moiety of
IPCyD in the binding with the DNA sequences, NMR titrations were

1
0
S. SUGANTHI ET AL.
performed doing a gradual addition of various DNAs into the IPCyD solu-
tion. On the addition of the nucleic acids, the protons signals get broad-
ened (Fig. 6 (b)). Nevertheless, the aromatic proton signals of IPCyD do
Figure 6. NMR spectra of various DNA-bound IPCyD: (a) free IPCyD; IPCyD bound to (b) CT-
DNA, (c) kit22, (d) telo24, and (e) myc22.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
not disappear completely in the case of CT-DNA binding. It means that
[
2]
the pyrenyl protons are not strongly interacting with the DNA duplex.
Contrary to this, the signals nearly disappear on binding to the G-quartets
viz., kit 22, telo 24, and myc 22 (Fig. 6 (c), (d), and (e)). The above results
apparently establish the binding of IPCyD to the duplex and the quadru-
plexes in different modes with the involvement of pyrenyl moiety in the
latter cases.
3
.4. IPCyD–Ber complex formation
By and large, the modes of interaction between IPCyD and the oligomers
in their typical conformations are electrostatic. We carried out the loading
of a known G-quadruplex binding agent, Ber in the host molecule (refer-
ring to IPCyD). The complex formation of IPCyD with Ber was studied by
ROESY NMR, which provided information on the protons of the host and
the guest molecules being in close proximity. An indication of the encapsu-
lation of the guest molecule in the cavity of b-CD moiety of IPCyD would
result in off-diagonal peaks. In the spectrum (Fig. 7 (a)) of the peak corre-
sponding to the H-5 protons is observed at around 3.32 ppm and the H-2
and H-6 protons have resonated at 3.1 ppm. The H-5 protons of b-CD are
[
38]
lined at the inner rim of the b-CD molecule.
The spectrum shows
cross-peaks for the 3.32 ppm and the 7–8 ppm peaks. The latter peaks are
attributed to the aromatic protons of Ber since these protons come to close
proximity of H-5 protons of b-CD, and it is an evidence for the formation
of the host: guest complex of b-CD: Ber in a fashion depicted in Fig. 7 (b).
The remaining part of the Ber molecule resides outside the b-CD molecule.
3
.5. IPCyD: Ber complex–DNA binding
Ber, being an established G-quadruplex binder, is chosen as a guest mol-
ecule for the IPCyD: Ber (host: guest) complex formation, which in turn is
tested for its DNA binding. The Ber molecule forms an inclusion complex
[
39]
[40]
with native b-CD
and its dimer binds to G-quarduplexes.
The bind-
ing titrations were performed by addition of the host: guest complex of
IPCyD: Ber to the DNA samples.
In the IPCyD: Ber vs. CT-DNA binding titration, the absorption spec-
trum shows hyperchromic shifts of the 275 and 345 nm bands (SI 2 (a)). In
addition, there is a fluorescence quenching at the stepwise addition of CT-
DNA (SI. 2 (b)). These results are similar to those observed in the case of
free IPCyD–CT-DNA binding. Therefore, the mode of IPCyD–CT-DNA
binding in free and Ber-complexed forms should be the same. Nevertheless,
3
5
ꢂ1
3
the KSV value drops down to 9.31 ꢁ 10 from 2.02 ꢁ 10 mol dm in the

1
2
S. SUGANTHI ET AL.
Figure 7. ROESY spectrum of IPCyD : Ber complex and the optimized mode of binding of Ber
with CD cavity.
case of free IPCyD. This is attributed to the role of Ber, which is an
‘internal competitor’ for binding to DNA and the operation of steric com-
plexity in the ter-molecular interaction (IPCyD: Ber: CT-DNA).
Similar results of hyperchromism of absorbance and fluorescence
quenching are observed in the case of IPCyD: Ber binding to kit 22. In the
case of telo 24 there is enhancement of fluorescence (see SI 3 and 4). The
KSV value of the fluorescence quenching due to kit 22 and the binding con-
6
5
ꢂ1
3
stant of telo 24 are 2.96 ꢁ 10 and 1.52 ꢁ 10 mol dm respectively. The
larger binding constant values for kit 22 means that the binding of IPCyD:

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
Table 1. Data derived on the binding interactions of IPCyD–DNA and IPCyD: Ber–DNA.
-3
-3
DNA
Ligand
Fluorescence response
K
b
mol dm
KSV mol dm
5
3
5
6
CT-DNA
Free IPCyD
IPCyD–Ber
Free IPCyD
IPCyD–Ber
Free IPCyD
IPCyD–Ber
Free IPCyD
IPCyD–Ber
Quenching
Quenching
Quenching
Quenching
Enhancement
Enhancement
Enhancement
Enhancement
2.02 ꢁ 10
9.32 ꢁ 10
3.71 ꢁ 10
2.96 ꢁ 10
kit22
5
telo24
myc22
1.31 ꢁ 10
1.52 ꢁ 10
5.22 ꢁ 10
1.21 ꢁ 10
5
5
5
Ber to the G-quadruplex is strong. The binding titration of the IPCyD: Ber
complex against myc 22 (see SI 5) shows a quenching of the shorter wave-
length fluorescence band and an enhancement of the longer wavelength band.
There is a distinct isoemissive point implying the existence of one binding
equilibrium, host: guest complex–myc 22. Put differently, the quenching of
fluorescence is attributed to one event i.e., binding of the complex to the G-
5
ꢂ1
3
quadruplex. The binding constant value is 1.21 ꢁ 10 mol dm . The previous
results suggest that the mode and strength of binding of the Ber-loaded IPCyD
to duplex and quadruplexes differ between them. Further, the host: guest com-
plexation contrasts the binding properties of free host molecule to DNA. The
data for all the binding events are presented in Table 1.
4
. Conclusions
An iminopyrenyl-b-cyclodextrin derivative is synthesized by coupling 6-deoxy-
-aminoethylamino-b-cyclodextrin with 1-pyrene-2-carboxaldehyde. Berberine,
6
which is a known G-quadruplex binder, forms a host-guest complex with the
iminopyrenyl derivative. The dioxolo moiety of the fused ring of berberine is
housed in the cavity of b-CD, as revealed by 2 D ROESY. A portion of the
berberine is exposed to the exterior of the b-CD molecule when it is part of
the inclusion complex. Among quadruplex DNAs, telo24 and myc22 show
enhancement of fluorescence on binding to the b-CD derivative whereas kit22
demonstrates quenching. Berberine demonstrates affinity for G-quadruplexes
even when residing in b-CD cavity. The iminopyrenyl-b-CD derivative shows
a competition with berberine for preferentially binding to DNAs. Of the three
G-quartet-forming oligomers, kit22 shows a more significant strength of bind-
ing to Berberine-loaded iminopyrenyl-b-cyclodextrin. A delicate design of lig-
and is found essential for ensuring the specific interaction of the host: guest
complexes with G-quadruplexes.
Acknowledgements
The authors thank the Department of Atomic Energy (DAE)–Board of Research in Nuclear
Sciences (BRNS), Government of India, for financial assistance through the project 37(2)/
14/17/2018-BRNS.

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Disclosure statement
The authors declare that they have no conflict of interest.
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