Crystal structure and DNA binding properties of khellin oxime

Article abstract of DOI:10.1016/j.molstruc.2019.07.019

The dried fruits of Ammi visnaga were subjected to Soxhlet extraction with water, followed by hexane and purified by column chromatography to yield Khellin (1). Oximation of 1 yielded khellin oxime (2), crystallized by slow evaporation from CH₂Cl₂:EtOAc (6:3), characterized by single-crystal X-ray diffraction method for the first time. The DNA binding affinities of the 1 and 2 have been appraised by examining their ability to bind to dsFSDNA with ultraviolet-visible and fluorescence spectroscopies, thermal denaturation and square wave voltammetry techniques. The binding constants of the both compounds with dsFSDNA are calculated by UV-vis measurements. The square wave voltammograms proved that both compounds bind to dsFSDNA by intercalative binding mode. Competitive studies with ethidium bromide (EB) have shown that 1 and 2 exhibit the ability to displace the DNA-bound EB indicating 1 and 2 bind to DNA with intercalation with EB for the intercalative binding site as a challenging displacement.

Full text of DOI:10.1016/j.molstruc.2019.07.019

Accepted Manuscript
Crystal structure and DNA binding properties of khellin oxime
Neslihan Beyazit, Kerem Kaya, Pelin Şenel, Ayşe Daut Özdemir, Ayşegül Gölcü
PII:
S0022-2860(19)30853-1
DOI:
https://doi.org/10.1016/j.molstruc.2019.07.019
MOLSTR 26772
Reference:
To appear in: Journal of Molecular Structure
Received Date: 24 April 2019
Revised Date: 18 June 2019
Accepted Date: 4 July 2019
Please cite this article as: N. Beyazit, K. Kaya, P. Şenel, Ayş.Daut. Özdemir, Ayşü. Gölcü, Crystal
structure and DNA binding properties of khellin oxime, Journal of Molecular Structure (2019), doi: https://
doi.org/10.1016/j.molstruc.2019.07.019.
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ACCEPTED MANUSCRIPT
Crystal structure and DNA binding properties of Khellin oxime
Neslihan Beyazita, Kerem Kayab, Pelin Şenelb, Ayşe Daut Özdemir*b and
Ayşegül Gölcüb
aFaculty of Sciences and Letters, Department of Chemistry, Hatay Mustafa Kemal University,
Hatay 31060, Turkey;
bFaculty of Sciences and Letters, Department of Chemistry, Istanbul Technical University,
Istanbul 34469, Turkey
*Corresponding author
E-mail address: daut@itu.edu.tr (Ayşe Daut Özdemir)

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Crystal structure and DNA binding properties of Khellin oxime
ABSTRACT
The dried fruits of Ammi visnaga were subjected to Soxhlet extraction with water,
followed by hexane and purified by column chromatography to yield Khellin (1).
Oximation of 1 yielded khellin oxime (2), crystallized by slow evaporation from
CH2Cl2:EtOAc (6:3), characterized by single-crystal X-ray diffraction method for the
first time. The DNA binding affinities of the 1 and 2 have been appraised by examining
their ability to bind to dsFSDNA with ultraviolet-visible and fluorescence
spectroscopies, thermal denaturation and square wave voltammetry techniques. The
binding constants of the both compounds with dsFSDNA are calculated by UV-vis
measurements. The square wave voltammograms proved that both compounds bind to
dsFSDNA by intercalative binding mode. Competitive studies with ethidium bromide
(EB) have shown that 1 and 2 exhibit the ability to displace the DNA-bound EB
indicating 1 and 2 bind to DNA with intercalation with EB for the intercalative binding
site as a challenging displacement.
Keywords: Ammi Visnaga; Khellin; Khellin oxime; Crystal structure; DNA binding
1. Introduction
In recent years, the study of the interaction of small molecules with DNA has been an
interesting area of interest [1-5]. In order to understand the synthesis of DNA-targeted drugs
and the mechanism of this interaction, it is extremely important to determine the type of
binding of these molecules to DNA. Small molecules can be linked to DNA by covalent
bonding as well as through non-covalent intermolecular interactions. Non-covalent binding
types are in the form of intercalation, electrostatic and major and minor grooves. When small
molecules interact with DNA through non-covalent interactions, many changes have
observed, such as gap formed between the base pairs of DNA, elongation of the helical
structure, angle tension, and the deterioration of the double-stranded structure. These changes
in the DNA structure affect many of the usual processes [6, 7]. Additionally, since most of the
pharmacological activities of a molecule is due to its molecular recognition with different
macromolecules such as DNA, RNA and proteins, in the literature, there are many detailed
studies related with the interactions between phytochemicals and DNA [8-10]. Therefore, it is
very important to examine this interaction.

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Ammi visnaga (L.) Lamarck, a member of Umbelliferae (Apiaceae) family has been used
since ancient times as an antispasmodic and in the treatment of angina pectoris [11]. Recent
pharmacological studies confirmed that physiological action A. visnaga is due principally to
the pyranocoumarins and furanochromones. Khellin (1) (Scheme), a natural furanochromone,
is one the major active principles found in A. visnaga. It is proposed to use in the
photochemotherapy of vitiligo due to its photosensitizing properties and lesser phototoxic
effects compared to the closely related psoralens [12,13]. The results of a computational and
experimental studies of 1 excited states and photobinding to DNA show that 1 predominantly
forms furan monoadducts in DNA, as opposed to psoralen compounds which can form
diadducts and lead to interstrand crosslinks of the DNA duplex [14-16]. Khellin oxime (2),
oximation product of 1, was first synthesized and characterized by Beugelmans et al. [17,18].
A literature survey revealed that there is no any study on the biological activity and DNA
binding properties of 2. However oxime containing natural or synthetic materials are
important group of bioactive compounds and they have received a great deal of attention due
to their pharmacological properties. Recent studies showed that oxime derivatives exhibit
insectisidal [19,20], fungicidal [21], antibacterial [22,23], antitubercular [24], anticancer
activities [25] and there are some reports on their DNA binding [26] and DNA cleavage
ability [27-29]. Recently Thanigaimalai et al. investigated the inhibitory activity of some
chromone oxime derivatives against interleukin-5 [30] and there is one patent describing the
potential using of chromone oxime derivatives as allosteric modulator of metabotropic
glutamate receptors [31].
According to the literature reviewed, since there is no any previous report on biological
activity of 2, in this study we evaluated dsFSDNA binding properties of 2 obtained as single
crystal by the oximation reaction of a naturally occuring furanochromone 1, which is isolated
and purified from the fruits of Ammi visnaga L. In order to evaluate the DNA binding ability
of 2, UV-vis and fluorescence spectroscopies, thermal denaturation and square wave
voltammetry (SWV) techniques were used.
2. Experimental
2.1. Reagents and Solutions
Solvents were obtained from commercial suppliers were purified and dried by standard
procedures. The chromatography solvents were technical grade and distilled prior to use.
Column chromatography was carried out using silica gel (Kieselgel 60, 70–230 mesh, Merck).
Kieselgel 60 F-254 plates (Merck) were used for thin-layer chromatography. dsFSDNA, tris-

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HCI, NaCI and ethydium bromide (EB) were purchased from Sigma-Aldrich Co and all
solvents were purchased from Merck. All chemicals and solvents were reagent grade and
were used as purchased without any further purification. 205 µM dsFSDNA stock solution
was prepared by dilution of dsFSDNA to buffer solution (containing 0.2 M tris-HCI and 150
mM NaCl at pH 7.4) followed by robust stirring for two days at room temperature and kept at
4 °C for no longer than seven days. The dsFSDNA solution thus yielded a UV absorbance
ratio of 1.85 at 260 and 280 nm (A260 / A280), indicating that dsFSDNA was sufficiently
purified from protein contamination. In this way, the molar concentration of DNA was
determined using the molar absorption coefficient (ε = 6600 M-1cm-1). In all experimental
studies 1x10-3 M stock 1 and 2 solutions were prepared by using ethanol and each batch was
prepared fresh.
2.2. Apparatus
UV-Vis (T80+ UV/VIS) Spectrometer and Agilent Technologies Spectrofluorometer
have been used in all measurements and samples that contain dsFSDNA and 1 or 2 were
mixed in vortex before the spectra were taken. Agilent VNMRS 500 MHz Nuclear Magnetic
Resonance Spectrometer have been used for the structure determination. The electrochemical
measurements were executed with an Autolab potentiostat/galvanostat (PGSTAT 302N, Eco
Chemie, the Netherlands). The experimental conditions were managed with General Purpose
Electrochemical System (GPES) and Nova 2.2 software packages. Glassy carbon electrode
(BAS; U: 3 mm diameter) was used in voltammetry studies and all measurements were made
by using BAS 100 W (Bioanalytical System, USA) potentiometer. For the triple electrode
system, an Ag/AgCl reference electrode (BAS; 3 M KCI in all experiments) and a platinum
wire counter electrode and a 10 mL standard single compartment three-electrode cell were
used. Before each measurement, the glassy carbon electrode surface is manually polished and
cleaned with a slurry of alumina powder (U: 0.01 cm) with regular circular motions on a
specific suede soft polishing cloth (BAS suede polishing pad). All measurements were carried
out at room temperature. For single crystal X-ray analysis, the crystal was mounted on a
micromount and attached to a goniometer head on a Bruker D8 VENTURE diffractometer
equipped with PHOTON100 detector and was measured with graphite monochromated Mo-
Kα radiation (λ = 0.71073 Å) using 1.0° ꢀ rotation frames at room temperature. The structure
was solved by direct method using the SHELXS [32] program and refined by full-matrix
least-squares methods with SHELXL-2014 [33]. All non-hydrogen atoms were refined

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anisotropically. All the hydrogen atoms were placed in their calculated positions and refined
in the riding model SADABS [34] was used to perform absorption correction. Molecular
drawings are generated using OLEX2. Ver. 1.2-dev [35].
2.3. Plant Material
The fruits of Ammi visnaga L. were harvested in September 2015, near Hatay, Turkey and
a voucher specimen have been deposited in the Herbarium of Biology Department, Hatay
Mustafa Kemal University.
2.3.1. Extraction and Isolation of Khellin (1)
The dried, riped and grounded fruits (1 kg) of Ammi visnaga L. were extracted with 2
L of boiling water in a Soxhlet apparatus for 40 h and the extract was concentrated to 250 mL
by rotary evaporator. The concentrated residue was then extracted with 100 mL n-hexane
repeatedly at room temperature by using separation flask and the collected n-hexane solution
was dried over Na2SO4 and evaporated to dryness under vacuum. The presence of 1 in the
hexane extract was detected by thin layer chromatography. It gave yellowish brown
fluorescence under 254 nm UV light with Rf values 0.56 in solvent system EtOAc:CHCl3
(60:40) [36] and its molar absorptivity constant 5140 cm-1M-1 in ethanol. The hexane residue
was submitted to silica gel G 60 column and eluted with dichloromethane followed by
dichloromethane-ethyl acetate in increasing polarity. The fraction eluted with
dichloromethane-ethyl acetate (90:10) was concentrated and purified by crystallization from
MeOH-water to obtain 1.9 g of 1.The structure of 1 was confirmed by comparing the melting
point of the compound and 1H- and 13C-NMR data reported in the literature [18,37,38].
2.3.2. Khellin Oxime (2)
(Z-4, 9-dimethoxy-7-methyl-5H-furo-[3,2-g]-chromene-5-one oxime): 3 mmol (0.52 g) of
p-toluenesulfonic acid was dissolved in 50 mL methanol and equimolar amount of 1 (3 mmol,
0.78 g) in methanol was added. The mixture was stirring and heating at boiling point of
methanol under reflux conditions for 1h. Then, 0.012 mol (0.8 g) NH2OH.HCl was added to
the reaction mixture and it was refluxed for 4 h. The progress of the reaction was monitored
by thin layer chromatography. After completion of the reaction, 2 M NaOH solution was
added dropwise until the white solid product precipitated from the solution. It was then
extracted with CHCl3, dried over Na2SO4, filtered and evaporated to dryness under vacuum.
The residue was submitted to silica gel G 60 column and eluted with dichloromethane -

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ethylacetate (6:3) mixture to obtain 630 mg of 2 with a yield of 76% (Scheme). Its molar
absorptivity constant is calculated as 12133 cm-1M-1 in ethanol solvent. The structure of 2 was
1
confirmed by comparing the melting point of the compound and H- and 13C-NMR data
reported in the literature [18].
3. Results and Discussion
2 was crystallized by slow evaporation from dichloromethane - ethylacetate (6:3) mixture
and it is investigated by single-crystal X-ray diffraction method.
3.1. Single Crystal X-Ray Analysis of 2
The single crystal of 2 with dimensions 0.01 x 0.06 x 0.20 was grown by slow evaporation
of its CH2Cl2:EtOAc (6:3) solution. Crystal data and structure refinement parameters are given
in Table 1. Table 2 shows the selected bond lengths, bond and torsion angles for the
compound. Thermal ellipsoids are plotted in Figures 1, face-to-face π−π stacking occuring
between pyran and benzene rings along a-axis are given in Figure 2, and intermolecular
hydrogen bonding between oxime moieties in Figure 3. Packing of the molecule can be found
in Figure 4. Further details on crystal data, data collection and refinements are included in the
supporting information and can also be found by downloading CCDC 1882506 via
https://www.ccdc.cam.ac.uk/structures/ .
2 was crystallized in a triclinic crystal system with P-1 space group. The asymmetric unit
contains two independent molecules. The rotation around the bonds of these independent
molecules are listed in Table 2. The crystal lattice is strongly stabilized by intermolecular
hydrogen bonding between OH---N of different oxime moieties and π-π face-to-face stacking
occurring between pyran and benzene rings with a centroid-to-centroid distance of 3.87 Å
with a shift angle of 20.5o which can be seen in Figure 2. O10-H11---N1 distance is 2.06 Å.
All the other bond distances, bond and torsion angles are in close agreement with the
literature concerning khellin and oxime derivatives. It is typical for oximes to form dimers as
shown in Figure 3. [39-41] The sum of the angles around the atoms of fusion C(5), C(6), C(9)
and C(10) is 361o which reflects the approximate planarity of the structure. The least-squares
planes calculations show that the three rings consisting of the molecule are coplanar with
mean atomic deviation 0.04 Å, the atoms deviating less than 0.08 Å from the mean plane.
Central benzene ring has equivalent bonds which are within 3σ of the average value 1.39 Å.
In the five- and six-membered rings the C-C single bonds vary between 1.39 Å and 1.37 Å.

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3.2. Interaction with dsFSDNA
3.2.1. DNA-binding Study with UV spectroscopy
Ultraviolet absorption spectroscopy is one of the most common and valid methods
used to investigate DNA interactions. The interaction of double helix DNA with any molecule
depends on the structure of that molecule. In this study, the interaction of the samples with
dsFSDNA are investigated by examining the absorption spectra of the mixture solutions
prepared at the appropriate concentration. Thus, a number of solutions have prepared by
adding the higher amounts of the determined dsFSDNA solution (20-175 µM) to the reagent
solutions in a certain concentration prepared from stock solutions. Spectrum analysis,
absorption and absorption peaks occurring in the wavelength (λmax) of the changes are shown
in Figure 5. These changes unveiled that information about the type of molecule-DNA
interaction and the DNA binding constant (Kb) of the compounds are calculated based on the
titration data. The intrinsic binding constant Kb of two compounds with dsFSDNA represents
the binding constant per DNA base pair, can be obtained by monitoring the changes in
absorbance between 250-400 nm with extended concentrations of dsFSDNA from plots
[DNA] / εa -εf versus [DNA] and is given by the ratio of slope to the y-intercept, according to
the following equation [42, 43].
ꢀ
ꢄ
ꢀ
ꢄ
ꢁꢂꢃ
ꢁꢂꢃ
ꢋ
=
+
ꢌ
ꢅƐ ꢇ Ɛ ꢉ
ꢅƐ ꢇ Ɛ ꢉ
× ꢅƐ ꢇ Ɛ ꢉ
ꢆ
ꢈ
ꢆ
ꢈ
ꢊ
ꢈ
ꢊ
where εa = Aobsd/[compound], εa = Aobsd/[compound], εa = extinction coefficient for the free
compound and εb=extinction coefficient for compound in the fully bound form. The high
value of Kb obtained for compounds suggest a strong binding of two compounds to
dsFSDNA. The Kb values of compounds are represented in Table 3.
When the dsFSDNA solution was added to the increasing concentrations in the
constant concentration of 1 or 2 solutions, the absorbance value of the samples decreased
(hypochromic effect) and the wavelength increased (red shift; batochromic effect). This result
indicates that both molecules bind to dsFSDNA by intercalation, as given by Radi's
voltammetric publication [14].
3.2.2. Thermal Denaturation Studies
The stability of the secondary structure of the dsFSDNA resulting from the binding of
compounds 1 and 2 to the DNA helix is usually determined by the thermal denaturation
technique. The melting temperature of dsFSDNA is an important parameter for the interaction

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of molecules with nucleic acids. As the temperature of the double-stranded DNA solution
increases, the hydrogen bonds between the base pairs of the dsFSDNA (in the double-
stranded structure) are broken and dsFSDNA begins to decompose into single strands slowly.
The temperature at which half of the total base pair separates is defined as the melting
temperature (Tm) of the DNA [44]. Tm is an extremely important parameter for the balance of
the double helix and the molecules change their Tm values depending on their binding power
to DNA. As can be seen from Figure 6, denaturation process for compounds 1 and 2 is
completed in one step. In the absence of compound 1 or 2, the Tm value of dsFSDNA was
measured at 72.2 oC. Also, as shown in Figure 6, the Tm value of the dsFSDNA solutions was
significantly increased by the addition of compound 1 or 2 (at a given concentration). For
example, at r = 0.083, ∆Tm values of 1 (6.4 oC) and 2 (3 oC) suggest that the insertion of these
compounds augmented the stability of the DNA double helix conformation, improved and
enhanced the DNA melting temperature. The Tm value of the compound + dsFSDNA solution
was greater than the Tm0 value of the solution containing only dsFSDNA indicating that the 1
or 2 compounds interacted by intercalation with dsFSDNA.
3.2.3. Fluorescence Spectroscopy Studies
The other important spectroscopic technique on the examination of the interaction of
compounds 1 and 2 with DNA, is the replacement with ethidium bromide (EB) using
fluorescence spectroscopy. According to the literature data given, EB is a planar molecule and
interferes with DNA by intercalation [45]. While EB is showing weak fluorescence alone, the
intensity of the emission increases considerably when bonded to DNA. Fluorescence
measurements have shown that the addition of intercalating compounds to an increasing
amount of EB-DNA solutions lead to significant reductions in the emission severity of these
solutions [42]. The reduction in emission intensities in the displacement studies with EB,
since the new compound removes the EB molecules from the DNA strand by intercalation
and replacing it with its locus. The compounds that cause a reduction in the emission intensity
of EB-dsFSDNA solutions are called quenchers. Replacement of compounds 1 and 2 with EB
was performed by fluorescence spectroscopy. In the EB replacement experiments, the
compounds were treated with 50 ꢁM dsFSDNA and 5 ꢁM EB-containing solutions in
increasing concentrations (0-400 ꢁM). The final volume of the solutions was completed to 5
mL with Tris-HCl / NaCl buffer solution (pH 7.4). The prepared EB-dsFSDNA-1 or EB-
dsFSDNA-2 solutions were maintained at 25°C for approximately half an hour before the

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measurement to ensure thermal equilibrium. The fluorescence spectra of the solutions were
then recorded in the range of 500-900 nm (Figure 7 a and b).
In order to determine the fluorescence quenching coefficient (Ksv), the plots drawn
between the I0/I values of the compounds against the different concentration values are shown
on the same figure (Fig. 7). The emission spectra of the EB–dsFSDNA solutions gradually
decreases in the presence of added compounds. The fluorescence quenching capabilities of
each compound are given by the Stern-Volmer equation I0 / I = 1 + Ksv [Compound] where I0
and I are the emission intensities in the absence and presence of the compounds, respectively,
and Ksv is the Stern-Volmer quenching coefficient. Fluorescence data were used to obtain the
Stern-Volmer lines shown in Figure 7. The values of Ksv obtained from the slopes of these
lines are given in Table 3. The values of Ksv were 2.15x104 for 1 and 3.66x103 for 2,
respectively. High Ksv value means that the quenching effect of EB-DNA emission of
compound 1 is higher than compound 2 [46]. However, the variations on absorption
intensities due to the increased 1 or 2 concentration, the apparent dsFSDNA binding constant
(Kapp) values of the compounds were calculated with the help of the following equation:
K
app [Compound] = KEB [EB] [47]
in which the compound concentration is the value at a 50% reduction of the fluorescence
intensity of EB and [EB]=5 ꢁM and KEB = 1x107 M-1 [48]. The Kapp values are given in Table
3, and compound 1 with a higher Kapp value than compound 2, explains that 1 more strongly
bounded to dsFSDNA by intercalation.
3.2.4. Electrochemical Method
The first electrochemical study of khellin was made in 1965 by Orlov et al. [49]. Later,
Kauffmann et al. published a second electrochemical study with carbon paste and polymeric
carbon electrodes [50]. The pre-concentration and voltammetric detection of khellin in
phosphate buffer (pH 7.0) at a DNA-coated carbon paste electrode is described by Radi [14].
This paper is the first study between khellin and DNA interaction but the electrochemical
binding constant is not calculated throught the text [14]. Electrochemical methods are
extremely sensitive, inexpensive, simple, easy to use, fast and advantageous techniques which
can be compatible with microfabrication technology. It also requires a small amount of
sample, so it is widely used in biological and chemical analysis. The electrochemical methods
of DNA interactions of small molecules are followed in two ways: (1) The change in the
current molecule signal is measured before and after interaction or (2) The change in the

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signal of the electroactive bases (either guanine or adenine or both) in the DNA is measured
and interpretations are made according to these changes. In this study, dsFSDNA solution was
added to the sample concentrations and the changes in the voltammogram of the sample
solution were examined. The typical square wave voltammograms (anodic direction) of 100
µM 1 and 200 µM 2 without and with dsFSDNA at GCE in tris-HCl/NaCl (0.2 M/0.2 M, pH
7.4) are shown in Figure 8a and b. For both compounds, further screening was carried out in
the direction of oxidation, but no peak current was observed. The compound 1 had an anodic
peak potential at 1.1330 V, whereas for the oxime compound (2) this potential shifted to
0.57404 V (more negative peak potential). The more negative peak potential of 2 value is due
to the displacement of the quinone oxygen in the para position with the nitrogen atom. When
dsFSDNA is added to a solution of 1 or 2 solutions, marked decreases in the peak current
values and shifts of peak potentials to more positive (the peak potential in 1 did not change
much) values are observed. The reduction in the signals of the compounds after interaction
with dsFSDNA can be attributed to damage to the elevated groups (-C=N, and the -C=O
group) in the structure of the compounds. It is also contemplated that the samples interact
with dsFSDNA and may interfere with the double helix structure of dsFSDNA. Furthermore,
according to a general rule, when the small molecules interact with DNA, the change in the
peak potential (shift to more negative or more positive peak potential) indicates that it is
bound by electrostatic and/or intercalation.
Furthermore, dsFSDNA binding constants (Kb) of each compound were calculated by
equation, the log (1 / [dsFSDNA]) = logKb + log [Scompound-dsFSDNA / Scompound – Scompound-DNA
from DNA + Compound → DNA –Compound reaction as in the other techniques. The terms
used in the equation are as follows; [dsFSDNA]: DNA concentration alone, Scompound-dsFSDNA
Current signal received from dsFSDNA after interaction with dsFSDNA (ꢁA), Scompound
]
:
:
Current signal received from the compound alone (ꢁA) [51]. The Kb and logKb values
calculated from these data are given in Table 3. For all three techniques, Kb values are close to
each other.
4. Conclusions
In the literature search that we conducted according to the Web of Science database, we
have not yet found any UV-Vis spectrophotometric dsFSDNA study on 1 and khellin oxime

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2, and also any single-crystal X-ray diffraction results on 2. The obtained single crystal X-Ray
data on this oxime derivative of 1 structure showed that the intermolecular hydrogen-bonding
interactions are the dominant forces present in the crystal lattice. This forms the dimers of
oximes via O ˗ H --- N interaction. In addition, biological studies of 1 and 2 were investigated
using four (UV, thermal denaturation, fluorescence spectroscopy, SWV) techniques for their
ability to bind dsFSDNA. The binding of both compounds to dsFSDNA by the way of
electrostatic and/or intercalation, is encouraging for all techniques. The reason of having
smaller DNA binding constant for 2 than the DNA binding constant of 1 is due to the N-OH
functional group of oxime compound 2, preventing more stronger intercalation with DNA.
In future, these compounds can be used in the medical field in further in vivo studies.
Supplementary Material
Supplementary material relating to this article is available online, alongside Table S1-
S13.
Disclosure Statement
No potential conflict of interest was reported by the authors.
Acknowledgements
The authors wish to thank Dr. Bunyamin Karagoz (for fluorescence spectrometer) and
Istanbul Technical University for all experimental conditions. N. Beyazit wishes to thank
Mustafa Kemal University Scientific Research Projects (HMKU BAP) through for the
financial support via project number: 9560.
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Table 1. Crystal data and structure refinement parameters for 2
Parameters
Units
2
Empirical Formula
C14H13NO5
275.25
292(0)
0.71073
Triclinic
P - 1
Formula Weight
g/mol
K
T
λ
Å
Crystal system
Space group
Unit cell dimensions
(Å, º)
a
5.2453(8)
15.571(2)
16.761(3)
1276.8(3)
69.062(4)
87.547(4)
87.476(4)
4
b
c
V(Å3)
α
β
γ
Z
Absorption coefficient
Dcalc
mm-1
g/ cm3
0.110
1.432
F(000)
576
Crystal size
mm
º
0.01×0.06×0.20
2.23 to 25.00
-6£h£6
θ range for data collection
Index ranges
-18£k£17
-19£l£19
9379
Reflections collected
Independent reflections
Coverage of independent reflections
Data/parameters
4408
%
98.0
4408 / 369
0.987 - 0.886
R1 = 0.0594
wR2 = 0.1413
R1 = 0.1041
wR2 = 0.1685
0.981
Max. and min. transmission
Final R indices [I≥2s(I)]
R indices (all data)
Goodness-of-fit on F2

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Table 2. Selected bond lengths (Å), bond and torsion angles (º) for 2
Bond Distances (Å)
Value
N1
O4
C4
1.401(4)
1.285(3)
1.434(5)
1.321(4)
1.366(3)
2.763(4)
1.400(4)
1.391(4)
1.371(4)
1.371(4)
1.366(3)
Value
N1
C4
C3
C3
C2
C2
O5
N1
C11
C10
C10
O5
C2
O4
C7
C11
O2
C5
O5
Bond Angles (°)
O4
N1
C4
111.4(3)
119.2(2)
104.7(2)
107.0(3)
124.8(3)
123.8(3)
Value
C2
O5
C5
C10
O2
C13
C11
C9
C13
C12
C10
C5
C11
C9
C6
Torsion Angles (°)
O4
C3
C5
O2
N1
C2
C4
C3
0.3(4)
O5
C5
5.7(4)
C9
C10
C12
O2
C11
179.3(3)
0.1(4)
C13
C14
C15
C8
O3
O8
O1
O7
N1
N2
O3
O8
C9
C16
C7
C5
-87.5(4)
-92.2(4)
12.6(5)
35.3(4)
0.3(4)
C24
C11
C20
C3
C010
O4
C21
C4
O10
C14
C15
C26
C9
C27
C10
C17
-1.2(4)
95.4(4)
88.8(4)
C16

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Table 3. The binding constans of compounds 1 and 2
UV M e t h o d
Fluorescence Method
Electrochemical Method
Kb
log Kb
% Hypo*
Ksv
logKsv
Kapp
logKapp
Kb
log Kb
Compound
6.02x104
5.89x103
4.78
3.77
13.81
14.84
2.15x104 4.29
3.66x103 3.54
1.00x106 6.00
1.43x105 5.15
1.74x104
2.82x103
4.24
3.45
1
2
*Hypo: Hypochromism (%Hypo = A-A0 /A0), n=7

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Scheme. Synthesis of khellin oxime (2) from khellin (1)
Figure 1. Thermal ellipsoids of 2 are drawn at 50% probability. Only one of the two
molecules from asymmetric unit is shown.

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Figure 2. Face-to-face π−π stacking occuring between pyran and benzene rings along a-
axis.
Figure 3. Intermolecular hydrogen bonding between oxime moieties.

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Figure 4. Crystal Packing of the structure 2 along a-axis.
Figure 5. UV-Vis absorption spectra for titration of the increasing amounts of
dsFSDNA with (1) (a) and (2) (b) in phosphate buffer solution at pH 7.4.

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dsFSDNA
1,1
1,0
A
260
0,9
0,8
20
40
60
80
100
o
temperature, C
1,20
1,18
1,16
1,14
1,12
1,10
1,08
1,06
1,04
dsFSDNA+1
A
260
20
40
60
80
100
o
temperature, C
1,20
1,15
1,10
1,05
1,00
0,95
0,90
0,85
0,80
dsFSDNA+2
A
260
20
40
60
80
100
o
temperature, C
Figure 6. Thermal denaturation profiles of dsFSDNA (120 µM) in the absence and in
the presence of compound 1 and 2 (10 µM) in a Tris/HCl buffer solution.

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Figure 7. Emission spectra variations when increasing amounts of 1 (a) and 2 (b) are
added to dsFSDNA solutions, [EB] = 5.0 µM, [dsFSDNA] 50.0 µM. The arrows show
the changes in intensity upon increasing amounts of 1 and 2. Insets: Stern–Volmer plot
of the fluorescence data. The r values on the graph show the ratio of [1 or 2] /
[dsFSDNA].

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Figure 8. The square wave voltammograms (anodic direction) of 100 µM 1 (a) and 200
µM 2 (b) without and with dsFSDNA at GCE in tris-HCl/NaCl at room temperature.

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Highlights
•
•
Khellin extraction with water from the dried fruits of Ammi visnaga.
Oximation of khellin yielded khellin oxime which is crystallized by slow evaporation from
CH2Cl2:EtOAc (6:3).
•
•
Characterization of khellin oxime by single-crystal X-ray diffraction method for the first time.
The DNA binding affinities of the khellin and khellin oxime have been appraised by
examining their ability to bind to dsFSDNA.