Supporting Information
Unexpected regiospecific formation and DNA binding of new 3(acridin-9-yl)methyl-2-iminothiazolidin-4-ones
JÁN IMRICHa*, DANICA SABOLOVÁb, MÁRIA VILKOVÁa and JÚLIA KUDLÁČOVÁb
a
Department of Organic Chemistry
b
Department of Biochemistry
Institute of Chemistry, Faculty of Science, P J Šafárik University, Moyzesova 11, 041 01
Košice, Slovakia
Table of Contents:
1. Experimental – Materials
2. Experimental – NMR measurements and CHN elemental analysis
3.
1
H, 13C, and 15N NMR spectra, Figures S1 – S10
4. Methods – UV-vis absorption spectroscopy (Figures S11 – S13)
5. Methods – Fluorescence measurements
6. Methods – Tm measurements
7. Methods – CD spectroscopy
8. Methods – DNA cleaving activity
9. UV-vis titrations of 5a-c by DNA (Figures S11 – S13)
10. Fluorescence titrations of 5c-d by DNA (Figures S14 – S16)
11. DNA cleaving activity, Figure S17
12. References
1
Experimental
Materials
All chemicals, calf thymus DNA, plasmid pUC 19 (2761 bp, DH 5α), agarose (type II No-A6877), EDTA, tris[hydroxymethyl]aminomethane (Tris), and dimethyl sulfoxide (DMSO)
were purchased from Sigma-Aldrich and ethidium bromide from Boehringer (Mannheim,
Germany). CT DNA was dissolved in the Tris-EDTA buffer at pH 7.3 and its purity was
determined by absorption measurement at 260 and 280 nm. The concentration was determined
using an extinction coefficient of CT DNA = 6600 M−1cm−1 at 260 nm. The A260/A280 ratio of
1.82 indicated that CT DNA was sufficiently free of protein.
NMR measurements and CHN elemental analysis
NMR spectra were recorded at room temperature on a Varian Mercury Plus spectrometer
operating at 400.13 MHz for 1H, and 100.61 MHz for
13
C, and on a VNMRS spectrometer
(Varian, USA) operating at 599.87 MHz for 1H, 150.84 MHz for 13C, and 60.79 MHz for 15N
in CDCl3 or DMSO-D6 at room temperature and referenced to a TMS (0.0 ppm) or a DMSO
signal (2.50 ppm for 1H and 39.5 ppm for 13C), or internally to neat nitromethane for 15N (0.00
ppm). Two-dimensional gCOSY, NOESY, gHSQC, and gHMBC methods were employed to
assign proton and carbon signals of the products. For
15
N NMR measurements, 1H,15N–
gHSQC and 1H,15N–gHMBC methods were used (Figures S1–S10). Melting points were
determined on a Boetius hot-stage apparatus and are uncorrected. Elemental analysis was
performed on a Perkin–Elmer CHN 2400 elemental analyzer. The reactions were monitored
using pre-coated ALUGRAM® SIL G/UV254 TLC sheets (Macherey-Nagel, Germany).
Preparative column chromatography was performed on a Merck silica gel, 230400 mesh.
2
1
H, 13C, and 15N NMR spectra
3-(Acridin-9-yl)methyl-2-(4-methoxyphenyl)imino-1,3-thiazolidin-4-one (5a):
Figure S1: 1H NMR spectrum of compound 5a
Figure S2: 13C NMR spectrum of compound 5a
3
Figure S3: 15N NMR spectrum of compound 5a
3-(Acridin-9-yl)methyl-2-allylimino-1,3-thiazolidin-4-one (5b):
Figure S4: 1H NMR spectrum of compound 5b
4
Figure S5: 13C NMR spectrum of compound 5b
3-(Acridin-9-yl)methyl-2-phenylimino-1,3-thiazolidin-4-one (5c):
Figure S6: 1H NMR spectrum of compound 5c
5
Figure S7: 13C NMR spectrum of compound 5c
Figure S8: 15N NMR spectrum of compound 5c
6
3-(Acridin-9-yl)methyl-2-methylimino-1,3-thiazolidin-4-one (5d):
Figure S9: 1H NMR spectrum of compound 5d
Figure S10: 13C NMR spectrum of compound 5d
7
Methods
UV-vis absorption spectroscopy
The DNA binding affinity was also measured by the UV-Vis spectroscopy (Figure 2, S11 –
S13). UV-vis spectra were measured on a Varian Cary 100 UV-vis spectrophotometer in a
0.01 M Tris buffer (pH 7.3). The compounds 5a–d were dissolved in DMSO from which
working solutions were prepared by dilution in the 0.01 M Tris buffer to 3 mM concentration
of the drugs. All measurements were performed in the range of 300–500 nm at 24°C. The
intrinsic binding constant Kb was calculated from an equation1 [DNA] / (εa - εf) = [DNA] / (εb εf) + 1 / Kb(εb - εf), in which [DNA] is the DNA concentration applied and εa, εb, and εf are the
extinction coefficients of the ligand 5 at a given DNA concentration, the ligand free in
solution and the ligand fully bound to DNA, respectively, as an intercept at zero DNA
concentration.
Fluorescence measurements
The binding of thiazolidinones 5a–d to calf thymus DNA was examined by a competitive
fluorescence experiment using a complex of CT DNA with ethidium bromide (EB) (Figure 3,
S14 – S16). The complex was titrated with increasing concentration of 5a–d acting as a
fluorescence quencher. Fluorescence quenching indicates the displacement of EB from the
DNA–EB complex with thiazolidinones 5a–d and can be quantitatively expressed by a
classical Stern-Volmer equation (1)24
I0/I = 1 + KSV [Q],
(1)
in which I0 and I are fluorescence intensities at emission wavelength 610 nm in the absence
and presence of the quencher, respectively, KSV is a Stern-Volmer quenching constant, and
[Q] is the concentration of the quencher 5a–d.3 Upon plotting (I0 /I) against [Q], the slope
gives the value of KSV. The DNA–EB complex was prepared by mixing EB and CT DNA in
the 0.01 M Tris buffer (pH 7.3) to achieve 4 μM and 17 μM concentrations, respectively. The
8
concentration of stock solution of the ligands 5a–d was 30 μM. Fluorescence spectra of
DNA–EB were measured at excitation wavelength of 510 nm using a slit width 10 nm for
excitation and emission beams. All measurements were performed at 24°C. The spectra were
recorded in 550–750 nm range and analyzed according to the Stern-Volmer equation.
Fluorescence measurements were performed on a Varian Cary Eclipse spectrofluorimeter.
Tm measurements
Thermal denaturation studies were conducted on the Varian Cary 100 UV-vis
spectrophotometer equipped with a thermostatic cell holder. The measurements were taken in
a 0.01 M BPE buffer (2 mL, pH 7.2) at 23°C. The absorbance at 260 nm was monitored for
either CT DNA (80 µM) or a mixture of CT DNA with 5a–d (70 µM) in the BPE buffer with
increasing heating rate of 1°C/min. Melting temperatures were determined as a maximum of
the first derivative of the melting curves.
CD spectroscopy
CD spectra of CT DNA (17 μM) and CT DNA with the ligands 5a–d (515 μM) were
recorded on a Jasco J-810 spectrophotometer in a 1 mm cuvette. All measurements were
performed in the 0.01 M Tris buffer (pH 7.4) at 24°C.
DNA cleaving activity
DNA cleavage experiments were performed by agarose gel electrophoresis as follows: pUC19
DNA (0.5 µg/µL) in Tris-HCl (pH = 7.0) was treated with the compounds 5ad in the 5, 15,
30, and 40 µM concentrations, respectively, in a total volume of 20 µL of distilled water.
After incubation at 37°C for the time indicated, the electrophoresis was carried out for2 h at
78 V in a TBE buffer (89 mM Tris-borate pH 8.3, 1 mM EDTA). Gels were stained with EB
(1 mg/mL), then distained in water prior to being photographed under UV light (Figure S17).
9
Figure S11. Spectrophotometric titration of 2-(4-methoxyphenylimino)-thiazolidinone 5a (6×10-6 M) in the 0.01
M Tris buffer (pH 7.3, 24C). The absorption descended with increasing concentration of CT DNA from top to
bottom (0–20 μM bp, in 2 μM intervals).
Figure S12. Spectrophotometric titration of 2-(allylimino)-thiazolidinone 5b (6×10-6 M) in the 0.01 M Tris
buffer (pH 7.3, 24C). The absorption descended with increasing concentration of CT DNA from top to bottom
(0–18 μM bp, in 2 μM intervals).
10
Figure S13. Spectrophotometric titration of 2-(phenylimino)-thiazolidinone 5c (6×10-6 M) in the 0.01 M Tris
buffer (pH 7.3, 24C). The absorption descended with increasing concentration of CT DNA from top to bottom
(0–16 μM bp, in 2 μM intervals).
Figure S14. Fluorescence emission spectra of EB bound to DNA in the absence and presence of 5b; (0–40 µM,
in 4 μM intervals), λex = 510 nm, DNA (17 µM), EB (4 µM) in 10 mM Tris (pH 7.3). Inset: Stern-Volmer plot
for quenching of EB by 5b. The fluorescence decreased with increasing concentration of 5b from top to bottom.
11
Figure S15. Fluorescence emission spectra of EB bound to DNA in the absence and presence of 5c; (0–40 µM,
in 4 μM intervals), λex = 510 nm, DNA (17 µM), EB (4 µM) in 10 mM Tris (pH 7.3). Inset: Stern-Volmer plot
for quenching of EB by 5c. The fluorescence decreased with increasing concentration of 5c from top to bottom.
Figure S16. Fluorescence emission spectra of EB bound to DNA in the absence and presence of 5d; (0–40 µM,
in 4 μM intervals), λex = 510 nm, DNA (17 µM), EB (4 µM) in 10 mM Tris (pH 7.3). Inset: Stern-Volmer plot
for quenching of EB by 5d. The fluorescence decreased with increasing concentration of 5d from top to bottom.
12
Figure S17. Cleavage of the supercoiled pUC19 DNA (0.5 μg/μL) with derivatives 5a–d in the Tris-HCl, 10
mM NaCl buffer at pH 7.0, 37°C. Lane 0: DNA alone; lane 1–4: DNA + 5a (5, 15, 30, 40 μM); lane 5–8: DNA +
5b (5, 15, 30, 40 μM); lane 9–12: DNA + 5c (5, 15, 30, 40 μM); lane 13–16: DNA + 5d (5, 15, 30, 40 μM). The
Forms I, II, III are supercoiled DNA, nicked DNA, and linear open circular DNA, respectively. The
electrophoresis was carried out for2 h at 78 V.
References
1. Wang H F, Shen R and Tang N 2009 Eur. J. Med. Chem. 44 4509
2. Lakowicz J R and Weber G 1973 Biochemistry 12 4161
3. Jamshidi M, Yousefi R, Nabavizadeh S M, Rashidi M, Haghighi M G, Niazi A and
Moosavi-Movahedi A A 2014 Int. J. Biol. Macromol. 66 86
4. Mansouri-Torshizi H, Saeidifar M, Rezaei-Behbehani G R, Divsalar A and Saboury A A
2010 J. Chinese Chem. Soc. 57 1299
13