syntheses: syntheses of (r)-/(s)-[c h n o...
TRANSCRIPT
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Syntheses:
Syntheses of (R)-/(S)-[C18H26N2O3Cu]Cl2
To a methanolic solution (20 mL) of (R)-/(S)-2-amino-2-phenylethanol (1.37 g, 10
mmol) was added drop wise 1,2-dibromoethane (0.43 g, 5 mmol) in 2:1 molar ratio.
The resulting solution was heated under reflux for ca. 20h. To the resulting solution
was added CuCl2.2H2O (0.85 g, 5 mmol) and was continued on reflux for 8h. The
reaction mixture was reduced to half of its volume on rotary evaporator and left
overnight at room temperature to obtain dark green colored crystalline product which
was filtered off and washed with hexane and dried in vacuo. (R-enantiomer)- Yield,
68%, m.p. 131 oC, []25D = -125, Anal. Calc. for [C18H26N2O3Cu]Cl2 (%): C,47.89;
H,5.81; N,6.21. Found: C,47.93; H,6.29; N,6.23. IR (KBr, cm-1, νmax) 3285(N-H),
1452(C-N), 1195(C-O), 733(Ar), 553(Cu-N), 458(Cu-O), UV-vis [MeOH; λmax/nm]
263nm. ESI-MS (m/z) [C18H26N2O3CuCl2+2H], 453. Λm (MeOH) 129.4 Ω-1cm2mol-1
(1:2 electrolyte).
(S-enantiomer)- Yield, 63%, m.p. 143oC, []25D = +95, Anal. Calc. for
[C18H26N2O3Cu]Cl2 (%):C,47.89; H,5.81; N,6.21. Found: C,47.95; H,6.31; N,6.19. IR
(KBr, cm-1, νmax) 3285(N-H), 1452(C-N), 1195(C-O), 733(Ar), 553(Cu-N) 458 (Cu-
O), UV-vis [MeOH; λmax/nm] 263nm. ESI-MS (m/z) [C18H26N2O3CuCl2+2H], 453.
Λm (MeOH) 119.4 Ω-1cm2mol-1 (1:2 electrolyte).
Both enantiomeric metal complexes exhibited identical molar conductance, IR and
UV-vis spectra.
Syntheses of (R)-/(S)-[C18H26N2O3Ni]Cl2
These complexes were synthesized by the procedure as described for (R)-/(S)-
[C18H26N2O3Cu]Cl2 where light green colored crystalline product was obtained which
was filtered off and washed with hexane and dried in vacuo. (R-Enantiomer) Yield,
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62%, []25D = -132, m.p. 151 oC. Anal. Calc. for [C18H26N2O3Ni]Cl2 (%): C,48.42;
H,5.87; N,6.28. Found: C,48.27; H,5.82; N,6.29. IR (KBr, cm-1, νmax) 3508(N-H),
1478(C-N), 1179(C-O), 1093(Ar C-H), 576(Ni-N), UV-vis [MeOH;λmax/nm] 262,
321. 1H NMR, (400MHz, DMSO, 25oC): δH 7.6-7.97(Ar-10H); 3.18(2H); 2.82(4H).
13CNMR, (100MHz,DMSO,25oC, ppm) δ 160.92(C-O);139-116(Ar-C);63.4 (C-H).
ESI-MS (m/z) [C18H26N2O3NiCl2]- 448. Λm (MeOH) 112.8 Ω-1cm2mol-1 (1:2
electrolyte).
(S-Enantiomer) Yield, 62%, []25D = +193, m.p. 153 oC Anal. Calc. for
[C18H26N2O3Ni]Cl2 (%): C,48.42; H, 5.87; N, 6.28. Found: C,48.29; H,5.81; N,6.26.
IR (KBr, cm-1, νmax), 3508(N-H), 1478(C-N),1179 (C-O), 1093(Ar C-H), 576(Ni-N),
UV-vis [MeOH;λmax/nm] 262, 321. 1H NMR, (400MHz, DMSO, 25 oC): δH 7.6-
7.97(Ar-10H); 7.4(4H); 3.18(2H); 2.82(4H). 13C NMR (100MHz, DMSO, 25 oC): δ
160.92(C-O); 139-116(Ar-C); 63.4 (C-H). ESI-MS (m/z) [C18H26N2O3Ni]Cl2, 448.
Λm (MeOH) 110.8 Ω-1cm2mol-1 (1:2 electrolyte).
Syntheses of (R)-/(S)-[C18H24N2O2Zn]Cl2
These complexes were synthesized by the procedure as described for (R)-/(S)-
[C18H26N2O3Cu]Cl2 where white crystalline product was obtained which was filtered
off and washed with hexane and dried in vacuo. (R-Enantiomer)- Yield, 57%, []25D
= -109, m.p. 144oC, Anal. Calc. for [C18H24N2O2Zn]Cl2 (%): C,49.51; H,5.54; N,6.41.
Found: C,49.67; H,5.62; N,6.47. IR (KBr, cm-1, νmax) 3450(N-H), 1455(C-N), 1134
(C-O), 1053(Ar C-H), 554(Zn-N), UV-vis [MeOH; λmax/nm] 263, 322. 1H NMR,
(400MHz, DMSO, 25oC): δH 7.6-7.97(Ar-10H); 7.4(4H); 3.18(2H); 2.82(4H).
13CNMR (100MHz, DMSO, 25oC): δ 160.92(C-O); 139-116(Ar-C); 77-75(C-H). ESI-
MS (m/z) [C18H24N2O2ZnCl2], 436. Λm (MeOH) 128.3Ω-1cm2mol-1 (1:2 electrolyte).
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(S-Enantiomer) Yield, 55%, []25D = 208, m.p. 134oC, Anal. Calc. for
[C18H24N2O2Zn]Cl2 (%):C,49.51; H,5.54; N,6.41. Found: C,49.61; H,5.64; N,6.46.IR
(KBr, cm-1, νmax), 3445(N-H), 1453(C-N), 1131(C-O), 1056(Ar C-H), 551(Zn-N),
UV-vis [MeOH; λmax/nm] 263 ,321. 1H NMR, (400MHz,DMSO,25oC): δH 7.6-
7.98(Ar-10H); 7.38(4H); 3.19(2H); 2.85(4H). 13CNMR, (100MHz,DMSO,25oC): δ
160.82(C-O); 139-117(Ar-C); 77-79(C-H). ESI-MS (m/z) [C18H24N2O2ZnCl2], 436.
Λm (MeOH) 126.3Ω-1cm2mol-1 (1:2 electrolyte).
Results and discussion
The (R)- and (S)- enantiomeric forms of complexes [C18H26N2O3Cu]Cl2,
[C18H26N2O3Ni]Cl2 and [C18H24N2O2Zn]Cl2 were synthesized as depicted in scheme
5.
Scheme 5. Schematic representation of the complexes(R)-/(S)-[C18H26N2O3Cu]Cl2, (R)-/(S)-[C18H26N2O3Ni Cl2 and (R)-/(S)-[C18H24N2O2Zn]Cl2
Empirical formulae and proposed structure were ascertained by elemental analysis,
polarimetry, molar conductivity measurements, UV-vis, ESI-MS and NMR
spectroscopy (in case of complexes (R)-/(S)-[C18H26N2O3Ni]Cl2 and (R)-/(S)-
[C18H24N2O2Zn]Cl2. The molar conductance measurements of the complexes in
MeOH suggest their ionic nature. All complexes are soluble in organic polar solvents,
MeOH, DMSO and DMF and [α]D values of the complexes reveal their R- and S-
stereochemistry. On the basis of UV-vis., mass spectroscopy and EPR data, the
proposed geometry of the complexes [C18H26N2O3Cu]Cl2 and [C18H26N2O3Ni]Cl2
HC NH2
OH+
BrBr+ MCl2
(1) MeOH(2) Reflux 18h
HC NH2
O O
HCNH2
M
OH2
M= Cu(II), Ni(II),Zn(II)
Cl2
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were assigned to be square pyramidal (five-coordinated environment) with apical H2O
molecule and four-coordinate preferably distorted tetrahedral in case of zinc complex
[C18H24N2O2Zn]Cl2.
Infrared Spectroscopy
The IR spectrum of the free phenyl glycinol exhibits characteristic bands of the
amine (-NH2) and the aliphatic (-OH) groups with typical values at 3200 cm-1 and
3400 cm-1 respectively [221]. The other ligand skeletal bands observed in the range
763, 1042, 1155, 1458, 2900 and 3028 cm-1 are ascribed to the out of plane -CH
bending of aromatic rings, C-O group, -CH2, and -CH group, respectively [200].
However upon complexation, the (NH2) stretching band was shifted towards lower
wave number (3158 cm-1) while the -OH absorption band disappeared, suggestive of
chelation of the ligand through the amine nitrogen atoms as well as through hydroxyl
oxygen deprotonation. Presence of a new medium intensity band at 2834 cm-1 in the
spectra of the complexes supports the dimerization of the phenyl glycinol moieties
through -CH2-CH2- spacer. The coordination of water molecules to the Cu (II) metal
was supported by the appearance of non-ligand band in the region 840-851cm-1
attributed to rocking mode of water. The FT-IR spectra of (R)-/(S)-
[C18H26N2O3CuCl2], (R)-/(S)-[C18H26N2O3NiCl2] and (R)-/(S)-[C18H24N2O2ZnCl2]
revealed (M-N) and (M-O) stretching vibrations in the range 430-450 and 535-580
cm-1, respectively [200, 245].
Nuclear magnetic Resonance spectroscopy The 1H and 13C NMR spectra of (R)-/(S)-[C18H26N2O3Ni]Cl2 and (R)-/(S)-
[C18H24N2O2Zn]Cl2 are consistent with the formation of metal complexes of phenyl
glycinol with ethane linker. The absence of characteristic -NH and -OH proton signals
in the range of 4-6 ppm reveals the coordination of -NH2 group [246] to the metal
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centre and subsequent deprotonation of -OH group [247] with the release of two HBr
molecules. A sharp signal which appears at 3.81 ppm was attributed to the proton
attached to the chiral carbon. Other characteristic signatures of methylene and
aromatic protons were observed at 2.8 ppm and 7.2-7.9 ppm, respectively [224]. The
broadening of the spectrum in the aromatic region was due to the merging of -NH2
protons in the same region as shown in figure 65 and 66.
Figure 65. 1H NMR spectrum of complex [C18H26N2O3Ni]Cl2
Figure 66.1H NMR spectrum of complex [C18H26N2O3Ni]Cl2
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Figure 67. 13C NMR spectrum of complex [C18H26N2O3Ni]Cl2
The 13C spectra of both the enantiomers of (R)-/(S)-[C18H26N2O3Ni]Cl2 and (R)-/(S)-
[C18H24N2O2Zn]Cl2 confirm the 1H NMR data. Various characteristic resonances due
to chiral CH, CH2-CH2 linkage and -CH2 and aromatic carbons were observed at 67
ppm, 75-77 ppm, and 116-139 ppm, respectively as depicted in figure 67 and 68
[248].
Figure 68. 13C NMR spectrum of complex [C18H24N2O2Zn Cl2
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Mass spectral analysis
The complexes (R)-/(S)-[C18H26N2O3Cu]Cl2, (R)-/(S)-[C18H26N2O3Ni]Cl2 and (R)-
/(S)- [C18H24N2O2Zn]Cl2 have been unambiguously characterized through mass
spectral analysis. The ESI mass spectrum of complex (R)-/(S)-[C18H26N2O3Cu]Cl2,
exhibits the molecular ion peak m/z at 453 which was assigned to [C18H26N2O3CuCl2
+1H+]. The complex (R)-/(S)-[C18H26N2O3Cu]Cl2, showed the prominent peaks m/z at
191.2 with a relative abundance of 90% which was assigned to [C18H26N2O3Cu] 2+.
The fragmentation peaks obtained at m/z 364, 300, 272 and 244 by the successive
expulsion of H2O; copper metal, two -CH2 groups and -CH2O group, respectively.
The relatively 60% abundant peak m/z at 138 corresponding to isotopic peak of free
phenyl glycinol was observed.
Similar pattern of isotopic peaks was obtained for the complexes (R)-/(S)-
[C18H26N2O3Ni]Cl2 and (R)-/(S)-[C18H24N2O2Zn]Cl2.
Electron paramagnetic resonance spectroscopy
The X-band electron paramagnetic resonance spectrum of complex (R)-
[C18H26N2O3Cu]Cl2, was recorded at a frequency of 9.1 GHz under the magnetic
field strength 3000 ± 1000 gauss with tetracyanoethylene (TCNE) as field marker (g =
2.0027) at LNT. The spectrum of the complex (R)-[C18H26N2O3Cu]Cl2, consists of a
very broad axial symmetrical line shape with g ||=2.19 and g = 2.073 and gav = 2.64
computed from the formula gav2 = g||
2+2g2/3, consistent with the square pyramidal
geometry as shown in figure 69 [249]. These parameters were in good agreement to
the values reported for other related square pyramidal Cu (II) systems and are typical
of axially symmetrical d9 Cu (II) complexes [196]. The trend g|| > g > 2 revealed that
the unpaired electron is present in the dx2
-y2
orbital [250].
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Figure 69. X-band polycrystalline powder EPR spectrum of complex (R)-[C18H26N2O3Cu]Cl2 at room temperature.
For a Cu (II) complex, g|| is a parameter sensitive enough to indicate covalence. For a
covalent complex, g|| < 2.3 and for an ionic environment, g|| = 2.3 or more. In the
present complex (R)-[C18H26N2O3Cu]Cl2, g|| > 2.3 indicates an appreciable metal-ionic
character [251].
Electronic absorption spectra
The electronic spectra of the metal complexes (R)-/(S)-[C18H26N2O3Cu]Cl2, (R)-/(S)-
[C18H26N2O3Ni]Cl2 and (R)-/(S)-[C18H24N2O2Zn]Cl2 were recorded in MeOH at room
temperature in the region 190-1100 nm. The UV region of the electronic spectra of
the complexes (R)-/(S)-[C18H26N2O3CuCl2], exhibited the sharp band at 263 nm
assigned to π-π* transition followed by a shoulder at 338-340 nm [226], attributed to
ligand to metal charge transfer (LMCT) bands and low energy band at 317nm
assigned to n→π* transitions. In the visible region, complexes (R)-/(S)-
[C18H26N2O3Cu]Cl2, display the bands at 636 nm and 644 nm, respectively which
have been assigned to (dxz, dyz→dx2-y2) transition, respectively. These results are
typical of a square pyramidal geometry around copper metal ion [252] which further
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authenticate square pyramidal geometry around the copper metal ion, as deduced by
EPR studies. The electronic spectra of the complexes (R)-/(S)-[C18H26N2O3Ni]Cl2
exhibit a similar spin allowed d-d transitions at 662 and 638 nm assigned to the 3B1
(F) →3E (F) and 3B1 (F) →3A2, 3E (P) transitions, respectively. These values are
consistent with penta-coordinate geometry around Ni2+ ion [253]. The electronic
spectra of both the enantiomers of complex (R)-/(S)-[C18H24N2O2Zn]Cl2 reveals the
distorted tetrahedral geometry.
DNA binding studies
Absorption titration studies
Upon addition of CT DNA to R- and S- enantiomeric complexes (R)-/(S)-
[C18H26N2O3Cu]Cl2, (R)-/(S)-[C18H26N2O3Ni]Cl2 and (R)-/(S)-[C18H24N2O2Zn]Cl2 of
fixed concentration (0.066 X 10-4 M), an increase in the molar absorptivity,
hyperchromism, 23-37% of the π-π* absorption band with concomitant red shift was
observed as depicted in figure 70, which reflects greater binding propensity of the
complexes for DNA via coordinate covalent or non-covalent groove binding mode.
The spectral ‘hyperchromic effect’ results from the contraction and overall damage
caused to the secondary structure of DNA double helix [206, 207], while the red shift
has been associated with the decrease in the energy gap between the highest and
lowest molecular orbitals (HUMO and LUMO) after binding of the complexes to
DNA [254]. Hyperchromism with no shift in absorbance is consistent with groove
binding, therefore in these complexes it can be attributed to external contact (surface
binding) with the duplex or through coordination of replaceable or labile H2O
molecules to N7 residue of guanine [255]. The differences in binding of two
enantiomeric forms of complexes (R)-/(S)- [C18H26N2O3Cu]Cl2, (R)-/(S)-
[C18H26N2O3Ni]Cl2 and(R)-/(S)-[C18H24N2O2Zn]Cl2 are quite evident as there is
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greater increase in molar extinction coefficient values attributed to hyperchromism,
37% in case of R-form of complexes with red shift of 4 nm in comparison to S-
complexes which exhibit a relatively lower hyperchromism, 28% and a red shift of 1-
2 nm as depicted for complex (R)-/(S)-[C18H26N2O3CuCl2], in figure 70.
Figure 70. Variation of UV-vis absorption for complex (R)-/(S)-[C18H26N2O3Cu]Cl2, with increase in the concentration of CT DNA (0.067 X10-4 - 0.466 X10-4 M) in buffer (5 mM Tris-HCl/50 mM NaCl, pH= 7.2) at room temperature. Inset: plot of [DNA]/(εa- εf) vs [DNA] for the titration of CT DNA.(■), experimental data points; full linear, linear fit of the data. [Complex]= 0.33X10-4 M. To further illustrate the enantioselective approach of the complexes, the quantitative
comparison of the DNA binding affinities of (R)-/(S)-[C18H26N2O3Cu]Cl2, (R)-/(S)-
[C18H26N2O3Ni]Cl2 and (R)-/(S)-[C18H24N2O2Zn]Cl2 with CT DNA, the intrinsic
Figure 71. Variation of UV-vis absorption for complex (R)-/(S)-[C18H26N2O3Ni]Cl2 with increase in the concentration of CT DNA (0.067 X10-4 - 0.466 X10-4 M) in buffer (5 mM Tris-HCl/50 mM NaCl, pH= 7.2) at room temperature. Inset: plot of [DNA]/(εa- εf) vs [DNA] for the titration of CT DNA.(■), experimental data points; full linear, linear fit of the data. [complex]= 0.33X10-4M.
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Figure 72. Variation of UV-vis absorption for complex (R)-/(S)-[C18H24N2O2Zn]Cl2 with increase in the concentration of CT DNA (0.067 X10-4 - 0.466 X10-4 M) in buffer (5 mM Tris–HCl/50 mM NaCl, pH= 7.2) at room temperature. Inset: plot of [DNA]/(εa- εf) vs [DNA] for the titration of CT DNA.(■), experimental data points; full linear, linear fit of the data. [complex]= 0.33X10-4 M.
binding constants Kb values of the complexes were determined with equation 1, by
monitoring the change in the absorbance of the π-π* bands with increasing
concentration of CT DNA [170]. The binding constant (Kb) values are given in table
3, which follows the order (R)-[C18H26N2O3Cu]Cl2 > (R)-[C18H26N2O3Ni]Cl2 > (S)-
[C18H26N2O3Cu]Cl2 > (S)-[C18H26N2O3Ni]Cl2 > (R)-[C18H24N2O2Zn]Cl2 > (S)-
[C18H24N2O2Zn]Cl2 as depicted in figure 71 and 72.
Furthermore, the Kb values clearly indicate the enantioselective approach of the
complexes emphasizing the stronger binding affinity of R-complexes for DNA in
comparison to S- complexes.
Table 3. The binding constant (Kb) values of all complexes with the DNA (± 0.08 mean standard deviation). Complex λmax KbX104 (M-1) % Hyperchromism Red Shift(nm) (R)-[C18H26N2O3Cu]Cl2 263 4.6 37 4 (S)-[C18H26N2O3Cu]Cl2 262 3.2 28 2 (R)-[C18H26N2O3Ni]Cl2 262 3.8 33 3 (S)-[C18H26N2O3Ni]Cl2 263 2.2 30 1 (R)-[C18H24N2O2Zn]Cl2 264 2.1 28 2 (S)-[C18H24N2O2Zn]Cl2 263 1.5 23 2
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Enantiomeric binding of R-form of complexes is evident with right handed B-DNA
helix which has distinct right- handed major and minor grooves of well-defined width
and depth [256].
Since Kb values are quite lower than the Kb values of classical intercalators such as
ethidium bromide (1.4 X 106 M-1); therefore, intercalative mode of binding was ruled
out. The copper complex (R)-[C18H26N2O3Cu]Cl2 is relatively a very strong and avid
DNA binder than the rest of the complexes.
To obtain the concrete information and to determine the coordination of metal
complexes to a specific site on DNA polymers; low molecular building blocks of
large nucleic acid DNA viz. mononucleotides or dinucleotides-metal complex
interaction becomes mandatory. Therefore, spectral titrations of R- and S-
enantiomeric complexes of (R)-/(S) -[C18H26N2O3Cu]Cl2, were carried out with 5’-
GMP. The observed spectral pattern was similar to CT DNA reflecting hyperchromic
effect with concomitant moderate red shift (1-2 nm) at π-π* as depicted in Figure 73
(i,ii). The intrinsic binding constant Kb values were found to be 3.16 X 104 M-1 and
2.68 X 104 M-1, respectively which are consistent with the Kb values of complexes
Figure73. Variation of UV-vis absorption for complex (R)-[C18H26N2O3CuCl2], and (S) - [C18H26N2O3CuCl2] with increase in the concentration of 5′ GMP (0.067 X10-4 - 0.466 X10-4 M) in buffer (5mM Tris–HCl/50 mM NaCl, pH= 7.2) at room temperature. Inset: plot of [5′ GMP]/(εa- εf) vs [DNA] for the titration of 5′ GMP.(■), experimental data points; full linear, linear fit of the data. [Complex]= 0.33X10-4, [5′ GMP] =1.12 X 10-4M.
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with CT DNA followed by UV-visible titrations. The enantioselective binding of R-
form with 5′-GMP is also clearly accentuated. These observations implicate that N7
position of the guanine residue is the most probable coordinating site. Moreover,
simultaneous interaction with O6 atom of the phosphate group is also likely as in 5′-
GMP as amino group and phosphate moiety lie in the same plane [257].
Fluorescence spectral studies
Both the enantiomeric complexes (R)-/(S)-[C18H26N2O3Cu]Cl2, (R)-/(S)-
[C18H26N2O3Ni]Cl2 emit strong luminescence at 332-337 nm region and complexes
(R)- /(S)-[C18H24N2O2Zn]Cl2 exhibit luminescence at 665 nm region in Tris-HCl
buffer at room temperature when excited at 263 nm. On addition of increasing
concentration of CT DNA to the fixed amount of complexes, there is an enhancement
of the emission intensity as shown in figure 74-76, indicative of strong interaction of
the complexes with CT DNA via coordinate covalent or electrostatic binding mode.
The enhancement of the emission intensity is largely due to the change in the
environment of metal complex and related to extent to which the complex gets into a
Figure 74. Emission spectra of complex (R)-/(S)-[C18H26N2O3Cu]Cl2, in Tris-HCl buffer in presence of DNA. [DNA] (0-0.466) X 10-4 M. Arrow shows the intensity change upon increasing concentration of the DNA. Inset: plot of r/cf versus r.
hydrophobic environment inside the DNA [210]. The hydrophobic environment inside
the DNA helix reduces the accessibility of the solvent H2O to the complex, which as a
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consequence restricts the complex mobility at the binding site; and results in a
decrease of the vibrational mode of relaxation and thus higher emission intensity
[258]. Hydrophobic interactions between the enantiomeric complexes and
polyelectrolyte may induce changes in the excited state properties either due to
electrostatic association or intercalation [259]. The intercalative mode of binding will
be sensitive to ligand characteristics such as planarity of ligand, extent of aromatic π
system available for stacking and depth of ligand which can penetrate into the double
helix. On the other hand, electrostatic interaction would be more sensitive to the
charge of the metal ion, ligand hydrophobicity and size of the complex [260]. An
observed increase in emission intensity is associated with electrostatic interaction.
Figure 75.Emission spectra of complex (R)-/(S)-[C18H26N2O3Ni]Cl2 in tris HCl buffer in presence of DNA.[DNA] (0-0.466) X 10-4M. Arrow shows the intensity change upon increasing concentration of the DNA. Inset:plot of r/cf versus r
Figure 76. Emission spectra of complex (R)-/(S)-[C18H24N2O2Zn]Cl2 in Tris-HCl buffer in presence of DNA. [DNA] (0-0.466) X 10-4M. Arrow shows the intensity change upon increasing concentration of the DNA. Inset: plot of r/cf versus r.
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Furthermore, the binding constant ‘K’ for the complexes (R)-/(S)-[C18H26N2O3Cu]Cl2,
(R)-/(S)-[C18H26N2O3Ni]Cl2 and (R)-/(S)-[C18H24N2O2Zn]Cl2 was determined by
using Scatchard equation [177]. The ‘K’ and the n values with excitation and emission
wavelengths of the complexes are given in table 4.
Table 4. Emission properties of complexes bound to CT DNA. Complex Binding Constant
(K) M-1 No. of Binding sites (n)
(R)-[C18H26N2O3Cu]Cl2 4.33 X 105 0.55 (S)-[C18H26N2O3Cu]Cl2 1.24 X 105 0.28 (R)-[C18H26N2O3Ni]Cl2 1.20 X 104 0.35 (S)-[C18H26N2O3Ni]Cl2 0.75 X 104 0.22 (R)-[C18H24N2O2Zn]Cl2 1.80 X 104 0.69 (S)-[C18H24N2O2Zn]Cl2 0.80 X 104 0.52
To evaluate the interacting strength of enantiomeric complexes of (R)-/(S)-
[C18H26N2O3CuCl2] emission quenching experiments using [Fe(CN)6]-4 as a quencher
Figure77. Emission quenching curves of complex (R)-[C18H26N2O3Cu]Cl2 (i) and (S)-[C18H26N2O3Cu]Cl2 (ii) in absence and presence of DNA with the increasing concentration of the quencher[Fe(CN)6]4-. were also performed. In the absence of DNA, emission intensity of the complex of
(R)-/(S)-[C18H26N2O3Cu]Cl2 were efficiently quenched by [Fe(CN)6]-4. The plots of
the complexes of (R)-/(S)-[C18H26N2O3Cu]Cl2 gave the value of Ksv = 9.2 X 104 M-1
and 8.6 X 104 M-1, respectively. In presence of DNA, the slope was remarkably
Io/I
[Fe(CN)6]4- X10-4 M
[Fe(CN)6]4- X10-4 M
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decreased to 3.2 X 104 and 4.3 X 104 M-1 for the complex of (R)-/(S)-
[C18H26N2O3CuCl2], respectively as shown in Figure77. (i,ii). The greater decrease of
the Ksv value for the complex of (R)- [C18H26N2O3CuCl2] in comparison to complex
of (S)-[C18H26N2O3Cu]Cl2, indicate higher DNA binding propensity of (R)-
[C18H26N2O3Cu]Cl2. These results are consistent with the electronic absorption
titration.
Cyclic voltammetry
The application of cyclic voltammetry to the study of metal complex-DNA interaction
provides a useful complement to the previously used methods of investigations, such
as UV-visible spectroscopy. Equilibrium constant (Kb) for the interaction of the metal
complexes with DNA can be obtained from the shifts in peak potentials, the number
of base pair sites involved in binding via intercalative, electrostatic or hydrophobic
interactions and from the dependence of the current passed during oxidation or
reduction of the bound species on the amount of the added DNA [234]. In the present
study, it was used to understand and underline the effect of enantiomers on the DNA
binding of the copper complexes. Previously, this technique was also employed to
probe the enantioselective interaction [261] of [Ru(phen)3]+2 and [Cu(phen)3]+2 and
other copper complexes with CT DNA [262].
The CV of the complexes (R)-/(S)-[C18H26N2O3Cu]Cl2 in the absence of DNA reveals
CuL22+ + e- CuL2
+
CuL2+ -DNACuL2
2+ -DNA + e-
E0f'
E0b'
115
fairly quasireversible wave involving Cu (II) / Cu (I) redox couple as depicted in
voltammogram in Figure 78 (i,ii). For both the enantiomers, the peak current ratio
approaches unity revealing quasireversible one electron redox process i.e. diffusion
controlled. However, the redox potential of the enantiomers (0.522-0.497mV) did not
display apparent variation due to the orientations of the enantiomers. On addition of
CT DNA to the complexes (R)-/(S)- [C18H26N2O3Cu]Cl2, there was a significant shift
in formal electrode potential E1/2= 0.294 mV and 0.243 mV for the complex
Figure 78. Cyclic voltammogram of (R)-[C18H26N2O3Cu]Cl2 (i) and (S)-[C18H26N2O3Cu]Cl2 (ii) (scan rate 0.2 Vs-1, MeOH, 25 °C) of (a) metal complex (b) metal complex in presence of CT DNA.
(R)-[C18H26N2O3Cu]Cl2 and (S)-[C18H26N2O3Cu]Cl2, respectively. In addition to
changes in formal potential, voltammetric current Ipa / Ipc and separation of peak
potential ΔEp also decreased as given in (table 5).There was a significant reduction in
cathodic peak current in case of (R)-[C18H26N2O3Cu]Cl2, which implies strong
binding of R- enantiomers with DNA duplex.
The ratio of the equilibrium constants for binding of the Cu (II) and Cu (I) species to
DNA has been estimated from the net shift in E1/2 on the addition of DNA using the
equation.
εbº ─ εfº = 0.059 log (K+1/K+2)
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Table 5. Electrochemical properties of copper complex in the absence and presence of CT DNA.
Circular dichroism
Circular dichoric studies are useful in diagnosing changes in the morphology of DNA
during complex-DNA interactions [263]. The CD spectrum of CT DNA exhibits a
positive band at 275 nm (UV, λmax, 260 nm) due to the base stacking and a negative
band at 245 nm caused by helicity, which is characteristic of right-handed B-DNA
form [264]. Simple groove binding and electrostatic interaction of the complexes with
DNA show less or no perturbation on the base stacking and helicity bands while
intercalation causes a characteristic decrease in both positive and negative bands
[265]. Figure79 (i-iii) displays (a) CD spectrum of CT DNA alone (b) CT DNA in
presence of (R)-[C18H26N2O3Cu]Cl2, (R)- [C18H26N2O3Ni]Cl2 and (R)-
[C18H24N2O2Zn]Cl2 and (c) CT DNA in presence of (S)-[C18H26N2O3Cu]Cl2, (S)-
[C18H26N2O3Ni]Cl2 and (S)-[C18H24N2O2Zn]Cl2. The addition of R-enantiomeric
complexes (R)-[C18H26N2O3Cu]Cl2, (R)- [C18H26N2O3Ni]Cl2 and (R)-
[C18H24N2O2Zn]Cl2 to the solution of CT DNA induced slight changes in intensity for
both positive and negative bands suggesting that complexes may interact in an
electrostatic mode; their perturbation on the base-stacking and helicity bands of CT
Complex Epc (mV)
Epa (mV)
Ipc (mA) X 10-4
Ipa (mA) X 10-4
ΔEp (mV)
E1/2 (mV)
(R)-[C18H26N2O3Cu]Cl2 -0.5229 -0.2289 6.821 7.353 -0.2940 -0.3759
(R)-[C18H26N2O3Cu]Cl2 + DNA
-0.4811 -0.2460 5.423 6.452 -0.2351 -3635
(S)-[C18H26N2O3Cu]Cl2 (S)-[C18H26N2O3Cu]Cl2 + DNA
-0.4977 -0.4696
-0.2541 -0.2765
4.767 5.525
4.422 5.133
-0.2436 -0.1931
-0.3759 -0.3731
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DNA follows an order of (R)-/(S)-[C18H26N2O3Cu]Cl2 > (R)-/(S) -(R)/(S)-
[C18H24N2O2Zn]Cl2 > (R)-/(S)-([C18H26N2O3Ni]Cl2. S- enantiomeric complexes (S)-
[C18H26N2O3Cu]Cl2, (S)-[C18H26N2O3Ni]Cl2 and (S)-[C18H24N2O2Zn]Cl2 perturbed
the negative helicity band considerably in comparison to R-enantiomeric analogs
(R)-[C18H26N2O3Cu]Cl2, (R)-[C18H26N2O3Ni]Cl2 and (R)-[C18H24N2O2Zn]Cl2
exhibiting an overall decrease of DNA ellipticity band. The CD conformational
changes of S-enantiomeric complex (S)-[C18H26N2O3Cu]Cl2, (S)-[C18H26N2O3Ni]Cl2
and (S)-[C18H24N2O2Zn]Cl2 are also consistent with its lower Kb values as quantified
by UV-vis titrations. Furthermore, the intensity of the positive and negative bands was
significantly diminished suggesting a conformational transition. Therefore, striking
differences were observed in the CD spectra of two enantiomeric forms.
Figure 79. (i) (a) CD spectrum of CT-DNA alone (b) CT-DNA in presence of (R)-[C18H26N2O3Cu] Cl2 and (c) CT-DNA in presence of (S)-[C18H26N2O3Cu]Cl2,(ii) (a) CD spectrum of CT-DNA alone (b) CT-DNA in presence of (R)-[C18H26N2O3Ni]Cl2 and (c) CT-DNA in presence of (S)-[C18H26N2O3Ni]Cl2 and (iii) (a) CD spectrum of CT-DNA alone (b) CT-DNA in presence of (R)-[C18H24N2O2Zn]Cl2 and (c) CT-DNA in presence of (S)-[C18H24N2O2Zn]Cl2
(i) (ii)
(iii)
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DNA cleavage activity The DNA cleavage activity of enantiomers of (R)-/(S)-[C18H26N2O3Ni]Cl2 was
studied by gel electrophoresis using supercoiled plasmid pBR322 DNA as a substrate.
The DNA cleavage activity was assessed by the conversion of supercoiled form of
DNA (Form I, SC form) to nicked circular (Form II, NC form) or linear open circular
DNA (Form III, LC). A concentration dependent DNA cleavage by (R)-/(S)-
[C18H26N2O3Cu]Cl2 was first performed. At 8 µM concentration, both the enantiomers
of [C18H26N2O3Cu]Cl2 exhibited DNA cleavage by the conversion of SC Form (I) into
NC Form (II). At a slightly higher concentration 24 µM (lane 4), DNA cleavage was
complete into Form II. NC Form was observed in case of (S)-[C18H26N2O3Cu]Cl2
whereas (R)-[C18H26N2O3Cu]Cl2 produced 90% NC Form II and rest 10% (LC form
III) as shown in figure 80 (i,ii). All these Forms are visible on gel of (R)-
[C18H26N2O3Cu]Cl2 indicating that R- enantiomer of complex [C18H26N2O3Cu]Cl2 is
involved in double strand DNA cleavage to generate the LC Form before converting
all of the SC form to NC DNA, through single strand breaking [215]. This distinct
pattern of gel electrophoresis discriminates clearly DNA cleavage activity by R- and
S- enantiomers of the complex; S- enantiomer reveals single strand breaks and less
efficient DNA cleavage while R- enantiomer cleaves DNA with much higher
efficiency to give both NC and LC forms indicative of double strand cleavage. The
ligand scaffold phenyl glycinol (partial intercalation) as a recognition element tunes
the DNA binding affinity and cleaves DNA effectively.
The cleavage efficiency of copper (II) complexes is usually dependent on activators
[266]. Thus, besides H2O2, other activators such as ascorbate (Asc), 3-
mercaptopropionic acid (MPA), singlet oxygen scavengers and radical scavengers like
sodium Azide (NaN3) and superoxide scavengers (SOD) were also used to investigate
119
the DNA cleavage activity [267] of (R)-[C18H26N2O3Cu]Cl2 and (S)-
[C18H26N2O3Cu]Cl2. As shown in figure 81 (i, ii),the cleavage activity of both
enantiomers of [C18H26N2O3Cu]Cl2 was significantly enhanced by the activators and
activating efficiency follows the order for (R)-[C18H26N2O3Cu]Cl2, Asc> H2O2
>MPA; surprisingly, reverse order was observed for (S)-[C18H26N2O3Cu]Cl2, due to
the differences in enantioselectivity and conformation scavengers like NaN3 and SOD
inhibited the DNA cleavage , suggesting that (1O2), O2·¯ radical or singlet oxygen like
entities is likely to be reactive species responsible for the cleavage reaction which
proceeds via oxidative pathway mechanism [238]. DNA cleavage in presence of
minor groove binding agent, DAPI [236] and major groove
(i)
(ii)
Figure 80. Gel Electrophoresis diagram showing the cleavage of pBR322 supercoiled DNA (300 ng) with metal complexes (R)-[C18H26N2O3Cu]Cl2 (i) and (S)-[C18H26N2O3Cu]Cl2 (ii): Lane 1, DNA alone; Lane 2, 8µl metal complex + DNA; Lane 3, 16µl metal complex + DNA; Lane 4, 24µl metal complex + DNA; Lane 5,32µl metal complex + DNA Lane 6, 40µl metal complex + DNA. binding agent, methyl green [237,268] were used to probe the potential interacting site
of complex (R)-[C18H26N2O3Cu]Cl2 with plasmid pBR322 DNA. The DNA was
120
treated with DAPI or methyl green prior to the addition of (R)-[C18H26N2O3Cu]Cl2.
The gel patterns presented in figure 83 revealed inhibition in presence of methyl green
(lane 3) suggesting that (R)-[C18H26N2O3Cu]Cl2 prefers major groove binding.
(i)
(ii)
Figure 81. (i,ii): Gel Electrophoresis diagram showing the cleavage of pBR322 supercoiled DNA (300 ng) with metal complexes (R)-[C18H26N2O3Cu]Cl2 (i) and (S)-[C18H26N2O3Cu]Cl2 [24 µl] in presence of different scavengers; Lane 1, DNA alone; Lane 2, metal complex + DNA+ (0.4mM) µl H2O2; Lane 3, metal complex + DNA + (0.4mM) MPA; Lane 4, metal complex + DNA+(0.4mM) Ascorbate ; Lane 5, metal complex + DNA+ (0.4mM) MPA; Lane 6, metal complex + DNA+ SOD (15 Units). 1 2 3
Figure 82: The cleavage patterns of the agarose gel electrophoresis of pBR322 DNA(300ng) by (R)-[C18H26N2O3Cu]Cl2 (24 µM) in presence of DNA minor groove binding agent DAPI and major groove binding agent methyl green .Lane 1, pBR322 DNA alone; Lane 2, DNA+(R)-[C18H26N2O3Cu]Cl2 + Methyl green(1µl of 0.01mg/ml); Lane3, DNA+(R)-[C18H26N2O3Cu]Cl2 +DAPI (8µM).
Form II Form I
Form II Form I
1 2 3 4 5 6
1 2 3 4 5 6
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Topoisomerase II activity
Topoisomerase II catalyzes DNA decatenation, a process essential for replication and
transcription of DNA. DNA double strand passage assay of the reaction mixture
(20µl) was used to distinguish the effect of (R)-[C18H26N2O3Cu]Cl2 on topoisomerase
II function employing method of Lee et al. [269]. As depicted in figure 84, complex
(R)-[C18H26N2O3Cu]Cl2 inhibited the activity of topoisomerase II at different
concentrations but highest complete inhibition was observed at 24µM concentration
(lane 4) which is very low concentration in comparison to reported topoisomerase II
poisioned drugs (>300M). These findings suggest that complex (R)-
[C18H26N2O3Cu]Cl2 is indeed, catalytic inhibitor (or poison) of human topoisomerase
II and the complex has an ability to form the non covalent cleavage complex similar
to other topoisomerase II poisons.
Figure 83. The cleavage patterns of the agrose gel electrophoresis diagram showing effect of different concentration of (R)-[C18H26N2O3Cu]Cl2 on the activity of DNA topoisomerase II α (5units); Lane 1, pBR322 DNA alone; Lane 2, pBR322 DNA+ topoisomerase II (5units) ; Lane 3, pBR322 DNA+ topoisomerase II (5units)+ 24µM (R)-[C18H26N2O3Cu]Cl2; Lane 4, pBR322 DNA+ topoisomerase II (5units)+ 32µl (R)-[C18H26N2O3Cu] Cl2.
This is evident from appearance of linear Form III in gel pictures (figure11) which
reveal permanent double stranded nicks. The cleavage complex formation is an
important feature of topoisomerase II poisons. As accumulation of sufficient double
Form I
Form II
1 2 3 4
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strand breaks in DNA brings about numerous adverse genetic aberrations, which
ultimately force the affected tumor cells to undergo apoptosis or necrosis [270].
Conclusion
New chiral enantiomeric metal complexes [C18H26N2O3Cu]Cl2, [C18H26N2O3Ni]Cl2
and [C18H24N2O2Zn]Cl2 derived from (R)- and (S)- 2-amino-2-phenylethanol with –
CH2-CH2- linker have been synthesized and thoroughly characterized. In vitro DNA
binding studies of (R)- and (S)- enantiomeric complexes [C18H26N2O3Cu]Cl2,
[C18H26N2O3Ni]Cl2 and [C18H24N2O2Zn]Cl2 were carried out to establish whether they
demonstrated any enantioselectivity in DNA binding profile. The intrinsic binding
constant values indicate that [C18H26N2O3Cu]Cl2 binds more avidly to DNA than rest
of the complexes. A subtle but detectable difference was observed in the interaction of
(R)- and (S)-enantiomers with DNA. Interaction between complex (R)-/(S)-
[C18H26N2O3Cu]Cl2 and pBR322 DNA was evaluated by agarose gel electrophoresis,
noticeably, the complex exhibits effective DNA cleavage and proceeds via oxidative
pathway. Furthermore, (R)-[C18H26N2O3Cu]Cl2 exhibits significant inhibitory effects
on topo II activity at a very low concentration ~24µM, which suggest that complex
(R)-[C18H26N2O3Cu]Cl2 is indeed catalytic inhibitor (or poison) of human
topoisomerase II. Indeed, complex (R)-[C18H26N2O3Cu]Cl2 is one of the most
effective chiral cancer chemotherapeutic candidates designed in terms of its selective
activity, and it warrants further vigorous in vivo investigations.