99

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,

100

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).

101

(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

102

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

103

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

104

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

105

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].

106

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

107

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

108

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.

109

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

110

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.

111

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

112

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.

113

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

114

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

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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.