Ternary copper complexes for photocleavage of DNA by red light: Direct evidence for sulfur-to-copper charge transfer and d-d band involvement

Article abstract of DOI:10.1021/ja036681q

A new class of ternary copper(II) complexes of formulation [Cu(L ⁿ)B](ClO₄) (1-4), where HLⁿ is a NSO-donor Schiff base (HL¹, HL²) and B is a NN-donor heterocyclic base viz. 1, 10-phenanthroline (phen) and 2,9-dimethyl-1,10-phenanthroline (dmp), are prepared, structurally characterized, and their DNA binding and photocleavage activities studied in the presence of red light. Ternary complex [Cu(L³)(phen)](ClO₄) (5) containing an ONO-donor Schiff base and a binary complex [Cu(L²)₂] (6) are also prepared and structurally characterized for mechanistic investigations of the DNA cleavage reactions. While 1-4 have a square pyramidal (4 + 1) CuN₃OS coordination geometry with the Schiff base bonded at the equatorial sites, 5 has a square pyramidal (4 + 1) geometry with CuN₃O₂ coordination with the alcoholic oxygen at the axial site, and 6 has a square planar trans-CuN₂O₂ geometry, Binding of the complexes 1-4 to calf thymus DNA shows the relative order: phen ? dmp. Mechanistic investigations using distamycin reveal minor groove binding for the complexes. The phen complexes containing the Schiff base with a thiomethyl or thiophenyl moiety show red light induced photocleavage. The dmp complexes are essentially photonuclease inactive. Complexes 5 and 6 are cleavage inactive under similar photolytic conditions. A 10 μM solution of 1 displays a 72% cleavage of SC DNA (0.5 μg) on an exposure of 30 min using a 603 nm Nd: YAG pulsed laser (60 mJ/P) in Tris-HCl buffer (pH 7.2). Significant cleavage of 1 is also observed at 694 nm using a Ruby laser. Complex 1 is cleavage inactive under argon or nitrogen atmosphere. It shows a more enhanced cleavage in pure oxygen than in air. Enhancement of cleavage in D₂O and inhibition with sodium azide addition indicate the possibility of the formation of singlet oxygen as a reactive intermediate leading to DNA cleavage. The d-d band excitation with red light shows significant enhancement of cleavage yield. The results indicate that the phen ligand is necessary for DNA binding of the complex. Both the sulfur-to-copper charge transfer band and copper d-d band excitations helped the DNA cleavage. While the absorption of a red photon induces a metal d-d transition, excitation at shorter visible wavelengths leads to the sulfur-to-copper charge transfer band excitation at the initial step of photocleavage. The excitation energy is subsequently transferred to ground state oxygen molecules to produce singlet oxygen that cleaves the DNA.

Full text of DOI:10.1021/ja036681q

Published on Web 09/12/2003
Ternary Copper Complexes for Photocleavage of DNA by Red
Light: Direct Evidence for Sulfur-to-Copper Charge Transfer
and d-d Band Involvement
Shanta Dhar, Dulal Senapati, Puspendu K. Das, Pabitra Chattopadhyay,
Munirathinam Nethaji, and Akhil R. Chakravarty*
Contribution from the Department of Inorganic and Physical Chemistry,
Indian Institute of Science, Bangalore-560012, India
Received June 14, 2003; E-mail: arc@ipc.iisc.ernet.in
Abstract: A new class of ternary copper(II) complexes of formulation [Cu(Ln)B](ClO4) (1-4), where HLn is
a NSO-donor Schiff base (HL1, HL2) and B is a NN-donor heterocyclic base viz. 1,10-phenanthroline (phen)
and 2,9-dimethyl-1,10-phenanthroline (dmp), are prepared, structurally characterized, and their DNA binding
and photocleavage activities studied in the presence of red light. Ternary complex [Cu(L3)(phen)](ClO4) (5)
containing an ONO-donor Schiff base and a binary complex [Cu(L2)2] (6) are also prepared and structurally
characterized for mechanistic investigations of the DNA cleavage reactions. While 1-4 have a square
pyramidal (4 + 1) CuN3OS coordination geometry with the Schiff base bonded at the equatorial sites, 5
has a square pyramidal (4 + 1) geometry with CuN3O2 coordination with the alcoholic oxygen at the axial
site, and 6 has a square planar trans-CuN2O2 geometry. Binding of the complexes 1-4 to calf thymus
DNA shows the relative order: phen . dmp. Mechanistic investigations using distamycin reveal minor
groove binding for the complexes. The phen complexes containing the Schiff base with a thiomethyl or
thiophenyl moiety show red light induced photocleavage. The dmp complexes are essentially photonuclease
inactive. Complexes 5 and 6 are cleavage inactive under similar photolytic conditions. A 10 µM solution of
1 displays a 72% cleavage of SC DNA (0.5 µg) on an exposure of 30 min using a 603 nm Nd:YAG pulsed
laser (60 mJ/P) in Tris-HCl buffer (pH 7.2). Significant cleavage of 1 is also observed at 694 nm using a
Ruby laser. Complex 1 is cleavage inactive under argon or nitrogen atmosphere. It shows a more enhanced
cleavage in pure oxygen than in air. Enhancement of cleavage in D2O and inhibition with sodium azide
addition indicate the possibility of the formation of singlet oxygen as a reactive intermediate leading to
DNA cleavage. The d-d band excitation with red light shows significant enhancement of cleavage yield.
The results indicate that the phen ligand is necessary for DNA binding of the complex. Both the sulfur-to-
copper charge transfer band and copper d-d band excitations helped the DNA cleavage. While the
absorption of a red photon induces a metal d-d transition, excitation at shorter visible wavelengths leads
to the sulfur-to-copper charge transfer band excitation at the initial step of photocleavage. The excitation
energy is subsequently transferred to ground state oxygen molecules to produce singlet oxygen that cleaves
the DNA.
Introduction
ties are found to be useful as highly sensitive diagnostic agents
as exemplified by the bleomycins or cis-platin in chemothera-
Metallointercalators play an important role in nucleic acids
chemistry for their diverse applications such as foot printing,
sequence specific binding and reactions, as new structural probes
and therapeutic agents.1-7 Transition metal complexes with their
versatile structures, redox behavior and physicochemical proper-
peutic applications. The impetus in this area of non-porphyrinic
chemistry draws from the pioneering work of Lippard and co-
workers on the intercalative chemistry of platinum complexes
as anticancer drugs.8 The chemistry of metallointercalators has
further been enriched from the significant contributions of
Barton and co-workers on polypyridyl complexes of 3d-5d
transition metals.1,9 Other notable contributions have come from
the work of Sigman and co-workers on copper-phenanthroline
complexes.6,10 Cleavage of DNA by metal complexes generally
(1) (a) Erkkila, K. E.; Odom, D. T.; Barton, J. K. Chem. ReV. 1999, 99, 2777.
(b) Pyle, A. M.; Barton, J. K. Prog. Inorg. Chem. 1990, 38, 413. (c) Barton,
J. K. Science 1986, 233, 727. (d) Barton, J. K.; Raphael, A. L. Proc. Natl.
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Res. 1993, 26, 98. (b) Sigman, D. S.; Mazumder, A.; Perrin, D. M. Chem.
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(8) (a) Jannette, K. W.; Lippard, S. J.; Vassiliades, G. A.; Bauer, W. R. Proc.
Natl. Acad. Sci. U.S.A. 1974, 71, 3839. (b) Lippard, S. J.; Berg, J. M.
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CA, 1994.
(9) Delaney, S.; Pascaly, M.; Bhattacharya, P. K.; Han, K.; Barton, J. K. Inorg.
Chem. 2002, 41, 1966 and references therein.
9
12118
J. AM. CHEM. SOC. 2003, 125, 12118-12124
10.1021/ja036681q CCC: $25.00 © 2003 American Chemical Society

Ternary Copper Complexes for Photocleavage of DNA
A R T I C L E S
Chart 1. Ligands Used and the Formulations of the Copper(II)
follows three major pathways, viz. oxidative cleavage by
abstraction of a sugar hydrogen atom, hydrolytic cleavage
involving a phosphate group, and damage of DNA by oxidation
of bases, primarily at the guanine site. Among several types of
reactions, those cleaving DNA on photoactivation under physi-
ological conditions have received considerable current attention2
due to their utility in highly targeted chemotherapeutic applica-
tions over the other types cleaving DNA hydrolytically or in
an oxidative manner in the presence of a reducing agent and/or
H2O2.
Complexes 1-6
Among the copper-based synthetic nucleases, bis(phen)copper
complexes are directed toward the oxidative cleavage of DNA
in the presence of a reducing agent.4,6,7,10-12 There are also some
reports of copper complexes cleaving DNA hydrolytically.13,14
The chemistry of photoactive copper complexes is, however,
virtually limited to only copper-porphyrin species.5 It is thus
highly desirable to develop the non-porphyrinic chemistry of
copper complexes15 for photodynamic therapeutic use. The
copper-porphyrin complexes are known to be significantly less
efficient in comparison to the free base due to significant
complexes in detail using red light sources and to explore the
mechanistic aspects of the cleavage activity for understanding
the role of different ligands and copper in the photodynamic
processes (Chart 1). In our efforts of designing copper com-
plexes suitable for PDT, we have chosen planar chelating
heterocyclic bases as groove binders to DNA and sulfur
containing tridentate Schiff base ligands as photosensitizers. The
rationale for choosing the sulfur containing Schiff base as an
ancillary ligand is due to the fact that compounds containing
thio or thione moieties generally show efficient intersystem
crossing leading to the formation of singlet oxygen.21,22 The
significant aspect of this work is the observation of an efficient
red light-induced cleavage of DNA by the ternary complexes
containing a phen ligand and the Schiff base with a thiomethyl
or thiophenyl moiety, thus making them potentially viable for
photodynamic therapeutic studies. This work also provides the
first direct evidence for the involvement of the d-d band in
the photoexcitation processes resulting in the activation of a
oxygen molecule that leads to DNA cleavage.
3
quenching of the porphyrin ππ* lifetime by the copper(II)
center.16,17 Again, the copper-based pseudo-nucleases are likely
to show significantly different structural and photophysical
properties in comparison to those of the polypyridyl complexes
of 3d-5d transition metals reported by Barton and co-workers.1,9
The photodynamic therapeutic process requires a photosen-
sitizer, a visible light source of around 630 nm (red light), and
oxygen to generate cytotoxic singlet oxygen.18 Porphyrin-based
photosensitizers are currently being used in photodynamic
therapy (PDT).19 The present work stems from our interest to
design copper-based synthetic nucleases that will satisfy the
basic requirements of drugs/agents used in PDT. Copper being
a bioessential transition metal ion, its complexes with tunable
coordination geometry in a redox active environment could find
better application in the cellular level. We have recently
communicated a ternary mono-phen copper(II) complex con-
taining an NSO-donor Schiff base showing DNA cleavage on
irradiation with visible light at 532 nm.20 This observation has
prompted us to study the nuclease activity of this class of
Experimental Section
Materials and Measurements. All reagents and chemicals were
purchased from commercial sources and used without further purifica-
tion. Solvents used for electrochemical and spectroscopic measurements
were purified by standard procedures. The calf thymus DNA and
supercoiled pUC19 DNA (cesium chloride purified) were purchased
from Bangalore Genei (India). Agarose (molecular biology grade),
distamycin, and ethidium bromide were from Sigma (USA). Tris-HCl
buffer solution was prepared using deionized, sonicated triple distilled
water. Schiff base HL1 was prepared by initially reacting salicylaldehyde
with 2-mercaptoethylamine hydrochloride (cysteamine hydrochloride)
in a 1:1 molar ratio in MeOH in the presence of 1 equiv of NaOH and
finally reacting the resulting Schiff base with NaOMe and MeI in
methanol at 25 °C for 3 h. Schiff base HL2 was prepared by
condensation of H2NCH2CH2SPh with salicylaldehyde in a 1:1 molar
ratio in MeOH. Schiff base HL3 was obtained by reacting salicylalde-
hyde with H2NCH2CH2OH in MeOH. Heterocyclic bases 1,10-
phenanthroline (phen) and 2,9-dimethyl-1,10-phenanthroline (dmp) were
purchased from Aldrich, USA.
(10) (a) Sigman, D. S.; Graham, D. R.; D′Aurora, V.; Stern, A. M. J. Biol.
Chem. 1979, 254, 12269. (b) Zelenko, O.; Gallagher, J.; Sigman, D. S.
Angew. Chem., Int. Ed. Engl. 1997, 36, 2776.
(11) (a) Veal, J. M.; Merchant, K.; Rill, R. L. Nucleic Acids Res. 1991, 19,
3383. (b) Veal, J. M.; Rill, R. L. Biochemistry 1991, 30, 1132. (c) Pitie´,
M.; Horn, J. D. V.; Brion, D.; Burrows, C. J.; Meunier, B. Bioconjugate
Chem. 2000, 11, 892. (d) Baudoin, O.; Teulade-Fichou, M.-P.; Vigneron,
J.-P.; Lehn, J.-M. Chem. Commun. 1998, 2349.
(12) (a) Humphreys, K. J.; Johnson, A. E.; Karlin, K. D.; Rokita, S. E. J Biol.
Inorg. Chem. 2002, 7, 835. (b) Pitie´, M.; Boldron, C.; Gornitzka, H.;
Hemmert, C.; Donnadieu, B.; Meunier, B. Eur. J. Inorg. Chem. 2003, 528.
(13) Chand, D. K.; Schneider, H.-J.; Bencini, A.; Bianchi, A.; Giorgi, C.; Ciattini,
S.; Valtancoli, B. Chem.-Eur. J. 2000, 6, 4001.
(14) Hegg, E. L.; Burstyn, J. N. Coord. Chem. ReV. 1998, 173, 133.
(15) (a) Patra, A. K.; Dhar, S.; Nethaji, M.; Chakravarty, A. R. Chem. Commun.
2003, 1562. (b) Eppley, H. J.; Lato, S. M.; Ellington, A. D.; Zaleski, J. M.
Chem. Commun. 1999, 2405.
(16) Praseuth, D.; Gaudemer, A.; Verlhac, J. B.; Kraljieˇ, I.; Sissoe¨ff, I.; Guile´,
E. Photochem. Photobiol. 1986, 44, 717.
The elemental analyses were done using a Heraeus CHN-O Rapid
instrument. The electronic, infrared, and EPR spectral data were
obtained from Hitachi U-3400, Bruker Equinox 55, and Varian E-109
X-band spectrometers, respectively. Room temperature magnetic
susceptibility data were obtained from a George Associates Inc.
(17) Somer, S.; Rimington, C.; Moan, J. FEBS Lett. 1984, 172, 267.
(18) (a) Ackroyd, R.; Kelty, C.; Brown, N.; Reed, M. Photochem. Photobiol.
2001, 74, 656. (b) Henderson, B, W.; Dougherty, T. J. Photochem.
Photobiol. 1992, 55, 145. (c) del C. Batlle, A. M. J. Photochem. Photobiol.
B 1993, 20, 5. (d) De Rosa, M. C.; Crutchley, R. J. Coord. Chem. ReV.
2002, 233-234, 351.
(19) (a) Sternberg, E. D.; Dolphin, D.; Bru¨ckner, C. Tetrahedron 1998, 54, 4151.
(b) Sessler, J. L.; Hemmi, G.; Mody, T. D.; Murai, T.; Burrell, A.; Young,
S. W. Acc. Chem. Res. 1994, 27, 43. (c) Henderson, B. W.; Busch, T. M.;
Vaughan, L. A.; Frawley, N. P.; Babich, D.; Sosa, T. A.; Zollo, J. D.; Dee,
A. S.; Cooper, M. T.; Bellnier, D. A.; Greco, W. R.; Oseroff, A. R. Cancer
Res. 2000, 60, 525.
(21) Qian, X.; Huang, T.-B.; Wei, D.-Z.; Zhu, D.-H.; Fan, M.-C.; Yao, W. J.
Chem. Soc., Perkin Trans. 2 2000, 715.
(22) (a) Jakobs, A.; Piette, J. J. Photochem. Photobiol. B 1994, 22, 9. (b) Jakobs,
A.; Piette, J. J. Med. Chem. 1995, 38, 869. (c) Seret, A.; Piette, J.; Jakobs,
A.; Van de Vorst, A. Photochem. Photobiol. 1992, 56, 409.
(20) Dhar, S.; Chakravarty, A. R. Inorg. Chem. 2003, 42, 2483.
9
J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003 12119

A R T I C L E S
Dhar et al.
(Berkeley, CA) Lewis-coil force magnetometer using Hg[Co(NCS)4]
as a standard. Electrochemical measurements were done at 25 °C on
an EG & G PAR model 253 Versa Stat potentiostat/galvanostat with
electrochemical analysis software 270 for cyclic voltammetric work
using a three electrode setup comprising a glassy carbon working,
platinum wire auxiliary, and a saturated calomel reference (SCE)
electrode. Potassium chloride (0.1 M) was used as a supporting
electrolyte for the electrochemical measurements in Tris-HCl buffer
medium (pH, 7.2).
layering hexane on the top of the dichloromethane solution of the
complexes. Intensity data for 1‚0.5MeOH, 2, and 4-6 were obtained
from a Bruker SMART APEX CCD diffractometer, equipped with a
fine focus 1.75 kW sealed tube Mo KR X-ray source, with increasing
ω (width of 0.3° per frame) at a scan speed of 18, 15, 10, 12, and 4
s/frame, respectively. The intensity data were corrected for absorption.23
Structures were solved and refined with SHELX programs.24 The
hydrogen atoms, either located or placed in the fixed positions, were
refined using a riding model. All non-hydrogen atoms were refined
anisotropically. Crystal structures of 1 and 2 showed the presence of
two molecules in the crystallographic asymmetric unit giving different
trigonality parameter (τ) values,25 possibly due to the crystal packing
effect. Perspective views of the molecules are obtained by ORTEP.26
Preparation of [Cu(L1)B](ClO4) (B ) phen, 1; dmp, 2), [Cu(L2)-
(dmp)](ClO4) (4), and [Cu(L3)(phen)](ClO4) (5). Ternary copper(II)
complexes 1, 2, 4, and 5 were prepared by a general procedure in which
a 0.2 g (0.5 mmol) quantity of dimeric copper(II) acetate hydrate in 10
mL of methanol was reacted with the heterocyclic base (1.0 mmol)
while stirring at 25 °C for 0.5 h followed by the addition of the
respective Schiff base (1.0 mmol) taken in 10 mL of MeOH. The
reaction mixture was stirred for 1 h, and the product was isolated as a
green solid in ∼65% yield on addition of a methanolic solution of
NaClO4 (1.0 mmol). The solid was isolated, washed with cold methanol,
and finally dried in vacuo over P4O10. Anal. Calcd for C22H20N3O5-
SClCu (1): C, 49.16; H, 3.72; N, 7.82. Found: C, 49.09; H, 3.96; N,
7.59. FTIR, cm-1 (KBr phase): 3431w, 1627s, 1529w, 1448m, 1427m,
1317w, 1091vs, 849m, 626m (vs, very strong; s, strong; m, medium;
w, weak). X-band EPR with g ) 2.21 (A ) 156 × 10-4 cm-1) and
DNA Binding and Cleavage Experiments. The concentration of
calf thymus (CT) DNA was determined from the absorption intensity
at 260 nm with an ꢀ value of 6600 M-1 cm-1 27
Relative binding of
.
the ternary complexes to CT DNA with respect to the bis-phen copper-
(II) complex, used as a standard, was studied by fluorescence spectral
method using ethidium bromide (EB) bound CT DNA solution in Tris-
HCl/NaCl buffer (pH, 7.2). The fluorescence intensities at 601 nm (510
nm excitation) of EB with an increasing amount of the ternary complex
concentration were recorded. Ethidium bromide was nonemissive in
Tris-buffer medium due to fluorescence quenching of the free EB by
the solvent molecules. In the presence of DNA, EB showed enhanced
emission intensity due to its intercalative binding to DNA. A competi-
tive binding of the copper complexes to calf thymus DNA resulted in
the displacement of bound EB, and as a consequence, the emission
intensity decreased. The relative order of binding was obtained from
the comparison of the slopes of the lines in the fluorescence intensity
versus complex concentration plot. The cleavage of DNA was studied
by agarose gel electrophoresis. For the cleavage reactions in the
presence of a reducing agent, supercoiled pUC19 DNA (6 µL, 0.5 µg)
in 50 mM tris-(hydroxymethyl)methane-HCl (Tris-HCl) buffer (pH,
7.2) containing 50 mM NaCl was treated with the metal complex (40
µM) and 3-mercaptopropionic acid (MPA, 5 mM) under dark conditions
followed by dilution with the Tris-HCl buffer to a total volume of 18
µL. For photocleavage studies, the reactions were carried out under
illuminated conditions using a UV source at 312 nm (96 W) or visible
monochromatic light source at 532 nm (125 W, mercury vapor lamp)
and 632 nm (250 W halogen lamp). The flash photolytic cleavage
experiments were carried out using a pulsed Nd:YAG pumped laser
Dye laser system (Spectra Physics, 10 Hz, 5-7 ns) and a pulsed Ruby
||
||
g
) 1.99 in DMF glass at 77 K; µeff ) 1.97µB. Anal. Calcd for
C24H24N3O5SClCu (2): C, 50.93; H, 4.24; N, 7.43. Found: C, 50.64;
H, 4.47; N, 7.21. FTIR, cm-1 (KBr phase): 3420w, 1627s, 1412m,
1086vs, 853w, 760m, 628s, 475w. X- band EPR with g ) 2.23 (A )
||
||
175 × 10-4 cm-1) and g ) 2.00 in DMF glass at 77 K; µeff ) 1.78µB.
Anal. Calcd for C29H26N3O5SClCu (4): C, 55.45; H, 4.14; N, 6.69.
Found: C, 55.19; H, 4.09; N, 6.37. FTIR, cm-1 (KBr phase): 3437w,
1614vs, 1528w, 1445m, 1352w, 1081vs, 864w, 760m, 611w. X-band
EPR with g ) 2.19 (A ) 120 × 10-4 cm-1) and g ) 2.03 in DMF
||
||
glass at 77 K; µeff ) 1.98µB. Anal. Calcd for C21H18N3O6ClCu (5): C,
49.67; H, 3.55; N, 8.28. Found: C, 49.43; H, 3.81; N, 8.47. FTIR,
cm-1 (KBr phase): 3461w, 1629vs, 1522m, 1311m, 1106vs, 837m,
751m, 718s, 617m, 423w. X-band EPR with g ) 2.18 (A ) 130 ×
||
||
10-4 cm-1) and g ) 2.03 in DMF glass at 77 K; µeff ) 2.05µB.
Preparation of [Cu(L2)(phen)](ClO4) (3). Complex 3 was prepared
by reacting a 0.38 g (1.0 mmol) quantity of Cu(ClO4)2‚6H2O in 10 mL
of MeOH with 0.26 g (1.0 mmol) of HL2 under stirring for 30 min at
25 °C. The resulting solution was then treated with phen (0.2 g, 1.0
mmol) taken in 10 mL of MeOH. The reaction mixture was stirred for
1 h, and the product was isolated as a green solid in ∼70% yield on
slow evaporation of the solvent at an ambient temperature. Anal. Calcd
for C27H22N3O5SClCu (3): C, 54.09; H, 3.67; N, 7.01. Found: C, 53.83;
H, 3.95; N, 6.86. FTIR, cm-1 (KBr phase): 3434w, 1605vs, 1510s,
1418s, 1394m, 1311w, 1105vs, 846w, 760m, 718m, 617m. X-band EPR
with g ) 2.18 (A ) 150 × 10-4 cm-1) and g ) 2.04 in DMF glass
1
laser (Lumonics, /6 Hz, 20 ns) under dark conditions. The laser light
at 532 nm was generated by frequency-doubling of the Nd:YAG laser
fundamental. Light at 560, 603, 640, 662, and 698 nm was generated
using various dyes (Rhodamine 590 chloride for 560 nm; Rhodamine
640 for 603 nm; DCM for 640 and 662 nm; LDA 698 for 698 nm) in
the dye laser pumped by the 532 nm output from the YAG laser. It
was possible to generate high power up to 60 mJ/P at 603 nm. The
dye used for 660 nm could give power only up to 20 mJ/P. Radiation
at 632.8 nm was generated in a low output (3 mW) CW He-Ne laser
(Scientifica-Cook Ltd make, U. K.). In each experiment, sample DNA
and the complex were premixed before being exposed to the laser light
in the dark. After exposure to the laser light, each sample was incubated
for 1 h at 37 °C and analyzed for the photocleaved products using gel
electrophoresis as discussed below. For the laser power dependence
studies, several quartz plates at the Brewster’s angle were stacked on
||
||
at 77 K; µeff ) 1.99µB.
Preparation of [Cu(L2)2] (6). This complex was prepared by
reacting 0.5 g (1.0 mmol) of copper(II) perchlorate hexahydrate with
0.7 g (2.0 mmol) of the Schiff base HL2 in CH2Cl2. The reaction mixture
was stirred for 0.5 h, and the product was isolated as a brownish green
solid in 85% yield on evaporation of the solvent. Anal. Calcd for
C30H28N2O2S2Cu (6): C, 62.55; H, 4.87; N, 4.87. Found: C, 62.78; H,
5.02; N, 5.01. FTIR, cm-1 (KBr phase): 2951m, 1605s, 1531w, 1436s,
1311w, 1254w, 1168w, 834w. X-band EPR with g ) 2.25 (A ) 163
||
||
× 10-4 cm-1) and g ) 2.02 in DMF glass at 77 K; µeff ) 1.75µB.
Solubility and Stability. The complexes are moderately soluble in
common organic solvents such as MeCN, CH2Cl2, DMF, and MeOH;
less soluble in water; and insoluble in hydrocarbons. They are stable
in the solid as well as in the solution phase.
(23) (a) Walker, N.; Stuart, D. Acta Crystallogr. 1983, A39, 158. (b) Blessing,
R. H. Acta Crystallogr. 1995, A51, 33.
(24) Sheldrick, G. M. SHELX-97, A Computer Program for Crystal Structure
Solution and Refinement; Universita¨t Go¨ttingen: Go¨ttingen, Germany, 1997.
(25) Addison, A. W.; Rao, T. N.; Reedijk, J.; Rijn. J. V.; Verschoor, G. C. J.
Chem. Soc., Dalton Trans. 1984, 1349.
(26) Johnson, C. K. ORTEP III, Report ORNL-5138; Oak Ridge National
Laboratory: Oak Ridge, TN, 1976.
(27) Reichmann, M. E.; Rice, S. A.; Thomas, C. A.; Doty, P. J. Am. Chem.
Soc. 1954, 76, 3047.
X-ray Crystallography. Single crystals of 1, 2, and 5 were obtained
by slow evaporation of aqueous methanolic solutions of the complexes.
Single crystals of 4 and 6 were grown by a diffusion technique by
9
12120 J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003

Ternary Copper Complexes for Photocleavage of DNA
A R T I C L E S
Table 1. Spectral and Cyclic Voltammetric Data for the Complexes 1-6
λ,b nm (ꢀ, M-1 cm-1
)
IR,a cm-1
no.
complex
ν(ClO )
d−d
CT
E
1/2,c V(∆E , mV)
4
p
1
2
3
4
5
6
[Cu(L1)(phen)](ClO4) (1)
[Cu(L1)(dmp)](ClO4) (2)
[Cu(L2)(phen)](ClO4) (3)
[Cu(L2)(dmp)](ClO4) (4)
[Cu(L3)(phen)](ClO4) (5)
[Cu(L2)2] (6)
1091
1086
1105
1081
1106
662 (120)
643 (110)
619 (200)
680 (195)
641 (130)
657 (130)
394 (1290),d 374 (1530)
457 (1280), 369 (3800)
392 (2000), 347 (2600)
377 (2900), 339 (3800)
369 (4900)
-0.15 (220)
-0.05 (480)
-0.03 (140)
-0.04,e 0.46f
-0.18 (182)
-0.31,e 0.03f
383 (1880), 326 (3600)
a In KBr phase. b In MeCN. c Corresponds to Cu(II)/Cu(I) couple at scan rate 50 mV s-1 in DMF-Tris-buffer (1:4 v/v)/0.1 M KCl. E1/2 ) 0.5(Epa + Epc).
∆Ep ) Epa - Epc, where Epa and Epc are anodic and cathodic peak potentials, respectively. d Additional broad peak near 476 nm (ꢀ, ∼93 M-1 cm-1
)
e Broad
f
Epc. Epa with a reduced current height.
the light path in front of the sample and the power was measured by
a laser power meter.
The inhibition reactions for the chemical nuclease reactions were
carried out under dark conditions by adding reagents (distamycin, 75
µM; DMSO, 4 µL; mannitol, 85 µM; ethanol, 4 µL) prior to the addition
of the complexes. The inhibition reactions for the photonuclease studies
were carried out at 532 nm using reagents (NaN3, 90 µM; DMSO, 4
µL; mannitol, 85 µM; ethanol, 4 µL; distamycin 75 µM) prior to the
addition of the complex. For the D2O experiment, this solvent was used
for dilution to 18 µL. Eppendorf and glass vials were used for the UV
and visible light experiments, respectively, at 25 °C in a dark room.
The samples after incubation for 1 h at 37 °C in a dark chamber were
added to the loading buffer containing 25% bromophenol blue, 0.25%
xylene cyanol, and 30% glycerol (3 µL), and the solution was finally
loaded on 0.8% agarose gel containing 1.0 µg/mL ethidium bromide.
Figure 1. Electronic spectrum of 1 in MeCN showing different wavelengths
Electrophoresis was carried out in a dark chamber for 3 h at 40 V in
TAE (Tris-acetate EDTA) buffer. Bands were visualized by UV light
and photographed. The extent of DNA cleavage was measured from
the intensities of the bands using UVITEC Gel Documentation System.
Due corrections were made for the low level of nicked circular (NC)
form present in the original supercoiled (SC) DNA sample and for the
low affinity of EB binding to SC compared to NC and linear forms of
DNA.28 Cleavage reactions for mechanistic investigations using NaN3,
pure oxygen, or inert atmosphere were also carried out using an Nd:
YAG laser at 603 nm.
used for pulsed laser irradiations (by arrow).
Results and Discussion
General Aspects and Crystal Structures. Our major objec-
tive in the synthesis of five ternary (1-5) complexes and one
binary (6) copper(II) complex is to explore the mechanistic
aspects of the photocleavage reactions using red light. Com-
plexes 1-4 have NSO-donor Schiff bases with a thiomethyl or
thiophenyl moiety. First, to explore the effect of the heterocyclic
base on the nuclease activity, we have prepared 1 and 2 using
planar heterocyclic DNA binding ligands phen and dmp in the
presence of a common Schiff base HL1. Again, to investigate
the role of the Schiff base on the photonuclease activity, phen
complexes 1 and 3 having two different NSO-donor Schiff bases
are synthesized. While the Schiff base HL1 has a thiomethyl
moiety, HL2 has a thiophenyl group. The photosensitizing
abilities of two thio moieties are expected to vary. For a
comparative study related to the role of sulfur in the photo-
cleavage reaction, we have prepared a ternary complex 5
containing a ONO-donor Schiff base and a phen ligand. To
probe the importance of the ternary structure and the heterocyclic
base on the photonuclease activity, a binary complex [Cu(L2)2]
(6) is prepared and its nuclease activity studied along with the
known binary species, viz. [Cu(phen)2(H2O)](ClO4)2.
Figure 2. A representative view of the structures of 1, 2, and 4 with a
CuN3OS core along with the perspective views of 5 having a CuN3O2 unit
and the square planar complex 6.
The complexes 1-6 are characterized from the analytical and
physicochemical data (Table 1). They exhibit a d-d band near
650 nm in the electronic spectra (Figure 1). An intense band
near 390 nm with a weak shoulder around 480 nm is assignable
to the sulfur-to-copper(II) charge transfer (LMCT) transition.29
Complexes 1-5 are redox active, and the Cu(II)/Cu(I) couple
is observed near -0.1 V (vs SCE) in DMF-Tris buffer
medium.30,31 All the complexes except 3 have been structurally
characterized (Figure 2). Crystal structures of 1, 2, and 4 consist
of a discrete monomeric cationic complex containing a tridentate
NSO-donor Schiff base and a didentate NN-donor heterocyclic
base (phen or dmp) along with a noncoordinating perchlorate
anion. These complexes are structurally similar having the Schiff
(28) Bernadou, J.; Pratviel, G.; Bennis, F.; Girardet, M.; Meunier, B. Biochem-
istry 1989, 28, 7268.
(29) Kita, T.; Miura, I.; Nakayama, N.; Kawata, T.; Kano, K.; Hirota, S.; Kodera,
M. J. Am. Chem. Soc. 2001, 123, 7715.
9
J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003 12121

A R T I C L E S
Dhar et al.
Table 2. Photocleavage Data of SC pUC19 DNA (0.5 µg) by
base bonded at the basal plane and the heterocyclic base
displaying axial-equatorial bonding. The crystal structure of
[Cu(L3)(phen)](ClO4) (5) shows a CuN3O2 core in which the
phenoxo oxygen and the imine nitrogen atoms of the Schiff
base occupy the basal plane while the alcoholic oxygen atom
binds at the axial site. The binary complex [Cu(L2)2] (6) has a
square planar structure with a trans geometry in a CuN2O2 core.
The sulfur atoms in this structure do not show any bonding
with the metal ion.
[Cu(L1)(phen)](ClO4) (1) and [Cu(L2)(phen)](ClO4) (3) Using an
Nd:YAG Pulsed Laser
laser
peak
power
(mJ/P)
exposure
time
(min)
Sl
no.
reaction
condition
complex
(µM)
λ
(nm)
form-I,
%
form-II,
%
1
2
3
4
5
6
7
8
9
DNA control
DNA + 1
DNA + 1
DNA + 1
DNA + 3
DNA + 3
DNA + 3
DNA control
DNA + 1
532
532
532
532
532
532
532
603
603
603
603
603
603
603
603
603
603
603
603
40
10
15
20
30
30
40
50
40
50
60
20
40
40
50
60
30
30
40
30
15
15
15
15
30
30
30
30
30
30
15
15
30
30
30
15
30
30
95
74
65
48
93
50
45
92
45
37
28
75
45
22
13
2
5
26
35
52
7
50
55
8
55
63
72
25
55
78
87
98
7
50
50
50
50
100
100
DNA Binding and Photoinduced Cleavage. The binding
of the complexes to calf thymus DNA has been studied by
fluorescence spectral technique using emission intensity of
ethidium bromide (EB). The bis-phen complex of copper(II)
has been used as a standard to determine the relative binding
propensities of the complexes. It is observed that the monoca-
tionic complexes having phen as a heterocyclic base show
greater binding to CT DNA than dmp complexes. The steric
constraint imposed by the methyl groups at 2 and 9 positions
in planar dmp seem to reduce the binding ability of this
heterocyclic base to the CT DNA. Among the phen complexes,
the one with a thiophenyl group displays greater binding
propensity than the one with a thiomethyl moiety. It is likely
that the phenyl group could make favorable contact(s) in the
DNA groove.6,11 The binary complex 6 shows poor binding to
DNA. The DNA cleavage activity of the complexes has been
studied under different reaction conditions. The complexes are
nuclease inactive in the absence of any reducing agent when
the reactions are carried out in a dark chamber. In the presence
of mercaptopropionic acid (5 mM) as a reducing agent, the
complexes (40 µM) containing phen ligands show efficient
cleavage of supercoiled pUC19 DNA (0.5 µg), while the dmp
species remain nuclease inactive. The results suggest that the
DNA binding of the complexes utilizing the planar heterocyclic
base phen is a necessity for observing any nuclease activity
among this class of ternary complexes.32
10
10
10
50
50
50
50
50
50
10 DNA + 1
11 DNA + 1
12 DNA + 1
13 DNA + 1
14 DNA + 1
15 DNA + 1
16 DNA + 1
17 DNA + 3
18 DNA + 3
19 DNA + 3
93
39
30
100
100
61
70
shows greater cleavage activity than that of 3 with a thiophenyl
group. The photonuclease activities of 1 and 3 have been studied
using different visible light sources such as a mercury vapor
(532 nm, 125 W), a halogen lamp (632 nm, 250 W), and a CW
laser (632.8 nm, 3 mW). A 150 µM quantity of complex 1 shows
53% cleavage of SC DNA on irradiation at 632 nm for 60 min
using a halogen lamp. A similar experiment using CW laser (3
mW) of 632.8 nm displays significantly higher cleavage. The
photonuclease activities of 1 and 3 have been studied in detail
using a pulsed dye laser at different wavelengths (Table 2, Figure
3). A 10 µM quantity of 1 shows 72% cleavage of SC DNA
for an exposure time of 30 min at 603 nm (60 mJ/P power).
Complex 3 displays cleavage activity at higher concentration
and with more exposure time. The laser setup used for the
experiments is displayed in Figure 4. The observed photocleav-
age activity of 1 compares well with those reported for the
porphyrin species, viz. PHOTOFRIN, an anticancer drug, which
is known to cleave DNA at 630 nm.18,19 A significant observa-
tion of this study is the efficient cleavage activity of complex
1 at 698 nm using the pumped dye laser or at 694 nm using a
Ruby laser source.
The utility of the complexes 1-6 in photoinduced DNA
cleavage reactions is initially probed using monochromatic UV
radiation of 312 nm in the absence of mercaptopropionic acid.
The phen complexes with a CuN3OS core are found to be
photonuclease active.33 The results indicate the necessity of the
phen as well as the NSO-donor Schiff base in the ternary
structure for observing the photonuclease activity. Among the
phen complexes with a CuN3OS core, 1 with a thiomethyl group
Mechanistic aspects of the photocleavage reaction have been
studied at 532 nm (mercury vapor lamp, 125 W) and 603 nm
(Nd:YAG laser) irradiation using different reagents (Figure 5).
The minor groove binder complex 1 is nuclease inactive under
argon or nitrogen atmosphere. This indicates the necessity of
oxygen in the cleavage reactions. Enhancement of cleavage in
D2O or pure oxygen and inhibition in the presence of azide
anion or histidine suggest the possible involvement of singlet
oxygen (O2, 1∆g) as the reactive species. In the presence of pure
oxygen, the extent of cleavage increases by 18% compared to
that in air. This indicates that the presence of oxygen is
necessary for the cleavage to be effective. Addition of KI,
DMSO, mannitol, or ethanol does not show any significant effect
on the photocleavage activity. The photoinduced DNA cleavage
data indicate the role of 1,10-phenanthroline as a binder to DNA,
while the NSO-donor Schiff base acts as a photosensitizer.
Photoexcitation of the phen complexes with a CuN3OS core
(30) Santra, B. K.; Reddy, P. A. N.; Nethaji, M.; Chakravarty, A. R. Inorg.
Chem. 2002, 41, 1328.
(31) Santra, B. K.; Reddy, P. A. N.; Nethaji, M.; Chakravarty, A. R. J. Chem.
Soc., Dalton Trans. 2001, 3553.
(32) Complex 6 is nuclease inactive under similar experimental conditions. The
cleavage efficiency of 3 with a thiophenyl group is found to be more than
1 having a thiomethyl moiety. This may be related to the greater binding
ability of 3 to DNA than 1. Complex 5 with an ONO-donor Schiff base
exhibits chemical nuclease activity. Mechanistic investigations reveal minor
groove binding of the complexes and possible involvement of hydroxyl
radical or reactive copper-oxo species in the cleavage reactions as the
complexes do not show any significant DNA cleavage activity in the
presence of inhibiting reagents such as DMSO, mannitol, ethanol, and the
minor groove binder distamycin.
(33) When SC DNA (0.5 µg) is irradiated in the presence of the complexes
1-6 with a wavelength of 312 nm for 10 min, the phen complexes (80
µM) having NSO-donor Schiff bases show significant cleavage activity.
The dmp complexes do not show any photonuclease activity. Similarly,
the phen complex 5 containing ONO-donor Schiff base, the binary Schiff
base complex 6, and the bis(phen)copper(II) complex are cleavage inactive
on photoirradiation. Control experiments using the Schiff base ligands alone
or other copper(II) compounds such as CuCl2‚2H2O, [Cu(phen)2(H2O)]-
(ClO4)2, and [Cu(TPP)] (H2TPP ) tetraphenylporphyrin) do not show any
apparent cleavage activity on photoirradiation at 638.2 nm using He-Ne
CW laser.
9
12122 J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003

Ternary Copper Complexes for Photocleavage of DNA
A R T I C L E S
Table 3. Photocleavage Dataa of SC pUC19 DNA (0.5 µg) in the
Presence of 1 on Irradiation with Wavelengths at the d-d Band
Using an Nd:YAG Pulsed Laser of 560, 640, 662, and 698 nm
pulse
laser
power
(mJ/P)
exposure
time
(min)
Figure 3. (a) DNA cleavage activity of 1 and 3 using an Nd:YAG pulsed
laser of wavelength 532 nm (lanes 1-9) and of 1 at 560 nm (lanes 10-
14). Lane 1, DNA control (20 mJ/P, 15 min); lane 2, DNA + 1 (50 µM, 5
mJ/P, 15 min); lane 3, DNA + 1 (50 µM, 15 mJ/P, 15 min); lane 4, DNA
+ 1 (50 µM, 18 mJ/P, 15 min); lane 5, DNA + 1 (50 µM, 20 mJ/P, 15
min); lane 6, DNA + 3 (50 µM, 10 mJ/P, 15 min); lane 7, DNA + 3 (50
µM, 20 mJ/P, 15 min); lane 8, DNA + 3 (100 µM, 30 mJ/P, 30 min); lane
9, DNA + 3 (100 µM, 40 mJ/P, 30 min); lane 10, DNA control (50 mJ/P,
15 min); lane 11, DNA + 1 (50 µM, 20 mJ/P, 15 min); lane 12, DNA +
1 (50 µM, 30 mJ/P, 15 min); lane 13, DNA + 1 (50 µM, 40 mJ/P, 15 min);
lane 14, DNA + 1 (50 µM, 50 mJ/P, 15 min). (b) DNA cleavage activity
of 1 and 3 using an Nd:YAG pulsed laser of wavelength 603 nm. Lane 1,
DNA control (50 mJ/P, 30 min); lane 2, DNA + 1 (10 µM, 40 mJ/P, 15
min); lane 3, DNA + 1 (10 µM, 40 mJ/P, 30 min); lane 4, DNA + 1 (10
µM, 50 mJ/P, 15 min); lane 5, DNA + 1 (10 µM, 50 mJ/P, 30 min); lane
6, DNA + 1 (10 µM, 60 mJ/P, 25 min); lane 7, DNA + 1 (10 µM, 60
mJ/P, 30 min); lane 8, DNA + 1 (50 µM, 20 mJ/P, 15 min); lane 9, DNA
+ 1 (50 µM, 20 mJ/P, 30 min); lane 10, DNA + 1 (50 µM, 40 mJ/P, 15
min); lane 11, DNA + 1 (50 µM, 40 mJ/P, 30 min); lane 12, DNA + 1 (50
µM, 60 mJ/P, 25 min); lane 13, DNA + 1 (50 µM, 60 mJ/P, 30 min); lane
14, DNA + 3 (50 µM, 30 mJ/P, 15 min); lane 15, DNA + 3 (100 µM, 30
mJ/P, 30 min); lane 16, DNA + 3 (100 µM, 40 mJ/P, 30 min).
Sl
no
reaction
condition
complex
(µM)
λ
(nm)
form-I,
%
form-II,
%
1
2
3
4
5
6
7
8
9
DNA control
DNA + 1
560
560
560
640
640
640
662
662
662
662
698
698
698
698
50
20
50
35
20
35
20
5
15
20
25
15
20
25
15
15
15
15
15
15
15
15
15
15
15
15
15
15
92
80
45
98
59
32
94
75
58
48
98
66
54
43
8
20
55
2
41
68
6
25
42
52
2
34
46
57
50
50
DNA + 1
DNA control
DNA + 1
50
50
DNA + 1
DNA control
DNA + 1
50
50
50
DNA + 1
10 DNA + 1
11 DNA control
12 DNA + 1
13 DNA + 1
14 DNA + 1
50
50
50
a A similar experiment using complex 5 (50 µM) having phen as DNA
binder and ONO donor ancillary Schiff base shows no apparent photo-
cleavage on irradiation with a pulsed laser of 662 nm (20 mJ/P) for 15 min
(form-I, 90%; form-II, 10%).
Figure 4. Pulsed laser setup for the photoirradiation of complexes 1 and
3 (PDL, pulsed dye laser; first, P1, and second, P2, 90° prisms; I, iris; DM,
dichroic mirror).
Figure 6. Gel diagrams showing the effect of d-d band on photosensi-
tizations at different wavelengths. (a) DNA cleavage activity of 1 (50 µM)
using pulsed Nd:YAG laser of wavelength 640 nm. Lane 1, DNA control
(35 mJ/P, 30 min); lane 2, DNA + 1 (30 mJ/P, 10 min); lane 3, DNA +
1 (30 mJ/P, 15 min); lane 4, DNA + 1 (30 mJ/P, 20 min); lane 5, DNA +
1 (30 mJ/P, 25 min); lane 6, DNA + 1 (30 mJ/P, 30 min); lane 7, DNA +
1 (15 mJ/P, 15 min); lane 8, DNA + 1 (20 mJ/P, 15 min); lane 9, DNA +
1 (35 mJ/P, 15 min). (b) DNA cleavage activity of 1 and 5 (50 µM) using
an Nd:YAG pulsed laser source of wavelength 662 nm. Lane 1, DNA control
(20 mJ/P, 15 min); lane 2, DNA + 1 (5 mJ/P, 15 min); lane 3, DNA + 1
(10 mJ/P, 15 min); lane 4, DNA + 1 (13 mJ/P, 15 min); lane 5, DNA +
1 (15 mJ/P, 15 min); lane 6, DNA + 1 (20 mJ/P, 15 min); lane 7, DNA +
5 (20 mJ/P, 15 min). (c) DNA cleavage activity of 1 (50 µM) using an
Nd:YAG pulsed laser source of wavelength 698 nm. Lane 1, DNA control
(25 mJ/P, 15 min); lane 2, DNA + 1 (5 mJ/P, 15 min); lane 3, DNA + 1
(10 mJ/P, 15 min); lane 4, DNA + 1 (15 mJ/P, 15 min); lane 5, DNA +
1 (20 mJ/P, 15 min); lane 6, DNA + 1 (25 mJ/P, 15 min). (d) DNA cleavage
activity of 1 using a pulsed Ruby laser of wavelength 694 nm. Lane 1,
DNA control (80 mJ/P, 30 min); lane 2, DNA + 1 (10 µM, 80 mJ/P, 30
min); lane 3, DNA + 1 (50 µM, 60 mJ/P, 30 min). The percentage SC
DNA cleavage for lanes 1-3 are 3%, 42%, and 50%, respectively.
Figure 5. Percentage of SC pUC19 DNA cleavage upon irradiation with
a mercury vapor lamp (125 W) of 532 nm for 10 min and Nd:YAG pulsed
laser of 603 nm for 15 min (40 mJ/P) in the presence of complex 1 having
concentrations of 80 and 50 µM, respectively, at different reaction
conditions. The control experiments in the absence of 1 show a cleavage
of 5% at 532 nm and 8% at 603 nm of SC DNA.
known to occur with porphyrin bases and their metal complexes
used in PDT.2,34
The laser wavelengths for cleavage experiments were chosen
based on the absorption spectrum of the complex (Figure 1).
Several wavelengths (603, 632.8, 640, 662, and 698 nm) under
the d-d transition band were selected to probe the involvement
of the long wavelength band in the photocleavage activity (Table
3, Figures 3 and 6). The wavelengths at 532 and 560 nm were
chosen since they are near the midpoint of the weak d-d band
(λmax, 661.5 nm) and the strong ligand-to-metal (S-to-Cu) charge
transfer band peaking at around 390 nm. The absorption
seems to sensitize the thio alkyl/aryl group of the complexes to
an excited state followed by an efficient energy transfer to the
triplet state which presumably activates oxygen from its stable
triplet (O2, Σg-) to the highly toxic singlet (O2, ∆g) state.
Although we have not made any attempt to isolate the cleaved
products, photocleavage of nucleic acids is known to involve
an initial oxidative reaction involving either a nucleobase or a
sugar residue. Among the four nucleobases, guanine is most
susceptible for oxidation by singlet oxygen for its low oxidation
potential, and guanine-selective photocleavage reactions are
3
1
(34) Croke, D. T.; Perrouault, L.; Sari, M. A.; Battioni, J. P.; Mansuy, D.; He´le`ne,
C.; Le Doan, T. J. Photochem. Photobiol. B 1993, 18, 41.
9
J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003 12123

A R T I C L E S
Dhar et al.
Conclusions
A series of ternary copper(II) complexes containing tridentate
Schiff bases and didentate heterocyclic bases and a binary
copper(II) complex have been prepared and structurally char-
acterized, and their photoinduced nuclease activities studied
under laser light illumination. The ternary copper(II) complexes
containing phen and NSO-donor Schiff base ligands show red
light induced photonuclease activity. Mechanistic investigations
reveal the role of phen as a minor groove binder to DNA and
the NSO-donor Schiff base as a photosensitizer. Photoinactivity
of the complexes under inert atmosphere, inhibition with azide
addition, and enhancement of cleavage in D2O or pure oxygen
suggest the involvement of singlet oxygen as a reactive species.
The pulsed laser photolysis data show significant cleavage of
DNA at 603 nm using a 10-50 µM quantity of the complex 1.
This complex also shows significant cleavage near 700 nm and
generally satisfies the basic requirements for a sensitizer to be
useful in photodynamic therapy. Consequently, this class of
ternary copper(II) complexes offer scopes for studies related to
clinical investigations along with the currently used porphyrin
derivatives and their metal complexes in PDT. This work
presents the first direct evidence for a dual involvement of the
sulfur-to-copper charge transfer (LMCT) and the d-d band in
the photosensitization process leading to an efficient DNA
cleavage activity near 660 nm.
Figure 7. Photocleavage activity of complex 1 after 9000 laser shots at
20 mJ/pulse at different wavelengths using an Nd:YAG PDL system.
Acknowledgment. The authors thank the Department of
Science and Technology, Government of India, for the financial
support (SP/S1/F01/2000) and for the CCD diffractometer
facility. We are thankful to Mr. P. A. N. Reddy for the
crystallographic studies and the Alexander von Humboldt
Foundation, Germany, for donation of an electroanalytical
system. We also thank the Convener, Bioinformatic Center, and
the Chairman, Department of Chemical Engineering, of our
Institute for database search and the Ruby Laser equipment,
respectively.
Figure 8. Variation of percentage DNA cleavage with laser power at
different wavelengths using an Nd:YAG PDL system.
coefficient at 532 and 560 nm is significant but much lower
than that at g600 nm. If only the d-d band is involved in the
initial step of the photocleavage mechanism at these two
wavelengths, we would expect no or very little cleavage of the
DNA. However, at 532 nm where the absorption is mostly due
to the tail of the LMCT band, 52% cleavage is observed. In
fact, the observation that the extent of cleavage is the same as
that at 662 nm under similar conditions leads us to believe that
the LMCT band is also contributing to the initial step of
photocleavage of the DNA (Figure 7). Initially the photon from
the laser is believed to excite the metal d-d transition and the
LMCT transition in the complex, which then transfers the energy
to a ground state oxygen molecule and excites it to the 1∆g state.
The variation of the percentage cleavage of SC DNA with
laser power has been studied at several visible wavelengths,
and the results are shown in Figure 8. The plots show linear
behavior, and the slopes of the straight lines at different
wavelengths are similar within experimental errors. The percent
cleavage increasing linearly with laser power indicates that the
initial photoexcitation step is a single photon process. The extent
of cleavage at different wavelengths under similar conditions
using a laser power of 20 mJ/P follows the absorption spectral
pattern closely (Figure 7). In fact, this strengthens our claim
that both the Cu(II) d-d band and sulfur-to-metal charge transfer
band are involved in the primary step of the photocleavage
process.
Supporting Information Available: Selected crystallographic
data, bond distances and angles for 1, 2, and 4-6 (Tables S1-
S3); trigonality parameter values for 1, 2, 4, and 5 (Table S4);
chemical nuclease data in the presence of mercaptopropionic
acid (Table S5); photonuclease data at 312 nm (Table S6);
photonuclease data at 532, 632, and 632.8 nm using a mercury
vapor lamp (125 W), halogen lamp (250 W), and CW laser (3
mW), respectively (Table S7); cleavage data in the presence of
different inhibiting reagents (Table S8); ORTEP diagrams with
50% probability thermal ellipsoids for the complexes 1, 2, and
4-6 with atom labeling schemes (Figures S1-S5); the DNA
binding plots for 1-6 (Figures S6 and S7); gel diagram showing
chemical nuclease activity in the presence of mercaptopropionic
acid (Figure S8); gel diagram displaying photocleavage data at
312 nm (Figure S9); gel diagram exhibiting photonuclease
activity at 532 and 632 nm (Figure S10); gel diagrams showing
inhibition reactions (Figure S11); gel diagram showing control
experiments using CuCl2‚2H2O, bis(phen)copper(II), and Cu-
(TPP) (Figure S12); full list of bond distances and bond angles,
anisotropic thermal parameters, and hydrogen atom coordinates
for complexes 1, 2, and 4-6 (CIF). This material is available
free of charge via the Internet at http://pubs.acs.org.
JA036681Q
9
12124 J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003