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Synthetic analogue approach for the functional domains of copper(II)
Dalton
bleomycins and its DNA cleavage activity†
Shanta Dhar, Munirathinam Nethaji and Akhil R. Chakravarty*
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore,
560012, India. E-mail: arc@ipc.iisc.ernet.in
Received 21st September 2004, Accepted 27th October 2004
First published as an Advance Article on the web 15th November 2004
The dicopper(II) complex [Cu2 (R SSR)2 (SO4 )2 ] (1), where R SSR is a Schiff base, has been prepared from the reaction
of CuSO4 ·5H2 O with the Schiff base N,N -1,1 -dithiobis(ethylenesalicylaldimine) (H2 RSSR) and structurally
characterized by X-ray crystallography. The crystal structure of 1 shows two {Cu(R SSR)}2+ units linked by two
sulfate ligands each showing a g3 ,l2 -binding mode. The Cu · · · Cu distance is 4.562(2) Å with each copper having a
square pyramidal (4 + 1) CuNO4 coordination geometry. The monoanionic Schiff base R SSR has a pendant cationic
amine –SCH2 CH2 NH3 + group which is presumably formed from the hydrolysis of one imine bond of
H2 RSSR. Complex 1 models the N- and C-terminus domains of bleomycins. The metal centers in 1 are essentially
magnetically non-interacting giving a −2J value of 3 cm−1 with the singlet as the ground state. Using complex 1 as a
precursor, ternary copper(II) complexes [Cu(R SSR)B(SO4 )] (2–4) are prepared, characterized and their DNA
binding and cleavage properties studied (B: kanamycin A, 2; 2,2 -bipyridine, 3; 1,10-phenanthroline, 4). IR spectral
data suggest a square pyramidal (4 + 1) geometry for the one-electron paramagnetic ternary complexes with the
sulfate bound to copper. The complexes are non-conducting in DMF but show conductivity in aqueous medium due
to dissociation of the sulfate ligand. They bind to calf thymus DNA in the minor groove giving the relative order:
4 > 2 > 1 ∼ 3 (K app = 5.4 × 105 M−1 for 4). The precursor complex 1 does not show any apparent chemical nuclease
activity when treated with supercoiled (SC) DNA in the presence of 3-mercaptopropionic acid (MPA). The
kanamycin A and phen adducts as such or generated under in situ reaction conditions using 1 and the ligand display
efficient chemical nuclease activity in the presence of MPA, while the bpy species shows poor cleavage activity. The
ternary kanamycin A complex presents the first synthetic model for three functional domains of bleomycins.
Introduction
Bleomycins (BLMs) are the glycopeptide antitumor antibiotics
that cleave DNA in an oxidative manner and are clinically used
for the treatment of squamous cell and malignant lymphomas.1–3
Bleomycins, in the presence of ferrous ion and molecular oxygen,
cause sequence-selective DNA strand scission. The structure of
bleomycins consists of three major domains playing different
functional roles (Scheme 1). The N-terminus domain has bind-
ing sites for the metal, viz. iron or copper. Binding of molecular
oxygen to the metal occurs in this domain. The C-terminus
domain with a bithiazole unit and a cationic terminal amine
moiety show DNA binding affinity and sequence selectivity.
The carbohydrate domain facilitates cell permeability of BLMs
and oxygen binding. Since activation of bleomycins requires a
metal ion cofactor such as Fe2+ (or Cu2+ in the presence of
dithiothreitol as reductant), the coordination chemistry of low
molecular weight transition metal complexes as synthetic models
assume paramount importance for gaining better insights into
different structural and functional aspects of BLMs.
BLM–DNA interactions have been studied using various Scheme 1 (a) Bleomycins with three functional domains. (b) Proposed
ternary structure of the kanamycin A copper(II) complex (2).
analogues/conjugates to probe specific roles played by each
domain.1–6 In comparison, synthetic model iron or copper
structure that mimics the N- and C-terminus domains.9 They
complexes are few and they do not model all the domains of the
suggested that methidiumpropyl-EDTA in presence of Fe2+
elephantine structure of BLMs.7–11 The structural and chemical
forms an iron–EDTA complex with a pendant cationic DNA
complexities associated with BLMs have been addressed by
binding site and the complex cleaves DNA in an oxidative
Mascharak and co-workers in a series of papers on synthetic
manner. Using a similar synthetic analogue approach, we are
model iron and copper complexes.7,8 The complexes generally
successful in forming ternary copper(II) complexes containing
model the metal binding N-terminus domain. Hertzberg and
carbohydrate or heterocyclic base (B) using a precursor cop-
DOI: 10.1039/b414639e
4180 Dalton Trans., 2004, 4180–4184 This journal is © The Royal Society of Chemistry 2004
cleavage activity of the kanamycin A complex in the presence of 1144vs, 1130vs, 1115vs, 1052w, 1000m, 936m, 912w, 760m, 656w,
mercaptopropionic acid (MPA). 620m, 586w, 486w, 456w (vs, very strong; s, strong; m, medium;
Ternary copper(II) complexes [Cu(R SSR)B(SO4 )] (2–4) are w, weak). leff per Cu = 2.0 at 300, 1.9 lB at 18 K [theoretical fit:
prepared for the DNA binding and cleavage studies from the −2J = 3 cm−1 , g = 2.18, g1 = 2.2, q = 0.004].
reaction of 1 with kanamycin A or heterocyclic bases such as 2,2 -
bipyridine (bpy) and 1,10-phenanthroline (phen) (B: kanamycin Synthesis of [Cu(R SSR)B(SO4 )] (B: kanamycin A, 2). The
A, 2; bpy, 3; phen, 4). Our choice of 1 as a precursor is based on ternary copper(II) kanamycin A adduct was prepared from
its novel structural features showing the presence of a cationic reaction of 1 (1.0 mmol, 0.83 g) with kanamycin A (2.0 mmol,
DNA-binder arm making it a potential synthetic model for the 1.16 g) in water (20 cm3 ) at 25 ◦ C on magnetic stirring for 12 h.
N- and C-terminus domains of BLMs. Herein we report the The solution was filtered and the filtrate on slow concentration
synthesis, structure and DNA cleavage properties of 1–4. gave brownish-green solid which was isolated, washed with
aqueous methanol (1 : 1 v/v) and dried in vacuum over P4 O10
giving ∼62% (0.55 g) yield (Found: C, 39.1; H, 3.9; N, 9.5, C29 H37
Experimental CuN6 O16 S3 (2) requires C, 39.3; H, 4.2; N, 9.5%). Conductivity
Materials (KM /X−1 cm2 mol−1 ) in DMF–H2 O (1 : 4 v/v) = 145. UV-
vis in DMF [kmax /nm (e/dm3 mol−1 cm−1 )]: 630 (175), 369
All reagents and chemicals were procured from commercial (1090), 310 (3970). IR (KBr phase, cm−1 ): 3337br, 3054w, 1622s,
sources and used without further purifications. Solvents used 1538m, 1465m, 1450m, 1400m, 1345w, 1315m, 1282w, 1205m,
were purified by standard procedures.13 Calf thymus (CT) 1145vs, 1109vs, 1000m, 939m, 906w, 756m, 613s, 483w, 453w
DNA and supercoiled (SC) pUC19 DNA (caesium chloride (br, broad).
purified) were purchased from Bangalore Genie (India). Agarose
(molecular biology grade) and ethidium bromide (EB) were Synthesis of [Cu(R SSR)B(SO4 )] (B: bpy, 3; phen, 4). Com-
from Sigma (USA). The disulfide Schiff base ligand (H2 RSSR) plexes 3 and 4 were prepared by following a general procedure
was prepared by a literature procedure using cysteamine in which the heterocyclic base (2.0 mmol: 0.31 g bpy or 0.4 g
hydrochloride (Lancaster, UK) and salicylaldehyde (Aldrich, phen) was reacted with an aqueous solution (20 cm3 ) of 1
USA).14 (1.0 mmol, 0.83 g). The solution was magnetically stirred for
5 h at 25 ◦ C. The solution was filtered. The product as a green
Physical measurements solid was isolated on slow concentration of the filtrate on a
rotary evaporator. The solid was washed with aqueous ethanol
The elemental analyses were done using a Thermo Finnigan (1 : 1 v/v) and dried in vacuum over P4 O10 (Yield: 74%, 0.42 g
Flash EA 1112 CHNSO analyser. The IR, electronic, and for 3; 68%, 0.4 g for 4). Characterization data for 3: Found:
fluorescence spectral data were obtained from Bruker Equinox C, 44.3; H, 4.0; N, 9.6, C21 H24 CuN4 O5 S3 requires C, 44.1; H,
55, Hitachi U-3400, and Perkin Elmer LS-50B spectrometers, re- 4.2; N, 9.8%. Conductivity (KM /X−1 cm2 mol−1 ) in DMF–H2 O
spectively. Conductivity measurements were made using Control (1 : 4 v/v) = 140. UV-vis in DMF [kmax /nm (e/dm3 mol−1 cm−1 )]:
Dynamics Conductivity Meter. Electrochemical measurements 611 (150), 371 (830), 274 (2300). IR (KBr phase, cm−1 ): 3700br,
were done at 25 ◦ C on an EG & G PAR model 253 Versa Stat 3037w, 1618s, 1595m, 1565w, 1532m, 1470m, 1442s, 1320m,
potentiostat/galvanostat with electrochemical analysis software 1242w, 1116vs, 1022m, 980w, 906w, 769s, 730m, 660w, 620s,
270 for cyclic voltammetric work using a three-electrode setup 470w, 456w. Characterization data for 4: Found: C, 46.2; H,
consisting of a glassy carbon working, platinum wire auxiliary 3.7; N, 9.5, C23 H24 N4 CuO5 S3 requires C, 46.3; H, 4.0; N, 9.4%.
and saturated calomel reference electrode. Variable temperature Conductivity (KM /X−1 cm2 mol−1 ) in DMF–H2 O (1 : 4 v/v) =
magnetic susceptibility data in the temperature range 18–300 K 135. UV-vis in DMF [kmax /nm (e/dm3 mol−1 cm−1 )]: 666 (105),
were obtained for polycrystalline samples using a George Asso- 372 (1820), 318 (1350), 268 (3260). IR (KBr phase, cm−1 ): 3740br,
ciates Inc. Lewis-coil-force magnetometer system (Berkeley, CA) 3050w, 1621s, 1541m, 1515m, 1473m, 1450m, 1424m, 1348m,
equipped with a closed-cycle cryostat (Air Products) and a Cahn 1330w, 1147vs, 1103vs, 1040w, 987w, 912w, 855s, 760m, 722s,
balance. Hg[Co(NCS)4 ] was used as a standard. Experimental 616s.
susceptibility data were corrected for diamagnetic contributions
and temperature independent paramagnetism. The corrected
molar magnetic susceptibilities for the dicopper(II) complex were Crystal structure determination of 1
theoretically fitted by the modified Bleaney–Bowers expression Crystal data. C22 H32 Cu2 N4 O10 S6 , M r = 831.96, green rect-
based on the isotropic form of the Heisenberg–Dirac–van Vleck angular (0.50 × 0.32 × 0.28 mm), monoclinic, space group
(HDvV) model giving H = −2JS 1 S 2 , where S1 = S2 = 12 .15,16 The P21 /c, a = 12.771(7), b = 15.664(9), c = 8.544(5) Å, b =
susceptibility equation used for fitting was: vCu = [Ng2 b2 /kT][3 + 105.971(9)◦ , U = 1643.2(16) Å3 , Z = 2, Dc = 1.682 g cm−3 ,
exp(−2J/kT)]−1 (1 − q) + (Ng1 2 b2 /4kT)q + N a, where q is the l = 1.732 mm−1 , min./max. transmission = 0.48/0.64, 2hmax =
fraction of monomeric impurity. 50◦ , k(Mo-Ka) = 0.71073 Å, T = 293(2) K.
Scheme 2 The reaction pathways for the synthesis of [Cu(R SSR)(SO4 )]2 (1) and ternary complexes (B; kanamycin A, 2; bpy, 3; phen, 4). Reagents:
(i) solvent: H2 O (2); H2 O–EtOH 5 : 1 v/v (3, 4).