Synthesis, structure, and phosphatase-like activity of a new trinuclear Gd complex with the unsymmetrical ligand H3L as a model for nucleases

Article abstract of DOI:10.1021/ic100276z

The new trinuclear gadolinium complex [Gd₃L₂(NOa) ₂(H₂O)₄]NO₃-SH₂O (1) with the unsymmetrical ligand 2-[A/-bis-(2-pyridylmethyl)aminomethyl]-4-methyl-6-[A/ -bis(2-hydroxy-2-oxoethyl)aminomethyl] phenol (H₃L) was synthesized and characterized. The new ligand H₃L was obtained in good yield. Complex 1 crystallizes in an orthorhombic cell, space group Pcab. Kinetic studies show that complex 1 is highly active in the hydrolysis of the substrate 2,4-bis(dinitrophenyl)phosphate (Km = 4.09 mM, V_max = 2.68 x 10 ^-2 mM s^-1 and k_cat = V_max/[1] = 0.67 s^-1). Through a Potentiometrie study and determination of the kinetic behavior of 1 in acetonitrile/water solution, the species present in solution could be identified, and a trinuclear monohydroxo species appears to be the most prominent catalyst under mild conditions. Complex 1 displays high efficiency in DNA hydrolytic cleavage, and complete kinetic studies were carried out (K_m = 4.57 x 10^-4M, k'_Cat=3.42h ^-1, and k'_Cat/K_m= 7.48 x 10³M ^-1 h^-1). Studies with a radical scavenger (dimethyl sulfoxide, DMSO) showed that it did not inhibit the activity, indicating the hydrolytic action of 1 in the cleavage of DNA, and studies on the incubation of distamycin with plasmid DNA suggest that 1 is regio-specific, interacting with the minor groove of DNA.

Full text of DOI:10.1021/ic100276z

Inorg. Chem. 2010, 49, 3057–3063 3057
DOI: 10.1021/ic100276z
Synthesis, Structure, and Phosphatase-Like Activity of a New Trinuclear Gd
Complex with the Unsymmetrical Ligand H3L As a Model for Nucleases
Maryene A. Camargo,† Ademir Neves,*,† Bruno Szpoganicz,† Adailton J. Bortoluzzi,† Franciele L. Fischer,‡
‡
§
ꢀ
Hernan Terenzi, and Eduardo E. Castellano
†
ꢀ
^
´
Laboratorio de Bioinorganica e Crystalografia (LABINC), Departamento de Quımica, Universidade Federal de
‡
ꢀ
ꢀ
~
^
Santa Catarina, 88040-900 Florianopolis, Santa Catarina, Brazil, Laboratorio de Expressao Genica,
´
ꢀ
Departamento de Bioquımica, CCB, Universidade Federal de Santa Catarina, 88040-900 Florianopolis, Santa
§
´
~
~
~
Catarina, Brazil, and Instituto de Fısica, Universidade de Sao Paulo,13560-970 Sao Carlos, Sao Paulo, Brazil
Received February 10, 2010
The new trinuclear gadolinium complex [Gd3L2(NO3)2(H2O)4]NO3 8H2O (1) with the unsymmetrical ligand 2-[N-bis-(2-
3
pyridylmethyl)aminomethyl]-4-methyl-6-[N-bis(2-hydroxy-2-oxoethyl)aminomethyl] phenol (H3L) was synthesized and
characterized. The new ligand H3L was obtained in good yield. Complex 1 crystallizes in an orthorhombic cell, space group
Pcab. Kinetic studies show that complex 1 is highly active in the hydrolysis of the substrate 2,4-bis(dinitrophenyl)phosphate
(Km = 4.09 mM, Vmax = 2.68 ꢀ 10-2 mM s-1, and kcat = Vmax/[1] = 0.67 s-1). Through a potentiometric study and
determination of the kinetic behavior of 1 in acetonitrile/water solution, the species present in solution could be identified,
and a trinuclear monohydroxo species appears to be the most prominent catalyst under mild conditions. Complex 1
displays high efficiency in DNA hydrolytic cleavage, and complete kinetic studies were carried out (Km = 4.57 ꢀ 10-4 M,
k0cat = 3.42h-1, and k0cat/Km =7.48 ꢀ 103 M-1 h-1). Studies with a radical scavenger (dimethyl sulfoxide, DMSO) showed
that it did not inhibit the activity, indicating the hydrolytic action of 1 in the cleavage of DNA, and studies on the incubation of
distamycin with plasmid DNA suggest that 1 is regio-specific, interacting with the minor groove of DNA.
Introduction
ions in a dinuclear complex which is more reactive toward
phosphate diester hydrolysis than the similar mononuclear
species.6 Dinuclear complexes are able to produce nucleo-
phile hydroxo groups at lower pH values, making these
species prominent catalysts in hydrolysis reactions.
Several systems are used in the coordination with
lanthanide ions, such as polyhydroxyl ligands,7 crown
ethers and azacrowns,8,9 and polyaminocarboxylate deri-
vatives,10 and so forth. These systems avoid the precipita-
tion of Ln(OH)3(s) and provide compounds with a positive
overall charge favoring, consequently, the catalytic action
toward the hydrolysis of phosphate esters. Branum and
Que reported LaIII and CeIV ions combined with the
dinucleating ligand HPTA, which represent the first metal
complexes capable of carrying out hydrolysis of double-
stranded DNA.11
Phosphodiesters are well-known for both their importance as
biomolecules (as components of DNA and RNA, for exam-
ple) and their stability. Thus, considerable efforts are being
made to develop catalytic systems (artificial nucleases) capable
of cleaving efficiently phosphodiester bonds.1-4 As with natu-
ral nucleases, a hydrolytic rather than oxidative cleavage
mechanism is desirable for molecular biology and clinical appli-
cations. Thus, there is an open field for the development of
lanthanide-based nucleases: even the CeIV ion, the lanthanide
with the highest redox activity, acts as a hydrolytic catalyst.5
In nature, a large number of metalloenzymes able to
catalyze phosphate ester hydrolysis are activated by two or
more metal ions (at the active site of the enzyme). Jurek and
co-workers have reported cooperativity between two LaIII
*To whom correspondence should be addressed. E-mail: ademir@
qmc.ufsc.br.
(6) Jurek, P. E.; Jurek, A. M.; Martell, A. E. Inorg. Chem. 2000, 39, 1016–
1020.
(7) Gomez-tagle, P.; Yatsimirsky, A. K. Inorg. Chem. 2001, 40, 3786–
3796.
(8) Peluffo, F.; Torres, J.; Kremer, C.; Dominguez, S.; Mederos, A.;
Kremer, E. Inorg. Chim. Acta 2006, 359, 2107–2114.
(9) Belousoff, M. J.; Ung, P.; Forsyth, C. M.; Tor, Y.; Spiccia, L.;
Graham, B. J. Am. Chem. Soc. 2009, 131, 1106–1114.
(10) Torres, J.; Brusoni, M.; Peluffo, F.; Kremer, C.; Dominguez, S.;
Mederos, A.; Kremer, E. Inorg. Chim. Acta 2005, 358, 3320–3328.
(11) Branum, M. E.; Que, L., Jr. J. Biol. Inorg. Chem. 1999, 4, 593–600.
(1) Liu, C.; Wang, M.; Zhang, T.; Sun, H. Coord. Chem. Rev. 2004, 248,
147–168.
ꢀ
(2) Mitic, N.; Smith, S. J.; Neves, A.; Guddat, L. W.; Gahan, L. R.;
Schenk, G. Chem. Rev. 2006, 106, 3338–3363.
(3) Gahan, L. R.; Smith, S. J.; Neves, A.; Schenk, G. Eur. J. Inorg. Chem.
2009, 2745–2758.
(4) Jarenmark, M.; Carlsson, H.; Nordlander, E. C. R. Chim. 2007, 10,
433–462.
(5) Shigekawa, H.; Ishida, M.; Miyake, K. Appl. Phys. Lett. 1999, 74, 460–
463.
r
2010 American Chemical Society
Published on Web 02/19/2010
pubs.acs.org/IC

3058 Inorganic Chemistry, Vol. 49, No. 6, 2010
Camargo et al.
In this paper, we present the synthesis of the new 2-[N-bis-(2-
pyridylmethyl)aminomethyl]-4-methyl-6-[N-bis(2-hydroxy-2-
oxoethyl)aminomethyl]phenol ligand (H3L) and the synthe-
sis and structure of its trinuclear [Gd3L2(NO3)2(H2O)4]-
Ligand 3 (2.65 mmol; 1.30 g) was dissolved in 40 mL of
methanol, and then KOH (18.52 mmol; 1.04 g) was added. This
mixture was stirred for 4 days at room temperature. The
resultant solution was dried under vacuum. The residual solid
obtained was dissolved in 10 mL of deionized water solution and
neutralized with HCl (4 mol L-1) to pH 7. Subsequent chroma-
tographic purification with basic DOWEX (1 ꢀ 2:200) eluted
with acetic acid led to H3L. Yield: 90%. Selected IR data (KBr):
ν (O-H, C-HAr and C-HAliph) 3551-2924; ν (CdC and CdN)
NO3 8H2O (1) complex, which displays high activity toward
3
the hydrolysis of the activated substrate 2,4-bis(dinitro-
phenyl)phosphate (BDNPP). Complex 1 was found to be
active toward the hydrolytic cleavage of plasmid DNA,
interacting with regio-specificity, indicating its potential ac-
tion as a chemical nuclease.
1600-1430; ν (COO-) 1631 and 1398; δ (C-HAr) 764 in cm-1
.
1H NMR δH (400 MHz; D2O): 1.85 (s, 3 H, CH3-residue of
CH3COOH); 1.98 (s, 3 H, CH3); 3.54 (s, 4 H, CH2); 3.72 (s, 2H,
CH2); 4.01 (4H, s, CH2); 4.09 (2H, s, CH2); 6.82 (2H, d, CHAr);
7.39 (4H, d, CHAr); 7.87 (2H, t, CHAr); 8.41 (2H, d, CHAr) in
ppm. 13C NMR δ (100 MHz; D2O): 19.27; 21.05; 55.63; 56.24;
56.86; 57.74; 116.94; 122.64; 124.96; 125.97; 130.56; 133.19;
133.94; 142.48; 144.90; 152.75; 153.24; 170.39; 177.71 (residue
of CH3COOH) in ppm.
Experimental Section
Abbreviations. The following abbreviations are used in this
paper: H3L, 2-[N-bis-(2-pyridylmethyl)aminomethyl]-4-methyl-
6-[N-bis(2-hydroxy-2-oxoethyl)aminomethyl]phenol; HPTA,
1,3-diamino-2-hydroxypropane-N,N,N0,N0-tetraacetate; BDNPP,
2,4-bis(dinitrophenyl)phosphate; DNPP, 2,4-dinitrophenyl phos-
phate; DNP, 2,4-dinitrophenolate; ESI-MS, electrospray ioniza-
tion-mass spectrometry; BTP, 1,3-bis[tris(hydroxymethyl)methyl-
amino]propane; bp, base pairs; DMSO, dimethyl sulfoxide.
Materials and Measurements. BDNPP and ligand 2 were
synthesized by previously described methods.12-14 All other
chemicals and solvents were of analytical or spectroscopic grade
purchased from commercial sources, and used without further
purification. Infrared spectra were recorded on a Perkin-Elmer
model 16PC spectrometer, in KBr pellets in the 4000-400 cm-1
range. 1H NMR and 13C NMR spectra were recorded on a
Varian Mercury Plus 400 spectrometer (400 and 100 MHz,
respectively) in CDCl3 or D2O and using tetramethylsilane
(TMS, δ = 0.00 ppm) as the internal standard. Elemental
analysis was performed on a Carlo Erba E-1110 instrument.
The mass spectrometry (MS) experiments were carried out using
a Q-TOF mass spectrometer (Micromass, Manchester, U.K.)
and electrospray ionization in the positive mode (ESI(þ)-MS).
Typical MS conditions were as follows: source temperature of
100 °C, desolvation temperature of 100 °C, capillary voltage of 3
kV, and cone voltage of 50 V. The sample was injected using a
syringe pump (Harvard Apparatus) at a flow rate of 5 μL/min.
Mass spectra were acquired within the range of 50-2000 m/z.
Preparation of 2-[N-bis-(2-pyridylmethyl)aminomethyl]-4-
methyl-6-[N-bis(2-methoxy-2-oxoethyl)aminomethyl]phenol (3).
Ligand 213,14 (0.01 mol; 4.21 g), dimethyl iminodiacetate (0.01
mol; 1.68 g), and triethylamine (0.02 mol; 2.90 mL) were
dissolved in 100 mL of dichloromethane in a 125 mL flask.
The reaction mixture was refluxed for 24 h and then stirred for
72 h at room temperature. The resultant solution was washed
with a saturated solution of NaHCO3 (6 ꢀ 30 mL), dried with
Na2SO4, and evaporated under vacuum to yield product 3 as a
viscous liquid. Yield: 91%. Selected IR data (KBr): ν (C-HAr
and C-HAliph) 3052-2842; ν (CdC and CdN) 1590-1434; δ
(O-H) 1372; ν (C-Ophenol and C-Oester) 1296-1150; ν (CdO)
1743; δ (C-HAr) 764 in cm-1. 1H NMR δH (400 MHz; CDCl3):
2.23 (3 H, s, CH3); 3.60 (4 H, s, CH2); 3.71 (6H, s, CH3); 3.80 (2
H, s, CH2); 3.90 (4 H, s, CH2); 3.96 (2 H, s, CH2); 6.95 (2H, d,
CHAr); 7.14-7.17 (2H, m, CHAr); 7.43-7.45 (2H, m, CHAr);
Synthesis of [Gd3L2(NO3)2(H2O)4]NO3 8H2O (1). Complex
3
1 was prepared by adding Gd(NO3)3 6H2O to a methanolic
3
solution containing the ligand H3L and NaOH (3:2:6
stoichiometry), under magnetic stirring at 40 °C for 40 min.
After total solvent evaporation, the solid obtained was recrys-
tallized in a methanol/ethanol/acetone (1:1:1) solution mixture,
yielding colorless monocrystals suitable for X-ray analysis.
Yield: 20%. Found: C, 32.21; H, 4.18; N, 8.50. Calcd for
[Gd3(C25H25N4O5)2(NO3)2(H2O)4]NO3 8H2O: C, 33.42; H,
3
4.15; N, 8.57. Selected IR data (KBr): ν (O-H, C-HAr and
C-HAliph) 3550-2922; ν (CdC and CdN) 1602-1450; ν
(C-Ophenol) 1256 and 1228; ν (COO-) 1654 and 1450; ν
(NO3-) 1482, 1386, and 1295; δ (C-HAr) 786 in cm-1. ESI-
MS: [Gd3L(NO3)3(CH3CN)6(H2O)6]3þ m/z 492; [Gd3L(NO3)3-
(CH3CN)5(H2O)6]3þ m/z 478; [Gd3L(NO3)3(CH3CN)4-
(H2O)6]3þ m/z 464 (only fragment ions were observed, probably
because of the gas-phase conditions).
Single-Crystal X-ray Structure Determination. A fragment
with dimensions of 0.26 ꢀ 0.44 ꢀ 0.47 mm3 was selected from a
crystalline sample of complex 1 for crystallographic analysis. X-
ray diffraction data were measured on a Kappa-CCD diffracto-
˚
meter with graphite-monochromated Mo KR (λ = 0.71073 A)
radiation, at room temperature. Diffraction data were collected
(j and ω scans with K-offsets) with COLLECT.15 Integration
and scaling of the reflections were performed with HKL DEN-
ZO-SCALEPACK suite of programs. The unit cell parameters
were obtained by least-squares refinement based on the angular
settings for all collected reflections using HKL SCALEPACK.16
Gaussian absorption correction was applied to the collected
reflections with PLATON,17 with maximum and minimum
transmission factors of 0.496 and 0.278, respectively. The
structure was solved by direct methods and refined by full-
matrix least-squares on F2.18 All non-hydrogen atoms were
refined with anisotropic displacement parameters. Hydrogen
atoms bond to C atoms were placed at their idealized positions
using standard geometric criteria. H atoms of the coordinated
and uncoordinated water molecules could not be located from
the Fourier difference map. Further crystallographic informa-
tion is presented in Table 1.
7.63-7.64 (2H, m, CHAr); 8.54-8.56 (2H, m, CHAr) in ppm. 13
C
Full tables containing the crystallographic data (except struc-
ture factors) were deposited at Cambridge Structural Database
(CCDC 752704) and these data are available free of charge at
www.ccdc.cam.ac.uk.
NMR δ (100 MHz; CDCl3): 20.69; 51.84; 53.36; 54.44; 55.83;
59.48; 122.42; 123.23; 123.49; 127.99; 130.69; 130.83; 136.97;
149.10; 153.86; 158.44; 171.97 in ppm.
Preparation of 2-[N-bis-(2-pyridylmethyl)aminomethyl]-4-me-
thyl-6-[N-bis(2-hydroxy-2-oxoethyl)aminomethyl]phenol (H3L).
Potentiometric Titrations. The potentiometric studies were
carried out with a Corning-350 research pH meter fitted with
blue-glass and Ag/AgCl reference electrodes calibrated to read
(12) Bunton, C. A.; Farber, S. J. J. Org. Chem. 1969, 34, 767–772.
(13) Karsten, P.; Neves, A.; Bortoluzzi, A. J.; Lanznaster, M.; Drago, V.
Inorg. Chem. 2002, 41, 4624–4626.
(14) Lambert, E; Chabut, B.; Chardon-Noblat, S; Deronzier, A; Chot-
tard, G.; Bousseksou, A; Tuchagues, J.-P.; Laugier, J.; Bardet, M.; Latour,
J.-M. J. Am. Chem. Soc. 1997, 119, 9424–9437.
(15) COLLECT; Enraf-Nonius BV: Delft, The Netherlands, 2000.
(16) Otwinowski, Z.; , Minor, W. In Methods Enzymol.; Carter, C. W., Jr.,
Sweet, R.M., Eds.; Academic Press: New York, 1997; Vol. 2, p 307.
(17) Spek, A. L. Acta Crystallogr. 2009, D65, 148–155.
(18) Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112–122.

Article
Inorganic Chemistry, Vol. 49, No. 6, 2010 3059
Table 1. Crystallographic Data and Structure Refinement for Complex 1
dependence of the reaction rate on the substrate concentration
(5 ꢀ 10-4 to 5 ꢀ 10-3 mol L-1) were carried out at 25 °C, pH 6.0,
with complex 1 (4 ꢀ 10-5 mol L-1). Because of the lower
solubility of the reagents, these were the best concentration
conditions for obtaining the kinetic parameters. To establish the
number of molecules of substrate which are hydrolyzed per
molecule of complex, the reaction was monitored at 445 nm,
under a 50-fold substrate excess at pH 6.0 and 25 °C.
empirical formula
formula weight
temperature
C50H74Gd3N11O31
1796.95
296(2) K
0.71073 A
orthorhombic
˚
wavelength
crystal system
space group
unit cell dimensions
Pbca
a = 27.0964(7) A
˚
R = 90°.
β = 90°.
γ = 90°.
˚
b = 17.7944(5) A
DNA Cleavage. The plasmid pBSK II (2961 bp), used for the
DNA cleavage assays, was purchased from Stratagene, trans-
formed into DH5R Escherichia coli competent cells and amplified
as previously described.21 The plasmid DNA was extracted from
E. coli and purified using the Qiagen Plasmid Maxi Kit proto-
col.22 The DNA cleavage activity of 1 was determined by follow-
ing the conversion of supercoiled plasmid DNA (FI) to open
circular DNA (FII) and/or linear DNA (FIII) using agarose gel
electrophoresis to separate the cleavage products. In general, 520
ng of pBSK II (40 μM in pb) in 25 mM PIPES buffer (pH 6.5, 7.0
and 7.5) or HEPES buffer pH 8.0 were treated with different
concentrations of 1 (10-125 μM) in CH3CN (25% in reaction
volume) for 7 h at50 °C (complex 1 displayedlow activity at25°C
and, consequently, the kinetic parameters could not be obtained
at this temperature). Thereafter, each reaction was quenched
adding 5 μL of a loading buffer solution (0.01% bromophenol
blue, 50% glycerol, and 250 mM EDTA pH 8.0) and then
subjected to electrophoresis on a 0.8% agarose gel containing
0.3 μg.mL-1 of ethidium bromide in 0.5 ꢀ TBE buffer (44.5 mM
Tris, 44.5 mM boric acid, and 1 mM EDTA) at 90 V for
approximately 1.5 h. The resulting gels were visualized and
digitized with a photo documentation system (UVP Inc., Upland,
CA, U.S.A.). The ratio of DNA in each band was quantified
using LabWorks software version 4.0 (UVP). The ratio of super-
coiled DNA was increased by a factor of 1.47, since the ability of
ethidium bromide to intercalate into this topoisomeric form is
lower relative to circular and linear forms.23 To study the
involvement of hydroxyl radicals in the mechanism of the DNA
cleavage performed by 1, 10% DMSO was added to the reaction
mixturesprior toadditionofthe complex. Todetermine the DNA
major or minor groove selectivity of 1, assays were performed
using the minor groove binder distamycin (50 μM), or the major
groove binder methyl green (50 μM). The plasmid DNA was
pretreated with the groove binder for 30 min at room tempera-
ture, and then the complex was added and the reaction was
˚
c = 27.4867(5) A
3
˚
13253.1(6) A
volume
Z
density (calculated)
absorption coefficient
F(000)
8
1.801 Mg/m3
3.063 mm-1
7128
crystal size
θ range for data collection 2.61 to 26.00°.
0.47 ꢀ 0.44 ꢀ 0.26 mm3
index ranges
-17 e h e 33
-21 e k e 10
-33 e l e 27
38608
reflections collected
independent reflections
refinement method
12995 [R(int) = 0.0592]
full-matrix least-squares on F2
data/restraints/parameters 12995/0/856
goodness-of-fit on F2
1.091
final R indices [I > 2σ(I)] R1 = 0.0445, wR2 = 0.1238
R indices (all data)
largest diff. peak and hole 1.897 and -2.432 e A
R1 = 0.0615, wR2 = 0.1353
˚
-3
-log [Hþ] directly. The electrode was calibrated using the data
obtained from the potentiometric titration of a known volume
of a standard 0.10 mol L-1 HCl solution with a standard 0.10
mol L-1 KOH solution. The ionic strength of the HCl solution
was maintained at 0.10 mol L-1 by addition of KCl. Measure-
ments were carried out in a thermostatted cell containing a
solution of the complex (0.05 mol/50 mL) with ionic strength
adjusted to 0.10 mol L-1 by addition of KCl, at 25.00 ( 0.05 °C.
The experiments were performed under argon to eliminate the
presence of atmospheric CO2. All of the experimental solutions
were prepared in acetonitrile/water (1:1, v/v) owing to the low
solubility of the complex in water. The pKw of the acetonitrile/
water containing 0.10 mol L-1 of KCl used was 15.40.19 Compu-
tations were carried out with the BEST7 program, and species
diagrams were obtained with SPE and SPEPLOT programs.20
Reactivity. Kinetic experiments for the hydrolysis of the
model substrate bis(2,4-dinitrophenyl)phosphate (BDNPP)
were followed spectrophotometrically for the absorbance in-
crease at 400 nm (ε = 12100 L mol-1 cm-1) because of the
formation of 2,4-dinitrophenolate (DNP) over time, under
conditions of excess substrate at 25 °C. In these experiments,
all of the solution mixtures were in an acetonitrile/water (3:1 v/v)
medium, owing to the low solubility of the complex in water
under the concentration conditions used. The systems showed
high catalytic activity, and their study was only possible through
the stopped-flow technique. The stopped-flow apparatus
(model SX-18MV) and the associated computer system were
manufactured by Applied Photophysics. The data were ana-
lyzed with the SX-18MV operating software. A total of 150 μL
per sample was transferred to a 1 cm thermostatically controlled
cell compartment. The experiments were carried out in tripli-
cate. Reactions were monitored during the first seconds, and the
data treated using the initial rate method. Initial rates were
obtained directly from the plot of the DNP concentration versus
time under the same experimental conditions. Studies on the
effects of pH on the hydrolysis reaction were performed in the
pH range of 3.5-10.0 (MES pH 3.5-6.5; HEPES pH 7.0-8.0;
CHES pH 9.0-10.0; I = 0.1 mol L-1 with LiClO4), under a 50-
fold excess of substrate, at 25 °C. Experiments to determine the
incubated as described above. The DNA cleavage rates (kobs
)
promoted by 1 were determined treating the plasmid DNA at
different complex concentrations (10-250 μM) for different time
intervals (0-6 h). The kobs were calculated for each complex
concentration assuming a pseudo-first-order kinetics and then
analyzed following the pseudo-Michaelis-Menten formalism.24
The reaction conditions were as described above.
Results and Discussion
Syntheses. The synthetic route used for the preparation
of the unsymmetrical ligand H3L is shown in Scheme 1.
The nucleophilic substitution of the chloride 213,14 with
dimethyl iminodiacetate (a) results in the intermediate 3,
and its consequent basic hydrolysis and purification with
basic DOWEX eluted in acetic acid (b) results in the
ligand H3L, in good yields.
(21) Ausubel, F. M.; Brent, R.; Kingston, R. E.; Moore, D. D.; Seidman,
J. G.; Smith, J. A.; Struhl, K. In Short Protocols in Molecular Biology: A
Compendium of Methods from Current Protocols in Molecular Biology; Wiley:
New York, 1999.
(22) HiSpeed Plasmid Purification Handbook; Qiagen: Hilden, Germany,
2001; p 46.
(23) Jin, Y.; Cowan, J. A. J. Am. Chem. Soc. 2005, 127, 8408–8415.
(24) Sreedhara, A.; Freed, J. D.; Cowan, J. A. J. Am. Chem. Soc. 2000,
122, 8814–8824.
ꢀ
(19) Herrador, M. A.; Gonzalez, A. G. Talanta 2002, 56, 769–775.
(20) Martell, A.; Motekaitis, R. J. Determination and Use of Stability
Constants, 2nd ed.; VCH: New York, 1992.

3060 Inorganic Chemistry, Vol. 49, No. 6, 2010
Camargo et al.
Figure 1. Molecular structure of the cation [Gd3(C50H58N10O20)]þ (right) and ORTEP plot of the coordination environment around the metal centers in
complex 1 (left, other atoms are omitted for clarity). Ellipsoid at the 40% probability level.
Scheme 1. Synthetic Route Used for the Preparation of the Unsymmetrical Ligand H3L
Two H3L ligands are able to coordinate three GdIII ions
held together by phenolate and carboxylate pendant arms
forming bridges. Infrared spectroscopy was essential for the
preliminary characterization of the ligand and the complex.
nitrogen atoms N121 and N111 (or N211 and N221), one
phenolate oxygen atom O131 (or O261), and one carboxy-
late oxygen atom O231 (or O181), in addition to the two
oxygen atoms of the bidentate nitrate O141 and O143 (or
O241 and O243) and two water molecules coordinated to
a terminal metal. The central metal, Gd3, is octacoordi-
nated to two tertiary amine nitrogen atoms N12 and N22,
two phenolate oxygen atoms O231 and O131, and four
carboxylate oxygen atoms O281, O261, O181, and O161.
Since the two terminal metal ions, Gd1 and Gd2, show
the same coordination environment, we can observe
similar lengths and bond angles. For example, the bond
The [Gd3L2(NO3)2(H2O)4]NO3 8H2O complex (1) shows
3
stretching frequencies typical of bidentate nitrates (1482 and
1295 cm-1) and stretching bands of free (non-coordinated)
anionic nitrate (1386 cm-1), which were assigned by the
difference between the spectra of complex 1 and H3L.
X-ray Structure Characterization. Complex 1 crystal-
lizes in an orthorhombic cell, space group Pcab. An Oak
Ridge thermal-ellipsoid plot (ORTEP)25 view of the cation
complex is presented in Figure 1. The crystallographic data
and the main bond distances/angles are given in Tables 1
and 2, respectively. The resolution of the crystal structure of
1 shows an asymmetric unit composed of a cation complex
[Gd3L2(NO3)2(H2O)4]þ and a nitrate anion as the counter-
ion with eight water molecules as the crystallization solvent.
Figure 1 shows the structure of the monocationic tri-
nuclear complex which is composed of three GdIII ions,
two L3- ligands (with phenolate and carboxylate pendant
arms forming bridges), four coordinated water molecules
and two coordinated bidentate nitrates. The two terminal
metal ions, Gd1 and Gd2, show the same coordination
environment, where each ion is nona-coordinated to one
tertiary amine nitrogen atom N11 (or N21), two pyridine
˚
distance Gd1-N11 (tertiary amine) is 2.653 A, which is
˚
similar to that of Gd2-N21 (2.651 A) and the angle
N121-Gd1-N111 is 129.30°, slightly greater than the
corresponding angle N221-Gd2-N211 (128.21°).
The central metal Gd3 is coordinated to the four car-
boxylate groups from the two ligands L3-, and it is possi-
ble to observe the differences between the average bond
˚
lengths: 2.419 A for Gd3-O(monodentate carboxylate)
˚
and 2.408 A for Gd3-O(carboxylate bridge).
The separations Gd1 Gd3 and Gd3 Gd2 (4.0482
3 3 3
3 3 3
˚
A and 4.0643 A, respectively) are similar to the Gd Gd
˚
3 3 3
separations found in the homotrinuclear complexes with
amine phenolate, multidentate ligands.26
(26) Setyawati, I. A.; Liu, S.; Rettig, S. J.; Orvig, C. Inorg. Chem. 2000, 39,
496–507.
(25) Spek, A. L. J. Appl. Crystallogr. 2003, 36, 7–13.

Article
Inorganic Chemistry, Vol. 49, No. 6, 2010 3061
˚
Table 2. Selected Bond Lengths [A] and Angles [deg] for Complex 1
Table 3. Logarithm K Values for Protonations of the Complex and Dissociation
of Coordinated Water Molecules, I = 0.1 mol L-1 (KCl)
Gd1-O131
Gd1-O12W
Gd1-O11W
Gd1-O143
Gd1-O141
Gd1-N121
Gd1-N111
Gd1-O261
Gd1-N11
Gd1-Gd3
Gd2-O231
Gd2-O22W
Gd3-O261
Gd3-O181
2.389(3)
2.404(4)
2.446(4)
2.489(4)
2.529(4)
2.539(5)
2.608(5)
2.645(3)
2.653(5)
4.0643(4)
2.401(3)
2.404(4)
2.417(3)
2.422(3)
Gd2-O21W
Gd2-O243
Gd2-O241
Gd2-N221
Gd2-N211
Gd2-N21
Gd2-O181
Gd2-Gd3
Gd3-O231
Gd3-O131
Gd3-O281
Gd3-O161
Gd3-N12
Gd3-N22
2.416(4)
2.444(4)
2.550(4)
2.551(5)
2.594(5)
2.655(4)
2.670(3)
4.0481(4)
2.377(3)
2.380(3)
2.406(4)
2.409(3)
2.536(4)
2.541(4)
equilibria
log K
[Gd3HL24þ]/[Gd3L23þ] [Hþ]
4.78
4.09
-5.61
-7.21
-9.31
-9.36
[Gd3H2L25þ]/[Gd3HL24þ] [Hþ]
[Gd3(OH)L22þ] [Hþ]/[Gd3L2
]
3þ
[Gd3(OH)2L2þ] [Hþ]/[Gd3(OH)L2
]
2þ
[Gd3(OH)3L2] [Hþ]/[Gd3(OH)2L2
]
þ
[Gd3(OH)4L2-] [Hþ]/[Gd3(OH)3L2]
O131-Gd1-O12W
O131-Gd1-O11W
140.92(13) O131-Gd1-O12W
85.52(13)
140.92(13)
85.52(13)
O131-Gd1-O11W
O12W-Gd1-O11W 82.33(14)
O12W-Gd1-O11W 82.33(14)
O131-Gd1-O143
O12W-Gd1-O143
O11W-Gd1-O143
O131-Gd1-O141
O12W-Gd1-O141
O11W-Gd1-O141
O143-Gd1-O141
O131-Gd1-N121
O12W-Gd1-N121
O11W-Gd1-N121
O143-Gd1-N121
O141-Gd1-N121
O131-Gd1-N111
O12W-Gd1-N111
O11W-Gd1-N111
O143-Gd1-N111
O141-Gd1-N111
N121-Gd1-N111
O131-Gd1-O261
O12W-Gd1-O261
O21W-Gd2-O241
O243-Gd2-O241
O231-Gd2-N221
O22W-Gd2-N221
O21W-Gd2-N221
O243-Gd2-N221
O241-Gd2-N221
O231-Gd2-N211
O22W-Gd2-N211
O21W-Gd2-N211
O243-Gd2-N211
O241-Gd2-N211
N221-Gd2-N211
O231-Gd2-N21
O22W-Gd2-N21
O21W-Gd2-N21
O243-Gd2-N21
O241-Gd2-N21
N221-Gd2-N21
N211-Gd2-N21
O231-Gd2-O181
O22W-Gd2-O181
O21W-Gd2-O181
O243-Gd2-O181
O131-Gd3-N22
O281-Gd3-N22
O161-Gd3-N22
143.11(13) O131-Gd1-O143
74.53(14)
143.11(13)
O12W-Gd1-O143
74.53(14)
115.88(14)
135.81(13)
70.07(14)
65.51(13)
50.45(13)
75.68(13)
139.08(15)
84.34(14)
77.00(15)
69.18(14)
98.95(13)
73.89(15)
147.36(15)
79.20(15)
123.66(14)
128.19(16)
65.62(11)
75.31(12)
69.60(14)
51.19(12)
75.15(13)
81.39(14)
140.00(14)
79.03(14)
70.41(14)
96.67(13)
148.76(14)
74.50(15)
78.62(14)
123.50(14)
129.33(15)
75.74(13)
145.46(14)
128.28(14)
68.33(13)
109.88(13)
66.14(14)
63.41(15)
66.43(11)
69.99(12)
74.07(12)
147.22(12)
69.31(12)
131.84(12)
123.08(13)
115.88(14) O11W-Gd1-O143
135.81(13) O131-Gd1-O141
70.07(14)
65.51(13)
50.45(13)
75.68(13)
O12W-Gd1-O141
O11W-Gd1-O141
O143-Gd1-O141
O131-Gd1-N121
139.08(15) O12W-Gd1-N121
84.34(14)
77.00(15)
69.18(14)
98.95(13)
73.89(15)
O11W-Gd1-N121
O143-Gd1-N121
O141-Gd1-N121
O131-Gd1-N111
O12W-Gd1-N111
Figure 2. Solid lines represent the species distribution curves of the
3þ
Gd3L2 system for the dissolution of 0.015 mmol of complex 1 in
acetonitrile/water solution, μ = 0.1 mol L-1 (KCl) at 25 °C. The dashed
line corresponds to the variation in the observed initial rates for the
hydrolysis of BDNPP as a function of pH in acetonitrile/water solution.
Conditions: [1] =4.0ꢀ 10-5 mol L-1; [BDNPP] = 2 ꢀ 10-3 mol L-1; at 25
147.36(15) O11W-Gd1-N111
79.20(15) O143-Gd1-N111
123.66(14) O141-Gd1-N111
128.19(16) N121-Gd1-N111
°C. (A) [Gd3H2L2]5þ;(B) [Gd3HL2]4þ;(C) [Gd3L2]3þ;(D) [Gd3(OH)L2]2þ
;
(E) [Gd3(OH)2L2]þ; (F) [Gd3(OH)3L2]; (G) [Gd3(OH)4L2]-.
65.62(11)
75.31(12)
69.60(14)
51.19(12)
75.15(13)
81.39(14)
O131-Gd1-O261
O12W-Gd1-O261
O21W-Gd2-O241
O243-Gd2-O241
O231-Gd2-N221
O22W-Gd2-N221
equilibrium constants were determined (Table 3) using
the BEST7 program,20 and the values obtained were used
to calculate the species distribution curves (Figure 2) and,
consequently, the species involved in catalysis.
140.00(14) O21W-Gd2-N221
Through the potentiometric results, it is possible to observe
protonations of the complex at low pH values, forming pro-
tonated [Gd3HL2]4þ and [Gd3H2L2]5þ species. At pH > 5
dissociation of coordinated water molecules is observed,
forming the corresponding hydroxide species [Gd3(OH)-
L2]2þ,[Gd3(OH)2L2]þ, [Gd3(OH)3L2],and[Gd3(OH)4L2]-.
Reactivity Studies. The catalytic activity of complex 1
in the hydrolysis of the activated aryl diester phosphate
BDNPP was investigated under conditions of excess
substrate, at 25 °C, by following spectrophotometrically
the absorbance increase at 400 nm until the conversion of
around 1 equiv of the liberated 2,4-dinitrophenolate
anion (DNP), using the stopped-flow technique.
The pH dependence of the catalytic activity for BDNPP
hydrolysis was investigated in the pH range 3.50-11.00,
activity being observed across the whole pH range stu-
died, and a bell-shaped profile was observed with a
maximum efficiency at around pH 6. The pH:V0 profile
for complex 1 could be better analyzed when associated
with the distribution curves of the species formed in
solution (Figure 2), in which the species involved in the
catalysis are proposed.
79.03(14)
70.41(14)
96.67(13)
148.76(14) O22W-Gd2-N211
74.50(15)
78.62(14)
O243-Gd2-N221
O241-Gd2-N221
O231-Gd2-N211
O21W-Gd2-N211
O243-Gd2-N211
123.50(14) O241-Gd2-N211
129.33(15) N221-Gd2-N211
75.74(13)
O231-Gd2-N21
145.46(14) O22W-Gd2-N21
128.28(14) O21W-Gd2-N21
68.33(13)
109.88(13) O241-Gd2-N21
O243-Gd2-N21
66.14(14)
63.41(15)
66.43(11)
69.99(12)
74.07(12)
N221-Gd2-N21
N211-Gd2-N21
O231-Gd2-O181
O22W-Gd2-O181
O21W-Gd2-O181
147.22(12) O243-Gd2-O181
138.07(12) O261-Gd3-N22
65.58(13)
75.45(13)
O181-Gd3-N22
N12-Gd3-N22
Potentiometric Equilibrium Studies. To determine the
possible species present in solution, potentiometric titra-
tion experiments of complex 1 were carried out and
equilibria involving protonations of the complex and
dissociation of coordinated water molecules were pro-
posed (Supporting Information, Figure S1). The results
obtained for 1 show the neutralization of 6 mol KOH per
mol of complex in the pH range 3.50-11.00. Thus, the
The results in Figure 2 show that at low pH values
protonated [Gd3H2L2]5þ (A) and [Gd3HL2]4þ (B) species
are formed, with no dissociated water molecule and,
consequently, no nucleophile to attack the diester bond,
which may explain their low catalytic activities. In the pH

3062 Inorganic Chemistry, Vol. 49, No. 6, 2010
Camargo et al.
range of around 5.0 to 7.5, higher activity toward BDNPP
hydrolysis is observed, where the following equilibria and
constants were determined.
2þ
½Gd3ðLÞ ꢁ3þ h ½Gd3ðOHÞðLÞ ꢁ þ Hþ
pK1 ¼ 5:61
2
2
2þ
½Gd3ðOHÞðLÞ2ꢁ h ½Gd3ðOHÞ2ðLÞ2ꢁþ þ Hþ
pK2 ¼ 7:21
These pKa values are in reasonable agreement with those
obtained from the sigmoidal fit of the curve of pH versus V0
(superimposed curve in Figure 2). These results suggest that
the deprotonation of one coordinated water molecule to a
metal center in the [Gd3L2]3þ (C) compound produces the
most prominent catalyst in the hydrolysis of BDNPP, the
[Gd3(OH)L2]2þ (D) species. Finally, the decrease in reacti-
vity at pH > 6.5 most probably arises because of the
disappearance of the most active species (D), and the for-
mation of the hydroxide species [Gd3(OH)2L2]þ (E), [Gd3-
(OH)3L2] (F), and [Gd3(OH)4L2]- (G). The increase in the
negative charges around these species (E-G) may inhibit
the nucleophilic attack on the BDNPP substrate (negatively
charged) or even lower the binding affinity of the complexes
to the substrate, resulting in their low catalytic activity.
Thus, complete kinetic studies were performed at the opti-
mum pH value of 6.0 at which it is believed that the tri-
nuclear [Gd3(OH)L2]2þ (D) species is the principal species
present in solution. The determination of the initial rates as a
function of the concentration of the substrate reveals satura-
tion kinetics with Michaelis-Menten-like behavior27 (Sup-
porting Information, Figure S2). The non-linear least-
squares fit of V0 versus [BDNPP] gives the following values:
Km=4.09 mM, Vmax=2.68 ꢀ 10-2 mM s-1, and the cataly-
tic constant kcat=Vmax/[1]=0.67 s-1. Under these condi-
tions, complex 1 shows a 5 million-fold acceleration in the
hydrolysis rate of BDNPP in comparison to the spontane-
ous hydrolysis.12 In addition, the catalytic activity of 1 in the
hydrolysis is around 4 ꢀ 103 times faster than the reaction
Figure 3. Plasmid DNA cleavage promoted by complex 1 at pH 6.5, 7.0,
7.5, and 8.0 (A, B, C, and D, respectively), at 50 °C and incubation for 7 h.
Lane 1: pBSK II DNA (40 μM bp). Lanes 2-7: DNA þ complex 1 10, 20,
30, 60, 90, and 125 μM, respectively. Reactions were performed in 25 mM
PIPES buffer (pH 6.5, 7.0 and 7.5) or HEPES buffer pH 8.0 in CH3CN:
H2O 25:75%.
and the BDNPP substrate was monitored. It was observed
that 2 equiv of DNP are released in 2 h, at 25 °C, which
indicates the hydrolysis of both the mono- and the diester.
As our main goal was to obtain an effective catalyst, a
hydrolysis reaction of BDNPP (2 ꢀ 10-3 M) promoted by
complex 1 (4 ꢀ 10-5 M) at 445 nm, pH 6, and 25 °C was
also monitored. These data revealed that the complex is
able to hydrolyze 6 molecules of substrate in 1 h and can
be considered as an efficient catalyst.
DNA Interaction. The influence of complex concentra-
tion and pH on the nucleic acid cleavage activity were
investigated at pH 6.5, 7.0, 7.5, and 8.0 with selected com-
plex 1 concentrations (0-125 μM), at 50 °C (Figure 3).
These results show that complex 1 effectively promotes the
cleavage of supercoiled (form I) DNA to nicked circular
(form II) and linear (form III) DNA with higher activity at
pH 7, a value which is one pH unit higher than that found in
the hydrolysis of the BDNPP substrate. However, the
species suggested as the most catalytic [Gd3(OH)L2]2þ
(D) is still the principal species present in solution at around
this pH range (Figure 2).
Thus, kinetic studies were performed at pH 7, under con-
ditions of excess complex over substrate (DNA), at 50 °C
(Supporting Information, Figure S3), and the following
kinetic parameters were obtained: k0cat = 3.42 h-1, Km =
4.57 ꢀ 10-4 M and k0cat/Km=7.48 ꢀ 103 M-1 h-1, provid-
ing a 100 million-fold rate enhancement over the uncatalyzed
DNA cleavage.30 Furthermore, complex 1 shows a higher
catalytic constant in comparison to the value of kcat=0.47 h-1
observed for the binuclear gadolinium species previously
reported by us,28 indicating different mechanisms of action
for the complexes with regard to DNA cleavage.
To investigate the mechanism of action of complex 1
toward DNA cleavage, experiments were performed in
the presence of dimethyl sulfoxide, a hydroxyl radical
scavenger. The cleavage activity of 1 was not affected by
the presence of DMSO (Supporting Information, Figure
S4), suggesting that diffusible HO• radicals are not in-
volved in the cleavage of plasmid DNA. These results are
in agreement with the known hydrolytic action of lantha-
nide ions toward the cleavage of phosphate esters.31,32
catalyzed by Gd(NO3)3 6H2O, under the same experimen-
3
tal conditions (Supporting Information, Table T1).
In a recent publication,28 we reported a dinuclear gado-
linium species (in solution) with a high efficiency in the
hydrolysis of BDNPP, which is around 25 times faster than
that of complex 1, indicating that the trinuclear gadolinium
complex probably displays a different action mechanism
toward BDNPP hydrolysis compared to the dinuclear
species. However, further studies are necessary to identify
the mechanism involved when complex 1 is the catalyst. On
the other hand, complex 1 has a catalytic constant around
20 times higher than the value observed for the hydrolysis
reaction of the same substrate using different LnIII com-
plexes with bis-tris propane (BTP) as the catalyst.29
To assess the possible hydrolysis of the monoester 2,4-
dinitrophenylphosphate (DNPP), one of the products
formed from the hydrolysis reaction of the diester
BDNPP, the stoichiometric reaction between complex 1
´
(27) Stryer, L. Bioquımica. 4nd ed.; Guanabara Koogan; Rio de Janeiro,
Brazil, 1996; pp 180-185.
(28) Camargo, M. A.; Neves, A.; Bortoluzzi, A. J.; Szpoganicz, B.;
Martendal, A.; Murgu, M.; Fischer, F. L.; Terenzi, H.; Severino, P. C.
Inorg. Chem. 2008, 47, 2919–2921.
(30) Radzicka, A.; Wolfenden, R. Science 1995, 267, 90–93.
(31) Kulkarni, A.; Patil, S. A.; Badami, P. S. Eur. J. Med. Chem. 2009, 44,
2904–2912.
(29) Longhinotti, E.; Domingos, J. B.; da Silva, P. L. F.; Szpoganicz, B.;
Nome, F. J. Phys. Org. Chem. 2005, 18, 167–172.
(32) Baykal, U.; Akkaya, E. U. Tetrahedron Lett. 1998, 39, 5861–5864.

Article
Inorganic Chemistry, Vol. 49, No. 6, 2010 3063
(H2O)4]NO3 8H2O (1) with the new unsymmetrical li-
3
gand H3L. Complex 1 shows efficient hydrolase-like
activity toward the BDNPP substrate and DNA, with
regio-specificity in the DNA binding, indicating its po-
tential action as a chemical nuclease. Finally, the syn-
thetic routes described for the preparation of the ligand
H3L and the trinuclear Gd complex can be used as general
methods for the preparation of trinuclear lanthanide
complexes with H3L. The synthesis of the corresponding
trinuclear TbIII and EuIII complexes, along with kinetic
and luminescent studies, is in progress to help elucidate
the mechanism involved in the catalytic cycle and un-
ambiguously identify the active species. Furthermore,
attempts to synthesize heterotrinuclear lanthanide com-
plexes will also be the subject of future research to exploit
their unique luminescent properties.
Acknowledgment. Financial support was received
ꢀ
Figure 4. Analysis of plasmid DNA cleavage (pBSK-II, 40 μM bp)
catalyzed by complex 1, in the absence and presence of distamycin and
methyl green (50 μM), after incubation for 6 h at 50 °C, at pH 7.0 (PIPES
buffer 25 mM).
from CNPq, INCT-catalise, INCT-Biologia Estrutural
e Bioimagem, FAPESC, and M.A.C. is grateful to CNPq
for a postdoc grant (PNPD-151665/2008-7). Prof. Dr.
^
Antonio C. Joussef and Sandro L. Mireski kindly helped
To obtain a greater understanding of the specific con-
tact surfaces between the catalytic species and the DNA
helix, studies were carried out with distamycin, a specific
minor groove binder and methyl green, a major groove
binder. The catalytic activity of complex 1 appears not to
be partially influenced by the presence of distamycin
(Figure 4), and not at all by methyl green, suggesting its
specificity for the minor groove of DNA.
with the organic purification methods and Prof. Dr.
Marcos N. Eberlin and Vanessa G. Santos with the ESI
analyses.
Supporting Information Available: X-ray crystallographic file
in CIF format; Figures S1-S4 and Table T1 in PDF format.
This material is available free of charge via the Internet at http://
pubs.acs.org. Full tables of crystallographic data of complex 1
(except structure factors) were deposited at Cambridge Struc-
tural Database (CCDC 752704), and these data are available
free of charge from: CCDC, 12 Union Road, Cambridge, CB2
1EZ, UK (Fax: þ44-1223-336-003; e-mail: deposit@ccdc.
cam.ac.uk or http://www.ccdc.cam.ac.uk.
Conclusions
In summary, we have synthesized and characterized
the new trinuclear gadolinium complex [Gd3L2(NO3)2-