Synthesis, crystal structure and superoxide dismutase activity of a five-co-ordinated manganese(II) complex

Article abstract of DOI:10.1039/a707433f

A five-co-ordinate manganese(II) complex [Mn(ntb)(Hsal)][ClO₄], where ntb is tris(benzimidazol-2-ylmethyl)-amine and Hsal^- is the anion of salicyclic acid (H₂sal), has been prepared and characterized by X-ray crystallography. The manganese adopts a distorted trigonal-bipyramidal co-ordination geometry with a N₄O ligand donor set and bears structural similarities to the active site of manganese superoxide dismutase. The electrochemical properties of the complex were studied in MeCN by cyclic voltammetry. The complex can catalyse the dismutation of superoxide (O₂^-) effectively in the riboflavin-methionine-nitro blue tetrazolium assay.

Full text of DOI:10.1039/a707433f

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Synthesis, crystal structure and superoxide dismutase activity of a
five-co-ordinated manganese(II) complex
Dao Feng Xiang, Chun Ying Duan, Xiang Shi Tan, Qin Wei Hang and Wen Xia Tang*
Coordination Chemistry State Key Laboratory, Coordination Chemistry Institute,
Nanjing University, Nanjing 210093, P.R. China
A five-co-ordinate manganese() complex [Mn(ntb)(Hsal)][ClO4], where ntb is tris(benzimidazol-2-ylmethyl)-
amine and HsalϪ is the anion of salicyclic acid (H2sal), has been prepared and characterized by X-ray crystal-
lography. The manganese adopts a distorted trigonal-bipyramidal co-ordination geometry with a N4O ligand
donor set and bears structural similarities to the active site of manganese superoxide dismutase. The electro-
chemical properties of the complex were studied in MeCN by cyclic voltammetry. The complex can catalyse the
dismutation of superoxide (O2Ϫ) effectively in the riboflavin–methionine–nitro blue tetrazolium assay.
The superoxide radical anion, a product of cellular respiration,
activated polymorphonuclear leukocytes and endothelial cells,
has been demonstrated to be a mediator of ischemia, reper-
fusion injury, inflammatory and vascular diseases.1 The main
lines of defence in mammalian organisms for controlling extra-
and intra-cellular superoxide radical anions are the Cu/Zn-,
Mn- and Fe-containing superoxide dismutase enzymes.2 Super-
oxide dismutase (SOD) catalyses the dismutation of superoxide
radical anions to the non-radical products oxygen and hydro-
gen peroxide3 [equation (1)] and protects living cells against the
Sigma Co. and used as received. Riboflavin was analysed by the
literature method.18 The salts KH2PO4 (A.R.) and K2HPO4
(A.R.) were recrystallized twice from deionized water. All other
chemicals were reagent grade used without further purification.
Preparations
Tris(benzimidazol-2-ylmethyl)amine (ntb) was synthesized
following a procedure reported previously19 with a yield of
53% and a m.p. of 270 ЊC.
[Mn(ntb)(Hsal)][ClO4]. To a stirring solution of ntb (204
mg, 0.5 mmol) in methanol (20 cm3) was added Mn(ClO4)2ؒ
6H2O (181 mg, 0.5 mmol) and then a solution of NaHsal (80
mg, 0.5 mmol) (H2sal = salicylic acid) in methanol (10 cm3).
The resulting clear solution was stirred for 3 h and then allowed
to stand at room temperature. Light yellow crystals suitable for
X-ray diffraction studies were obtained after 1 week (196 mg,
yield 56%) (Found: C, 53.34; H, 3.93; Mn, 7.91; N, 14.21. Calc.
for C31H26ClMnN7O7: C, 53.26; H, 3.75; Mn, 7.86; N, 14.03%).
CAUTION: although we experienced no difficulties with the
compounds isolated as their perchlorate salts, the unpredictable
behaviour of such salts necessitates extreme caution in their
handling.
Ϫ
᝽
ϩ
ؒ
O2 ϩ HO2 ϩ H → O2 ϩ H2O2
(1)
toxicity of hyperoxia. The application of superoxide dismutase
as a pharmaceutical has attracted considerable attention.4–6
However, SODs have molecular weights too high to cross cell
membranes7 and can only provide extracellular protection.8
In order to circumvent this difficulty, a stable non-toxic,
low-molecular-weight metal complex that catalyses the dis-
mutation of superoxide anion might be a suitable alternative
to superoxide dismutase in clinical applications9–11 with the
desirable qualities of low cost, cell permeability and non-
immunogenicity. In the past decades many Cu᎐Zn SOD model
compounds have been reported, while very little reported work
deals with iron SOD model compounds and the manganese
SOD model compound12–15 for which crystal structures have
been determined. The crystal structure of the native (oxidized
form) manganese SOD, reveals that the manganese adopts a
trigonal-bipyramidal co-ordination geometry with N3O2
ligand-donor sets,16 the three nitrogens being provided by three
histidine residues in the protein. Among reported structur-
ally characterized manganese complexes which exhibit SOD
activity, only [Mn(OCH2Ph)(N2C3H2Pri2-3,5){HB(N2C3HPri2-
3,5)3}] possesses trigonal-bipyramidal geometry.14 In our efforts
to search for manganese SOD mimics, we succeeded in prepar-
ing and characterizing a new five-co-ordinate manganese()
complex which bears structural similarities to the active sites of
manganese SOD and found that it is effective for superoxide
dismutation.
Crystallography
Parameters for data collection and refinement are given in Table
1. The intensities were collected at 295 K on a Siemens P4
four-circle diffractometer with monochromated Mo-Kα (λ =
0.710 73 Å) radiation using the θ–2θ scan mode with a vari-
able scan speed 5.0–50.0Њ minϪ1 in ω. The data were corrected
for Lorentz-polarization effects during data reduction using
XSCANS.20
The structure was solved by direct methods and refined on F2
by full-matrix least-squares methods using SHELXTL.21 All
the non-hydrogen atoms were refined anisotropically. The
hydrogen atoms were placed in calculated positions (C᎐H 0.96,
N᎐H 0.90 and O᎐H 0.85 Å) assigned fixed isotropic thermal
parameters 1.2 times the equivalent isotropic U of the atoms to
which they are attached (1.5 times for the OH and methyl
groups) and allowed to ride on their respective parent atoms.
Their contributions were included in the structure-factor
calculations.
Experimental
Materials
All computations were carried out on a Pc-586 computer
using the SHELXTL PC package.21 Analytical expressions of
neutral-atom scattering factors were employed and anomalous
dispersion corrections were incorporated.22
Tetra-n-butylammonium perchlorate was twice recrystallized
from absolute ethanol and dried in vacuum at 40 ЊC prior to
use. Acetonitrile was purified as previously described.17 Nitro
blue tetrazolium and methionine (B.R.) were obtained from
CCDC reference number 186/865.
J. Chem. Soc., Dalton Trans., 1998, Pages 1201–1204
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Table 1 Crystal data and summary of data collection for the complex
[Mn(ntb)(Hsal)][ClO4]
Formula
Mr
C31H26ClMnN7O7
698.98
Colour/habit
Crystal dimensions/mm
Crystal system
Space group
a/Å
Pale yellow prism
0.40 × 0.40 × 0.30
Triclinic
P1
10.030(3)
¯
b/Å
c/Å
12.563(4)
14.189(7)
α/Њ
β/Њ
99.54(3)
96.96(3)
γ/Њ
112.46(2)
1595.6(10)
2
1.455
718
U/Å3
Z
D/Mg mϪ3
F(000)
µ/mmϪ1
Index ranges
Fig. 1 Molecular structure and atom numbering of the complex with
hydrogen atoms omitted for clarity. Thermal ellipsoids are drawn at the
30% probability level
0.556
0 р h р 10, Ϫ14 р k р 13,
Ϫ16 р l р 16
5763
Reflections collected
Independent reflections
Absorption correction
Data, restraints, parameters
R, RЈ (observed data) [I > 2σ(I )]
wϪ1
5391 (Rint = 0.0244)
Semiempirical from ψ scans
5391, 0, 433
0.0600, 0.1641
2
σ2(Fo ) ϩ (0.080P)2 ϩ 0.15P*
Goodness of fit on F 2
1.035
1.112, Ϫ0.618
ρmax, ρmin/e ÅϪ3
* P = (Fo2 ϩ 2Fc2)/3.
Table 2 Selected bond lengths (Å) and angles (Њ)
Mn᎐N(1)
Mn᎐N(2)
Mn᎐N(4)
2.508(4)
2.146(4)
2.176(4)
Mn᎐N(6)
Mn᎐O(1)
Mn ؒ ؒ ؒ O(2)
2.124(4)
2.035(3)
3.181(8)
N(1)᎐Mn᎐N(2)
N(2)᎐Mn᎐N(4)
N(2)᎐Mn᎐N(6)
N(1)᎐Mn᎐N(4)
N(1)᎐Mn᎐N(6)
73.76(13)
107.05(13)
104.02(14)
71.58(12)
73.04(13)
N(4)᎐Mn᎐N(6)
N(1)᎐Mn᎐O(1)
N(4)᎐Mn᎐O(1)
N(2)᎐Mn᎐O(1)
N(6)᎐Mn᎐O(1)
123.02(14)
173.46(13)
101.94(14)
109.7(2)
110.8(2)
Fig. 2 Packing of the unit cell of the complex
important hydrogen-bonding parameters in Table 3. The
molecular structure of the complex is shown in Fig. 1. The
crystal structure of the complex consists of discrete
[Mn(ntb)(Hsal)]ϩ cations and perchlorate. The MnII is five-co-
ordinate with a N4O ligand donor set. The ligand ntb acts as a
tetradentate N-donor and the carboxylate group of the salicyl-
ate is co-ordinated to the manganese unidentately. The co-
ordination geometry of the manganese may be best described
as distorted trigonal bipyramid. The trigonal plane is occupied
by the three ligating nitrogen atoms of the benzimidazolyl
groups. The Mn atom protrudes toward O(1) and is 0.639 Å out
of the plane. The angles N(4)᎐Mn᎐N(6), N(2)᎐Mn᎐N(4),
N(2)᎐Mn᎐N(6) are 123.02(14), 107.05(13) and 104.02(14)Њ,
respectively. The axial ligating atoms are N(1) and O(1) with
Mn᎐N(1) 2.508(4) Å, Mn᎐O(1) 2.035(3) Å and N(1)᎐Mn᎐O(1)
173.46(13)Њ. The N(1)᎐Mn᎐N(2) 73.76(13), N(1)᎐Mn᎐N(4)
71.58(12) and N(1)᎐Mn᎐N(6) 73.04(13)Њ angles appear essen-
tially imposed by the stereochemistry of the ligand ntb. The
distance between Mn and O(2) is 3.181(8) Å, i.e. O(2) is not
co-ordinated. The perchlorate anions and the solvent water
molecules are disordered. The distorted trigonal-bipyramidal
geometry of the complex is close to that known for the active
site of manganese SOD.16 However, the donor set and the oxid-
ation state of manganese are different. The manganese in the
complex has a N4O ligand donor set and is in the divalent state,
while that in native manganese SOD has a N3O2 ligand donor
set and is trivalent but can be reduced to the divalent state
without any loss of enzymatic activity.24
Other physical techniques
Elemental analyses were performed on a Perkin-Elmer 240C
analytical instrument. The room-temperature magnetic sus-
ceptibility data on powder samples were measured at 6000 G
(0.6 T) by using a CAHN-2000 Faraday-type magnetometer.
The apparatus was calibrated with [Ni(en)3][S2O3] (en = ethane-
1,2-diamine). Diamagnetic corrections were made using
Pascal’s constants. Cyclic voltammetry was performed on a
BAS-100B electrochemical analyzer, in conjunction with a
purged dinitrogen gas inlet and outlet, a glassy-carbon working
electrode, a Ag–AgCl reference electrode and a platinum
auxiliary electrode. All electrochemical experiments were
carried out under dry and purified dinitrogen atmosphere at
298 K. The solution was 1.5 × 10Ϫ3  in complex and 0.1  in
supporting electrolyte, NBun ClO4.
4
Assay of SOD activities
The SOD activities were measured according to the previously
reported method.23 The reduction of nitro blue tetrazolium was
monitored at 560 nm on a Shimadzu UV-240 spectro-
photometer. All photoinduced reactions were performed at
30 ЊC.
Results and Discussion
Crystal structure
Selected bond distances and angles are listed in Table 2,
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Table 3 Hydrogen-bonding parameters
X᎐H ؒ ؒ ؒ Y
X ؒ ؒ ؒ Y/Å
X᎐H᎐Y/Њ
N(3)᎐H(3) ؒ ؒ ؒ O(12A)
N(7)᎐H(7) ؒ ؒ ؒ O(14)
O(3)᎐H(3) ؒ ؒ ؒ O(2)
N(5)᎐H(5) ؒ ؒ ؒ O(2B)
2.939
2.963
2.597
2.838
145.7
147.6
146.4
154.6
Symmetry codes: A 2 Ϫ x, 2 Ϫ y, 1 Ϫ z; B 1 Ϫ x, 2 Ϫ y, Ϫz.
Fig. 4 The SOD activity of the complex in the riboflavin–methionine–
nitro blue tetrazolium assay
Ϫ
1O2᎐O2 and O2Ϫ᎐H2O2 because the reference electrodes and
solvents are different, the redox potential of the complex in
water solution should be in the allowed range of a SOD mimic
since the results of SOD activity assay obtained showed that it
has relatively high activity (see below).
Fig. 3 Cyclic voltammogram of a 1.5 × 10Ϫ3  solution of the complex
in MeCN (0.1  NBun ClO4) at a glassy-carbon electrode and Ag–AgCl
as a reference electrod4e. Sweep rate: (a) 50, (b) 100 and (c) 200 mV sϪ1
.
Superoxide dismutase assay
Temperature 25 ЊC
The superoxide dismutase activity of the complex was examined
indirectly using the nitro blue tetrazolium assay.23 Illumination
of reaction mixtures which contained 3.4 × 10Ϫ6  riboflavin,
0.01  methionine, 4.6 × 10Ϫ5  nitro blue tetrazolium, and
0.05  potassium phosphate at pH 7.8 and at 30 ЊC caused an
increase in absorbance at 560 nm of 0.091 minϪ1 in aerobic
solutions. When the complex was added the rate of increase
was reduced with increasing concentration (Fig. 4). The
complex shows an IC50 value of 0.70 µ which indicates that
it is a potent superoxide dismutase mimic. The IC50 value is
the concentration of the complex which exerts activity equal
to one unit of that of native SOD. The present value is one
of the lowest for a manganese superoxide dismutase mimic
and is close to the 0.75 µ reported by Kitajima et al.14 for a
manganese() benzoate tris(pyrazolyl)borate complex. It is
worth noting that the present complex and the benzoate tris-
(pyrazolyl)borate complex both have a N4 co-ordination sphere
and lower than Oh symmetry with azine and azol types of
ligands, and both possess a distorted trigonal-bipyramidal
geometry which is similar to the active site of native manganese
SOD. It is probable that the co-ordination of such nitrogen
bases and their particular geometry leads to high superoxide
dismutase activity.
There is significant intermolecular hydrogen-bonding associ-
ation. Two mononuclear molecules form a dimer through a
couple of hydrogen-bonding bridges [N(5)᎐H(5) ؒ ؒ ؒ O(2)].
Besides, each perchlorate is hydrogen bonded to N(7) of
the ligand ntb in one dimer and N(3) of ntb in another
[N(7)᎐H(7) ؒ ؒ ؒ O(14) and N(3)᎐H(3) ؒ ؒ ؒ O(12)], forming
a
bridge between two dimers (not shown in Fig. 2). These inter-
actions lead to a zigzag chain structure running parallel to the
crystallographic a axis. Additionally, the unidentate salicylate
possesses an intramolecular hydrogen bond [O(3)H(3) ؒ ؒ ؒ O(2)]
and this intramolecular hydrogen bond may be an important
factor in stabilizing the trigonal-bipyramidal geometry because
the carbonyl group of the carboxylate is hydrogen bonded to
the α-hydroxy group, thus preventing direct co-ordination of
O(2).
Magnetic properties and cyclic voltammetry
The effective magnetic moment of the complex at room tem-
perature is 5.88 µB (µB ≈ 9.27 × 10Ϫ24 J TϪ1) which is close to the
value expected for high-spin manganese().
The electrochemical properties of the complex have been
studied by cyclic voltammetry (CV) in dry and degassed MeCN.
The cyclic voltammogram is shown in Fig. 3. There is a steeply
sloping baseline due to the relatively large background current
which was not deducted. The anodic peak potential Epa does
not vary with scanning rate and the peak-to-peak separation
(∆Ep = Epa Ϫ Epc) of 60 mV indicates a reversible one-electron
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J. Chem. Soc., Dalton Trans., 1998, Pages 1201–1204