New conjugates of β-cyclodextrin with manganese(iii) salophen and porphyrin complexes as antioxidant systems

Article abstract of DOI:10.1039/c0dt01480j

Oxidative stress is the hallmark of several pathologies like arthritis, hypertension and many neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. In this scenario, antioxidant compounds can play a pivotal role in treating these severe pathologies. The synthesis of molecules able to mimic physiologically-relevant proteins is nowadays of particular interest. Several transition metal complexes, especially manganese(iii) complexes with porphyrin and salen-type ligands, have been reported to be superoxide scavengers. Here we report the synthesis and spectroscopic characterization of manganese(iii) complexes of 5[4-(6-O-β-cyclodextrin)-phenyl],10,15,20- tri(4-hydroxyphenyl)-porphyrin and of 6^A-deoxy-6^A[(S- cysteamidobenzoyl(3,4-diamino)-N,N′-bis(salicylidene))] -β-cyclodextrin. The superoxide dismutase activity of the metal complexes was investigated by indirect methods. The catalase and peroxidase activities were tested using ABTS assays. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c0dt01480j

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Dalton
Transactions
Cite this: Dalton Trans., 2011, 40, 2913
www.rsc.org/dalton
PAPER
New conjugates of b-cyclodextrin with manganese(III) salophen and porphyrin
complexes as antioxidant systems†
Valentina Oliveri, Antonino Puglisi and Graziella Vecchio*
Received 27th October 2010, Accepted 14th January 2011
DOI: 10.1039/c0dt01480j
Oxidative stress is the hallmark of several pathologies like arthritis, hypertension and many
neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. In this scenario, antioxidant
compounds can play a pivotal role in treating these severe pathologies. The synthesis of molecules able
to mimic physiologically-relevant proteins is nowadays of particular interest. Several transition metal
complexes, especially manganese(III) complexes with porphyrin and salen-type ligands, have been
reported to be superoxide scavengers. Here we report the synthesis and spectroscopic characterization of
manganese(III) complexes of 5[4-(6-O-b-cyclodextrin)-phenyl],10,15,20-tri(4-hydroxyphenyl)-porphyrin
and of 6A-deoxy-6A[(S-cysteamidobenzoyl(3,4-diamino)-N,N¢-bis(salicylidene))]-b-cyclodextrin. The
superoxide dismutase activity of the metal complexes was investigated by indirect methods. The catalase
and peroxidase activities were tested using ABTS assays.
from Eukarion have also been shown to protect from ischemia-
Introduction
reperfusion injury of the brain,17 heart24 and kidney25,26 in animal
Oxidative stress has become widely viewed as an underlying condi-
models. On this basis some complexes are used in cosmetics as
tion of a number of diseases, such as ischemia-reperfusion injury,1
anti-wrinkle agents.
chronic inflammation,2 diabetes,3 neurodegenerative disease,4,5
Manganese(III) porphyrin complexes have been widely inves-
cancer6 and aging.7 Therefore, natural and synthetic antioxidants
have been actively sought.
tigated and their activities with regard to the nature of the
side chain have been recently discussed in a comprehensive
review.27 Manganese porphyrins have been shown to be SOD
mimics, ONOO- scavengers and redox modulators of cellular
transcriptional activity. These compounds could also be mimics of
the cyt P450 family of enzymes. Metalloporphyrin SOD/catalase
mimetics have also been shown to protect against radiation
injury.28–30 In addition, metalloporphyrins should be effective
in treating peripheral diseases such as diabetes31 and heart
disease.32 Manganese-containing mesoporphyrins have demon-
strated efficacy in cellular and animal models of neurodegenerative
diseases.33 Clinical trials are now under way for manganese(III)
tetrakis(N,N¢-diethylimidazolium-2-yl)porphyrin (AEOL 10150),
an injectable Mn porphyrin, for treatment of ALS (amyotrophic
lateral sclerosis).34 Metalloporphyrins may be advantageous over
other types of compounds because of their stability.
We have already reported some SOD mimetics based on metal
complexes of cyclodextrin (CD) conjugates and have found that
the CD cavity, in some cases greatly, improves the SOD activity of
the complexes.35–39
CDs can be used either as drug solubilizers by forming inclusion
complexes or to form covalent conjugates. The chemical conjuga-
tion could confer on the linked moiety new properties: improved
stability, solubility and site specificity. CD–drug bioconjugates
are reported in the literature as very promising site specific anti-
inflammatory pro-drugs for colon diseases.40 The synthesis of a
SOD synzyme containing CDs could be a novel approach to deliver
Superoxide dismutase is the first line of defence against oxidative
stress under physiological and pathological conditions. Therefore,
mimetics of antioxidant enzymes such as superoxide dismutase
(SOD) and catalase are reported as potential new drugs able to
reduce oxidative stress damage.2,8,9 Among these systems, metal
complexes of manganese(II) or (III) are the most widely investigated
molecules.10 One reason for favouring manganese is that catalyst
decomposition can release free metal; in the case of metal ions
such as copper10 and iron11 this can give rise to severe toxicity
in vivo.
Particularly, the manganese(III) complexes of porphyrins12,13 or
salen type ligands9,14–16 have been reported as both SOD and
catalase mimetics. Generally the SOD-like activity investigated in
vitro is considered to be the operative mechanism in vivo.9,17,18 It has
been shown that salen complexes exert beneficial effects in several
in vitro and in vivo animal models of human diseases which involve
oxidative stress ranging from Alzheimer’s,19,20 Parkinson’s,21 prion
disease22 and motor neuron disease to multiple sclerosis and
excitotoxic neuronal injury.23 The salen-type systems investigated
Dipartimento di Scienze Chimiche, University of Catania, V. le A. Doria
6, 95125, Catania, Italy. E-mail: gr.vecchio@unict.it; Fax: +39095580138;
Tel: +390957385064
† Electronic supplementary information (ESI) available: NMR spectra
of ligands and intermediates, CD spectra of the MnCDL1. See DOI:
10.1039/c0dt01480j
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 2913–2919 | 2913
©

View Article Online
∑
1
O2 - scavenger to the colon.41 Reactive Oxygen Species (ROS) such
at 499.883 MHz. The H NMR spectra were obtained by using
standard pulse programs from a Varian library. In all cases the
length of the 90◦ pulse was 7 ms. The 2D experiments wereacquired
using 1 K data points, 256 increments and a relaxation delay of
1.2 s. T-ROESY spectra were obtained using a 300 ms spin-lock
time. DSS was used as external standard.
∑
∑
-
as O2 and OH have been reported to have a role in mediating
intestinal damage in inflammatory bowel diseases. The CD ability
to quench the ∑OH radicals could render the conjugates able to act
against a cocktail of radicals.42
In this paper we report the synthesis of two new manganese(III)
complexes obtained conjugating two members of the most in-
vestigated SOD mimics such as porphyrin and salophen with
b-cyclodextrin in order to further investigate the effects of the
oligosaccharidic moiety. In particular, we report the synthesis and
spectroscopic characterization of the manganese(III) complexes of
5[4-(6-O-b-cyclodextrin)-phenyl],10,15,20-tri(4-hydroxyphenyl)-
porphyrin (CDL1) and of 6A-deoxy-6A[(S-cysteamidobenzoyl(3,4-
diamino)-N,N¢-bis(salicylidene))]-b-cyclodextrin (CDL2) (Chart
1). The SOD-like activity of the metal complexes was investigated
by indirect methods.43 The catalase and peroxidase activities were
tested using the ABTS assays.44
UV/Visible and circular dichroism spectroscopy. UV/Visible
spectra were recorded with an Agilent 8452A diode array spec-
trophotometer. The spectra were recorded at 25 ◦C, on freshly
prepared aqueous solutions. Circular dichroism measurements
were performed at 25 ◦C under a constant flow of nitrogen on
a JASCO model J-810 spectropolarimeter. The spectra represent
the average of 5 or 10 scans.
Mass spectrometry. The ESI-MS measurements were per-
formed on a Finnigan LCQ-Duo ion trap electrospray mass
spectrometer.
Superoxide dismutase assay
SOD-like activity was determined by the indirect method of
cytochrome c (cyt c) or Nitro Blue Tetrazolium (NBT) reduction.43
Superoxide radical anion was enzymatically generated by the
xanthine–xanthine oxidase (XO/XOD) system and spectropho-
tometrically detected by monitoring the formation of reduced cyt
c or NBT at 550 nm. The reaction mixture was composed of cyt
c 50 mM or NBT 250 mM, xanthine 50 mM, catalase 30 mg ml-1 in
phosphate buffer 10 mM, at pH 7.4. An appropriate amount of
xanthine oxidase was added to 2.0 ml reaction mixture to produce
∑
a DA550 nm min-1 of 0.024. This corresponded to a O2 production
rate of 1.1 mmol min-1. The cytochrome or NBT reduction rate was
measured in the presence and in the absence of the investigated
complex for 500 s. Stock solutions of complexes were prepared in
water or in methanol, depending on solubility. Where present, at a
final concentration that did not exceed 7%, methanol did not affect
-
Chart 1
◦
assay activity. All measurements were carried out at 25 0.2 C
using 1 ¥ 1 cm thermostated cuvettes in which solutions were
magnetically stirred. In separate experiments urate production by
xanthine oxidase was spectrophotometrically monitored at 295
nm, ruling out any inhibition of xanthine oxidase activity. The
I50 (the concentration which causes the 50% inhibition of cyt c
or NBT reduction) of manganese(III) complexes at pH 7.4 was
determined. Three determinations were carried out.
Experimental
General
b-Cyclodextrin (Fluka), anhydrous N,N-dimethylformamide
(DMF) (Aldrich) and 5,10,15,20-tetra(4-hydroxyphenyl)por-
phyrin (p-THPP, Porphyrin Systems) were used without further
purification.
3,4-diaminobenzoic acid (Fluka) was purified by chromatogra-
phy on silica gel with acetone as the eluent.
Merck Lichroprep RP-8 (40–60 mm) was used for reversed phase
column chromatography. Analytical and preparative HPLC was
carried out on a Varian Prepstar instrument equipped with a
Prostar 330 photodiode array detector. Preparative (5 mm, 22 ¥
250 mm) and analytical (5 mm, 2.0 ¥ 250 mm) Econosphere ODS
(Alltech) columns were used. CD derivatives were detected on
TLC by UV and by an anisaldehyde test. TLC was carried out
on silica gel plates (Merck 60-F254). 5[4-(6-O-b-cyclodextrin)-
phenyl],10,15,20-tri(4-hydroxyphenyl)-porphyrin was synthesised
as reported elsewhere.45
Catalase activity assay
Catalase activity was measured by the method described
elsewhere.37 A calibration curve for absorbance against hydrogen
peroxide concentration was obtained. Complexes were dissolved
in phosphate buffer (10 mM, pH 7.4). Hydrogen peroxide solution
(30 mM) was incubated at various concentrations of the complexes
(ranging from 10-5 to 10-6 M) in 0.010 M phosphate buffer at pH
7.4 at 25 ◦C.
After 50 min, ABTS (2,2¢-azino-bis(3-ethylbenzothiazoline-6-
sulfonic acid) (50 mM) and horseradish peroxidase (1.3 mg ml-1)
were added. After 5 min, the reaction went to completion and
the absorbance at 735 nm was read. The data analysis gives the
amount of hydrogen peroxide removed per minute per mmole of
compound. Three determinations were carried out.
NMR spectroscopy. 1H NMR spectra were recorded at 25 ◦C
in D2O or CD3OD with a Varian UNITY PLUS spectrometer
2914 | Dalton Trans., 2011, 40, 2913–2919
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Peroxidase activity assay
in cysteamine moiety), 2.70 (2H, m, H b to the amide group in
cysteamine moiety).
Peroxidase activity was assessed by monitoring the hydrogen
peroxide-dependent oxidation of ABTS spectrophotometrically.46
The assay mixture consisted of ABTS (0.2 mM), hydrogen
peroxide (0.5 mM) and manganese(III) complex in ph◦osphate
buffer (50 mM) at pH 7.4. Assays were conducted at 25 C. The
ABTS oxidation was monitored at 735 nm to eliminate interference
with the manganese complex absorption. The amount of oxidized
ABTS was estimated using the value e (at 735 nm) of 1.5 ¥ 104 M-1
cm-1. No ABTS oxidation due to the complexes or the hydrogen
peroxide was observed in the conditions of the assay.
6A -deoxy-6A[(S-cysteamidobenzoyl(3,4-diamino)-N,N¢-bis(sali-
cylidene))]-b-cyclodextrin (CDL2). Salicylaldehyde (3.2 ml,
0.030 mmol) was added to CDL0 (20 mg, 0.015 mmol) in anhy-
drous methanol (about 25 ml) under stirring at room temperature.
The mixture rapidly became yellow. After 4.5 h, the solvent was
evaporated in vacuo. The residue was suspended in a small volume
of acetonitrile and centrifuged off. The supernatant was taken
away with a pipette and this washing procedure was repeated twice
more. Yield: 19 mg (82%).
ESI-MS: m/z = 1536.3 (CDL2+H)+, 1558.4 (CDL2+Na)+.
1H NMR (500 MHz, CD3OD) (ppm) 8.90 (1H, s, iminic proton
meta to the amidic bond), 8.86 (1H, s, other iminic proton), 7.89–
7.87 (2H, m, H ortho to the iminic group and H ortho to the
carbonylic group and iminic group), 7.59 (1H, d, J1,2 = 8.0 Hz, H
Ligand synthesis
N,N¢-Di-Boc-3,4-diaminobenzoic acid. 3,4-diaminobenzoic
acid (2.0 g, 13.1 mmol) was dissolved in methanol (30 ml).
Di-tert-butyl pyrocarbonate (11.21 g, 51.4 mmol) was added and
the mixture was stirred at room temperature for 2 h. The solvent
was evaporated to dryness in vacuo, the solid was purified by
column chromatography on silica gel (ethyl acetate–n-hexane
6 : 4). The appropriate fractions were concentrated to give the
product. Yield: 1.48 g (32%).
ortho to the iminic group and meta to the OH), 7.57 (1H, d, J1,2
=
8.0 Hz, other proton ortho to the iminic group and meta to the
OH), 7.48 (1H, d, J1,2 = 9.0 Hz, H meta to the carbonylic group),
7.42 (1H, d, J1,2 = 7.0 Hz, H ortho to the OH), 7.40 (1H, d, J1,2
=
7.0 Hz, other proton ortho to the OH), 7–6.96 (4H, m, Hs meta
and para to the OH), 4.96 (7H, d, other H-1 of CD), 3.97 (H, m,
H-5A), 3.86–3.75 (25 H, m, H-3, H-5, H-6 of CD), 3.61 (2H, m,
CH2 a to the amide group), 3.49 (7H, m, H-2 of CD), 3.44 (7H,
m, H-4 of CD), 3.18 (1H, m, H-6¢-A of CD), 2.97(1H, t, H-6A of
CD), 2.93 (2H, t, CH2 b to the amide group).
TLC: Rf 0.78 (AcOEt/n-hexane 3 : 2).
ESI-MS: m/z = 374.9 [M+Na]+, 726.9 [2M+Na]+.
1H NMR: (CD3OD, 500 MHz) d (ppm): 7.85 (1H, s, H-2 of
aromatic moiety), 7.62 (1H, dd, J5–6 = 9.0 Hz, H-6 of aromatic
ring), 6.75 (1H, dd, J5–6 = 9.0 Hz, H-5 of aromatic ring), 1.29 (18
H, s, H of Boc).
13C NMR (125 MHz, CD3OD) 171.0 (C O), 166.5 (C N),
166.3 (other C N), 161.6 (C–OH), 161.8 (other C–OH), 143.2
(C–C N), 144.0 (C–C N), 135.7 (C ortho to OH), 135.4 (other
C ortho to OH), 134.2 (C ortho to C N), 133.8 (other C ortho to
6A -deoxy-6A[(S-cysteamidobenzoyl(3,4-diamino))]-b-cyclodex-
trin (CDL0). Di-Boc-3,4-diaminobenzoic acid (104 mg,
0.29 mmol) was dissolved in dry DMF. 1-hydroxybenzotriazole
(HOBt) (40 mg, 0.30 mmol) and O-(benzotriazol-1-yl)-
C
N), 134.0 (C meta to C O), 126.3 (C ortho to N C and meta
to C O), 120.5–116.6 (other Cs of aldehyde moieties), 104.0 (C-1
of cyclodextrin), 103.7 (C-1A 85.4 (C-4A), 83.0 (C-4A), 74.8 (C-
3), 74.4 (C-2), 73.8 (C-5), 61.6 (C-6), 40.8 (CH2 alpha to NHCO),
34.4 (CH2 beta to NHCO), 34.24 (C-6A).
N,N,N¢,N¢-bis(tetramethylene)uronium
hexafluorophosphate
(HBPyU) (130 mg, 0.30 mmol) were added. This mixture
was stirred at room temperature for 30 min. 6A-deoxy-
6A[(S-cysteamine)]-b-cyclodextrin38 (350 mg, 0.29 mmol) was
added to the solution and the reaction mixture was stirred
at 35 ◦C under nitrogen for 22 h and was followed by TLC
(PrOH/AcOEt/H2O/NH3 4 : 3 : 2 : 1 Rf 0.43). The solvent was
evaporated in vacuo and the solid was purified by chromatography
on RP-8 column (gradient H2O–CH3CN 0→50%). The
isolated product was deprotected by stirring with CF3COOH
(10 ml) for 1.5 h. The reaction was followed by TLC analysis
(PrOH/AcOEt/H2O/NH3 4 : 3 : 2 : 1 Rf 0.20). The CF3COOH
was evaporated and the obtained solid was purified by CM-
Elemental analysis for C65H89N3O37S·4H2O: calc. C 48.54; H
6.08; N 2.61; found C, 48.41; H, 6.12; N, 2.55.
Preparation of manganese(III) complexes
Manganese(III) complex of L1 (MnL1). L1 (70 mg,
0.10 mmol) was dissolved in anhydrous methanol (10 ml).
Mn(CH3COO)3·2H2O (27 mg, 0.10 mmol) was added and the mix-
ture was stirred for 4 h under reflux. The solvent was evaporated
to dryness in vacuo, the solid was purified by chromatography on
silica gel with AcOEt/CH3OH (4 : 1) as the eluent. Yield: 54 mg
(68%).
+
Sephadex C-25 (NH4 form), eluting with a linear gradient of a
NH4HCO3 solution 0→0.2 M.
TLC: Rf 0.39 (AcOEt/CH3OH 4 : 1).
ESI-MS: m/z = 731.4 [MnL1]+.
Appropriate fractions were collected, (Rf 0.23, PrOH/H2O/
AcOEt/NH3, 4 : 3 : 2 : 1) and the solvent was evaporated. Yield:
77 mg (20%).
UV-Vis: (MeOH) l/nm (e/M-1 cm-1) 225 (35616), 320 (13377),
347 (16743), 384 (27313), 402 (26859), 427 (26996), 471 (46732),
573 (5669), 610 (6345).
ESI-MS: m/z = 1350.5 (M+Na)+.
1H NMR (500 MHz, D2O) d (ppm): 7.15 (1H, s, H ortho to
the amide and amine groups), 7.07 (1H, dd, H ortho to the amide
group), 6.80 (1H, dd, H meta to the amide group), 5.01–4.89 (7H,
m, H-1 of CD), 3.95–3.63 (26H, m, H-3, H-5 and H-6 of CD),
3.59–3.36 (14H, m, H-2 and H-4 of CD), 3.29 (1H, t, H-4A of
CD), 2.87 (3H, m, H-6A of CD, and 2H a to the amide group
Manganese(III) complex of CDL1 (MnCDL1). CDL1
(100 mg, 0.056 mmol) was dissolved in dry DMF (5 ml).
Mn(CH3COO)3·2H2O (15 mg, 0.056 mmol) was added and
the mixture was stirred for 1 h under reflux. The solvent
was evaporated to dryness in vacuo, the solid was purified by
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 2913–2919 | 2915
©

View Article Online
preparative HPLC (linear gradient H2O→CH3CN). Yield: 96 mg
(90%).
TLC: Rf 0.62 (PrOH/H2O/AcOEt/NH3 5 : 3 : 1 : 2).
ESI-MS: m/z = 1848 [Mn(CDL1)]+.
UV-Vis: (MeOH) l/nm (e/M-1 cm-1) 224 (38791), 319 (15993),
348 (18195), 384 (25949), 404 (26220), 425 (30784), 471 (41630),
572 (5964), 609 (6391).
Elemental analysis for MnC88H100N4O40·4H2O: calc. C, 53.36;
H, 5.50; N, 2.83; found C, 53.01; H 5.55, N, 2.79.
Manganese(III) complex of CDL2 (MnCDL2). CDL0 (17.8 mg,
0.0134 mmol) was dissolved in anhydrous methanol (5 ml).
Mn(CH3COO)3·2H2O (3.6 mg, 0.0134 mmol) and salicylaldehyde
(2.9 ml, 0.0268 mmol) were added. The colourless solution turned
to brown immediately and was refluxed for 3 h. The mixture was
left at room temperature, the precipitated solid was filtered off and
washed with acetonitrile. Yield: 17.7 mg (80%).
ESI-MS: m/z = 1588.3 [Mn(CDL2)]+, 805.8 [Mn(CDL2)+Na]2+.
UV-Vis: (H2O) l/nm (e/M-1 cm-1): 194 (34828), 246 (29333),
306 (18469), 335 (20509), 434 (7577), 547 (1351).
Fig. 1 1H NMR spectrum of CDL2 (CD3OD, 500 MHz).
MnCDL1 were characterised by ESI-MS, UV-Vis and circular
dichroism. The ESI-MS spectrum of MnL1 shows one peak at
731.4 m/z due to [MnL1]+.
The ESI-MS spectrum of MnCDL1 shows two peaks at 1848.3
and 935.9 m/z due to [Mn(CDL1)]+ and the double charged species
[Mn(CDL1)+Na]2+. No peaks were observed at m/z lower than
400. The peaks in this region have been assigned to manganese
clusters due to manganese impurities.48
Elemental analysis for MnC67H91N3O39S·4H2O: calc. C, 46.75;
H, 5.80; N, 2.44; found C, 46.51; H, 5.71; N, 2.39.
Results and discussion
The UV-Vis spectrum (Fig. 2) shows typical absorptions of
manganese(III) porphyrin complexes.49
Ligands
In order to prepare Mn(III) complexes, porphyrin and salen-
type ligands were synthesised. 10,15,20-tri(4-hydroxyphenyl)-
porphyrin (CDL1) was synthesised for comparison as reported
elsewhere.45
CDL0 was synthesised starting from N,N¢-di-Boc-3,4-
diaminobenzoic acid and 6A-deoxy-6A[(S-cysteamidobenzoyl(3,4-
diamino))]-b-cyclodextrin (CDMEA), in the presence of activat-
ing and condensating agents typically used for these kinds of
reactions.47 After activation of the carboxylic group of the benzoic
acid, the CDMEA forms an amide bond by a nucleophilic attack
via its amino group. In order to prevent uncontrolled amide bond
formation, the amino groups of 3,4-diaminobenzoic acid were
protected with Boc groups.
The Boc groups were removed by CF3COOH. The final product
CDL0 was fully characterised. NMR spectra confirm the identity
of the ligand. The H-1 protons of the CD moiety are divided into
six groups as often observed in the case of other derivatives.38
In addition to the signals due to the CD protons, the signals of
the chain are evident: the protons of the ethylenic chain of the
cysteamine moiety appear at 2.87 ppm and at 2.70 ppm, while the
protons of the aromatic ring resonate at 7.15, 7.07 and 6.80 ppm.
The new CDL2 ligand was characterised in methanol by ESI-
MS and NMR.
On the 1H NMR spectrum (Fig. 1), the signals due to the chain,
the aromatic rings and CD moiety are evident. The signals due to
the cysteamine moiety are shifted to downfield compared to those
of CDL0. The iminic protons appear as different signals at 8.90
and 8.86 ppm for the asymmetry of the salophen moiety.
Fig. 2 UV-Vis spectra of MnCDL2 (---) and MnCDL1 (–) (2.8 ¥ 10-5 M).
The circular dichroism spectrum shows two weak negative
bands in the Soret region, due to the dipole–dipole coupling of
the porphyrin moiety and the CD cavity. This behaviour has been
generally reported for functionalised CDs and is due either to
the inclusion of the functionalizing moiety in the cavity or its
proximity.
In the case of the [Mn(CDL1)]+, the circular dichroism spectrum
is not modified by the addition of various amounts of 1-
adamantanol, suggesting that the porphyrin is out of the cavity.
The negative sign of the induced circular dichroism band suggests
that the porphyrin moiety is near to the cavity, oriented with the
plane of the porphyrin parallel to the symmetry axis of CD.50 In
comparison with the ligand CDL1, the complex shows a different
behaviour. In line with previous results,45 the complexation
of the manganese(III) ion destroys the supramolecular dimeric
Complexes
The manganese complex of L1 and CDL1 were synthesised starting
from the ligands and manganese acetate under reflux. MnL1 and
2916 | Dalton Trans., 2011, 40, 2913–2919
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Table 1 I50 and kcat values of manganese complexes
structure of the ligand. The manganese(III) complex of CDL2 was
synthesised at room temperature as reported elsewhere for similar
compounds.51 It was characterised by UV-Vis and ESI-MS.
The ESI-MS spectrum of MnCDL2 shows three peaks at 1588.3,
805.8 and 542.8 m/z due to [Mn(CDL2)]+, [Mn(CDL2)+Na]2+ and
[Mn(CDL2)+H+K]3+ species, respectively.
The UV-Vis spectrum of MnCDL2 (Fig. 2) is similar to the
MnL2 one.52 This suggests that the coordination of manganese(III)
in the CD–salophen conjugate is similar to that of manganese(III)
salophen. The circular dichroism spectrum of MnCDL2 does not
show any dichroic band, ruling out any interaction of the aromatic
moiety with the CD cavity.
Complex
MnCDL2
MnL2
I50 (mM)
kcat (M-1s-1)
Reference
1.11( 0.05)a
1.4( 0.2)b
1.3a
1.32( 0.06) ¥ 107a
1.0( 0.1) ¥ 107b
1.2 ¥ 107a
This paper
This paper
52
This paper
This paper
This paper
27
1.7( 0.2)b
7.5( 0.9)b
8( 1) ¥ 106b
1.7( 0.2) ¥ 106b
< 1.0 ¥ 105b
6.8 ¥ 104b
MnCDL1
MnL1
MnTPP+
MnTPFP+
1.0 ¥ 105b
27
a Determined by NBT, b Determined by cyt c.
SOD-like activity
MnCDL2 is only slightly higher than that of the simple analogue
without CD (MnL2).52 Unlike the other CD-conjugates in which
the proximity of the cavity and the complex significantly improves
the catalytic activity,39 in this system the catalytic moiety is rigid
and too far away from the cavity to show any improvement in
activity.
MnCDL1 also shows a good IC50 (Table 1) even if it is less
effective than the MnCDL2 complex. This behaviour is in line
with the general trend observed for neutral porphyrin complexes
which are less active than salen complexes. In order to assess the
effect of the cavity, we synthesised the MnL1 complex and tested
its SOD-like activity. The MnL1 showed a kcat <1 ¥ 105 M-1s-1. A
more accurate determination of SOD-like activity was not possible
due to its low solubility under assay conditions.
The kcat value for this system is in the range of values reported
for other neutral porphyrins.27 It is interesting to note that the kcat
value for MnCDL1 is significantly higher than those reported for
neutral porphyrin complexes27 (Table 1). The improvement of the
SOD activity as a consequence of the conjugation of the CD has
been already observed in other metal complexes of functionalised
CDs.39 In the case of MnCDL1, the improvement of SOD activity
is higher than that observed for salen CD-conjugates.38
On the basis of the data in the literature36,37,57 we can hypothesize
that the CD cavity improves the catalytic activity for the presence,
in proximity to the catalytic moiety, of a microenvironment which
can trap radical species58–61 so favouring their reaction with the
catalytic centre. The hydroxylic groups of the CD might play
a synergic role in the catalytic mechanism. The increased SOD
activity for MnCDL1 compared to MnL1 might therefore be
explained by the spatial proximity of the porphyrin moiety to
the CD cavity.
SOD-like activity of the manganese(III) complexes was determined
by competition kinetics using NBT or cytochrome c target as
reported elsewhere.53
Possible interferences of the tested compounds with the xan-
thine/xanthine oxidase reaction were assessed by following the
rate of urate accumulation at 295 nm in the absence of the target.
The complexes, at the concentration used in the assay, had no
effect on the reaction of the XO/XOD system. Inhibition of
target reduction by the complex, when plotted as V0/Vcomplex-1
against the [complex] yielded a straight line (Fig. 3). V0 is the
∑
-
uninhibited reduction rate of cyt c or NBT by O2 and Vcat is the
rate of reduction of cyt c or NBT inhibited by the complex. When
[complex] is equal to I50, kcat[complex] = kdetector[detector] and kcat
can be determined. kdetector is the constant for the reaction between
∑
-
the O2 and the detector molecule and it is 2.6 ¥ 105 and 5.88 ¥
104 M-1 s-1 when the detector is cyt c and NBT, respectively.54 In
the case of MnCDL2 we reported data obtained using both cyt
c and NBT as targets to rule out interferences with the target
as reported by us for similar systems.38 The results obtained
with NBT are higher than those obtained with cyt c as typically
observed in the case of simple Mn-salophen complex.55 Reduced
NBT is reoxidated by O2 and this determines higher antioxidant
activity56
Peroxidase activity
Peroxidase-like activity of synthesised manganese complexes was
evaluated. The peroxidase activity of MnL2 was also determined
for comparison.
The following mechanism is reported in the literature15 for the
peroxidase activity, where A is a typical substrate:
Mn(III) + H2O2 ꢀ Mn(V) O + H2O
Fig. 3 The SOD-like activity of MnCDL2. V is the reduction rate of NBT
(Vo) alone and (Vc) in the presence of the complex.
Mn(V) O + AH2 ꢀ Mn(IV)–OH + AH+
The I50 values are reported in Table 1. MnCDL2 shows a good
I50, similar to the values reported for this class of compounds. I50 of
Mn(IV)–OH + AH2 ꢀ Mn(III) + H2O + AH+
Dalton Trans., 2011, 40, 2913–2919 | 2917
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Table 2 Catalase and peroxidase activities of manganese complexes
porphyrinic system leads us to assume that the saccharidic residue
of the CD is somehow involved and improves the reaction with
superoxide radicals.
Complex
mM ABTS·min-1
mmol of H2O2/min/mmole
The functionalisation with the CD is also an efficient strategy to
target the antioxidant bioconjugates to the colon for orally usable
drugs such as the metalloporphyrins.62
MnCDL1
MnL2
MnCDL2
—
18( 1)
15( 3)
8.9( 0.4)
40.2( 1.6)
36.0( 0.9)
In order to assess the peroxidase activity of manganese com-
plexes, ABTS was used as substrate. The reaction of ABTS with
H2O2 in the presence of complex generates radical cation:
Acknowledgements
The authors wish to thank Tiziana Campagna for technical
assistance. This research is funded by MIUR (2008R23Z7K).
2 ABTS + H2O2 + 2H+ ꢀ 2ABTS∑+ + 2H2O
The reduced form of ABTS is colourless, while the oxidized
ABTS, the ABTS∑+ radical cation, is green and its visible spectrum
shows several characteristic absorption bands at 415, 650, 735 and
815 nm. In the absence of the complex, a solution of ABTS and
H2O2 is stable for several hours at 25 ◦C, in the dark, without
showing any formation of radical cation.
Notes and references
1 H. Urakamia and M. B. Grisham, Pathophysiology, 2006, 13, 183.
2 D. Salvemini, T. M. Doyle and S. Cuzzocrea, Biochem. Soc. Trans.,
2006, 34, 965.
3 C. Maritim, R. A. Sanders and J. B. Watkins, J. Biochem. Mol. Toxicol.,
2003, 17, 24.
In order to compare our results with the data in the literature,
the amount of ABTS∑+ formed per minute was determined at the
complex concentration of 1.0 ¥ 10-5 M. MnL2 and MnCDL2
exhibit similar peroxidase activity and their activities (Table 2)
are very similar to those of the Eukarion compounds.15 MnCDL1
did not show peroxidase activity at the investigated concentrations
(1.0 ¥ 10-5 M and 1.0 ¥ 10-4 M).
4 U. Singh and I. Jialal, Pathophysiology, 2006, 13, 129.
5 V. Chauhan and A. Chauhan, Pathophysiology, 2006, 13, 195.
6 C. J. Weydert, T. A. Waugh, J. M. Ritchie, K. S. Iyer, J. L. Smith, L. Li,
D. D. R. Spitz and L. W. Oberley, Free Radical Biol. Med., 2006, 41,
226.
7 J. A. Vinson, Pathophysiology, 2006, 13, 151.
8 B. J. Day, Antioxid. Redox Signaling, 2008, 10, 355.
9 S. Melov, S. R. Doctrow, J. A. Schneider, J. Haberson, M. Patel, P. E.
Coskun, K. Huffman, D. C. Wallace and B. Malfroy, J. Neurosci., 2001,
21, 8348.
10 D. P. Riley, Chem. Rev., 1999, 99, 2573.
11 S. D. Aust, L. A. Morehouse and C. E. Thomas, J. Free Radicals Biol.
Med., 1985, 1, 3.
12 Duke University PCT Int. Appl. WO9609053. 1996, Duke University
WO9640223, 1996, Duke University U.S. Patent US5227405, 1993.
13 B. J. Day, I. Fridovich and J. D. Crapo, Arch. Biochem. Biophys., 1997,
347, 256.
14 M. A. Sharpe, R. Olloson, V. C. Stewart and J. B. Clark, Biochem. J.,
2002, 366, 97.
15 S. R. Doctrow, K. Huffman, C. B. Marcus, W. Musleh, A. Bruce,
M. Baudry, B. Malfroy, in Antioxidants in Disease Mechanisms and
Therapeutic Strategies, ed. H. Sies, Academic Press, New York, 1997,
pp. 247–270.
Catalase activity
The catalase activity was determined by an indirect assay (Table 2).
The mechanism by which the Mn complex acts as a catalase
mimetic involves the oxidation of Mn(III) to oxomanganese by
H2O2, releasing water. The oxomanganese is then reduced to
Mn(III) by another hydrogen peroxide molecule to form oxygen
and water:
Mn(III) + H2O2 ꢀ Mn(V) O + H2O
16 R. Liu, I. Y. Liu, X. Bi, R. F. Thompson, S. R. Doctrow, B. Malfroy
and M. Baudry, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 8526.
17 S. R. Doctrow, K. Huffman, C. B. Marcus, G. Tocco, E. Malfroy, C. A.
Adinolfi, H. Kruk, K. Baker, N. Lazarowych, J. Mascarenhas and B.
Malfroy, J. Med. Chem., 2002, 45, 4549.
18 K. Baker, C. B. Marcus, K. Huffman, H. Kruk, B. Malfroy and S. R.
Doctrow, J. Pharmacol. Exp. Ther., 1998, 284, 215.
19 I. Anderson, C. Adinolfi, S. Doctrow, K. Huffman, K. A. Joy, B.
Malfroy, P. Soden, H. T. Rupniak and J. C. Barnes, Biochem. Soc.
Symp., 2002, 67, 141.
Mn(V)O + H2O2 ꢀ Mn(III) + H2O + O2
The catalase activity of MnL2 was also determined for com-
parison. Both salophen derivatives showed higher activity than
MnCDL1. This trend is reported in the literature62 and shows that
neutral porphyrins are not particularly efficient H2O2 scavengers.
For all these new systems the values are significantly higher
than those obtained with the method reported for some Eukarion
compounds based on salens and porphyrins.62
20 J. Bruce, B. Malfroy and M. Baudry, Proc. Natl. Acad. Sci. U. S. A.,
1996, 93, 2312.
21 J. Pong, S. R. Doctrow and M. Baudry, Brain Res., 2000, 881, 182.
22 M. W. Brazier, S. R. Doctrow, C. L. Masters and S. J. Collins, Free
Radical Biol. Med., 2008, 45, 184.
Conclusions
23 Y. Rong, S. R. Doctrow, G. Tocco and M. Baudry, Proc. Natl. Acad.
Sci. U. S. A., 1999, 96, 9897.
24 S. Pucheu, F. Boucher, T. Sulpice, N. Treasallet, Y. Bonhomme, B.
Malfroy and J. de Leiris, Cardiovasc. Drug Ther., 1996, 10, 331.
25 M. Sami, M. A. Sharpe, P. R. Gard and P. K. Chatterjee, Free Radical
Biol. Med., 2007, 43, 528.
26 P. K. Chatterjee, N. S. Patel, E. O. Kvale, P. A. Brown, K. N. Stewart,
H. Mota-Filipe, M. A. Sharpe, R. Di Paola, S. Cuzzocrea and C.
Thiemermann, Am. J. Nephrol., 2004, 24, 165.
27 I. Batinic-Haberle, J. S. Reboucas and I. Spasojevic, Antioxid. Redox
Signaling, 2010, 13, 877 and reference therein.
In this paper new manganese(III) porphyrin and salophen conju-
gates with b-cyclodextrin are reported.
Apart from significantly improving the aqueous solubility of
both systems compared to the simple manganese-complexes, the
cyclodextrin might play a synergic role in the catalytic process.
In the case of the salophen derivative the functionalisation gives
rise to a slight increased antioxidant activity of the system in
vitro compared to the system not linked to the cyclodextrin. The
new SOD mimic based on porphyrin–CD conjugate shows SOD
activity about 15 times higher than the reference compounds. The
greater proximity of the catalytic centre to the CD cavity for the
28 B. Gauter-Fleckenstein, K. Fleckenstein, K. Owzar, C. Jiang, I.
Batinic-Haberle and Z. Vujaskovic, Free Radical Biol. Med., 2008, 44,
982.
2918 | Dalton Trans., 2011, 40, 2913–2919
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
29 Z. Vujaskovic, I. Batinic-Haberle, Z. N. Rabbani, Q. F. Feng, S. K.
Kang, I. Spasojevic, T. V. Samulski, I. Fridovich, M. W. Dewhirst and
M. S. Anscher, Free Radical Biol. Med., 2002, 33, 857.
30 R. A. Rosenthal, E. Vorotnikova, L. Fisette, S. Braunhut, K. Huffman,
M. Tries and S. R. Doctrow, Free Radical Biol. Med., 2006, 41, S159.
31 J. D. Piganelli, S. C. Flores, C. Cruz, J. Koepp, I. Batinic-Haberle,
J. Crapo, B. Day, R. Kachadourian, R. Young, B. Bradley and K.
Haskins, Diabetes, 2002, 51, 347.
47 C. Donze’, E. Rizzarelli and G. Vecchio, J. Inclusion Phenom. Mol.
Recognit. Chem., 1998, 31, 27.
48 S. Reboucas, I. Spasojevic and I. Batinic-Haberle, JBIC, J. Biol. Inorg.
Chem., 2008, 13, 289.
49 K. M. Kadish, K. M. Smith, Roger Guilard (Eds), Handbook of
porphyrin, vol. 4, Academic Press, Boston, 2003.
50 M. Kodaka, J. Phys. Chem., 1991, 95, 2110.
51 J. Boucher, J. Inorg. Nucl. Chem., 1974, 36, 531.
52 V. Oliveri and G. Vecchio, Eur. J. Med. Chem., 2011,
DOI: 10.1016/j.ejmech.2010.12.023, in press.
32 N. L. Lubbers, J. S. Polakowski, J. D. Crapo, C. D. Wegner and B. F.
Cox, J. Cardiovasc. Pharmacol., 2003, 41, 714.
33 B. J. Day and M. Patel, Drugs Future, 2008, 33, 1025.
34 R. W. Orrell, Curr. Opin. Investig. Drugs, 2006, 7, 70.
35 E. Rizzarelli and G. Vecchio, Coord. Chem. Rev., 1999, 188, 343.
36 R. P. Bonomo, V. Bruno, E. Conte, G. De Guidi, D. La Mendola,
G. Maccarrone, F. Nicoletti, E. Rizzarelli, S. Sortino and G. Vecchio,
Dalton Trans., 2003, 4406.
37 A. Puglisi, G. Tabb`ı and G. Vecchio, J. Inorg. Biochem., 2004, 98, 969.
38 V. Lanza and G. Vecchio, J. Inorg. Biochem., 2009, 103, 381.
39 F. Bellia, D. La Mendola, C. Pedone, E. Rizzarelli, M. Saviano and G.
Vecchio, Chem. Soc. Rev., 2009, 38, 2756.
40 F. Hirayama and K. Uekama, Adv. Drug Delivery Rev., 1999, 36, 125.
41 K. Emerit, S. Pelletier, D. Tosoni-Verligue and M. Mollet, Free Radical
Biol. Med., 1989, 7, 145.
42 D. La Mendola, S. Sortino, G. Vecchio and E. Rizzarelli, Helv. Chim.
Acta, 2002, 85, 1633.
53 I. Spasojevc, I. Batinic-Haberle, D. R. Stevens, P. Hambright, N. A.
Thorpe, J. Grodkowski, P. Neta and I. Fridovich, Inorg. Chem., 2001,
40, 726.
54 I. Baudry, S. Etienne, A. Bruce, M. Palucki, E. Jacobsen and B. Malfroy,
Biochem. Biophys. Res. Commun., 1993, 192, 964.
55 Z. X. Liu, G. B. Robinson and E. M. Gregory, Arch. Biochem. Biophys.,
1994, 15, 74.
56 S. Liochev and I. Fridovich, Arch. Biochem. Biophys., 1995, 318,
408.
57 F. Bellia, D. La Mendola, G. Maccarrone, P. Mineo, D. Vitalini, E.
Scamporrino, S. Sortino, G. Vecchio and E. Rizzarelli, Inorg. Chim.
Acta, 2007, 360, 945.
58 M. Voicescu, G. Ionita, M. Vasilescu and A. Meghea, J. Inclusion
Phenom. Macrocyclic Chem., 2006, 54, 217.
59 H. Kariu and P. Tordo, Tetrahedron Lett., 2004, 45, 1043.
60 M. Hardy, D. Bardelang, H. Karoui, A. Rockenbauer, J.-P. Finet, L.
Jicsinszky, R. Rosas, O. Ouari and P. Tordo, Chem.–Eur. J., 2009, 15,
11114.
43 C. Beauchamp and I. Fridovich, Anal. Biochem., 1971, 44, 276.
44 R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang and C.
Rice-Evans, Free Radical Biol. Med., 1999, 26, 1231.
45 A. Puglisi, R. Purrello, E. Rizzarelli, S. Sortino and G. Vecchio, New J.
Chem., 2007, 31, 1499.
46 S. R. Doctrow, K. Huffman, C. B. Marcus, G. Tocco and E. Malfroy,
J. Med. Chem., 2002, 45, 4549.
61 I. Snyrychova`, Free Radical Biol. Med., 2010, 48, 264.
62 R. A. Rosenthal, K. D. Huffman, L. W. Fisette, C. A. Damphousse,
W. B. Callaway, B. Malfroy and S. R. Doctrow, JBIC, J. Biol. Inorg.
Chem., 2009, 14, 979.
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 2913–2919 | 2919
©