New artificial biomimetic enzyme analogues based on Iron(II/III) schiff base complexes: An effect of (Benz)imidazole organic moieties on phenoxazinone synthase and DNA recognition

Article abstract of DOI:10.3390/molecules24173173

Elucidation of the structure and function of biomolecules provides us knowledge that can be transferred into the generation of new materials and eventually applications in e.g., catalysis or bioassays. The main problems, however, concern the complexity of the natural systems and their limited availability, which necessitates utilization of simple biomimetic analogues that are, to a certain degree, similar in terms of structure and thus behaviour. We have, therefore, devised a small library of six tridentate N-heterocyclic coordinating agents (L¹–L⁶), which, upon complexation, form two groups of artificial, monometallic non-heme iron species. Utilization of iron(III) chloride leads to the formation of the 1:1 (Fe:Ln) ‘open’ complexes, whereas iron(II) trifluoromethanosulfonate allows for the synthesis of 1:2 (M:Ln) ‘closed’ systems. The structural differences between the individual complexes are a result of the information encoded within the metallic centre and the chosen counterion, whereas the organic scaffold influences the observed properties. Indeed, the number and nature of the external hydrogen bond donors coming from the presence of (benz)imidazole moieties in the ligand framework are responsible for the observed biological behaviour in terms of mimicking phenoxazinone synthase activity and interaction with DNA.

Full text of DOI:10.3390/molecules24173173

molecules
Article
New Artificial Biomimetic Enzyme Analogues Based
on Iron(II/III) Schiff Base Complexes: An Effect of
(
Benz)imidazole Organic Moieties on Phenoxazinone
‡
Synthase and DNA Recognition
Aleksandra Bocian 1
,†
, Martyna Szyma n´ ska , Daria Brykczy n´ ska , Maciej Kubicki
1,†
1
1
,
1
2
1,
Monika Wał e˛ sa-Chorab
Adam Gorczy n´ ski * and Violetta Patroniak 1
, Giovanni N. Roviello
, Marta A. Fik-Jaskółka *,
1,
1
Faculty of Chemistry, Adam Mickiewicz University, Uniwersytetu Pozna n´ skiego 8, 61-614 Pozna n´ , Poland
Institute of Biostructures and Bioimaging—CNR, via Mezzocannone 16, 80134 Napoli, Italy
Correspondence: martafik@amu.edu.pl (M.A.F.-J.); adam.gorczynski@amu.edu.pl (A.G.)
These authors contributed equally to the experimental results given in this work.
Dedicated to Professor Jean-Marie Lehn on the occasion on his 80th birthday.
2
*
†
‡
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Antonella Dalla Cort and Maura Pellei
Received: 28 July 2019; Accepted: 29 August 2019; Published: 31 August 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Elucidation of the structure and function of biomolecules provides us knowledge that can
be transferred into the generation of new materials and eventually applications in e.g., catalysis or
bioassays. The main problems, however, concern the complexity of the natural systems and their
limited availability, which necessitates utilization of simple biomimetic analogues that are, to a certain
degree, similar in terms of structure and thus behaviour. We have, therefore, devised a small library
1
6
of six tridentate N-heterocyclic coordinating agents (
L –L ), which, upon complexation, form two
groups of artificial, monometallic non-heme iron species. Utilization of iron(III) chloride leads to
the formation of the 1:1 (Fe:Ln) ‘open’ complexes, whereas iron(II) trifluoromethanosulfonate allows
for the synthesis of 1:2 (M:Ln) ‘closed’ systems. The structural differences between the individual
complexes are a result of the information encoded within the metallic centre and the chosen counterion,
whereas the organic scaffold influences the observed properties. Indeed, the number and nature of the
external hydrogen bond donors coming from the presence of (benz)imidazole moieties in the ligand
framework are responsible for the observed biological behaviour in terms of mimicking phenoxazinone
synthase activity and interaction with DNA.
Keywords: iron; Schiff base; DNA binding; 2-aminophenol; biomimetic activity
1
. Introduction
Nature exhibits an astonishing ability to construct sophisticated molecular machineries to
target selected, otherwise hardly approachable by synthetic chemists, molecular transformations [1].
Metalloenzymes were recognized as one class of such species, with judiciously chosen transition metal
ions and enzyme active sites being able to facilitate various redox processes in a catalytic and selective
manner [
renders chemistry the hallmark of modern science [
necessitates the use of artificially constructed biomimetic analogues for further advancement of this
discipline [ ]. This solution provides access to simple, readily accessible species that retain certain
2]. Knowledge gained from studies of their structure and function is of prime importance that
3
]. Nevertheless, the natural systems’ complexity
4
structural features of enzymes, and so their chemical behaviour and function may become mimicked.
From transition metal cations that constitute the redox-active part of enzymes like copper,
manganese, or iron, the latter one is of particular importance due to its low biotoxicity, wide availability
Molecules 2019, 24, 3173; doi:10.3390/molecules24173173
www.mdpi.com/journal/molecules

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in nature and electronic states that favour binding of O [
5,
6]. In particular, one may discriminate
2
heme [
framework of its active site. The seminal, most widely studied examples of the former group involve
studies on the families of horseradish peroxidases [ ] and cytochromes P450 [10]. The second group
is a much broader class of molecular species, which can be divided in accordance to the number of
iron centres [ 11]. Notable representative examples include the bimetallic methane monooxygenase
MMO), which catalyses oxidation of hydrocarbons [12] or the monometallic ‘2-His-1-carboxylate facial
7] and non-heme [8] Fe-based enzymes, classification, and functions that depend on the structural
9
8,
(
triad’ of relevance for various oxidative transformations [13].
These oxidations are a result of the reductive dioxygen activation, which are dependent on the
structural features of the artificial biomimetic analogue, specifically the coordination environment
around the metallic centre(s) and its redox characteristics [14]. It is thus possible to generate small
groups of structurally similar coordinating agents, where one could incorporate the chosen structural
motif into the ligands scaffold and evaluate its effect on the biological outcome. Schiff base ligands
were recognized as very potent candidates for the construction of such libraries due to their robustness
and stability, facile synthetic methodologies and rich complexation behaviour [15
iron(II/III) complexes were investigated as DNA binding agents [17 19] or as phenoxazinone synthase
PHS) artificial analogues [16 20 25]. Interestingly, in Nature it is the copper(II) ion that catalyses
oxidation of o-aminophenols to phenoxazinones [26 27], which means that PHS-active iron systems
,16]. Specifically,
–
(
, –
,
could be used to gain additional insight into the oxidative biological mechanisms [20,28].
Owing to our experience in the construction of the imine-scaffolded metallosupramolecular
architectures [29–37] and bioassays [30,36,38–40], we designed and synthesized a small library of
Schiff base ligands (Scheme 1), where the main structural difference involves the different number and
disposition of the hydrogen bond donors.
Scheme 1. Schematic representation of ligands synthesized for the purpose of this work; green frame
denotes the family of benzimidazole-scaffolded ligands whereas blue frame shows the family of
pyridine-scaffolded coordinating agents.
Coordination with iron(II)/(III) metallic centres led to the formation of two families of complexes
(‘open’ and ‘closed’ species) depending on the applied counterion, which were in turn investigated as
artificial phenoxazinone synthase analogues and DNA-binding agents. To the best of our knowledge,
this is the first report that studies the effect of H-bonding on the multifunctional behaviour of library of
Schiff base iron agents in terms of bioassays.

Molecules 2019, 24, 3173
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2
. Results and Discussion
2
.1. Synthesis and Characterization
A series of six hydrazone Schiff base ligands L1
–
L6 was successfully synthesized in a two-step
1
3
synthetic protocol. [31] Commercially available 2-chlorobenzimidazole (in
L
–L
) or 2-bromopyridine
) were subjected to nucleophilic substitution reaction with an excess of methylhydrazine and
the product was reacted with the corresponding imidazolecarboxaldehyde. As a result, two sets of
4
6
(
in L –L
coordinating agents were obtained in 41.1–90% yields, with the common N -tridentate meridional
3
binding subunit which differed in the number (0–2) and topology (axial vs. equatorial) of donor
hydrogen bonds (Scheme 1).
Their UV-Vis and NMR (when possible) solution studies (Figure 1b, Figures S11–S13) indicate
stability of synthesized complexes in solution, indicating that the organic Schiff base ligands do not
decoordinate. Interestingly, from the family of ‘closed’ species, the ones with the pyridine moiety
10–12 were found to be diamagnetic, whereas the benzimidazole-scaffolded ones 7–9 are paramagnetic
at room temperature.
Figure 1. a) Schematic representation of compounds
A
: [Fe(Lx)Cl ] ‘open’ (left) and B: [Fe(Lx)2](OTf)2
3
‘
closed’ systems (right). b) UV-Vis spectra of ‘open’ 1–6 (left) and ‘closed’ system complexes 7–12 (right)
−
5
recorded in MeOH at c = 2 × 10 M.
2
.2. Description of Crystal Structures
The structures of iron complexes with six different ligands (L1 L6, cf. Scheme 1) were obtained.
–
The crystal structures confirmed that these complexes can be formed either with one or two tridentate
ligand molecules, therefore confirming our initial assumptions. Out of 12 possible compounds
studied, we were able to determine crystal structures for nine complexes (cf. Experimental Section,
Electronic Supplementary Information Figures S32–S35.
The first group consists of neutral complexes with the general formula [Fe( x
exception of , which is ionic and contains one coordinated methanol molecule instead of one of
L )Cl ] (with one
3
2
the chloride anions, additional Cl acts as a counterion). Figure 2 shows a representative example of
these complexes.
In all these complexes Fe(III) cations are six coordinated, by three nitrogen atoms from ligand
−
−
2, two Cl and methanol oxygen, Figure 2b) in a slightly
molecule and three Cl anions (in case of
distorted octahedral fashion. The distortion is mainly caused by trans-coordination of two nitrogen
x
◦
atoms of L molecule, appropriate N1-Fe-N15 angles differ significantly from 180 , they are in a
range 147–148 . Table S1 lists the relevant geometrical features for all the complexes. In this group of
◦

Molecules 2019, 24, 3173
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complexes, the crystal structures are determined mainly by N-H···Cl (C-H···Cl) hydrogen bonds, and
often by secondary π···π or C-H···π interactions (hydrogen bond data are listed in Table S2).
Figure 2. Representative perspective views of ‘open’ (4—a, 2—b)) and ‘closed’ complexes (9—c);
ellipsoids are drawn at the 50% probability level, hydrogen atoms are shown as spheres of arbitrary
−
radii. Thin blue line in 2 represents the O–H···Cl hydrogen bond.
Second group contains cationic complexes of general formula [Fe(Lx) ]2+, with two triflate anions
2
balancing the charge. Also in these cases Fe(II) is six coordinated, by six nitrogen atoms from two
ligand molecules, but distortions from ideal octahedral geometry are larger than in former group
(cf. Table 1). Figure 2c shows example of the member of this group (9). In the crystal structures of
this group the N-H···O (triflate) or C-H···O hydrogen bonds are always present. Probably due to
more complicated packing of different moieties, in almost all examples the voids filled by solvent
molecules. Table S1 contains also information of the size of the voids, determined as a part of the
unit cell. All ligand molecules are almost or approximately planar, as may be seen by analyzing the
dihedral angles between planar fragments of the molecules (Table S1).
Table 1. Kinetic parameters for complexes 1–12.
Vmax (10 3Ms
−
−1
)
−3
−1
kcat (h )
Catalyst
KM (10 M)
1
[
[
[
[
[
[
Fe(L )Cl ] (1)
3.71
2.55
3.45
2.68
2.07
3.01
3.98
1.96
3.68
0.86
1.30
2.13
1.45
2.16
1.95
2.02
2.02
2.19
1.56
2.08
1.86
1.79
1.89
2.08
185.25
127.30
172.30
134.05
103.34
150.49
199.40
97.68
3
2
Fe(L )Cl ] (2)
3
3
Fe(L )Cl ] (3)
3
4
Fe(L )Cl ] (4)
3
5
Fe(L )Cl ] (5)
3
6
Fe(L )Cl ] (6)
3
1
[
[
[
[
[
[
Fe(L ) ](OTf) (7)
2
2
2
Fe(L ) ](OTf) (8)
2
2
3
Fe(L ) ](OTf) (9)
183.80
42.76
65.14
2
2
4
Fe(L ) ](OTf) (10)
2 2
5
Fe(L ) ](OTf) (11)
Fe(L ) ](OTf) (12)
2
2
6
106.53
2
2
In the crystal structures, Coulombic interactions between ions and directional N-H···O hydrogen
bonds between cations and anions (Table S2) are important factors for final crystals architectures.
2
.3. Catalysis and DNA Binding Affinity
The structural similarities present within each group of iron complexes represent a great starting
point for determination of the structure/properties dependencies, specifically an influence of the
number and disposition of H-bonding within the ligands framework on their biomimetic behavior as:
(i) artificial phenoxazinone synthase analogues; (ii) DNA-recognition binders.

Molecules 2019, 24, 3173
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2
.3.1. Phenoxazinone Synthase (PHS) Activity
Oxidation of 2-aminophenol can be done using dioxygen or hydrogen peroxide [21], using different
solvent as an environment [24] and what is the most important using a wide variety of complexes.
Examples in the literature include: cobalt(II) [21 23], manganese(II) [21] or iron(III) compounds [20 28].
–
,
At room temperature and in the presence of air, the present series of iron(II)/(III) complexes
catalyze the aerobic oxidative dehydrogenation of o-aminophenol (OAPH) to the corresponding
light-absorbing 2-aminophenoxazine-3-one chromophore (APX) (Scheme 2) and the process is strongly
dependent on the chosen complex.
Scheme 2. Schematic representation of oxidation of 2-aminophenol (OAPH) to 2-aminophenoxazine-
3
-one chromophore (APX).
−
4
First of all, a ten-fold excess of o-aminophenol solution (2
×
10 M) was added to methanolic
−
5
solutions of the chosen iron complex (2
×
10 M), so that an excess of substrate is present and
first-order kinetic reaction mechanism could be maintained. The reaction was carried out at room
temperature in the presence of air, without any base to minimize the possibility of auto-oxidation
of OAPH [41] and in methanol for solubility issues. The catalytic properties of the complexes were
monitored spectrophotometrically by observing the increasing intensity of the absorption band at
433 nm, which corresponds to the formation of APX chromophore. Measurements were made
separately for the complex, o-aminophenol and also immediately after mixing of the substrates as a
function of time. The chosen measurement time intervals (1 h, 24 h, 48 h, 72 h) are a result of minimal
spectroscopic changes at first hours, most probably associated with high iron(III) oxidation state for
‘
open’ complexes and sterically blocked ‘closed’ complexes. Spectra registered for complexes 1–12
are gathered in the Supplementary Information (Figures S37–S46) and the most catalytically active
complexes from each of the two groups (1 for ‘open’ and 7 for ‘closed’), are shown in Figure 3.
Thanks to observation of changes in the intensity of bands, it is possible to confirm the catalytic
properties of the studied complexes, since blank tests without catalyst (Figure S36) confirm that the
latter is necessary to obtain the observed conversion of OAPH to APX.
To gain deeper mechanistic insight into oxidation of OAPH with studied iron(II/III) complexes,
kinetic studies were performed at room temperature for each system by varying the relative
−
4
−6
concentration of complexes from 2
×
10 M–2
×
10 M and measurement of the reaction initial rates
at 433 nm. By ensuring an excess of the substrate with regard to the complex, the initial rates method
exhibits a first-order dependence on complex concentration and was treated with the Michaelis–Menten
model, in which linearization affords a double reciprocal Lineweaver–Burk plot allowing analysis of
the following parameters: the maximum velocity (Vmax), binding constant (K ), and rate constant
M
(kcat) (Figure 3b,c) and Figures S37–S46, Supplementary information). Indeed, rate saturation kinetics
was observed, which confirms the validity of the applied kinetic model and also indicates that the
reaction involves the formation of complex-substrate intermediate in a pre-equilibrium stage and its
subsequent irreversible oxidation is associated as the rate-determining step of the catalytic cycle [25].

Molecules 2019, 24, 3173
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Figure 3. a) The spectral profile showing the growth of 2-aminophenoxazine-3-one at 433 nm due to
addition of complex (left) and (right) to 2-aminophenol dissolved in methanol. The spectra were
recorded under aerobic conditions during the three days. ) Plot of rate vs. concentration for complex
(left) and (right). ) Linewear–Burk plot of phenoxazinone synthase-like activity for complex (left)
and 7 (right).
1
7
b
1
7
c
1
Table 1 includes all kinetic parameters determined for twelve iron complexes under study used
−3
−1
in oxidation of OAPH, with the range of corresponding values being: Vmax = 0.86–3.98 [10 Ms ],
KM = 1.45–2.08 [10 M] and kcat = 42.76–199.40 [h ].
−3
−1
Comparison of different catalysts can be done by kcat, which is also expressed as the turnover
number and can be related to its activity in a given time unit. Table 2 compares our compounds with
different ones found in the literature and shows that our systems can be regarded as functional models
of phenoxazinone synthase (PHS) which is involved in the biosynthesis of Actinomycin D, the latter
of which is a naturally occurring nontrivial APX derivative exhibiting anti-cancer properties [42–44].
We did observe however that activity of each complex is an interplay of its ‘primary’ (open vs. closed)
and ‘secondary’ (number and disposition of H-bonds) structural features.

Molecules 2019, 24, 3173
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Table 2. Catalytic oxidation of o-aminophenol with transition metal complexes.
kcat (h 1)
−
Catalyst
Solvent
Ref.
1
[
Fe(L )Cl ] (1)
Methanol
Methanol
DMF
185.25
199.40
137.0
56.0
32.36
196.18
33.26
54.0
This work
This work
[28]
3
1
[
Fe(L ) ](OTf) (7)
2
2
a
[
[
L
FeCl (L )]
Fe(L )Cl ]
)(4,4 -byp)ClO }]n
2
b
Methanol
Methanol
Methanol
Methanol
Methanol
Methanol
Methanol
b
[20]
[42]
[42]
[23]
[45]
[46]
[47]
3
c
0
[
[
{Fe(
4
c
[
FeL Cl]
2
d
[
[
Co(L )(N ) ]
3
3
e
Co(L )(N ) ]
3
2
f
Co(
L
)Cl(H O)]Cl
L Co(L ) ]ClO
·
H O
2
13.68
11.48
2
g
h
[
2
4
a
0
0
L = 1,3-bis(5 -methyl-2 -thiazolylimino)isoindoline; L = N,N’-Bis(2-Methylbenzimidazolyl) pyridinediamide;
c
e
f
d
L
L
L
=
N,N’-Disalicylidene-1,2-propylenediamine;
L
=
(2-pyridylmethyl)(2-pyridylethyl)amine;
=
2-{[3-(3-Dimethylaminopropylamino)-propylimino]-methyl}-6-methoxy-phenol;
g
=
N,N’-bis(pyridin-2-ylmethylene)-2,2-dimethylpropane-1,3-diamine;
N,N’-bis(3-methoxysalicylidehydene)cyclohexane-1,2-diamine; L = 4-aminopyridine.
L
=
h
Supplementary Table S3, which helps in following the structural features as a function of catalytic
parameters is provided in Supplementary Information. It must be noted that whereas higher kcat
denotes higher activity of a given complex, lower KM values indicate stronger binding of complex to
OAPH which is related to TON, yet might be limited by further reaction processes.
6
> ~ 2
L5
L4
Within the group of ‘open’ complexes
1
–
6
, KM decreases in the order of
L
L
>
=
>
L3
>
L
1
and seems to be affected by the ligands H-bonding scaffold. Indeed, whereas the highest
6
value is found for
L with no H-bond donors, ca. 10% decrease was observed for complexes with
5
4
scaffold comprising one NH moiety. Interestingly, its relative disposition (
L
vs. L ) does not seem to
have relevant influence herein. Further decrease in the series exhibits iron(III) complex coordinated to
1
benzimidazole-imidazole ligand
L
of a mixed axial/equatorial H-bond disposition. Quite unexpectedly,
2
6
isomeric ligand
L
from iron complex interacts with OAPH in a similar fashion as
L
, though their
TON values differ. Since coordination number 6 of Fe(III) metal ion can be safely termed as saturated,
its interaction with OAPH is possible by assuming exchange of at least one coordinated chloride
anion with solvent molecules. Indeed, X-ray structure that we provide for complex
2 proves that
2
it was possible to isolate such species in the solid state as [Fe(
L
)Cl (MeOH)]Cl. Consequently,
2
OAPH can be assumed to coordinate to iron(III) via phenoxo anion, with concomitant exchange of
coordinated methanol molecule. This is where non-binding NH moiety from coordinated OAP group
2
could additionally interact with NH units of ligand scaffold. (compare with proposed mechanism
1
3
6
4
2
5
in Section 2.3.2). TONs change within the order of
L
>
L
>
L
>
L
~L
>
L
and seem to not be
dependent on H-bonding moieties (specifically compare KM and TONs of
4
and 6), which also prove
that the rate-determining step would be associated with irreversible oxidation of the OAP-catalyst
complex, not the binding of OAPH with catalyst per se.
Interesting comparison comes with studies of the ‘closed’ complexes. One could expect that they
would not be catalytically active, since it should be hard to dissociate one of the tridentate imine
1
ligands. In addition, HNMR studies of diamagnetic 10
–
12 complexes do not show formation of
1
3
6
multiple species in solution. It turns out that TONs decrease in the following order
L > L > L >
L ligands – similarly as in the
2
5
4
1
3
L
~
L
>
L
, with the most active complexes being ones based on
L
and
‘open’ class. Intriguingly, KM decreases in exactly the same order, which would imply that appropriate
number and disposition of H-bonding between OAPH and coordinated ligand should be responsible
for generation of OAPH···complex interactions. NH on imidazole moiety seems to influence such an
interaction more significantly than the benzimidazole unit, however since TONs and K follow the
M
same trend, redox Fe(II/III) oxidation changes would explain oxidation of OAPH in spite of binding of
the latter one via H—bonding and not cationic centre. All in all, ‘closed’ species are somewhat less
efficient for oxidation of OAPH than ‘open’ species.

Molecules 2019, 24, 3173
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2
.3.2. Proposed Mechanism of OAPH Oxidation
On the basis of obtained results and literature data [25
,
41], we would like to show proposed
mechanism of oxidation of 2-aminophenol (OAPH) to 2-aminophenoxazine-3-one (APX) with ‘open’
iron complexes investigated herein (Scheme 3).
Scheme 3. Proposed mechanism of phenoxazinone synthase like activity of complex 1 in methanolic
environment of reaction.
As was noted earlier, ‘open’ species are prone to ligand exchange and this is what happens in
the first step between chloride and methanol molecule. Such a configuration allows the molecule
of complex to interact with molecule of 2-aminophenol (OAPH), which results in coordination of
the latter one to the metallic centre in its deprotonated form, with simultaneous extrusion of the
methanol solvent molecule to the environment and additional interactions from the ligand/H-bond
moieties. Subsequent oxidation is presumed to be the rate-determining step, which could explain
certain time-lag associated with the initial reaction times. Obtained 2-aminophenoxazinone (APX) is
formed on the basis of quinone imine intermediate, reacting with second molecule of OAPH and a
series of subsequent redox transformations [25,41].
2
.3.3. DNA Binding Affinity
The compounds may interact with DNA in a variety of ways: they can combine by electrostatic
interaction with a phosphate sugar backbone, intercalate between pairs of bases, covalently bind with
nucleobases or attach in major or minor grooves [48]. Binding ability of metal complexes to DNA can
be studied by using spectroscopic methods such as: electronic absorption titration [49], fluorescence
competitive binding with ethidium bromide (EB) [50] and circular dichroism (CD) measurements [51].
Based on the observed changes in the spectrum the type of interaction may be specified.
Absorption Titration
The absorption titration experiment provides basic information concerning the presence and
strength of interactions between compounds and DNA. The most important and straightforward

Molecules 2019, 24, 3173
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indication is hypochromism in the spectrum of compound that increases together with the increase of
concentration of CT-DNA in the sample solution [52,53]. Monitoring of this phenomenon allows one
to evaluate the ability of compounds to interact with DNA double helix.
Absorption spectra of all complexes in the presence of CT-DNA were measured in the range
of 250–600 nm (Figures S47–S49). Interestingly, neither of ‘open’ complexes interact with CT-DNA,
and only three ‘closed’ complexes did show spectral changes attributable to specific complex-nucleic
acid interactions (10, 11, 12—Figure 4). It shows the significance of the “secondary” structure of
potential metallodrugs (if we say that the ligand framework and metal salt are the “primary” structure,
then their mutual arrangement in the space may be called the “secondary” structure of the system).
Interestingly, 10–12 employ pyridine moiety in the ligands “primary” structure, which would imply
that introduction of additional H-bonding through benzimidazole moiety hampers such process.
However, not only the pyridine/benzimidazole bias in the ligand scaffold is visible, since the topology
and substitution pattern of imidazole is relevant for DNA binding as well. Compounds 11 and 12
with 2-imidazole substituent interact with DNA much stronger (hypochromism of MLCT band at
ca. 485 nm of 96% and 79%, respectively) than 10 with 4-imidazole substituent (hypochromism of
MLCT band of 45%). At the same time blocking of hydrogen bonding donor site N-H as N-CH
3
5
6
in imidazole unit facilitates the DNA binding (comparison of
L
and
L
in complexes 11 and 12).
One may say that two-level recognition in the presented case is observed and is crucial for the DNA
binding phenomenon: (i) lack of hydrogen bonding donors in the “primary” structure and (ii) ‘closed’
composition of the “secondary” structure of complex.
Figure 4. Absorption titration of 12 (left), 11 (middle) and 10 (right) with increasing concentrations of
CT-DNA (0–100 µM). Arrows show hypsochromic and hypochromic changes upon increasing CT-DNA
concentration. Inset: plot of [DNA]/(
linear fitting of the data.
ε
a
− εf) versus [DNA];
I
, experimental data points; solid line,
As mentioned before, interaction of complexes 10
of the intensity of the MLCT band at ca. = 485 nm. In general, a strong hypochromism in the
absorption spectra is an indication of intercalative binding mode, since the distance between intercalated
compound and DNA bases decreases and the electrons of both combine [54]. Only for 11 and 12 the
, 11 and 12 with DNA is visible as a decrease
λ
π
binding constants (K ) were calculated, since the diminution of the MLCT band in case of 10 did not
b
reach 50%. In the face of this fact, we focused in our further discussion on 11 and 12. Both complexes
5
6
1
1
and 12, as well as the corresponding ligands (
employed in this study (10 mM TrisHCl, 5 mM NaCl, 50 mM pH 7.5) as shown in Figure S50. In general,
binds to the DNA twice more efficiently than 11 what is reflected in the intrinsic binding constants
L and L ), are stable in the biological medium
1
2
4
2
4
2
K = 2.8797 × 10 (R = 0.98602 for 6 points, inset Figure 4) and K = 1.1820
×
10 (R = 0.99087 for
b
b
8
points, inset Figure 4), for 12 and 11, respectively. In case of both complexes, a hypsochromic shifts
of ca. 8 nm of ILCT bands (ca. 335 nm) were observed. Changes in the intensity and position of the
ligand-derived bands may suggest electrostatic interaction with CT-DNA. On the other hand, there is
an isobestic point at 353 nm for complex 11 and 354 nm for complex 12 as well as hypochromism of
MLCT bands at 488 nm and 493 nm for complexes 11 and 12, respectively, indicating intercalative
kJ
binding to DNA [15
,
55]. The standard Gibb’s free energy for DNA binding is
−
25.03
and 22.86
−
mol
kJ
for 12 and 11, respectively, which indicates the spontaneous binding of both compounds with
mol

Molecules 2019, 24, 3173
10 of 24
DNA. Also, the ability of ligands L5 and L6 to bind with CT-DNA was examined (Figure S51). Since no
significant changes in the intensity of bands as increasing amounts of DNA were added, one may
assume that they do not interact with DNA in non-coordinated form.
Competitive Binding Fluorescence Experiment
In order to further investigate the strength of intercalation mode of synthesized complexes with
DNA the competitive binding experiments with ethidium bromide (EB) as a probe were carried out.
EB is a weak luminescent compound exhibiting high affinity to the DNA helix. Formation of a DNA–EB
complex enhances its emission at λmax = 590 nm (λexc = 467 nm) due to strong intercalation of EB planar
phenanthridine rings between adjacent base pairs of the DNA helix. Displacement of EB from its
DNA–EB complex due to gradual titration by a competing molecule results in subsequent quenching
of its emission band.
Competitive binding experiments with DNA-EB complex showed that compounds 11 and 12 are
able to bind with the DNA scaffold and release EB from its complex. Significant decrease in emission
intensity was observed for both complexes, which indicates that complexes bind with DNA in spaces
occupied by EB or EB is released due to conformational changes in the double helix. The quenching
4
4
constants are: KSV = 1.32
quenching constants, it can be concluded that complex 12 is slightly better binder than complex
, which is consistent with the results obtained in the electronic absorption titration experiments.
×
10 and K = 1.06
×
10 for 12 and 11 (Figure 5), respectively. Based on
SV
1
1
This could be explained by the fact that lack of hydrogen bonds in the “primary” structure of 12 results
in easier insertion between the base pairs of the DNA helix than 11. Both complexes act on the principle
of intercalation with DNA due to the “secondary” ’closed’ structure.
Figure 5. Emission spectra of ethidium bromide (EB) bound to CT DNA in the presence of increasing
amount of 12 (left) and 11 (right). Arrow shows the hypochromic changes upon increasing 12 or 11
concentration. Inset: Stern–Volmer plot of I /I
−
1 versus [12] or [11] for the titration of EB-CT DNA
0
complex;
I
, experimental data points; solid line, linear fitting of the data.
DNA Binding Investigation via CD
The study of morphological changes of DNA structure caused by the interaction of DNA
with compounds can be observed using circular dichroism (CD) measurements. This technique
is a very sensitive and informative method for studying the structural changes of the secondary
structure of nucleic acids and proteins. The B-DNA helix exhibits two conservative bands due to
its helicity—negative one at ca. 250 nm and a positive one at ca. 270 nm [50]. CD experiments
were performed on the 12-mer oligo DNA of composition d(GTTAATCGCTGG) in the presence of
different amounts of compounds 11 and 12 in the 1–30 eq. range. No significant CD perturbation
◦
under the above conditions was observed. However, after thermal treatment (70 C) and subsequent
slow cooling down of the sample solution, DNA underwent conformational changes in the presence of
12 corresponding to the variations observed in the positive band at 270 nm and are more pronounced

Molecules 2019, 24, 3173
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for this ‘hydrogen bond free’ complex than in the case of 11. Based on CD studies one may hypothesize
that 12 is more effective than 11 (Figure 6) in interacting with DNA by preventing its correct annealing.
Such findings from CD spectra correspond well with the results from DNA titration and EB competitive
binding. Moreover, the differences observed in the CD DNA spectrum due to 12, and in particular,
the decrease of intensity of the positive band at 270 nm, resemble similar effects previously associated
to well-characterized ligand-induced DNA conformational perturbations [16].
Figure 6. CD spectra relative to DNA duplex (2 µM) in the presence of 30 equiv. of 12 (left) and 11
◦
◦
(
(
right) in Tris HCl 5 mM NaCl 50 mM (pH 7.5) at 20 C (blue line) and after thermal treatment at 70 C
◦
5 min.) and subsequent cooling to 20 C over 1 h in the same buffer (green line).
3
. Experimental Section
3
.1. Materials and Methods
All reagent and solvent were purchased form commercial sources and used as received.
Elemental analyses for C, H, and N were carried out using an Elementar Analyser Vario EL
III (Elementar Analysensysteme GmbH, Hanau, Germany). Infrared spectra were recorded on an
iS50 FT-IR, ThermoScientific Nicolet (Thermo Fisher Scientific wissenschaftliche Geräte GmbH, Wien,
Austria). Electrospray ionisation mass spectroscopy (ESI-MS) was performed using Mass Spectrometer
ZQ Waters (Miromass&Waters; Milford, USA) by dissolving the powder samples of compounds in
−
4
methanol at ~10 M. The cone voltage values are depicted in each spectrum in Electronic Supplementary
1
13
Material. H and C-NMR spectra were run on a Bruker Ultrashield 300 MHz spectrometer (Bruker,
Karlsruhe, Germany) or Varian Gemini 400 MHz spectrometer (L ) (Varian, Darmstadt, Germany) and
1
were calibrated against the residual protonated solvent signals with chemical shifts represented in
ppm ((CD ) SO: 2.50 ppm, water: 3.33 ppm; CD CN: 1.94 ppm, water: 2.13 ppm; CDCl : 7.26 ppm,
3
2
3
3
water: 1.56 ppm; CD OD: 3.31 ppm, water: 4.87 ppm). Each time ca. 2 mg of sample was dissolved in
3
0
.6 mL of deuterated solvent.
CT-DNA, ethidium bromide, Tris and NaCl were supplied from Sigma Aldrich (Sigma Aldrich,
Pozna n´ , Poland) and used without further purification. CT-DNA was dissolved in Tris Buffer (5 mM
TrisHCl, 50 mM NaCl, pH 7.25) prior to use. The CT-DNA solution gave a ratio of UV absorbance of
1
.82:1 at 260 and 280 nm, indicating that the CT-DNA sample was sufficiently free from protein [56
,57].
CT-DNA concentration per nucleotide was determined from the UV absorbance at 260 nm using the
= 6600 dm3
60
·mol
−1
·cm
−1
[58]. Oligo DNA was supplied from Genomed S.A.
extinction coefficient
(
ε
2
Genomed S.A., Warsaw, Poland) and used without further purification. Electronic absorption titrations
of compounds were performed on Jasco V-770 spectrophotometer (Jasco Europe S.R.L., Cremella, Italy)
◦
equipped with a Peltier at a temperature of 20 C between 600 and 250 nm, in 10 × 10 mm quartz
cells. Emission spectra in the competitive fluorescence titration experiments were measured at room
temperature on a JASCO FP-6200 spectrofluorimeter (Jasco, Tokyo, Japan) in the range 540–720 nm
with excitation and emission slits of 10 nm and excitation wavelength λexc = 467 nm. CD experiments

Molecules 2019, 24, 3173
12 of 24
were performed by a Jasco J-810 spectropolarimeter (Jasco Europe S.R.L., Cremella, Italy) equipped
with a Peltier PTC-423S/15 (Jasco Europe S.R.L., Cremella, Italy) with a response time of 4 s, a scanning
speed of 50 nm/min and a bandwidth of 1.0 nm. Freshly prepared stock solutions of complexes
−
3
(
at concentration 2
×
10 M) were taken for all spectroscopic investigations of the binding mode with
DNA. It needs to be emphasized that compounds are stable in this medium for several days. After this
time some precipitate starts to occur.
3
.2. Synthesis of Ligands
.2.1. Synthesis of Ligands L –L3
1
3
Synthesized ligands (Schemes 4 and 5) were obtained as yellow, crystalline solids and their
1
13
identity and purity were confirmed by routine analytical methods ( H-NMR, C-NMR, melting points,
ESI-MS, Figures S1–S10, S14–S19). These species were subsequently reacted in equimolar ratio with
x
iron(III) chloride and 2:1 (
L
:M) iron(II) trifluoromethanesulfonate salts, respectively to obtain two
x
x
classes of iron-based coordination compounds:
A: [Fe(L )Cl ] ‘open’ and B: [Fe(L ) ](OTf) ‘closed’
3 2 2
systems (Figure 1a). In general, weakly coordinating triflates do not form coordination bonds with
Fe(II) ions, therefore two, tridentate Schiff base ligands fill its coordination sphere to form octahedrally
coordinated ‘closed’ species. In the presence of chlorides the octahedral binding mode is preserved,
nonetheless Fe(III) ions are bound by only one chelate imine ligand; the remaining coordination sites are
potentially labile and are filled by three chlorides, hence ‘open’ species. Names refer to the coordination
sites of the central metal ion, since chlorides are in fact more susceptible to exchange in the solvent
medium than the chelating Schiff base ligand, which is also readily visible upon comparison of X-ray
structures (e.g.,
2 vs. 4). We managed to successfully synthesize and characterize all 12 complexes
(Figures S20–S31), 9 of which also by means of X-ray crystallography, and confirmed their structural
character within each group, that is amenable to both the oxidation state of iron and the coordinative
strength of the anion chosen during the synthesis. Details are included in Section 3.
1
3
Scheme 4. Schematic representation of ligands L –L .

Molecules 2019, 24, 3173
13 of 24
4
6
Scheme 5. Schematic representation of ligands L –L .
The first step:
At two-necked round-bottomed flask 2-chlorobenzimidazole (8.00 g, 0.05 mol) was weighed and
placed under argon atmosphere. Methylhydrazine (11.9 g, 0.25 mol) in five-fold excess was dissolved
◦
in a anhydrous ethanol and was added to the reaction mixture, which was warmed to 80 C and
stirred for 2 h. The white, crystalline product was filtered on Büchner funnel and dried under vacuum.
Yield 68.2% (5.8 g, 0.036 mol).
The next step:
-
for L1: condensation of 4-imidazolecarboxyaldehyde with 2-(1-methyl-hydrazine)-benzimidazole
was performed. At two-necked round-bottomed flask 2-(1- methylhydrazine)benzimidazole
(
(
1.00 g, 6.16 mmol) was placed under argon atmosphere. The 4-imidazolecarboxyaldehyde
0.591 g, 6.16 mmol) was dissolved in a anhydrous ethanol and was added to the reaction mixture.
◦
Yellow suspension was formed and the reaction mixture was stirred for 24 h at 60 C. Yellow clear
solution was cooled to room temperature and the precipitate appeared, which was filtered by
vacuum filtration, washed with anhydrous ethanol and dried under vacuum. Yield 80% (1.184 g,
4
.9 mmol.)
ESI-MS(+) m/z (%): 241 (100) [H 1
L
]+, 263 (10) [Na
L
1]+; ESI-MS(
−
): 239 (100) [L1 − H] . IR:
−
ν(N-H)br
3
6
7
300–2500;
ν
1
(C=C)ar 1596, 1560;
ν
(C-N) 1451; (C=N)ar 1277;
ν
ρ(C-H)ar. 1162, 1091;
γ
(C-H)ar. 942, 737,
δ (ppm) 12.38 (s, 0.7H, NH(i/j)); 11.53 (s, 0.7H, NH(i/j));
.89 (s, 1H, H ); 7.78 (s, 1H, H ); 7.31 (s, 3H, Hb,c,g); 7.01 (s, 2H, Ha,d); 4.37 (s, 0.7H, NH(i/j)); 3.56 (s,
−1
23 cm . H-NMR ((CD ) SO, 300 MHz):
3
2
h
f
1
Me(e)). H-NMR ((CD ) SO + K CO , 400 MHz):
δ (ppm) 7.80 (s, 1H, H ); 7.77 (s, 1H, H ); 7.38 (s, 1H,
h f
3 2
2
3
g
H ); 7.32–7.28 (dd, 2H, J = 5.8, 3.2 Hz, Hb, c); 7.00–6.96 (dd, 2H, J = 5.9, 3.2 Hz, Ha,d); 3.56 (s, Me(e)).
1
3
C-NMR ((CD ) SO + K CO , 75 MHz):
δ
(ppm) 154.5, 138.7, 137.3, 131.9 (2C), 128.9, 125.2, 119.9 (2C),
3
2
2
3
◦
1
-
12.7 (2C), 31.1. Melting temperature: 176–179 C.
for L2: condensation of 2-imidazolecarboxyaldehyde with 2-(1-methyl-hydrazine)-benzimidazole
was performed. At two-necked round-bottomed flask 2-(1- methylhydrazine)benzimidazole (1.00 g,
6.16 mmol)was weighed and placed under argon atmosphere. 2-Imidazolecarboxyaldehyde
(
0.591 g, 6.16 mmol) was dissolved in anhydrous ethanol and was added to the reaction mixture.
◦
Yellow suspension was formed, and the reaction mixture was stirred for 24 h at 60 C. Yellow clear
solution was cooled to room temperature and evaporated to dryness. The resulting solid was
dissolved in methanol and boiling acetonitrile was added to the flask. Cooling of the reaction
mixture resulted in formation of pale pink crystalline product. The precipitate was filtered
by vacuum filtration, washed with anhydrous ethanol and dried under vacuum to give 1.24 g
(
5.16 mmol) of ligand. The supernatant was concentrated to minimal amount of volume and the
next part of precipitate was obtained 0.140 g (0.58 mmol). Total yield is 91% (1.38 g, 5.74 mmol).

Molecules 2019, 24, 3173
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ESI-MS(+) m/z (%): 241 (40) [H 2
L
]+, 263 (100) [Na
L
2]+; ESI-MS(
−
): 239 (100) [L2 − H] . IR:
−
ν(N-H)
br
(C-H)ar. 945, 737 cm 1.
−
3
400–2400;
ν
(C=C)ar. 1552;
ν
(C-N) 1452; (C=N)ar 1271;
ν
ρ(C-H)ar. 1145, 1040;
γ
1
H-NMR ((CD ) SO, 300 MHz):
δ (ppm) 12.33 (s, 1H, NH ); 11.66 (s, 1H, NH ); 7.70 (s, 1H, H );
(i) (j) f
.45–7.28 (m, 3H, Hb, c, g); 7.10–6.99 (m, 3H, Ha, d, h); 3.59 (s, Me(e)). C-NMR ((CD ) SO, 75 MHz): δ
3
2
13
7
3 2
(
2
ppm) 154.2, 144.9, 143.3, 134.1, 129.8, 127.8, 121.2, 120.4, 118.2, 116.9, 109.7, 31.7. Melting temperature:
95–306 C. At 295 C ligand started to become darker and decomposed at 306 C.
◦
◦
◦
-
for L3: condensation of 1-methyl-2-imidazolecarboxyaldehyde with 2-(1-methyl-hydrazine)
-benzimidazole was performed. At two-necked round-bottomed flask 2-(1- methylhydrazine)
benzimidazole (1.00 g, 6.16 mmol)was weighed and placed under argon atmosphere.
The 1-methyl-2-imidazolecarboxyaldehyde (0.704 g, 6.16 mmol) was dissolved in anhydrous
ethanol and was added to the reaction mixture. Yellow suspension was formed and the reaction
◦
mixture was stirred for 24 h at 60 C. Clear yellow solution was cooled to room temperature and
kept in fridge. Yellow crystals were filtered by vacuum filtration, washed with anhydrous ethanol
and dried under vacuum. Yield is 80% (1.26 g, 4.96 mmol).
ESI-MS(+) m/z (%): 255 (30) [H 3
L
]+, 277 (100) [Na
L
3]+; ESI-MS(
−
): 253 (100) [L3 − H] . IR:
−
ν(N-H)
br
(C-H)ar. 944, 743 cm 1.
−
3
500-2450;
ν
(C=C)ar. 1573;
ν
(C-N) 1445; (C=N)ar 1284;
ν
ρ(C-H)ar. 1170, 1038;
γ
1
H-NMR ((CD ) SO, 300 MHz):
δ (ppm) 11.25 (s, 1H, H ), 7.81 (s, 1H, H ), 7.36–7.29 (m, 3H, Hb, c, h),
(j) (f)
.04–6.99 (m, 3H, Ha, d, i), 4.01 (s, Me(g)), 3.65 (s, Me(e)). C-NMR ((CD ) SO, 75 MHz): δ (ppm) 153.4,
3 2
3
2
13
7
1
42.6, 144.1, 134.1, 129.8, 128.4, 124.3, 120.8, 119.8, 116.2, 110.1, 35.1, 31.6.
.2.2. Synthesis of Ligands L –L6
4
3
The first step:
At two-necked round-bottomed flask 2-bromopyrridine (8.00 g, 0.05 mmol) was weighed and
placed under argon atmosphere. Methylhydrazine (11.6 g, 0.25 mmol) was dissolved in anhydrous
◦
ethanol and was added to the reaction mixture. Then the reaction mixture was warmed to 80 C and
stirred for 24 h. The oil residue was purified by column chromatography (SiO ; 5% MeOH in CH Cl ).
2
2
2
Yield 52% (3.24 g).
The next step:
-
for L4: condensation of 4-imidazolecarboxyaldehyde with 2-(1-methylhydrazine)pyridine was
performed. At two-necked round-bottomed flask the 2-(1-methylhydrazine)pyridine (0.510g,
4.15 mmol) was weighed and placed under argon atmosphere. 4-imidazolecarboxyaldehyde
(
0.399 g, 4.15 mmol) was dissolved in anhydrous ethanol and was added to the reaction mixture.
◦
Yellow solution formed immediately and the reaction mixture was stirred for 24 h at 60 C.
The clear and orange solution was concentrated to minimal amount of volume, then Et O was
2
added and it was left in the refrigerator. The subsequent pale pink precipitate was filtered by
vacuum filtration, washed with anhydrous ethanol and dried under vacuum to give 0.607 g
(
3.01 mmol) of ligand. 10 mL of Et O was added to the supernatant and next part of precipitate
2
was obtained 0.053 g (0.27 mmol). Total yield is 79.2% (0.660 g, 3.28 mmol).
ESI-MS(+) m/z (%): 202 (20) [H 4
300–2600; (C-H)imin. 3011; (CH ) 2951;
309; (C-H)ar. 1199, 1121;
d, 1H, J = 4.3 Hz, H ); 7.66 (s, 1H, H ); 7.61 (s, 1H, H ); 7.56–7.47 (m, 2H, Hc, d); 7.23 (s, 1H, H
1
1
L
]+, 224 (25) [Na
L
4]+; ESI-MS(
−
): 200 (100) [L4 − H] . IR:
−
ν
(N-H)br
(C=N)ar
(ppm) 8.18
); 6.73 (t,
(ppm) 157.4,
3
1
(
ν
ν
ν
(C=C)ar. 1588, 1565, 1542;
ν
(C-N) 1475, 1440;
ν
3
−
γ(C-H)ar. 891, 768, 644, 594 cm . H-NMR (CDCl , 300 MHz): δ
3
1 1
ρ
a
f
h
g
13
H, J = 5.0 Hz, H ); 6.40 (s, broad, NH); 3.59 (s, Me ). C-NMR (CDCl , 75 MHz): δ
b (e) 3
◦
47.0, 137.7, 136.0, 131.6, 125.6, 125.4, 115.6, 109.7, 29.7. Melting temperature: 163–165 C.
-
for L5: condensation of 2-imidazolecarboxyaldehyde with 2-(1-methylhydrazine)pyridine was
performed. At two-necked round-bottomed flask the 2-(1-methylhydrazine)pyridine (0.525 g,
4.26 mmol) was weighed and placed under argon atmosphere. 2-Imidazolecarboxyaldehyde

Molecules 2019, 24, 3173
15 of 24
(
0.469 g, 4.26 mmol) was dissolved in anhydrous ethanol and was added to the reaction mixture.
◦
Yellow solution formed immediately and the reaction mixture was stirred for 24 h at 60 C.
The clear and yellow solution was concentrated to minimal amount of volume and left in the
refrigerator. The subsequent yellow precipitate was filtered by vacuum filtration, washed with
anhydrous ethanol and dried in the vacuum to give 0.301 g (1.49 mmol). The supernatant was
concentrated to minimal amount of volume and left in the refrigerator. The next part of precipitate
was obtained 0.052 g (0.26 mmol). Total yield is 41.1% (0.353 g, 1.75 mmol).
ESI-MS(+) m/z (%): 202 (30) [H 5
149; (C-H)imin. 3075; (CH ) 3002;
213, 1124;
.0 Hz, H ); 7.83 (d, 1H, J = 8.6 Hz, H ); 7.67 (t, 1H, J = 7.9 Hz, H
.87 (t, 1H, J = 5.5 Hz, H ); 3.61 (s, Me ). C-NMR (CD OD, 75 MHz):
L
]+, 224 (100) [Na
L
5]+; ESI-MS(
−
): 200 (100) [L5 − H] . IR:
−
ν
(N-H)
3
1
5
6
ν
ν
ν
(C=C)ar. 1591, 1566;
ν
(C-N) 1475, 1448;
ν
(C=N)ar. 1288;
ρ(C-H)ar
3
−
1 1
γ
(C-H)ar. 852, 773, 751, 738 cm . H-NMR (CD OD, 300 MHz):
δ
(ppm) 8.18 (d, 1H, J =
); 7.62 (s, 1H, H ); 7.10 (s, 2H, H , Hh);
(ppm) 157.2, 149.9, 146.5,
3
a
d
c
f
g
13
δ
b
(e)
3
◦
1
45.1, 137.6, 124.2, 115.9, 109.9, 99.9, 28.6. Melting temperature: 191–193 C.
6
-
for L : synthesized according to our previous work [31]
3
.3. Synthesis of Complexes
3
.3.1. Synthetic Method for ‘Open’ Complexes
For complexes 1, 4
–
6
,
[
L
Fe
(
Lx)]Cl3 the molar ratio of the ligand to the corresponding salt was 1:1.
1
4
5
6
To a solution of
L
,
L
,
L
in MeOH the methanolic solution of FeCl3 6H O was added. Mixture
·
2
was stirred for 24h at room temperature. Then solvents were evaporated under vacuum to minimal
amount of them and Et O was added. Obtained precipitate was filtrated and washed twice with Et O
2
2
(10 mL in total) and dried in vacuum. Yields based on ligands are shown in Table 3.
Table 3. The amount of substrate used to prepare complexes and information about yield and crystals
of them.
Ligand
mg)
Fe(II/III)
Salt (mg)
Yield a
Crystals
From
Complex
Precipitation Color/Solid
(
mg
%
1
2
3
4
5
6
7
8
9
26.7
402.9
60.0
22.3
23.9
21.9
70.0
70.0
70.0
70.0
70.0
55.0
30.0
453.6
63.8
30.0
30.0
30.0
51.6
51.6
48.7
61.6
61.6
45.3
very dark green
dark green
dark green
dark green
black
dark green
cinnamon
hot chocolate
raspberry
50.4
693.7
66.2
46.8
47.3
88.9
81
iPr O
tBuOMe
-
2
67.3
89.4
87.7
76.8
72.3
72.3
75.0
91.9
88.8
89.2
iPr O
2
-
39.8
87.9
iPr O
2
Et O
2
104.0
89.0
121.0
117.0
90.0
iPr O
2
tBuOMe
1
1
1
0
1
2
red
Et O
2
warm brown
dark chocolate
-
PhMe
a
yield based on powder.
For complexes
2
and
3
reaction system was evacuated on the vacuum/gas line. To a solution of L2
6H O was added. Dark-green solution formed
3
and
L
in dry MeCN (dried on molecular sieves) FeCl3
·
2
instantly and the reaction mixture was stirred for 48 h at room temperature. The green precipitate was
filtered via suction filtration and dried in the vacuum. Yields based on ligands are shown in Table 3.
Crystals suitable for X-ray characterization were grown by vial-to-vial diffusion of different
◦
external precipitating co-solvents into the methanolic solutions of complexes at 4 C. Please note that
thoroughly dried samples were used for catalytic and biological studies, hence elementary analysis
does not account for crystallization solvents that were found in the X-ray structures.

Molecules 2019, 24, 3173
16 of 24
1
1]+; ESI-MS(
−
) m/z (%): 239 (100) [L1 − H] . Anal. calc.
−
1
[FeL Cl ]: ESI-MS(+) m/z (%): 241 (100) [H
L
3
for [Fe(C H N )(Cl) ] (402.48): C: 35.81; H, 3.01; N, 20.88; found: C: 35.77; H, 3.31; N, 20.30%. IR:
12
12
6
3
ν
(C-H)arom 3125, 3093; νas(C-H)alif 2992, 2924; νs(C-H)alif 2849; ν(C=C) 1788, 1578, 1473; ν(C=N) 1437,
1
7
350
δ
(CH ) 1327,
ν
(C-O) 1289, 1245, 1214, 1148
γ
(C-H)arom 1085, 1048, 1008, 972, 931, 907, 850, 806,
3
−
1
56, 675, 615 cm .
2
2]+, 366 (30) [Fe 2
+
2
1
1
[FeL Cl (MeOH)]Cl: ESI-MS(+) m/z (%): 241 (70) [H
L
L
Cl ] ; ESI-MS(
−
) m/z (%):
2
2
−
98 (100) [FeCl ] . Anal. calc. for [Fe(C H N )(Cl) (MeOH)]Cl (434.52): C: 35.93, H: 3.71, N:
4
12 12
6
2
9.34%; found: C: 35.70, H: 3.45, N: 19.77%. IR: v(N-H) 3419,
(C=C) 1611, 1577, 1552, 1476, 1462; (C=N) 1438
287, 1255, 1216, 1180 γ(C-H)arom 1119, 1083, 1054, 1008, 971, 883, 810, 753, 710, 651, 603 cm .
ν
(C-H)arom 3145, 3057; νas(C-H)alif 2973;
νs(C-H)alif 2938;
ν
ν
δ
(CH3) 1421, 1364 (C-O) 1314,
ν
−
1
1
3
3]+, 277 (5) [Na
3]+, 344 (30) [Fe(L3 − H)Cl] . Anal. calc.
+
3
[FeL Cl ]: ESI-MS(+) m/z (%): 255 (40) [HL L
3
for [Fe(C H N )(Cl) ] (416.49): C: 37.49, H: 3.39, N: 20.18%; found: C: 37.75, H: 3.39, N: 20.41%. IR:
13
14
6
3
ν
(N-H) 3546,
ν
(C-H)ar 3011;
ν
(C=C) 1575, 1495, 1476, 1463;
ν
(C=N) 1315
ρ(C-H) 1258, 1057, 1043, 1004,
−1
9
79; γ(C-H) 869, 756 cm .
4
4]+; ESI-MS(
) m/z (%): 200 (100) [L4 − H] . Anal. calc.
−
4
[FeL Cl ]: ESI-MS(+) m/z (%): 202 (100) [H
L
−
3
for [Fe(C H N )(Cl) ] (363.44): C, 33.05; H, 3.05; N, 19.27; found: C, 32.96; H, 3.09; N, 19.90%. IR:
10
11
5
3
ν
(C-H)arom 3125, 3096, 3026; νas(C-H)alif 2913; νs(C-H)alif 2862;
ν(C=C) 1596, 1560, 1470; ν(C=N) 1430
−1
δ(CH ) 1321, ν(C-O) 1261, 1217, 1174 γ(C-H)arom 1084, 995, 958, 918, 906, 846, 774, 648, 610 cm .
3
5
5]+, 327 (20) [Fe 5
+
5
(
[FeL Cl ]
·
CH CN: ESI-MS(+) m/z (%): 202 (40) [H
L
L
Cl ] ; ESI-MS( ) m/z (%): 198
−
3
3
2
−
40) [FeCl ] . Anal. calc. for [Fe(C H N )(Cl) ]
·
CH CN (404.49): C, 35.63; H, 3.49; N, 20.78; found:
(C-H)arom 3154, 3128, 3078, 3044; νas(C-H)alif 2917;
4
10 11
5
3
3
C, 35.55; H, 3.40; N, 20.65%. IR: v(N-H) 3505
νs(C-H)alif 2892; (C=C) 1608, 1574, 1481;
γ(C-H)arom 1083, 1015, 869, 848, 767, 710, 626 cm .
ν
ν
ν
(C=N) 1420, 1388
δ
(CH ) 1310, (C-O) 1255, 1224, 1165
ν
3
−1
6
6 +
6
[FeL Cl ]: ESI-MS(+) m/z (%): 216 (100) [HL ] . Anal. calc. for [Fe(C H N )(Cl) ] (377.46): C,
3 11 13 5 3
3
5.00; H, 3.47; N, 18.55; found: C, 33.96; H, 3.51; N, 18.30%. IR: v(N-H) 3552
ν
(C-H)arom 3149, 3097;
(CH ) 1325 (C-O)
νas(C-H)alif 2917; νs(C-H)alif 2846;
ν
(C=C) 1636, 1593, 1520, 1488; (C=N) 1442
ν
δ
ν
3
−
1
1
300, 1219, 1166 γ(C-H)arom 1116, 1016, 871, 778, 710, 621 cm .
3
.3.2. Synthesis Method for ‘Closed’ Complexes
x
For complexes
7
–
12 [Fe(L ) ](OTf) the molar ratio of the ligand to the corresponding salt was 2:1.
2
2
1
6
1
To a solution of
L
–
L
in MeOH (MeCN/MeOH; 1:1, v:v; in case of ligand
L
) the methanolic solution of
Fe(OTf) was added. Mixture was stirred for 48h at room temperature. Then solvents were evaporated
2
under vacuum to the minimal amount and Et O was added. Obtained precipitate was filtrated via
2
suction filtration and washed twice with Et O (10 mL in total) and dried under vacuum. Yields based
2
on ligands are shown in Table 3.
Crystals suitable for X-ray characterization were grown by vial-to-vial diffusion of different
◦
external precipitating co-solvents into the methanolic 7–10, 12 solution of complexes at 4 C.
1
1 +
1]+, 268 (80)
7
[Fe(L ) ](CF SO )
·
C H O: ESI-MS(+) m/z (%): 241 (40) [H
L
] , 263 (5) [Na
L
2
3
3 2
4
10
1
1
2+
1
+
−
[Fe
L
] , 535 (35) [Fe
L
(
·
L
−
H)] ; ESI-MS(
−
): 149 (100) [CF SO ] . Anal. calc. for
2
3
3
[
1
1
Fe(C H N ) ](CF SO )
9.05%. IR:
225; νs(CF SO ) 1180; ρ(C-H) 1134, 1104; γ(C-H) 750, 640 cm .
C H O (908.66): C: 39.66, H: 3.77, N: 18.50%; found: C: 39.59, H: 3.84, N:
12
12 6 2
3
3 2
4
10
ν
(C-H)ar 3200, 3056;
ν
(C=C) 1640, 1575, 1480, 1475;
ν
1
(C=N) 1354, 1340; νas(CF SO ) 1240,
3
3
−
3
3
2
2 +
L L
2]+, 268 (80) [Fe 22]2+, 535 (95)
8
[
[Fe(L ) ](CF SO ) ESI-MS(+) m/z (%): 241 (75) [H
L
]
,
263 (5) [Na
2
2
3
+
3 2:
2
Fe
C: 37.60, H: 2.98, N: 20.70%. IR:
(C=N) 1390; νas(CF SO ) 1248, 1225; νs(CF SO ) 1160;
L (
L − H)] . Anal. calc. for [Fe(C H N ) ](CF SO ) (834.54): C: 37.42, H: 2.90, N: 20.14%; found:
12
12 6 2
3
3 2
ν
(N-H) 3492;
ν
(C-H)ar 3154, 3100, 2948;
ν
(C=C) 1640, 1575, 1480, 1475;
(C-H) 750,
ν
ρ(C-H) 1118, 1052, 1030, 1008; γ
3
3
3
3
−
1
6
40 cm .

Molecules 2019, 24, 3173
17 of 24
3
C H O: ESI-MS(+) m/z (%): 282 (95) [FeL3 ]2+, 563 (70) [Fe 3
3
+
9
[Fe(L ) ](CF SO )
·
L
(
L − H)] ; ESI-MS(
−
):
2
3
3 2
−
5
12
2
1
1
1
6
49 (100) [CF SO ] . Anal. calc. for [Fe(C H N ) ](CF SO )
·
C H O (950.74): C: 41.69, H: 4.24, N:
(C-H)ar 3120, 2954; ν(C=C) 1640, 1560,
(C-H) 1054, 1030;
3
3
3
13 14 6 2
3
3 2
5
12
7.68%; found: C: 41.52, H: 4.41, N: 17.25%. IR:
480, 1475;
ν
(N-H) 3475;
ν
ν
(C=N) 1370; νas(CF SO ) 1250, 1238; νs(CF SO ) 1152;
ρ
γ
(C-H) 750,
3
3
3
−
1
42 cm .
4
C H O: ESI-MS(+) m/z (%): 229 (100) [FeL4 ]+, 457 (35) [Fe 4
L − H)]+;
4
1
0
[Fe(L ) ](CF SO )
·
L
(
2
3
3 2
4
10
2
−
ESI-MS(
−
): 149 (100) [CF SO ] . Anal. calc. for [Fe(C H N ) ](CF SO ) C H O (830.56): C: 37.60,
·
3
3
10 11 5 2
3
3 2 4 10
H: 3.88, N: 16.86 %; found: C: 37.71, H: 3.57, N: 16.54%. IR:
ν
(N-H) 3208, 3185;
ν
(C-H)ar 3118, 2910;
(C-H) 1090,
−
032; γ(C-H) 765, 647 cm . H-NMR (CD CN, 300 MHz): δ (ppm) 10.85 (s, 2H); 9.12 (s, 2H); 7.64–7.40
3
ν
(C=C) 1608, 1575, 1550, 1500; (C=N) 1315; νas(CF SO ) 1248, 1230; νs(CF SO ) 1151; ρ
ν
3
3
3
3
1 1
1
(m, 6H); 7.10 (s, 2H); 7.01 (d, 2H); 6.71 (t, 2H); 4.18 (s, 6H).
[Fe(L ) ](CF SO ) ESI-MS(+) m/z (%): 229 (100) [FeL5 ]2+, 457 (20) [Fe 5
5
L
(
L − H)] ; ESI-MS(
5
+
−):
1
1
1
2
3
3 2:
2
−
49 (100) [CF SO ] . Anal. calc. for [Fe(C H N ) ](CF SO ) (756.44): C: 34.93, H: 2.93, N: 18.52 %;
3 3 10 10 5 2 3 3 2
found: C: 34.10, H: 3.09, N: 18.02%. IR:
ν
(N-H) 3540, 3480;
ν
(C-H)ar 3175, 3110, 3055;
ν
(C=C) 1610,
(C-H) 850,
−
1
7
2
576, 1540, 1500, 1418;
59 cm . H-NMR (CD CN, 300 MHz):
H); 6.77 (t, 2H); 6.37 (s, 2H); 4.28 (s, 6H).
ν
(C=N) 1352; νas(CF SO3 ) 1248; νs(CF SO ) 1151;
ρ(C-H) 1030; γ
3
3
3
−1 1
δ
(ppm) 9.28 (s, 2H); 7.69–7.52 (m, 4H); 7.06 (d, 2H); 7.01 (s,
3
6
CH OH: ESI-MS(+) m/z (%): 243 (100) [FeL6 ]2+, 635 (30) [Fe 6
+
1
2
[Fe(L ) ](CF SO )
·
L
(CF SO )] ;
2
3
3 2
3
2
2
3
3
−
ESI-MS(
−
): 149 (100) [CF SO ] . Anal. calc. for [Fe(C H N ) ](CF SO ) CH OH (816.56): C: 36.77,
·
3
3
11 13 5 2
3
3 2 3
H: 3.70, N: 17.15%; found: C: 36.66, H: 3.38, N: 17.50%. IR:
ν
(C-H)ar 3120, 2958;
(C=N) 1338; νas(CF SO ) 1254, 1237; νs(CF SO ) 1151; (C-H) 1125, 1035;
(ppm) 9.26 (s, 2H); 7.69 (t, 2H); 7.56 (d, 2H); 7.03 (d,
ν
(C=C) 1612, 1570,
1
7
2
546, 1500, 1448;
60, 648 cm . H-NMR (CD CN, 300 MHz):
H); 6.89 (s, 2H); 6.75 (t, 2H); 6.29 (s, 2H); 4.28 (s, 6H); 3.92 (s, 6H).
ν
ρ
γ
(C-H)
3
3
3
3
−1 1
δ
3
3
.4. X-ray Crystallography
Diffraction data were collected by the
four-circle diffractometer with EOS CCD detector and graphite-monochromated MoK
ω
-scan technique at 100(1)K (
2
,
6
–
10
,
12) on Rigaku XCalibur
radiation
λ = 0.71073 Å) and at 130(1)K (1, 4) on Rigaku SuperNova four-circle diffractometer with Atlas CCD
α
(
detector and mirror-monochromated CuKα radiation (λ = 1.54178 Å). The data were corrected for
Lorentz-polarization as well as for absorption effects [59]. Precise unit-cell parameters were determined
by a least-squares fit of the reflections of the highest intensity, chosen from the whole experiment.
The structures were solved with SHELXT-2013 [60] and refined with the full-matrix least-squares
procedure on F2 by SHELXL-2013 [61]. All non-hydrogen atoms were refined anisotropically,
hydrogen atoms were placed in idealized positions and refined as ‘riding model’ with isotropic
displacement parameters set at 1.2 (1.5 for CH ) times Ueq of appropriate carrier atoms. The crystals
3
of
1 were of very poor quality, and the diffraction was measurable only for low angles. Therefore
the number of data is low and the C and N atoms were refined isotropically. However, as this was
the only example of the complex of its class, and the geometry looks quite reasonable, we decided
to include this structure in the manuscript. Structures of five other complexes (generally, with large
voids with unidentified electron density) were deposited in the CDB and attached as Supplementary
material. Alerts B in 7, 8, 9, 10 and 12 are related to the lack of certain (small) number of reflections,
caused by the experimental conditions, or (for 12) by high value of Flack parameter. In this case,
we have tried the twin/basf refinement but the results are slightly inferior (even though the Alert
vanishes). Crystallographic data for the structural analysis has been deposited with the Cambridge
Crystallographic Data Centre, deposition numbers are included in Table 4, which is shown below.
Copies of this information may be obtained free of charge from: The Director, CCDC, 12 Union Road,
Cambridge, CB2 1EZ, UK; e-mail: deposit@ccdc.cam.ac.uk, or www: www.ccdc.cam.ac.uk.

Molecules 2019, 24, 3173
18 of 24
Table 4. Crystal data, data collection and structure refinement.
Compound
1
2
4
6
7
8
9
10
12
24FeN122
+
·
2(CF
SO
)
−
C
24
H
24FeN122+
·
2(CF
SO
)
−
C26H28FeN12
2+
C
20
H
22FeN102+
·
2(CF
SO
)
−
C
22
H
26FeN102+
·
2(CF
−
O+
−
C
24
H
3
3
3
3
3
3
3
3
SO )
Formula
C
12
H
12Cl
3
FeN
6
C
13
H
16Cl
2
FeN
6
·
Cl
C10H11Cl3FeN5
C11H13Cl3FeN5
−
·
C4H10O
·solvent
·2(CF3SO3) ·C5H12O
·C4H10O
·CH4O
(
FeL12) ·2(CF3SO3)
2+
−
(FeL22) ·2(CF3SO3)
2+
−
(FeL 2) ·2(CF3SO3)
3
2+
−
(FeL42) ·2(CF3SO3)
2+
−
(FeL62) ·2(CF3SO3)
2+
−
1
[Fe 2
OH)]+
−
4
6
FeL Cl3
L
Cl
2
(CH
3
·
Cl
FeL Cl3
FeL Cl3
·
C4H10O
·solvent
834.54
·C5H12O
950.74
·C4H10O
830.58
·CH3OH
816.56
Formula
weight
Crystal
system
Space group
a (Å)
4
02.48
434.52
363.44
377.46
908.66
triclinic
monoclinic
triclinic
monoclinic
monoclinic
monoclinic
triclinic
monoclinic
orthorhombic
P-1
7.459(2)
9.055(2)
12.460(3)
75.69(2)
87.94(2)
66.12(3)
743.7(4)
2
P21/n
7.6840(4)
13.9429(6)
16.8684(6)
90
98.281(4)
90
1788.39(14)
4
P-1
7.5198(4)
8.1040(6)
12.4702(10)
75.205(6)
84.655(5)
66.519(6)
673.87(9)
2
P21/c
7.2144(2)
12.4684(3)
16.7244(4)
90
99.730(2)
90
1482.75(7)
4
C2/c
23.1912(8)
17.0021(5)
22.3107(8)
90
116.163(4)
90
7895.8(5)
8
P21/c
12.9879(7)
13.0532(8)
21.9024(19)
90
91.711(6)
90
3711.5(4)
4
P-1
12.4297(8)
13.0407(7)
13.4814 (7)
88.870(4)
74.131(5)
75.262(5)
2029.9(2)
2
P21/c
15.5382(2)
12.55315(14)
18.1225 (2)
90
91.2230(13)
90
3534.05(7)
4
Pca21
22.3563(6)
8.3830(2)
17.5420(6)
90
b (Å)
c (Å)
◦
α ( )
◦
β ( )
90
90
◦
γ ( )
3
V(Å )
3287.60(16)
4
1.650
Z
Dx(g cm 3)
−
1.797
1.614
1.791
1.691
1.529
1.493
1.556
1.561
F(000)
406
13.136
884
1.305
366
14.392
764
1.553
3728
0.577
1696
0.604
980
0.565
1704
0.634
1672
0.680
µ (mm 1)
Reflections:
collected
unique
−
1450
7518
4550
2666 (0.054)
2319
6161
3106 (0.022)
2688
18337
7987 (0.031)
6243
13346
6526 (0.059)
3623
30279
7129 (0.083)
5726
15200
7095 (0.015)
6301
17885
5695 (0.029)
5323
9
17 (0.042)
716
3576 (0.024)
2958
(Rint)
with I>2σ(I)
R(F)
0
0
0
0
.078
.233
.104
.233
0.033
0.075
0.029
0.058
0.081
0.059
0.030
0.063
[
I>2σ(I)]
2
wR(F )
0.081
0.044
0.196
0.084
0.059
0.037
0.174
0.077
0.153
0.146
0.156
0.070
0.072
0.035
0.165
0.067
[
I>2σ(I)]
R(F) [all
data]
2
wR(F ) [all
0.087
0.204
0.063
0.188
0.174
0.169
0.075
0.169
data]
Goodness of
fit
1
.05
1.05
1.14
1.04
1.24
1.02
1.07
1.02
1.08
max/min ∆ρ
0
.77/−0.98
851825
0.50/−0.29
1851824
1.75/−0.79
1851829
0.32/−0.31
1851826
1.68/−0.67
1871758
0.87/−0.45
1871759
1.14/−0.62
1871760
0.34/−0.39
1871763
0.83/−0.92
1871761
−
3
(
e·Å
)
CCDC
1
number

Molecules 2019, 24, 3173
19 of 24
3
.5. Catalytic Oxidation of 2-Aminophenol
Catalytic oxidation of o-aminophenol Phenoxazinone synthase like activity of our synthesized
−5
‘
open’ and ‘close’ system complexes was investigated by the reaction of 2.0
×
10 M methanolic
−4
solutions of the complexes with 2.0
×
10 M methanolic solution of o-aminophenol (OAPH) at
room temperature and in the presence of air. The reactions were monitored on spectrophotometer
by increasing absorbance at ca. 433 nm which correspond to band from the product of reaction,
phenoxazinone chromophore (2-aminophenoxazine-3-one). Each measurement was performed three
times and given values represent a representative average.
Determination of different kinetic parameters for the catalytic activity was done using the
procedure reported earlier [25,41] based on Michaelis– Menten model. It gave a double reciprocal
Lineweaver–Burk plots on which values of Vmax were interpreted, values of KM were from 1
Vmax on
2
x-axis, while values of kcat is results of Vmax/[catalyst].
3
.6. DNA Binding Assays
3
.6.1. Absorption Titration
The absorbance titrations were performed in fixed concentration of metal complexes (20 µM) while
gradually increasing the concentration of CT-DNA within the range from 0 to 100
solution was allowed to equilibrate 5 min. before the spectra were recorded. Using the absorption
titration data, the binding constant K was determined according to the equation [62]:
µM. Each sample
b
[
DNA]/(εa − ε ) = [DNA]/(ε − ε ) +1/K (ε − ε )
f
b
f
b
b
f
where [DNA] is the concentration of CT-DNA in the base pairs, εa corresponds to the extinction
coefficient observed (Aobsd/[M]), corresponds to coefficient of free compound, is the extinction
ε
ε
b
f
coefficient of the compound fully bound to CT-DNA, and K is the intrinsic binding constant. The Kb
b
value was given by the ratio of slope to intercept in the plot of [DNA]/(εa − ε ) versus [DNA].
f
The standard Gibb’s free energy for DNA binding was calculated based on the equation [63]:
0
h
G = −RTlnK
b
where ∆G 0h is the standard Gibb’s free energy, R corresponds to gas constant, T is temperature and Kb
stands for binding constant of the appropriate complex.
3
.6.2. Competitive Binding Fluorescence Experiment
The competitive binding of compounds with ethidium bromide (EB) has been investigated by the
fluorescence emission spectroscopy in order to evaluate its ability to displace EB from its DNA–EB
fluorescent complex. The solution of EB (20
for 30 min. at 25 C before the measurements were taken. While increasing the concentration of
µ
M) and CT-DNA (26
µ
M) was allowed to equilibrate
◦
investigated complexes, the fluorescence spectra were recorded in the range of 540–720 nm at λexc
67 nm. The ability of compounds to quench the emission of DNA-EB complex was evaluated by the
Stern–Volmer equation [64]:
=
4
I0/I = 1 + K
SV
were I and I are fluorescence intensities in absence and presence of compound, respectively, and KSV
0
is the Stern–Volmer constant. KSV depends on the ratio of bound EB to the concentration of CT-DNA.
3
.6.3. Circular Dichroism Studies
For the CD experiments 2 M oligo DNA d(GTTAATCGCTGG) as a solution in Tris Buffer (10 mM
µ
TrisHCl, 5 mM NaCl, 50 mM pH 7.5) was used. 1–30 eq. of complexes were added to the solution 5 min.
◦
before measurements. At 1:30 molar ratio the samples were annealed at 70 C for 5 min. and then

Molecules 2019, 24, 3173
20 of 24
slowly cooled down to room temperature. Quartz cuvettes with a path length of 1 cm were used
◦
with the sample volumes of 2 mL. Spectra were collected in the range 320–220 nm at 20 C and were
averaged over three scans.
4
. Conclusions
Synthesis of a small library of Schiff base ligands is presented, which were designed so as to behave
as efficient tridentate chelators with simultaneous incorporation of different number and disposition of
non-coordinating NH moieties. Information encoded within iron(II/III) metallic centres and counterions
x
allowed for generation of two families of complexes, which we term as ‘open’: [Fe(L )Cl ] and ‘closed’:
3
x
[
[Fe(L ) ](OTf) . Complexes were studied as phenoxazinone synthase activity mimics and DNA-binders,
2
2
with the aim to see if there is an effect of hydrogen bonding on the studied biological activity.
Catalytic studies and derived kinetic parameters of 2-aminophenol (OAPH) oxidation with
synthesized complexes show that the number and disposition of NH moieties influences the initial
binding of OAPH to the complex. Nonetheless, its subsequent oxidation to 2-aminophenoxazine-3-one
chromophore (APX) and thus catalytic activity is an outcome of additional factors. Consequently,
attributing their activity solely to H-bonding is inaccurate. We postulate however that the mechanism
of action differs within the ‘open’ and ‘closed’ families, and effect of non-covalent interactions is more
important in the latter case.
The more straightforward effect of employing benzimidazole and imidazole moieties in the
x
ligands scaffold was observed for DNA-binding studies. Intriguingly, the family of ‘open’ [Fe(L )Cl ]
3
complexes do not interact with studied nucleic acid, which can be attributed to the effect of negatively
charged chlorides, which may repel the complex from the sugar-phosphate backbone of CT-DNA.
x
In contrast, ‘closed’ [Fe(L ) ](OTf) complexes exhibit the behavior of bulky cations that are able to
2
2
interact with the negatively charged backbone of DNA. Electronic absorption titration, fluorescence
competitive binding with EB and CD titration allowed one to confirm that from the group of triflate
analogues 7–12, only the pyridine-scaffolded ‘closed’ species 11 and 12 effectively bind with DNA,
most probably by the intercalation-type mode. This shows that the benzimidazole moiety effectively
precludes the remaining species from intercalation or any other significant interaction with nucleic
acids and the imidazole substituent can be used for tuning of the interaction strength. In our previous
works [38,40,65], a similar phenomenon was observed, where complexes without hydrogen bonding
donors in the “primary” structure bind to DNA scaffold—such as helical and “open” complexes with
terpyridine-type and quaterpyridine-type ligands [66].
We are currently working on ways to introduce H-bonding moieties in the immediate vicinity of
the compounds’ coordination sphere as well as for utilization of the above-demonstrated phenomenon
on aspects of crystal engineering and spin-crossover magnetic properties.
Supplementary Materials: The following are available online: Ms PDF document with all supplementary Figures
and Tables.
Author Contributions: A.B.: repeated synthesis and full characterization of ligands, synthesis and characterization
of ‘open’ 1–6 and ‘closed’ 7–12 iron complexes, writing—original draft preparation, catalytic measurements;
M.S. and G.N.R.: biological measurements and their description, writing—original draft preparation; D.B.:
catalytic measurements; M.K.: X-ray measurements and description of crystal structures; M.W.-C.: first synthesis
4
5
of ligands L and L ; M.A.F.-J.: planning of DNA binding assays, biological measurements and their description,
writing—final version of paragraphs concerning DNA binding, review and editing; A.G.: conceptualization,
1
3
first synthesis of ligands L –L and complexes
writing—review and editing.
1–6, writing—final manuscript, review and editing, V.P.: supervision,
Funding: A.G. thanks to the National Science Centre in Cracow, Poland (grant PRELUDIUM
no. 2015/17/N/ST5/01973) for financial support. Supported by the Foundation for Polish Science (FNP).
M.A.F.J. thanks to the National Science Centre in Cracow, Poland (grant SONATINA no. 2017/24/C/ST5/00181) for
financial support. A.B.: the work was supported by the grant no. POWR.03.02.00-00-I023/17 co-financed by the
European Union through the European Social Fund under the Operational Program Knowledge Education
Development. M.S.: the work was supported by the grant no. POWR.03.02.00-00-I026/16 co-financed
by the European Union through the European Social Fund under the Operational Program Knowledge
Education Development.

Molecules 2019, 24, 3173
21 of 24
Acknowledgments: We thank Justyna Pachla for assistance in synthesis of ligand L6.
Conflicts of Interest: The authors declare no conflict of interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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