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

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

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

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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.

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2

. Results and Discussion

2

.1. Synthesis and Characterization

A series of six hydrazone Schiﬀ base ligands L¹

–

L⁶was 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(L^x)Cl ] ‘open’ (left) and B: [Fe(L^x)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 (L¹L⁶, 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

◦

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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(L^x) ]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

k_c_a_t(h )

Catalyst

K_M(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

2.19

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

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

Fe(L ) ](OTf) (8)

2

3

Fe(L ) ](OTf) (9)

183.80

42.76

65.14

2

4

Fe(L ) ](OTf) (10)

2 2

5

Fe(L ) ](OTf) (11)

Fe(L ) ](OTf) (12)

2

6

106.53

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.

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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].

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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 ],

K_M= 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.

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Table 2. Catalytic oxidation of o-aminophenol with transition metal complexes.

k_c_a_t(h ¹)

−

Catalyst

Solvent

Ref.

1

[

Fe(L )Cl ] (1)

Methanol

DMF

185.25

199.40

137.0

56.0

32.36

196.18

33.26

54.0

This work

[28]

3

1

[

Fe(L ) ](OTf) (7)

2

a

[

L

FeCl (L )]

Fe(L )Cl ]

)(4,4 -byp)ClO }]n

2

b

Methanol

b

[20]

[42]

[23]

[45]

[46]

[47]

3

c

0

[

{Fe(

4

c

[

FeL Cl]

2

d

[

Co(L )(N ) ]

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

L = 1,3-bis(5 -methyl-2 -thiazolylimino)isoindoline; L = N,N’-Bis(2-Methylbenzimidazolyl) pyridinediamide;

c

e

f

d

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 K_Mvalues 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

, K_Mdecreases in the order of

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 K_Mand 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, K_Mdecreases 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.

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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

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

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 L⁵and L⁶to 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

constants are: K_S_V= 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

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

11 of 24

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

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

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)_b_r

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, H_b_,_c_,_g); 7.01 (s, 2H, H_a_,_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, H_b_,_c); 7.00–6.96 (dd, 2H, J = 5.9, 3.2 Hz, H_a_,_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

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

14 of 24

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, H_b_,_c_,_g); 7.10–6.99 (m, 3H, H_a_,_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, H_b_,_c_,_h),

(j) (f)

.04–6.99 (m, 3H, H_a_,_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

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)_i_m_i_n_.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, H_c_,_d); 7.23 (s, 1H, H

1

L

]⁺, 224 (25) [Na

L

⁴]⁺; ESI-MS(

−

): 200 (100) [L⁴− H] . IR:

−

ν

(N-H)_b_r

(C=N)ar

(ppm) 8.18

); 6.73 (t,

(ppm) 157.4,

3

1

(

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)_i_m_i_n_.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

(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 , H_h);

(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

(

L^x)]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 FeCl₃6H 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

(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

55.0

30.0

453.6

63.8

30.0

51.6

48.7

61.6

45.3

very 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

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

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

6

3

ν

(C-H)arom 3125, 3093; νas(C-H)_a_l_i_f2992, 2924; νs(C-H)_a_l_i_f2849; 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

[FeL Cl (MeOH)]Cl: ESI-MS(+) m/z (%): 241 (70) [H

L

Cl ] ; ESI-MS(

−

) m/z (%):

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)_a_l_i_f2973;

νs(C-H)_a_l_i_f2938;

ν

δ

(CH3) 1421, 1364 (C-O) 1314,

ν

−

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)_a_l_i_f2913; νs(C-H)_a_l_i_f2862;

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

Cl ] ; ESI-MS( ) m/z (%): 198

−

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)_a_l_i_f2917;

4

10 11

5

3

C, 35.55; H, 3.40; N, 20.65%. IR: v(N-H) 3505

νs(C-H)_a_l_i_f2892; (C=C) 1608, 1574, 1481;

γ(C-H)arom 1083, 1015, 869, 848, 767, 710, 626 cm .

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)_a_l_i_f2917; νs(C-H)_a_l_i_f2846;

ν

(C=C) 1636, 1593, 1520, 1488; (C=N) 1442

ν

δ

ν

3

−

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

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

2+

1

+

−

[Fe

L

] , 535 (35) [Fe

L

(

·

L

−

H)] ; ESI-MS(

−

): 149 (100) [CF SO ] . Anal. calc. for

2

3

[

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

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

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

−

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

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

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

(C=N) 1370; νas(CF SO ) 1250, 1238; νs(CF SO ) 1152;

ρ

γ

(C-H) 750,

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

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

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

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

−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

3

−

ESI-MS(

−

): 149 (100) [CF SO ] . Anal. calc. for [Fe(C H N ) ](CF SO ) CH OH (816.56): C: 36.77,

·

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

−1 1

δ

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

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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 K_Mwere 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

f

where [DNA] is the concentration of CT-DNA in the base pairs, εa corresponds to the extinction

coeﬃcient observed (A_o_b_s_d/[M]), corresponds to coeﬃcient of free compound, is the extinction

ε

b

f

coeﬃcient of the compound fully bound to CT-DNA, and K is the intrinsic binding constant. The K_b

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 ∆G⁰_h is the standard Gibb’s free energy, R corresponds to gas constant, T is temperature and K_b

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 K_S_V

0

is the Stern–Volmer constant. K_S_Vdepends 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

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

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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Sample Availability: Samples of the compounds: L1–L6, 1–12 are available from the authors.

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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

DOI: 10.3390/molecules24173173

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