Comparison of nonheme manganese‐ and iron‐containing flavone synthase mimics

Article abstract of DOI:10.3390/molecules26113220

Heme and nonheme‐type flavone synthase enzymes, FS I and FS II are responsible for the synthesis of flavones, which play an important role in various biological processes, and have a wide range of biomedicinal properties including antitumor, antimalarial, and antioxidant activities. To get more insight into the mechanism of this curious enzyme reaction, nonheme structural and functional models were carried out by the use of mononuclear iron, [Fe^II(CDA‐BPA*)]²⁺ (6) [CDA‐BPA = N,N,N’,N’‐tetrakis‐(2‐pyridylmethyl)‐cyclohexanediamine], [Fe^II(CDA‐BQA*)]²⁺ (5) [CDA‐BQA = N,N,N’,N’‐tetrakis‐(2‐quinolilmethyl)‐cyclohexanediamine], [Fe^II(Bn‐TPEN)(CH3CN)]²⁺ (3) [Bn‐ TPEN = N‐benzyl‐N,N’,N’‐tris(2‐pyridylmethyl)‐1,2‐ diaminoethane], [Fe^IV(O)(Bn‐TPEN)]²⁺ (9), and manganese, [Mn^II(N4Py*)(CH3CN)]²⁺ (2) [N4Py* = N,N‐bis(2‐pyridylmethyl)‐1,2‐di(2‐pyridyl)ethyl-amine)], [Mn^II(Bn‐TPEN)(CH3CN)]²⁺ (4) complexes as catalysts, where the possible reactive inter-mediates, high‐valent Fe^IV(O) and Mn^IV(O) are known and well characterised. The results of the catalytic and stoichiometric reactions showed that the ligand framework and the nature of the metal cofactor significantly influenced the reactivity of the catalyst and its intermediate. Comparing the reactions of [Fe^IV(O)(Bn‐TPEN)]²⁺ (9) and [Mn^IV(O)(Bn‐TPEN)]²⁺ (10) towards flavanone under the same conditions, a 3.5‐fold difference in reaction rate was observed in favor of iron, and this value is three orders of magnitude higher than was observed for the previously published [Fe^IV(O)(N2Py2Q*)]²⁺ [N,N‐bis(2‐quinolylmethyl)‐1,2‐di(2‐pyridyl)ethylamine] species.

Full text of DOI:10.3390/molecules26113220

molecules
Article
Comparison of Nonheme Manganese- and Iron-Containing
Flavone Synthase Mimics
2
Dóra Lakk-Bogáth 1, Natalija Pantalon Juraj 2 , Bashdar I. Meena 1, Berislav Peric´
, Srec´ko I. Kirin 2
and József Kaizer 1,
*
1
2
Research Group of Bioorganic and Biocoordination Chemistry, University of Pannonia,
H-8201 Veszprém, Hungary; lakkd@almos.uni-pannon.hu (D.L.-B.); bashdarismael@gmail.com (B.I.M.)
Division of Materials Chemistry, Rud¯er Boškovic´ Institute, Bijenicˇka c. 54, HR-10000 Zagreb, Croatia;
Natalija.Pantalon.Juraj@irb.hr (N.P.J.); Berislav.Peric@irb.hr (B.P.); srecko.kirin@irb.hr (S.I.K.)
Correspondence: kaizer@almos.uni-pannon.hu; Tel.: +36-88-62-4720
*
Abstract: Heme and nonheme-type flavone synthase enzymes, FS I and FS II are responsible for
the synthesis of flavones, which play an important role in various biological processes, and have a
wide range of biomedicinal properties including antitumor, antimalarial, and antioxidant activities.
To get more insight into the mechanism of this curious enzyme reaction, nonheme structural and
functional models were carried out by the use of mononuclear iron, [FeII(CDA-BPA*)]2+
(
6
) [CDA-
BPA = N,N,N’,N’-tetrakis-(2-pyridylmethyl)-cyclohexanediamine], [FeII(CDA-BQA*)]2+
(5) [CDA-
BQA = N,N,N’,N’-tetrakis-(2-quinolilmethyl)-cyclohexanediamine], [FeII(Bn-TPEN)(CH3CN)]2+
[Bn-TPEN = N-benzyl-N,N’,N’-tris(2-pyridylmethyl)-1,2-diaminoethane], [FeIV(O)(Bn-TPEN)]2+
(9),
(3)
and manganese, [MnII(N4Py*)(CH3CN)]2+
ethylamine)], [MnII(Bn-TPEN)(CH3CN)]2+
(
(
2
) [N4Py* = N,N-bis(2-pyridylmethyl)-1,2-di(2-pyridyl)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
4) complexes as catalysts, where the possible reactive
intermediates, high-valent FeIV(O) and MnIV(O) are known and well characterised. The results of the
catalytic and stoichiometric reactions showed that the ligand framework and the nature of the metal
cofactor significantly influenced the reactivity of the catalyst and its intermediate. Comparing the reac-
Citation: Lakk-Bogáth, D.; Juraj, N.P.;
Meena, B.I.; Peric´, B.; Kirin, S.I.;
Kaizer, J. Comparison of Nonheme
Manganese- and Iron-Containing
Flavone Synthase Mimics. Molecules
2021, 26, 3220. https://doi.org/
10.3390/molecules26113220
tions of [FeIV(O)(Bn-TPEN)]2+ ) and [MnIV(O)(Bn-TPEN)]2+
(9 (10) towards flavanone under the same
conditions, a 3.5-fold difference in reaction rate was observed in favor of iron, and this value is three
orders of magnitude higher than was observed for the previously published [FeIV(O)(N2Py2Q*)]2+
[N,N-bis(2-quinolylmethyl)-1,2-di(2-pyridyl)ethylamine] species.
Academic Editors: Jadwiga Handzlik
and Katarzyna Kucwaj-Brysz
Keywords: flavone synthase; iron(IV)-oxo; manganese(IV)-oxo; oxidation; kinetic studies
Received: 7 May 2021
Accepted: 26 May 2021
Published: 27 May 2021
1. Introduction
Flavones are low molecular weight phytochemicals that play an important role
in various biological processes and have a positive impact on our health [1]. Due to
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
their wide range of biological activities (malaria, anti-cancer, anti-diabetes, asthma, an-
tiviral, antioxidant, anti-microbial, anti-ulcer, anti-inflammation, cardiovascular activity,
neuroprotection, etc.) their syntheses have become important goals of medicinal and
bioorganic chemists [
Baker-Venkataraman-rearrangement from o-hydroxyacetophenone [
flavanones using various stoichiometric reagents such as DMSO/I2 [
2
–
8
]. Flavones can be synthesised by various methods including
], and oxidation of
], SeO2 [10], 2,3-
1
9
dichloro-5,6-dicyano-1,4-benzoquinone [11], thallium salts [12] and manganese acetate [13].
The oxidation of flavanones by heme and nonheme iron-dependent enzymes is one of
the most important steps during the biosynthesis of flavones. High-valent oxoiron(IV)
intermediates as key oxidants are well-established in both heme and nonheme enzymes,
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
including cytochrome P450, bovine liver catalase (BLC) [14
II) [16 20], pterin-dependent phenylalanine hydroxylase [21], and
dioxygenases (taurine dioxygenase, TauD [22 24] and flavone synthase I, FS I) [25
,
15], flavone synthase II (FS
-keto acid-dependent
28],
–
α
–
–
Molecules 2021, 26, 3220. https://doi.org/10.3390/molecules26113220
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3220
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and their synthetic biomimetic models. Interestingly, two main classes of flavone syn-
thase enzymes are known, FS I and FS II, with totally different active sites and catalytic
mechanisms (Scheme 1). The majority of flavone synthase enzymes (FS II) contain iron(III)-
protoporphyrin (PFeIII) as a prosthetic group with (P+•)FeIV=O oxidant (CYP93B), and
the reaction proceeds via the formation of 2-hydroxyflavanone (monooxygenase activity)
and its subsequent dehydration into the flavones [29]. In contrast, FS I enzymes utilise
nonheme mononuclear iron(II)-2-oxoglutarate (FeII/2-OG) as a prosthetic group where the
reaction can be described by oxoiron(IV) mediated, direct non-concerted 2,3-desaturation
without 2-hydroxyflavanone formation [30].
Scheme 1. Oxidation of flavanone by heme and nonheme flavone synthases, FS I and FS II.
Since flavanone itself is a chiral molecule, oxidative kinetic resolution (OKR) of racemic
flavanones can also be performed with a chiral iron catalyst and oxoiron(IV) intermediates.
Increasing interest in the region and stereoselective metal-based reactions to generate new
stereogenic centres in a highly diastereoselective and/or enantioselective fashion inspires
the search for biomimetic oxidation catalysts. Intermediates of this type were observed
in catalytic oxidation systems and synthetised and identified indirectly by the use of iron
precursor complexes with various chiral and achiral aminopyridine ligands [31–36].
In the present work, we carried out stoichiometric and catalytic flavanone oxidation re-
actions with spectroscopically well-characterised nonheme oxoiron(IV) intermediates com-
pared to their analogous oxomanganese(IV) compounds, [FeIV(O)(Bn-TPEN)]2+
[FeIV(O)(CDA-BPA)]2+ 11), [MnIV(O)(N4Py*)]2+ ) [39], [MnIV(O)(Bn-TPEN)]2+
and their precursor complexes, [FeII(Bn-TPEN)(CH3CN)]2+ ), [FeII(CDA-BQA*)]2+
[FeII(CDA-BPA*)]2+ ) [41], [MnII(N4Py*)(CH3CN)]2+
(
9
) [37
,38],
(
(8
(10) [40
]
(
3
(
5),
(6
(2
) [39], [MnII(Bn-TPEN)(CH3CN)]2+
(4) (Scheme 2) [40]. To the best of our knowledge, this study provides the first mechanistic
details of oxomanganese(IV)-mediated flavanone oxidation compared to their analogous
oxoiron(IV)-mediated systems, which may serve as a functional model of FS enzymes.
Based on the detected intermediary products, the catalysis of double-bond formation is
suggested to take place in two steps, namely by the monohydroxylation of the substrate,
and then the elimination of water from the intermediary 2-hydroxyflavanone. This mech-
anism is different from the hitherto known FS I enzyme, but it is consistent with other
2-oxoglutarate-dependent enzymes, and the heme iron-dependent flavone synthase II.

Molecules 2021, 26, 3220
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Scheme 2. Oxoiron(IV) and oxomanganese(IV) complexes with their iron(II) and manganese(II)
precursor complexes were used in this study.
2. Results and Discussion
2.1. Nonheme Iron and Manganese-Containing Biomimics of the Flavone Synthase Enzyme
The use of well-chosen ligands made it possible to prepare, spectroscopically charac-
terise, and study the reactivity of the putative intermediates in enzymatic processes. In
the last 20 years, a number of precursor iron(II) complexes with their high-valent oxo-
iron(IV) intermediates have been prepared by the use of multidentate N-donor ligands
such as TPA, N4Py, Py5 [2,6-(bis-(bis-2-pyridyl)methoxymethane)pyridine] and TPEN
(N,N,N0,N0-tetrakis(2-pyridylmethyl)- ethylenediamine) [37
–42]. These complexes as cata-
lysts and/or intermediates catalyse all types of oxidation reactions such as epoxidations,
heteroatom oxidations, and even C-H oxidations including hydrogen-atom transfer (HAT)
and oxygen-atom transfer (OAT) [43–45].
The primary aspect of ligand selection, as a continuation of our previous work, was to
increase the reactivity of the catalyst and its intermediates towards flavanone [46]. For this
purpose, new ([FeII(CDA-BQA*)]2+
(
5
), [FeIV(O)(CDA-BPA*)]2+
(
11)) and spectroscopically
), [FeII(Bn-TPEN)(CH3CN)]2+ ) [FeIV(O)(Bn-
9)) nonheme iron(II) and oxoiron(IV) complexes, were chosen. Since nonheme
well-characterised ([FeII(CDA-BPA*)]2+
TPEN)]2+
(
6
(3
(
oxomanganese (IV) complexes have proven to be versatile oxidants [40], in addition to
iron-containing models we also aimed to elucidate the role of the metal cofactor through
the comparison of well-defined iron- and manganese-containing systems. Previously
reported [MnII(N4Py*)(CH3CN)]2+
[MnIV(O)(N4Py*)]2+ ), [MnIV(O)(Bn-TPEN)]2+
tion reactions were chosen for these measurements [39
of flavanone was performed with , and . Catalytic oxidation of ethylbenzene was
performed with 5 and 6, and stoichiometric oxidation reactions were performed with 7, 8,
10, and 11. N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) is a well-known
(
2
), [MnII(Bn-TPEN)(CH3CN)]2+
(
4
) as catalysts and
10) as possible intermediates in the oxida-
40]. In this work, catalytic oxidation
(8
(
,
2,
3
,
4,
5
6
9,
metal chelator. TPEN complexes are often used as Zn(II) and Cd(II) indicators, and in this
case, substituting the pyridine for quinoline results in enhancement of fluorescence inten-
sity and use of these ligands as fluorescent probes [47
,48]. Fe(II) complexes of the TPEN
group of ligands have interesting electronical properties, where conformational changes

Molecules 2021, 26, 3220
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are linked to different spin-state interconversion processes [41]. Due to the interesting
redox behaviour of these Fe(II) complexes they have been studied as superoxide dismutase
mimics [49] and for their reactivity towards hydrogen peroxide [50].
In this work, a single-crystal structure was obtained for the complex [FeII(
CDA-
±
BQA)](CF3SO3)2 (5). The complex was prepared with the racemic version of ligand CDA-
BQA*. In the CSD database, there are 29 structures of Fe(II) complexes of these types of
ligands, with four pyridyl or quinoline groups connected by an ethylenediamine or cyclo-
hexanediamine linker [51
linker is [FeII(CDA-BPA*)](ClO4)2 (
newly synthesised [FeII(CDA-BQA*)](CF3SO3)2 (
) is compared in Figure 2 and Table 1. Both complexes are prepared with racemic ligands,
however, 5 crystallised as a racemate, while 6 has spontaneously resolved into its optical
isomers, containing only the (R,R) enantiomer. While complex has a regular octahedral
geometry, the Fe-N bonds in are elongated, forming a pentagonal bipyramidal geometry
with an equatorial vacancy, as determined using the program FindGeo [53]. The reason
for this is likely the steric crowding of the quinoline groups in . The significantly longer
Fe-N bond lengths (>2.2 Å) are in agreement with a high spin Fe(II) centre in . The
UV-Vis spectrum of in acetonitrile is dominated by the intense * band at 307 nm
(=12,800 M−1 cm−1), and an additional broad feature of low intensity between 330 and
460 nm ( = 356 with
= 2080 M−1 cm−1) can be assigned to an MLCT transition. The
weak intensity is consistent with the high spin state of the iron(II) centre [46].
,
52]. The only reported Fe(II) complex with a cyclohexanediamine
) (CSD refcode YAMXAL) [41] The geometry of the
) (Figure 1) and [FeII(CDA-BPA*)](ClO4)2
6
5
(6
6
5
5
5
5
π-π
λ
max
ε
Figure 1. X-ray structure of FeII(
CDA-BQA*)](CF3SO3)2 (5), showing a 30% probability of ther-
±
mal ellipsoids.

Molecules 2021, 26, 3220
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Figure 2. Overlay of structures FeII(CDA-BQA*)](CF3SO3)2 ( ) (red) and [FeII(CDA-BPA*)](ClO4)2 (
5 6) (blue) containing
only (R,R)-enantiomers of the ligands.
Table 1. Selected bond lengths of complex [FeII(
CDA-BQA*)](CF3SO3)2 (5) compared to the literature example of
±
[FeII(CDA-BPA*)](ClO4)2 (6) complex. The geometry was determined using the program FindGeo [53].
Fe-N1/Å
Fe-N2/Å
Fe-N3/Å
Geometry/RMSD/Å
Pentagonal bipyramid with a vacancy
[FeII(±CDA-BQA*)](CF3SO3)2 (5)
[FeII(CDA-BPA*)](ClO4)2 (6) [41]
2.194
2.238
2.252
(equatorial) (regular)
0.271
Octahedron (regular)
0.369
1.993
2.003
2.012
2.038
1.994
1.993
2.2. Catalytic Oxidation Reactions
The catalytic activities of the three ferrous and two manganese complexes [FeII(Bn-
TPEN)(CH3CN)]2+ ), [FeII(CDA-BQA*)]2+ ), [FeII(CDA-BPA*)]2+ ), [MnII(N4Py*)
(CH3CN)]2+ ) and [MnII(Bn-TPEN)(CH3CN)]2+
) were studied in the oxidation of
(
3
(
5
(6
(2
(4
flavanone, utilising mCPBA (meta-chloroperoxybenzoic acid) as the co-oxidant. The oxi-
dation reactions were carried out under standard catalytic conditions (5:100:500 ratio for
the catalyst:substrate:oxidant) in acetonitrile at room temperature (Table 2 and Figure 3).
Similarly to our previous results, it took less than 10 min to have about 30–36% and 19–24%
yields (based on the substrate) for the iron(II)- and manganese(II)-catalysed reactions,
respectively. Much lower yields (0.36 and 2.8%) were observed for the Fe(ClO4)2 and
Mn(ClO4)2 salts, respectively. Both the iron(II) and manganese(II)-catalysed oxidations
of flavanone produced flavones (F) as a major product in all cases in addition to two
minor products, namely 2-hydroxy-2-phenyl-chroman-4-one (A, 2-hydroxy-flavanone) and
its open tautomeric form 1-(2-hydroxy-phenyl)-3-phenyl-propane-1,3-dione (D). It was
found earlier [46] that complex
1 together with mCPBA oxidizes flavanone, and a turnover
number (TON) of 6.93 for F, 0.11 for D and 0.02 for A was obtained with an overall yield of
9.42%. Its manganese analogue produced an overall yield of 18.8%, TON for F = 3.58, TON
for D = 0.19 and TON for A = ~0.01.

Molecules 2021, 26, 3220
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◦
Table 2. Catalytic oxidation of flavanone (FH2) carried out by 2, 3, 4, 5 and 6 with mCPBA in CH3CN at 25 C.
[H2O]
/mM
[MII]
/mM
[FH2]
/mM
[mCPBA]
/mM
Yield (F)
/%
Yield (D) c
/%
Yieldt
/%
MII
TON
TON
[FeII(CDA-BPA*)]2+
44
44
44
44
55
65
75
85
95
44
44
44
5
5
5
5
5
5
5
5
5
5
5
5
100
100
100
100
100
100
100
100
100
100 a
100 b
100
500
500
500
500
500
500
500
500
500
500
500
500
35.2
29
7.04
5.8
1.15
1.4
0.23
0.28
0.17
0.19
0.23
0.3
36.35
30.4
(6)
[FeII(Bn-TPEN)]2+
(3)
[FeII(CDA-BQA*)]2+
20.8
17.9
15.7
13.8
12.2
10.7
9
4.16
3.58
3.14
2.76
2.44
2.14
1.88
3.36
2.24
4.62
0.83
0.92
1.2
21.63
18.8
(5)
[MnII(N4Py*)]2+
(2)
[MnII(N4Py*)]2+
16.9
(2)
[MnII(N4Py*)]2+
1.45
2.4
15.25
14.64
13.9
(2)
[MnII(N4Py*)]2+
0.48
0.64
0.92
0.2
(2)
[MnII(N4Py*)]2+
3.2
(2)
[MnII(N4Py*)]2+
4.2
13.2
(2)
[MnII(N4Py*)]2+
16.8 a
11.2 b
1.1
17.9
(2)
[MnII(N4Py*)]2+
1.8
0.36
0.12
12.95
23.7
(2)
[MnII(Bn-TPEN)]2+
(4)
23.1
0.61
a
b
In the presence of 2,6-di-tert-butylphenol (2,6-DTBP), [2,6-DTBP] = 25 mM. In the presence of 2,6-di-tert-butylphenol (2,6-DTBP),
[2,6-DTBP] = 60 mM.c Yield of A is less than 0.1%.
Figure 3. Comparison of the flavones-synthase activity of iron(II) and manganese(◦II) complexes.
Conditions: [MII] = 5 mM, [FH2] = 100 mM and [mCPBA] = 500 mM in CH3CN at 25 C.

Molecules 2021, 26, 3220
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It is important to mention that the amount of 1,3-dione (D) can be increased from
0.92% (TON = 0.19) to 4.2% (TON = 0.92) in the presence of H2O, suggesting an equilibrium
step during the flavone formation (Figure 4B). However, when 2,6-di-tert-butylphenol
(DTBP) was added significantly less flavanone was converted, suggesting a free-radical
type mechanism as a parallel process of the metal-based oxidation (Figure 4A). Significantly
higher yields were observed for
3
(30.4%, TON for F = 5.80, TON for D = 0.28),
5 (21.6%,
TON for F = 4.16, TON for D = 0.17),
6
(36.35%, TON for F = 7.04, TON for D = 0.23) and 4,
suggesting clearly that the ligand framework and the nature of the metal influenced the
catalytic activities of these complexes. The relative reactivities of iron(II) and manganese(II)
complexes are in the order of [FeII(CDA-BPA*)]2+ ) > [FeII(Bn-TPEN)(CH3CN)]2+
(6 (3)
> [MnII(Bn-TPEN)(CH3CN)]2+ ) > [FeII(CDA-BQA*)]2+
(
4
(5
) > [MnII(N4Py*)(CH3CN)]2+
(
2
) > [FeII(N4Py*)(CH3CN)]2+
(1). It should be underlined that the presence of both the
theoretical tautomers (A and D) of the acid-labile intermediate of flavone strongly supports
the hypothesis that flavone biosynthesis proceeds via 2-hydroxylation of flavanone. A
similar mechanism was proposed for the cytochrome P450 monooxygenase (FS II) and
other 2-oxoglutarate-dependent dioxygenases.
Figure 4.
(A
) Comparison of the product formation in the [MnII(N4Py*)(CH3CN)]2+
(
2) catalysed oxidation of flavanone
with mCPBA as co-oxidant and 2,6-DTBP as a radical trapping agent. (B) Comparison of the product formation in the
presence of added water.
Recently, chiral N4Py-type and L-proline derived aminopyridine containing oxo-
iron(IV) complexes were reported which could perform enantioselective oxidation of
various substrates such as thioanisole, hydrocarbons, alkenes and substituted cyclohex-
anones [31,33]. To the best of our knowledge, these are the first examples of chiral non-
heme oxoiron(IV) complexes tested in asymmetric C-H hydroxylation reactions. Since
flavanone is a chiral molecule including benzylic C-H bonds, oxidative kinetic resolution
(OKR) of racemic flavanones can in principle also be performed with a chiral iron cata-
lyst and oxoiron(IV) intermediates. The oxidation of ethylbenzene that can be used as a
model compound of flavanone, by chiral [FeIV(O)(N4Py*)]2+
(7) species showed moderate
enantioselectivities up to 33% ee as a result of a non-rebound mechanism including the
epimerization of the long-lived alkyl radical before the rebound step [36]. To increase the
enantioselectivity, we also examined the enantioselectivity of the [FeII(CDA-BPA*)]2+
(6)
catalyst and the in situ generated [FeIV(O)(CDA-BPA*)]2+
(11) intermediate from 6 and
PhIO in the asymmetric C-H hydroxylation of ethylbenzene utilizing TBHP and PhIO as
co-oxidants (Figure 5 and Table 3). In these probe reactions, moderate enantioselective
hydroxylation could be obtained in all cases, so the studies on oxidative kinetic resolution
of racemic flavanones were discarded.

Molecules 2021, 26, 3220
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Figure 5. Dependence of the K/A ratio and the enantiomeric excess (ee%) on the oxidant concen-
tration for the [FeII(CDA-BPA*)]2+
0 C.
(
6) catalysed oxidation of ethylbenzene with TBHP in CH3CN at
◦
Table 3. Catalytic oxidation of ethylbenzene carried out by (R,R)-(
co-oxidants a.
−
)-6
and (R,R)-(
−
)-
5
with various
Yield
/% d
ee
/%
TON (A) b
[6]:[PhEt]:[Co-oxidant]
TON (K) c
K/A
2:500:50 (TBHP)
2:500:100 (TBHP)
2:500:200 (TBHP)
2:1500:16 (PhIO)
1.38
1.82
2.06
0.96
2.83
5.20
6.17
2.14
16.84
14.04
8.23
2.05
2.86
3.00
2.23
7.25 (R)
4.13 (R)
2.68 (R)
12.21 (R)
38.75
Yield
/% d
ee
/%
[5]:[PhEt]:[Co-oxidant]
2:500:100 (TBHP)
TON (K c)
10.08
K/A
-
TON (A b)
-
20.16
-
a
b
c
d
Reaction conditions: see Experimental section. 1-Phenylethanol. Acetophenone. Based on oxidant.
2.3. Stoichiometric Oxidation Reactions
We have previously reported for a family of aminopyridine containing oxoiron(IV) com-
plexes, FeIV(O)(N4Py) [N4Py = N,N’-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine],
FeIV(O)(TPA) [TPA = tris(2-pyridylmethyl)amine] that the reactivity in HAT and OAT reac-
tions can be significantly enhanced by the introduction of 6-substituents on the pyridine
rings (TPA), or by the replacement of one or more pyridyl arms of TPA, N4Py or N4Py*
with quinolyl arms, despite their thermal stabilities [46,54,55]. Modification of the N4Py
ligand as described above resulted in the following reaction rate orders for the flavanone ox-
idation: [FeIV(O)(N2Py2Q*)]2+ > [FeIV(O)(N4Py)]2+ > [FeIV(O)(N4Py*)]2+ (Table 4), where
the most reactive complex reacts 80-fold faster than the slowest one [46]. The stability and
reactivity of the complexes are also greatly influenced by the number of nitrogen-donor
atoms and their hybrid state. For example the oxidation of ethylbenzene by pentadentate
[FeIV(O)(Bn-TPEN)]2+
faster than that of [FeIV(O)(N4Py)]2+
were performed using the previously published [FeIV(O)(Bn-TPEN)]2+
synthetised FeIV(O)(CDA-BPA)]2+
11) complexes for the stoichiometric oxidation of fla-
vanone. Complexes and 11 could be generated from their iron(II) precursor complexes by
literature methods using 2 equiv solid PhIO for 30–40 min [37 38]. The reaction of with
(9
) complex with two N(sp3) and 3 N(sp2) donor atoms react 20-fold
37]. Taking these results into account, further studies
) and the newly
[
(9
(
9
,
6

Molecules 2021, 26, 3220
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PhIO in acetonitrile at room temperature results in a green species (11), characterised by a
characteristic absorption band at 740 nm (ε ≈ 450 M−1 cm−1), which can be assigned as a
ligand-field (d-d) transition on the low-spin (S = 1) Fe(IV) centre. This species is much less
stable (t1/2 ≈ 2 h at 25 ◦C) than that was found for 9 (t1/2 ≈ 6 h at 25 ◦C). Their decay can
be significantly enhanced by the addition of flavanone.
Table 4. Rate constants and activation parameters for the oxidation of flavanone with oxometal(IV) complexes.
103k2
∆H‡
∆S‡
∆G‡
Solvent
/T
Complex
Refs.
1
/s−1 M−
/kJ mol−1
/J mol−1 K−1
/kJ mol−1
0.24 ± 0.01
0.57 ± 0.03
19.2 ± 1.1
6.50 ± 0.32
1340 ± 67
680 ± 27
70 ± 3
63 ± 4
48 ± 2
−75 ± 9
−93 ± 13
−121 ± 8
92.2
90.9
83.7
CH3CN (25 ◦C)
CH3CN (25 ◦C)
[46]
[46]
[46]
[46]
[FeIV(O)(N4Py*)]2+ (7)
[FeIV(O)(N4Py)]2+
CH3CN (25 ◦C)
[FeIV(O)(N2Py2Q*)]2+
CH3CN (10 ◦C)
[FeIV(O)(N2Py2Q*)]2+
28 ± 2
−150 ± 8
72.7
CH3CN (25 ◦C)
This work
This work
This work
This work
This work
This work
[FeIV(O)(Bn-TPEN)]2+ (9)
[FeIV(O)(Bn-TPEN)]2+ (9)
[FeIV(O)(Bn-TPEN)]2+ (9)
[FeIV(O)(CDA-BPA*)]2+ (11)
[MnIV(O)(N4Py*)]2+ (8)
[MnIV(O)(Bn-TPEN)]2+ (10)
CH3CN (10 ◦C)
1520 ± 90
970 ± 40
CH3CN/TFE (10 ◦C)
CH3CN (10 ◦C)
0.58 ± 0.03
420 ± 15
CH3CN/TFE (25 ◦C)
CH3CN/TFE (10 ◦C)
43 ± 3
−114 ± 10
76.7
The detailed kinetic measurements were carried out in CH3CN and CH3CN/CF3CH2OH
◦
(TFE) = 1:1 solutions at 10–25 C, and the decay of the oxoiron(IV) species
9 and 11 during
the flavanone oxidation was followed as a decrease in absorbance at ~740 nm (Figure 6 and
Tables S2–S5). The yields of flavone were around ~80% for both complexes. The reaction
rates in the presence of 10–50 times excess of substrate obeyed pseudo-first-order kinetics,
and the pseudo-first-order rate constants (kobs) were directly proportional to the concentra-
tion of flavanone, from which the reaction rate constants (k2) are 0.68 ± 0.027 M−1 s−1 and
−1 −1 ◦
for 9 and 11 at 10 C, respectively (Figure 7 and Table 4). These values
are three orders of magnitude higher than those observed for the previously published
[FeIV(O)(N2Py2Q*)]2+ species [46], showing clearly that the ligand framework significantly
influenced the reactivity of the oxoiron(IV) species.
0.97
±
0.04 M
s
Figure 6.
inset shows the time course of the decay of
addition of flavanone (50 mM) in CH3CN at 10 C. The inset shows the time course of the decay of 11 monitored at 742 nm.
(A) UV-Vis spectral changes of 9
(2 mM, red line) upon addition of flavanone (50 mM) in CH3CN at 10 ◦C. The
9
monitored at 739 nm. (
B
) UV-Vis spectral changes of 11 (2 mM, red line) upon
◦

Molecules 2021, 26, 3220
10 of 16
Figure 7. (A) Determination and comparison of second-order rate constants by plotting kobs values against flavanone
◦
concentration for a series of MIV(O) complexes in CH3CN at 10 C, [M]0 = 2 mM. (
B) Eyring plots of log k/T versus 1/T for
9 and 10, [9,10] = 2 mM, [FH2] = 50 mM.
The relative reactivity of oxoiron(IV) complexes is in the order of [FeIV(O)(CDA-
BPA*)]2+ 11) > [FeIV(O)(Bn-TPEN)]2+ ) > [FeIV(O)(N2Py2Q*)]2+ > [FeIV(O)(N4Py)]2+
[FeIV(O)(N4Py*)]2+
), which is consistent with our catalytic results. Based on the tempera-
ture dependence of the reactivity of
(
(
9
>
(
7
−1
−1
8 J mol
9
(with
∆
H‡ = 28
±
2 kJ mol
,
∆
S‡ =
−
150
∆
±
−1
K
,
∆
G‡ = 72.7 kJ mol−1), the value of -T
∆
S‡ determined was bigger than H‡, indicating
an entropy-controlled reaction, contrary to the previously reported enthalpy-controlled
reactions with N4Py-type ligands. As a result of a compensation effect increasing activa-
tion, enthalpies are offset by increasingly positive entropies yielding ∆H‡ = 114 kJ mol−1
at the intercept (Figure 8A). The experimentally determined difference between ∆
G‡ values
is 20 kJ mol−1, which is significant and consistent with the observed reaction rate order
(Figure 8B).
Figure 8.
and oxoiron(IV) complexes.
(A) Isokinetic plot and (B) plot of ∆
G‡ versus lnk2 for the oxidation of flavanone with various oxomanganese(IV)
To elucidate the role of the metal cofactor, such manganese and iron containing
systems have been chosen from the literature where the structure of the high valent metal
oxo intermediates are already known. Our choice fell on the [MnIV(O)(N4Py*)]2+
(
8
) and
10) complexes. These intermediates can be generated in TFE
and TFE/CH3CN by the use of PhIO as an oxidant, and the oxidation of flavanone can
[MnIV(O)(Bn-TPEN)]2+
(
be investigated following their decrease in absorbances at 944 nm (
respectively (Figure 9A).
8) and 1040 nm (10),

Molecules 2021, 26, 3220
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◦
Figure 9.
(
A
) UV-Vis spectral changes of
8
(2 mM, red line) upon addition of flavanone (1.8 M) in CH3CN/TFE at 25 C. The
monitored at 944 nm. ( ) Determination and comparison of second-order
inset shows the time nd course of the decay of
8
B
rate constants by plotting kobs values against flavanone concentration for complexes
25 C, [M]0 = 2 mM.
7 and 8 in CH3CN and CH3CN/TFE at
◦
Due to the correct comparison of the iron and manganese-containing system, we first
investigated the effect of the solvent on the reaction rate. The reaction of [FeIV(O)(Bn-
TPEN)]2+
(
9) with flavanone in CH3CN/TFE (1:1) resulted in a 2-fold increase in rate,
which is not considered significant. Considering the solvent effect, almost the same reaction
rate was observed for the [FeIV(O)(N4Py*)]2+
with k2 = 0.24(1)
10−3 M−1 s−1 and k2 = 0.58(3)
tively (Figure 9B). Comparing the reactions of [FeIV(O)(Bn-TPEN)]2+
TPEN)]2+
in favour of iron (Table 4 and Figure 7)). The difference in reaction rates and yields of
products (~80% flavone for and ~40% flavone for 10 based on the complex concentration)
(7
) and [MnIV(O)(N4Py*)]2+
(
8
) ◦complexes
×
×
10−3 M−1 s−1 at 25 C, respec-
(9
) and [MnIV(O)(Bn-
(10) under the same conditions, a 3.5-fold difference in reaction rate was observed
9
can be explained by a different mechanism based on the literature data. While in the case of
oxoiron(IV) complexes the reactions occur mostly via an oxygen-rebound mechanism [56],
in the case of manganese the process involving C-H activation can be described mainly by
a non-rebound mechanism with a smaller reaction rate (Scheme 3) [40].
Scheme 3. Proposed mechanism for the C-H activation by oxoiron and oxomanganese complexes.
3. Materials and Methods
Reactions were carried out in ordinary glassware and chemicals were used as pur-
chased from commercial suppliers without further purification. GC analyses were per-
formed on an Agilent 6850 gas chromatograph equipped with a flame ionization detector
and a 30 m SUPELCO BETA DEX225 (CHIRASIL-L-VAL) (Sigma-Aldrich, Budapest, Hun-
gary) column. ESI-MS samples were analysed using a triple quadruple Micromass Quattro
spectrometer (Waters, Milford, MA, USA) and an HPLC-MS system (Agilent Technologies
1200, Budapest, Hungary) coupled with a 6410 Triple-Quadrupole mass spectrometer,
operating in a positive ESI mode. Synthesis of the ligand was carried out in a microwave
reactor (CEM Discover), (CEM Inc, Scottsdale, AZ, USA) monitored by TLC on aluminium
oxide 60 F254 neutral plates and detected with a UV lamp (254 nm). NMR spectra were

Molecules 2021, 26, 3220
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obtained on a Bruker Avance 300 (Bruker Biospin AG, Fällanden, Switzerland) or 600 spec-
trometers, operating at 300 or 600 MHz for 1H and 75 or 150 MHz for 13C. The spectra are
recorded at room temperature. Chemical shifts,
δ (ppm), indicate a downfield shift from
the residual solvent signal (δH: 1.94 ppm, δC: 118.26 ppm for CD3CN and δH: 7.26 ppm, δC
:
77.16 ppm for CDCl3). Coupling constants, J, are given in Hz. The syntheses of most of
the complexes used in this study have been previously reported: these complexes and the
corresponding references are listed as follows: [FeII(Bn-TPEN)(CH3CN)]2+
(
3
), [FeIV(O)(Bn-
TPEN)]2+ 38], [MnII(Bn-TPEN)(CH3CN)]2+ ), [MnIV(O)(Bn-TPEN)]2+
(9) [37, (4 (10) [40],
[MnII(N4Py*)(CH3CN)]2+ (2), [MnIV(O)(N4Py*)]2+ (8) [39], [FeII(CDA-BPA*)]2+ (6) [41].
Synthesis of ligands CDA-BPA* and CDA-BQA*. The synthesis was performed accord-
ing to a modified previously reported procedure [47]. The amine (1 eq.), K2CO3 (12 eq.),
2-(chloromethyl)pyridine hydrochloride or 2-(chloromethyl)quinoline hydrochloride (4 eq.)
and KI (1 eq.) were suspended in 50 mL of acetonitrile. The reaction mixture was heated
in a microwave reactor (50 W, reflux) for 1 h. The solvent was evaporated in a vacuum,
the residue suspended in ethyl acetate and washed three times with brine and saturated
NaHCO3, the organic layer dried over anhydrous sodium sulphate, filtered and evaporated
in a vacuum. The crude ligand was purified by automated flash chromatography on a
pre-packed neutral alumina column (48 g).
±
CDA-BPA: (
12.0 mmol), 2-(chloromethyl)pyridine hydrochloride (656.2 mg, 4.0 mmol), KI (166.0 mg,
1.0 mmol). Automated flash chromatography 0 5% dichloromethane in methanol (Rf = 0.42,
5% dichloromethane in methanol). Yield: 303.0 mg (0.6 mmol, 63%), brown oil.
(R,R)-CDA-BPA: (1R,2R)-( )-1,2-Diaminocyclohexane (114.2 mg, 1.0 mmol), K2CO3
(1658.4 mg, 12.0 mmol), 2-(chloromethyl)pyridine hydrochloride (656.2 mg, 4.0 mmol),
KI (166.0 mg, 1.0 mmol). Automated flash chromatography 0 5% dichloromethane in
methanol. Yield: 337.5 mg (0.7 mmol, 71%), light brown powder. 1H NMR (300 MHz,
CD3CN) : 8.39 (d, J = 4.7 Hz, 4H), 7.61 (d, J = 7.8 Hz, 4H), 7.55–7.41 (m, 4H), 7.18–7.03
(m, 4H), 3.70 (d, J = 14.3 Hz, 4H), 3.58 (d, J = 14.3 Hz, 4H), 2.85–2.66 (m, 2H), 1.79–1.61 (m,
2H), 1.36–1.23 (m, 1H), 1.23–1.00 (m, 4H), 0.96–0.79 (m, 1H). 13C NMR (75 MHz, CD3CN)
161.6, 149.5, 136.7, 124.5, 122.6, 60.8, 56.5, 26.8, 25.7. ESI-MS (m/z): 479.2 (M + H+, 100%).
CDA-BQA: ( )-trans-1,2-Diaminocyclohexane (70.5 L, 0.6 mmol), K2CO3 (970.2 mg,
±
)-trans-1,2-Diaminocyclohexane (120.0 µL, 1.0 mmol), K2CO3 (1658.4 mg,
→
−
→
δ
δ
:
±
±
µ
7.0 mmol), 2-(chloromethyl)quinoline hydrochloride (500.0 mg, 2.3 mmol), KI (96.3 mg,
0.6 mmol). No further purification was required, (Rf = 0.28, ethyl acetate:hexane 1:1). Yield:
372.3 mg (0.5 mmol, 94%), yellow powder.
(R,R)-CDA-BQA: (1R,2R)-(
(651.6 mg, 4.7 mmol), 2-(chloromethyl)quinoline hydrochloride (378.9 mg, 1.6 mmol), KI
−
)-1,2-Diaminocyclohexane (44.5 mg, 0.4 mmol), K2CO3
(64.7 mg, 0.4 mmol). Automated flash chromatography 10
Yield: 202.3 mg (0.3 mmol, 76%), light yellow powder. H NMR (300 MHz, CDCl3)
→
40% ethyl acetate in hexane.
: 7.99
1
δ
(d, J = 8.3 Hz, 4H), 7.91 (d, J = 8.5 Hz, 4H), 7.77 (d, J = 8.5 Hz, 4H), 7.72–7.59 (m, 8H),
7.52–7.41 (m, 4H), 4.01 (d, J = 14.1 Hz, 4H), 3.85 (d, J = 14.1 Hz, 4H), 3.00–2.81 (m, 2H),
2.49–2.27 (m, 2H), 1.88–1.69 (m, 2H), 1.25–0.94 (m, 4H). 13C NMR (75 MHz, CDCl3)
δ: 161.3,
147.7, 135.8, 129.3, 129.1, 127.6, 127.4, 126.1, 122.2, 60.7, 56.6, 29.8, 26.0, 24.4. ESI-MS (m/z):
679.4 (M + H+, 100%).
FeII(CDA-BQA*) (
5): Ligand
±
CDA-BQA (14.2 mg, 0.02 mmol) and Fe(CF3SO3)2
(7.4 mg, 0.02 mmol) were dissolved in dry acetonitrile and mixed. The reaction vessel was
placed in a tank with dry diethyl ether for vapour diffusion. The complex preparation
was performed in a drybox in an atmosphere of argon. After 1 day, crystals suitable for
single crystal X-ray diffraction were obtained. UV-Vis [MeCN = acetonitrile] [
λmax, nm
logε
] = 203 (5.03), 231 (4.78), 307 (4.13). FT-IR bands (KBr pellet cm−1): 3429 m, 3068
w, 2933 w, 2859 w, 1701 s, 1653 s, 1600 w, 1279 s, 1166 s, 1029 s, 784 w, 667 m, 638 m,
522 w. Anal Calcd for C48H42F6FeN6O6S2: C, 55.82; H, 4.10; N, 8.14. Found: C, 55.79;
H, 4.15; N, 8.19. X-ray: Chemical formula: C50H45F6FeN7O6S2, Formula weight: 1073.90,
Crystal system: orthorhombic, Space group: Cmca, a (Å): 19.7874(9), b (Å): 42.5557(15), c
(Å): 12.4013(4), Volume (Å3): 10442.7(7), Z: 8, Calc. density (g cm−1): 1.366, Temperature

Molecules 2021, 26, 3220
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(K): 293(2), Abs. coeff. (mm−1): 3.715, F (0 0 0): 4432, Obs. reflections: 5532, Goodness-of-fit:
2
2
2
1.055, R1: 0.0761, wR2: 0.2287 (w=1⁄[σ
2(Fo )+(0.1721P)2+1.4205P] and P = (Fo +2Fc )/3).
CCDC deposit number 2082080 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/
retrieving.html (accessed on 30 April 2021) (or from the Cambridge Crystallographic Data
Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033) (Table S1).
Catalytic oxidation of ethylbenzene was carried out under thermostatic (273 K) condi-
tions. In a typical reaction, 500
was delivered by a syringe pump in air to a stirred solution (1 mL) of
ethylbenzene inside a vial. The final concentrations were 2 mM catalyst
µ
L of TBHP (diluted from 70% TBHP) solution in CH3CN
catalysts, and
, 50 mM (100,
6
6
200 mM) oxidant, and 500 mM substrate. The mixture was stirred for 15 min. The products
were identified by GC analysis, and their yields were determined by comparison with
authentic compounds using bromobenzene (25.00
the reactions.
×
10−3 M) as an internal standard in
Comp−le3x
6
(2.00
×
10−3 M) was dissolved in acetonitrile (2 mL), then iodosobenzene
−3
(16.00
×
10 M) and ethylbenzene (1500
×
10 M) were added to the solution in CH3CN
at 273 K. The mixture was stirred for 3 h and the products were identified by GC analysis,
and their yields were determined by comparison with authentic compounds using bro-
mobenzene (4.00
was determined with GC analysis on a chiral column: (ee = [R] − [S])/([R] + [S]).
×
10−3 M) as an internal standard in the reactions. Enantiomeric excess
Catalytic oxidation of flavanone was carried out under thermostatic (298 K) conditions.
In a typical reaction, 5.8 mg 2 (5 mM) and 33.6 mg flavanone (100 mM) was dissolved in
1 mL CH3CN, and 169 mg mCPBA (500 mM) in 500 µL CH3CN was delivered by syringe
pump to a stirred solution. After syringe pump addition (5 min the solution was stirred
for 5 min and a known amount of PhBr (0.315 mmol) was added as an internal standard.
The iron complex was removed by passing the reaction mixture through a silica column
followed by elution with ethyl acetate. The products (1,3-dione (D) and flavone) were
identified by GC/MS and confirmed by comparison with authentic samples. Flavone is
commercially available and it was purchased from Sigma-Aldrich. 1-(2-hydroxy-phenyl)-3-
phenyl-propane-1,3-dione (D): o-Benzoyloxyacetophenone has been prepared by the action
of benzoyl chloride on a pyridine solution of o-hydroxyacetophenone. Its rearrangement
to 1-(2-hydroxy-phenyl)-3-phenyl-propane-1,3-dione (D) by alkali has been described [57].
(F) m/z: 222 (85.92%), 194 (58.9%), 165 (18%) 120 (100%), 92 (95,2%). 63 (35.3%). 1,3-dione
(D): m/z: 240 (15.1%), 223 (8.3%), 121 (25.2%), 120 (7.3%), 106 (7.2%), 105 (100%), 77 (30%),
69 (6.0%), 65 (9.3%), 51 (4.5%), 39 (8.3%).
Stoichiometric reactions were carried out under thermostatic conditions at 10 ◦C in
1 cm quartz cuvettes. In a typical experiment, [FeII(Bn-TPEN)(CH3CN)]2+ (2
×
10−3 M)
was dissolved in acetonitrile(CH3CN-TFE) (2.0 cm3), then iodosobenzene (4
×
10−3 mM)
was added to the solution. The mixture was stirred for 50 min then excess iodosobenzene
was removed by filtration. Flavanone (50 mM) was added to the solution and the reaction
−1
was monitored by UV-Vis spectrophotometer (Agilent 8453) at 739 nm (ε
= 450 M cm−1).
4. Conclusions
In conclusion, we previously found that N4Py-based iron(II) complexes capable of
carrying out 2,3-desaturation of flavanone via 2-hydroxyflavanone intermediate formation
can act as a functional flavone synthase model. As a continuity of this study, efforts have
been made to enhance the catalytic activity by the use of TPEN-type ligands and investi-
gate the role and effect of the metal cofactor through manganese and iron complexes with
the same ligand framework. Comparing the reactions of [FeIV(O)(Bn-TPEN)]2+
(
9
) and
[MnIV(O)(Bn-TPEN)]2+
(10) towards flavanone under the same conditions, a 3.5-fold differ-
ence in reaction rate was observed in favour of iron, and this value is three orders of mag-
nitude higher than that was observed for the previously published [FeIV(O)(N2Py2Q*)]2+
species. The relative reactivity of oxoiron(IV) complexes is in the order of [FeIV(O)(CDA-
BPA*)]2+ 11) > [FeIV(O)(Bn-TPEN)]2+ ) > [FeIV(O)(N2Py2Q*)]2+ > [FeIV(O)(N4Py)]2+
( (9

Molecules 2021, 26, 3220
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> [FeIV(O)(N4Py*)]2+
(
7), which is consistent with our catalytic results, and shows that
addition of cyclohexanediamine as the chiral element successfully lead to increase in the
catalytic activity. Although we have reported the first example of efficient flavanone oxi-
dation by iron and manganese complexes, detailed studies are underway to elucidate the
mechanisms of flavone formation.
Supplementary Materials: The following are available online. Figure S1: Ligands 1a
( cda-bpa)–2b
±
(
±
cda-bqa); Figure S2: Packing of [FeII(CDA-BQA*)]2+ complexes in rac-5. Individual enantiomers
are shown in red and blue colours, respectively. Triflate anions and acetonitrile molecules are omitted
for clarity; Table S1: Experimental data for the X-ray diffraction studies; Table S2: The calculated
kobs values in the reaction of
9 and flavanone in MeCN; Table S3: The calculated kobs values in the
reaction of and flavanone in MeCN/TFE; Table S4: The calculated kobs values in the reaction of 11
9
and flavanone in MeCN; Table S5: The calculated kobs values in the reaction of 10 and flavanone in
MeCN/TFE.
Author Contributions: Conceptualization, J.K. and S.I.K.; resources, D.L.-B., N.P.J., B.I.M., and B.P.;
writing—original draft preparation, J.K., writing—review and editing, J.K. All authors have read and
agreed to the published version of the manuscript.
Funding: Financial support of the GINOP-2.3.2-15-2016-00049 are gratefully acknowledged. Part of
the work was supported by the Croatian Science Foundation (IP-2014-09-1461), and CAT Pharma
(KK.01.1.1.04.0013), a project co-financed by the Croatian Government and the European Union
through the European Regional Development Fund—the Competitiveness and Cohesion Opera-
tional Programme.
Data Availability Statement: Not available.
Conflicts of Interest: The authors declare no conflict of interest.
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