Novel riboflavin-inspired conjugated bio-organic semiconductors

Article abstract of DOI:10.3390/molecules23092271

Flavins are known to be extremely versatile, thus enabling routes to innumerable modifications in order to obtain desired properties. Thus, in the present paper, the group of bio-inspired conjugated materials based on the alloxazine core is synthetized using two efficient novel synthetic approaches providing relatively high reaction yields. The comprehensive characterization of the materials, in order to evaluate the properties and application potential, has shown that the modification of the initial alloxazine core with aromatic substituents allows fine tuning of the optical bandgap, position of electronic orbitals, absorption and emission properties. Interestingly, the compounds possess multichromophoric behavior, which is assumed to be the results of an intramolecular proton transfer.

Full text of DOI:10.3390/molecules23092271

molecules
Article
Novel Riboflavin-Inspired Conjugated
Bio-Organic Semiconductors
Jan Richtar 1, Patricie Heinrichova 1, Dogukan Hazar Apaydin 2 ID , Veronika Schmiedova 1,
Cigdem Yumusak 2, Alexander Kovalenko 1, Martin Weiter 1, Niyazi Serdar Sariciftci 2
ID
and Jozef Krajcovic 1,
*
1
Faculty of Chemistry, Materials Research Centre, Brno University of Technology, Purkynˇova 118,
612 00 Brno, Czech Republic; xcrichtar@fch.vut.cz (J.R.); heinrichova@fch.vut.cz (P.H.);
schmiedova.veronika@seznam.cz (V.S.); kovalenko.alx@gmail.com (A.K.); weiter@fch.vut.cz (M.W.)
Linz Institute for Organic Solar Cells (LIOS), Physical Chemistry, Johannes Kepler University Linz,
Altenbergerstraße 69, 4040 Linz, Austria; Dogukan.Apaydin@jku.at (D.H.A.);
Cigdem.Yumusak@jku.at (C.Y.); serdar.sariciftci@jku.at (N.S.S.)
2
*
Correspondence: krajcovic@fch.vut.cz
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 8 August 2018; Accepted: 31 August 2018; Published: 5 September 2018
Abstract: Flavins are known to be extremely versatile, thus enabling routes to innumerable
modifications in order to obtain desired properties. Thus, in the present paper, the group of
bio-inspired conjugated materials based on the alloxazine core is synthetized using two efficient novel
synthetic approaches providing relatively high reaction yields. The comprehensive characterization
of the materials, in order to evaluate the properties and application potential, has shown that
the modification of the initial alloxazine core with aromatic substituents allows fine tuning of the
optical bandgap, position of electronic orbitals, absorption and emission properties. Interestingly,
the compounds possess multichromophoric behavior, which is assumed to be the results of an
intramolecular proton transfer.
Keywords: bio-inspired material; conjugated materials; flavins; biomimetic energy storage;
oxygen evolution
1. Introduction
Natural and bio-inspired organic conjugated materials [1–7] have become a new wave. Given the
sustainability, biocompatibility, non-toxicity, versatility and potentially low-cost of bio-organic
bio-inspired materials, it is evident that such materials will be widely considered as candidates
for future bio-organic semiconductor science and technology, such as ultra-thin electronic platforms
for surgical and point-of-care devices, diagnostic implants [8–11], soft robotics [12,13], biodegradable
electronics [14,15] and food packaging [16].
Flavins, a group of naturally occurring materials, are especially remarkable due to their properties
and high versatility from a chemical point of view. They belong to the redox coenzymes, being able to
react as efficient one or two electron redox systems [17] accompanied by proton transfer at the nitrogen
atoms of diazabutadiene of the isoalloxazine moiety, which makes them suitable as a prosthetic
groups for a variety of enzymatic redox reactions [18
,19] occurring over a wide potential range
(>500 mV) [20 21]. An intriguing example is the role of flavin in the respiratory electron transport chain
,
where the electrons from the reduced form of flavin adenine dinucleotide (FADH2) are transported
along the group of proteins situated in the inner mitochondrial membrane carrying out proton flux
across the membrane. An electrochemically induced proton gradient is generated resulting in the
formation of high-energy adenosine triphosphate (ATP) [22]. Consecutive recycling of the flavin
Molecules 2018, 23, 2271; doi:10.3390/molecules23092271
www.mdpi.com/journal/molecules

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redox centers is furnished by FAD reduction in the Krebs cycle of mitochondria. On the basis of the
analogy with this electrochemical cellular metabolism, several flavin cofactors have been proposed
as lithium and sodium storing electrodes in rechargeable batteries [23–25]. Molecular tailoring of
the structure of flavins showed that the capability to act as an effective redox center during charge
transduction is preserved also by the structurally simpler lumichrome, alloxazine (tautomers of the
isoalloxazine moiety of the flavin cofactors) and lumazine [24]. Furthermore, a recent work indicated
that even slight branching of the alloxazine with electron-donating groups, inducing the lowering of the
reduction potential, should allow for further increases in battery voltage made thereof [26]. In addition,
flavin-containing donor-acceptor structures [27
gates or molecular switches [29 30] and in photovoltaics [30]. The photophysical properties and
redox potentials of flavins are determined by hydrogen-bonding [31 32], metal coordination [33 34],
stacking [35] or the local environment [36]. On the other hand, the influence of enlarging the
-conjugated system on the optoelectronic properties of flavin derivatives remains less investigated,
,28] have also found application in molecular logic
,
,
,
π
π
–π
although shift of the absorption maxima to a longer wavelengths and change of the redox properties
of the chromophore and of the extinction coefficient are expected [37 38]. Indeed, it was shown
that the enlarging of the fused -system from the basic N10-butyl substituted flavin core to the
,
π
pyrene-attached derivative was accompanied by progressive bathochromic shift in both the absorption
(up to 550 nm) and emission (up to 628 nm) spectra and all π-extended derivatives showed intensive
emission with quantum yields of up to 80% while they maintained their electrochemical redox behavior
analogous to parent flavin [39]. Despite the aforementioned viability of advanced alloxazine and
lumazine derivatives of flavin as the charge-carrier materials for organic electronics only relatively few
reports [40–43] about optoelectronic behavior are out there.
In the present paper, we report on the properties of novel alloxazine and lumazine derivatives
investigated by means of UV/VIS spectroscopy, quantum-chemical calculations, thermogravimetric
analysis with the intention of providing a comprehensive overview. We selected and synthesized a
series of 11 fused and non-fused aromatic alloxazine and lumazine derivatives. The elongation of the
π
-conjugated system would provide bathochromic shift in absorption and emission characteristics [44],
influence the redox potentials [45], while fusing of the aromatic rings further planarizes the molecule,
thus enhancing the π-conjugated overlap [45].
2. Results and Discussion
2.1. Synthesis
The synthetic routes to the target compounds are shown in Scheme 1. As a result, using novel
synthetic approaches, we synthetized a series of 11 alloxazine and lumazine derivatives. Even though
the synthesis of compounds 1–4 and 6–11 was previously reported in the literature in varying
detail [46–51], we envisaged two general and straightforward synthetic approaches (denoted as
Approaches A and B), both of them differing in the source of the uracil moiety of the final flavin
derivatives, improving the earlier procedures and providing the completely novel fused system
5
. The key step of the synthesis is represented by the formation of central pyrazine ring by the
condensation of a suitable 1,2-diketone with a 1,2-diamine. Both approaches are complementary and
enable the preparation of structurally advanced flavin derivatives (Figure 1) with relatively simple
synthetic methodologies.
Approach A is the most favorable for the preparation of fused aromatic flavins from aromatic
1,2-diamines. It comprises the condensation of a 1,2-diamine with alloxan under neutral or acidic
conditions (Scheme 1) [46]. The alloxan (monohydrate form) can be easily prepared by oxidation of
barbituric acid with CrO3 in an acidic milieu of glacial acetic acid [52]. The flavins
from commercially available 1,2-diamines (Figure 1) while the synthesis of the derivatives
required laboratory preparation of 1,2-diamines and . Pyrene-4,5-diamine was prepared by
three-step synthesis: oxidation [53] of pyrene by NaIO4 catalyzed by RuCl3 provided pyrene-4,5-dione
1–4
were prepared
A
–D
5
and
6
E
F
E

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(
L
) that was transformed to pyrene-4,5-dioxime (
reduced to
was obtained by the reaction of o-phenylenediamine
M
) by reaction with NH2OH.
M
was subsequently
E
by catalytic hydrogenation using a H2/Pd/C system [54]. 2,3-Diaminophenazine
F
A
and FeCl3 in dilute hydrochloric acid [55].
We also probed the preparation of non-fused flavins by Approach A. It embodied finding a suitable
means of synthesis of 1,2-diarylethane-1,2-diamines which could react with alloxan. Compared
to aromatic 1,2-diamines, the reaction would provide 2,3-dihydropyrazines that would have to be
oxidized to the final flavins [56]. We performed pilot experiments on the preparation of the flavin
with intermediate diamine ( ) being prepared in a similar fashion to . Benzil ( ) was transformed
to 1,2-dioxime ( ) and submitted to the reduction. Contrary to the reduction of simple oximes to
8
P
E
N
O
amines, reduction of 1,2-dioximes is probed in less detail. We tested several reduction methodologies
used for the reduction of simple oximes, i.e., reduction by LiAlH4 [57], SnCl2/HCl [58], Red-Al [59],
Pd/C/N2H4 [60], NaBH4/NiCl2 [61], NaBH3CN/TiCl3/NH4OAc [62] and NaBH4/TiCl4 [63] under
various reaction conditions. Among them, only reduction with NaBH4/TiCl4 provided us with desired
1,2-diphenyl-ethane-1,2-diamine
desired 2,3-dihydropyrazine or directly flavin
or acidic glacial AcOH conditions. We have thus envisaged another approach.
P
with good yields. On the other hand, we could not obtain the
Q
8
by condensation with alloxan under either neutral
Scheme 1. General scheme of the synthesis of flavins 1–11 by Approaches A and B. Reaction conditions:
(i) AcOH, H3BO3; (ii) AcOH, MeOH/H2O/THF.
Approach B is a suitable way to prepare non-fused aromatic flavins from 1,2-diketones and
5,6-diaminouracil, taking into consideration that from the synthetic point of view, 1,2-diketones are
more readily available than the corresponding 1,2-diamines. The synthesis embodies a condensation
of a 1,2-diketone with 5,6-diaminouracil under acidic conditions [64]. The procedure was improved
by altering the solvent system by using of THF which dissolves the corresponding 1,2-diketones
better than lower alcohols resulting in a broader spectrum of employable substrates. The intermediate
5,6-diaminouracil was obtained by a 3-step synthesis [65]. Condensation of ethyl cyanoacetate with
urea under basic (NaOEt) conditions yielded 6-aminouracil. The nitrosation of 6-aminouracil provided
5-nitroso-6-aminouracil that was subsequently reduced to 5,6-diaminouracil by sodium hydrosulfite.
Subsequently, the flavins
the flavin 11 from the prepared diketone
7
–
10 were prepared from commercially available 1,2-diketones
G–J whereas
K
. The 2,20-thenil
K
was obtained by benzoin condensation
of thiophene-2-carbaldehyde to 2,20-thenoin and its in situ oxidation by ambient oxygen [66].
The synthesis of flavin derivatives by both approaches required appropriate purification methods
in order to obtain high purity materials, suitable for further optical and electrochemical measurements.
Due to very limited solubility of the all prepared compounds digestion in boiling methanol was used
as the first stage of the purification. Vacuum sublimation was used as the final purification step.
Target compounds with the final purity were used for further experiments.
The structures of the prepared flavins (except for compound
6
which is practically insoluble in
where
common solvents) was confirmed by 1H-NMR and also by 13C-NMR in the case of compound
4
only 1H-NMR could be considered insufficient (see Supporting Information). A typical phenomenon of
the proton spectra for the target compounds is the appearance of two singlets in the 11–13 ppm region
belonging to strongly deshielded N–H protons along with a downfield shift of the aromatic protons
caused by their conjugation to the strongly electron-withdrawing diimide moiety. The purity of the
final compounds was further checked by elementary analysis. Nevertheless, it has to be stated that
additional quantitative and qualitative analysis of the purity of the final compounds by various either

Molecules 2018, 23, 2271
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normal or reverse-phase HPLC methods proved to be rather inconclusive as the compounds are poorly
soluble in all commonly employed solvents, including water. On the other hand, with the stepwise
purification process involving digestion and vacuum sublimation we obtained final compounds with
sufficiently high purity for the purposes of optical and electrochemical studies.
Figure 1. Compounds under investigation, synthetized by Approach A and Approach B.
◦
TGA measurements proved the high thermal stability of the flavin derivatives reaching 450 C for
compound
the decomposition temperature with the increasing number of fused aromatic rings (from 276.78 ◦C
for compound , see Supporting Information). On the other hand,
to 443.11 ◦C for the compound
the series of non-fused flavin derivatives (compounds 11) provided decomposition temperatures in
the range of 305.62 ◦C (compound 10) to 354.20 ◦C (compound 11). It should be stated that the most
6. The series of fused flavin derivatives (compounds 1–7) showed a progressive increase of
3
6
8
–
of derivatives are stable to temperatures higher than 300 ◦C and compounds
5 and 6 even more than
400 ◦C. Such thermal stability, not often observed at small molecules, may strongly contribute to the
suitability of the materials for the applications in organic electronics.

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2.2. Optical Measurements
Flavin-like compounds are well known to possess the formation of tautomers in the solution
by means of the proton transfer [67] resulting in the formation of the iso-form. Thus, in our case,
the influence of the conjugated functional “tail” on the proton transfer would be noticeable in the
absorption-emission optical spectra in DMSO. Depending on the electronegativity, conjugated length,
planarity and the absence/presence of the free rotation we expect to observe clear differences in the
optical behavior of the materials under study. Indeed, how it is shown below, the optical measurements
of the considered compounds, possessed interesting, but complex behavior.
The optical spectra of all compounds under study consisted of more than one fluorescence band
with different intensity, which on one hand can be an indication of impurities. Indeed a small amount
of impurities can drastically affect the emission spectra, however on the other hand, the assumption
that the multi-chromophoric behavior is based on the correspondence of the absorption spectra with
the excitation ones. In this case the possible impurities, which could remain after the recrystallization
should not affect the absorption spectra. Moreover, the formation of the tautomers in other alloxazine
derivatives, i.e., lumichrome [67] or 6,7-dicyanolumazine [40] (compound 4) are well known. As an
example, it has been reported, that in aqueous solution of the 6,7-dicyanolumazine at neutral pH,
it possesses two-chromophoric behavior with two different absorption bands at 350 and 400 nm.
However in the case of the whole family of materials under study, it is practically impossible to
perform the measurements in aqueous solution for all the derivatives, as most of the derivatives cannot
be dissolved in water. However, it has to be noted, that due to the high purity of our compounds the
second chromophore effects are unlikely to be caused by impurities and/or degradation products
(see TGA and NMR spectra in the Supporting Information). Thus, considering the formation of the
iso-form from the alloxazine derivatives, we presume the presence of two chromophores marked Ch.
A and Ch. B. Moreover, the equilibrium concentration of tautomers can be changed by conditions
like temperature or acidity of solvent. The effect of the environment of pH is common for the flavins,
thus additionally we demonstrate the effect of acidity of the environment on the fluorescence emission
spectra of the compound
1 (see Supporting Information). There is clear evidence that long wavelength
emission of the chromophore (Ch. B) is missing in acidic environments in contrast to the basic ones,
which indicates that the proton transfer is hindered and one of the tautomers is eliminated in the
acidic solvent.
It has to be noted that the intensity ratio, in some cases differs by up to a few orders of magnitude
(especially for compounds 2, 4, 7, 9). When, the emission band of the chromophore with higher energy
gap (Ch. A) has smaller intensity due to reabsorption in all the cases, which results from spectral
overlap between emission and absorption spectra as depicted in Figure 2. Simultaneously these spectral
overlaps indicate a resonance energy transfer from the higher energy chromophore (Ch. A) to the lower
energy chromophore (Ch. B), which amplifies the fluorescence intensity of chromophore Ch. B.
Figure 2. Cont.

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Figure 2. Cont.

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Figure 2. UV-Vis absorption, excitation and emission spectra of the compounds considering two-
chromophore model with the marked optical band gaps of observed chromophores.
In the case of compounds 2, 5, 8, 10, 11, the overlap between emission spectra of the observed
two chromophores was seen. This situation makes the resolution of chromophores in spectra more
speculative, but the presence of two chromophores was confirmed by measurement of the excitation
spectra. If the emission spectrum were originated from only one chromophore the excitation spectrum
would follow the absorption spectrum. However this condition is not fulfilled for the compounds
under investigation. On the other hand, superposition of excitation spectra of Ch. A and Ch. B
chromophores well fitted to the absorption spectrum in the solution.
Exception occurs for solution with compounds
6 and 10. Seemingly, in case of compound 6
the appearance of a third chromophore can be observed. Moreover excitation and emission spectra
attributed to chromophore Ch. B have resolved vibronic structures with overlap of zero-phonon
peaks. This small Stokes shift is characteristic of very rigid structures with very small reorganization
energy. The compound 10 in DMSO solution provides only one fluorescence band attributed to Ch. B
according to its position. Nevertheless, in the acidic environment the emission band of Ch. A occurs.
Thus, in neutral environment we presume that the fluorescence of Ch. A is effectively reabsorbed by
and/or quenched by energy transfer to Ch. B.
The optical band gap values (Figure 3) were determined as the intersection point between
normalized excitation (absorption) and emission spectra of each chromophore. Generally, the optical
measurements have shown that the short-wavelength chromophores (Ch. A) have an absorption band
in the range of 2.7–3.3 eV and the long-wavelengths chromophores (Ch. B) in the range from 2.4 to
2.8 eV. Therefore, the band gap difference between long- and short-wavelength chromophores is about
0.5
±
0.1 eV for the most of the cases, however, in case of compounds 5 and 11 the difference of 0.3 eV
was observed.
Figure 3. Optically measured bandgap and Stoke’s shift values for the compounds 1–11.

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Values of Stoke’s shift (Figure 3 and Table 1) for the shorter linear compounds
1–
4
were in the
range of 0.3 to 0.8 eV for the alloxazine form and in the range from 0.4 to 0.7 eV for the iso-form. In this
case the deviation of the Stoke’s shift is caused by the reorganization of the electron density along
the linear molecule during the excitation depending on its length (e.g., compounds
the presence/strength of the electron withdrawing substitutions competing with the alloxan ending
functional group (e.g., compounds and ). In case of more complex structure, as for compounds
, we observed smaller Stoke’s shift values for both alloxazine and isoalloxazine forms, this can
be explained that in case of planar non-linear rigid structures the reorganization energy is lower
(compounds and ) or the electron density is equally distributed due to the acceptor groups
equilibrium (compound ) as it will be shown later. On the other hand the presence of the free
1 and 2) and/or
3
4
5–7
5
7
6
rotation in the non-fused compounds 8–11 is resulting in relatively large Stoke’s shift.
Table 1. Main optical parameters of the compounds.
#
ABSmax, nm
ABSmax*, nm
Eg(opt), eV
Eg*(opt), eV ∆EStoke’s, eV ∆EStoke’s, eV
1
2
3
4
5
6
7
8
9
386
390
390
334
434
356
410
363
363
358
393
413
450
450
425
455
417
448
410
407
419
477
3.0
2.9
3.1
3.1
2.7
3.3
3.0
3.2
3.2
3.1
2.7
2.6
2.4
2.5
2.6
2.5
2.7
2.6
2.8
2.7
2.6
2.4
0.3
(0.5)
0.6
0.8
(0.1)
0.4
0.1
(0.6)
0.4
(0.6)
(0.6)
0.6
0.7
0.4
0.5
0.3
0.3
0.3
0.6
0.6
0.6
0.3
10
11
ABSmax, absorption maximum of the short-wavelength chromophore; ABSmax*, absorption maximum of the
long-wavelength chromophore (if applicable); Eg(opt) optical band gap of the short-wavelength chromophore;
Eg*(opt) optical band gap of the long-wavelength chromophore (if applicable);
brackets are approximate.
∆EStoke’s—Stoke’s shift. Values in
Nevertheless, to understand the complex optical behavior of the materials we should take a deep
insight into the molecular structures if the compounds. Compounds are fused, having donor
—benzene, naphthalene, pyrene and phenanthrene moiety, respectively) or acceptor
—difluorobenzene, dicyano and phenazine moiety, respectively) “tails” anchored to the
and 11—phenyl
and 10—pyridine and furan moieties,
1–7
(
(
1
3
,
,
2, 5, 7
4, 6
central pyrazine ring, similarly to non-fused compounds, having electron poor (
and thiophene moieties, respectively) and electron rich (
8
9
respectively) conjugated groups. Nevertheless, it would be feasible, that the electronegativity of the
conjugated part will affect the proton transfer. Thus, for materials, having both electron donating
(fused aromatic rings for the materials
5 and 7) and electron accepting (pyridine and furan for the
materials 9 and 10) we assume a multi-chromophore model and performed DFT modeling.
2.3. DFT Modeling
Geometry optimization was performed using the standard B3LYP functional expanded with the
6-311G** basis set, which has proved its accuracy for organic conjugated systems. The solvent effect was
simulated using the SCRF procedure. The computational protocol was firstly to find the energy minima
of the alloxazine form and then to optimize isoalloxazine derivative: direct comparison of the energy
difference between the initial and iso-form is intended to give a general overview about the stability
of the given tautomer. To get an overview about the electronic transitions the absorption spectra of
the compounds in both alloxazine and isoalloxazine forms were calculated for the 10 lowest states.
The comparison between the simulated absorption spectra of alloxazine and isoalloxazine tautomers
has shown that the spectra of the iso-form are bathochromically shifted (see Supporting Information).
Regarding the energy difference between the initial and iso- form, the optimized structures were
compared to each other, considering the compounds are dissolved in the DMSO. Theoretical approach

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has shown that in all the cases alloxazine form was found to be favorable. The lowest isomerization
energies were found for the compounds 10 and (0.02, 0.09, 0.10 and 0.16 eV respectively), for the
compounds and the energy difference between the alloxazine and isoalloxazine form was in
the range of 0.18 to 0.22 eV and the highest isomerization energy was computed for the compounds
and 11 (0.28 and 0.27 eV, respectively). Thus we assume that the alloxazine form is dominant, and
7
,
8,
9
1
,
2,
3,
5
6
4
as in the absorption spectra, the short-wavelength form is prevalent, however higher absorption of
the chromophore does not indicate a higher ratio of the chromophore A. In the present case the ratio
of the various chromophores will be highly affected by the solvent and needs further detailed study.
Moreover we do not exclude that depending on the environment, additional chromophores can appear
due to protonation/deprotonation, carbonyl group reduction, etc.
In Figures 4 and 5, the HOMO and LUMO orbitals of the compounds are depicted (for the
iso- form see the SI). All of the linear planar fused molecules were found to have a homogenous
electron density distribution, which results in a relatively low band gap, unusual for molecules of
such size, especially for compound
results in a nearly homogeneous electron density distribution equally “stretched” along the molecule.
In the case of non-linear fused compounds (molecule and ) a slight asymmetry in the LUMO was
6, where the presence of the electron-rich pyrazine building block
5
7
observed—the electron density is delocalized on one of the aromatic rings, thus the optical gap for the
bigger molecules e.g., 5 and 7 is similar to that of the smaller ones (e.g., 1, 3 and 4).
Figure 4. DFT calculated HOMO and LUMO orbitals of the fused alloxazine form compounds.

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Figure 5. DFT calculated HOMO and LUMO orbitals of the non-fused alloxazine form compounds.
It has to be noted that like the non-linear fused compounds
5 and 7, non-fused systems in the
excited state (LUMO orbitals) possess an electron density selectively distributed near only one of the
aromatic rings in the 7th position which, however, can be explained by the asymmetrical alloxazine
group, where strongly electronegative carboxyl groups cause a redistribution of the electron density,
resulting in unequal rotation angles between the alloxazine core and the non-fused aromatic rings.
Generally, in the optimized ground state the dihedral rotation angles between the core and the aromatic
substituents for the compounds 8–11 are stated in Table 2. Interestingly, the isomerization of the
compound results in the redistribution of the electron clouds due to the interaction of the transferred
proton in the position 8th and the aromatic rings, especially in the case of the non-fused compounds.
This is the result of the free rotation between the isoalloxazine core and the abovementioned ending
groups. Electropositive hydrogen in the iso-tautomers repulses the aromatic rings, especially with the
electron-poor thiophene, resulting in the large difference in the dihedral angles of the two tautomers.
Table 2. Dihedral rotation angle between the alloxazine/isoalloxazine core and the aromatic rings.
Alloxazine
Isoalloxazine
◦(pos7)
◦(pos6)
◦(pos7)
◦(pos6)
α ,
4
#
α ,
1
α ,
2
α ,
3
8
9
10
11
34.4
36.6
49.5
54.5
31.0
88.6
32.7
31.5
14.3
17.7
31.7
19.6
27.2
18.5
15.3
2.1
α1,◦ dihedral rotation angle in the X position, α2,◦ dihedral rotational angle in the Y position.

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2.4. Electrochemical Measurements
Lumichrome derivatives usually exhibit a redox reaction associated with the transfer of two
electrons and two protons, which is highly important when the application of such materials in
high-energy-density redox-flow batteries is considered [68]. Almost all of the derivatives, except
3
,
8
, and
which makes them possible candidates for catalytic reactions like oxygen evolution reaction conditions.
Upon reduction, the compounds and showed reversible 2-electron reduction while exhibited
1-electron reversible reduction. Compound showed on the other hand, revealed 2-electron irreversible
9, showed promising electrochemical behavior in the positive part of the voltammogram
1
8
3
5
reduction behavior. These compounds might be suitable candidates for hydrogen evolution reaction
in water. Table 3 shows the energy levels (HOMO and LUMO) of flavin derivatives calculated using
electrochemical methods.
Table 3. HOMO and LUMO levels obtained by the electrochemical measurements.
Compound
HOMO (eV)
LUMO (eV)
1
2
3
4
5
6
7
8
9
−6.30
−4.20
−4.80
−4.30
−4.00
−3.90
−4.50
−4.05
−3.90
−3.93
−4.00
−4.10
n.a.
−6.10
−6.30
−6.50
−6.20
n.a.
n.a.
n.a.
n.a.
−6.50
10
11
Comparing the electrochemical measurements with the values obtained from the optical
measurements we can assume that the measured values are related to the low band-gap isoalloxazine
form for the derivatives 1, 3, 4, 5 and 11. For compound 6 the band gap obtained by the electrochemical
measurement was found to be significantly smaller than the one measured optically, and further
measurements are needed to explain this phenomenon.
All of the compounds suffer from high solubility in organic solvents upon reduction and oxidation.
Their electrochemical behavior in aqueous media, as well as their catalytic performance, is currently
under investigation.
3. Conclusions
The novel synthesis approaches, and comprehensive characterization, including optics,
DFT modeling and electrochemistry of various novel Flavin-inspired derivatives are described in the
present paper. The compounds possess high thermal stability and chemical versatility, which indicates
their potential as building blocks in the future (e.g., as copolymers or metal-organic complexes).
Extension of the conjugated tail of the alloxazine group bathochromically shifts the absorption and
emission spectrum. However, as was shown, in solution the compounds possess multi-chromophoric
behavior due to proton transfer and the formation of the isoalloxazine form. The appearance of
the tautomeric forms can be affected by many factors, including the molecular structure, solvent,
pH, etc., which requires the comprehensive study of each individual compound. According to the
electrochemical measurements the materials under investigation show promising electrochemical
behavior for catalytic reactions like oxygen evolution. Moreover these compounds can be successfully
applied as a redox centers in biomimetic energy storage [25].

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4. Experimental Section
4.1. Materials and Synthesis
All solvents and reagents were obtained commercially and used as received unless stated
otherwise. All moisture-sensitive reactions were performed in dry flasks fitted with glass stoppers or
rubber septa under a positive pressure of argon. Air- and moisture-sensitive liquids and solutions were
transferred by syringe or stainless steel cannula. Anhydrous Na2SO4 was used to dry organic solutions
during workup, and evaporation of the solvents was performed under reduced pressure using a rotary
evaporator. Flash column chromatography was performed using 220–440 mesh silica gel. Thin-layer
chromatography was conducted on Supelco 60 TLC plates (Sigma Aldrich, St. Louis, MO, USA) with
1
254 nm fluorescent indicator. Spots were observed under UV irradiation (254 nm or 354 nm). H- and
13C-NMR spectra were recorded in CDCl3 or DMSO-d6 using an Avance III 300 MHz spectrome◦ter
(Bruker, Billerica, MA, USA) with working frequencies of 300 MHz and 75 MHz, respectively, at 30 C.
Chemical shifts are expressed in parts per million (
referenced to residual protons in the NMR solvent (CHCl3:
δ
scale) downfield from tetramethylsilane and are
7.25 ppm, DMSO: 2.50 ppm). Coupling
δ
δ
constants (J) are given in Hz with coupling expressed as s—singlet, bs—broad singlet, d—dublet,
dd—doublet of doublet, t—triplet, tdd—doublet of triplet of doublets, ddd—doublet of doublet of
doublets, m—multiplet. Elemental analysis was performed using an EuroEA3000 Elemental Analyser
(Eurovector, Pavia, Italy). Melting points were determined using a Kofler apparatus equipped with a
Nagema PHMK 05 microscope (Nagema, Dresden, Germany) and the temperatures were not corrected.
Thermogravimetric analysis was performed using TGA Q50 instrument (TA Instruments, New Castle,
DE, USA) with nitrogen as the carrier gas. Mass spectra were recorded on a GC–MS spectrometer ITQ
700 (DEP) (Thermo Fisher Scientific, Waltham, MA, USA) IR spectra were recorded on an ALPHA
FT-IR Spectrometer (Bruker).
4.2. General Procedure for the Preparation of the Fused Flavins 1–6 by Approach A
The corresponding diamine (1.5 mmol, 1 eq.) was mixed with alloxan monohydrate (0.21 g,
1.5 mmol, 1 eq.) and boric acid (0.092 g, 1.5 mmol, 1 eq.) in glacial acetic acid (10 mL). The mixture
was stirred under an Ar atmosphere at 60 ◦C for 15 h. The reaction mixture gradually turned into a
suspension. The solid fraction was collected by filtration, washed with acetic acid (20 mL) and diethyl
ether (20 mL). The crude product was purified by digestion in boiling methanol. Digested flavin
derivative was further subjected to vacuum sublimation to yield the final target compound.
4.3. Benzo[g]pteridine-2,4(1H,3H)-dione (1)
Compound
1 was synthesized according to the General Procedure, Approach A. Reaction of
diamine
A
(0.500 g, 4.62 mmol) with alloxan monohydrate (0.741 g, 4.62 mmol) and boric acid (0.291 g,
◦
4.62 mmol) provided compound
1
as a light green solid (yield 0.56 g, 86%). M.p. > 330 C (lit.
◦
1
>400 C) [69]. H-NMR (DMSO-d6, TMS): δ 11.92 (s, 1H), 11.73 (s, 1H), 8.17 (d, J = 4.8 Hz, 1H), 7.96–7.91
(m, 2H), 7.81–7.73 (m, 1H). Elemental analysis calcd (%) for C10H6N4O2: C 56.08, H 2.82, N 26.16;
found: C 56.04, H 2.85, N 26.12.
4.4. Naphtho[2,3-g]pteridine-2,4(1H,3H)-dione (2)
Compound
2 was synthesized according to the General Procedure, Approach A. Reaction of
diamine (0.402 g, 2.53 mmol) with alloxan monohydrate (0.402 g, 2.53 mmol) and boric acid (0.159 g,
B
◦
◦
2.53 mmol) provided compound
torr, decomp.) [70]. H-NMR (DMSO-d6, TMS):
2
as a red solid (yield 0.517 g, 73%). M.p. > 330 C (lit. 385 C at 0.001
11.93 (s, 1H), 11.73 (s, 1H), 8.90 (s, 1H), 8.52 (s, 1H),
1
δ
8.26 (d, J = 4.8 Hz, 1H), 8.19 (d, J = 4.8 Hz, 1H), 7.70–7.65 (m, 1H), 7.63–7.58 (m, 1H). Elemental analysis
calcd (%) for C14H8N4O2: C 63.64, H 3.05, N 21.20; found: C 63.61, H 2.99, N 21.25.

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4.5. 8,9-Difluoronaphtho[2,3-g]pteridine-2,4(1H,3H)-dione (3)
Compound
3 was synthesized according to the General Procedure, Approach A. Reaction of
diamine
C
(0.499 g, 3.47 mmol) with alloxan monohydrate (0.561 g, 3.47 mmol) and boric acid (0.211
◦
g, 3.47 mmol) provided compound
(lit. 310–312 C) [46]. H-NMR (DMSO-d6, TMS):
3
as a light purple solid (yield 0.504 g, 58%). M.p. 310–311 C
◦
1
δ 12.01 (s, 1H), 11.78 (s, 1H), 8.29 (dd, J = 10.9, 8.6 Hz,
1H), 8.00 (dd, J = 11.4, 8.2 Hz, 1H). Elemental analysis calcd (%) for C10H4F2N4O2: C 48.01, H 1.61, N
22.04; found: C 48.07, H 1.68, N 22.09.
4.6. 2,4-Dioxo-1,2,3,4-tetrahydropteridine-6,7-dicarbonitrile (4)
Compound
diamine (0.500 g, 4.63 mmol) with alloxan monohydrate (0.740 g, 4.63 mmol) and boric acid (0.291 g,
4.63 mmol) provided compound
as a light grey solid (yield 0.319 g, 32%). M.p. > 330 ◦C. 1H-NMR
(DMSO-d6, TMS): 158.55, 149.98, 149.35,
12.87 (br s, 1H), 12.17 (s, 1H). 13C-NMR (DMSO-d6, TMS):
4 was synthesized according to the General Procedure, Approach A. Reaction of
D
4
δ
δ
134.72, 131.41, 125.39, 114.09, 113.55. Elemental analysis calcd (%) for C8H2N6O2: C 44.87, H 0.94, N
39.25; found: C 48.84, H 1.02, N 39.21.
4.7. Pyreno[4,5-g]pteridine-11,13(10H,12H)-dione (5)
Compound
diamine (0.270 g, 1.16 mmol) with alloxan monohydrate (0.186 g, 1.16 mmol) and boric acid (0.072 g,
1.16 mmol) provided compound
(DMSO-d6, TMS): 12.19 (s, 1H), 11.82 (s, 1H), 9.22–9.14 (m, 2H), 8.48 (d, J = 7.5 Hz, 1H), 8.39
5 was synthesized according to the General Procedure, Approach A. Reaction of
E
◦
as a yellow solid (yield 0.293 g, 75%). M.p. > 330 C. 1H-NMR
5
δ
(d, J = 7.5 Hz, 1H), 8.26–8.12 (m, 4H). Elemental analysis calcd (%) for C20H10N4O2: C 71.00, H 2.98, N
16.50; found: C 71.08, H 2.95, N 16.44. EI/MS (m/z): 338.080. Found 338.081 (100). IR (neat, cm−1):
3182, 3064, 2937, 2825, 1720, 1689, 1570, 1555, 1447, 1422, 1385, 1364, 1304, 1279, 1234, 1176, 1163, 1033,
928, 859, 829, 812, 752, 718, 653, 617, 590, 525, 487, 459, 445, 434.
4.8. Pteridino[6,7-b]phenazine-2,4(1H,3H)-dione (6)
Compound
6 was synthesized according to the General Procedure, Approach A. Reaction of
diamine
F
(0.101 g, 0.480 mmol) with alloxan monohydrate (0.077 g, 0.480 mmol) and boric acid
◦
(0.030 g, 0.480 mmol) provided compound
6
as a dark purple solid (yield 0.080 g, 57%). M.p. > 330 C.
Elemental analysis calcd (%) for C16H8N6O2: C 60.76, H 2.55, N 26.57; found: C 60.74, H 2.52, N 26.50.
4.9. General Procedure for the Preparation of the Non-Fused Flavins 7–11 by Approach B
5,6-Diaminouracil sulphate (1.31 mmol, 1 eq.) was suspended in water (35 mL). The mixture was
made alkaline using a methanolic solution of NaOH (0.65M, 3 eq.) and heated to 90 ◦C for 15 min.
A solution of diketone (1.96 mmol, 1.5 eq.) in a mixture of THF (40 mL) and water (15 mL) was added
and the mixture was stirred at 90 ◦C for 45 min. The reaction mixture was acidified glacial acetic acid
(15 mL) and heated at 90 ◦C for another 20 h. The reaction mixture was subsequently concentrated to
approximately 1/3 of the volume, cooled in the freezer and the as-obtained suspension was filtered
and the solid fraction was washed with cold water (30 mL) and cold ethanol (20 mL) to yield the crude
flavin derivative. The crude product was purified by digestion in boiling methanol. The digested
flavin derivative was further subjected to vacuum sublimation to yield the final target compound.
4.10. Phenanthro[9,10-g]pteridine-11,13(10H,12H)-dione (7)
Compound
diketone (0.817 g, 3.92 mmol) with 5,6-diaminouracil sulphate (1.00 g, 2.62 mmol) provided
compound
12.21 (s, 1H), 11.84 (s, 1H), 9.09–8.99 (m, 2H), 8.91–8.81 (m, 2H), 7.98–7.90 (m, 1H), 7.89–7.80 (m, 3H).
Elemental analysis calcd (%) for C8H2N6O2: C 68.79, H 3.21, N 17.83; found: C 68.66, H 3.11, N 17.75.
7 was synthesized according to the General Procedure, Approach B. Reaction of
G
◦
1
7
as a yellow solid (yield 0.304 g, 37%). M.p. > 330 C. H-NMR (DMSO-d6, TMS):
δ

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4.11. 6,7-Diphenylpteridine-2,4(1H,3H)-dione (8)
Compound
diketone
compound
(DMSO-d6, TMS):
8 was synthesized according to the General Procedure, Approach B. Reaction of
H
8
(0.577 g, 2.75 mmol) with 5,6-diaminouracil sulphate (0.700 g, 1.83◦mmol) provided
◦
1
as a bright grey solid (yield 0.262 g, 30%). M.p. 321–323 C (lit. 320–325 C) [43]. H-NMR
δ
12.06 (s, 1H), 11.73 (s, 1H), 7.44–7.29 (m, 10H). Elemental analysis calcd (%) for
C18H12N4O2: C 68.35, H 3.82, N 17.71; found: C 68.24, H 3.74, N 17.62.
4.12. 6,7-Di(pyridin-2-yl)pteridine-2,4(1H,3H)-dione (9)
Compound
diketone (0.416 g, 1.96 mmol) with 5,6-diaminouracil sulphate (0.500 g, 1.31 mmol) provided
compound
TMS): 12.11 (s, 1H), 11.73 (s, 1H), 8.31 (d, J = 4.5 Hz, 1H), 8.24 (d, J = 4.5 Hz, 1H), 7.92 (tdd, J = 7.6, 4.2,
9 was synthesized according to the General Procedure, Approach B. Reaction of
I
◦
1
9
as a bright beige solid (yield 0.105 g, 25%). M.p. 277–288 C. H-NMR (DMSO-d6,
δ
1.7 Hz, 2H), 7.79 (dd, J = 14.5, 7.8 Hz, 2H), 7.38 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.31 (ddd, J = 7.4, 4.8,
1.2 Hz, 1H). Elemental analysis calcd (%) for C16H10N6O2: C 60.38, H 3.17, N 24.40; found: C 60.29, H
3.08, N 24.48.
4.13. 6,7-Di(furan-2-yl)pteridine-2,4(1H,3H)-dione (10)
Compound 10 was synthesized according to the General Procedure, Approach B. Reaction
of diketone
J (0.373 g, 1.96 mmol) with 5,6-diaminouracil sulphate (0.500 g, 1.31 mmol) provided
compound 10 as a yellow solid (yield 0.168 g, 43%). Purification of the crude product also involved
flash column chromatography with gradient elution (EtOAc
→
EtOAc/MeOH 50/50
→
MeOH)
1
followed by vacuum sublimation. M.p. 285–286 ◦C (lit. 285–287 ◦C) [47]. H-NMR (DMSO-d6, TMS):
δ
12.05 (s, 1H), 11.65 (s, 1H), 7.95 (d, J = 1.0 Hz, 1H), 7.85 (d, J = 1.0 Hz, 1H), 6.78-6.75 (m, 1H), 6.72–6.68
(m, 2H), 6.55 (d, J = 3.1 Hz, 1H). Elemental analysis calcd (%) for C14H8N4O4: C 56.76, H 2.72, N 18.91;
found: C 56.69, H 2.99, N 19.01.
4.14. 6,7-Di(thiophen-2-yl)pteridine-2,4(1H,3H)-dione (11)
Compound 11 was synthesized according to the General Procedure, Approach B. Reaction of
diketone
K
(0.654 g, 2.94 mmol) with 5,6-diaminouracil sulphate (0.750 g, 1.96 mmol) provided
1
compound 11 as a yellow solid (yield 0.264 g, 40%). M.p. > 330 ◦C (lit. 355–357 ◦C) [47]. H-NMR
(DMSO-d6, TMS):
δ 12.02 (s, 1H), 11.69 (s, 1H), 7.84 (d, J = 4.9 Hz, 1H), 7.79 (d, J = 4.9 Hz, 1H), 7.22
(d, J = 3.5 Hz, 1H), 7.18–7.07 (m, 3H). Elemental analysis calcd (%) for C14H8N4O2S2: C 51.21, H 2.46,
N 17.06, S 19.53; found: C 51.27, H 2.51, N 17.01, S 19.61.
4.15. Optical Measurements
Absorption spectra of solution were measured using a Varian Cary Probe 50 UV-Vis
spectrophotometer (Agilent Technology, Santa Clara, CA, USA). The measured spectra were processed
using the CaryWinUV software. The absorption spectra were corrected to a baseline, which was
determined by measurement of absorption spectra of pure solvent. Solutions were studied in Hellma
QS quartz cuvette with an optical path of 10 mm. Solution concentrations of sublimed materials for
optical measurements were adjusted to a level of light absorption less than 1. Concentration of all
compounds/samples was 1
×
10−5 mol −1. All optical characteristics were measured in ambient
L
·
air. Excitation and emission spectra of solution were measured using a Horiba Fluorolog (Horiba
Jobin Yvon, Kyoto, Japan). The measured spectra were processed using the FluorEssence software.
Samples were excited at absorption maxima and varied according to the material.
4.16. Electrochemical Measurements
In order to determine the energetic levels for each compound we have conducted electrochemical
experiments. Cyclic voltammetry for the materials under investigation was performed using

Molecules 2018, 23, 2271
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a Jaissle Potentiostat-Galvanostat IMP 88 PC (Jaissle Elektronik GmbH, Waiblingen, Germany).
The electrochemical setup consisted of 3-electrode system. A Cr-Au coated (5/80 nm respectively)
glass slide coated with the respective small molecule served as the working electrode while a Pt plate
was used as the counter electrode. A silver wire coated with silver chloride was used as the quasi
reference electrode. Electrolyte solution contained 0.1 M TBAPF6 in acetonitrile and the potential
was cycles between
−
1.2 V and 1.2 V vs. Ag/AgCl quasi-reference electrode. The potentials were
calibrated internally against ferrocene/ferrocenium couple (Fc/Fc+) and the following formula is used
to estimate the HOMO and LUMO levels:
EHOMO = −(4.75 eV + Eoxonset vs. NHE)
ELUMO = −(4.75 eV + Eredonset vs. NHE)
(1)
(2)
4.17. DFT Calculations
Molecular structures were simulated using the Gaussian 09 software package [71], for all the
compounds presented in the paper the electronic structures of the above-mentioned systems were
optimized using the 6-311G** split-valence polarized triple-ζ basis set was combined with Becke’s
three-parameter hybrid functional with the Lee, Yang, and Parr correlation functional (B3LYP).
Absorption spectra were calculated using TD-DFT procedure for 10 lowest excited states.
Author Contributions: J.R.: synthesis of intermediates and final products, structural identification, analytical
methods, M.W., P.H., V.S.: optical measurement and interpretation of experimental data, A.K.: Quantum chemical
calculation, Ideas supporting, C.Y.: thin film preparation, thin layers characterisation, D.H.A.: electrochemical
measurement, N.S.S.: supervising of physical chemistry methods, interpretation of measurement results,
J.K.: synthesis of final products, analytical methods, supervising of manuscript progress. The manuscript was
written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Funding: This research was funded by the Czech Science Foundation grant No. 17-24707S. Research infrastructure
was supported by project No. REG LO1211 from the National Programme for Sustainability I (MEYS CR), projects
Nos. FCH-S-18-003 and FCH-S-18-5331. Austrian Science Foundation, FWF is gratefully acknowledged for
financial support within the Wittgenstein Award (Solare Energie Umwandlung Z222-N19 for N.S.S.). The APC
was funded by Decision 38/2017—Open Access Fund, Brno University of Technology.
Conflicts of Interest: There are no conflicts to declare.
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Sample Availability: Samples of the compounds 1–11 are available from the authors.
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