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, Purkyňova 118,
612 00 Brno, Czech Republic; [email protected] (J.R.); [email protected] (P.H.);
[email protected] (V.S.); [email protected] (A.K.); [email protected] (M.W.)
2 Linz Institute for Organic Solar Cells (LIOS), Physical Chemistry, Johannes Kepler University Linz,
Altenbergerstraße 69, 4040 Linz, Austria; [email protected] (D.H.A.);
[email protected] (C.Y.); [email protected] (N.S.S.)
* Correspondence: [email protected]




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

Molecules 2018, 23, 2271 2 of 18

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 [27,28] have also found application in molecular logic
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],
π–π stacking [35] or the local environment [36]. On the other hand, the influence of enlarging the
π-conjugated system on the optoelectronic properties of flavin derivatives remains less investigated,
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 π-system from the basic N10-butyl substituted flavin core to the
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 1–4 were prepared
from commercially available 1,2-diamines A–D (Figure 1) while the synthesis of the derivatives 5 and
6 required laboratory preparation of 1,2-diamines E and F. Pyrene-4,5-diamine E was prepared by
three-step synthesis: oxidation [53] of pyrene by NaIO4 catalyzed by RuCl3 provided pyrene-4,5-dione
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(L) that was transformed to pyrene-4,5-dioxime (M) by reaction with NH2 OH. M was subsequently
M wastosubsequently
reduced E by catalyticreduced to E by using
hydrogenation catalytic
a Hhydrogenation
2 /Pd/C system using
[54]. a2,3-Diaminophenazine
H2/Pd/C system [54]. F
was obtained by the reaction of o-phenylenediamine A and FeCl3 in dilute A
2,3-Diaminophenazine F was obtained by the reaction of o-phenylenediamine and FeCl3 inacid
hydrochloric dilute
[55].
Wehydrochloric
also probed acid [55]. We also
the preparation of probed
non-fusedthe flavins
preparation of non-fused
by Approach A. It flavins
embodied by Approach A. It
finding a suitable
embodied
means finding aofsuitable
of synthesis means of synthesis of 1,2-diarylethane-1,2-diamines
1,2-diarylethane-1,2-diamines which could
which could react with alloxan. react
Compared
with alloxan. Compared to aromatic 1,2-diamines, the reaction would provide 2,3-dihydropyrazines
to aromatic 1,2-diamines, the reaction would provide 2,3-dihydropyrazines that would have to be
that would
oxidized to thehave
finaltoflavins
be oxidized to performed
[56]. We the final flavins
pilot [56]. We performed
experiments on the pilot experiments
preparation of theon the 8
flavin
preparation of the flavin 8 with intermediate diamine (P) being prepared in a similar fashion to E.
with intermediate diamine (P) being prepared in a similar fashion to E. Benzil (N) was transformed
Benzil (N) was transformed to 1,2-dioxime (O) and submitted to the reduction. Contrary to the
to 1,2-dioxime (O) and submitted to the reduction. Contrary to the reduction of simple oximes to
reduction of simple oximes to amines, reduction of 1,2-dioximes is probed in less detail. We tested
amines, reduction of 1,2-dioximes is probed in less detail. We tested several reduction methodologies
several reduction methodologies used for the reduction of simple oximes, i.e., reduction by LiAlH4
used for the reduction of simple oximes, i.e., reduction by LiAlH4 [57], SnCl2 /HCl [58], Red-Al [59],
[57], SnCl2/HCl [58], Red-Al [59], Pd/C/N2H4 [60], NaBH4/NiCl2 [61], NaBH3CN/TiCl3/NH4OAc [62]
Pd/C/N2 H4 [60], NaBH4 /NiCl2 [61], NaBH3 CN/TiCl3 /NH4 OAc [62] and NaBH4 /TiCl4 [63] under
and NaBH4/TiCl4 [63] under various reaction conditions. Among them, only reduction with
various
NaBHreaction conditions. Among them, only reduction with NaBH4 /TiCl
4/TiCl4 provided us with desired 1,2-diphenyl-ethane-1,2-diamine 4 provided
P with us with
good yields. Ondesired
the
1,2-diphenyl-ethane-1,2-diamine
other hand, we could not obtain P withthe good yields.
desired On the other hand,
2,3-dihydropyrazine Q orwedirectly
could not obtain
flavin 8 bythe
desired 2,3-dihydropyrazine
condensation with alloxan Q or directly
under flavin 8or
either neutral byacidic
condensation with alloxan
glacial AcOH under
conditions. Weeither
have neutral
thus
or envisaged
acidic glacial AcOH
another conditions. We have thus envisaged another approach.
approach.

Scheme
Scheme 1. 1. Generalscheme
General schemeof
ofthe
thesynthesis
synthesis of
of flavins
flavins 1–11
1–11by
byApproaches
ApproachesAAand
andB.B.
Reaction conditions:
Reaction conditions:
(i) AcOH, H 3BO3; (ii) AcOH, MeOH/H2O/THF.
(i) AcOH, H3 BO3 ; (ii) AcOH, MeOH/H2 O/THF.

Approach B is a suitable way to prepare non-fused aromatic flavins from 1,2-diketones and
Approach B is taking
5,6-diaminouracil, a suitable
into way to prepare
consideration thatnon-fused aromaticpoint
from the synthetic flavins from 1,2-diketones
of view, 1,2-diketonesare and
5,6-diaminouracil,
more readily available than the corresponding 1,2-diamines. The synthesis embodies a condensationare
taking into consideration that from the synthetic point of view, 1,2-diketones
more
of areadily available
1,2-diketone withthan the corresponding
5,6-diaminouracil under1,2-diamines.
acidic conditions The[64].
synthesis embodieswas
The procedure a condensation
improved
of by
a 1,2-diketone
altering the with 5,6-diaminouracil
solvent system by usingunder of THF acidic
whichconditions
dissolves[64]. The procedure1,2-diketones
the corresponding was improved
bybetter
altering
thanthe solvent
lower system
alcohols by using
resulting of THF
in a broader which dissolves
spectrum of employable the corresponding 1,2-diketones
substrates. The intermediate
better than lower alcohols
5,6-diaminouracil resulting
was obtained byina a3-step
broader spectrum
synthesis [65].ofCondensation
employable substrates. The intermediate
of ethyl cyanoacetate with
5,6-diaminouracil was obtained
urea under basic (NaOEt) by a yielded
conditions 3-step synthesis
6-aminouracil. [65]. The
Condensation
nitrosation of of6-aminouracil
ethyl cyanoacetate
provided with
5-nitroso-6-aminouracil
urea under basic (NaOEt) that was subsequently
conditions reduced to 5,6-diaminouracil
yielded 6-aminouracil. The nitrosation of by6-aminouracil
sodium hydrosulfite.
provided
Subsequently, the flavins
5-nitroso-6-aminouracil that was7–10subsequently
were prepared from to
reduced commercially available
5,6-diaminouracil 1,2-diketones
by sodium G–J
hydrosulfite.
whereas thethe
Subsequently, flavin 11 from
flavins 7–10 thewereprepared
prepareddiketone K. The 2,2′-thenil
from commercially availableK was obtained by
1,2-diketones G–J benzoin
whereas
condensation of thiophene-2-carbaldehyde to 2,2′-thenoin
0 and its in
the flavin 11 from the prepared diketone K. The 2,2 -thenil K was obtained by benzoin condensationsitu oxidation by ambient
of oxygen [66].
thiophene-2-carbaldehyde to 2,20 -thenoin and its in situ oxidation by ambient oxygen [66].
The synthesis of flavin
The synthesis of flavin derivatives derivatives by approaches
by both both approaches requiredrequired appropriate
appropriate purification
purification methods
methods in order to obtain high purity materials, suitable for further optical and electrochemical
in order to obtain high purity materials, suitable for further optical and electrochemical measurements.
measurements. Due to very limited solubility of the all prepared compounds digestion in boiling
Due to very limited solubility of the all prepared compounds digestion in boiling methanol was used
methanol was used as the first stage of the purification. Vacuum sublimation was used as the final
as the first stage of the purification. Vacuum sublimation was used as the final purification step.
purification step. Target compounds with the final purity were used for further experiments.
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
The structures of the prepared flavins (except for compound 6 which is practically insoluble in
common solvents) was confirmed by 1H-NMR and also by 13C-NMR in the case of compound 4
common 1 13 C-NMR in the case of compound 4 where
where solvents)
only 1H-NMR was confirmed
could be by H-NMR insufficient
considered and also by (see Supporting Information). A typical
only 1 H-NMR could
phenomenon of thebeproton
considered
spectrainsufficient
for the target(seecompounds
SupportingisInformation).
the appearance A typical phenomenon
of two singlets in the of
the11–13
proton ppm region belonging to strongly deshielded N–H protons along with a downfield shift ofregion
spectra for the target compounds is the appearance of two singlets in the 11–13 ppm the
belonging
aromaticto strongly
protons deshielded
caused by theirN–H protonstoalong
conjugation with a downfield
the strongly shift of thediimide
electron-withdrawing aromatic protons
moiety.
caused by their
The purity conjugation
of the to the strongly
final compounds electron-withdrawing
was further checked by elementary diimide moiety.
analysis. The purity
Nevertheless, of the
it has
final compounds was further checked by elementary analysis. Nevertheless,
to be stated that additional quantitative and qualitative analysis of the purity of the final compoundsit has to be stated that
additional
by various quantitative
either normaland orqualitative analysis
reverse-phase HPLC of the purity proved
methods of the final
to becompounds by various
rather inconclusive as either
the
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Molecules 2018, 23, x FOR PEER REVIEW 4 of 18

normal or reverse-phase
compounds are poorlyHPLC methods
soluble proved toemployed
in all commonly be rather inconclusive as the compounds
solvents, including water. On the areother
poorly
soluble
hand,inwith
all commonly
the stepwiseemployed solvents,
purification including
process water.
involving On theand
digestion other hand, with
vacuum the stepwise
sublimation we
obtained process
purification final compounds with sufficiently
involving digestion highsublimation
and vacuum purity forwethe purposes
obtained finalofcompounds
optical andwith
electrochemical
sufficiently studies.
high purity for the purposes of optical and electrochemical studies.

Figure 1. Compounds under investigation, synthetized by Approach A and Approach B.

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
forTGA measurements
compound provedofthe
6. The series highflavin
fused thermal stability of
derivatives the flavin derivatives
(compounds 1–7) showed reaching 450 ◦ C for
a progressive
compound Thedecomposition
6. the
increase of series of fusedtemperature
flavin derivatives
with the(compounds 1–7) showed
increasing number of fuseda progressive
aromatic ringsincrease
(from of
the276.78
decomposition temperature with the ◦
°C for compound 3 to 443.11 °C increasing number6,ofsee
for the compound fused aromaticInformation).
Supporting rings (from 276.78
On the C
forother
compound 3 toseries
443.11 ◦ C for the compound 6, see Supporting
hand, the of non-fused flavin derivatives (compoundsInformation).
8–11) provided Ondecomposition
the other hand,
thetemperatures
series of non-fused
in the range of 305.62 °C (compound 10) to 354.20 °C (compound 11). It should be in
flavin derivatives (compounds 8–11) provided decomposition temperatures
thestated
rangethat
of 305.62 ◦ C (compound 10) to 354.20 ◦ C (compound 11). It should be stated that the most
the most of derivatives are stable to temperatures higher than 300 °C and compounds 5
of and
derivatives are stable
6 even more than to temperatures
400 higherstability,
°C. Such thermal than 300not◦ Coften
and compounds 5 and molecules,
observed at small 6 even more than
may
◦ C. Such
400strongly contribute
thermal to the suitability
stability, of the
not often materials
observed for themolecules,
at small applications in organic
may stronglyelectronics.
contribute to the
suitability of the materials for the applications in organic electronics.
Molecules 2018, 23, 2271 5 of 18

Molecules 2018, 23, x FOR PEER REVIEW 5 of 18

2.2. Optical Measurements
2.2. Optical Measurements
Flavin-like compounds are well known to possess the formation of tautomers in the solution
by means Flavin-like compounds
of the proton are well
transfer [67] known
resulting to possess the formation
in the formation of tautomers
of the iso-form. inThus, the solution
in our bycase,
means of the proton transfer [67] resulting in the formation of the
the influence of the conjugated functional “tail” on the proton transfer would be noticeable in 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,
absorption-emission optical spectra in DMSO. Depending on the electronegativity, conjugated
planarity and the absence/presence of the free rotation we expect to observe clear differences in the
length, planarity and the absence/presence of the free rotation we expect to observe clear differences
optical behavior of the materials under study. Indeed, how it is shown below, the optical measurements
in the optical behavior of the materials under study. Indeed, how it is shown below, the optical
of the considered compounds, possessed interesting, but complex behavior.
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
The optical spectra of all compounds under study consisted of more than one fluorescence band
with
withdifferent
different intensity, which
intensity, on one
which on onehandhandcan be cananbe indication
an indicationof impurities. Indeed
of impurities. a small
Indeed a amount
small
of impurities can drastically affect the emission spectra, however on
amount of impurities can drastically affect the emission spectra, however on the other hand, the the other hand, the assumption
that the multi-chromophoric
assumption behavior is based
that the multi-chromophoric behavioron the correspondence
is based of the absorption
on the correspondence spectra with
of the absorption
thespectra
excitation
withones. In this case
the excitation the In
ones. possible
this case impurities,
the possible which could remain
impurities, whichafter
couldthe recrystallization
remain after the
should not affect the absorption spectra. Moreover, the formation of the
recrystallization should not affect the absorption spectra. Moreover, the formation of the tautomers tautomers in other alloxazine
derivatives, i.e., lumichrome
in other alloxazine derivatives,[67]i.e.,
or 6,7-dicyanolumazine [40] (compound 4)[40]
lumichrome [67] or 6,7-dicyanolumazine are (compound
well known. 4) As
arean
example,
well known.it has been As reported,
an example, that init aqueous
has been solution
reported,of thethat
6,7-dicyanolumazine
in aqueous solution at neutral
of the pH,
6,7-dicyanolumazine
it possesses two-chromophoricat neutralbehavior
pH, it possesses
with twotwo-chromophoric
different absorption behavior
bandswith at 350 twoanddifferent
400 nm.
absorption
However bands
in the caseat of
350the
and 400 nm.
whole familyHowever in the case
of materials underof the whole
study, it isfamily of materials
practically under to
impossible
study,the
perform it is practically impossible
measurements in aqueoustosolution
performforthe all measurements
the derivatives,inasaqueous
most of the solution for allcannot
derivatives the
derivatives, as most of the derivatives cannot be dissolved in water.
be dissolved in water. However, it has to be noted, that due to the high purity of our compounds theHowever, it has to be noted, that
due tochromophore
second the high purity of our
effects compounds
are unlikely to the besecond
caused chromophore
by impurities effects
and/orare unlikely
degradation to be products
caused
by impurities and/or degradation products (see TGA and
(see TGA and NMR spectra in the Supporting Information). Thus, considering the formation of the NMR spectra in the Supporting
Information). Thus, considering the formation of the iso-form from the alloxazine derivatives, we
iso-form from the alloxazine derivatives, we presume the presence of two chromophores marked Ch.
presume the presence of two chromophores marked Ch. A and Ch. B. Moreover, the equilibrium
A and Ch. B. Moreover, the equilibrium concentration of tautomers can be changed by conditions
concentration of tautomers can be changed by conditions like temperature or acidity of solvent. The
like temperature or acidity of solvent. The effect of the environment of pH is common for the flavins,
effect of the environment of pH is common for the flavins, thus additionally we demonstrate the
thus additionally we demonstrate the effect of acidity of the environment on the fluorescence emission
effect of acidity of the environment on the fluorescence emission spectra of the compound 1 (see
spectra of the compound 1 (see Supporting Information). There is clear evidence that long wavelength
Supporting Information). There is clear evidence that long wavelength emission of the chromophore
emission
(Ch. B)ofisthe chromophore
missing in acidic (Ch. B) is missing
environments in acidictoenvironments
in contrast the basic ones, inwhich
contrast to the basic
indicates ones,
that the
which indicates that the proton transfer is hindered and one
proton transfer is hindered and one of the tautomers is eliminated in the acidic solvent. 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
It has to be
magnitude noted that
(especially forthe intensity ratio,
compounds 2, 4, 7,in9).some
When, cases
thediffers
emission by band
up to ofa few orders of magnitude
the chromophore with
(especially for compounds 2, 4, 7, 9). When, the emission band of the chromophore
higher energy gap (Ch. A) has smaller intensity due to reabsorption in all the cases, which results with higher energy
gapfrom
(Ch. spectral
A) has smaller
overlap intensity
between due to reabsorption
emission and absorption in all the cases, as
spectra which resultsinfrom
depicted spectral
Figure 2.
overlap between emission
Simultaneously and absorption
these spectral spectra as
overlaps indicate depicted in
a resonance Figuretransfer
energy 2. Simultaneously
from the higher theseenergy
spectral
chromophore
overlaps indicate(Ch. A) to theenergy
a resonance lowertransfer
energy chromophore
from the higher (Ch. B), which
energy amplifies(Ch.
chromophore the A)fluorescence
to the lower
intensity of chromophore Ch. B.
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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Molecules 2018, 23, x FOR PEER REVIEW 7 of 18

Figure
Figure 2. UV-Vis absorption, 2. UV-Vis
excitation and absorption, excitation
emission spectra and
of the emission spectra
compounds of the
considering compounds considering
two-
two-chromophore model with the marked optical band
chromophore model with the marked optical band gaps of observed chromophores. gaps of observed chromophores.

Figure 2. UV-Vis absorption, In the excitation

case of compounds
and emission 2, 5, 8, 10,of11,thethecompounds
spectra overlap between
considering emission spectra of the observ
In the case of compoundswith
two-chromophore model
2, 5, the overlap
8, 10, 11,optical
the marked was bandThis
between
gapssituation
of observed
emission spectra of the observed
chromophores.
two chromophores seen. makes the resolution of chromophores in spectra mo
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 excitati
speculative, butcase
In the the of
presence
compounds of two
spectra. 2, chromophores
If 5,the8, 10, was confirmed
11, the overlap
emission spectrum between by measurement
were emission
originated spectra
fromofonly of the
the oneexcitation
observedchromophore the excitati
twoIfchromophores
spectra. the emission was seen. This
spectrum were situation
originated makesfromtheonly
resolution
one of chromophores
chromophore the in spectra more
excitation spectrum
spectrum would follow the absorption spectrum. However this condition is not fulfilled for t
would speculative,
follow the but the presence
absorption of two chromophores
spectrum. However was condition
this confirmed by is measurement
not fulfilled of the
for theexcitation
compounds
compounds under investigation. On the other hand, superposition of excitation spectra of Ch. A a
under spectra. If the emission
investigation. OnCh.thespectrum were superposition
originated fromof only one chromophore the excitation
Bother hand,
chromophores well fitted to theexcitation
absorptionspectraspectrum of in
Ch. theA and Ch. B
solution.
spectrum would follow the absorption spectrum. However this condition is not fulfilled for the
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 t
compounds under investigation. On the other hand, superposition of excitation spectra of Ch. A and
Exception occurs forappearance solution with a compounds
of absorption
third chromophore 6 and 10. Seemingly,
cansolution.
be observed. in Moreover
case of compoundexcitation 6and emission spect
Ch. B chromophores well fitted to the spectrum in the
the appearance of a third chromophore can be observed. Moreover excitation
Exception occurs for solution with compounds 6 and 10. Seemingly, in case of compound 6with
attributed to chromophore Ch. B have resolved vibronic and emission
structures spectra
the overlap of zero-phon
attributed to chromophore peaks.
Ch. This
B small
have Stokes
resolved shift is
vibroniccharacteristic
structures
appearance of a third chromophore can be observed. Moreover excitation and emission spectra of very
with rigid
overlap structures
of with
zero-phonon very small reorganizati
peaks. This small
attributed energy.
Stokes shift isCh.
to chromophore The compound
characteristic
B have resolved 10 in
of very DMSO solution
rigid structures
vibronic provides
structureswith only
withoverlap
very smallone fluorescence
reorganization attributed to Ch
of zero-phonon band
energy. TheThis
peaks. compound 10according
small Stokes inshift
DMSO tosolution
its position.
is characteristic Nevertheless,
provides
of very oneinfluorescence
onlystructures
rigid thewith
acidic environment
very band
small attributed the emission
reorganization to Ch. Bband of Ch. A occu
energy. The compound Thus,
10 in in neutral
DMSO environment
solution provides we
only presume
one that
fluorescence
according to its position. Nevertheless, in the acidic environment the emission band of Ch. A occurs. the fluorescence
band attributed of Ch.
to Ch.A is effectively reabsorbed
B
according to its and/or
position. quenched
Nevertheless, inby
the energy
acidic transfer
environment to Ch.theB.
Thus, in neutral environment we presume that the fluorescence of Ch. A is effectively reabsorbed by emission band of Ch. A occurs.
and/orThus, in neutral
quenched byenvironment The
energy transferweoptical
presume
to Ch. band
B. gap
that values (Figure
the fluorescence of Ch.3)Awere determined
is effectively as theby
reabsorbed intersection point betwe
and/or quenched by energy transfer
normalized to Ch.
excitation B.
The optical band gap values (Figure 3) (absorption)
were determined and emission
as thespectra of eachpoint
intersection chromophore.
betweenGenerally, the optic
The optical bandmeasurements
gap values (Figure have 3) were that
shown determined as the intersection
the short-wavelength point between
chromophores (Ch. A) have an absorpti
normalized excitation (absorption) and emission spectra of each chromophore. Generally, the optical
normalized excitation band (absorption)
in the andrange emission
of 2.7–3.3spectra
eV andof eachthechromophore.
long-wavelengths Generally, the optical (Ch. B) in the range fro
chromophores
measurements
measurements havehaveshownshownthatthat
the short-wavelength
theTherefore,
short-wavelength chromophores (Ch. A)A)have ananabsorption band
2.4 to 2.8 eV. the bandchromophores
gap difference(Ch. between have
long- absorption
and short-wavelength chromophor
in theband
range of 2.7–3.3
in the range ofeV and the
2.7–3.3 eV0.5long-wavelengths
and theeVlong-wavelengths chromophores (Ch.(Ch. B) in thethe range from
from2.4 to
is about ± 0.1 for the most of chromophores
the cases, however,B)inincase range
of compounds 5 and 11 the differen
2.8 eV.
2.4Therefore, the band gap
to 2.8 eV. Therefore, the difference
band gap betweenbetween
difference long- and long- short-wavelength
and short-wavelength chromophores
chromophores is about
of 0.3 eV was observed.
0.5 ±is0.1 eV for
about 0.5 ±the
0.1most
eV forofthe
themost
cases, however,
of the in case of
cases, however, incompounds
case of compounds 5 and 511and the11difference of 0.3 eV
the difference
of 0.3 eV was observed.
was observed.

Figure 3. Optically measured bandgap and Stoke’s shift values for the compounds 1–11.
Figure
Figure 3. Optically
3. Optically measuredbandgap
measured bandgap and
and Stoke’s
Stoke’s shift
shiftvalues
valuesforfor
thethe
compounds 1–11.1–11.
compounds
Molecules 2018, 23, 2271 8 of 18

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 1 and 2) and/or
the presence/strength of the electron withdrawing substitutions competing with the alloxan ending
functional group (e.g., compounds 3 and 4). In case of more complex structure, as for compounds
5–7, 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 5 and 7) or the electron density is equally distributed due to the acceptor groups
equilibrium (compound 6) as it will be shown later. On the other hand the presence of the free
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

1 386 413 3.0 2.6 0.3 0.6
2 390 450 2.9 2.4 (0.5) 0.7
3 390 450 3.1 2.5 0.6 0.4
4 334 425 3.1 2.6 0.8 0.5
5 434 455 2.7 2.5 (0.1) 0.3
6 356 417 3.3 2.7 0.4 0.3
7 410 448 3.0 2.6 0.1 0.3
8 363 410 3.2 2.8 (0.6) 0.6
9 363 407 3.2 2.7 0.4 0.6
10 358 419 3.1 2.6 (0.6) 0.6
11 393 477 2.7 2.4 (0.6) 0.3
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); ∆EStoke’s —Stoke’s shift. Values in
brackets are approximate.

Nevertheless, to understand the complex optical behavior of the materials we should take a deep
insight into the molecular structures if the compounds. Compounds 1–7 are fused, having donor
(1, 2, 5, 7—benzene, naphthalene, pyrene and phenanthrene moiety, respectively) or acceptor
(3, 4, 6—difluorobenzene, dicyano and phenazine moiety, respectively) “tails” anchored to the
central pyrazine ring, similarly to non-fused compounds, having electron poor (8 and 11—phenyl
and thiophene moieties, respectively) and electron rich (9 and 10—pyridine and furan moieties,
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
 Molecules 2018, 23, x FOR PEER REVIEW 9 of 18
Molecules 2018, 23, 2271 9 of 18
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
hasapproach
shown that has in
shown
all thethat in all
cases the casesform
alloxazine alloxazine
was foundformto wasbe found to beThe
favorable. favorable. The lowest
lowest isomerization
isomerization
energies were foundenergies were
for the found for 7,the
compounds 8, compounds
10 and 9 (0.02, 7, 8, 10 0.10
0.09, and and
9 (0.02,
0.160.09, 0.10 and 0.16for
eV respectively), eVthe
respectively), for the compounds 1, 2, 3, 5 and 6 the energy difference between
compounds 1, 2, 3, 5 and 6 the energy difference between the alloxazine and isoalloxazine form was in the alloxazine and
theisoalloxazine
range of 0.18 form
to 0.22waseV in and
the range of 0.18isomerization
the highest to 0.22 eV and the highest
energy isomerization
was computed for theenergy was
compounds
computed
4 and 11 (0.28forandthe0.27
compounds 4 and 11 Thus
eV, respectively). (0.28 and 0.27 eV,that
we assume respectively). Thusform
the alloxazine we assume that theand
is dominant,
alloxazine form is dominant, and as in the absorption spectra, the
as in the absorption spectra, the short-wavelength form is prevalent, however higher absorption short-wavelength form is of
the chromophore does not indicate a higher ratio of the chromophore A. In the present case of
prevalent, however higher absorption of the chromophore does not indicate a higher ratio thethe
ratio
chromophore A. In the present case the ratio of the various chromophores will be highly affected by
of the various chromophores will be highly affected by the solvent and needs further detailed study.
the solvent and needs further detailed study. Moreover we do not exclude that depending on the
Moreover we do not exclude that depending on the environment, additional chromophores can appear
environment, additional chromophores can appear due to protonation/deprotonation, carbonyl
due to protonation/deprotonation, carbonyl group reduction, etc.
group reduction, etc.
In Figures 4 and 5, the HOMO and LUMO orbitals of the compounds are depicted (for the
In Figures 4 and 5, the HOMO and LUMO orbitals of the compounds are depicted (for the iso-
iso-
form seesee
form thethe
SI). SI).
All ofAlltheoflinear
the linear
planarplanar fused molecules
fused molecules were foundwereto found
have a to have a homogenous
homogenous electron
electron density distribution, which results in a relatively low band
density distribution, which results in a relatively low band gap, unusual for molecules gap, unusual forofmolecules
such size, of
such size, especially
especially for compoundfor compound
6, where the6, where
presence theofpresence of the electron-rich
the electron-rich pyrazine
pyrazine building building
block resultsblock
in
results
a nearly homogeneous electron density distribution equally “stretched” along the molecule.molecule.
in a nearly homogeneous electron density distribution equally “stretched” along the In the
In case
the case of non-linear
of non-linear fused fused compounds
compounds (molecule
(molecule 5 and5 and a slight
7) a7)slight asymmetry
asymmetry in the
in the LUMO
LUMO was was
observed—the
observed—the electron
electrondensity
densityisisdelocalized
delocalizedon onone
oneofofthe
thearomatic
aromaticrings,
rings,thus
thusthetheoptical
opticalgapgapfor
forthe
bigger molecules
the bigger e.g., 5e.g.,
molecules and5 7and
is similar to that
7 is similar of the
to that smaller
of the ones
smaller (e.g.,
ones 1, 31,and
(e.g., 3 and4).4).

Figure 4. DFT calculated HOMO and LUMO orbitals of the fused alloxazine form compounds.
Figure 4. DFT calculated HOMO and LUMO orbitals of the fused alloxazine form compounds.
Molecules 2018, 23, 2271 10 of 18
Molecules 2018, 23, x FOR PEER REVIEW 10 of 18

Figure 5. DFT calculated HOMO and LUMO orbitals of the non-fused alloxazine form compounds.
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
It hasstate
excited to be notedorbitals)
(LUMO that likepossess
the non-linear
an electronfused compounds
density and 7, non-fused
selectively5 distributed near onlysystems in the
one of the
excited state (LUMO orbitals) possess an electron density selectively distributed
aromatic rings in the 7th position which, however, can be explained by the asymmetrical alloxazinenear only one of the
aromatic rings in
group, where the 7thelectronegative
strongly position which, however,
carboxyl cancause
groups be explained by the asymmetrical
a redistribution alloxazine
of the electron density,
group, where
resulting stronglyrotation
in unequal electronegative carboxyl
angles between thegroups cause
alloxazine a redistribution
core of thearomatic
and the non-fused electronrings.
density,
resulting in unequal rotation angles between the alloxazine core and the non-fused aromaticthe
Generally, in the optimized ground state the dihedral rotation angles between the core and rings.
aromaticin
Generally, substituents
the optimizedfor the compounds
ground state the8–11 are stated
dihedral in Table
rotation angles 2. between
Interestingly, the and
the core isomerization
the aromatic
of the compound
substituents for theresults in the redistribution
compounds 8–11 are stated of in
theTable
electron clouds due tothe
2. Interestingly, theisomerization
interaction of ofthethe
transferred proton in the position 8th and the aromatic rings, especially in the case of
compound results in the redistribution of the electron clouds due to the interaction of the transferred the non-fused
compounds.
proton This is8th
in the position theand
result of the free
the aromatic rotation
rings, between
especially in thethe
caseisoalloxazine
of the non-fusedcore compounds.
and the
abovementioned ending groups. Electropositive hydrogen in the iso-tautomers repulses
This is the result of the free rotation between the isoalloxazine core and the abovementioned ending the aromatic
rings, especially with the electron-poor thiophene, resulting in the large difference in the dihedral
groups. Electropositive hydrogen in the iso-tautomers repulses the aromatic rings, especially with the
angles of the two tautomers.
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.
Table 2. Dihedral rotation angle between the alloxazine/isoalloxazine core and the aromatic rings.
Alloxazine Isoalloxazine
# α1Alloxazine
,°(pos7) α2,°(pos6) α3,°(pos7)Isoalloxazine
α4,°(pos6)
8 ◦ (pos7)
34.4 36.6 49.5 32.7
# α1 , α2 ,◦ (pos6) α3 ,◦ (pos7) α4 ,◦ (pos6)
9 31.5 31.7 54.5 18.5
8 10 34.414.3 36.6
19.6 31.049.5 15.3 32.7
9 11 31.517.7 31.7
27.2 88.654.5 2.1 18.5
10 14.3 19.6 31.0 15.3
α1,° dihedral rotation angle in the X position, α2,° dihedral rotational angle in the Y position.
11 17.7 27.2 88.6 2.1
α1 ,◦ dihedral rotation angle in the X position, α2 ,◦ dihedral rotational angle in the Y position.
Molecules 2018, 23, 2271 11 of 18

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 9, showed promising electrochemical behavior in the positive part of the voltammogram
which makes them possible candidates for catalytic reactions like oxygen evolution reaction conditions.
Upon reduction, the compounds 1 and 8 showed reversible 2-electron reduction while 3 exhibited
1-electron reversible reduction. Compound 5 showed on the other hand, revealed 2-electron irreversible
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 −6.30 −4.20
2 n.a. −4.80
3 −6.10 −4.30
4 −6.30 −4.00
5 −6.50 −3.90
6 −6.20 −4.50
7 n.a. −4.05
8 n.a. −3.90
9 n.a. −3.93
10 n.a. −4.00
11 −6.50 −4.10

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].
Molecules 2018, 23, 2271 12 of 18

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 Na2 SO4 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
254 nm fluorescent indicator. Spots were observed under UV irradiation (254 nm or 354 nm). 1 H- and
13 C-NMR spectra were recorded in CDCl or DMSO-d using an Avance III 300 MHz spectrometer
3 6
(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 (δ scale) downfield from tetramethylsilane and are
referenced to residual protons in the NMR solvent (CHCl3 : δ 7.25 ppm, DMSO: δ 2.50 ppm). Coupling
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.
>400 ◦ C) [69]. 1 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 C10 H6 N4 O2 : 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 B (0.402 g, 2.53 mmol) with alloxan monohydrate (0.402 g, 2.53 mmol) and boric acid (0.159 g,
2.53 mmol) provided compound 2 as a red solid (yield 0.517 g, 73%). M.p. > 330 ◦ C (lit. 385 ◦ C at 0.001
torr, decomp.) [70]. 1 H-NMR (DMSO-d6 , TMS): δ 11.93 (s, 1H), 11.73 (s, 1H), 8.90 (s, 1H), 8.52 (s, 1H),
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 C14 H8 N4 O2 : C 63.64, H 3.05, N 21.20; found: C 63.61, H 2.99, N 21.25.
Molecules 2018, 23, 2271 13 of 18

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 3 as a light purple solid (yield 0.504 g, 58%). M.p. 310–311 ◦ C
(lit. 310–312 ◦ C) [46]. 1 H-NMR (DMSO-d6 , TMS): 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 C10 H4 F2 N4 O2 : 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 4 was synthesized according to the General Procedure, Approach A. Reaction of
diamine D (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 4 as a light grey solid (yield 0.319 g, 32%). M.p. > 330 ◦ C. 1 H-NMR
(DMSO-d6 , TMS): δ 12.87 (br s, 1H), 12.17 (s, 1H). 13 C-NMR (DMSO-d6 , TMS): δ 158.55, 149.98, 149.35,
134.72, 131.41, 125.39, 114.09, 113.55. Elemental analysis calcd (%) for C8 H2 N6 O2 : 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 5 was synthesized according to the General Procedure, Approach A. Reaction of
diamine E (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 5 as a yellow solid (yield 0.293 g, 75%). M.p. > 330 ◦ C. 1 H-NMR
(DMSO-d6 , TMS): δ 12.19 (s, 1H), 11.82 (s, 1H), 9.22–9.14 (m, 2H), 8.48 (d, J = 7.5 Hz, 1H), 8.39
(d, J = 7.5 Hz, 1H), 8.26–8.12 (m, 4H). Elemental analysis calcd (%) for C20 H10 N4 O2 : 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 C16 H8 N6 O2 : 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 7 was synthesized according to the General Procedure, Approach B. Reaction of
diketone G (0.817 g, 3.92 mmol) with 5,6-diaminouracil sulphate (1.00 g, 2.62 mmol) provided
compound 7 as a yellow solid (yield 0.304 g, 37%). M.p. > 330 ◦ C. 1 H-NMR (DMSO-d6 , TMS):
δ 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 C8 H2 N6 O2 : C 68.79, H 3.21, N 17.83; found: C 68.66, H 3.11, N 17.75.
Molecules 2018, 23, 2271 14 of 18

4.11. 6,7-Diphenylpteridine-2,4(1H,3H)-dione (8)

Compound 8 was synthesized according to the General Procedure, Approach B. Reaction of
diketone H (0.577 g, 2.75 mmol) with 5,6-diaminouracil sulphate (0.700 g, 1.83 mmol) provided
compound 8 as a bright grey solid (yield 0.262 g, 30%). M.p. 321–323 ◦ C (lit. 320–325 ◦ C) [43]. 1 H-NMR
(DMSO-d6 , TMS): δ 12.06 (s, 1H), 11.73 (s, 1H), 7.44–7.29 (m, 10H). Elemental analysis calcd (%) for
C18 H12 N4 O2 : 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 9 was synthesized according to the General Procedure, Approach B. Reaction of
diketone I (0.416 g, 1.96 mmol) with 5,6-diaminouracil sulphate (0.500 g, 1.31 mmol) provided
compound 9 as a bright beige solid (yield 0.105 g, 25%). M.p. 277–288 ◦ C. 1 H-NMR (DMSO-d6 ,
TMS): δ 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,
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 C16 H10 N6 O2 : 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)
followed by vacuum sublimation. M.p. 285–286 ◦ C (lit. 285–287 ◦ C) [47]. 1 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 C14 H8 N4 O4 : 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
compound 11 as a yellow solid (yield 0.264 g, 40%). M.p. > 330 ◦ C (lit. 355–357 ◦ C) [47]. 1 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 C14 H8 N4 O2 S2 : 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·L−1 . All optical characteristics were measured in ambient
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 15 of 18

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 + Eox onset vs. NHE) (1)

ELUMO = −(4.75 eV + Ered onset vs. NHE) (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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