Photo-chemically induced dynamic nuclear polarization
(photo-CIDNP) was used to observe the light-induced disproportionation
reaction of 6,7,8-trimethyllumazine starting out from its triplet state to
generate a pair of radicals comprising a one-electron reduced and a
one-electron oxidized species. Our evidence is based on the measurement of
two marker proton hyperfine couplings, Aiso(H(6α)) and
Aiso(H(8α)), which we correlated to predictions from density
functional theory. The ratio of these two hyperfine couplings is reversed in
the oxidized and the reduced radical species. Observation of the dismutation
reaction is facilitated by the exceptional C–H acidity of the methyl group
at position 7 of 6,7,8-trimethyllumazine and the slow proton exchange
associated with it, which leads to NMR-distinguishable anionic (TML-)
and neutral (TMLH) protonation forms.
Introduction
The first intentional synthesis of pteridine-2,4(1H,3H)-dione (1; see
Scheme 1) was described by Kühling in 1894 (Kühling,
1894, 1895). Due to its blueish-green fluorescence even in very dilute
aqueous solutions, the compound was named lumazine (Kuhn and Cook,
1937). More than 70 years after its first synthesis, 1 was
recognized as a natural product and isolated from male ants of the species
Formica polyctena (Schmidt and Viscontini, 1967) and more recently also from
the leaves of Brassica juncea L. (brown mustard) (Sharma et al., 2018).
Unsubstituted lumazine (1) is not widely distributed in nature
(Daniels et al., 2019); related compounds substituted at
positions 6, 7 and 8 are of greater biological significance. Importantly,
6,7-dimethyl-8-ribityllumazine (2) is the direct biosynthetic
precursor of riboflavin (4; vitamin B2) (Masuda, 1957;
Maley and Plaut, 1959; Plaut, 1960, 1963) whose derivatives flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are universally
distributed and are involved in an amazing variety of essential biological
processes; for recent reviews, see Walsh and Wencewicz (2013) and Piano et al. (2017). The biosynthetic precursor 2 affords
riboflavin by a mechanistically unique dismutation that is catalyzed by the
enzyme riboflavin synthase and does not require any cosubstrates or
cofactors (Plaut and Harvey, 1971). Even more surprisingly, the
dismutation affording 4 from 2 can also proceed
non-enzymatically in neutral or acidic aqueous solution under an inert gas
atmosphere (Rowan and Wood, 1963; Kis et al., 2001).
Lumazine (1), 6,7-dimethyl-8-ribityllumazine
(2), 6,7,8-trimethyllumazine (3) and riboflavin (4).
6,7-Dimethyl-8-ribityllumazine (2) acts as cofactor of lumazine
protein (LumP), an optical transponder involved in bioluminescence of
certain marine bacteria (Koka and Lee, 1979; Small et al., 1980). More
recently, 2 has also been found in two members of the
photolyase/cryptochrome protein family, namely cryptochrome B (CryB) from
Rhodobacter (R.) sphaeroides (Geisselbrecht et al., 2012) and the photolyase-related
protein B (PhrB) from Agrobacterium (A.) tumefaciens (Zhang et al., 2013). Both proteins
belong to the new subclass of FeS bacterial cryptochromes and photolyases
(BCP), also called CryPro. R. sphaeroides CryB controls light-dependent and
singlet-oxygen-dependent gene expression of the photosynthetic apparatus
(Frühwirth et al., 2012). Similar to A. tumefaciens PhrB, CryB has also repair
activity for (6–4) photoproducts in photodamaged DNA (von
Zadow et al., 2016). It has been speculated that 2 acts as antenna
chromophore in this protein class (Geisselbrecht et al., 2012).
This additional cofactor absorbs at shorter wavelengths (λmax= 420 nm) than the essential FAD cofactor (λmax around 450 nm), which is the origin of light-induced one-electron transfer that
initiates radical-pair spin chemistry in photolyases and cryptochromes
(Biskup et al., 2009; Sheppard et al., 2017). The precise role of
2 in this photolyase/cryptochrome subclade needs to be evaluated,
in particular in its interplay with the FAD cofactor.
6,7-Dimethyl-8-ribityllumazine and certain structural analogs, e.g.,
6,7,8-trimethyllumazine (3), exhibit anomalously high C–H acidity
of the methyl group at position 7. For 2 and 3, pKa
values of 8.3 (Pfleiderer et al., 1966; Bown et
al., 1986) and 9.9 (Pfleiderer et al., 1966; McAndless and Stewart,
1970; Bown et al., 1986) have been reported, respectively. Using 1H and 13C NMR,
compound 3 has been found to form an anionic species under alkaline
conditions, which has been assigned a 7α-exomethylene motif. Compound
2 forms additionally several tricyclic ether anion species under
the participation of the OH groups of the ribityl side chain attached at
position 8 (Bown et al., 1986). Interestingly, riboflavin
synthase selectively binds the 7α-exomethylene anion of 2,
which is believed to be crucial for the dismutation of 2 affording
a stoichiometric mixture of riboflavin (4) and
5-amino-6-ribitylaminouracil. This reaction has been shown to proceed via a
pentacyclic intermediate which was isolated using an inactive mutant of
riboflavin synthase (Illarionov et al., 2001). Various pathways
have been proposed for the riboflavin synthesis from 2
(Truffault et al., 2001; Gerhardt et al., 2002; Kim et al., 2010). For
the non-enzymatic reaction, a quantum mechanical simulation favors a
nucleophilic addition mechanism, which was calculated as the lowest energy
pathway yielding riboflavin (Breugst et al., 2013).
In this contribution, we report on a process between the neutral and the
anionic 6,7,8-substituted lumazine species 3. Studies along these
lines may ultimately shed light on the role of the related compound
2 in light-induced redox reactions of proteins from the CryPro
subclade of photolyases and cryptochromes.
Experimental partSample preparation
6,7,8-Trimethyllumazine was prepared using a procedure described previously
(Masuda, 1957) and purified using high-performance liquid chromatography (HPLC). For details, see
the Supplement.
NMR and photo-CIDNP spectroscopy
NMR and photo-chemically induced dynamic nuclear polarization (photo-CIDNP) experiments were performed as described previously
(Pompe et al., 2019), using a Bruker Avance III HD 600 MHz
NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) operating at
14.1 T. Light excitation was achieved by coupling the output of a
nanosecond-pulsed laser system, comprising an Nd:YAG laser source (Surelite I, Continuum, Santa Clara, CA, USA) in combination with a broadband optical
parametric oscillator (OPO) (Continuum OPO PLUS), into an optical fiber with
a diameter of 1 mm (Thorlabs, Dachau, Germany). The optical fiber was
inserted into the NMR tube via a coaxial insert (Wilmad WGS-5BL).
Photo-CIDNP difference spectra were recorded directly by using a
pre-saturation pulse train to destroy thermal polarization prior to the
laser flash (Goez et al., 2005). This avoids errors involved
with the subtraction of light and dark spectra from separate experiments. A
destructive phase cycle was additionally applied in which every second scan
contained light excitation to avoid residual thermal NMR signals, especially
contributions from the solvent peak (HDO) at 4.8 ppm.
Quantum chemical calculations
Molecular structures were drawn in Avogadro 1.2.0
(Hanwell et al., 2012) and were subsequently pre-optimized
using the MMFF94 force field (Halgren, 1996b, a). Geometry
optimizations were performed in Orca 4.0.1.2 (Neese,
2012, 2018) using the B3LYP functional (Stephens et al.,
1994) and a TZVP basis set (Schäfer et al., 1994). The CPCM
model (Barone and Cossi, 1998) was used to simulate water solvation.
Calculations of hyperfine coupling constants and g matrices were
then carried out by using a B3LYP functional together with the EPR-II basis
set (Barone, 1996).
Results and discussions
Deprotonation of 6,7,8-trimethyllumazine affords a structurally unusual
7α-exomethylene anion (Beach and Plaut, 1970;
Bown et al., 1986) subsequently designated TML- (see Fig. 1). With a
reported pKa around 9.9 (Pfleiderer et al., 1966),
6,7,8-trimethyllumazine exhibits extraordinarily strong C–H acidity. For
photo-CIDNP experiments, samples with a predetermined ratio of
6,7,8-trimethyllumazine (the neutral molecule form is subsequently
designated TMLH; see Fig. 1) and the cognate anion designated TML- were
prepared by the addition of NaOD to a 6,7,8-trimethyllumazine solution (1–4 mM range) in 99 % D2O. The TMLH : TML- ratio was monitored using
1H NMR at 600 MHz. In 6,7,8-trimethyllumazine, the three methyl groups
at positions 6, 7 and 8 and the hydrogen at N(3) afford 1H resonances.
In D2O, H(3) and the 7-methyl group exchange protons with the bulk
solvent (Beach and Plaut, 1970); the latter exchange follows
first-order kinetics on a double-digit minutes timescale (McAndless
and Stewart, 1970). For the neutral TMLH species, we observed resonances of
equal intensity at chemical shift values of 3.91 and 2.52 ppm, which we
assigned as H(8α) and H(6α), respectively, based on reported
values in the literature (4.02 ppm, H(8α); 2.61 ppm, H(6α))
(McAndless and Stewart, 1970); see NMR spectra in Fig. 2. For
TML- in alkaline solution, chemical shift values have been reported for
H(8α) (3.15 ppm) and H(6α) (2.10 ppm) (Beach and
Plaut, 1970). Accordingly, we assigned the resonances at 3.09 and 2.06 ppm to H(8α) and H(6α), respectively. Based on the integrals
of the resonance lines of the two species, photo-CIDNP experiments were
conducted at TML--to-TMLH ratios of 1 : 11, 1 : 2, 1 : 1, 1.5 : 1, 7 : 1 and
10 : 1 at various pH values below, near or above the pKa.
CH acidity of the methyl group attached to position 7 in
6,7,8-trimethyllumazine. The deprotonated (anionic) form TML- can be
drawn in various mesomeric structures, two of which are depicted on the
right-hand side.
1H NMR and photo-CIDNP data of alkaline solutions of
6,7,8-trimethyllumazine: (a) TMLH : TML- ratio of 1 : 10;
6,7,8-trimethyllumazine concentration: 4 mM; excitation wavelength: 425 nm;
(b) TMLH : TML- ratio of 1 : 1; 6,7,8-trimethyllumazine concentration: 4 mM; excitation wavelength: 470 nm. Signals marked with “×” arise from sample impurities.
Photoexcitation of the TMLH / TML- solutions for 1H photo-CIDNP
experiments was performed using 6 ns pulses of an Nd:YAG-laser-pumped OPO
adjusted to 425 or 470 nm (pulse energies of 8 mJ at 425 nm and 30 mJ at 470 nm). TMLH absorbs preferentially at these wavelengths because the
long-wavelength absorbance of TML- (364 nm) is blue-shifted with
respect to that of TMLH (402 nm) (Pfleiderer et al., 1966); see
Fig. 3. Typically, 8 free induction decays (FIDs) were collected and
averaged. Selected photo-CIDNP data are shown in Fig. 2. Three resonances
exhibit substantial nuclear spin polarization: the position 6 and 8 methyl
group signals of TML- and the position 8 methyl group signal of TMLH.
The protons of the position 6 methyl group of TMLH do not show appreciable
hyperpolarization. As in NMR, the position 7 methyl group does not exhibit
any resonances in photo-CIDNP due to proton exchange with deuterons from the
solvent.
UV/vis spectra of TMLH and TML- recorded in water and in 1 M
NaOH, respectively. The vertical dashed lines indicate the wavelengths
chosen for sample irradiation (425 and 470 nm).
The occurrence of nuclear hyperpolarization upon photoexcitation of alkaline
6,7,8-trimethyllumazine samples provides clear evidence for a photochemical
reaction involving radical pair intermediates. Since 6,7,8-trimethyllumazine
is the only organic species present that absorbs light in the visible range,
we suggest disproportionation to take place under the given conditions; see
Fig. 4: light-initiated electron transfer from the anionic TML- to the
neutral TMLH generates the neutral radical TMLox⚫ and initially
the anionic radical TMLHred⚫- as short-lived products. In the
dark, backward electron transfer takes place to regenerate the initial
species TML- and TMLH, respectively.
Photo-induced reversible disproportionation of
6,7,8-trimethyllumazine. ISC, intersystem crossing.
To corroborate this notion, we analyzed intensities and signs of the
hyperpolarized NMR resonances. In the high-field approximation, the applied
time-resolved photo-CIDNP scheme using a pulsed laser as a light source
(Closs et al., 1985; Goez et al., 2005; Kuhn, 2013) renders signal
intensities that are proportional to the isotropic hyperfine coupling
constant Aiso of the respective nucleus
(Adrian, 1971; Morozova et al., 2011). In the
present case with only two weakly coupled 1H spins per species, the
relative enhancement factors can, in principle, be extracted by signal
integration. Kaptein (1971) introduced a simple rule for the net polarization
Γi of a hyperpolarized resonance.
Γi results from the product of four signs and yields either
“+” or “-” for an absorptive or an emissive signal, respectively:
Γi=μ×ε×sgn(Δg)×sgn(Aiso,i).
The parameter μ is either “+” in case the radical pair is formed
from a triplet precursor or “-” in case of a singlet precursor. The
reaction route following the formation of the intermediate radical pair
determines the sign of ε, either “+” for
recombination/re-encounter or “-” for dissociation, the latter leading to
so-called escape products. The sign of the difference of the two (isotropic)
g factors of the two involved radicals, Δg=g1-g2,
depends on which of the two radical moieties comprising the radical pair is
observed (see below). Finally, the sign of the isotropic hyperfine coupling
constant (Aiso,i) of the respective nucleus i is of relevance.
To apply Kaptein's sign rule for rationalizing the polarization of a
particular resonance in the photo-CIDNP spectrum of 6,7,8-trimethyllumazine,
we consider the following aspects: (i) since the time delay between pulsed
laser excitation and the radio-frequency pulse applied for the detection of
the FID was chosen rather short (∼ 80 ns), the detection of
recombination products is supposed to be more likely than that of escape
products, thus ε→ “+”. (ii) Little is known about the
g factors of paramagnetic lumazine species. Early EPR studies were focused
on analyses of hyperfine patterns in EPR spectra from radicals of
6,7,8-trimethyllumazine (Ehrenberg et al., 1970) and derivatives
thereof (Westerling et al., 1977), but they did not report on their
g values. A more recent high-field EPR study considered
6,7-dimethyl-8-ribityllumazine bound to lumazine protein
(Paulus et al., 2014). This cofactor was photo-reduced to
yield the neutral 6,7-dimethyl-8-ribityllumazine radical, from which the
isotropic g factor of 2.0032 ± 0.0001 was obtained by averaging the
principal values of the g matrix. The lack of respective
experimental data for the specific 6,7,8-trimethyllumazine radicals involved
in disproportionation (see Fig. 4) prompted us to perform quantum-chemical
computations at the density-functional theory (DFT) level to calculate the necessary values. Starting
point for geometry optimization was a previously published structure model
of 6,7,8-trimethyllumazine along with six coordinating water molecules
(Schreier et al., 2011). Calculated giso values
were 2.0031 for the neutral radical TMLox⚫ and 2.0034 for the
anionic radical TMLHred⚫-. Tentatively, the giso value of
TMLHred⚫- may be compared to the measured value of protein-bound
6,7-dimethyl-8-ribityllumazine radical because both species result from
one-electron reduction of their aromatic moiety; nevertheless, the
substituents bound to the 8-position and the protonation states of both
radicals are different. Neglecting the unequal substitution at position 8,
the neutral 6,7-dimethyl-8-ribityllumazine radical may be considered as the
species obtained by protonation of TMLHred⚫- to yield
TMLH2red⚫ (see Fig. 4). Extrapolated to the realm of the
related flavins that share similar hydrogen-bonding motifs with the
respective lumazines (see Scheme 1), the couple TMLHred⚫-/ TMLH2red⚫ is expected to behave like anionic (Fl⚫-) / neutral (FlH⚫) flavin semiquinone radicals. For the
latter, slightly larger giso values were observed for anion radicals
than for neutral radicals (Schleicher and Weber, 2012): ∼ 2.0035 (Barquera et al., 2003; Okafuji et al., 2008) versus ∼ 2.0034 (Fuchs et al., 2002; Barquera et al., 2003; Schnegg et al.,
2006), respectively, but the difference is quite small. This seems to also
hold for the one-electron reduced lumazine radicals:
giso(TMLHred⚫-) >giso(neutral
6,7-dimethyl-8-ribityllumazine radical). (iii) Absolute values of most
proton hyperfine couplings have been determined for a cationic (one-electron
reduced) 6,7,8-trimethyllumazine radical species (Ehrenberg et
al., 1970) and for the neutral 6,7-dimethyl-8-ribityllumazine radical
(Paulus et al., 2014). However, the signs of the Aiso
values have not been determined experimentally. Since the available
hyperfine data from the literature are only of limited value for the
interpretation of our photo-CIDNP spectra, we performed quantum-chemical
computations also of the hyperfine structure of the 6,7,8-trimethyllumazine
radicals under discussion. The 1H hyperfine couplings relevant for the
interpretation of the photo-CIDNP NMR data are compiled in Table 1, together
with the respective giso values. A full set of hyperfine couplings from
all other protons as well as from 13C and 15N nuclei can be found
in the Supplement (Tables S2 to S5). Additionally, the
respective data for two further one-electron reduced species have been
included that potentially result from protonation of the anionic
TMLHred⚫- at N(1) or N(5): TMLH2red⚫(H(1)) and
TMLH2red⚫(H(5)), respectively. Importantly, for all
one-electron reduced 6,7,8-trimethyllumazine radicals, DFT predicts
isotropic hyperfine couplings of the H(8α) protons that are much
larger than those of H(6α): Aiso(H(8α)) ≫Aiso(H(6α)) (see Table 1); EPR data are consistent
with this finding (Ehrenberg et al., 1970). For the
TMLox⚫ radical that results from TML- by withdrawal of one
electron, DFT predicts Aiso(H(6α)) >Aiso(H(8α)).
Isotropic g values and selected isotropic methyl proton hyperfine
couplings for various oxidized and reduced 6,7,8-trimethyllumazine radicals.
Photo-excitation of an alkaline 6,7,8-trimethyllumazine solution (4 mM;
TMLH : TML- ratio of 1 : 10) with 425 nm laser pulses resulted in a
photo-CIDNP spectrum with both TML- resonances in emission (see Fig. 2a). The ratio of the integrals of the signals assigned to H(6α)
and H(8α) is 1 : 0.439. The situation is different for the signals
assigned to TMLH: whereas the H(8α) resonance exhibits enhanced
absorption, the one of H(6α) does not show significant
hyperpolarization. Virtually the same polarization pattern is observed for a
less alkaline 6,7,8-trimethyllumazine solution (4 mM) with a TMLH : TML-
ratio of 1 : 1, i.e., pH ≈ pKa (see Fig. 2b). However, to obtain
a discernible photo-CIDNP spectrum in this case, the excitation wavelength
of our laser system had to be tuned to 470 nm. The higher wavelength was
useful because of the rather high TMLH concentration and the high absorbance
associated therewith, which did not allow for sufficient photo-excitation of
the active sample volume given the available output power of our laser
source at 425 nm. Nevertheless, the obtained signal-to-noise ratio remained
rather low and it even decreased upon further decreasing the amount of
TML- relative to that of TMLH. Observation of the photo-CIDNP effect at
470 nm is clear evidence for photo-excitation of TMLH rather than TML-.
The latter has very low absorbance at 425 nm and does not virtually absorb
at 470 nm (Pfleiderer et al., 1966; see Fig. 3).
By far the highest signal-to-noise ratio of the photo-CIDNP data was
obtained in the alkaline range at about one pH unit above the pKa of
TMLH / TML-. We could not conduct NMR experiments under more basic pH
conditions because the high ion strength of our sample precluded proper
tuning of the probe head. Additionally, we varied the 6,7,8-trimethyllumazine
concentration in a range between 1.0 and 4.0 mM. In all cases, photo-CIDNP
revealed a hyperpolarization pattern similar to the one shown in Fig. 2a.
To rationalize our findings, we correlated the relative intensities of the
hyperpolarized NMR resonances obtained by photo-CIDNP to DFT predictions of
hyperfine couplings of the various paramagnetic one-electron oxidized or
reduced lumazine species involved in the suggested disproportionation scheme
along a procedure introduced by Ivanov and Yurkovskaya
(Morozova et al., 2011; see Table 1, Fig. 5 and
Supplement). Upon backward electron transfer in the dark, i.e.,
radical pair recombination (ε= “+”), hyperpolarization
generated on the intermediate oxidized species TMLox⚫ and the
reduced species TMLHred⚫- (or a protonated neutral species
TMLH2red⚫ thereof) is transferred to the diamagnetic
products TML- and TMLH, respectively.
Correlations of relative photo-CIDNP intensities to predicted
Aiso values from DFT. TMLox⚫: slope = 0.0672 MHz-1, R2= 0.9996; TMLHred⚫-: slope = 0.0277 MHz-1,
R2= 0.7827; TMLH2red⚫(H(5)): slope = 0.0248 MHz-1, R2= 0.9863; linear combination of TMLH2red⚫(H(5)) and TMLHred⚫- with a ratio of 0.772 : 0.228: slope = 0.0262 MHz-1, R2= 1. CIDNP intensities were multiplied by the
respective sgn(Δg).
Considering the hyperpolarized NMR resonances of TML- leads to Δg=g(TMLox⚫)-g(TMLHred⚫-)<0 (see Table 1); i.e., the sign entering Eq. (1) becomes negative: sgn(Δg)= “-”. Furthermore, the hyperfine couplings from the precursor state
TMLox⚫ are relevant. The calculated isotropic H(6α) and
H(8α) hyperfine couplings of TMLox⚫ are positive; hence
sgn(Aiso,i)= “+” for i∈(H(6α),H(8α)).
Given the fact that both resonances are emissively polarized, i.e.,
Γi= “-”, requires μ= “+” for the precursor
multiplicity. Consequently, radical pair formation must proceed from a
triplet-state precursor of radical pair formation, i.e., from 3TMLH,
which is generated by intersystem crossing from an excited singlet state
(1∗TMLH) of TMLH (see Fig. 4). Plotting the photo-CIDNP intensities
with respect to the hyperfine couplings obtained using DFT (see Fig. 5)
reveals nearly perfect correlation: a linear regression fit constrained to
go through the origin yields a slope of 0.0672 MHz-1 and R2= 0.9996. Observation of hyperpolarized resonances of H(6α) and
H(8α) from TML- both in emission and in an intensity ratio that
correlates well with hyperfine coupling computations from DFT provides clear
evidence for the existence of the oxidized TML species TMLox⚫,
which is a redox state of lumazine that up to now had not been substantiated
by experiments.
If we retain the signs of ε (“+”, recombination) and μ
(“+”, triplet precursor) and reverse the sign of Δg to “+”
(because Δg=g(TMLred⚫-)-g(TMLox⚫)>0), then we expect for the H(8α) resonance of TMLH
because of Γi= “+” (absorptive resonance) a positive
isotropic hyperfine coupling constant of its paramagnetic precursor state. A
very small hyperfine coupling near or equal to zero is expected for
H(6α) as hardly any hyperpolarization is observed for these nuclei
in the photo-CIDNP spectrum (Fig. 2). This situation is reversed as compared
to that of TML-, for which the H(6α) resonance experiences much
stronger hyperpolarization than H(8α). Our DFT calculations confirm
a large and positive value for Aiso(H(8α)) of TMLHred⚫- but also predict a negative hyperfine coupling of substantial absolute
value for Aiso(H(6α)). This latter finding is clearly not
supported by the photo-CIDNP data shown in Fig. 2. Therefore, we have
extended our DFT studies of 6,7,8-trimethyllumazine radicals to protonated
variants of TMLHred⚫-, namely the neutral species
TMLH2red⚫(H(1)) (protonated at N(1)) and
TMLH2red⚫(H(5)) (protonated at N(5)); see Tables 1, S3 and S4.
Protonation of TMLHred⚫- at N(1) to yield
TMLH2red⚫(H(1)) does not significantly alter the isotropic
hyperfine couplings of H(6α) and H(8α); also giso is
virtually unaffected (see Tables 1 and S4). However, addition of a
proton at N(5) to yield TMLH2red⚫(H(5)) does shift both
hyperfine couplings to more positive values. Aiso(H(6α)) even
changes its sign and assumes a small positive value, but more than 10 times
smaller than that of Aiso(H(8α)); see Tables 1 and S3. Our
photo-CIDNP data thus support rapid protonation of TMLHred⚫- at
N(5) to yield the neutral radical species TMLH2red⚫(H(5)).
When considering a linear combination of the hyperfine data of
TMLH2red⚫(H(5)) and TMLHred⚫- (see TMLH2red⚫(H(5))/TMLHred⚫- in Fig. 5) with a
ratio of 0.772 : 0.228, we obtain Aiso(H(6α)) = 0 and
Aiso(H(8α)) = 18.1 MHz and consequently, of course, perfect
correlation of DFT-calculated hyperfine couplings and relative photo-CIDNP
intensities with R2= 1 and a slope of 0.0262 MHz-1. Such an
approach has been successfully applied previously to photo-CIDNP studies of
other systems; see, e.g., Morozova et al. (2018) and Torres et al. (2021).
The obtained ratio of TMLH2red⚫(H(5)) to TMLHred⚫- should be treated with caution as the photo-CIDNP intensities were
correlated with hyperfine data predicted from DFT because experimental
values are unavailable. The accuracy of DFT hyperfine predictions however
strongly depends on the choice of functional basis set and molecular
geometry (Kirste, 2016; Witwicki et al., 2020).
The slopes of the straight lines through the origin in the correlation plots
of TML- and TMLH in Fig. 5 are clearly different: 0.0672 MHz-1
versus 0.0262 MHz-1 (for TMLH2red⚫(H(5))/TMLHred⚫-), respectively. Furthermore, even though
Aiso of H(8α) in TMLH2red⚫ has a larger value
than Aiso of H(6α) in TMLox⚫, the corresponding
photo-CIDNP intensity of the resonance in the recombination product is
significantly smaller than that of the most intense signal of the respective
counter radical. These findings may have several reasons: (i) introduction
of a proton at N(5) adds a further large hyperfine
coupling (Aiso(H(5)) of substantial anisotropy (see Table S3).
Protonation dynamics of H(5) could enhance relaxation by which
hyperpolarized spin-state population decays to the population at thermal
equilibrium. (ii) Hyperpolarization could also be dissipated into the
solvent upon radical-pair recombination. Backward electron transfer from
TMLH2red⚫ yields the diamagnetic TMLH2+, a
species that will certainly deprotonate quickly to regenerate TMLH,
especially given the alkaline conditions. Hence, intermediate electron-spin
redistribution leading to buildup of hyperpolarization at H(5) will likely
be transferred to the surroundings on release of this proton. (iii) Despite
the fact that electron exchange has been observed in other systems leading
to a decay of hyperpolarization (Closs and Sitzmann, 1981), we
consider such a mechanism along the scheme #TMLHred⚫-+TMLH→#TMLH+TMLHred⚫- (“#” denotes
nuclear spin polarization) less likely given that at the elevated pH values
under consideration neutral TMLH is present only at rather low
concentrations.
Figure 6 shows the singly occupied molecular orbitals (SOMOs) of the neutral
radicals TMLH2red⚫(H(5)) and TMLox⚫. Positive
and negative signs of the frontier orbitals are depicted with light and dark
grey shading, respectively. Mere inspection gives a hint for the quite
different ratios of isotropic hyperfine couplings of H(8α) and
H(6α) in both species: the amplitude of the SOMO at the 6-methyl
group of TMLH2red⚫(H(5)) is small as compared to that of
TMLox⚫. For the 8-methyl group the opposite trend is observed.
Considerable SOMO amplitudes are observed for H(5) in
TMLH2red⚫(H(5)), which leads to a strong and anisotropic
hyperfine coupling of this exchangeable proton. This explains the
dissipation of hyperpolarization into the solvent on backward electron
transfer leading from TMLH2red⚫(H(5)) to TMLH and H+.
SOMOs of observed 6,7,8-trimethyllumazine radical species. Dark
and light grey contours denote negative and positive signs of the molecular
wavefunctions. (a) TMLH2red⚫(H(5)); (b) TMLox⚫.
The SOMOs of TMLH2red⚫(H(5)) and TMLox⚫
significantly differ in terms of wavefunction amplitudes and signs, in
particular at the respective π system of the pyrazine ring. Very high
amplitude is observed at C(7α) of TMLox⚫. This gives a
hint that the electronic structure of the related one-electron reduced
TML- may be better represented by a C(7α)-carbanion rather than
a 7α-exomethylene motif at position 7 (see Fig. 1). Clearly, the two
observable proton resonances per radical species and their hyperpolarization
detected using photo-CIDNP NMR are insufficient to draw a precise picture of
the delocalization of the unpaired electron spin density over the carbons
and nitrogens in the heterocyclic cores. To learn more about the electron-spin
distributions in the two radicals generated by disproportionation of
3 and to further corroborate the existence of the oxidized species
TMLox⚫, we plan further photo-CIDNP experiments on specifically
designed 13C and 15N isotopologs of 2 and 3.
Conclusions
Using photo-CIDNP NMR we have discovered a disproportionation reaction upon
photoexcitation of alkaline solutions of 6,7,8-trimethyllumazine. In its
classical definition, disproportionation refers to a “reversible or
irreversible transition in which species with the same oxidation state
combine to yield one of higher oxidation state and one of lower oxidation
state” (McNaught and Wilkinson, 1997). This includes redox
reactions of the following type: 2X→Xox⚫++Xred⚫-.
From the perspective of oxidation states, this scheme applies to
6,7,8-trimethyllumazine, which upon photo-induced electron transfer
generates a pair of radicals comprising a species devoid of one electron
(TMLox⚫) and another with an excess electron (TMLHred⚫-). However, our proposed mechanism deviates from the classical
disproportionation scheme in so far as (i) the reaction is initiated
directly by light and (ii) that a redox reaction takes place between two
different protonation states of 6,7,8-trimethyllumazine, i.e., between TMLH and
TML-. Clearly, disproportionation starts out from photoexcitation of
TMLH (notably, only TMLH has significant absorption at 470 nm). Once the
triplet state of TMLH is formed by intersystem crossing from an excited
singlet state of this molecule, it abstracts an electron from TML-,
thereby generating a pair of interacting radicals (see Fig. 4).
Interestingly, one quite unusual radical species is generated by this
disproportionation that has not been reported before: TMLox⚫.
The existence of a species in such a high redox state was speculated upon in
triplet quenching of the unsubstituted lumazine (1)
(Denofrio et al., 2012); one should however keep in mind
that the aromatic moiety of 6,7,8-trimethyllumazine (3) differs
from that of the unsubstituted lumazine. A similar species was proposed for
triplet quenching of the related flavins (Görner, 2007), but
convincing experimental evidence on the existence of a species in such a
high oxidation state was still lacking for both cases until now. By
detecting two important hyperfine couplings, we provide strong evidence for
the existence of 6,7,8-trimethyllumazine in a further high redox state.
Clearly, we owe this success to two peculiarities of
6,7,8-trimethyllumazine: (i) the extraordinary acidity of its 7-methyl group
which compares to that of the ammonium ion and (ii) its proton exchange on
a timescale that is slow compared to that of NMR and which consequently
leads to distinguishable anionic (TML-) and neutral (TMLH) protonation
forms in terms of NMR properties. Flavins by comparison do not exhibit a
corresponding acidity of their methyl groups. Therefore, our photo-CIDNP
detection scheme is not readily extendable to the realm of flavins for a
proof of the existence of the speculative FADox⚫+ species.
Our data on 6,7,8-trimethyllumazine provide evidence for an extended range
of redox states of lumazines in general. Further studies on
lumazine-mediated photocatalysis will show whether the existence of species
of the type of TMLox⚫ will be involved that shed new light on
the role of 6,7-dimethyl-8-ribityllumazine as chromophore, e.g., in the new
class of recently discovered CryB cryptochromes (Geisselbrecht
et al., 2012) and PhrB photolyases (Oberpichler et al., 2011; Zhang et
al., 2013) of the CryPro subclade. Further studies will be conducted on the
suitability of TML as a photosensitizer.
Code availability
For information regarding the correlation plot, please contact the corresponding author.
Data availability
Raw data are available at https://freidok.uni-freiburg.de/data/194780 (Wörner et al., 2021).
The supplement related to this article is available online at: https://doi.org/10.5194/mr-2-281-2021-supplement.
Author contributions
AB and SW designed the research. Compounds were synthesized by JC and samples prepared by JW. NMR experiments were carried out by JW. Data analysis was performed by JW and SW, and the manuscript was written by SW and JW with input from all the authors.
Competing interests
The authors declare that they have no conflict of interest.
Special issue statement
This article is part of the special issue “Robert Kaptein Festschrift”. It is not associated with a conference.
Acknowledgements
Stefan Weber thanks the SIBW/DFG for financing NMR instrumentation that is operated within the MagRes Center of the Albert-Ludwigs-Universität Freiburg. Jing Chen is thankful for a fellowship from the China Scholarship Council. We thank Ursula Friedrich for HPLC purification of 6,7,8-trimethyllumazine.
Financial support
This open-access publication was funded by the University of Freiburg.
Review statement
This paper was edited by Rolf Boelens and reviewed by two anonymous referees.
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