Multimodal Carbon Monoxide Photorelease from Flavonoids

Photooxygenation of flavonoids leads to the release of carbon monoxide (CO). Our structure–photoreactivity study, employing several structurally different flavonoids, including their 13C-labeled analogs, revealed that CO can be produced via two completely orthogonal pathways, depending on their hydroxy group substitution pattern and the reaction conditions. While photooxygenation of the enol 3-OH group has previously been established as the CO liberation channel, we show that the catechol-type hydroxy groups of ring B can predominantly participate in photodecarbonylation.

F lavonoids are polyphenolic secondary metabolites found essentially in all plant tissues.Due to their antioxidant, anti-inflammatory, antimutagenic, and anticarcinogenic properties and their generally no or low toxicity, they are valuable in many biotechnological, pharmaceutical, or medical applications. 1Their general structure consists of two phenyl rings (A and B) and one heterocyclic ring (C) bearing H, OH, or OCH 3 substituents in all available positions (Figure 1).
Flavonoids are natural photoprotectants 2 and scavengers of radicals and reactive oxygen species, 3 and their excited states offer rich photochemistry thanks to the diversity of functional groups. 4xygenation is a characteristic reaction of flavonol (1, 3hydroxyflavone; Figure 1) derivatives.Quercetin (2, 3,3′,4′,5,7-pentahydroxyflavone) is readily degraded by fungi, accompanied by the formation of carbon monoxide (CO), 5 and is even slowly oxidized by air O 2 in a basic aqueous solution in the dark. 6It has been shown that the photoinduced oxygenation of flavonols involves several reaction pathways influenced by pH, as they can exist in acid and base forms 7 (Scheme 1, in blue).Matsuura proposed that photooxygenation of the acid form proceeds via reaction of a triplet excited state, formed by excited-state intramolecular proton transfer (ESIPT) 8 and intersystem crossing (ISC), with ground-state O 2 via an endoperoxide intermediate that rearranges to give CO and salicylic acid ester (Scheme 1, path A). 9,10 We have shown that the conjugate base of flavonol derivatives undergoes an analogous oxidative CO release in polar protic solvents (path B). 7 In addition, singlet oxygen ( 1 O 2 ) produced by triplet sensitization efficiently oxidizes the conjugate base, yielding the same products (path C), 7,9,11 whereas the acid form is essentially unreactive. 7O, formed endogenously by oxidative heme degradation, is one of the essential cell signaling molecules that participates in various physiological processes in mammals. 12CO is also produced in plants during photorespiratory metabolism 13 and shows signaling effects by increasing plant resistance to abiotic

Scheme 1. Photoinduced Oxygenation of Flavonol
Letter pubs.acs.org/OrgLettstress. 14Given the widespread occurrence of flavonoids in the plant world and their putative potential to release CO (as photoactivatable CO-releasing molecules, photoCORMs 15 ), it seems logical to consider the potent and versatile functions of CO-mediated flavonoids in plant biology and medicine.
The polyphenolic complex structures of natural flavonoids carry several hydroxy groups in all A, B, and C rings.Because mechanistic studies have so far only been performed on simple flavonol structures, we decided to thoroughly study the photooxygenation of several naturally occurring as well as synthetic flavone derivatives to find out how individual functional groups influence their reactivity.The chosen methods included a detailed study of their spectroscopic and photochemical behavior using steady-state and time-resolved methods as well as tracking the photorelease of CO from isotopically labeled derivatives.
The OH groups of the studied compounds are not dissociated in pure methanol (Figures S27−S35).Upon the addition of 6 equiv of NaOCH 3 as a base, the absorption band maxima were bathochromically shifted (to give the corresponding base forms 2B, 3B, 4B (∼27−51 nm), 5B (55 nm), 6B (37 nm); Table 1 and Figures S27−S35), attributed to the deprotonation of at least one OH group.A mixture of the neutral and monoanionic forms was observed for 2 in PBS (5% DMSO, pH 7.4; Figure S36).
When irradiated directly (dir) in methanol at 395 nm, undissociated flavonols (acid forms) 2A, 3A, and 4A produced CO with similar chemical yields of 0.24−0.28equiv (Table 1).CO release from 3A and 4A was more efficient (the quantum yields of CO production (Φ CO ) were 0.0013 and 0.0018, respectively) than from 2A (0.0003) but much less efficient than from 7A (0.03). 7Such low quantum efficiencies are most probably connected to ESIPT, 8 responsible for the ultrafast nonradiation decay demonstrated for quercetin. 20,21Luteolin (5) was photostable under the same conditions, and taxifolin (6) had no absorption above 350 nm; thus, we did not study its photochemistry.Parent flavonol 1A released only 0.05 equiv of CO, while its naphthyl derivative 7A gave a larger chemical yield (0.80 equiv) and exhibited a higher efficiency (Φ CO = 0.03). 7We inspected the cause of this nonproductive photodegradation and found that an adduct of the nucleophilic attack of methanol on the C-2 carbon (ring C) of 2A was formed (Figure S44).On the other hand, thione 8A showed nearly quantitative CO production (0.98 equiv) with an exceptionally high quantum efficiency of 0.43. 17This excellent result thus reflects the compound's ability to suppress unwanted side processes, as also primarily observed for the π-extended flavonol 7, 7 and possibly enhanced intersystem crossing due to the heavy-atom effect of the sulfur atom.
The photochemical activities (including CO production) of flavonols 1 22 and 7 23 and flavone (5) 24,25 have been associated with their triplet excited states.We used nanosecond transient   absorption spectroscopy to determine the triplet lifetimes of compounds 2A, 3A, and 5A in degassed methanol.Compounds 2A and 5A have relatively short lifetimes (140 and 910 ns, respectively), whereas 3A without OH groups on ring A decayed remarkably slowly (77 μs) (Figures S45−S50).An efficient nonradiative deactivation pathway of the ESIPT state, as reported for 1, 26 and a solvent-mediated hydrogentransfer deactivation thanks to the increased number of OH functionalities seem to be the most reasonable explanations for such short lifetimes.Some triplet-excited flavonols in protic solvents were reported to sensitize singlet oxygen, 7,23 whereas their ground states are known to react with 1 O 2 . 7,10,11The quantum yield of 1 O 2 production (Φ Δ ) from triplet excited 2A in methanol was found to be very small (∼10 −4 ), indicating an inefficient process 3 orders of magnitude lower than Φ Δ found for 7A (0.14 23 ).Nevertheless, we investigated CO release in the reaction of selected flavonoids with 1 O 2 produced by an external 1 O 2 sensitizer (rose bengal; sens; Table 1).While both 1A and 7A in methanol reacted with 1 O 2 with a higher CO yield of ∼0.65 equiv, the yields from 2A, 3A, and 4A were relatively moderate (0.15−0.30equiv).Surprisingly, thione 8A was unreactive under the same conditions and was not investigated further.
Both 5A and 6A released even more CO upon sensitization (0.40 and 0.35 equiv, respectively).They lack an enol hydroxy group (3-OH, ring C) and yet photorelease CO, suggesting that different structural features were involved in photooxygenation.This partly contradicts the reported study on the efficiency of singlet oxygen quenching of selected flavonoids, which showed that the 1 O 2 physical quenching efficiency by ground-state flavonoids is mainly controlled by the presence of a catechol group (ring B), while the OH group on ring C is predominantly responsible for their chemical reactivity. 27O was also photoproduced from flavonoids with an excess of a base that dissociated the most acidic OH group(s) (NaOCH 3 , 6 equiv; Table 1).In general, the base forms of flavonols gave lower CO yields, which must be related to the alternative photodegradation pathways discussed above.However, much higher CO yields were obtained in the presence of 1 O 2 in PBS (pH 7.4, almost 1 equiv; Table 1).
In addition, we investigated the reaction kinetics of 2A in methanol with 1 O 2 (k Σ ), and with a rate constant of k Σ ∼ 10 6 M −1 s −1 and an estimated quantum yield of photodecarbonylation by self-sensitization of ∼10 −6 for 2A, CO production via 1 O 2 oxygenation is 300 times less efficient than the reaction of the triplet state with 3
Concomitant release of 12 CO and 13 CO was observed from 13 2 and 13 3 (Table 1), supporting the involvement of a new mechanistic pathway suggested by photolysis of 5 and 6 that does not bear the enol 3-OH group.Indeed, 13 5 and 13 6 were almost exclusive producers of 12 CO.
The 12 CO/ 13 CO ratios were markedly influenced by both the flavonoid structure and the reaction conditions.The specific behavior was very pronounced for quercetin ( 13 2), which produced predominantly 13 CO when directly irradiated in methanol, whereas 12 CO was the major product obtained upon sensitization, especially in PBS (pH 7.4), where both acid and base forms exist in a ratio of about 1:1 19 (Figure S36).(Note: CO was detected in small amounts (0.06) during the same period of time in the dark, as also reported for moderately basic media before; 6 therefore, the photodecarbonylation yield shown in Table 1 is corrected.)When 2 in PBS was irradiated in the presence of a large excess of a 1 O 2 trap (DABCO, 10 mM), essentially only 13 CO was released.This means that different rings/sites of the molecules were swapped as the CO source by reaction conditions, although irradiation always leads through a common intermediate, the excited triplet state.In addition, CO was not liberated in the presence of ascorbic acid as an unselective trap of reactive oxygen species (ROS) and oxidation intermediates, which most probably include peroxo compounds (e.g., Scheme 1).
In contrast to 13 3, which generates both 12 CO and 13 CO, 13 9 with the protected 3′,4′-hydroxy groups produced isotopically pure 13 CO under all reaction conditions.The hydroxy groups on ring B in 13 3 must thus be responsible for the release of 12 CO.This is also valid for all remaining flavonoids 13 2, 13 5, and 13 6 with the 3′,4′-hydroxy-substituted ring B. Another important fact that emerged from the measured data is the maximum yield of CO, which never exceeded 1 equiv.Therefore, we examined the reactivity of catechol-containing model compounds toward oxygenation.Substituted catechols are known to react with 1 O 2 via a type II photooxygenation, possibly via exoperoxide intermediates, which rearrange to oquinone derivatives and other oxidation products (Scheme 2). 32,33In addition, o-quinones were reported to undergo photodecarbonylation by visible-light irradiation, 34 and CO was shown to be generated from humic acid-containing catechol under irradiation. 35To prove that the catechol group releases CO upon 1 O 2 sensitization, photooxygenation of 1,2-dihydroxybenzene (catechol) with rose bengal as a sensitizer was carried out under different conditions (see the Supporting Information).The CO yield was found to be ∼0.1 equiv in methanol and increased to 0.38 equiv in the presence of a base (NaOCH 3 , 6 equiv; no CO is liberated in the dark).The yield obtained in PBS was ∼0.3 equiv.
In conclusion, this study changes our view of the photooxygenation of flavonoids that leads to the release of carbon monoxide.We found that the previously established mechanism involving the enol 3-OH group of ring C can be accompanied or even replaced by photodecarbonylation involving the catechol group of ring B. The extent of these orthogonal photooxygenation pathways depends on the pH, solvent, and photoinitiation type.Knowledge of the photooxygenation mechanism is of paramount importance when considering the application of flavonoids as photoCORMs, and it may help to elucidate the mechanisms of release of CO from flavonoids in living plants.

Figure 1 .
Figure 1.Flavonoid structures discussed in this work.