Numerous endogenous photosensitizers, among which riboflavin has attracted much attention, can photogenerate various reactive oxygen species (ROS, e.g. ¹O₂ and O₂^˙−) [1, 2]. It has been reported that riboflavin is an efficient ROS-generator [1, 3, 4] and can cause photosensitizing DNA damage [5, 6]. Polyphenolic compounds, e.g. rutin and catechin (Figure 1), are ideal antioxidants with strong free radical-scavenging ability. Recently, it was reported that rutin and catechin play dual roles in protecting from the photosensitizing damage caused by riboflavin, that is, as ROS scavengers and triplet excited (T₁) state riboflavin quenchers [7]. The free radical-scavenging mechanisms of rutin and catechin have been investigated before [8], however, more effort is needed to elucidate the deactivating mechanisms of T₁ state riboflavin by rutin/catechin. In recent years, density functional theory (DFT) calculations have been widely used to study both the photosensitization and deactivation mechanisms of excited state photosensitizers [4, 9–12]. Therefore, in the present study, we attempt to explore how T₁ state riboflavin was deactivated by rutin/catechin by means of theoretical calculations.

The calculation procedures are as follows. First, the geometries of riboflavin, rutin and catechin were fully optimized by DFT [14, 15] and B3LYP functional [16–18] with 6–31G(d,p) Gaussian basis set in vacuo. Then, the lowest T₁ excitation energies (E_T1) of the three molecules were estimated by time-dependent DFT (TD-DFT) [19–21] with the same basis set. Moreover, in view of the fact that the diffusion functions are crucial for treatment of anion and cation radicals, the vertical electron affinities (VEA) and vertical ionization potentials (VIP) of riboflavin, rutin and catechin were calculated by using a combined DFT method labeled as B3LYP/6–31+G(d,p)/B3LYP/6–31G(d,p), which means that B3LYP/6–31+G(d,p) was used to perform a single-point calculation using B3LYP/6–31G(d,p)-optimized geometries [10]. The O-H bond dissociation enthalpy (BDE) of rutin/catechin and H-atom affinity (HAA) of riboflavin were obtained by a hybrid method combining DFT and semiempirical method AM1, labeled as (RO)B3LYP/6–311+G(2d,2p)/AM1, which takes advantages of accuracy and economy [8]. The solvent (benzene and water) effects were taken into account by employing the self-consistent reaction field (SCRF) method with polarizable continuum model (PCM) [22–24] for the single point calculations. All the calculations were accomplished using the Gaussian 03 package of programs [25].

As we know, the ground state photosensitizer is initially excited to the singlet excited state upon irradiation and then may intersystem cross to the relatively long-lived T₁ state. T₁ state riboflavin can react with molecular oxygen to photogenerate various ROS [1, 3, 4] and at the same time, it can be deactivated by antioxidants through the following possible pathways:

The first deactivating pathway may proceed through the direct energy transfer between T₁ state riboflavin (RF) and polyphenols (PhOH) (Equation 1). (1) RF ( T 1 )   +   PhOH → RF + PhOH ( T 1 )

The second deactivating pathway involves the electron transfer between T₁ state riboflavin and polyphenols (Equation 2). (2) RF ( T 1 )   +   PhOH → RF . - + PhOH . +

Moreover, as the polyphenolic antioxidants are ideal H-atom donors [8], T₁ state riboflavin may be deactivated by polyphenols through a H-atom transfer process (Equation 3). (3) RF ( T 1 )   +   PhOH → RFH . + PhO .

Therefore, the corresponding electronic parameters of riboflavin, rutin and catechin, including E_T1, VEA, VIP, O-H BDE and HAA, were estimated and listed in Table 1, according to which, the deactivating reactions of T₁ state riboflavin by rutin/catechin were analyzed.

Primarily, the E_T1 of riboflavin, rutin and catechin have been calculated using TD-DFT methods, whose accuracy in estimating the T₁ state properties of various photosensitizers has been verified [4, 9–13]. It can be seen that the theoretical E_T1 of rutin/catechin is much higher than that of riboflavin (Table 1), implying that the direct energy transfer-based deactivating pathway (Equation 1) is not feasible on thermodynamic grounds in both solvents.

As to the direct electron transfer-based deactivating pathway (Equation 2), its feasibility depends on the VEA of T₁ state riboflavin (VEA_T1) and VIP of rutin/catechin. According to the theoretical results, the summation of VEA_T1 of riboflavin and VIP of rutin/catechin is positive both in benzene and water, implying that the electron transfer-based deactivating pathway is also not favorable from the thermodynamic point of view.

Thirdly, rutin and catechin are excellent H-atom donating substrates [8]. To explore whether the H-atom transfer reaction from rutin/catechin to T₁ state riboflavin (Equation 3) can occur or not, the O-H BDE of rutin and catechin, which has been successfully used to measure the molecular H-atom-donating ability [8], and the HAA of riboflavin, an appropriate theoretical parameter to characterize the molecular H-atom-abstracting ability [8], have been calculated. Despite the fact that rutin and catechin possess several phenolic hydroxyls that may donate H-atoms, previous studies demonstrated that the hydroxyl at position 4’ (Figure 1) is the most active one [26] and the corresponding O-H BDE in benzene and water is listed in Table 2. The theoretically estimated HAA of T₁ state riboflavin at N1 (Figure 1), which has been reported to be the thermodynamically favorable position to accept a H-atom [27], is –97.24 kcal/mol in benzene and –106.19 kcal/mol in water (Table 2). As the summation of HAA_T1 of riboflavin and the O-H BDE of rutin/catechin is negative in both solvents, the H-atom transfer-based quenching pathway is thermodynamically feasible. Therefore, the H-atom transfer-based T₁ state riboflavin deactivating mechanism by rutin/catechin is proposed as illustrated in Figure 2.

In summary, through comparing the electronic parameters of riboflavin, rutin and catechin, including E_T1, VEA, VIP, BDE and HAA, it can be inferred that the H-atom transfer pathway is more feasible on thermodynamic grounds relative to the direct energy transfer or direct electron transfer pathways responsible for the T₁ state riboflavin deactivation by rutin/catechin. The results have important implications to design/screen better polyphenolic antioxidants as protectors against the photo-oxidative damage induced by riboflavin.