A DFT study on the degradation mechanism of vitamin B2

Graphical abstract


Introduction
Riboflavin (RF) or vitamin B2 is a water-soluble vitamin that is widely present in a variety of foods, such as almonds, mushrooms and vegetables (Ball, 2006). Photo, thermal, and chemical degradation reactions of the vitamin are serious problems for its preservation (Uchida, et al., 2016). A general scheme for the degradation of RF is shown in Scheme 1 (Smith and Metzler, 1963;Ali, et al., 2014;Song, et al., 1965).
Aqueous solutions of riboflavin (RF) are unstable in light (hν) and degrade to give formylmethylflavin (FMF), lumiflavin (LF) and lumichrome (LC) (Ahmad, et al., 2004;Huang, et al., 2006). While FMF is the intermediate in the photo-degradation, it can also lead to LF and LC in the dark (non-photochemical) basic aqueous solution (Song, et al., 1965;Ahmad, et al., 1980). The kinetics of both photolysis of RF (Woodcock, et al., 1982;Allen, andParks, 1979: Ahmad, et al., 2019;Gul et al., 2014;Ahmad, et al., 2004;Ahmad, et al., 2010;Ahmad, et al., 2013;Ahmad, et al., 2008) and basic hydrolysis of FMF (Song, et al., 1965;Ahmad, et al., 1980) have been studied extensively including gas-phase photofragmentation studies of deprotonated riboflavin, [RF − H] − , using laser-interfaced mass spectrometry (Wong, et al., 2021). O-H bond dissociation energies of the phenolic compounds (rutin and catechin) and T 1 state H-atom affinity of RF were evaluated to examine the role of polyphenolic antioxidants against the photo-oxidative damage induced by RF (Ji and Shen, 2008). However, mechanisms of C -C and C -N covalent bond cleavage in RF and FMF are still unclear. Precise understanding of the mode of the bond scission in the ribityl side chain during photo-and thermal degradations would contribute to development of methods for stabilizing the vitamin B2 in products exposed to both light and heat during preparation and storage, or retail.
Since there have been no theoretical studies of RF degradation, in this work, DFT (density functional theory) calculations of reaction paths in Scheme 1 were carried out. Elementary processes were traced and the intervening species, which have not been detected experimentally, were investigated. The following questions were scrutinized carefully: (1) photo-irradiation of RF leads to the (π, π*) excited state of the tricyclic isoalloxazine ring (Sikorska, et al., 2005), because the HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied orbital) are π and π* orbitals and the state appears not to be concerned with cleavage of σ bonds in the ribityl side chain [-CH(OH)-CH(OH)-CH(OH)-CH 2 OH], thus it is not clear how (π → π*) excitation is related to the scission of C-C σ bond; and (2) whether photochemical and basecatalyzed reactions of FMF → LF + LC (in Scheme 1) are similar or not.

Methods of calculations
Riboflavin is a water soluble species, thus hydrogen bonds with water molecules that induce proton transfer need to be included explicitly in the reaction model. Thanthiriwatte et al. (2011) conducted extensive investigations of various hydrogen-bonded dimers, using DFT potential energy curves. Comparisons between these and CBS extrapolated limit CCSD(T) ones were made, where CBS stands for complete basis set and CCSD(T) stands for coupled cluster calculations, using both single and double substitutions from the Hartree-Fock determinant with triple excitations. The functional wB97X-D (Chai and Head-Gordon, 2008) which includes the dispersion correction provides very good potential energy curves, therefore it was used in this work. Geometry optimizations of reaction systems were carried out including the PCM (polarizable continuum model, Cances, et al., 1997;Cossi, et al., 1998;Mennucci and Tomasi, 1997) solvent effect. The adopted basis set was 6-311 + G(d,p). Reaction paths in the excited state were traced by the use of the unrestricted wB97X-D, i.e., UwB97X-D, in the triplet spin state. This is because reactions in Scheme 1 are known to occur in the spin state after the intersystem crossing from the singlet excited state (Ali, et al., 2014). In addition, the spin unrestricted DFT was reported to give the energy gap between the ground state and the lowest triplet state of RF, which was in good agreement with that derived from the emission spectra (Sikorska, et al., 2005).
First, transition states (TSs) were sought by partial optimizations at bond interchange regions. Second, by the use of Hessian matrices, TS geometries were optimized. They were characterized by vibrational analyses, which checked whether the obtained geometries had single imaginary frequencies (ν ‡ s). From TSs, reaction paths were traced by the intrinsic reaction coordinate (IRC) method (Fukui, 1981;Hratchian and Schlegel, 2005) to obtain the energy-minimum geometries.
Energy changes were shown by the use of Gibbs free energies (T = 298.15 K, P = 1 atm). All the calculations were carried out using the GAUSSIAN 16 (Frisch et al., 2016) program package. The computations were performed at the Research Center for Computational Science, Okazaki, Japan. All the Cartesian coordinates of the calculated species are shown in the Supplementary data.

The fragmentation of RF to FMF in the triplet spin state
Scheme 2 shows the fragmentation path of RF(T) → FMF(T), where (T) stands for the triplet spin state. Each optimized geometry is shown in Fig. S1.
Among various conformations of the ribityl side chain in RF, that with the O(32)-H(33)…N(3) intramolecular hydrogen bond was adopted as the precursor geometry ( Fig. S1-1). Along the hydrogen bond, the H(33) migration TS, TS1(T), is obtained ( Fig. S1-2). Noteworthy is that H(33) is not a hydrogen atom but a proton, and concomitantly one electron is shifted along O(32) → N(3). The shifts of the proton and one electron are illustrated in Scheme 3.

The reaction of FMF → LC and LF in the triplet state
Scheme 4 exhibits degradation paths of FMF(T) → LC(T) and LF(T). TS4(T) (Fig. S3-2) shows the H(32) migration TS for conversion of C(26)-H(32) to H(32)-N(3). The migration pattern is similar to that of TS1(T) in Fig. S1-2. H(32) is moving as a proton and concomitantly one electron is moving from C(26) to N(3) in the π electronic cloud. After TS4(T), Int3(T) (Fig. S3-3) is formed. From Int3(T), cleavage of the N(11)-C(18) bond is brought about at TS5(T) (Fig. S3-4). After TS5(T), triplet ketene (H 2 C = C = O(T)) is evolved and the ground state LC(S 0 ) is afforded as shown in Fig. S3-5. In spite of the stability of the triplet state in the tricyclic ring of LC, the ketene becomes the triplet state. In fact, the [LC(S 0 ) + ketene(T)] system is 8.03 kcal/mol more stable than the [LC(T) + ketene(S 0 )] one. TS6(T) (Fig. S4-1) is the C…C separation TS, which gives Int4(T) (Fig. S4-2) containing the formyl radical H-C•=O. From the radical pair Int4(T), the H(26) migration takes place at TS7(T) (Fig. S4-3), leading to LF(T) and CO (Fig. S4-4). The triplet state of Int4(T) has two distant Scheme 1. The photo-degradation of RF to LC and LF via FMF and the base catalyzed reaction of FMF to LC and LF. unpaired electrons (at the tricyclic ring and at the formyl radical), and the open singlet spin state would be formed readily. In this state, the H (26) migration forming the methyl group occurs without an energy barrier.
Abstraction of the α hydrogen of subst.THF was shown to be of the largest exothermicity (in Fig. 2 of the reference). Since the transition state of the abstraction was not reported in the reference, it was determined in this study by uwB97xd/6-311 + G(d,p) SCRF=(PCM, solvent = water). The calculated result is shown in Fig. S6. The ΔG ‡ value is small, +10.11 kcal/mol. Accordingly, LF(T) was confirmed to be of a high reactivity for the hydrogen abstraction.

The base-catalyzed reaction of FMF → LC and LF
To simulate the reaction, a model of FMF + HO -(H 2 O) 3 was adopted as illustrated in Scheme 5. In the scheme, the first step consisting of the HO addition to the terminal aldehyde group of FMF is expressed by the general base catalysis (Jencks and Carriuolo, 1961).
Scheme 6 shows the calculated results.
ΔG ‡ = +17.18 kcal/mol of TS10 leading to LF is much smaller than ΔG ‡ = +29.99 kcal/mol of TS9 leading to LC. In fact, the base-catalyzed reaction of FMF gave mainly LF with a less significant competing reaction yielding LC (Song, et al., 1965).
The reason why two fragmentation channels are present from Int5 is explained as follows. The tetrahedral intermediate Int5 has the unstable alkoxide moiety -(H)(OH)C-O − and its recovery to the carbonyl group C=O is required. There are two pathways for the recovery. One is TS9 where the N(11)-C(18) cleavage and OH migration occur

Concluding remarks
In this work, degradation reaction paths starting from riboflavin (RF) were investigated by DFT calculations for the first time. In the route from RF to FMF of the lowest triplet spin state (Scheme 2), two intermediates Int1(T) and Int2(T) were found. Int1(T) was formed by the concomitant transfer of one proton and one electron of the hydroxyl group of the ribityl side chain. The terminal aldehyde group of FMF (formylmethylflavin) was afforded in Int2(T) after the C…C cleavage. From FMF(T), there were two degradation channels (Scheme 4). Release of ketene(T) and carbon monoxide led to LC (lumichrome, S 0 ) and LF (lumiflavin, T), respectively.
Question (1) has been raised in the Introduction, i.e., the relation between the (π, π*) triplet state and the cleavage of σ bonds. As shown in TS1(T) (Fig. S1-2) and TS4(T) (Fig. S3-2), the in-plane migration of a proton and the out-of-plane one of an electron occurred simultaneously.
The migration weakened a σ bond in the ribityl chain, which gave rise to the degradation. The base-catalyzed (ground state) degradation of FMF was investigated with HO -(H 2 O) 3 (Scheme 6). The first step was formation of the tetrahedral intermediate Int5 (Fig. S5-3). Scission of N-C and C-C bonds in the -CH 2 -CH(OH)-O − group of Int5 led to [LC-H + ] − and [LF-H + ] − , respectively.
Question (2) in the Introduction is about the similarity or the difference between the photochemical reaction of FMF to LC and LF and the ground base-catalyzed one. While the driving force was different, cleavage of N-C and C-C bonds occurred in a similar way.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.