Synthesis of rigidified flavin–guanidinium ion conjugates and investigation of their photocatalytic properties

Flavin chromophores can mediate redox reactions upon irradiation by blue light. In an attempt to increase their catalytic efficacy, flavin derivatives bearing a guanidinium ion as oxoanion binding site were prepared. Chromophore and substrate binding site are linked by a rigid Kemp’s acid structure. The molecular structure of the new flavins was confirmed by an X-ray structure analysis and their photocatalytic activity was investigated in benzyl ester cleavage, nitroarene reduction and a Diels–Alder reaction. The modified flavins photocatalyze the reactions, but the introduced substrate binding site does not enhance their performance.


Introduction
Flavins are redox-active chromophores [1][2][3][4][5][6] and represent one of the most abundant classes of natural enzyme co-factors [7][8][9]. Recently, the photo redox properties of flavins have been used to catalyze chemical reactions . A general drawback of photochemical processes in homogeneous solution is the limited preorganization of the reactants and the chromophore, which may lead to low selectivities and slow conversions in diffusion controlled reactions. To overcome this problem, Kemp's acid [31] derivatives have been used as sterically defined templates enhancing the efficiency and selectivity of photoreactions [32][33][34][35][36][37][38][39][40][41][42][43]. Flavins with geometrically defined substrate binding sites have not been reported so far and we expected that the close vicinity of substrate and flavin should enhance the rate of photoinduced electron transfer processes, which strongly depend on distance [44]. We present here the synthesis of geometrically defined flavin-guanidinium ion conjugates based on a Kemp's acid skeleton (Scheme 1). The guanidinium moiety should serve as a hydrogen bonding site for oxoanions or carbonyl groups [45][46][47][48][49]. The structure of the new flavins was determined in solid state and in solution and their photocatalytic properties were tested.

Synthesis
The synthesis of the potential photocatalysts 1 and 2, consisting of the flavin chromophore, the guanidinium substrate binding site and a Kemp's acid derived rigid linker, starts from Kemp's acid anhydride (5) [50][51][52]. The anhydride 5 was allowed to react with previously prepared flavins 4 and 8 [21] in the presence of DMAP as catalyst. The amide formation of the carboxyl group with Boc-protected guanidine was achieved using standard peptide coupling conditions. Boc-deprotection with hydrogen chloride in diethyl ether yielded the guanidinium chloride salts 1 and 2 (Scheme 2). The guanidinium salts are soluble in water and methanol, but also in chloroform and acetonitrile.

Structural investigations
The structure of compounds 1, 2, 6, and 9 was examined in the solid state and in solution. Figure 1 shows the X-ray crystal structures of 6 and 9. The planar flavin chromophore is turned outward relative to the Kemp's acid. Intermolecular π-π-interactions between the flavin heteroarenes are observed.
The structure of compound 1 in the solid state ( Figure 2) shows an almost identical orientation of the flavin group to that of the acid 6. The acyl guanidinium ion group is almost planar and in a parallel orientation relative to the Kemp's acid imide group. 2-D NMR spectra of compounds 1 and 2 revealed several NOE contacts, but the flexibility of the molecule did not allow the determination of preferred conformations.
The most stable conformer of compound 1 in the gas phase was determined by computational methods (semi-empirical AM1, Spartan program package, Figure 3, see also Supporting Information File 1) [53]. In this structure the flavin is turned towards the guanidinium ion forming a hydrogen bond between the flavin carbonyl oxygen atom and the guanidinium moiety (distance ~2.1 Å). However, simple gas phase calculations overestimate the effect of hydrogen bonds [54][55][56][57][58] and in solution the flavin chromophore is expected to rotate freely around the C-C single bonds of the ethane linker.

Photocatalytic reactions
Compounds 1 and 2 were tested as photocatalysts in three different reactions and their performance was compared to tetraacetyl riboflavin 3 or compound 8. Dibenzyl phosphate esters are oxidatively cleaved by blue light irradiation (440 nm) in the presence of compounds 1 and 2 (Scheme 3). The acceleration of the reaction in acetonitrile by 1 and 2, bearing a guan-idinium ion binding site with phosphate affinity, is significantly larger ( Table 1, entries 1+2) in comparison to the ammonium salt 8 (entry 3). In water, however, the accelerating effect is not observed (entries [5][6][7][8]. The presence of the photocatalyst is essential in all cases, as the non-catalyzed hydrolysis is slow under the reaction conditions (<5% conversion).  In the presence of sacrificial electron donor substrates, such as aliphatic amines, flavins can photoreduce nitro arenes to anilines under blue light irradiation (Scheme 4). 4-Nitrophenyl phosphate was used as a substrate for photoreduction in water and in acetonitrile. The results summarized in Table 2 show that 10 mol% of flavin 2, the same amount of tetraacetyl riboflavin (3) or compound 8 catalyze the photoreaction equally well. The guanidinium ion binding site of 1 and 2 does not lead to a more effective conversion. was probed by UV/vis and emission spectroscopy in acetonitrile and buffered aqueous solution. The emission intensity of the chromophore of 2 decreased slightly in the presence of the anions in acetonitrile indicating a weak interaction. In aqueous solution the presence of the anions did not induce significant changes of the emission properties suggesting affinity constants smaller than 10 3 L/mol.
Photo Diels-Alder reactions in the presence of a sensitizer and light have been described [59][60][61][62][63][64]. Therefore flavins 1 and 2 were tested as catalyst for the cycloaddition of maleimide to anthracene in toluene (Scheme 5). Table 3 summarizes the results. A significantly higher yield of the cycloaddition product was obtained after 8 h at 40 °C in the presence of compound 2 (entry 3), if compared to the control reaction (entry 6). Upon irradiation with blue light the yield after 8 h reaction time increased further (entry 2) and was significantly higher as in the absence of a photocatalyst (entry 5). However, a comparison with tetraacetyl riboflavin (3) under identical reaction conditions showed an even more pronounced acceleration of the reaction (entry 4). Blue light irradiated flavins accelerate the anthracene maleimide cycloaddition significantly, but flavins 1 and 2 do not provide additional benefit if compared to tetraacetyl flavin 3.

Conclusion
We have prepared new flavin derivatives that bear an acyl guanidinium group, which is linked to the chromophore via a rigid Kemp's acid spacer. The connectivity and expected relative geometry of 1 and of the carboxylic acids 6 and 9 was confirmed by X-ray structure analysis. Guanidinium cations are known to bind oxoanions, such as phosphates, via hydrogen bonds. Therefore a benefit to the photocatalytic activity of 1 and 2 was expected, as the binding site could keep reaction substrates in close proximity to the redox active chromophore, facilitating photoinduced electron transfer processes. Initial exemplary photocatalytic experiments showed that flavin-derivatives 1 and 2 catalyze oxidative benzyl ether cleavage, nitro arene reductions and Diels-Alder reactions. However, no significant gain in photocatalytic performance by the guanidinium ion substrate binding site was observed in comparison to flavins lacking the binding site and the rigid Kemp's acid skeleton. The primary interaction between the aromatic substrates and the heteroaromatic flavin chromophore seems to dominate the formation of the substrate-catalyst aggregate. Hydrogen bonds between the substrate and the acylguanidinium group are not decisive for their interaction. The rigidity of the Kemp's triacid skeleton is not effectively transferred in 1 and 2 to the relative flavin-guanidinium ion orientation, which is due to the flexible ethane linker between imide and flavin. Derivatives with a more constrained conformation of the flavin chromophore and the substrate binding sites may lead to chemical photocatalysts with better performance.

Experimental General
The      , 1 H, N-H