Catalytic Amine Oxidation under Ambient Aerobic Conditions: Mimicry of Monoamine Oxidase B**

The flavoenzyme monoamine oxidase (MAO) regulates mammalian behavioral patterns by modulating neurotransmitters such as adrenaline and serotonin. The mechanistic basis which underpins this enzyme is far from agreed upon. Reported herein is that the combination of a synthetic flavin and alloxan generates a catalyst system which facilitates biomimetic amine oxidation. Mechanistic and electron paramagnetic (EPR) spectroscopic data supports the conclusion that the reaction proceeds through a radical manifold. This data provides the first example of a biorelevant synthetic model for monoamine oxidase B activity.


I GENERAL INFORMATION
All reagents were purchased from commercial suppliers: Acros Organics, Alfa Aesar, Sigma Aldrich or Fluorochem and used without further purification. Flash chromatography was performed on chromatography grade, silica, 60 Å particle size 35-70 micron from Sigma Aldrich using the solvent system as stated. 1 H and 13  Flavins were synthesised by the previously published procedures [1] , with the modification that for 7-CF3 substituted flavin 2a recrystallization was performed with hot filtration from CF3CH2OH instead of HCO2H in order to ensure complete removal of alloxan monohydrate (3a).

2,2-dideuterobenzylamine (7)
According to the published procedure, [5] 1M THF solution of lithium aluminium deuteride (9.4 mL, 9.4 mmol) was diluted with dry THF (5 mL) and cooled to 0 ˚C . Benzonitrile (0.88 mL, 8.5 mmol) was added dropwise as a solution in dry THF (5 mL). The solution was warmed to r.t. then refluxed for 16 h. The reaction was cooled in ice, and quenched sequentially with Et2O (5 mL), H2O (0.5 mL) and 10% NaOH (0.7 mL). The mixture was filtered through celite and water (5 mL) was added and the mixture was extracted with ether (3 x 10 mL). The solution was dried with MgSO4 and solvent was removed in vacuo. Data in accordance with that previously published. [6] N,N-dimethylalloxan (3b) In a modification of the published procedure, [7] to a solution of selenium dioxide (0.75 g, 6.8 mmol) in dioxane (3 mL) and water (180 µL) was added N,N-dimethylbarbituric acid (1.06 g, 6.4 mmol) as a suspension in dioxane (5 mL) using an addition funnel. The mixture was refluxed for 72 h, then cooled and filtered through celite, solvent was removed in vacuo, and the crude product was recrystallized from benzene/hexane to give N,N-dimethylalloxan 3b as a white powder (226 mg, 21%), and was observed in DMSO as a 2:1 ratio of its monohydrate to the free carbonyl. 1  Data in accordance with that previously published. [8] S15      Benzylamine 1a (55 μL, 0.5 mmol) was added, and the solution was then used for EPR analysis. Alternatively, for analysis in deuterated solvent, d3-2,2,2-trifluoroethanol was instead used. For experiments with deuterated substrate, PhCD2NH2 7 was used in place of benzylamine.

Preparation of anaerobic aminyl radical 4a'
To a solution of flavinium chloride x (20 mg, 50 μmol) and alloxan monohydrate (4 mg, 25 μmol) in 2,2,2-trifluoroethanol (1 mL) was added dimethyl sulfide (370 μL, 5 mmol), and the solution was purged with nitrogen for 10 minutes. Benzylamine (55 μL, 0.5 mmol) was added, and the solution was then taken up into a 4 mm X-band EPR tube and opened inside a double-ended Schlenk tube. The solution was frozen and then subjected to four freezepump-thaw cycles, then sealed under vacuum and used for EPR analysis. Alternatively, for analysis in deuterated solvent, d3-2,2,2-trifluoroethanol was instead used. For experiments with deuterated substrate, PhCD2NH2 7 was used in place of benzylamine.

Further EPR discussion
At X-band (~ 9.7 GHz), the continuous wave EPR experiments allow us to characterize the parameters defining the strongest interaction terms of the time-independent spin-Hamiltonian. [9] The fact that the g factor reflects the global electronic structure, while the hyperfine structure is determined by the local spin density distribution in the vicinity of the nuclei such as EPR-active 14 N, 1 H, 19 F and EPR-silent 12 C, 16

S27
Other evidence was further required to demonstrate the interacting 14 N nuclei. For this purpose, the pulsed EPR experiment, namely hyperfine sublevel correlation spectroscopy (HYSCORE), [11] is used to obtain information about the surrounding magnetic nuclei around the radical unpaired electron spin. This 2D ESEEM method has the ability to resolve the weak hyperfine interactions (<5 MHz) of the remote nuclei with the unpaired electron spin, which are masked or suppressed by EPR broadening in the hyperfine structure. ESEEM appears only if the allowed and forbidden EPR transitions are simultaneously induced by the high power microwave pulses. [12] In the case of the 14 N nucleus with the spin I = 1, the timeindependent spin-Hamiltonian includes an additional nuclear quadrupole interaction term. [12] The ESEEM spectrum expected from 14   Accordingly, the X-band 14 N HYSCORE spectrum resolves the three contour-peaks from the three nuclear frequencies, nearly satisfying the "cancellation condition" | 14 N -14 AN/2| ≈ 0 in one of the electron spin manifolds (Figure S12). These frequencies, with the property + = 0 + -, are slightly broadened here as ef±/K deviates from 0. However, these frequencies can appear in the spectrum up to a ratio ef±/K ~ 0.75-1. [13] In orientation disordered samples, Upon addition of alloxan and amine, a completely new EPR spectrum was observed which appears to correspond to the dibenzylaminyl radical, consistent with an H-abstraction mechanism. This radical demonstrated long-term stability and therefore is consistent with an oxygen-starved resting state. These EPR experiments strongly suggested a reaction mechanism with inherent radical character. For this radical, several proton nuclei (I = 1/2) are nearly equally coupled, i.e. possessing almost the same isotropic couplings. The radical contains protons on its two rings that are grouped into the two nearly equivalent sets but with slightly different isotropic couplings of ~ 11 MHz and ~ 9 MHz, respectively. Again, the spin polarization mechanism is responsible for the resolved hyperfine splittings from these ring protons. The central nitrogen atom carries a significant spin density distribution to contribute to a slight asymmetry of the absorption derivative; as a result its neighbouring two protons also possess the same order of the isotropic coupling ~ 13 MHz.

COMPUTATIONAL AND THEORETICAL DETAILS
All quantum-chemical calculations were performed using the AIMS package. [15] Within this all-electron approach the electronic wavefunctions are constructed using numeric atomcentred basis functions. A converged `tight' basis set was employed, which includes d, f and g functions on the N atoms, and scalar relativistic effects were included.
Local structure optimisations were performed using the forces from density functional theory (DFT) using the PBE exchange-correlation potential. [16] Additional tests were performed using the hybrid PBE0 and B3LYP exchange-correlation functionals, [17] which were found not to change the localisation of the radical spin.
The inclusion of a polarisable continuum would not change the qualitative trends reported here. Indeed the current model has been shown to reproduce other similar experiments to within 3.5% of the measured IP. [18] Whilst solvation of radicals in aqueous media has been observed, [19]