Introduction of flavin anions into photoredox catalysis: Acid-base equilibria of lumichrome allows photoreductions with an anion of an elusive 10-unsubstituted isoalloxazine

Introduction of flavin anions into photoredox catalysis: Acid-base equilibria of lumichrome allows photoreductions with an anion of an elusive 10-unsubstituted isoalloxazine Dorota Prukała,* Ekaterina Zubova, Eva Svobodová, Ludmila Šimková, Naisargi Varma, Josef Chudoba, Jiří Ludvík, Iwona Gulaczyk, Marek Sikorski,* Radek Cibulka* Supplementary Information Content S1. Materials and methods ............................................................................................................... 2 S2. Synthesis and characterization of flavin derivatives ................................................................... 3 S3. Photoredox catalysis ................................................................................................................... 7 S4. UV-Vis, fluorescence and excitation spectra and fluorescence lifetime measurements ............ 9 S5. CV measurements ..................................................................................................................... 16 S6. Estimation of E*ox of 2ain singlet excited state ....................................................................... 17 S7. Experimental and theoretical NMR data in the presence of base ............................................ 18 S8. NMR data for synthesized flavin derivatives ............................................................................. 31 S9. Cartesian coordinates ................................................................................................................ 36 S10. References ................................................................................................................................. 41 2 S1. Materials and methods Starting materials were purchased from Merck (Sigma-Aldrich) and Fluorochem. The solvents were purified and dried using standard procedures. Commercially obtained reagents were used as received without further purification unless otherwise stated. The compound structures were drawn and named using ChemDraw. Nuclear magnetic resonance (NMR) spectra were recorded in CD3CN, DMSOd6 , DMF-d6, TFA-d3 or in their mixtures on an Agilent 400-MR DDR2 (399.94 MHz for 1H, 100.58 MHz for 13C), JEOL-ECZL400G (400 MHz for 1H, 101 MHz for 13C), or Bruker Avance III 600 MHz (600 MHz for 1H and 151 MHz for 13C) at 298 K. Data for 1H NMR are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, dt = doublet of triplets, br = broad etc.), coupling constant (Hz), and integration. All NMR spectra were processed and assigned using MestreNova. High-resolution mass spectra were obtained on Q-Tof Micro (Waters), equipped with a quadrupole and time-offlight (TOF) analyser and a multichannel plate (MCP) detector. The melting points were measured on a Boetius melting point apparatus and are not corrected. Photoredox catalysis was performed using Luxeon Star LED 470 nm (65 lm @ 700mA) with dominant peak wavelengths 460–485 nm in tempered aluminium block (for arrangement, see S3) and Luxeon Star LED 505 nm (76 lm @ 350mA) with dominant peak wavelengths 490–515 nm. UV-Vis absorption spectra were recorded on a UV-2550 spectrophotometer (Schimadzu). Steady-state emission spectra and fluorescence excitation spectra were recorded on a Jobin Yvon-SpexFluorolog 3-22 spectrofluorometer. Note that spectra were recorded using the same excitation and emission slits for uniform spectral resolution and sensitivity. Fluorescence lifetime measurements of all compounds were performed using the time-correlated single-photon counting (TCSPC) method. Decays were measured with the TCSPC Triple Illuminator as an accessory for the Fluorologs 3-22 steady-state spectrofluorometer that adds lifetime-capability in the time domain. The excitation source were NanoLED diodes (λexc = 368 nm and 389 nm) from IBH. The instrument in this hardware configuration is capable of measuring lifetimes as short as 400 ps. Deconvolution of fluorescence decay curves was performed using IBH Consultants software. Electrochemical measurements (cyclic voltammetry) were managed by a computer driven potentiostat PGSTAT101 (Autolab-Metrohm) using NOVA 1.11 software, for details see Chapter 5. Quantum chemical calculations: Geometries of all the structures were fully optimized to minima, confirmed by frequency calculations having no imaginary frequencies, using Gaussian 16 software1 package employing a DFT method with a B3LYP functional in the calculation together with cc-pVTZ and aug-cc-pVTZ basis set. Acetonitrile as a solvent was approximated by CPCM method. Absorption spectra were calculated at the same level of theory using TD-DFT approach (the first 15 excited states were calculated). The optimized geometries were used for energy calculation. The energies were calculated at DFT-B3LYP level of theory together with cc-pVTZ and aug-cc-pVTZ basis set in Gaussian 16 software package. Acetonitrile as a solvent was simulated using CPCM approach. The NMR calculations were also performed using the NMR GIAO method at the same level of theory (DFT-B3LYP with cc-pVTZ and aug-cc-pVTZ basis set) and with acetonitrile as a solvent in the CPCM model. 3 S2. Synthesis and characterization of flavin derivatives 3-Methyllumichrome (1a-H) To the solution of 3,4-dimethylaniline (2.410 g, 19.88 mmol) in acetic acid (3.5 mL) acetic anhydride (3.5 ml, 31.74 mmol, 1.6 equiv.) was added. The reaction mixture was heated to reflux for 15 min and then added dropwise to water with ice. Precipitate (old pink color) was filtered off using Büchner funnel. The filtration cake was dissolved in acetic acid (6 mL) and added dropwise to the mixture of 65% nitric acid (12 ml, 266 mmol, 13 equiv.) and acetic acid (10 mL) cooled to +5 °C. The reaction mixture was allowed to warm to room temperature (90 min) and poured into water with ice (100 mL). Resulting precipitate was filtered off using Büchner funnel and washed with water (approximately 500 mL). After drying on air was obtained 2.337 g (56 %) of bright yellow crystals of 2-nitro-4,5dimethylacetanilide (m.p. 99 – 103 °C, lit.2 101 - 104 °C) 1H NMR (400 MHz, CDCl3) δ 10.29 (s, 1H), 8.53 (s, 1H), 7.97 (s, 1H), 2.34 (s, 3H), 2.28 (s, 3H), 2.27 (d, J = 0.7 Hz, 3H). HRMS APCI pos m/z for C10H13N2O3 [M+H+] calculated 209.09027, found 209.09158. 2-Nitro-4,5-dimethylacetanilide was dissolved in 96% sulfuric acid (15 mL) and heated to 90 °C for 30 min. After cooling to r.t., the reaction mixture was poured dropwise on ice. The precipitated orange crystals were filtered off using Büchner funnel and dried on air. It was obtained 1.380 g (74 %) of 3,4dimethyl-6-nitroaniline, m.p. 136 – 139 °C, lit.3 140-141 °C. 1H NMR (400 MHz, CDCl3) δ 7.87 (s, 1H), 6.59 (s, 1H), 2.22 (s, 3H), 2.18 (s, 3H). HRMS APCI pos m/z for C8H11N2O2 [M+H+] calculated 167.08150, found 167.08138. 3,4-Dimethyl-6-nitroaniline (1.380 g, 8.30 mmol) was dissolved in acetic acid (60 mL) and palladium on carbon (10%, 0.150 g) was added. Hydrogen (balloon) was bubbled through the reaction mixture until the color disappear (20 hours). Residual of palladium was filtered off through celite and the filtrate was added to the hot solution of N-methylalloxane (1.310 g, 8.39 mmol) and boric acid (0.620 g, 10.03 mmol) in acetic acid (60 mL). Reaction mixture was stirred in dark at r.t. for 4 hours. Bright yellow crystals were filtered off through frit (S4), washed with diethyl ether (10 ml) and dried on vacuo. It was obtained 1.270 g (60 %) of 1a-H, m.p. 354 – 359 °C. 1H NMR (400 MHz, DMSO-D6) δ 12.12 (s, 1H), 7.95 – 7.91 (m, 1H), 7.73 – 7.68 (m, 1H), 3.29 (s, 3H), 2.48 (d, J = 1.0 Hz, 3H), 2.47 – 2.44 (m, 3H). 13C NMR (101 MHz, DMSO-D6) δ 160.46, 150.34, 145.16, 144.84, 141.78, 139.05, 138.58, 129.50, 128.75, 125.89, 27.74, 20.30, 19.67. HRMS APCI pos m/z for C13H13N4O2 [M+H+] calculated 257.10330, found 257.10336. 4 1-Methyllumichrome (3a-H) Mixture of 3,4-dimethylaniline (0.900 g, 5.60 mmol) and 1-methyl-6-chlorouracil (2.410 g, 19.90 mmol) was heated to 150 °C under stopper with calcium chloride for 2 hours. To the cooled mixture, diethyl ether (30 mL) was added and the mixture was sonicated. Product was filtered off, washed with diethyl ether, methanol and again with diethyl ether. Filtrate was evaporated, diethyl ether was added (100 mL) and precipitated crystals were filtered off. Crystals were collected. It was obtained 1.390 g (99 %) of 1-methyl-6-(3,4-dimethylanilino)uracil, m.p. 316 – 320 °C. 1H NMR (400 MHz, DMSO-D6) δ 10.62 (s, 1H), 7.20 (dd, J = 12.7, 8.1 Hz, 2H), 7.06 (d, J = 11.7 Hz, 3H), 7.03 (d, J = 2.3 Hz, 1H), 6.96 (dd, J = 7.9, 2.3 Hz, 1H), 4.39 (s, 1H), 2.22 (dd, J = 5.3, 2.8 Hz, 13H), 2.08 (s, 7H). HRMS APCI pos m/z for C13H16N3O2 [M+H+] calculated 246.12370, found 246.12352. To the mixture of anilinouracil (1.390 g, 5.60 mmol) and acetic acid (30 mL), sodium nitrite (1.89 g, 26.40 mmol) was added. The mixture was stirred at r.t. for 2 hours and after addition of water (20 mL), the mixture was stirred 10 minutes. Precipitated crystals were filtered off through Büchner funnel and washed with diethyl ether. It was obtained 0.990 g of orange crystals as mixture of alloxazine and alloxazinium-N-oxide (3:1, according 1H NMR). Part (0.275 g) of the mixture of alloxazin and alloxazinium-N-oxide was dissolved in the mixture of solvents DMF (8 mL) and acetic acid (45 mL). Palladium on carbon was added (10%, 70 mg) and the mixture was stirred under hydrogen (6 atm) in autoclave at r.t. for 46 hours. The reaction mixture was heated until dissolving all solids and palladium on carbon was filtered off. After evaporation of solvents, it was obtained 0.255 g (64 %) of 3a-H, m.p. 338-342 °C, lit.4 >300 °C. 1H NMR (400 MHz, CD3CN / DMSO-D6) δ 11.49 (s, 1H), 7.93 (d, J = 1.3 Hz, 1H), 7.77 (s, 1H), 3.59 (s, 3H), 2.51 (d, J = 1.0 Hz, 3H), 2.48 (d, J = 1.1 Hz, 3H). 1H NMR (600 MHz, DMSO-d6) δ 7.93 (d, J = 1.2 Hz, 1H), 7.80 (d, J = 1.2 Hz, 1H), 3.52 (s, 3H), 2.48 (s, 3H), 2.46 (d, J = 1.0 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 159.73, 150.31, 146.61, 144.85, 141.00, 139.22, 137.60, 130.73, 128.56, 126.33, 28.27, 20.21, 19.62. HRMS APCI pos m/z for C13H13N4O2 [M+H+] calculated 257.10330, found 257.10294. 5 Lumiflavin (7,8,10-trimethylisoalloxazine, 4a-H) Lumiflavin 4a-H was obtained starting from 3,4-dimethyl-6-nitroaniline using procedure described in literature.5 M.p. > 370 °C. 1H NMR (400 MHz, CD3CN) δ 10.73 (s, 1H), 7.88 (s, 1H), 7.64 (s, 1H), 3.98 (s, 3H), 2.52 (s, 3H), 2.41 (s, 3H). 13C NMR (151 MHz, CD3CN / DMSO-D6) δ 161.07, 156.67, 154.38, 138.10, 134.91, 132.87, 132.16, 117.09, 32.62, 21.12, 19.24. HRMS APCI pos m/z for C13H13N4O2 [M+H+] calculated 257.10330, found 257.10355. 1,3-Dimethyllumichrome (5a) Lumichrome (0.080 g, 0.33 mmol) was dissolved in dry DMF (4 mL), potassium carbonate (0.300 g, 2.17 mmol) and methyl iodide (0.270 g, 0.525 mmol) were added. The mixture was stirred under argon atmosphere for 2 hours, then poured into water and extracted with ethyl acetate. Combined extracts were washed with water, brine and dried over magnesium sulfate. After evaporation of solvent was obtained 0.090 g (99 %) of 1,3-dimethyllumichrome, m.p. 242 – 245 °C, lit.6 246 – 248 °C. 1H NMR (400 MHz, CD3CN / DMSO-D6) δ 7.94 (d, J = 1.3 Hz, 1H), 7.79 – 7.76 (m, 1H), 3.68 (d, J = 0.5 Hz, 3H), 3.43 (d, J = 0.5 Hz, 3H), 2.52 (d, J = 1.0 Hz, 4H), 2.51 – 2.48 (m, 4H). 13C NMR (151 MHz, CD3CN / DMSO-D6) δ 160.84, 151.81, 146.53, 146.47, 142.74, 140.77, 139.44, 130.56, 129.68, 127.35, 29.67, 29.09, 20.75, 20.09. HRMS APCI pos m/z for C14H15N4O2 [M+H+] calculated 271.11895, found 271.11838. Lumichrome To the mixture of 1,2-dimethyl-4,5-dinitrobenzene (0.600 g, 3.06 mmol) in acetic acid (20 mL), palladium on carbon (10%, 160 mg) was added. The mixture was stirred under hydrogen atmosphere (balloon) for 24 hours. Palladium on carbon was filtered off through celite, washed with acetic acid and filtrate was immediately added to the hot solution of alloxane hydrate (0.490 g, 3.06 mmol, 1 equiv.) and boric acid (0.208 g, 3.36 mmol, 1.1 equiv.) in acetic acid (30 mL). The mixture was stirred at r.t. in 6 dark overnight. Precipitated orange crystals were filtered off through frit (S4), washed with acetic acid and diethyl ether. It was obtained 0.580 g (78 %) of lumichrome, m.p. 358 - 362°C., lit.6 368 – 370 °C. 1H NMR (400 MHz, CD3CN / DMSO-D6) δ 11.32 (s, 2H), 7.90 (s, 1H), 7.69 (s, 1H), 2.50 (s, 3H), 2.47 (d, J = 1.0 Hz, 3H). 13C NMR (151 MHz, CD3CN / DMSO-D6) δ 161.84, 151.13, 147.53, 146.19, 143.09, 140.38, 139.87, 130.99, 129.81, 126.94, 20.68, 20.04. HRMS APCI pos m/z for C12H11N4O2 [M+H+] calculated 243.08765, found 243.08786. 7 S3. Photoredox catalysis Dehalogenation – inert conditions Substrate (0.038 mmol), alloxazine or flavin derivative (10 mol%; 4×10-3 mmol), -terpinene (2 equiv.; 0.076 mmol), and Cs2CO3 (2 equiv.; 0.076 mmol) were suspended in DMF (0.5 mL), placed into vial or Schlenk tube and bubbled with argon or degassed using the freeze-pump-thaw technique (3 × 5 min), respectively. The reaction mixture was stirred and irradiated with LED 470 nm (65 lm @ 700mA) or LED 505 nm for 3-bromo-9H-fluoren-9-one at 25 °C for 16 h. Conversion was determined by GC-MS. Dehalogenation – under air 4-Bromobenzonitrile (0.038 mmol), alloxazine derivative (10 mol%; 4×10-3 mmol), -terpinene (2 equiv.; 0.076 mmol), and Cs2CO3 (2 equiv.; 0.076 mmol) were suspended in DMF (0.5 mL) and placed into vial. The reaction mixture was stirred and irradiated with LED 470 nm (65 lm @ 700mA) at 25 °C for 16 h. Conversion was determined by GC-MS. Dehalogenation – other bases 4-Bromobenzonitrile (0.038 mmol), 1a-H (10 mol%; 4×10-3 mmol), -terpinene (2 equiv.; 0.076 mmol), and CsOAc or TBAH2PO4 (2 equiv.; 0.076 mmol), were dissolved/suspended in DMF (0.5 mL), placed into vial or Schlenk tube and bubbled with argon or degassed using the freeze-pump-thaw technique (3 × 5 min), respectively. The reaction mixture was stirred and irradiated with LED 470 nm (65 lm @ 700mA) at 25 °C for 16 h. Conversion was determined by GC-MS. Coupling with P(OMe)3 – inert conditions 4-Bromobenzonitrile (0.038 mmol), trimethyl phosphite (5 equiv.; 0.190 mmol), 1a-H (10 mol%; 4×103 mmol), -terpinene (2 equiv.; 0.076 mmol), and Cs2CO3 (2 equiv.; 0.076 mmol), were suspended in DMF (0.5 mL), placed into vial or Schlenk tube and bubbled with argon or degassed using the freezepump-thaw technique (3 × 5 min). The reaction mixture was stirred and irradiated at 470 nm (65 lm @ 700mA) at 25 °C for 48 h. Conversion was determined by GC-MS. 8 Coupling with P(OMe)3 – under air 4-Bromobenzonitrile (0.038 mmol), trimethyl phosphite (5 equiv.; 0.190 mmol), 1a-H (10 mol%; 4×103 mmol), -terpinene (2 equiv.; 0.076 mmol), and Cs2CO3 (2 equiv.; 0.076 mmol) were suspended in DMF (0.5 mL) and placed into vial. The reaction mixture was stirred and irradiated at 470 nm (65 lm @ 700mA) at 25 °C for 48 h. Conversion was determined by GC-MS. FigureS3.1 Typical set-up of photochemical experiment 9 S4. UV-Vis, fluorescence and excitation spectra and fluorescence lifetime measurements Anions of 3-methyllumichrome (1a-H), 1-methyllumichrome (3a-H) and lumiflavin (4a-H) were obtained in situ, adding salt tetrabutylammonium acetate (TBAOAc). TBAOAc was of ≥ 99% of purity and was from Sigma-Aldrich. Before use, TBAOAc was dried under vacuum and stored in a desiccator. Flavin derivatives were dissolved in dry acetonitrile (ACN). Only freshly prepared solutions were used in order to minimize possible photolytic reactions or hydrolysis. All experiments were carried out at room temperature. 200 300 400 500 600 0,0 0,2 0,4 0,6 342 nm Absorbance l / nm 0 1.96x10-5 3.92x10-5 5.88x10-5 7.83x10-5 9.78x10-5 1.47x10-4 1.95x10-4 2.43x10-4 2.92x10-4 442 nm 4a-H c(TBAOAc) mol L-1 Figure S4.1. The absorption spectra of lumiflavin (4a-H) in ACN (8.49×10-6 M) with TBAOAc. 400 450 500 550 600 650 700 750 0 1x106 2x106 3x106 4x106 5x106 Rel. Intensity l / nm 0 1.96x10-5 3.92x10-5 5.88x10-5 7.83x10-5 9.78x10-5 1.47x10-4 1.95x10-4 2.43x10-4 2.92x10-4 527 nm 528 nm 4a-H c(TBAOAc) mol L-1 Figure S4.2. The emission spectra of lumiflavin (4a-H) in ACN (8.49×10-6 M) with TBAOAc, excited at λ = 393 nm. 16 S5. CV measurements An undivided 10 ml cell with three-electrode system: work - GCE ø 3 mm, reference - SCE, auxiliary - Pt plate, scan rate 100 mV·s–1 in ACN deoxygenated by argon. 0.1 mol L-1. Tetrabutylammonium hexafluorophosphate (TBAPF6) as the supporting electrolyte; samples concentrations 0.5 x 10-3 mol L1. The concentration of added base change from 0.25·10-3 mol L-1 to 25·10-3 mol L-1. The maximum concentration of added base (TBAOAc, respective TBAH2PO4, 25·10-3 mol L-1) correspond to 50 equivalents. A B Figure S5.1 Cyclic voltammograms of (A) 4a-H and (B) 5a in the absence and in the presence of TBAOAc (50 equiv.). Data obtained in acetonitrile in the presence of TBAPF6 (c = 0.1 mol L-1) using glassy carbon working, saturated calomel reference and platinum auxiliary electrodes. 17 S6. Estimation of E*ox of 2ain singlet excited state E*ox of 2ain single excited state was estimated from ground state redox potential (Ep/2 = 1.02 V; i.e. value from CV measurement of 1a-H in the presence of TBAOAc) and the value E0−0 = 2.52 eV corresponding to intersection of the normalized absorption and emission bands (see Figure S6.1). E*ox(2a-) = Ep/2 - E0−0 = 1.02 – 2.52 = -1.5 V 300 400 500 600 700 0.00 0.02 0.04 0.06 0.08 0.10 0.12 l / nm Absorbance 0 1x106 2x106 3x106 4x106 Relative intensity 493 nm = 2.52 eV Figure S6.1 Normalized UV-VIS and fluorescence spectra of anion 2a-. 18 S7. Experimental and theoretical NMR data in the presence of base NMR analysis of mixture 3-methyllumichrome (1a-H) with tetrabutylammonium acetate in mixture of solvents CD3CN/DMSO-d6 85/15 Stock solution (c = 17 mM) of 1a-H was prepared from 6.58 mg of 3-methyllumichrome (1a-H) and 1500 l of DMSO-d6. Stock solution (c = 34 mM) of tetrabutylammonium acetate (TBAOAc) was prepared from 30.50 mg of TBAOAc and 3000 l of CD3CN. Into NMR tube was added solution of flavine, TBAOAc and CD3CN in different ratio. Table S7.1 Preparation and NMR analysis of mixture 1a-H with TBAOAc 1:0 1:0.5 1:1 1:2 1.3 1:4 1:5 1:6 1:7 1:8 1.9 1:10 1:100 V(1a-H) 100 100 100 100 100 100 100 100 100 100 100 100 c(1a-H) 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.7 V(TBAOAc) 0 25 50 100 150 200 250 300 350 400 450 500 C(TBAOAc) 0 1.2 2.4 4.8 7.2 9.6 12.0 14.4 16.8 19.2 21.6 24 300 V(CD3CN) 600 575 550 500 450 400 350 300 250 200 150 100 ppm 7.918 7.901 7.864 7.847 7.837 7.833 7.820 7.816 7.815 7.806 7.807 7.815 7.749 ppm 7.702 7.687 7.653 7.637 7.628 7.624 7.612 7.609 7.618 7.600 7.608 7.600 7.547 ppm 2.499 2.493 2.479 2.472 2.468 2.467 2.462 2.460 2.461 2.457 2.457 2.461 2.432 pm 2.476 2.470 2.456 2.452 2.447 2.446 2.440 2.438 2.438 2.435 2.436 2.437 2.411 Figure S7.1 Changes in chemical shifts of aromatic protons H6 and H9 after addition of TBAOAc 19 0 2 4 6 8 10 100 7,55 7,60 7,65 7,70 7,75 7,80 7,85 7,90 7,95 Chemical shift (ppm) Equivalent of base Model Exponential Equation y = y0 + A*exp(R0*x) Plot B y0 7,81054 ± 0,00337 A0,10708 ± 0,00547 R0 -0,50436 ± 0,07004 Reduced Chi-Sqr 3,78738E-5 R-Square (COD) 0,97721 Adj. R-Square 0,97215 Model Exponential Equation y = y0 + A*exp(R0*x) Plot B y0 7,60565 ± 0,0035 A0,09644 ± 0,00587 R0 -0,52319 ± 0,08556 Reduced Chi-Sqr 4,32628E-5 R-Square (COD) 0,96813 Adj. R-Square 0,96105 Figure S7.2 Changes in chemical shifts of aromatic protons H6 and H9 after addition of TBAOAc NMR analysis of mixture 3-methyllumichrome (1a-H) with tetrabutylammonium dihydrogenphosphate in mixture of solvents CD3CN/DMSO-d6 85/15. Stock solution (c = 17.5 mM) of 1a-H was prepared from 6.75 mg of 3-methyllumichrome (1a-H) and 1500 l of DMSO-d6. Stock solution (c = 36 mM) of tetrabutylammonium dihydrogenphosphate (TBAH2PO4) was prepared from 36.70 mg of TBAH2PO4 and 3000 l of CD3CN. Into NMR tube was added solution of flavine, TBAH2PO4 and CD3CN in different ratio. Table S7.2 Preparation and NMR analysis of mixture 1a-H with TBAH2PO4 1:0 1:0.5 1:1 1:2 1.3 1:4 1:5 1:6 1:7 1:8 1.9 1:10 1:85 V(1a-H) 100 100 100 100 100 100 100 100 100 100 100 100 c(1a-H) 2.5 2.5ř 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 V(TBAH2PO4) 0 25 50 100 150 200 250 300 350 400 450 500 C(TBAH2PO4) 0 1.25 2.5 5.1 7.7 10.3 12.85 15.4 18.0 20.6 23.1 25.7 210.4 V(CD3CN) 600 575 550 500 450 400 350 300 250 200 150 100 ppm 7.918 7.890 7.863 7.827 7.799 7.778 7.765 7.750 7.738 7.730 7.724 7.719 7.652 ppm 7.702 7.676 7.652 7.619 7.595 7.575 7.563 7.549 7.538 7.531 7.525 7.520 7.449 ppm 2.500 2.488 2.479 2.466 2.457 2.448 2.444 2.438 2.433 2.430 2.428 2.426 2.395 ppm 2.476 2.465 2.456 2.443 2.433 2.426 2.420 2.415 2.410 2.407 2.405 2.403 2.373 20 Figure S7.3 Changes in chemical shifts of aromatic protons H6 and H9 after addition of TBAH2PO4. 0 2 4 6 8 10 84 86 7,45 7,50 7,55 7,60 7,65 7,70 7,75 7,80 7,85 7,90 7,95 Chemical shift (ppm) Equivalent of base Model Exponential Equation y = y0 + A*exp(R0*x) Plot B y0 7,70607 ± 0,00263 A0,20967 ± 0,0025 R0 -0,26814 ± 0,00942 Reduced Chi-Sqr 4,93816E-6 R-Square (COD) 0,99913 Adj. R-Square 0,99894 Model Exponential Equation y = y0 + A*exp(R0*x) Plot B y0 7,50761 ± 0,0026 A0,19186 ± 0,00245 R0 -0,26217 ± 0,0098 Reduced Chi-Sqr 4,51133E-6 R-Square (COD) 0,99904 Adj. R-Square 0,99883 Figure S7.4 Changes in chemical shifts of aromatic protons H6 and H9 after addition of TBAH2PO4. 21 Table S7.3: Chemical shifts of selected protons (C7-CH3, C8-CH3, H6, H9) of flavin derivatives in mixture of solvents CD3CN/DMSO-d6 85/15 or in common solvents after addition of base (and acid). Flavine derivative ppm ppm ppm ppm 3-Methyllumichrome (1a-H) 2.476 2.500 7.701 7.917 3-Methyllumichrome (1a-H) + TBAOAc 2.429 2.451 7.588 7.793 3-Methyllumichrome (1a-H) + TBAOAc + CF3COOD 2.474 2.497 7.699 7.913 3-Methyllumichrome (1a-H) + TBAH2PO4 2.400 2.423 7.511 7.710 3-Methyllumichrome (1a-H) + TBAH2PO4 + CF3COOD 2.479 2.502 7.706 7.920 3-Methyllumichrome (1a-H) + TBAOH 2.392 2.415 7.499 7.703 3-Methyllumichrome (1a-H) + excess of TBAOAc 2.411 2.432 7.547 7.749 3-Methyllumichrome (1a-H) + excess of TBAH2PO4 2.373 2.395 7.449 7.652 3-Methyllumichrome (1a-H) + TBAOAc in DMF-D6 2.427 2.449 7.532 7.753 3-Methyllumichrome (1a-H) + TBAOAc + CF3COOD in DMF-D6 2.515 2.538 7.740 7.950 3-Methyllumichrome (1a-H) in CD3CN 2.502 2.525 7.714 7.947 3-Methyllumichrome (1a-H) in DMSO-d6 2.454 2.478 7.705 7.930 3-Methyllumichrome (1a-H) in CD3COOD 2.550 2.590 7.903 8.087 3-Methyllumichrome (1a-H) in DMF-D6 2.517 2.539 7.742 7.955 1-Methyllumichrome (3a-H) 2.484 2.514 7.768 7.929 1-Methyllumichrome (3a-H) + TBAOAc 2.470 2.497 7.728 7.902 1-Methyllumichrome (3a-H) + TBAOAc + CF3COOD 2.483 2.513 7.767 7.929 1-Methyllumichrome (3a-H) + TBAH2PO4 2.453 2.478 7.673 7.863 1-Methyllumichrome (3a-H) + TBAH2PO4 + CF3COOD 2.481 2.511 7.753 7.917 1-Methyllumichrome (3a-H) + TBAOH 2.436 2.457 7.637 7.844 1-Methyllumichrome (3a-H) + NaH n.v. n.v. 7.625 7.856 Lumiflavin (4a-H) 2.413 2.516 7.642 7.881 Lumiflavin (4a-H) + TBAOAc 2.412 2.515 7.645 7.879 Lumiflavin (4a-H) + TBAOAc + CF3COOD 2.413 2.517 7.646 7.882 Lumiflavin (4a-H) + TBAH2PO4 2.411 2.517 7.647 7.881 Lumiflavin (4a-H) + TBAOH Aduct and decomposition 1,3-Dimethyllumichrome (5a) 2.493 2.522 7.776 7.944 1,3-Dimethyllumichrome (5a) + TBAOAc 2.486 2.515 7.776 7.937 1,3-Dimethyllumichrome (5a) + TBAOAc + CF3COOD 2.492 2.516 7.779 7.941 1,3-Dimethyllumichrome (5a) + TBAH2PO4 2.482 2.511 7.765 7.930 1,3-Dimethyllumichrome (5a)+ TBAH2PO4 + CF3COOD 2.484 2.512 7.766 7.931 1,3-Dimethyllumichrome (5a)+ TBAOH – ADUCT! 2.109 2.133 6.591 6.812 Lumichrome 2.469 2.495 7.689 7.901 22 Figure S7.5 NMR spectra of flavin and formation of their anions after addition of TBAOAc, TBAH2PO4 or TBAOH. 1a-H 1a-H + TBAOAc 1a-H + TBAH2PO4 1a-H + TBAOH 3a-H 3a-H + TBAOAc 3a-H + TBAH2PO4 3a-H + TBAOH 4a-H 4a-H + TBAOAc 4a-H + TBAH2PO4 4a-H + TBAOH 5a 5a + TBAOAc 5a + TBAH2PO4 5a + TBAOH 23 Table S7.4: Changes in chemical shift 3-methyllumichrome (1a-H) in mixture of solvents CD3CN/DMSOd6 85/15 after addition of different bases (10 eq) and CF3COOD (10 eq). ppm ppm ppm ppm 3-Methyllumichrome 1a-H 2.476 2.499 7.702 7.918 + TBAOAc 2.429 2.451 7.588 7.793 + TBAOAc + CF3COOD 2.474 2.497 7.699 7.913 + excess (100 eq) TBAOAc 2.411 2.432 7.546 7.749 + NaOH 2.467 2.490 7.680 7.899 + NaOH + CF3COOD 2.479 2.502 7.708 7.926 + TBAHCO3 2.474 2.298 7.700 7.912 + TBAHCO3 + CF3COOD 2.474 2.499 7.702 7.914 + DBU 2.392 2.413 7.473 7.693 + DBU + CF3COOD 2.477 2.500 7.703 7.916 + K2CO3 2.455 2.479 7.655 7.867 + K2CO3 + CF3COOD 2.475 2.499 7.703 7.919 + NaH 2.387 2.408 7.484 7.710 + NaH + CF3COOD 2.478 2.508 7.710 7.941 + TBAH2PO4 2.400 2.423 7.511 7.710 + TBAH2PO4 + CF3COOD 2.479 2.502 7.706 7.920 +excess (85 eq) TBAH2PO4 2.373 2.395 7.449 7.652 + TBAOH 2.392 2.415 7.499 7.703 24 Figure S7.6 Changes in chemical shifts of aromatic protons H6 and H6 of 3-methyllumichrome (1aH) after addition of different bases and CF3COOD. 1a-H 1a-H + TBAOAc 1a-H + TBAOAc + CF3COOD 1a-H + TBAOAc (excess) 1a-H + NaOH 1a-H + NaOH + CF3COOD 1a-H + TBAHCO3 1a-H + TBAHCO3 + CF3COOD 1a-H + DBU 1a-H + DBU + CF3COOD 1a-H + K2CO3 1a-H + K2CO3+ CF3COOD 1a-H + NaH 1a-H + NaH + CF3COOD 1a-H + TBAH2PO4 1a-H + TBAH2PO4 + CF3COOD 1a-H + TBAOH 25 Table S7.5 Theoretical NMR chemical shifts calculated using GIAO/B3LYP/cc-pVTZ (with solvent - acetonitrile) 1a-H 1a2a-H 3a-H 3a4a-H 4aisotropic (shielding constant) sigma (chemical shift) isotropic (shielding constant) sigma (chemical shift) isotropic (shielding constant) sigma (chemical shift) isotropic (shielding constant) sigma (chemical shift) isotropic (shielding constant) sigma (chemical shift) isotropic (shielding constant) sigma (chemical shift) isotropic (shielding constant) sigma (chemical shift) TMS 31,7369 H6 23,4623 8,2746 23,793 7,9439 23,5404 8,1965 23,4604 8,2765 23,6383 8,0986 23,5485 8,1884 23,7998 7,9371 H7 23,6817 8,0552 24,0339 7,703 24,1789 7,558 23,5878 8,1491 23,8006 7,9363 23,9368 7,8001 24,2554 7,4815 N1-CH3 29,0408 2,6961 29,123 2,6139 N1-CH3 29,0954 2,6415 29,2074 2,5295 N1-CH3 29,0407 2,6962 29,1232 2,6137 N3-CH3 27,3091 4,4278 27,1245 4,6124 27,2646 4,4723 N3-CH3 28,6593 3,0776 28,8716 2,8653 28,7607 2,9762 N3-CH3 28,6611 3,0758 28,8715 2,8654 28,7626 2,9743 N10-CH3 29,0444 2,6925 29,1599 2,577 N10-CH3 29,1153 2,6216 29,2597 2,4772 N10-CH3 29,0446 2,6923 29,1599 2,577 C8-CH3 29,0544 2,6825 29,1682 2,5687 29,0981 2,6388 29,0878 2,6491 29,1535 2,5834 29,1832 2,5537 29,2647 2,4722 C8-CH3 29,1154 2,6215 29,2699 2,467 29,1969 2,54 29,0878 2,6491 29,1537 2,5832 29,1834 2,5535 29,2648 2,4721 C8-CH3 29,0548 2,6821 29,1684 2,5685 29,098 2,6389 29,1164 2,6205 29,2131 2,5238 29,2073 2,5296 29,33 2,4069 C7-CH3 29,0945 2,6424 29,1982 2,5387 29,1897 2,5472 28,4337 3,3032 28,7001 3,0368 28,3682 3,3687 28,7133 3,0236 C7-CH3 29,095 2,6419 29,1982 2,5387 29,1898 2,5471 28,4338 3,3031 28,6993 3,0376 28,3692 3,3677 28,7139 3,023 C7-CH3 29,1263 2,6106 29,284 2,4529 29,2129 2,524 27,1606 4,5763 26,9696 4,7673 25,9269 5,81 26,0117 5,7252 N1-H 23,8598 7,8771 N3-H 23,8013 7,9356 23,9056 7,8313 N10-H 22,6767 9,0602 32 Figure S8.2 1H and 13C NMR spectra of 1-methyllumichrome (1,7,8-trimethylalloxazine (3a-H)) 33 Figure S8.3 1H and 13C NMR spectra of lumiflavine (7,8,10-trimethylisoalloxazine (4a-H)) 34 Figure S8.4 1H and 13C NMR spectra of 1.3-dimethyllumichrome (1,3,7,8-tetramethylalloxazine (5a)) 35 Figure S8.5 1H and 13C NMR spectra of lumichrome (7,8-dimethylalloxazine) 36 S9. Cartesian coordinates Cartesian coordinates (in Å) of the ground state singlet equilibrium structure of anion from 1a-H. C -2.406395 1.359199 -0.000116 C -1.204684 0.612161 0.000027 C -1.264716 -0.813869 0.000167 C -2.543762 -1.423617 0.000278 C -3.701528 -0.684512 0.000106 C -3.635269 0.750106 -0.000127 H -2.327054 2.438991 -0.000213 H -2.585626 -2.505283 0.000508 C 1.068619 0.494604 0.000050 C 1.032212 -0.944853 0.000075 N -0.150574 -1.570651 0.000187 C 2.386743 1.164149 0.000098 C 3.358700 -1.120323 -0.000199 N 2.150098 -1.696631 -0.000033 N 3.464448 0.313704 0.000161 O 2.533211 2.385448 0.000158 O 4.416942 -1.766559 -0.000646 C 4.788431 0.933833 0.000214 H 5.526391 0.142924 0.000719 H 4.908837 1.560233 -0.882473 H 4.908387 1.561003 0.882395 C -5.037483 -1.375001 0.000139 H -5.627856 -1.097644 -0.875902 H -4.918492 -2.456553 0.000471 H -5.628254 -1.097055 0.875680 C -4.896701 1.570250 -0.000386 H -5.513143 1.357533 -0.876664 H -5.513496 1.357733 0.875657 H -4.667365 2.634181 -0.000506 N -0.010908 1.242157 0.000011 Cartesian coordinates (in Å) of the ground state singlet equilibrium structure of 1a-H. C -2.435661 -1.368890 -0.000053 C -1.231071 -0.627081 -0.000011 C -1.293964 0.801253 0.000028 C -2.559312 1.422880 0.000006 C -3.719935 0.686561 -0.000038 C -3.658506 -0.749538 -0.000068 H -2.361799 -2.448208 -0.000073 H -2.593861 2.504026 0.000036 C 1.043238 -0.526480 0.000031 C 0.970684 0.890120 0.000035 N -0.164365 1.546811 0.000051 C 2.369575 -1.188418 0.000081 C 3.407094 1.061477 0.000001 N 2.146832 1.602561 0.000000 N 3.470499 -0.334739 -0.000059 O 2.521137 -2.396391 0.000191 O 4.400385 1.767781 0.000004 37 C 4.793913 -0.968118 -0.000178 H 5.542190 -0.186796 -0.000844 H 4.901136 -1.591043 0.884554 H 4.900476 -1.591970 -0.884313 H 2.096032 2.610987 0.000082 C -5.051521 1.380791 0.000011 H -5.641165 1.102401 0.875884 H -4.929819 2.461568 -0.000119 H -5.641336 1.102181 -0.875676 C -4.923785 -1.560809 -0.000041 H -5.536706 -1.340580 0.876303 H -5.536909 -1.340350 -0.876181 H -4.702620 -2.625920 -0.000193 N -0.043294 -1.264618 0.000030 Caresian coordinates (in Å) of the ground state singlet equilibrium structure of 2a-H. C -2.405274 -1.368265 0.000072 C -1.216673 -0.612790 0.000115 C -1.312599 0.799293 0.000059 C -2.563308 1.417056 -0.000065 C -3.720318 0.658476 -0.000112 C -3.642773 -0.767302 -0.000017 H -2.312631 -2.445690 0.000116 H -2.623252 2.497237 -0.000155 C 1.076642 -0.517277 0.000190 C 1.081531 0.924912 0.000145 N -0.141265 1.509080 0.000099 C 2.401796 -1.185743 0.000170 C 3.383049 1.092656 0.000095 N 2.149995 1.679991 0.000193 N 3.480893 -0.328451 -0.000268 O 2.540315 -2.399210 0.000428 O 4.410996 1.757699 0.000313 C 4.807842 -0.950531 -0.000872 H 5.548345 -0.162314 -0.003769 H 4.924111 -1.572977 0.884040 H 4.921190 -1.576950 -0.883297 C -5.055903 1.342087 -0.000312 H -5.642013 1.055836 0.875317 H -4.946381 2.423923 -0.000380 H -5.641770 1.055677 -0.876055 C -4.895055 -1.599152 -0.000011 H -5.511184 -1.390208 0.876711 H -5.511382 -1.389941 -0.876525 H -4.655817 -2.660292 -0.000176 N -0.007872 -1.235500 0.000181 H -0.165688 2.521432 0.000074 38 Caresian coordinates (in Å) of the ground state singlet equilibrium structure of anion from 3a-H. C -2.393936 -1.364772 0.000058 C -1.118360 -0.757369 -0.000050 C -1.024555 0.660755 -0.000197 C -2.217611 1.415235 -0.000165 C -3.453716 0.811129 -0.000040 C -3.547311 -0.619010 0.000065 H -2.437250 -2.446387 0.000163 H -2.135726 2.494453 -0.000216 C 1.163505 -0.916327 -0.000078 C 1.252000 0.514008 -0.000114 N 0.180463 1.281488 -0.000235 C 2.433219 -1.711304 -0.000070 C 3.673464 0.316231 0.000144 N 2.492200 1.100441 -0.000026 N 3.595062 -1.034007 0.000060 O 2.388693 -2.953290 -0.000196 O 4.761423 0.908508 0.000364 C -4.702737 1.648341 0.000049 H -5.320590 1.439306 0.876000 H -4.462471 2.709507 -0.000107 H -5.320857 1.439087 -0.875659 C -4.892162 -1.293370 0.000249 H -5.480390 -1.012198 0.876479 H -5.480536 -1.012353 -0.875937 H -4.783340 -2.376152 0.000336 N 0.001530 -1.520475 -0.000025 C 2.581433 2.558369 0.000014 H 2.092853 2.968447 -0.882762 H 2.092597 2.968459 0.882628 H 3.630617 2.822452 0.000135 Caresian coordinates (in Å) of the ground state singlet equilibrium structure of 3a-H. C -2.426442 -1.375742 0.000019 C -1.149692 -0.767631 0.000008 C -1.054310 0.655895 0.000012 C -2.243519 1.413076 -0.000003 C -3.478027 0.807916 -0.000004 C -3.574381 -0.626114 0.000022 H -2.470527 -2.456708 0.000022 H -2.159706 2.491515 -0.000019 C 1.124996 -0.912201 -0.000005 C 1.219401 0.511923 0.000011 N 0.149909 1.272874 0.000017 C 2.360674 -1.726148 -0.000019 C 3.640213 0.417439 0.000002 N 2.462248 1.133423 0.000016 N 3.511175 -0.965172 -0.000011 O 2.405263 -2.942377 -0.000037 O 4.746331 0.930526 0.000000 C -4.725827 1.643457 -0.000069 H -5.342494 1.430902 0.875610 H -4.487242 2.704558 -0.000042 H -5.342360 1.430927 -0.875855 39 C -4.920780 -1.294023 0.000043 H -5.505952 -1.007956 0.876369 H -5.506051 -1.007858 -0.876182 H -4.817201 -2.376907 -0.000017 N -0.035437 -1.524923 -0.000004 C 2.525556 2.598359 0.000022 H 2.029389 2.989650 -0.884593 H 2.029420 2.989647 0.884658 H 3.569320 2.884016 0.000006 H 4.388220 -1.467613 -0.000018 Caresian coordinates (in Å) of the ground state singlet equilibrium structure of anion from 4a-H. C -2.128338 -1.491367 0.000089 C -0.912857 -0.787373 -0.000035 C -0.935762 0.624298 -0.000102 C -2.173921 1.278947 -0.000092 C -3.365178 0.569203 -0.000022 C -3.348744 -0.849905 0.000119 H -2.073624 -2.571972 0.000127 H -2.215843 2.356720 -0.000131 C 1.380588 -0.832464 -0.000051 C 1.463329 0.620322 -0.000014 N 0.267643 1.304482 -0.000186 C 2.694731 -1.568328 -0.000163 C 3.780797 0.528750 0.000231 N 2.590335 1.268919 0.000197 N 3.818558 -0.839495 -0.000019 O 2.690972 -2.813726 -0.000402 O 4.843824 1.176905 0.000509 C -4.670693 1.313813 -0.000035 H -5.270815 1.057507 0.875650 H -4.511936 2.390033 0.000089 H -5.270635 1.057678 -0.875892 C -4.630016 -1.638549 0.000335 H -5.240021 -1.410679 0.877062 H -5.240541 -1.410471 -0.875950 H -4.427358 -2.707757 0.000183 N 0.263034 -1.486431 -0.000081 C 0.272822 2.769019 -0.000316 H -0.234746 3.142395 -0.888187 H -0.233476 3.142602 0.888217 H 1.303075 3.097989 -0.001066 Caresian coordinates (in Å) of the ground state singlet equilibrium structure of 4a-H. C -2.164315 1.500543 -0.000033 C -0.945907 0.794363 0.000012 C -0.965069 -0.622117 0.000034 C -2.200227 -1.279175 0.000041 C -3.389614 -0.569068 -0.000002 C -3.377886 0.855475 -0.000061 H -2.109267 2.580543 -0.000064 H -2.242700 -2.356271 0.000074 C 1.337257 0.823435 0.000046 40 C 1.427866 -0.622279 0.000000 N 0.238841 -1.300104 0.000050 C 2.616198 1.584694 0.000080 C 3.741024 -0.628160 -0.000098 N 2.556110 -1.297222 -0.000063 N 3.722369 0.777736 -0.000022 O 2.693669 2.801766 0.000174 O 4.830594 -1.190485 -0.000194 C -4.692956 -1.312694 0.000042 H -5.291363 -1.052707 -0.875529 H -4.535778 -2.388640 0.000037 H -5.291284 -1.052689 0.875661 C -4.662366 1.635963 -0.000155 H -5.269547 1.401956 -0.876782 H -5.269580 1.402103 0.876492 H -4.466021 2.705922 -0.000240 N 0.220382 1.485333 0.000037 C 0.238136 -2.769493 0.000116 H -0.272489 -3.132676 0.889258 H -0.272159 -3.132768 -0.889181 H 1.264455 -3.107327 0.000330 H 4.624870 1.233202 -0.000039 41 S10. References 1. Gaussian 16, Revision C.01, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, G. A. Petersson, H. Nakatsuji, X. Li, M. Caricato, A. V. Marenich, J. Bloino, B. G. Janesko, R. Gomperts, B. Mennucci, H. P. Hratchian, J. V. Ortiz, A. F. Izmaylov, J. L. Sonnenberg, D. Williams-Young, F. Ding, F. Lipparini, F. Egidi, J. Goings, B. Peng, A. Petrone, T. Henderson, D. Ranasinghe, V. G. Zakrzewski, J. Gao, N. Rega, G. Zheng, W. Liang, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, K. Throssell, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. J. Bearpark, J. J. Heyd, E. N. Brothers, K. N. Kudin, V. N. Staroverov, T. A. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. P. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, J. M. Millam, M. Klene, C. Adamo, R. Cammi, J. W. Ochterski, R. L. Martin, K. Morokuma, O. Farkas, J. B. Foresman, and D. J. Fox, Gaussian, Inc., Wallingford CT, 2016. 2. P. Chattopadhyay, R. Nagpal and P. S. Pandey, Aust. J. Chem., 2008, 61, 216-222. 3. R. Kuhn, K. Reinemund and F. Weygand, Ber. Deutsch. Chem. Ges., 1934, 67, 1460-1462. 4. F. Müller and K. H. Dudley, Helv. Chim. Acta, 1971, 54, 1487-1497. 5. I. Jhulki, P. K. Chanani, S. H. Abdelwahed and T. P. Begley, Journal of the American Chemical Society, 2016, 138, 8324-8327. 6. S. Chen and F. W. Foss, Org. Lett., 2012, 14, 5150-5153.