CHEMICAL REGENERATION METHOD OF OXIDIZED COENZYME NAD (P)+

Abstract

It discloses a chemical regeneration method of oxidized coenzyme NAD(P).sup.+ which is under an oxygen or air atmosphere condition, adding a catalytic amount of bridged flavin, and oxidizing NAD(P)H to obtain NAD(P).sup.+. The catalyst for regeneration of cofactor is cheap and easily available small organic molecule having no noble metal; this regeneration system can regenerate NADH and NADPH; this regeneration system has a wide pH range and temperature range, being applicable to various oxidation reactions catalyzed by nicotinamide-dependent oxidoreductase.

Claims

1. A method for regenerating chemically an oxidized coenzyme NAD(P).sup.+, characterized in that, under an oxygen or air atmosphere condition, adding a catalytic amount of a bridged flavin, and oxidizing NAD(P)H to obtain NAD(P).sup.+.

2. The method according to claim 1, characterized in that, the bridged flavin has a formula as follow: ##STR00007## wherein, R.sub.1 and R.sub.2 are independently selected from a group consisting of hydrogen, methyl, trifluoromethyl, methoxyl, halogen atom, nitro and amino; R.sub.3 is selected from a group consisting of hydrogen, C1-C5 alkyl, phenyl and benzyl; X.sup. is selected from a group consisting of halide ion, nitrate radical and trifluoromethanesulfonic acid radical.

3. The method according to claim 1, characterized in that, the bridged flavin is 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride, 8-chloro-1,10-ethyleneisoalloxazine chloride, or 1,10-ethyleneisoalloxazine chloride.

4. The method according to claim 1, characterized in that, the mole number of the catalytic amount of the bridged flavin is 0.1-5% of the mole number of the NAD(P)H.

5. The method according to claim 1, characterized in that, the condition for oxidizing NAD(P)H into NAD(P).sup.+ is in a pH 4-10 and at a temperature 30-70 C.

6. The method according to claim 1, characterized in that, the bridged Flavin is coupled with an oxidation reaction catalyzed by a NAD(P).sup.+-dependent oxidoreductase, forming a regeneration circulation system of the coenzyme NAD(P).sup.+.

7. The method according to claim 6, characterized in that, the NAD(P).sup.+-dependent oxidoreductase is one or more enzymes selected from a group consisting of EC1.1.1.X, EC1.2.1.X, EC1.3.1.X, EC1.4.1.X, EC1.5.1.X, EC1.6.1.X, EC1.7.1.X, EC1.8.1.X, EC1.10.1.X, EC1.12.1.X, EC1.13.1.X, EC1.16.1.X, EC1.17.1.X, EC1.18.1.X, EC1.20.1.X and EC1.22.1.X.

8. The method according to claim 7, characterized in that, the NAD(P).sup.+-dependent oxidoreductase is horse liver alcohol dehydrogenase, glucose dehydrogenase or glycerol dehydrogenase.

9. A process for an oxidation reaction by using a NAD(P).sup.+-dependent oxidoreductase as a catalyst, characterized in that, using a bridged flavin as a NAD(P).sup.+ regeneration catalyst, under oxygen or air atmosphere condition, oxidizing NAD(P)H into NAD(P).sup.+, forming a regeneration circulation system of coenzyme NAD(P).sup.+.

10. The process according to claim 9, characterized in that, the bridged flavin has a formula as follow: ##STR00008## wherein, R.sub.1 and R.sub.2 are independently selected from a group consisting of hydrogen, methyl, trifluoromethyl, methoxyl, halogen atom, nitro and amino; R.sub.3 is selected from a group consisting of hydrogen, C1-C5 alkyl, phenyl and benzyl; X.sup. is selected from a group consisting of halide ion, nitrate radical and trifluoromethanesulfonic acid radical.

11. The process according to claim 10, characterized in that, the bridged flavin is 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride, 8-chloro-1,10-ethyleneisoalloxazine chloride, or 1,10-ethyleneisoalloxazine chloride.

12. The process according to claim 9, characterized in that, the mole number catalyzed by the bridged flavin is 0.1-5% of the mole number of a substrate catalyzed by an enzyme.

13. The process according to claim 9, characterized in that, the condition for oxidizing NAD(P)H into NAD(P).sup.+ is in pH 4-10 and at temperature 30-70 C.

14. The process according to claim 9, characterized in that, the NAD(P).sup.+-dependent oxidoreductase is one or more enzymes selected from a group consisting of EC1.1.1.X, EC1.2.1.X, EC1.3.1.X, EC1.4.1.X, EC1.5.1.X, EC1.6.1.X, EC1.7.1.X, EC1.8.1.X, EC1.10.1.X, EC1.12.1.X, EC1.13.1.X, EC1.16.1.X, EC1.17.1.X, EC1.18.1.X, EC1.20.1.X and EC1.22.1.X.

15. The process according to claim 14, characterized in that, the NAD(P).sup.+-dependent oxidoreductase is horse liver alcohol dehydrogenase, glucose dehydrogenase or glycerol dehydrogenase.

16. A method for using a bridged flavin as a catalyst for oxidizing NAD(P)H into NAD(P).sup.+.

17. The method according to claim 16, characterized in that, the bridged flavin has formula as follow: ##STR00009## wherein, R.sub.1 and R.sub.2 are independently selected from a group consisting of hydrogen, methyl, trifluoromethyl, methoxyl, halogen atom, nitro and amino; R.sub.3 is selected from a group consisting of hydrogen, C1-C5 alkyl, phenyl and benzyl; X.sup. is selected from a group consisting of halide ion, nitrate radical and trifluoromethanesulfonic acid radical.

18. The method according to claim 17, characterized in that, the bridged flavin is 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride, 8-chloro-1,10-ethyleneisoalloxazine chloride, or 1,10-ethyleneisoalloxazine chloride.

19. The method according to claim 16, characterized in that, the mole number catalyzed by the bridged flavin is 0.1-5% of the mole number of NAD(P)H.

20. The method according to claim 16, characterized in that, the condition for oxidizing NAD(P)H into NAD(P).sup.+ is in pH 4-10 and at temperature 30-70 C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 is a synthesis route diagram of 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride;

[0048] FIG. 2 is a route diagram of a coupling reaction of 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride as a NAD.sup.+ regeneration catalyst with horse liver alcohol dehydrogenase and glucose dehydrogenase.

DESCRIPTION OF THE EMBODIMENTS

[0049] Based on the following examples, the present invention can be better understood. However, a person skilled in the art will readily understand that, the contents described in the examples are only used to illustrate the present invention, and should not and will not restrict the present invention described in detail in the claims.

Example 1: Synthesis of 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride

[0050] 4-bromo-3-nitrotrifluorotoluene (1.76 g, 6.53 mmol) and ethanolamine (1.19 g, 19.59 mmol) were dissolved in 20 mL ethanol, potassium carbonate (1.08 g, 7.83 mmol) was added and stirred under reflux for 5-6 hours, after thereaction solution was cooled, filtered, the filtrate was concentrated under reduced pressure, to obtain an orange solid, and 20 mL saturated brine was added, extracted with ethyl acetate (340 mL), the organic phases were combined, washed with a saturated brine, dried on anhydrous sodium sulfate, rotary evaporated, purified by silica gel column chromatography to obtain 4-trifluoromethyl-2-nitro-N-(2-hydroxyethyl)aniline (1.58 g, yield 96%).

[0051] To an anhydrous methanol solution of 4-trifluoromethyl-2-nitro-N-(2-hydroxyethyl)aniline (1.20 g, 4.80 mmol) (5 C.), ammonium formate (1.51 g, 24.0 mmol) and 5% Pd/C (1.60 g) were added, stirred and reacted at 0 to 25 C. for 1 hour, filtered, the filter residue was washed with methanol, the filtrate was concentrated under reduced pressure, producing a red solid, 30 mL saturated brine was added, extracted with ethyl acetate (330 mL), the organic phases were combined, dried on anhydrous sodium sulfate, filtered, concentrated under reduced pressure, to obtain a white solid (1.04 g, 94%).

[0052] To a 50 C. acetic acid solution of 4-trifluoromethyl-N-(2-hydroxyethyl)-o-phenylenediamine (1 g, 4.35 mmol) filled with argon, alloxan monohydrate (0.73 g, 4.35 mmol) and boric acid (0.29 g, 4.67 mmol) were added, reacted for 1 hour, cooled, the obtained yellow solid was filtered, washed with dichloromethane, vacuum dried, the obtained yellow solid was immediately placed into a round bottom flask filled with nitrogen gas, and thionyl chloride (20 mL) was slowly added meanwhile vigorously stirred, reacted for 16 hours under 50 C. nitrogen gas shielding, then cooled and filtered, the obtained solid was washed with dichloromethane, the crude product was dissolved in a minimal amount of 98% formic acid solution, and recrystallized with diethyl ether, to obtain a yellow solid (1.28 g, 95%). ESI-MS.sup.+: 309; .sup.1H NMR (CD.sub.3COOD-CF.sub.3COOH, 1:6) 4.96 (t, J=8.9 Hz, 2H), 5.58 (t, J=8.9 Hz, 2H), 8.20 (d, J=8.8 Hz, 1H), 8.47 (d, J=8.8 Hz, 1H), 8.81 (s, 1H), .sup.13C NMR (CD3COOD-CF.sub.3COOH, 1:6) 46.2, 52.1, 122.6 (q, J=272.6 Hz, CF.sub.3), 118.9, 131.4, 131.7, 133.9, 135.4 (q, J=35.5 Hz, C CF.sub.3), 136.8, 141.1, 145.2, 146.4, 158.8.

Example 2: Synthesis of 1,10-ethyleneisoalloxazine chloride

[0053] 2-fluoronitrobenzene (0.92 g, 6.53 mmol) and ethanolamine (1.19 g, 19.59 mmol) were dissolved in 20 mL ethanol, potassium carbonate (1.08 g, 7.83 mmol) was added and stirred under reflux for 5-6 hours, after the reaction solution was cooled, filtered, the filtrate was concentrated under reduced pressure, to obtain an orange solid, and 20 mL saturated brine was added, extracted with ethyl acetate (340 mL), the organic phases were combined, washed with a saturated brine, dried on anhydrous sodium sulfate, rotary evaporated, purified by silica gel column chromatography to obtain 2-nitro-N-(2-hydroxyethyl)aniline (0.98 g, yield 82.9%).

[0054] To a anhydrous methanol solution (5 C.) of 2-nitro-N-(2-hydroxyethyl)aniline (0.87 g, 4.80 mmol), ammonium formate (1.51 g, 24.0 mmol) and 5% Pd/C (1.60 g) were added, stirred and reacted at 0-25 C. for 1 hour, filtered, the filter residue was washed with methanol, the filtrate was concentrated under reduced pressure, producing a red solid, and 30 mL saturated brine was added, extracted with ethyl acetate (330 mL), the organic phases were combined, dried on anhydrous sodium sulfate, filtered, concentrated under reduced pressure, to obtain a white solid (0.67 g, 92%).

[0055] In a 50 C. acetic acid solution of N-(2-hydroxyethyl)-o-phenylenediamine (0.66 g, 4.35 mmol) filled with argon, alloxan monohydrate (0.73 g, 4.35 mmol) and boric acid (0.29 g, 4.67 mmol) were added, reacted for 1 hour, cooled, the resulting yellow solid was filtered, washed with dichloromethane, vacuum dried, the yellow solid obtained was immediately placed into a round bottom flask filled with nitrogen gas, and thionyl chloride (20 mL) was slowly added meanwhile vigorously stirred, reacted for 16 hours under 50 C. nitrogen gas shielding, then cooled and filtered, the solid obtained was washed with dichloromethane, the crude product was dissolved in a minimal amount of 98% formic acid solution, and recrystallized with diethyl ether, to obtain a yellow solid (0.88 g, 73%). ESI-MS+: 240.8, .sup.1H NMR (CD3COOD-CF.sub.3COOH, 1:6) 4.94 (t, J=8.9 Hz, 2H), 5.53 (t, J=8.9 Hz, 2H), 8.04-8.57 (4H); .sup.13C NMR (CD3COOD-CF.sub.3COOH, 1:6) 45.9, 51.9, 117.2, 130.3, 131.9, 133.2, 134.2, 142.1, 142.2, 144.1, 146.7, 159.4.

Example 3: Synthesis of 8-chloro-1,10-ethyleneisoalloxazine chloride

[0056] 2,4-dichloronitrobenzene (1.25 g, 6.53 mmol) and ethanolamine (1.19 g, 19.59 mmol) were dissolved in 20 mL ethanol, potassium carbonate (1.08 g, 7.83 mmol) was added and stirred under reflux for 5-6 hours, after the reaction solution was cooled, filtered, the filtrate was concentrated under reduced pressure, to obtain an orange solid, and 20 mL saturated brine was added, extracted with ethyl acetate (340 mL), the organic phases were combined, washed with saturated brine, dried on anhydrous sodium sulfate, rotary evaporated, purified by silica gel column chromatography to obtain 5-chloro-2-nitro-N-(2-hydroxyethyl)aniline (1.26 g, yield 89%).

[0057] To a 20 mL water solution (100 C.) containing 5-chloro-2-nitro-N-(2-hydroxyethyl)aniline (1.04 g, 4.80 mmol) and Sn (1.71 g, 14.40 mmol), 9 mL concentrated hydrochloric acid was slowly dripped in, then the reaction was placed at 5 C. and cooled for 30 minutes, neutralized with a 50% NaOH water solution, extracted with ethyl acetate (330 mL), the organic phases were combined, dried on anhydrous sodium sulfate, filtered, concentrated under reduced pressure, to obtain a white solid (0.47 g, 52%).

[0058] To a 50 C. acetic acid solution of 4-trifluoromethyl-N-(2-hydroxyethyl)-o-phenylenediamine (0.37 g, 2.0 mmol) filled with argon, alloxan monohydrate (0.32 g, 2.0 mmol) and boric acid (0.13 g, 2.14 mmol) were added, and reacted for 1 hour, cooled, the solid formed was filtered, washed with dichloromethane, vacuum dried, the solid obtained was immediately placed into a round bottom flask filled with nitrogen gas, and thionyl chloride (10 mL) was slowly added meanwhile stirred vigorously, reacted for 16 hours under 50 C. nitrogen gas shielding, then cooled and filtered, the solid obtained was washed with dichloromethane, the crude product was dissolved in a minimal amount of 98% formic acid solution, and recrystallized with diethyl ether, to obtain a solid (0.43 g, 69%). ESI-MS+: 274.9, .sup.1H NMR (CD3COOD-CF.sub.3COOH, 1:6) 4.92 (t, J=8.9 Hz, 2H), 5.49 (t, J=8.9 Hz, 2H), 8.06 (d, J=8.4 Hz, 2H), 8.50 (d, J=8.4 Hz, 1H); .sup.13C NMR (CD3COOD-CF.sub.3COOH, 1:6) 45.9, 51.9, 117.2, 130.9, 131.8, 134.2, 135.1, 140.6, 144.6, 146.6, 150.1, 159.1.

Example 4: 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride oxidizing NADH to NAD.SUP.+

[0059] At 30 C., in a 10 mL 50 mM potassium phosphate buffer of pH 7, the initial concentration of NADH was 0.2 mM, the concentration of 7-trifluoromethyl 1,10-ethyleneisoalloxazine chloride was 4 M, the reaction solution was connected with the outside air. The change of the light absorption value of the reaction system at 340 nm was detected every 2 minutes, the conversion rate was calculated, and the results were shown in Table 1.

TABLE-US-00001 TABLE 1 Conversion rate of 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride oxidizing NADH with time light absorption value time (min) at 340 nm conversion rate (%) 0 1.244 0.00 1 0.5991 51.84 2 0.2815 77.37 3 0.1425 88.55 4 0.0676 94.57 5 0.0313 97.48 6 0.0137 98.90 7 0.0056 99.55 8 0.002 99.84 9 0 100

Example 5: 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride oxidizing NADPH to NADP.SUP.+

[0060] At 30 C., in a 10 mL 50 mM potassium phosphate buffer of pH 7, the initial concentration of NADPH was 0.2 mM, the concentration of 7-trifluoromethyl 1,10-ethyleneisoalloxazine chloride was 4 M, the reaction solution was connected with the outside air. Change of light absorption value of the reaction system at 340 nm was detected every 2 minutes, the conversion rate was calculated, and the results are shown in Table 2.

TABLE-US-00002 TABLE 2 Conversion rate of 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride oxidizing NADPH with time light absorption value time (min) at 340 nm conversion rate (%) 0 1.244 0.00 1 0.5862 52.88 2 0.204 83.60 3 0.0983 92.10 4 0.0215 98.27 5 0.0098 99.21 6 0.0033 99.73 7 0 100

Example 6: 8-chloro-1,10-ethyleneisoalloxazine chloride oxidizing NADH to NAD.SUP.+

[0061] At 30 C., in a 10 mL 50 mM potassium phosphate buffer of pH 7, the initial concentration of NADH was 0.2 mM, the concentration of 8-chloro-1,10-ethyleneisoalloxazine chloride was 4 M, the reaction solution was connected with the outside air. Change of the light absorption value of the reaction system at 340 nm was detected every 2 minutes, the conversion rate of NADH was calculated, and the results are shown in Table 3.

TABLE-US-00003 TABLE 3 Conversion rate of 8-chloro-1,10-ethyleneisoalloxazine chloride oxidizing NADH with time time (min) light absorption value conversion rate (%) 0 1.244 0.00 1 0.721 42.04 2 0.5612 54.89 3 0.4401 64.62 4 0.3471 72.10 5 0.2751 77.89 6 0.2192 82.38 7 0.1752 85.92 8 0.1406 88.70 9 0.1133 90.89 10 0.092 92.60 11 0.0751 93.96 12 0.0615 95.06 13 0.0506 95.93 14 0.042 96.62 15 0.0352 97.17 16 0.0299 97.60 17 0.0255 97.95 18 0.0219 98.24 19 0.0192 98.46 20 0.0169 98.64

Example 7: 1,10-ethyleneisoalloxazine chloride oxidizing NADH to NAD.SUP.+

[0062] At 30 C., in a 10 mL 50 mM potassium phosphate buffer of pH 7, the initial concentration of NADH was 0.2 mM, the concentration of 1,10-ethyleneisoalloxazine chloride was 4 M, the reaction solution was connected with the outside air. Change of the light absorption value of the reaction system at 340 nm was detected every 2 minutes, the conversion rate of NADH was calculated, and the results are shown in Table 4.

TABLE-US-00004 TABLE 4 Conversion rate with time of 1,10-ethyleneisoalloxazine chloride oxidizing NADH time (min) light absorption value conversion rate (%) 0 1.244 0.00 1 0.7525 39.51 2 0.616 50.48 3 0.5059 59.33 4 0.4166 66.51 5 0.3436 72.38 6 0.2839 77.18 7 0.2354 81.08 8 0.1957 84.27 9 0.163 86.90 10 0.1363 89.04 11 0.1145 90.80 12 0.0964 92.25 13 0.0816 93.44 14 0.0693 94.43 15 0.0591 95.25 16 0.0506 95.93 17 0.0436 96.50 18 0.038 96.95 19 0.0334 97.32 20 0.0295 97.63

Example 8: 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride oxidizing NADH at different pHs

[0063] At 30 C., respectively in 10 mL 50 mM potassium phosphate buffer of pH 4-10, the initial concentration of NADH was 0.2 mM, the concentration of 7-trifluoromethyl 1,10-ethyleneisoalloxazine chloride being added was 0.5 M, the reaction solution was connected with the outside air, after being reacted for 10 minutes, the light absorption value at 340 nm was detected, the concentrations of NADH and NAD.sup.+ were calculated, TON and TOF at different pHs were calculated, and the results are shown in Table 5, when pH<7, TON and TOF were increased with increase in pH, when pH>7, TON and TOF were reduced with increase in pH.

TABLE-US-00005 TABLE 5 TON and TOF of 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride oxidizing NADH at different pHs pH TON TOF (min.sup.1) 4 36.01 3.60 5 38.91 3.89 6 53.06 5.31 7 95.18 9.52 8 72.67 7.27 9 49.84 4.98 10 19.29 1.93

Example 9: 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride oxidizing NADH at different temperatures

[0064] In a 50 mM potassium phosphate buffer of pH 7, the initial concentration of NADH was 0.2 mM, the concentration of 7-trifluoromethyl 1,10-ethyleneisoalloxazine chloride was 0.5 M, the reaction solution was connected with the outside air, after being reacted at 30 C., 40 C., 50 C., 60 C., and 70 C. respectively for 10 minutes, the light absorption value at 340 nm was detected, the concentrations of NADH and NAD+ were calculated, TON and TOF at different temperatures were calculated, and the results are shown in Table 6, when T<40 C., changes of TON and TOF with temperature was not large, when T>40 C., TON and TOF were reduced with increase in temperature, the reason is that solubility of air in water is reduced with increase in temperature.

TABLE-US-00006 TABLE 6 TON and TOF of 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride oxidizing NADH at different pHs T ( C.) TON TOF (min.sup.1) 30 96.78 9.68 40 96.14 9.61 50 56.27 5.63 60 48.23 4.82 70 42.77 4.28

Example 10: Substituted derivative of 1,10-ethyleneisoalloxazine chloride oxidizing NADH to NAD.SUP.+

[0065] At 30 C., in a 50 mM potassium phosphate buffer of pH 7, the initial concentration of NADH was 0.2 mM, the concentrations of 7-trifluoromethyl-3-methyl-1,10-ethyleneisoalloxazine chloride (Compound A), 7-methyl-1,10-ethyleneisoalloxazine chloride (Compound B), 8-methyl-1,10-ethyleneisoalloxazine chloride (Compound C), 7,8-dimethyl-1,10-ethyleneisoalloxazine chloride (Compound D), 3-benzyl-1,10-ethyleneisoalloxazine chloride (Compound E) were 0.5 M, the reaction solution was connected with the outside air, after being reacted for 10 minutes, the light absorption value at 340 nm was detected, the concentrations of NADH and NAD+ were calculated, TON and TOF at different temperatures were calculated, and the results are show in Table 7.

TABLE-US-00007 TABLE 7 TON and TOF of several bridged flavins catalytically oxidizing NADH Compound TON TOF (min.sup.1) A 89.34 8.93 B 70.53 7.05 C 69.98 7.00 D 56.88 5.69 E 75.27 7.53

Example 11: 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride as a catalyst for regenerating NAD.SUP.+ coupled with horse liver alcohol dehydrogenase catalyzing lactonization of 1,4-butanedioloxidize to -butyrolactone

[0066] At 30 C., in a 1 mL 50 mM potassium phosphate buffer (pH8) of 1,4-butanediol 20 mM, NAD.sup.+ 0.1 mM, 7-trifluoromethyl-1,10-ethyleneisoalloxazine 0.05 mM, catalase 50 U/mL, and horse liver alcohol dehydrogenase 20 U/mL, the reaction solution was connected with the outside air. 200 L reaction solution were taken out respectively at 1 h, 2 h, 4 h, 8 h of reaction, and 200 L ethyl acetate was added for extraction, the reaction process was detected by gas chromatography, and the results are shown in Table 8:

TABLE-US-00008 TABLE 8 Conversion rate with reaction time of 7-trifluoromethyl-1,10- ethyleneisoalloxazine chloride coupled with horse liver alcohol dehydrogenase catalyzing 1,4-butanediol time (h) conversion rate (%) 1 38 2 71.18 4 93 8 100

Example 12: 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride as a catalyst for regenerating NAD.SUP.+ coupled with horse liver alcohol dehydrogenase catalyzing oxidization of cyclohexanol to cyclohexanone

[0067] At 30 C., in a 1 mL 50 mM potassium phosphate buffer (pH8) of cyclohexanol 20 mM, NAD.sup.+ 0.1 mM, 7-trifluoromethyl-1,10-ethyleneisoalloxazine 0.05 mM, catalase 50 U/mL, horse liver alcohol dehydrogenase 20 U/mL, the reaction solution was connected with the outside air connect. 200 L reaction solution were taken out respectively at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h of the reaction, 200 L ethyl acetate was added for extraction, the reaction process was detected by gas chromatography, and the results are shown in Table 9:

TABLE-US-00009 TABLE 9 Conversion rate with reaction time of 7-trifluoromethyl-1,10- ethyleneisoalloxazine chloride coupled with horse liver alcohol dehydrogenase catalyzing cyclohexanol time (h) conversion rate (%) 1 31 2 39.23 4 51.36 8 62.3 24 90.72

Example 13: 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride as a catalyst for regenerating NAD.SUP.+ coupled with horse liver alcohol dehydrogenase catalyzing oxidization and lactonization of 3-methyl-1,5-pentanediol to 3-methylyvalerolactone

[0068] At 30 C., in a 1 mL 50 mM potassium phosphate buffer (pH8) of 3-methyl-1,5-pentanediol 20 mM, NAD.sup.+ 0.1 mM, 7-trifluoromethyl-1,10-ethyleneisoalloxazine 0.05 mM, catalase 50 U/mL, horse liver alcohol dehydrogenase 20 U/mL, the reaction solution was connected with the outside air. 200 L reaction solution were taken out respectively at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h of the reaction, 200 L ethyl acetate was added for extraction, the reaction process was detected by gas chromatography, and the results are shown in Table 10.

TABLE-US-00010 TABLE 10 Conversion rate with reaction time of 7-trifluoromethyl-1,10- ethyleneisoalloxazine chloride coupled with horse liver alcohol dehydrogenase catalyzing 3-methyl-1,5-pentanediol time (h) conversion rate (%) 1 59.83 2 71.18 4 82.76 8 93.77 12 98.64 24 100

Example 14: 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride as a catalyst for regeneration NAD.SUP.+ coupled with glucose dehydrogenase catalytically oxidizing glucose to gluconic acid

[0069] At 30 C., in a 10 mL reaction system of glucose 25 mM, NAD.sup.+0.5 mM, 7-trifluoromethyl-1,10-ethyleneisoalloxazine 0.5 mM, NaCl 50 mM, catalase 50 U/mL, glucose dehydrogenase 120 U/mol (glucose), the reaction solution was connected with the outside air. 200 L reaction solution were taken out respectively at 1 h, 2 h, 4 h, 6 h of the reaction, 800 L water was added for dilution, the reaction process was detected by liquid chromatography, and the results are shown in Table 11.

TABLE-US-00011 TABLE 11 Conversion rate with reaction time of 7-trifluoromethyl-1,10- ethyleneisoalloxazine chloride coupled with glucose dehydrogenase catalyzing glucose time (h) conversion rate (%) 1 38.1 2 55.0 4 89.6 6 100

Example 15: 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride as a catalyst for regenerating NAD.SUP.+ coupled with glucose dehydrogenase catalytically oxidizing xylose to xylonic acid

[0070] At 30 C., in a 10 mL reaction system of xylose 25 mM, NAD.sup.+0.5 mM, 7-trifluoromethyl-1,10-ethyleneisoalloxazine 0.5 mM, NaCl 50 mM, catalase 50 U/mL, glucose dehydrogenase 120 U/mol (xylose), the reaction solution was connected with the outside air. 200 L reaction solution were taken out respectively at 1 h, 2 h, 4 h, 6 h, 12 h, 24 h of the reaction, 800 L water was added for dilution, the reaction process was detected by liquid chromatography, and the results are shown in Table 12.

TABLE-US-00012 TABLE 12 Conversion rate with reaction time of 7-trifluoromethyl-1,10- ethyleneisoalloxazine chloride coupled with glucose dehydrogenase catalyzing xylose conversion rate time (h) (%) 1 17.5 2 32.3 4 52.1 6 65.9 8 76.8 12 82.9 24 100

Example 16: 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride as a catalyst for regenerating NAD.SUP.+ coupled with glycerol dehydrogenase catalytically oxidizing glycerol to 1,3-dihydroxyacetone

[0071] At 30 C., in 10 mL reaction system (potassium phosphate buffer of 50 mM and pH 10) of glycerol 25 mM, NAD.sup.+ 0.5 mM, 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride 0.5 mM, atalase 50 U/mL, glycerol dehydrogenase 120 U/mol, the reaction solution was connected with the outside air. 200 L reaction solution were taken out respectively at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h of the reaction, 800 L of water was added for dilution, the reaction process detected by liquid chromatography, and the results are shown in Table 13.

TABLE-US-00013 TABLE 13 Conversion rate with reaction time of 7-trifluoromethyl-1,10- ethyleneisoalloxazine chloride coupled with glycerol dehydrogenase catalyzing glycerol conversion rate time (h) (%) 1 235 2 56.3 4 51.7 8 72.6 12 86.5 24 100