Peroxidase-induced C–N bond formation via nitroso ene and Diels–Alder reactions

The formation of new carbon–nitrogen bonds is indisputably one of the most important tasks in synthetic organic chemistry. Here, nitroso compounds offer a highly interesting reactivity that complements traditional amination strategies, allowing for the introduction of nitrogen functionalities via ene-type reactions or Diels–Alder cycloadditions. In this study, we highlight the potential of horseradish peroxidase as biological mediator for the generation of reactive nitroso species under environmentally benign conditions. Exploiting a non-natural peroxidase reactivity, in combination with glucose oxidase as oxygen-activating biocatalyst, aerobic activation of a broad range of N-hydroxycarbamates and hydroxamic acids is achieved. Thus both intra- and intermolecular nitroso-ene as well as nitroso-Diels–Alder reactions are performed with high efficiency. Relying on a commercial and robust enzyme system, the aqueous catalyst solution can be recycled over numerous reaction cycles without significant loss of activity. Overall, this green and scalable C–N bond-forming strategy enables the production of allylic amides and various N-heterocyclic building blocks utilizing only air and glucose as sacrificial reagents.


Enzymatic synthesis & analytical data:
General procedure for the enzyme-mediated nitroso ene reactions To a 10 mM solution of benzyl hydroxycarbamate 3a in 7 mL phosphate buffer (pH 7.0, 100 mM) containing 20 vol% n-heptane was added 100 mg Brij 35, 70 U HRP, 70 U GOx and 5.0 eq. of olefin. The reaction was initiated by adding D-glucose (50 mM) and incubated at room temperature. After full conversion (followed by TLC), 8 mL MeCN was added, and the reaction mixture was extracted 4x with EtOAc. The combined organic phases were dried over Na2SO4, filtered and the solvent was removed under reduced pressure. The crude was purified via column chromatography on silica gel.

Enzyme screening
To a 10 mM solution of benzyl hydroxycarbamate 3a in 7 mL phosphate buffer (pH 7.0, 100 mM) containing 10 vol% EtOAc was added 100 U laccase or the HRP/GOx couple (70 U each). 1.5 eq. of 1,3-cyclohexadiene 10 was added and the reaction was initiated by adding Dglucose (50 mM). After full conversion (followed by TLC), the mixture was extracted 3x with DCM. The organic phase was filtered through a short silica plug and the solvent was removed under reduced pressure. The yield of 11a was determined by quantitative 1 H-NMR with dimethylsulfone as internal standard.  Reaction conditions: 70 U HRP, 14 U GOx and 5 equiv. D-Glucose were dissolved in 6 mL phosphate buffer (0.1 M, pH 7) and a solution of 0.14 mmol 11a in 1 mL EtOAc was added. After 20 h, the mixture was extracted three times with 20 mL EtOAc. The organic phase was filtered through a short silica plug and the solvent was removed under reduced pressure.

Synthesis of cis/trans-19 references
(2E,4Z)-6-((tert-butyldimethylsilyl)oxy)hexa-2,4-dien-1-yl hydroxycarbamate E,Z-18 (21 mg, 0.07 mmol, 1 eq.) was dissolved in 1 mL n-heptane. 1 mL phosphate buffer was added, and the mixture was cooled to 0 °C followed by the addition of sodium periodate (22.6 mg, 0.106 mmol, 1.45 eq.). The mixture was stirred vigorously at 0 °C for 10 min. The reaction was allowed to warm to room temperature and stirred for 1 h. The phases were separated, and the aqueous phase was extracted 4x with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4 and the solvent was removed under reduced pressure. The crude was purified via column chromatography

Enzymatic synthesis & analytical data
General procedure for the enzyme mediated nitroso Diels Alder reactions To a 10 mM solution of hydroxycarbamate in 7 mL phosphate buffer (pH 7.0, 100 mM) containing 20 vol% EtOAc was added 70 U HRP, 14 U GOx and 3.0 eq. of diene. The reaction was initiated by adding D-glucose (50 mM) and incubated at room temperature. After full conversion (followed by TLC), the reaction mixture was extracted 3x with EtOAc. The combined organic phases were dried over Na2SO4, filtered and the solvent was removed under reduced pressure. The crude was purified via column chromatography on silica gel.

17)
According to the general procedure benzyl hydroxycarbamate 3a and sorbic alcohol E,E-16 were reacted. Full conversion was observed after 2 h. The crude was purified via column chromatography on silica gel (6:1 Ten parallel reactions were performed. To a 10 mM solution of hydroxycarbamate E,Z-18 in 18 mL phosphate buffer (pH 7.0, 100 mM) containing 5.1 mL n-heptane, was added 180 U HRP and 180 U GOx. The reaction was initiated by adding D-glucose (50 mM) and incubated at 25 °C. Full conversion was reached after 1.5 h and all ten reactions were combined for the work-up. The reaction mixture was extracted 3x with EtOAc. The combined organic phases were dried over Na2SO4, filtered and the solvent was removed under reduced pressure. The crude was purified via column chromatography on silica gel