Regiospecific C-H amination of (-)-limonene into (-)-perillamine by multi-enzymatic cascade reactions

Abstract

Background: (-)-Limonene, one of cyclic monoterpenes, is an important renewable compound used widely as a key building block for the synthesis of new biologically active molecules and fine chemicals. (-)-Perillamine, as derived from (-)-limonene, is a highly useful synthon for constructing more complicated and functionally relevant chemicals.

Aim: We aimed to report a more sustainable and more efficient method for the regiospecific C-H amination of (-)-limonene into (-)-perillamine.

Results: Here, we report an artificial penta-enzymatic cascade system for the transformation of the cheap and easily available (-)-limonene into (-)-perillamine for the first time. This system is composed of cytochrome P450 monooxygenase, alcohol dehydrogenase and w-transaminase for the main reactions, as well as formate dehydrogenase and NADH oxidase for cofactor recycling. After optimization of the multi-enzymatic cascade system, 10 mM (-)-limonene was smoothly converted into 5.4 mM (-)-perillamine in a one-pot two-step biotransformation, indicating the feasibility of multi-enzymatic C7-regiospecific amination of the inert C-H bond of (-)-limonene. This method represents a concise and efficient route for the biocatalytic synthesis of derivatives from similar natural products.

Keywords: (−)-Limonene; (−)-Perillamine; CYP153A7; Multi-enzyme cascade; Regiospecific C–H amination; Transaminase.

Scheme 1

A multi-enzyme cascade designed for the regiospecific bioamination of (−)-limonene into (−)-perillamine. 2-PTAM 2-pentanamine, 2-PTON 2-pentanone

Fig. 1

Optimization of the temperature (a) and pH (b) of Module 2. Reaction conditions (0.5 mL): (−)-Perillyl alcohol 10 mM, LkADH 0.2 U/mL, [LkADH]/[NOX]/[ATA-117] = 1/5/10, 0.2 mM NAD⁺, 0.1 mM PLP, 50 mM 2-pentanamine, potassium phosphate buffer (pH 7.5, 100 mM) or Tris–HCl buffer (pH 8–9, 100 mM) or Gly-NaOH buffer (pH 9.5–10, 100 mM), 30℃, 35 ℃ or 40 ℃, 800 rpm, 12 h. Temp.: temperature; Concn.: concentration

Fig. 2

Optimization of the kind (a) and equivalents (b) of amino donor in Module 2. Reaction conditions (0.5 mL): shaken at 35 °C, 800 rpm for 12 h, KPB buffer (pH 7.5, 100 mM). The reaction mixture (0.5 mL) was composed of 10 mM (−)-perillyl alcohol (with 2% DMSO), 0.2 U/mL LkADH, 0.4 U/mL SmNOX, 2 U/mL ATA-117, 0.2 mM PLP, 0.2 mM NAD⁺, in addition to: a 50 mM DL-Ala/IPA/2-pentanamine; b 10−100 mM 2-pentanamine. IPA isopropanyl alcohol, Concn. Concentration, 2-PTAM 2-pentanamine, equiv. equivalent

Fig. 3

Time course of the 100-mL preparative-scale multi-enzyme cascade reaction. Step I: 10 mM (−)-limonene (with 2% DMSO), 15 g_cdw L⁻¹ A7F resting cells, 0.2 mM NAD⁺, 100 mM sodium formate, 1 mg/mL Triton X-100, KPB buffer (100 mM, pH 7.5), 25 °C, 200 rpm. Step II: supplement of 0.2 U mL⁻¹ LkADH, 1 U mL⁻¹ SmNOX, 2 U mL⁻¹ ATA-117, 80 mM 2-pentanamine, 0.2 mM NAD⁺ and 0.2 mM PLP, incubated at 35 °C and shaken at 200 rpm. Concn.: concentration