Chemoenzymatic Cascade Synthesis of Phenol Diarylamine Using Non-Heme Diiron N-Oxygenase

doi:10.21203/rs.3.rs-1970030/v1

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Chemoenzymatic Cascade Synthesis of Phenol Diarylamine Using Non-Heme Diiron N-Oxygenase

https://doi.org/10.21203/rs.3.rs-1970030/v1

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Diarylamines are important moieties in organic materials and bioactive molecules. The traditional synthetic approach to diarylamines is metal catalysis in organic solvents. Herein, we report the chemoenzymatic cascade synthesis of phenol diarylamines. Non-heme diiron N-oxygenase AzoC, which catalyzes the oxidization of amines to nitroso groups for azoxymycins biosynthesis in Streptomyces chattanoogensis, is engineered as a catalyst. This synthetic method bears a broad range of phenol and aniline substrates. Mechanistic study indicates that the enzymatically formed nitroso intermediate couple to phenoxide facilitates the non-enzymatic diarylamine synthesis. Our study suggests that diarylamines can be enzymatic synthesized in aqueous solutions and highlights the synthetic potential of non-heme diiron N-oxygenases.

Diarylamines

Biocascade

Enzymatic Synthesis

Non-heme diiron N-oxygenase

Phenols

Enzymes are ideal catalysts for green and sustainable chemistry. However, the natural catalytic activity of an enzyme is limited owing to its conformational specificity and in vivo biochemical environments.^1-3 Several methods, such as directed evolution^4-6, cofactor modification^7-9, and artificial enzyme design^10-11, have been developed to expand the catalytic repertoire of enzymes to afford new-to-nature reactions, bio-functions, and chemicals. Apart from these methods, artificial cascade reaction is also a powerful tool for enzyme activity expansion.^12-13 In this study, we designed a chemoenzymatic cascade reaction for the biosynthesis of phenol diarylamines using an engineered non-heme diiron N-oxygenase (AzoC-II, Figures 1D).

Introducing C-N bonds into arenes is a fundamental synthetic strategy for many valuable chemicals.^14-16 Diarylamines, in which two aryl rings are attached by a C–N–C bond, are versatile building blocks in pharmaceuticals, agrochemicals, dyes, and industrial materials (Figure 1A).^17-26 Besides, diarylamines are useful intermediates in the synthesis of carbazoles,²⁷ acridines,²⁸ indoles, dibenzazapines, etc.²⁹ Traditionally, diarylamines are synthesized via the oxidative C-N coupling of amines with aryl halides or aryl boronic acids in organic solvents using expensive metal catalysts at high temperatures (Figure 1B).^30-32Representative reactions for diamine synthesis include the Buchwald-Hartwig coupling and Cham-Lam cross-coupling reactions. Recently, electrochemical ³³ and metal-free diamine synthetic methods ³⁴ have also been reported. The direct conversion of aryl C-H to C-N is probably the most convenient method, but at the same time, remains a critical challenge in chemical synthesis. In this regard, enzymatic C-N bond formation for diarylamine synthesis, which has not been reported yet, might be a green and feasible solution.

Non-heme diiron N-oxygenase AzoC has been reported to catalyze the direct four-electron oxidation of aromatic amines to their nitroso analogs during azoxymycins biosynthesis in Streptomyces chattanoogensis (Figure 1C).^35-36Aromatic nitroso compounds are active intermediates in C-N and N-N bond synthesis.^37-42 During C-N bond formation, aromatic nitroso groups often serve as electrophiles;³⁴ while electron-rich benzenes such as phenol derivatives serve as nucleophiles; meanwhile, the reactivities are significantly influenced by the reaction conditions.⁴³ Hence, we interrogated whether the aromatic nitroso-producing activity of AzoC could be used for C-N bond construction in diarylamine synthesis by manipulating the reaction conditions.

Reaction design and optimization

We initiated our study using 2-naphthol (2) as a nucleophile, and p-aminobenzoic acid (1) as the amine source (Figure 2A). We first questioned whether there is a possibility of halting the activity of AzoC at the nitroso-producing step, which might be cascaded with electron-rich phenol compounds for the offspring reactions. Therefore, we expressed and purified AzoC, and performed the nitroso generating reaction and control reactions. With glucose dehydrogenase (GDH, supplementary methods) as the NADH regenerating system (to replace the 1 mM NADH usage of the source literature) and phenazine methosulfate (PMS) as an electron mediator to regenerate the iron center of diiron oxygenase (Figure 4B)⁴⁴, nitroso intermediate 3 was formed as the primary product (Figure 2B, i and ii). Interestingly, when 2 was added to the reaction system, a new peak with a retention time of 15 min was observed (Figure 2B, iii). The incubation of pro-boiled AzoC with 1 and 2 showed no production of 3, 4 and 5 (Figure 2B, iv). We then performed a scale-up reaction and isolated 5. NMR and single-crystal X-ray diffraction data revealed that 5 is a phenol-diarylamine product (Figure 2C). According to the literature, 5 is an efficient inhibitor of HIV integrase ⁴⁵ and amyloid aggregation⁴⁶.

We then optimized the reaction condition. Temperature, additive, pH, and buffer solution were analyzed on the yield of 5 (Table 1, entries 1–15, the yields were first determined using HPLC, and the best yield was confirmed by column isolation). The yield of 5 increased from 36% (Table 1, entry 2) to 82% (Table 1, entry 7) when the reaction was performed at 25 °C in PBS buffer (pH 8) for 1 h. No 5 was observed at pH values above 11 or below 6; thus, pH is regarded as the crucial factor in this reaction. Under this reaction system, 4 was also isolated. According to a previous study, 4 was non-enzymatically dimerized from 3 in a radical manner.³⁶ The non-enzymatic diarylamine and the azoxy synthesis are competing in the consumption of 3 (Figure S3). Hence, 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), a radical scavenger and polymerization inhibitor, was used to block the azoxy synthetic shunt and to improve the yield of 5. Indeed, the yield of 5 increased to 97% when 10 mol% of TEMPO was added to the reaction system (Table 1, entry 13).

Substrate scope

With the optimal reaction condition, we then tested the aniline substrate scope. We first tested the p-carboxy substituted aniline substrates, and found all of them were converted to diarylamine products with good to excellent yield (Figure 3, 5 and 5-1a to 5a-1e). We then tested p- substituted trifluoromethyl (strong electron-withdrawing group) and methoxy (strong electron-donating group) substrates, and obtained the diarylamine products 5-1f and 5-1g in good yields (Figure 3). However, the m- and o- sites substituted aniline substrates, are not accepted by AzoC. We then performed protein engineering to expand the substrate scope. According to the crystal structure of non-heme diiron N-oxygenase AurF (PDB ID: 3-CHT)⁴⁷, which shows a broader substrate scope and 39% identity to AzoC, the Y93, V97, T100, N200 and R302 residues are involved in substrate binding (Figure S2). Hence, we built a mutant library on the corresponding Y94, T98, L101, N203, Q304 residues of AzoC, and tested the activity against p-, m- and o- sites substituted aniline substrates. A mutant: AzoC-T98V-L101T (AzoC-II), which recognizes both the p- and m- substituted substrates, was obtained (Figure 3, 5, 5-1a to 5-1i). The bearing of broad electron inductive substitution on the p- and m- sites by AzoC-II suggest wide compatibility of the aniline substrates.

We then studied the phenol substrate scope using AzoC-II as the catalyst. We first investigated the inductive influence of 2 on this reaction. The strong electron-withdrawing bromine group- and the strong electron-donating methoxy group-modified analogs of 2 were compatible with the reaction, and good to excellent yields were obtained (Figure 3, 5a-5d). The yield of the bromine-modified substrates was lower than that of the methoxy-modified substrates, implying that the electronic state of phenols has a potential influence on this reaction. We then questioned whether other phenols were tolerable to this enzymatic diamine coupling reaction. We tested 1-naphthol, and 6 (CCDC 2124158) was successfully isolated in excellent yield. We further tested other bicyclic phenols. Quinolinol substrates 8-hydroxyquinoline and 6-hydroxyisoquinoline were compatible under our reaction conditions, and respective products 7 (CCDC 2124159) and 8 were generated in good yields. For the case of indole substrates as 4-hydroxyindole and 5-hydroxyindole, the reaction solutions turned red. However, unidentified insoluble sediments were generated instead of the diamine products 10 and 11, which might be caused by the active secondary amine on indole’s pyrrole ring. We thus used the N-Boc-protected 4-hydroxyindole as the phenol substrate, most of which was unconverted, and 10 was isolated in 23% yield (Boc is a deactivating group of phenol and was found deprotected during the silicon isolation), which indicates a compatibility of the indole as the phenol aromatic source. Single-ring phenols such as hydroquinone, 4-(dimethylamino)phenol, and phenol were also tested. Hydroquinone diamine product 9 was successfully isolated in good yield. For the case of 4-(dimethylamino)phenol and phenol, instead of isolating the expected diamines 12 and 13, only nitroso product 3 and azoxy product 4 were generated when the reactions time were prolonged up to 12 h. Since the electronic state of phenols influences the reaction, we presume that the relatively low electron density on the benzene ring hindered the formation of 12 and 13.

Mechanism studies

We then investigated the reaction mechanism. Previous study suggests that NADH can mediate the transformation of aromatic nitroso compound into its hydroxylamine analog, both of which are active nitrogen sources. ³⁶ Therefore, we first determined the actual nitrogen source for the diamine product. Commercially available nitrosobenzene and phenylhydroxylamine (1f and 1g in Figure 4A) were used to conduct non-enzymatic reactions (1f and 1g were incubated respectively with 50 μM 2, 1 mM NADH, and 5 μM TEMPO in 20 mM pH 8 PBS buffer for 1 h at 25 °C), and 5f was isolated in 98% yield (1f as nitrogen source) and 11% yield (1g as nitrogen source), indicating that the nitroso compound is the authentic nitrogen source. A radical reaction mechanism for the non-enzymatic nitroso-phenol coupling reaction could be ruled out because adding a radical scavenger improved the yield of 5 (Table 1). Considering that pH is a crucial factor in our reaction system, we then focused on the ionic reaction mechanism. The nitrogen atom of nitroso group is partly positively charged. Thus, phenols have two possible routes: either the electron-rich aromatic ring or the phenoxide negative ion serves as the negative charge center. We tested other electron-rich ring systems, such as naphthalene, 2-methoxynaphthalene, and anthracene; and no products other than nitroso and azoxy compounds were formed. Thus, the first possibility could be ruled out. While alkaline buffers facilitated the diarylamine formation (Table 1 and Figure S4), the phenoxide ion mechanism seems more reliable. The NADH produced by GDH system might serve as the reducing agent in N-O to N-H reduction. To confirm this, we synthesized 3 ⁴⁸ and conducted the non-enzymatic reaction in PBS buffer (pH 8) without using GDH or NADH. The yield of 5 was significantly reduced to 41%; fortunately, a new product 5’ was isolated in 11% yield (Figure 4A). Based on these studies and the previous enzymatic mechanism study, we proposed a mechanism in Figure 4B.^14b In the enzymatic process, the GDH system provides the persistent NADH to propel the cycling between PMS and reduced-PMS; then, the reduced PMS serves as a chemical electron mediator⁴⁴ to regenerate the active center of AzoC; and the cycled iron center of AzoC recruit oxygen and catalyzes the oxidation of aniline to nitroso intermediate in a cycling manner. In the non-enzymatic process, the partially positively charged nitroso nitrogen atom couples with the negatively charged carbon center of phenoxide ions to form C-N bonds, followed by the reduction of the N-O bond to N-H bond by NADH to afford the diarylamine product (Figure 4B). This mechanism study also reviews an efficient non-enzymatic synthetic route to diarylamine in aqueous solution with aromatic nitroso compound and phenol as substrates.

In this study, we expanded the biosynthetic capacity of non-heme diiron N-oxygenase for diamine synthesis by artificial cascade design. Traditionally, diarylamine syntheses are studied in the field of metal-organic chemistry, most of which involve multi-step reactions or reactions requiring volatile organic solvents. In this chemoenzymatic cascade reaction, the phenol hydroxyl group serves as both the directing and the aromatic ring-activating group. N-oxygenase activates amine to the active nitroso intermediate. The reaction proceeds well with a broad range of anillin and phenol substrates. Our study not only provides a one-step, atom-economic, green, and safe alternative for the synthesis of phenol diarylamines, but also demonstrates that the synthetic application of enzymes could be expanded by artificial cascade design.

General Experiment procedures: Unless otherwise noted, chemical reagents were purchased commercially and used as received (Sigma-Aldrich, Macklin Lab, and Aladdin Bio-Chem Technology). GDH was purchased from SyncoZymes Co., Ltd (Shanghai). Protein marker ，Bradford protein concentration quantification kit ， PCR mixture and dpnI enzyme were purchased from Yeasen Biotech (Shanghai). ¹H- and ¹³C- NMR spectra were recorded on a Bruker UltraShield 400 MHz and 600 MHz spectrometers. High resolution mass spectra were obtained using Bruker UHR ES TOF MS. Single crystal X-ray diffraction data was collected on SuperNova diffractometer.

AzoC (GenBank Accession Code: AKQ24642.1) sequence:

MSSRAPEELTGVQSPELPAYDPDDQAENAVIARLAGNWHRRAAVKREEPNLADLFELARDDY
PERILPFRDHPTFRALPPEDRARLLSWAWISYNRTTVLLEGQIVNPAFQLGLDGEFPQPVSELM
QRSLAQAMVDEQYHTLMHLNASAVTRRRRGEAFADAALPKPLVVREHEARLASCANERERR
LTTLAFATVAEISINAYLNLIADDKEIQPVNSATVRIHNRDEYCHASISAVLAEQVHHTLDDGE
RRYFLQSLVAGLEAFVGNDFMAWHRIMDEAGIRGGHEMLDDIQHAGGRKRLVQDFSGLRKL
VERLDAVDDLDFDWSRSVTGSDAVSPTR

Protein expression and purification: Escherichia coli BL21(DE3) containing the AzoC expressing plasmid pET28a-azoC was incubated into 1 L of LB liquid culture with the supplementation of with 1mM ammonium ferric sulfate and 50 μg/mL kanamycin and further cultivated at 37 °C, 220 r.p.m for 5 h until the OD 600 reached 0.6. Then, the culture was added with 0.1 mM isopropyl β-D-1-thiogalactopyranoside and further inducted at 25 °C for 24 h. Cells were collected and lysed with sonication in buffer A (20 mM Tris, pH 8.0, 250 mM NaCl, and 10 mM imidazole). Then, the cell lysate was centrifuged at 14000 rpm for 15 min, and the supernatant was collected and referred to Ni2+-nitrilotriacetic acid resin column for protein purification. After loading the sample, the Ni2+-nitrilotriacetic acid resin column was first eluted with buffer A solution containing 50mM imidazole to remove the undesired proteins, then eluted with buffer A solution containing 250mM imidazole to get the target enzyme. The recombinant protein was desalted on a 10-kDa YM-3 column with 20mM Tris buffer (pH 7.5). The resulting enzyme was analyzed with SDS-PAGE. Protein concentration was determined with the Bradford assay, and bovine serum albumin was employed as standard.

HPLC analysis: HPLC analysis was performed on a Thermal Ultimate 3000 system with a VWD Uv detector. A Thermal Hypersil Gold column (250*4.6 mm, 5 μm) was used. Mobile phase A was 0.1% trifluoroacetic acid (TFA) in water, and mobile phase B was acetonitrile. The flow rate was 1 mL/min. During the analysis, mobile phase B rose from 5% to 100% in 30 minutes. All reaction sample was mixed with an equal volume of methanol and filtrated with a 0.45 μm filter membrane (organic phase).

Protein engineering method: Mutation experiments were conducted by whole plasmid PCR using Pfu DNA polymerase. The plasmid pET28a-AzoC was PCR amplified with site saturation mutagenesis primers. The resulting PCR products were digested with DpnI to eliminate the template plasmid. Furthermore, 10 μL of digested products were transformed into E.coli BL21(DE3) for the single colony isolation on the kanamycin LB plate. And colonies grown on the kanamycin plate were further tested for the enzyme reactions. The positive mutant was referred to DNA sequencing to identify the mutant site. For AzoC II, primer F(cgatctgcccctcggtcagcaccacggtgcggttgtacg) and primer R (cgtacaaccgcaccgtggtgctgaccgaggggcagatcg) were used for the construction of the mutant.

GDH Stock solution: NAD⁺ (5 mg/mL), Glucose (180 mg/mL), GDH (10 mg/mL) dissolved in PBS buffer (pH 8). For AzoC enzymatic reaction, 0.1% v/v GDH stock solution can be used to replace the NADH.

Scale-up bioconversion method: E. coli BL21(DE3) containing pET28a-azoC were grown in LB liquid culture with the supplementation of with 1mM ammonium ferric sulfate and 50 μg/mL kanamycin and further cultivated at 37 °C, 220 r.p.m for 5 h until the OD 600 reached 1. Then, the culture was added with 0.1 mM isopropyl β-D-1-thiogalactopyranoside and further inducted at 25 °C for 24 h. Later, cells were collected and lysed, and referred to 14000 rpm centrifuge at 4 °C for 15 min. The supernatants were collected and diluted 10 times with HEPES buffer or Tris buffer, or PBS buffer, then the solution was added with 50 μM phenol substrates, 100 μM para- aminobenzoic acid, 1mM NADH, 10 μM PMS and 5 μM TEMPO, and incubated at 30 °C overnight.

Silica gel chromatography: Silica gel chromatography was performed with silica gel (200-300 meshes), and dichloromethane, methanol, hexane and ethyl acetate were used as eluents. Scale-up reactions were acidified to pH 5 with HCl, extracted with ethyl acetate and vacuum dried, then referred to silica gel chromatography.

Corresponding Author

*Dr. Yuan-Yang Guo - Collaborative Innovation Centre of Henan Province for Green Manufacturing of Fine Chemicals; Key Laboratory of Green Chemical Media and Reactions, Ministry of Education; NMPA Key Laboratory for Research and Evaluation of Innovative Drug; Henan Key Laboratory of Organic Functional Molecule and Drug Innovation; School of Chemistry and Chemical En-gineering, Henan Normal University, Xinxiang, Henan 453007, China.

E-mail: [email protected]

Funding Sources

This work was supported by the National Natural Science Foundation of China (31901014) and Doctoral Initiation Fund (qd18008) to Y.-Y. G., and funds from Central Plains Science and Technology Innovation Leader Project (214200510008) and Henan Provincial University Science and Technology Innovation Team (21IRTSTHN001) to L. L.

Acknowledgement

The authors thank Prof. Yi-Ling Du and Prof. Zhiguo Zhang for the helpful discussions.

Author Contributions

Yuan-Yang Guo conceived the idea, designed the experiments, and prepared the manuscript. Yuan-Yang Guo, Ze-Hua Tian, Luying Wang and Zheng-De Lai performed the enzymatic reactions and silica column isolations. Lingjun Li provided part of the financial support. Yong-Quan Li provided the experimental strain source. All authors have been involved in the discussion during the preparation of this manuscript and have given approval to the final version of the manuscript.

Data Availability

Data available on request from the authors

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Table 1 is available in the Supplementary Files section.

There is NO Competing Interest.

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Table 1. Optimization of reaction conditions for the synthesis of 5.
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Chemoenzymatic Cascade Synthesis of Phenol Diarylamine Using Non-Heme Diiron N-Oxygenase

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Chemoenzymatic Cascade Synthesis of Phenol Diarylamine Using Non-Heme Diiron N-Oxygenase