Paired electrolysis enables para-C–H amination of phenols with nitroarenes and mechanistic visualization via multifunctional electrochemical mass spectrometry

Domain: biology
Published: 2026-03-18
Editorial outcome: major_revision

Abstract
Direct C–N bond formation from nitroarenes offers an appealing alternative to conventional routes that rely on aniline intermediates, yet existing metal-catalyzed approaches often suffer from narrow substrate scope and the need for stoichiometric additives. Herein, we report a paired electrolysis strategy that enables highly selective para-C–H amination of phenols with nitroarenes, delivering unprotected p-hydroxy diphenylamines in a single step without transition-metal catalysts or external reductants. Central to this advance is a multifunctional automated injection electrochemical mass spectrometry (AIEC-MS) platform, which accelerates reaction screening, identifies productive electrochemical conditions, and reveals broad compatibility with diverse phenolic and nitroarene substrates. Real-time EC-MS analysis captures key transient intermediates—including a putative aryl-nitrene species—thus providing mechanistic visualization that clarifies how anodic and cathodic events cooperatively generate the reactive partners. Together, these insights demonstrate both the synthetic utility and mechanistic distinctiveness of paired electrolysis for direct C–N bond construction.

Introduction
C–N bond formation has long been a cornerstone of synthetic chemistry,1,2 traditionally achieved through methods like Buchwald-Hartwig,3-9 Ullmann-type,10-13 and Chan-Lam14-18 couplings. These approaches typically rely on amines derived from the reduction of nitro compounds. Direct utilization of nitro compounds as starting materials for C–N bond formation offers economic benefits and broad substrate compatibility. 19-28 Recent progress has shown the effectiveness of selective reductive amination of nitroarenes using iron28 and phosphorus catalysts29-32 , demonstrating their utility in C–N bond synthesis. This strategy was further refined by integrating electrochemistry with reductive amination, enabling the coupling of nitrobenzene with phenylboronic acid without the need for external reducing agents.33 Despite these advancements, these reactions still depend on functionalized aryl reagents as coupling partners for nitrobenzene. An emerging strategy is C–H bond amination, which provides a compelling route to construct C–N bonds from readily available substrates. 34-37

p-Hydroxy diphenylamine serves as a key building block for dyes, pesticides, and ethanol fuel antioxidants, and this scaffold is widely found in both pharmaceutical agents and natural products (Figure 1a).38-42 Typically, its synthesis from p-nitrophenol requires acetyl protection of the phenol hydroxyl group, adding complexity to the process (Figure 1b).43 Although methods for synthesizing hydroxyl-compatible compounds exist, they often involve expensive metal catalysts and Grignard reagents.44,45 The direct arylation of aminophenol from nitrobenzene offers significant advantages, while Weinmayr’s direct arylation from nitrobenzene demands high-pressure hydrogen and ultrapure hydrofluoric acid.46

Developing a mild and selective C–H bond amination of phenols using nitrobenzene therefore remains an attractive yet challenging goal.

Electrochemistry operates under mild conditions, eliminating the need for external oxidizing or reducing agents, and facilitates both oxidation and reduction within a single system.47-50 Paired electrolysis enables simultaneous oxidative and reductive transformations at the same electrode, effectively overcoming mass transfer limitations that typically hinder such reactions.51-54 While the electrochemical reduction of nitroarenes to generate reactive intermediates55 and the C–H activation of phenol under electrochemical conditions56-59 have been individually studied, their combined application remains underexplored.

Herein, a paired electrolysis method is developed for the para-C(sp²)–H amination of phenols with nitroarenes, enabling the efficient synthesis of p-hydroxy diphenylamines (Figure 1c). This approach is guided by a multifunctional EC-MS platform, which streamlines reaction optimization and demonstrates broad substrate compatibility. Moreover, Real-time EC-MS monitoring facilitates the real-time detection of key intermediates, including aryl nitrene, providing valuable mechanistic insights into electrochemically driven C–N bond formation. This integrated system not only enhances the understanding of the underlying reaction mechanisms but also expands the scope of electrochemical transformations in organic synthesis, demonstrating the versatility and potential of this methodology for constructing C–N bonds from readily available substrates.

Results
Electrochemical mass spectrometry (EC-MS) has emerged as a valuable technique60-66 for efficient reaction screening and reactive intermediates characterization in synthetic chemistry.67-76 In this work, we developed an onsite EC-MS strategy for rapid reaction screening and mechanistic investigation. This method combines an automated sample introduction system with in situ EC-MS analysis,77 allowing for batch detection of reaction samples with minimal material consumption. The rapid and cost-effective approach makes it ideal for identifying key products and potential reactants. Moreover, by utilizing high-resolution mass spectrometry (HR-MS) and tandem mass spectrometry (MS/MS), the onsite EC-MS strategy enables the identification and characterization of active intermediates during electrochemical reactions, providing compelling evidence for elucidating reaction mechanisms.

As illustrated in Figure 2a, we developed an in situ EC-MS system by integrating a coaxial flow electrolytic cell, composed of stainless-steel capillaries and platinum wires, with atmospheric pressure chemical ionization (APCI). During EC-MS detection, the reaction solution is introduced to flow through the electrolytic region, and any intermediates or products generated are immediately directed to the mass spectrometer by sonic spray for APCI-MS analysis. The APCI based in situ EC-MS system is also coupled with an autosampler capable of automated batch injection and mass spectrometric analysis (Figure 2b, further device details are provided by Figure S2).

By utilizing the automated injection in situ EC-MS system (AIEC-MS) for reaction development, we established an electrochemical reaction and mechanistic investigation strategy (Figure 2c). Initially, reaction solutions containing nmol-level candidate substrates were prepared and subjected to batch mass spectrometry screening using AIEC-MS. By monitoring specific m/z channels, we rapidly identified potentially reactive substrates. Furthermore, the integration of high-resolution and tandem mass spectrometry allowed for the in situ identification and structural elucidation of reaction intermediates, providing robust mechanistic insights. Specifically, to evaluate the reactivity of nitrobenzene, we tested a range of substrates with 4-nitrotoluene using a simple electrochemical system guided by our analysis strategy. Screening reactive candidates was performed by establishing m/z channels based on potential products from candidate substrates and nitroarenes, and matching these channels with sample ion flow diagrams (Figure 2d). Among ten potential reactants (S-P1 to S-10, as shown in Figure S3), phenol was identified as capable of undergoing electrochemical reductive coupling with nitrobenzene to yield the para-coupled product 4i, as confirmed by NMR analysis. We extended this approach to various substituted phenols (S-10-1 to S-10-4) and consistently observed mass spectrometric signals corresponding to these phenols and their coupling products. These results demonstrate that the reaction is compatible with a broad range of substituted phenols, showcasing the versatility and wide applicability of our electrochemical synthesis method.

Based on the AIEC-MS results, we optimized the reaction conditions to achieve selective C(sp²)–H bond amination of phenol using nitrobenzene as the amino source via paired electrolysis in an undivided cell (Table S1). We first examined various electrode combinations (Table S1, Entries 2-5) and found that electrode choice significantly affected the reaction outcome. Changing the anode reduced the product yield to 24%, while using nickel or platinum as cathodes resulted in yields of 53% and 44%, respectively. Modifying both anode and cathode materials further decreased the yield to 22%. Factors such as current intensity and electrolyte selection had a minimal impact on the yield of 4i (Table S1, Entries 6-8). Hexafluoroisopropanol (HFIP) proved essential for target product formation, whereas trifluoroethanol, a similar solvent, produced the product with a lower yield (Table S1, Entries 9-11). A control experiment confirmed that electrical input was essential for the reaction to proceed (Table S1, Entry 12).

We then investigated the performance of various nitroarenes to evaluate their suitability as substrates (Figure 3). Halogen-containing functional groups (Br, Cl, F) were well-tolerated, delivering the target products with good yields (4a-4c). Substrates with diverse substitution patterns and multiple halogen atoms (4e-4g) consistently gave positive results. Notably, even fluorinated nitrobenzenes, which are less commonly reported, achieved a 70% yield (4c). Substrates with multiple fluorine atoms and CF3 groups (4d, 4h) also readily produced the desired compounds. Electronic effect analysis revealed that substrates with electron-withdrawing groups (4i-4l) outperformed those with neutral or electron-donating groups. The position of the electron-withdrawing group (4p) had minimal impact on the reaction outcome. Substrates with electron-neutral groups (4m-4o) consistently yielded satisfactory results. Representative functional groups frequently employed in C–N bond formation were successfully accommodated (4q-4u). Substrates bearing long-chain ketones (4v) and sulfonylamine moieties (4w), commonly encountered in organic synthesis, delivered 41% and 74% yields, respectively. Benzoheterocyclic substrates (4x) and those bearing additional functional groups (4y) afforded the corresponding products in good yields. Even amides and aliphatic halides (4z, 4aa) were effectively converted into the target compounds in moderate yields. The inherent selectivity of electrochemistry enabled the reduction of a single nitro group in dinitro substrates (4ab-4ad), affording the desired products with high specificity.

Substrates substituted with deuterium also showed favorable reactivity. The reaction efficiently tolerated substrates with multiple deuterated sites (5a, 5b) without significant changes in yield. Deuteration studies on substrates with specific functional groups (5c-5e) further demonstrated that the reaction accommodates both the functional groups and deuteration with minimal impact on yields.

We further explored the compatibility of phenol substrates by examining halogenated derivatives (Figure 4a). Fluoro-, chloro-, and bromo-substituted phenols (6a-6c) consistently yielded the desired products with efficiencies ranging from 43% to 54%. Additionally, phenols bearing electron-donating groups (6d, 6e) produced favorable yields. Substrates with substituents at various positions on the phenolic ring (6f, 6g) demonstrated comparable efficiencies, achieving yields between 50% and 54%. Furthermore, unsaturated phenols (6h, 6i) produced moderate yields, suggesting their potential for further modification and functionalization. Additionally, phenols with amide substituents were also compatible with the reaction, and the desired product was afforded(6j).

Our study extended beyond phenolic substrates. Using a high-throughput screening system, we broadened the substrate scope to include ethylbenzene derivatives (6k) and phenyl methyl ether substrates (6l, 6m). This expansion facilitated the synthesis of identical products via two distinct synthetic pathways (4j), thereby demonstrating the versatility and broad applicability of our electrochemical amination methodology.

Subsequently, we focused on scaling up the reaction. After optimizing parameters such as flow rate, we performed the reaction in a flow cell at the gram scale, demonstrating compatibility with aldehydes (Figure 4b). Compared to conventional multi-step protocols, our approach eliminates the need for metal catalysts, reduces overall reaction time, and achieves higher yields. In certain instances, we observed aniline as a reduction byproduct and investigated its potential involvement in the reaction mechanism. Control experiments involving nitrosobenzene, phenylhydroxylamine, and aniline revealed that while nitrosobenzene slightly decreased the product yield, aniline did not facilitate the formation of the desired product (Figure 4c). Real-time EC-MS experiments corroborated these results, indicating that aniline does not participate in the primary reaction pathway.

Cyclic voltammetry (CV) experiments were performed to elucidate the electrochemical behavior of the reaction, revealing notable shifts in the reduction potentials of the substrates (Figure 5a). In the absence of hexafluoroisopropanol (HFIP), the reduction peak for substrate 1i appeared at a lower potential. Upon the initial addition of reagent 2a, no significant reduction peaks were observed. However, the subsequent introduction of HFIP resulted in the emergence of clear reduction peaks. A comparative analysis of the onset potential for nitrobenzene reduction revealed a value of −1.4 V under standard conditions, while a notably more positive onset potential of −0.7 V was observed in the presence of HFIP., indicating enhanced electron affinity. This shift suggests that HFIP facilitates the stabilization of the reduced species, thereby promoting the electron transfer necessary for the reaction. These findings highlight the critical role of HFIP in modulating the electrochemical environment to favor the desired reduction processes. Linear sweep voltammetry (LSV) under single-electrode conditions showed that compound 2a exerts only a negligible influence on the cathodic reduction of 1i (Figure 5b). By contrast, square-wave voltammetry (SWV)—which records both cathodic and anodic events—revealed that the presence of 2a markedly accelerates the formation of the reduced product of 1i (Figure 5c).

To assess whether the activation of the C–H bond in phenol controls the reaction rate, competitive intermolecular experiments were conducted between phenol and D3-phenol under standard conditions (Figure 5d). The analysis of the kinetic isotope effect (KIE) curve yielded a value of 1.18, indicating that C–H bond cleavage in phenol is not the rate-determining step. Further kinetic studies demonstrated that the reaction follows zero-order kinetics to nitrobenzene (1i) and first-order kinetics to phenol (2a) (Figure 5e, 5f).

To deepen our understanding of the coupling process between nitroaromatics and phenol, we utilized in situ EC-MS to study the reaction mechanism (Figure 6a). By comparing the state of 1i before and after electrolysis, we observed that 1i ([M+H]+, m/z 138.0550) was converted into several compounds, including nitrosobenzene (1i-1, [M+H]+, m/z 122.0600), N-(p-tolyl)hydroxylamine (1i-2, [M+H]+, m/z 124.0757), p-methylaniline (1i-3, [M+H]+, m/z 108.0808), the less common N-oxide (1i-4, [M+H]+, m/z 106.0651), an intermediate (1i-5, [M+H]+, m/z 198.0913), and the product (4i, [M+H]+, m/z 200.1070). For clarity, the HR-MS spectra of each substance before and after electrolysis are presented separately in Figure S4.

During a one-minute electrolysis experiment (Figure 6b), we observed temporal variations in the mass spectrometric signals of reaction-related intermediates and products. Approximately 0.15 minutes before initiating electrolysis, signals for the reactant 1i and trace amounts of the product 4i were detectable. After electrolysis commenced at 0.15 minutes, a slight decrease in the mass spectrometric signal for 1i was noted, along with a marked increase in signals for 4i and intermediates 1i-4 and 1i-5. These changes in mass spectrometric signals confirm their close association with the electrochemical reaction and suggest that nitrobenzene can be reduced to N-oxides during the process. To further investigate the role of intermediates 1i-1 and 1i-3, we introduced them directly into the reaction. Interestingly, while reactions involving 1i-1 resulted in a significant decrease in the yield of 4i, no 4i was detected in the reaction involving 1i-3, suggesting that 1i-3 has a limited role in the reaction. HR-MS/MS analysis was also conducted to identify intermediates 1i-1 to 1i-4 (Figure 6c), confirming that the structure of 1i-4 matches the nitrene structure identified in previous work.78

The quasimolecular ion of 198.0918 caught our attention, and we identified it as intermediate 1i-5. To determine the structure of intermediate 1i-5, we employed HR-MS/MS (Figure 7a), which revealed several neutral losses: 15.0235, 27.9949, 43.0184, and 55.0058. These losses matched exactly with the masses of CH3 (15.0235), CO (27.9949), C2H3O (43.0184), and C3H3ON (55.0053), confirming the presence of unsaturated alkanes containing N and O heteroatoms. The characteristic fragment ion peaks at 105.0533, 106.0649, and 107.0727 suggest that intermediate 1i-5 could have a p-methylaniline structure (Exact Mass = 107.0735), providing further evidence to validate its structure. Similar structures were also detected in isotope-labeling experiments; the analogous fragment peaks observed for D3-5 confirm the accuracy of the preceding data. The unique structure of intermediate 1i-5 has not been previously reported, indicating the presence of a novel mechanism in this reaction.

Moreover, product 4i ([M+H]+, m/z 200.1070) was detected by in situ EC-MS, revealing that the HR-MS/MS spectra of 4i obtained online and offline differed (Figure 7b). The fragment peaks at 106.0652, 107.0730, and 108.0445 were observed in the online mass spectrum, with 107.0730 being the base peak. In contrast, the offline spectrum showed a much weaker intensity for the fragment ion 107.0730. Using D3-1i instead of 1i in the reaction yielded similar results, confirming this phenomenon (Figure S6). This led us to hypothesize that intermediate 1i-6, an isomer of 4i, was detected by in situ EC-MS. To explore the relationship between these fragment ions and the parent ion, we employed Quantum Chemical Mass Spectrometry (QC-MS), an open-source tool for fully automated calculations of positive ion collision-induced dissociation mass spectra of singly charged molecular ions.79, 80 QC-MS simulations showed a strong intensity for the fragment ion 107.0735 of intermediate 1i-6, highlighting its contributions to the online tandem mass spectrum (Figure S23).

Using tritiated phenol D3-2a as the starting substrate, we identified a target product with an exact mass of 202.1195, incorporating two deuterium atoms. Tandem mass spectrometry confirmed the presence of structure D2-6, analogous to intermediate 1i-6 (Figure 7c). These findings indicate that product formation requires both reduction and hydrogenation, whereas direct coupling involving nitrosobenzene would necessitate reduction and dehydration. The differences suggest that nitrosobenzene does not directly participate in forming intermediate 1i-6 (Figure S24). Instead, intermediate 1i-6 is generated through the reduction and hydrogenation of intermediate 1i-5. Additionally, nearly all detected intermediates corresponding to 1i-5 lack three deuterium atoms, indicating that this step is the sole pathway to 1i-6. Among nitrobenzene reduction intermediates, only aryl nitrene 1i-4 can eliminate the ortho hydrogen of phenoxide via C–H bond insertion during coupling. Therefore, we propose that aryl nitrene 1i-4 directly contributes to the formation of intermediate 1i-5.

In subsequent experiments, we replaced nitrobenzene with nitrosobenzene and performed online MS analyses. As shown in Figure 8a, during the electrochemical reduction of nitrosobenzene (1I-1), the mass spectrum before electrolysis, obtained via EC-APCI-MS, revealed a characteristic peak for 1I-1 at [M+H]+, m/z 108.0442. Upon initiation of the electrolysis, mass spectral signals corresponding to a series of hydrogenation-dehydration products were detected, including phenylhydroxylamine ([M+H]+, m/z 110.0600), aniline ([M+H]+, m/z 108.0442), as well as the coupling intermediate 1I-5 ([M+H]+, m/z 184.0757) and the product 4I ([M+H]+, m/z 186.0913). These results confirm that nitrosobenzene undergoes reduction, analogous to nitrobenzene, without altering the intermediate profile of the reaction. Importantly, the post-electrolysis mass spectrum also revealed the generation of the nitrene intermediate 1l-4 ([M+H]+, m/z 92.0495), suggesting that nitrosobenzene acts as an intermediate in this system, undergoing further reduction to form the nitrene species.

As a prerequisite for this reaction pathway, we sought to determine whether nitrobenzene undergoes further reduction within the reaction system. According to the LSV experiment shown in Figure S16, the reduction potential of nitrotoluene (1i) is approximately -0.52 V, corresponding to the initial reduction potential required for converting 1i into nitroso-toluene. We maintained cathodic potentials of -0.40 V, -0.60 V, and -1.0 V for 4 hours, respectively (Figure S25). After the reaction, only the system with a cathodic potential of -1.0 V detected product formation. This result indicates that nitrobenzene must undergo further reduction to proceed with the subsequent reactions.

To confirm that the active intermediate has a sufficiently long lifetime in paired electrolysis, we employed a combination of a separation cell and alternating current (AC) to conduct the coupling reaction (Figure S26). Due to the alternating nature of AC, where the polarity switches between positive and negative, only one type of electrode reaction can occur at a time in each chamber of the separation cell. Consequently, the reduction of nitrobenzene and the oxidation of phenol cannot occur simultaneously. By increasing the AC frequency to 5 Hz, we observed significant product formation in the system. Considering that the electrode polarity switches every 0.01 seconds at this frequency, we conclude that in the original integrated system, nitrene possesses a sufficient lifespan to facilitate the coupling reaction.

To establish a direct correlation between nitrene formation and electrochemical reactions, we designed an intermediate capture experiment (Figure 8b). When 1,2-diphenylvinyl (7a) was used to replace phenol in the reaction, the formation of aziridine (7b, [M+H]+, m/z 286.1590) was detected through online mass spectrometry, and HR-MS/MS detected its neutral losses of 78.0481, 92.0638, 107.0749, and 178.0804 correspond to the exact masses of C6H6 (78.0470), C7H8 (92.0626), C7H9N (107.0735) and C14H10 (178.0783), suggesting that 7b has a structure of benzene ring and N atom. In the electricity on-off experiment, the extracted ion chronograms (EIC) demonstrated that the generation of both the nitrene intermediate and 7b was dependent on the applied current, confirming that the nitrene detected in our system arises from the electrochemical reaction and is not an artifact of the mass spectrometry analysis. Additionally, when benzaldehyde was used directly in the reaction instead of phenol, we successfully isolated the oxazolidine tricyclic product. This reaction was also monitored by online mass spectrometry, which further confirmed that the generation of the nitrene intermediate and the product was electricity-dependent.

The Cardogan reaction, an established method for carbazole synthesis, provides a useful model for demonstrating the involvement of nitrene intermediates.81-83 We adapted this reaction to a system comprising nitrobenzene and phenol (Figure 8c). Due to its intramolecular nature, we utilized a magnesium electrode as a sacrificial anode. Upon completion of the reaction, we isolated product 7f with a yield of 41%. In situ EC-MS analysis revealed that intermediates, including hydrogenation products 7e-1 ([M+H]+, m/z 184.0757) derived from nitrobenzene, were associated with the electrochemical process. This behavior, resembling that of the template reaction, supports the conclusion that our system effectively reduces nitrobenzene to generate nitrene 7e-2 ([M+H]+, m/z 168.0808), which then participates in the subsequent cyclization reaction to form product 7f ([M+H]+, m/z 168.0808). EIC showed that the generation of 7e-2 & 7f and other intermediates were dependent on electrolysis and confirmed the intermediates’ mass spectrum signals caused by electrochemistical process.

Based on the intermediates identified by in situ EC-MS and our experimental results, the proposed reaction mechanism is outlined in Figure 8d. Initially, substrates 1i and 2a undergo paired electrolysis at the electrodes. Substrate 1i is reduced and deoxygenated in two steps at the cathode via nitroso intermediate 1i-1, leading to the formation of aryl nitrene 1i-4 in a monoclinic state. Concurrently, phenol undergoes oxidation at the anode, forming a radical intermediate 2a-1, which is further oxidized to generate the cationic intermediate 2a-2. The monoclinic aryl nitrene 1i-4 then inserts into the C–H bond of cationic intermediate 2a-2, resulting in their coupling and the release of a hydrogen ion, forming intermediate 1i-5. Intermediate 1i-5 is subsequently reduced at the cathode to form intermediate 1i-6, which then re-aromatization to produce the target product 4i.

In conclusion, we have established a paired electrolysis strategy that enables efficient C(sp²)–H amination of phenols with nitroarenes, providing a straightforward route to p-hydroxy diphenylamines under mild and practical conditions. The integration of multifunctional EC-MS played a central role by accelerating substrate evaluation through rapid and reliable screening and by offering direct evidence for transient intermediates that clarify key steps of the coupling mechanism. This combination of electrochemical synthesis with real-time analytical capabilities enhances mechanistic understanding and supports the continued advancement of electrochemical reaction design. Looking ahead, further refinement of EC-MS–based screening and mechanistic visualization is expected to broaden the scope, efficiency, and applicability of future electrochemical transformations.

Methods
General information. All glass wares were oven dried at 110 oC for hours and cooled down under vacuum. Unless otherwise noted, materials were obtained from commercial suppliers and used without further purification. The carbon cloth, platinum wire and plugs were commercially purchased. The instrument for electrolysis was dual display potentiostat (DJS-292B) (made in China). Thin layer chromatography (TLC) employed glass 0.25 mm silica gel plates. Flash chromatography columns were packed with 200-300 mesh silica gel. Gradient flash chromatography was conducted eluting with a continuous gradient from petroleum to the indicated solvent, and they were listed as volume/volume ratios. MS data were acquired by LTQ Velos Orbitrap Elite mass spectrometer (Thermo Fisher Scientific Co., Ltd., USA). The temperature of the ion transmission tube was set lo 275 oC; the energy of the S-lens was set to 60 %; the number of micro scan was set to 1. MS/MS data were acquired by setting isolation window at 1.0 Da and normalized collision energies from 20 to 35 (units were defined by the instrument manufacturer, the collision gas was helium). The resolution of MS was set to 60000. All MS data were analyzed through the Qual browser (Xcalibur workstation, version 4.2.47). The high voltage power supply was from the mass spectrometer. Both the mass spectrometer and the external voltage power supply were well grounded. Unless otherwise stated, normalization is performed by dividing by the maximum value.

General procedures for the synthesis of p-hydroxy diphenylamines. In an oven-dried undivided two-necked cell (10 mL) equipped with a stir bar. Arylhydrazine 4-Nitrotoluene (27.4 mg, 0.2 mmol), Phenol (56.5 mg, 0.6 mmol), nBu4NBF4 (65.8 mg, 0.2 mmol) were combined and added. The cell was equipped with a piece of platinum (15 mm × 15 mm × 1 mm) as the anode and a piece of carbon cloth (15 mm × 15 mm × 1 mm) as the cathode, and then charged with argon to replace air atmosphere. DCE (3.0 mL) and HFIP (3.0 mL) were added to the tube through a syringe. The reaction mixture was stirred and electrolyzed at a constant current of 10 mA under room temperature (25 oC) for 5 h (2.9 F/mol). When the reaction finished, the desired products were obtained in the corresponding yields after purification by flash chromatography on 200-300 mesh silica gel (petroleum : Ethyl acetate = 4 : 1). 3m, 3s, 3t, 3u, 3v: Otherwise above general procedure, carbon cloth anode (15 mm × 15 mm × 0.1 mm), platinum plate cathode (15 mm × 15 mm × 0.1 mm), constant current = 10 mA, 8 h, 1a (0.10 mmol), 2a (0.60 mmol).

Mass Spectrometric Screening Process of Reaction Substrates. Device Setup. The schematic diagram of the EC-APCI-MS apparatus is depicted in Figure S2a. The flow EC cell, featuring a coaxial structure, comprises a platinum wire inserted into a stainless steel tube. The platinum wire is exposed for approximately 2 mm, with the rest insulated to prevent shorting. At the left outlet of the stainless steel tube, a T-connector fixes the platinum electrode and introduces the reaction solution to the flow EC cell. On the right side of the stainless steel tube, pneumatic nebulization of the reaction solution is realized by using a T-connector and sheath gas. A stainless steel needle with voltage of 3.5 kV is placed near the pneumatic nebulization nozzle for atmospheric chemical ionization. The spray nozzle, APCI electrode tip, and mass spectrometer inlet are aligned linearly with a 3 mm gap between each of them. All connections are sealed with PEEK tubing of matching sizes to ensure proper integration. By connecting the inlet of the Electrochemical Mass Spectrometry to the outlet of an autosampler (Ultimate 3000, Thermo Scientific) via a fitting, we established an Auto Injection EC-MS system capable of rapid electrochemical reaction screening. Utilizing the batch processing function of the interaction software (Thermo Xcalibur 3.0.63) between the autosampler and the mass spectrometer, Auto Injection EC-MS facilitates automated sample injection through flow injection. Unless otherwise specified, acetonitrile (HPLC grade, degassed via ultrasonication prior to use) was used as the mobile phase, with a sample volume set to 5 μL, an injection interval of 2 minutes, a reaction voltage of 15 V, the voltage of the corona electrode of +3.5 kV, the flow rate of 10 μL/min, and the sheath gas pressure of 0.4 MPa.Preparation of Reaction Solutions. To screen potential substrates for coupling with p-nitrotoluene, a solution containing 1 mM p-nitrotoluene, 1 mM candidate substrate, and 10 mM lithium triflate in a 9 : 1 acetonitrile/water mixture was prepared and placed in the vials. Additionally, a 1 mL solution of 10 mM lithium triflate in a 9 : 1 acetonitrile/water mixture was prepared as a blank sample and also placed in the vials.

Online electrochemical reactions monitoring. Online electrochemical reactions were monitored by EC-MS, and the electrochemical reaction conditions have been slightly modified for compatibility with mass spectrometry systems. The solution of acetonitrile: water = 9 : 1 (v : v) with 10 mM lithium triflate as supporting electrolyte containing 0.6 mM phenol and 0.2 mM p-nitrotoluene was loaded into EC-MS. The voltage was applied to that the current of electrolytic cell reaches 10 mA.

Deuterated nitrobenzene, Deuterated phenol, nitrosobenzene or Aniline was also used as a reaction substrate for online monitoring, and the experimental conditions were consistent with the above.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the manuscript titled “Paired Electrolysis enables para-C-H Amination of Phenols with Nitroarenes to p-Hydroxy Diphenylamines Guided by Multifunctional Electrochemical Mass Spectrometry”, the authors Yi，Lei and Wei investigate a paired electrolysis strategy for selective para-C–H amination of phenols with nitroarenes, enabling the one-step synthesis of unprotected p-hydroxy diphenylamines without relying on transition-metal catalysts or external reductants.. The study presents a comprehensive analysis of para-C-H amination of phenols with nitroarenes to p-hydroxy diphenylamines, which is of broad interest to the field of electrochemical organic synthesis. The findings are novel and potentially significant, as they provide new insights into forming the C-N bond. My recommendation is minor revision, and my specific comments are as follows.\nComment 1: Phenols are commonly found in natural products. To further demonstrate the practical utility of this methodology, it would be valuable to include one or two examples using structurally complex phenols, which could help illustrate the applicability of this approach in natural product synthesis.\nComment 2: In the substrate scope studies, various substituted phenols were examined. It would be helpful if the authors could comment on the relative reaction rates observed for phenols bearing electron-donating versus electron-withdrawing groups, along with a brief explanation of the electronic effects influencing reactivity.\nComment 3: The Supporting Information includes attempts with aromatic amine derivatives (page 7). Have the authors also explored the reactivity of Ts-protected anilines in this transformation?\nComment 4: There appears to be an inconsistency between the structural formula of intermediate 6 in Figure 6a and that in Figure 7. The authors should verify and correct this discrepancy.\nComment 5: The 13CNMR spectrum provided for compound 4d in the Supporting Information seems to be partially cut off. A complete spectrum should be provided.\nComment 6: In the Supporting Information, Section 10 (“Constant Potential Test”) states that reactions were performed at cathodic potentials of –0.4 V, –0.6 V, and –1.0 V. However, Figure S25 is labeled with “2.6 V”. This likely represents a typographical error and should be corrected to “0.6 V” for consistency.\nComment 7: There are several minor spelling and formatting errors that require attention. For example, on page 20 of the Supporting Information, “Arylhydrazine” should be removed and “Phenol” should be changed to “phenol”. A thorough proofreading of the manuscript and SI is recommended to correct similar inaccuracies.", "reviewer_2": "In this manuscript, the authors present an electrochemical, metal-free one-step strategy for C–N bond formation directly from nitroarenes, enabling the synthesis of unprotected p-hydroxy diphenylamines. This approach is further integrated with the AIEC-MS platform for rapid screening, demonstrating a broad substrate scope. In-depth mechanistic studies using in situ EC-MS uncover key intermediates, including aryl nitrenes and related species. However, I would like to recommend its publication in this journal after the following comments have been addressed.\n1. Since the reaction involves the generation of phenoxy radical species, which have the potential to undergo self-dimerization, did the authors observe such byproducts in the reaction mixture, or does this process interfere with the overall reaction?\n2. Why do nitroarenes bearing electron-donating substituents lead to lower product yields?\n3. What factors govern the selective reduction of only one nitro group while leaving the second nitro group intact, as observed in products 4ab–4ad?\n4. If the para position of the phenol ring is substituted with an alkyl group, does the reaction still proceed? Furthermore, what would be the expected outcome if both the para and ortho positions are sterically blocked?\n5. For completeness, the authors might consider citing “Org. Lett. 2022, 24, 12, 2310–2314”, where a related approach for the oxidation of phenol has been demonstrated.", "reviewer_3": "General Assessment\nThis manuscript describes a paired electrolysis strategy for para-C–H amination of phenols with nitroarenes, guided by multifunctional electrochemical mass spectrometry (EC-MS). The approach allows for one-step synthesis of p-hydroxy diphenylamines under mild, metal-free conditions, and provides mechanistic insight via in situ monitoring of reactive intermediates. While the work presents an innovative combination of electrochemistry and EC-MS analysis, several aspects of experimental design, mechanistic interpretation, and substrate scope require substantial clarification before it is suitable for publication. Therefore, I recommend major revision.\nMinor Comments\n• Line 245: The text refers to “Scheme 3c,” but no such scheme exists. This should be corrected.\n• Line 265: Figure 7b is said to show compound 4i, but it appears to correspond to 5i, which creates confusion.\n• Figure 3: The content resembles a table of compounds rather than a figure. It should be reformatted or consistently described.\n• In Fig. 5a, could the observed positive shift in peak potential in the presence of HFIP be attributed to proton involvement in the electrochemical reduction of nitrobenzene?\n• Figure 6a: Intermediates should be labeled consistently with the text. For example, the compound currently labeled “1” should be labeled as 1i-1 to avoid confusion with starting materials.\n• Line 232: The manuscript states that 1i-1 is p-nitrotoluene, but based on mass data and mechanistic context, this should be nitrosobenzene. Similarly, 1i-2 should be N-(p-tolyl)hydroxylamine instead of p-hydroxytoluene.\n• Figure S25 (Supporting Information): X-axis potentials should be reported as negative.\n• Figure 8c: The MS of 7e1 and 7f appear to be incorrect.\n• Several sentences are vague or incomplete. For example, Lines 210–212 (“Comparative analysis of nitrobenzene’s reduction potential…”) should be rewritten more clearly with quantitative electrochemical data.\nMajor Comments\n• The complete reduction of nitrobenzene is usually a six-electron process. Could the authors monitor the reaction progress using cyclic voltammetry and, ideally, perform constant-potential electrolysis to confirm the paired electrolysis mechanism?\n• Supporting Information: Electrode polarity switching frequency is reported inconsistently (1, 2, 5 Hz vs 0, 2, 5 Hz in Table X). This should be clarified. Also, indicate if the reaction was performed in a two-compartment cell under constant current without polarity switching.\n• Authors suggest a nitrene intermediate (based on mass spectrometry), but there is no direct evidence of its participation in C–H amination. Observing traces in MS does not confirm it as a key intermediate; the reaction may proceed via classical nitroso/hydroxylamine pathways.\n• The modified electrolysis conditions used for mass spectrometry are very different from bulk electrolysis conditions. It is unclear how the mechanism can be reliably studied under such differing conditions.\n• Many products could potentially be accessed more easily via anilines, so the synthetic advantage is unclear.\n• It is recommended to replicate NMR spectra of 4ab and 4ac to ensure reproducibility and accuracy.", "reviewer_4": "The manuscript by Prof Lei and colleagues have described Paired Electrolysis enables para-C-H Amination of Phenols with Nitroarenes to p-Hydroxy Diphenylamines Guided by Multifunctional Electrochemical Mass Spectrometry. There are several key aspects in mass spectrometry that need to be clarified and further verified to enhance the scientific rigor and impact of the research. I recommend accepting the paper after revisions.\n1. Mass spectra with a certain mass range should be provided to facilitate the reader's understanding of the actual situation of the entire reaction. The mass spectra should show isotopic peaks in Figure S4. By observing the intensity and position of the isotopic peaks, more information about the elemental composition of the sample can be observed.\n2. Discussion of fragmentation processes of tandem mass spectra in Figure 7, Figure S5 and Figure S6should be added in Supporting Information.\n3. Specific experimental operations for offline in Figure 7b should be added in Supporting Information.\n4. Central to this approach is a multifunctional automated injection electrochemical mass spectrometry (AIEC-MS) platform. Its ionization mode is described as APCI. But from the perspective of Device Setup, what is the difference from ESI? Theoretically, ESI is more reflective of the reaction species than APCI."}, "author_response": "Response: We thank the reviewer for the positive evaluation of our work and for recognizing the significance of the paired-electrolysis strategy developed for the para-C–H amination of phenols with nitroarenes. We are grateful for the reviewer’s acknowledgment that the method offers a transition-metal-free and reductant-free route to unprotected p-hydroxy diphenylamines, and that the mechanistic insights provided by multifunctional electrochemical mass spectrometry contribute meaningfully to the broader field of electrochemical organic synthesis.\nComment 1: Phenols are commonly found in natural products. To further demonstrate the practical utility of this methodology, it would be valuable to include one or two examples using structurally complex phenols, which could help illustrate the applicability of this approach in natural product synthesis.\nResponse: Thank you very much for your comments. Phenolic frameworks are indeed widespread in pharmaceutical agents and natural products, and protecting-group manipulations are often unavoidable in their conventional synthetic routes. To further demonstrate the practical utility and structural generality of our method, we applied the paired-electrolysis protocol directly to a commercially available drug precursor, achieving the one-step synthesis of the hydroxyl-containing ester R-3. This intermediate can be readily converted into 4′-hydroxy diclofenac, an established anti-inflammatory and analgesic compound.\nBy comparison, traditional synthetic routes to this target typically require four discrete steps, rely on stoichiometric nitrite and boron tribromide, and involve a copper(I) iodide catalyst, thereby increasing the operational burden and necessitating additional purification. Our method circumvents these limitations, underscoring its efficiency and applicability to structurally complex, pharmaceutically relevant phenols.\nFigure R1. Comparison between the synthesis of the drug-related molecule and the corresponding conventional synthetic route.\nComment 2: In the substrate scope studies, various substituted phenols were examined. It would be helpful if the authors could comment on the relative reaction rates observed for phenols bearing electron-donating versus electron-withdrawing groups, along with a brief explanation of the electronic effects influencing reactivity.\nResponse: Thanks very much for your comments. To evaluate the influence of electronic effects on reaction kinetics, we compared the consumption rates of m-cresol and m-bromophenol under the standard reaction conditions. Within the first forty minutes, m-cresol exhibited a significantly faster consumption rate than m-bromophenol.\nTo further probe the electronic origins of this reactivity difference, we performed cyclic voltammetry (CV) measurements on both substrates. The CV data show that m-cresol features an earlier onset potential and a more positive half-wave potential compared with m-bromophenol, along with a higher peak current. These results collectively indicate that phenols bearing weak electron-donating substituents are more easily oxidized than those containing weak electron-withdrawing groups.\nFigure R2. Conversion profiles and cyclic voltammograms of phenols bearing different substituents.\nComment 3: The Supporting Information includes attempts with aromatic amine derivatives (page 7). Have the authors also explored the reactivity of Ts-protected anilines in this transformation?\nFigure R3.Substrate evaluation involving Ts-protected anilines and related derivatives.\nResponse: Thanks very much for your comments. We conducted the suggested experiments, and the results are summarized in Figure R3. When Ts-protected anilines were subjected to the standard reaction conditions, no desired product (R-10) was obtained. Likewise, Ts-substituted phenols failed to deliver the corresponding coupling product (R-11). We attribute this lack of reactivity to the strong electron-withdrawing nature of the Ts group, which likely raises the oxidation potential of the substrates beyond the operative window of the system.\nIn contrast, when a nitroarene—bearing an electron-withdrawing group of comparable strength to Ts—was directly employed as the nitrogen source, the reaction proceeded smoothly to afford the desired product (R-12) in high yield. This outcome suggests that while electron-withdrawing substituents are not inherently incompatible, their position and participation in the redox processes critically determine reactivity.\nComment 4: There appears to be an inconsistency between the structural formula of intermediate 6 in Figure 6a and that in Figure 7. The authors should verify and correct this discrepancy.\nResponse: Thanks very much for your comments. We appreciate the reviewer’s careful examination of the mechanistic figures. Upon re-evaluation, we confirmed that the structural representation of intermediate 6 was indeed inconsistent between Figure 6a and Figure 7. We have now corrected the structure to ensure full consistency across all mechanistic schemes and figures.\nComment 5: The 13C NMR spectrum provided for compound 4d in the Supporting Information was indeed incomplete due to an export error. We have now provided the full, uncut spectrum in the revised Supporting Information.\nComment 6: In the Supporting Information, Section 10 (“Constant Potential Test”) states that reactions were performed at cathodic potentials of –0.4 V, –0.6 V, and –1.0 V. However, Figure S25 is labeled with “2.6 V”. This likely represents a typographical error and should be corrected to “0.6 V” for consistency.\nResponse: Thanks very much for your comments. The discrepancy in Section 10 of the Supporting Information was indeed caused by a typographical error. We have corrected the mislabeled potential in Figure S25 and revised the corresponding textual description to ensure full consistency throughout the document.\nComment 7: There are several minor spelling and formatting errors that require attention. For example, on page 20 of the Supporting Information, “Arylhydrazine” should be removed and “Phenol” should be changed to “phenol”. A thorough proofreading of the manuscript and SI is recommended to correct similar inaccuracies.\nResponse: Thanks very much for your comments. Thanks very much for your comments. We have carefully corrected the spelling and formatting issues noted by the reviewer.\nReviewer #2 (Remarks to the Author):\nIn this manuscript, the authors present an electrochemical, metal-free one-step strategy for C–N bond formation directly from nitroarenes, enabling the synthesis of unprotected p-hydroxy diphenylamines. This approach is further integrated with the AIEC-MS platform for rapid screening, demonstrating a broad substrate scope. In-depth mechanistic studies using in situ EC-MS uncover key intermediates, including aryl nitrenes and related species. However, I would like to recommend its publication in this journal after the following comments have been addressed.\n1. Since the reaction involves the generation of phenoxy radical species, which have the potential to undergo self-dimerization, did the authors observe such byproducts in the reaction mixture, or does this process interfere with the overall reaction?\nResponse: Thanks very much for your comments. We indeed observe trace amounts of phenoxy–phenoxy dimerization products at the end of the reaction. However, because phenol is employed in excess, this side process does not interfere with the formation of the desired product. In practice, these byproducts predominantly appear as deposits on the anode surface. Owing to the good solubility of these species in DCE, such deposition is barely detectable during the early stages of electrolysis. Monitoring the reaction over time indicates that the formation of these byproducts only leads to a slight increase in cell voltage, without affecting the overall efficiency of the transformation.\n2. Why do nitroarenes bearing electron-donating substituents lead to lower product yields?\nResponse: Thanks very much for your comments. We initially hypothesized that the diminished yields observed for nitroarenes bearing electron-donating substituents might originate from differences in their electronic properties. To examine this possibility, we performed cyclic voltammetry measurements to compare their reduction potentials. Surprisingly, the onset potentials of p-nitrotoluene and p-nitroanisole showed no significant difference, suggesting that redox thermodynamics alone do not account for the observed reactivity trend.\nFigure R4. Cyclic voltammetry experiments of nitrobenzenes bearing different substituents\n1.0\n0.8\n0.6\n0.4\n0.2\n0.0\n0.0 0.2 0.4 0.6 0.8 1.0 1.2\nFigure R5. Online electrochemical mass-spectrometric switching experiments for nitrobenzenes with different substituents\nTo gain deeper mechanistic insight, we conducted online electrochemical mass-spectrometric switching experiments. For p-nitrotoluene, the corresponding reduction intermediate R-17 (m/z 106.0659) displayed a clear current-dependent fluctuation in ion intensity, indicating a short-lived species. In contrast, the reduction intermediate derived from p-nitroanisole, R-18 (m/z 122.0606), exhibited no such fluctuation, consistent with a markedly longer lifetime.\necnadnubA\nevitaleR\n106.0659\nTime / min\n1.0\n0.8\n0.6\n0.2\n0.0\n0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5\necnadnubA\nevitaleR\nElectrode on Electrode off Electrode on Electrode off\n122.0606\nTime / min\nNO 2 N N\nR-13 R-15 R-17\nNO 2 N N\nO O O\nR-14 R-16 R-18\nFigure R6.Conversion pathways of the Nef reaction–related intermediates\nOur prior studies revealed that nitroso intermediates can undergo spontaneous ring expansion, which prolongs their lifetime while retaining reactivity. However, the p-nitroanisole–derived intermediate exhibits a significantly longer lifetime than other nitroarene intermediates. We propose that, due to its stronger electron-donating substituent, this species preferentially forms a nitrogen-containing seven-membered ring rather than interconverting between reactive intermediates. This structural divergence leads to loss of productive reactivity and, consequently, to the lower yield observed for reactions involving p-nitroanisole.\n3. What factors govern the selective reduction of only one nitro group while leaving the second nitro group intact, as observed in products 4ab–4ad?\nResponse: Thanks very much for your comments. We believe that two factors govern the selective reduction of only one nitro group in products 4ab–4ad. First, the selectivity is strongly time-dependent: in our standard protocol, the reaction is quenched before complete consumption of the dinitro substrate. When the reaction time is intentionally extended in control experiments, high-resolution mass spectrometry clearly detects the formation of the doubly aminated product.\nSecond, we propose that the diamination product possesses a significantly lower oxidation potential than the corresponding monoamination product. As a result, any small amount of diamine generated under the standard conditions is preferentially re-oxidized at the anode and thus removed from the reaction mixture. This electrochemical self-regulation accounts for the exclusive formation of monoaminated products under the optimized conditions.\nFigure R7. Formation of the diamination product upon extending the reaction time with dinitro substrates\n4. If the para position of the phenol ring is substituted with an alkyl group, does the reaction still proceed? Furthermore, what would be the expected outcome if both the para and ortho positions are sterically blocked?\nResponse: Thanks very much for your comments. We investigated both scenarios experimentally. When p-cresol (R-22) was used as the substrate, the reaction mixture became considerably more complex. Online electrochemical mass spectrometry revealed the formation of benzylamine (R-23) as well as an ortho-aminated product (R-24), indicating that blocking the para position redirects the reaction toward alternative pathways.\nFurthermore, when 2,4,6-trimethylphenol (R-25) was subjected to the standard conditions, we observed virtually no conversion of the phenol substrate. Instead, most of the p-nitrotoluene was reduced to aniline (R-26), accompanied by the formation of a small amount of azobenzene (R-27). These results suggest that when both the para and ortho positions are sterically hindered, C–N bond formation is effectively suppressed, and the reaction predominantly follows reductive pathways of the nitroarene.\nFigure R8.Reactions involving phenols with different substitution patterns\n5. For completeness, the authors might consider citing “Org. Lett. 2022, 24, 12, 2310–2314”, where a related approach for the oxidation of phenol has been demonstrated.\nResponse: Thanks very much for your comments. We agree that the referenced work is highly relevant to the background of our study. We have now included “Org. Lett. 2022, 24, 2310–2314” in the revised manuscript to provide a more complete contextual framework.\nReviewer #3 (Remarks to the Author):\nThis manuscript describes a paired electrolysis strategy for para-C–H amination of phenols with nitroarenes, guided by multifunctional electrochemical mass spectrometry (EC-MS). The approach allows for one-step synthesis of p-hydroxy diphenylamines under mild, metal-free conditions, and provides mechanistic insight via in situ monitoring of reactive intermediates. While the work presents an innovative combination of electrochemistry and EC-MS analysis, several aspects of experimental design, mechanistic interpretation, and substrate scope require substantial clarification before it is suitable for publication. Therefore, I recommend major revision.\nResponse: Thanks very much for your comments. We sincerely appreciate the reviewer’s careful evaluation of our work. In the revised manuscript, we have addressed all concerns regarding experimental design, mechanistic interpretation, and substrate scope. Specifically, we have expanded the mechanistic studies, provided additional control experiments, clarified key aspects of the electrochemical setup, and enriched the substrate scope with more structurally diverse examples. We believe these revisions significantly strengthen the robustness and clarity of our conclusions, and we hope the improved version will meet the standards required for publication.\nMinor Comments\n• Line 245: The text refers to “Scheme 3c,” but no such scheme exists. This should be corrected.\nResponse:Thanks very much for your comments. We have revised accordingly.\n• Line 265: Figure 7b is said to show compound 4i, but it appears to correspond to 5i, which creates confusion.\nResponse:Thanks very much for your comments. We have revised accordingly.\n• Figure 3: The content resembles a table of compounds rather than a figure. It should be reformatted or consistently described.\nResponse:Thanks very much for your comments. We have revised accordingly.\n。\n• In Fig. 5a, could the observed positive shift in peak potential in the presence of HFIP be attributed to proton involvement in the electrochemical reduction of nitrobenzene?\n0.0\n−4.0×10−4\n−8.0×10−4\n−1.2×10−3\n−3.0 −2.5 −2.0 −1.5 −1.0 −0.5 0.0\nFigure R9.CV experiments of the reaction under different solvent conditions\nResponse: Thanks very much for your comments. To clarify the origin of the positive shift in peak potential observed in the presence of HFIP, we conducted comparative CV experiments in which HFIP was replaced by ethanol (EtOH). As shown in Figure R9, although the absolute currents differ slightly between the two solvent systems, the key electrochemical features remain essentially unchanged: both electrolytes display nearly identical onset potentials (≈ –0.5 V) and reach the first cathodic peak at approximately –2.0 V. These results indicate that, for the cathodic reduction of nitrobenzene, the acidity or proton-donating ability of HFIP can indeed be substituted by other protic solvents without significantly altering the fundamental reduction behavior.\nA\n/\ntnerruC\nEtOH\nHFIP\nPontential / V\nFigure R10. Product distributions obtained after the reaction under different solvent conditions\nHowever, a clear divergence emerges during the reverse scan starting from –3.0 V. Under our standard HFIP-containing conditions, only trace amounts of p-toluidine are detected after electrolysis. By contrast, when HFIP is replaced with EtOH, the target amination product is not observed at all; instead, p-nitrotoluene is almost completely reduced to p-toluidine (Figure R10). These observations demonstrate that although HFIP and EtOH provide similar CV profiles in terms of reduction potentials, they exert markedly different influences on the overall reaction pathway and product distribution.\nTherefore, the positive shift in peak potential cannot be attributed to proton involvement in the reduction process. Rather, it likely originates from solvent–solute interactions or co-solvent effects unique to HFIP, which play a critical role in steering the downstream reactivity.\n• Figure 6a: Intermediates should be labeled consistently with the text. For example, the compound currently labeled “1” should be labeled as 1i-1 to avoid confusion with starting materials.\nResponse: Thanks very much for your comments. We have revised the labeling of intermediates in Figure 6a to ensure consistency with the text; for example, the compound previously labeled as “1” is now correctly labeled as 1i-1 to avoid confusion with the starting materials.\n• Line 232: The manuscript states that 1i-1 is p-nitrotoluene, but based on mass data and mechanistic context, this should be nitrosobenzene. Similarly, 1i-2 should be N-(p-tolyl)hydroxylamine instead of p-hydroxytoluene.\nResponse: Thanks very much for your comments. We have corrected the errors in the manuscript: 1i-1 is now properly identified as nitrosobenzene, and 1i-2 has been corrected to N-(p-tolyl)hydroxylamine.\n• Figure S25 (Supporting Information): X-axis potentials should be reported as negative.\nResponse:Thanks very much for your comments. We have revised accordingly.\n• Figure 8c: The MS of 7e1 and 7f appear to be incorrect.\nResponse:Thanks very much for your comments. We have revised accordingly.\n• Several sentences are vague or incomplete. For example, Lines 210–212 (“Comparative analysis of nitrobenzene’s reduction potential…”) should be rewritten more clearly with quantitative electrochemical data.\nResponse:Thanks very much for your comments. We have revised accordingly.\nMajor Comments\n• The complete reduction of nitrobenzene is usually a six-electron process. Could the authors monitor the reaction progress using cyclic voltammetry and, ideally, perform constant-potential electrolysis to confirm the paired electrolysis mechanism?\n0.0\nR-13\n−4.0×10−4\n−8.0×10−4\n−1.2×10−3\n−3.0 −2.5 −2.0 −1.5 −1.0 −0.5 0.0\nFigure R11. Nitrobenzene CV reduction curves under reaction conditions and possible explanations\nResponse: Thanks very much for your comments. The electrochemical reduction of nitrobenzene has been extensively studied. Although the overall process involves a six-electron transfer through two intermediates—nitrosobenzene and hydroxylamine—these steps are largely reversible. As a result, cyclic voltammetry (CV) monitoring typically shows a broad peak, analogous to catalytic current behavior.\nA\n/\ntnerruC\nPontential / V\n20\n16\n12\n8\n4\n0\n−1.0 −0.8 −0.6 −0.4\nFigure R12. Results of constant cathodic potential electrolysis\nWe also performed constant-potential electrolysis experiments, as described in the Supporting Information. At a cathodic potential of –0.4 V, no significant reduction of nitrobenzene or formation of the target product was observed, although phenol oxidation occurred, leading to detectable phenol dimer formation. At –1.0 V, nitrobenzene reduction commenced, and the target product R-23 was obtained in 17% yield. These results confirm that the reaction proceeds only when phenol oxidation and nitrobenzene reduction occur concurrently, providing strong evidence that the transformation follows a paired electrolysis mechanism.\n%\n/\ndleiY\n-1.0 V, 17%\n-0.6 V, N.D. -0.4 V, N.D.\nPontential / V\n• Supporting Information: Electrode polarity switching frequency is reported inconsistently (1, 2, 5 Hz vs 0, 2, 5 Hz in Table X). This should be clarified. Also, indicate if the reaction was performed in a two-compartment cell under constant current without polarity switching.\nResponse: Thanks very much for your comments. The correct electrode polarity switching frequencies are 1, 2, and 5 Hz; the 0 Hz reported in the text was a typographical error. Although all experiments reported in this study were performed under alternating polarity conditions, we also conducted constant-current electrolysis in a two-compartment cell. In these experiments, the target product was successfully detected at the end of the reaction, confirming the viability of the reaction under constant-current conditions.\n• Authors suggest a nitrene intermediate (based on mass spectrometry), but there is no direct evidence of its participation in C–H amination. Observing traces in MS does not confirm it as a key intermediate; the reaction may proceed via classical nitroso/hydroxylamine pathways.\nResponse: Thanks very much for your comments. Nitrosobenzene is a well-established intermediate in the electroreduction of nitrobenzene, and our experiments similarly confirm that it is formed during the reaction. However, existing deuterium-labeled online electrochemical mass spectrometry (EC-MS) experiments do not support the direct participation of nitrosobenzene in the C–H amination coupling. In our proposed mechanism, nitrobenzene is first reduced to nitrosobenzene, which must undergo further reduction to generate a nitrene intermediate before it can participate in the coupling reaction. This proposal is based on two key observations: first, nitrosobenzene can be further reduced to form the nitrene intermediate, and second, the formation of the target product requires this further reduction of nitrosobenzene.\nTo verify this, we performed the following control experiments (Fig. R13):\nA) Replacing nitrobenzene with authentic nitrosobenzene and monitoring the reaction by online EC-MS revealed the same series of intermediates, including the nitrene, confirming that nitrosobenzene can undergo further reduction to generate the nitrene species.\nB) Cyclic voltammetry determined that the nitroso → nitrene reduction occurs at –0.51 V vs Ag/AgCl. Constant-potential electrolysis at –0.40 V or –0.60 V for 4 h yielded no p-hydroxy diphenylamine, while holding the cathode at –1.0 V for 4 h afforded the amination product (17%), indicating that further reduction of nitrosobenzene is necessary for product formation.\nC) Taken together with our previous online EC-MS results, these findings confirm that nitrosobenzene cannot directly participate in the coupling reaction. In the reaction system, nitrobenzene is first reduced to nitrosobenzene, which is subsequently reduced to a nitrene intermediate that undergoes C–H amination to generate the target p-hydroxy diphenylamine.\nFigure R13.A) Intermediates for electrochemical nitrosobenzene coupling reactions by APCI-EC-MS; B) LSV and constant pressure experiments with nitrobenzene; C) Possible nitrobenzene reduction and coupling mechanism\n• The modified electrolysis conditions used for mass spectrometry are very different from bulk electrolysis conditions. It is unclear how the mechanism can be reliably studied under such differing conditions.\nResponse：Thanks for your comments. We acknowledge your concern regarding the modification of electrolysis conditions for compatibility with the mass spectrometry (MS) system and its potential impact on the reaction mechanism. To address this, we undertook a comprehensive investigation to ensure that the underlying mechanism remains consistent despite the altered conditions.\n1. Scale-Up Experiments: We conducted milligram-scale reactions using the modified conditions. Although there was a slight decrease in yield, we successfully obtained a substantial amount of the desired product, indicating that the core reaction remains effective under these adjusted parameters.\n2. Online Mass Spectrometry Detection: Target products were reliably detected using the online MS setup. This demonstrates that the modifications necessary for MS compatibility do not hinder the formation of the intended products.\nFigure R14.General and APCI-MS conditioned control experiments.\nBased on these findings, we conclude that the changes made to accommodate the MS system primarily influence the reaction yield and efficiency. Importantly, they do not alter the fundamental reaction pathway or mechanism. Therefore, we are confident that the mechanistic insights derived under the modified conditions are representative of those under the original reaction conditions.\n• Many products could potentially be accessed more easily via anilines, so the synthetic advantage is unclear.\nThank you for your constructive comments. We have carefully addressed the mechanistic concerns and clarified the presentation of our work. Below, we outline the key advantages of our approach, which we believe offer substantial value to synthetic chemistry:\nFigure R15.Mass Spectrometry Advantages and Expansion Prospects.\n1) Substrate Diversity\nOur reaction system accommodates a wide range of functional groups, including phenol, ethylbenzene, and benzyl ether. It also enables catalyst-free reduction amination of alkyl nitro compounds, which is rarely achieved under mild conditions.\n2) Scalability and Practical Application\nWe have demonstrated gram-scale reactivity, highlighting the robustness and practicality of our method. The preparation of p-hydroxydiphenylamine—an important intermediate and pharmaceutical precursor—illustrates its efficiency. Compared with conventional protocols, our approach lowers raw material costs and avoids halogen waste.\n3) Advanced Mass Spectrometry Integration\nAutomated mass spectrometry accelerates substrate screening while providing precise information on reaction intermediates. This strategy reduces experimental workload and offers clearer mechanistic insights than standard analytical methods.\n4) Real-time Mechanistic Clarifications\nOur study addresses longstanding questions in ortho amination of nitrobenzene and phenol. By optimizing reaction conditions, we expanded the range of compatible substrates without resorting to harsh protocols. This mechanistic understanding lays the groundwork for developing new reactions and refining established methodologies.\n• It is recommended to replicate NMR spectra of 4ab and 4ac to ensure reproducibility and accuracy.\nResponse:Thanks very much for your comments. We have revised accordingly.\nReviewer #4 (Remarks to the Author):\nThe manuscript by Prof Lei and colleagues have described Paired Electrolysis enables para-C-H Amination of Phenols with Nitroarenes to p-Hydroxy Diphenylamines Guided by Multifunctional Electrochemical Mass Spectrometry. There are several key aspects in mass spectrometry that need to be clarified and further verified to enhance the scientific rigor and impact of the research. I recommend accepting the paper after revisions.\n1. Mass spectra with a certain mass range should be provided to facilitate the reader's understanding of the actual situation of the entire reaction. The mass spectra should show isotopic peaks in Figure S4. By observing the intensity and position of the isotopic peaks, more information about the elemental composition of the sample can be observed.\nResponse: Thanks very much for your comments. We have now provided full-scan mass spectra covering the relevant mass ranges in the Supporting Information to offer a comprehensive view of the reaction progress. The updated spectra in Figure S4 clearly display the isotopic peak patterns, allowing readers to verify the elemental composition of the observed intermediates and products. By examining both the intensity and positions of these isotopic peaks, one can gain additional insight into the molecular identities and confirm the assignments made in the work.\nFigure R16. Detected tandem mass spectrometry (MS/MS) data\n2. Discussion of fragmentation processes of tandem mass spectra in Figure 7, Figure S5 and Figure S6should be added in Supporting Information.\nResponse: Thanks very much for your comments. In this study, we employed high-resolution tandem mass spectrometry (MS/MS) to characterize the structures of both products and intermediates.\nFirst, we analyzed and verified the structures of product 4i and intermediate 1i-5 in Figure 7a:\n1. Identification of product 4i: Product 4i undergoes characteristic C–N bond cleavage on the aromatic ring, producing fragment ions C7H7N (m/z 105.0578), C7H8N (106.0657), and C7H9N (107.0735), as well as neutral losses corresponding to methyl (C12H11ON, 185.0841), hydroxyl (C13H12N, 182.0970), and amino (C13H11O, 183.0807) substituents. These fragment ions are fully consistent with the MS/MS data in Figure 7, confirming the product structure.\n2. Identification of intermediate 1i-5: The intermediate 1i-5 exhibits similar C–N bond cleavage on the aromatic ring, indicating the presence of an aniline-type structure. The key differences from 4i are neutral losses at m/z 15.0235, 27.9949, 43.0184, and 55.0058, corresponding to CH3, CO, C2H3O, and C2HON, respectively. These losses are consistent with C–C bond cleavage in a quinone-type structure, suggesting that 1i-5 contains a quinone moiety. Based on the MS/MS spectrum in Figure 7b (left), the detected signal at m/z 198.0918 is attributed to intermediate 1i-5.\nTo further validate the structures of intermediates and products, deuterated reagents were employed:\n1. Validation of 1i-5 using D3-1i: As shown in Figure 7b (right), the deuterated intermediate D3-1i-5 produced fragment ions C7H4D3N (108.0721), C7H5D3N (109.0835), and C7H6D3N (110.0916) upon C–N bond cleavage, consistent with the proposed 1i-5 structure.\n2. Validation of 1i-6 and product 4i using D3-1i: Similar results were observed for D3-1i-derived intermediate D3-1i-6 and product D3-4i, showing the same aniline-type fragment ions, confirming the structures of 1i-6 and 4i (Figure S6).\n3. Validation of 1i-6 using D3-2a: The reaction of D3-2a produced intermediate D2-6, which showed fragment ions C7H8N (106.0657), C7H9N (107.0735), and C7H10N (108.0808) corresponding to the aniline core, along with deuterated methyl (C12H9D2ON, 187.0964), hydroxyl (C13H10D2N, 184.1093), and amino (C13H9D2O, 185.0933) substituents. These results further support the structure of intermediate 1i-6.\nFinally, as shown in Figure S5, we characterized intermediates and products formed during the electrochemical reduction of nitrobenzene:\n1. p-Tolylhydroxylamine: Fragment ions at m/z 109.0522, 107.0490, and 106.0651 correspond to neutral losses of CH3 (15.0235), NO (29.9980), and H2O (18.0106), consistent with the proposed structure (Figure S5a).\n2. Nitrosotoluene: Fragmentation produced m/z 92.0618 via neutral loss of NO (29.9980), consistent with the expected structure (Figure S5b).\n3. Aniline: Fragment ion at m/z 93.0572 corresponds to neutral loss of CH3 (15.0235), consistent with the structure (Figure S5c).\n4. Arylnitrenium rearrangement product: Detected m/z 106.0651 corresponds to a methyl-substituted N-hetero seven-membered ring formed via rearrangement, producing a fragment at m/z 79.0541 via HCN (27.0109) loss, consistent with the proposed N-hetero seven-membered ring structure (Figure S5c, reference: 10.1002/anie.202219302).\nIn summary, these MS/MS analyses, complemented by deuterium labeling, provide strong evidence supporting the structural assignments of both intermediates and products in our study.\n3. Specific experimental operations for offline in Figure 7b should be added in Supporting Information.\nResponse: Thanks very much for your comments. The detection of product 4i was performed offline using high-resolution mass spectrometry (HRMS). Specifically, a solution of acetonitrile:water = 9:1 (v:v) containing 0.2 mM 4i was loaded into the EC-MS system with electrolysis turned off. These experimental details have been added to Section 2.3 of the Supporting Information.\n4. Central to this approach is a multifunctional automated injection electrochemical mass spectrometry (AIEC-MS) platform. Its ionization mode is described as APCI. But from the perspective of Device Setup, what is the difference from ESI? Theoretically, ESI is more reflective of the reaction species than APCI.\nResponse: Thanks very much for your comments.Compared to electrosp ray ionization (ESI), atmospheric pressure chemical ionization (APCI) offers two key advantages for constructing an in situ electrochemical cell in the AIEC-MS platform. First, the APCI process is physically separated from the electrochemical cell, minimizing interference between the electrochemical reaction and the ionization process. Second, APCI is more suitable for detecting and analyzing less polar species, including the nitro and nitroso compounds investigated in this work."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The author has addressed all my concerns. I hereby approve the publication of this article in Nature Communications.", "reviewer_2": "The manuscript can be accepted.", "reviewer_3": "The authors have adequately addressed all of my previous comments, and the revised manuscript is now clearer and more complete. The methodology is sound and meets the expected standards for publication in Nature Communications.", "reviewer_4": "There are several key aspects in mass spectrometry that need to be clarified and further verified to enhance the scientific rigor and impact of the research. My specific comments are as follows.\n1. Mass spectra with a certain mass range should be provided to facilitate the reader's understanding of the actual situation of the entire reaction. This allows readers to have a comprehensive understanding of the entire response system. The mass range should be from 80 to 220 Da. This is because we found no reaction-related substances in these ranges during the initial data processing. Phenol ([M+H]⁺, m/z 95.0491), one of the starting materials, cannot be detected in the positive ion mode of APCI due to its electron-rich nature. To improve the completeness of our data, we hereby provide a comparison of mass spectra before and after the reaction in the range of 80–220 Da.\nFigure R16 should be primary mass spectra for both the electrolysis on and off and a local magnification. Figure R16 is not a tandem mass spectra. Figure R16 also does not give tandem mass spectrometry data.\n2. The ionization of APCI results in a significant increase in fragmentation of certain compounds relative to ESI. It is recommended to increase the contrast between APCI and ESI to exclude that some of these ions are generated in APCI."}, "author_response": "Response: We are grateful for your positive feedback on our work. Your constructive suggestions have significantly contributed to the improvement of our manuscript.\nResponse: We are grateful for your positive feedback on our work. Your constructive suggestions have significantly contributed to the improvement of our manuscript.\nResponse: We are grateful for your positive feedback on our work. Your constructive suggestions have significantly contributed to the improvement of our manuscript.\nResponse: We would like to thank you for your valuable suggestions. Your prior recommendations have provided significant guidance for the mass spectrometry part of our study. We fully endorse your commitment to the rigor of scientific research.\n1. Mass spectra with a certain mass range should be provided to facilitate the reader's understanding of the actual situation of the entire reaction. This allows readers to have a comprehensive understanding of the entire response system. The mass range should be from 80 to 220 Da.\nResponse: Thank you for your question. We indeed did not directly provide the mass spectrometry data in the range of 80–100 Da and 215–220 Da in the manuscript and supporting information. This is because we found no reaction-related substances in these ranges during the initial data processing. Phenol ([M+H]⁺, m/z 95.0491), one of the starting materials, cannot be detected in the positive ion mode of APCI due to its electron-rich nature. To improve the completeness of our data, we hereby provide a comparison of mass spectra before and after the reaction in the range of 80–220 Da.\nFigure R1. HR-MS before and after electrolysis\nFigure R16 should be primary mass spectra for both the electrolysis on and off and a local magnification. Figure R16 is not a tandem mass spectra. Figure R16 also does not give tandem mass spectrometry data.\nResponse: Thank you for your question. As you correctly pointed out, there is an error in the legend of Figure R16. We have now revised it to (HR-MS before and after eletrolysis of reactant 1i, p-nitrotoluene 1, p-hydroxytoluene 2, p-methylaniline 3, N-oxide 4, intermediate 5 and product 4i).\n2. The ionization of APCI results in a significant increase in fragmentation of certain compounds relative to ESI. It is recommended to increase the contrast between APCI and ESI to exclude that some of these ions are generated in APCI.\nResponse: Thank you for your question. We have supplemented the relevant data from the online mass spectrometry experiments using ESI, as shown in Figure R2. It is worth noting that no ion signals related to p-nitrotoluene and p-methylnitrosobenzene were detected by ESI either before or after the reaction. This indicates that such devices cannot effectively detect nitrobenzene and nitrosobenzene derivatives. In contrast, in the APCI data we provided, distinct ion signals were observed for both p-nitrotoluene in the reaction solution before electrolysis and p-methylnitrosobenzene formed after the reaction. Furthermore, for the relatively easily detectable amine intermediates and products, there was only a difference in ion intensity between ESI and APCI. These results confirm that the intermediates we detected are generated from the electrochemical reaction, rather than from the APCI process."}, {"round": 3, "reviewer_comments": {"reviewer_4": "The manuscript can be accepted."}, "author_response": ""}]

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