Nitroreductase-triggered indazole formation

Domain: neuroscience
Published: 2026-02-02
Editorial outcome: minor_revision

Abstract
Biocatalysis contributes significantly to the development of more sustainable synthetic pathways by using mild reaction conditions and water as a solvent. However, many relevant classes of compounds, including privileged groups in drug design, are not yet accessible via enzymatic pathways. In this context, the development of an enzymatic route to indazoles remains an unmet challenge. Here, we present a nitroreductase-triggered indazole formation, in which 2-nitrobenzylamine derivatives are converted to reactive nitrosobenzylamine intermediates that spontaneously cyclize and aromatize to indazoles. Two nitroreductases accept a series of 2-nitrobenzylamine derivatives with excellent conversions (up to >99%). In the case of N-substituted nitrosobenzylamines, 2H-indazoles are formed, whereas other derivatives led to 1H-indazoles. The synthetic value of the nitroreductase-triggered indazole formation is further demonstrated by successful coupling with an imine reductase in a sequential cascade reaction on a 50 mg scale. With this cascade, 2H-indazoles are accessible from cheap 2-nitrobenzaldehyde and primary amines, resulting in up to 85% conversion and 68% isolated yield.

Introduction
It is a well-known phenomenon in drug discovery that many bioactive compounds share the same scaffold 1,2. Among these “privileged groups”, bicyclic N-heterocycles such as benzimidazoles, benzoxazoles, and (tetrahydro)isoquinolines are overrepresented, making them very important targets for synthetic strategies. Consequently, many of these privileged groups have already been accessed via biocatalytic routes, which are often discussed in the context of sustainable synthesis 3–6. However, enzymatic routes have not been described for all N-heterocyclic groups, such as in the case of indazoles. These molecules are found in many commercially successful drugs, such as granisetron, axitinib, niraparib and pazopanib, which are used to treat nausea and cancer (Fig. 1A). The 1H- and 2H-tautomers of indazoles are equally important for drug development. In the case of the N-unsubstituted indazoles, the 1H-tautomer dominates because it is more stable 7–9. Due to the importance of both indazole forms, several synthetic routes have been developed for the preparation of 1H- and 2H-indazoles (Fig. 1B) 9. The most commonly used synthetic strategies for the synthesis of 1H-indazoles include acylation, nitrosation, and cyclization of o-toluidine 10 and intramolecular amination of 1-aryl-2-(2-nitrobenzylidene)hydrazines 11. The Cu-catalyzed sequential condensation of 2-bromobenzaldehyde, a primary amine and azide 12 and the palladium-catalyzed cyclization of N-aryl-N-(o-bromobenzyl)hydrazines are used to form 2H-indazoles 13. Furthermore, organophosphorus-catalyzed pathways for forming 2H-indazoles were developed starting from 2-nitrobenzaldehydes and primary amines 14,15. 3-Alkoxy-group-functionalized 2H-indazoles can be generated via the classical, base-catalyzed Davis-Beirut reaction. This reaction relies on an intramolecular, redox reaction of 2-nitrobenzylamines, followed by the addition of an alcohol at the 3-position of the indazole ring 16. A photochemical acid-catalyzed variation has been established that make these products accessible from 2-nitrobenzyl alcohols, primary amines, and alcohols 17. Additionally, a Lewis acid-catalyzed approach for the synthesis of 2H-indazoles from imines of 1-(2-aminophenyl)ethenone derivatives has been published recently 18. These traditional chemical and electrochemical methods to prepare indazoles suffer from drawbacks such as the need for hazardous hydrazine derivatives, the use of noble metal catalysts or redox cells, and the employment of often harsh reaction conditions (Fig. S1).

Enzyme catalysis can be an attractive alternative for the synthesis of 1H- and 2H-indazoles, especially to provide a synthetic route with a lower environmental impact. At the same time, it can facilitate the expansion of the indazole synthesis toolbox and enable enzymatic cascades, e.g., for further diversification of indazoles by enzymatic N-methylation 19. To date, however, the enzymatic synthesis of indazoles remains an unmet challenge, despite the presence of indazoles in natural products 20–23. Nitroreductases (NRs) are known to catalyze the reduction of nitro (-NO2) compounds to amines (-NH2) via nitroso (-NO) and hydroxylamine (-NHOH) intermediates 24. The reaction catalyzed by NRs occurs sequentially, which is why in some cases nitroso or hydroxylamine intermediates can be observed 25–30. Recently, a photoenzymatic system was developed allowing the NR-catalyzed reaction of aromatic nitro compounds to be directed selectively to amines, azoxy or azo compounds, depending on the reaction conditions 25. Without the assistance of the photocatalytic system, the nitroso intermediate was found to be most difficult to reduce by the NR BaNTR1 from Bacillus amyloliquefaciens, leading to a significant accumulation of this intermediate. Inspired by the possibility of generating reactive nitroso intermediates that can lead to spontaneous N-N bond formation 31, we hypothesize that an NR-based reaction could potentially lead to selective indazole formation. This requires the generation of reactive nitrosobenzylamine intermediates that spontaneously form the desired indazole scaffold when the cyclization reaction occurs faster than the enzymatic reduction.

Herein, we report a NR-triggered indazole formation based on the direct two-electron reduction of 2-nitrobenzylamines to reactive nitrosobenzylamine intermediates, which spontaneously cyclize and aromatize to the desired indazoles with excellent conversions (up to >99%, Fig. 1C). To further demonstrate the synthetic value of this route, we successfully coupled an imine reductase (IRED15) to the NR-triggered indazole formation in a sequential cascade reaction. With this cascade, 2H-indazoles were accessible from cheap 2-nitrobenzaldehyde and primary amines with up to 85% conversion and 68% isolated yield. This approach not only expands the reaction repertoire of flavin-dependent NRs, but also adds to the available enzyme toolbox for the challenging task of indazole synthesis via ring-closing N-N bond formation in a single enzyme-catalyzed reaction step.

Results
Setting up the NR-triggered indazole reaction
We began our investigations by screening an in-house panel of NRs (BaNTR1 from Bacillus amyloliquefaciens, NfsA from Escherichia coli, BmoNR from Bacillus mojavensis, and BpNR from Blautia producta) using 2-nitrobenzylamine (1a) as a substrate 25,27. In the first experiment using BaNTR1, it was found that within 1 hour substrate 1a was mostly depleted. At the same time, none of the potential products 2a, 3a, and 4a could be identified with gas chromatography-mass spectrometry (GC-MS) analysis. Instead, it was found that 1H-indazole (6a’) was formed (Figs. 2A and 2B right, Fig. S2). The same was found for NfsA and BmoNR, while BpNR was not active towards 1a (Fig. 2B left, Figure S3). 2H-indazole (6a), which is formed according to the proposed mechanism, was not detected due to its quick tautomerization to the more stable 1H-form 6a’ 7–9.

Control experiments confirmed that the reaction proceeded solely in the presence of BaNTR1, NfsA and BmoNR (Fig. 2B). In addition, we demonstrated that 2a cyclizes to indazole by titrating base to the reaction, thereby maintaining a stable pH value. This approach enabled us to monitor the gluconic acid formation from the cofactor regeneration system and the protons released by the reaction itself (Fig. S25 and Supporting Methods). It is known that the cyclization of 2a can occur spontaneously in an acidic aqueous/methanolic solution 32, and that NRs release their intermediates before the next reduction step 30. However, we cannot rule out the possibility that cyclization is catalyzed by the enzyme even though cyclization would most likely also occur spontaneously. The turnover number (moles × moles -1) of BaNTR1, NfsA and BmoNR was determined to be around 240, aligning well with the generally high catalytic activity of NRs 25,27. The two most active enzymes, BaNTR1 and NfsA, were used for all subsequent experiments. At the same time, BaNTR1 and NfsA were used as representatives of NADH-dependent and NADPH-dependent NRs, respectively, ensuring that an enzyme with a suitable cofactor preference was available for subsequent cascade development with other oxidoreductases.

Reaction optimization
Next, we further investigated the NR-triggered conversion of 1a to 6a’. First, we investigated whether the NR and glucose dehydrogenase (GDH) concentrations in the reaction with 1a could be lowered without prolonging the reaction time required for a good conversion (Fig. 3). Reducing the Bacillus megaterium GDH (BmGDH) concentration from 8 µM to 0.8 µM had no effect on the course of the reaction, which led us to reduce the BmGDH concentration to 1.6 µM for all further experiments. In contrast, reducing the concentration of NfsA or BaNTR1 resulted in a significant change in the product formation after 1 hour. Considering that some substrates might lead to lower activity when screening the substrate range, the standard concentration of NR was kept at 20 µM.

In addition, we found that the 50 mM phosphate buffer at pH 7.0 used in all previous experiments was not sufficient to prevent the buffer from going into the acidic range during the reaction. This was caused by the gluconic acid formed during the cofactor regeneration cycle. By increasing the buffer strength to 200 mM and raising the pH to 8.0, it was possible to achieve higher or even complete conversion to 6a’ with BaNTR1 and NfsA, respectively (Fig. S4).

To prove the concept, we performed the NR-triggered indazole formation reaction with resting cells and lysate (Figs. S22 and S23). The reactions using lysate reached 96% conversion after 4 hours, whereas the reactions with resting cells did not exceed 38% at the same time. This indicates that cofactor regeneration in resting cells or diffusion through the membrane limits the reaction rate. Although whole cell or lysate formulations have advantages for potential industrial applications, we continued using purified enzymes to ensure that any observed biotransformation originates from the NR and not the host background.

Substrate scope analysis
Having established and optimized the NR-triggered indazole formation reaction, we explored the substrate scope of NfsA and BaNTR1 for this transformation (Fig. 4 and Figs. S4–12). A conversion of >99% was achieved for substrates 1a-f and 1l in the case of NfsA. The enzymes generally tolerate polar, non-polar and bulky R3 substitutions, whereas a methyl group on R2 strongly reduces activity. Although the indazole products are not chiral, both enzymes seem to prefer the (S)-enantiomer as a substrate, indicated by the fact that the conversion of (rac)-1g is faster compared to (R)-1g. Nevertheless, at least in the case of NfsA, conversions of more than 50% were achieved in the reaction of (rac)-1g, proving that both enantiomers can be converted. This behavior is advantageous in this particular case because the product of both enantiomers is accessible and thus racemic mixtures can be used. The substrate 1h did not lead to the expected products, as only the decarboxylated product (6a’) was observed in this reaction. It is possible that decarboxylation is favored over deprotonation in the aromatization reaction of 5h. As expected, the R3-substituted substrates lead to 2H-indazoles, while the R3-unsubstituted substrates (1a, 1g and 1i-o) lead to 1H-indazoles. For substrates substituted at the aromatic ring (1i-o), steric hindrance seems to play an important role. Although NfsA and BaNTR1 accept the 4-chloro- and 4-methoxy-substituted substrates 1l and 1m, both enzymes are less active on the 3-chloro- and 3-methoxy-substituted substrates 1n and 1o. However, contrary to the general trend, BaNTR1 exhibits higher activity than NfsA with 1n and 1o. Interestingly, the 5-bromo- and 5-chloro substituted substrates (1i and 1j) are well converted to the corresponding 1H-indazoles. However, when 1k was used as the substrate, only small amounts of 6k’ were produced and the substrate disappeared overnight. At the same time, the reaction mixture and its extract turned violet; however, no new products were observed on the gas chromatography (GC) chromatogram. This indicates that reactive nitrogen species may have formed oligomers, which is also supported by the observation of precipitate formation over time. This indicates that cyclization is hindered by the strong electron-donating effect of the para-methoxy substitution, which reduces the electrophilicity of the nitroso group.

Gas chromatography (GC) and high-resolution mass spectrometry (HR-MS) were performed on all products (Figs. S27–40 and S42–51) to confirm their identity. In addition, the reactions were scaled up to a 20 mg scale for NMR analysis (Figs. S57–79). However, it was found that the products partially evaporated upon prolonged drying in vacuum. Therefore, it was decided to avoid extensive drying of the compounds, which led to solvent residues in the NMR analysis. The isolated yields shown in Fig. 4 were calculated taking into account the remaining solvents (Table S1).

We speculated that the NR-triggered cyclization could be extended towards the formation of 6-membered ring systems, leading us to investigate the formation of cinnoline to further demonstrate the synthetic potential of our strategy. Thus, we used 2-(2-nitrobenzyl)ethanamine (7) to explore the NfsA-triggered cinnoline formation. However, the 3,4-dihydrocinnoline (9) formed from the intramolecular condensation of 2-(2-nitrosobenzyl)ethanamine (8) must first undergo tautomerization to 1,4-dihydrocinnoline (10) and oxidation to the final product cinnoline (11) 33. The last step in particular was not fully completed after 24 hours, resulting in a mixture of 45% 10 and 40% 11 with only traces of 9 and 14 % 7 remaining (Fig. 5). In the reaction with BaNTR1, 89% of substrate 7 remained, while 6% 10 and 4% 11 were formed. This experiment demonstrated that cinnolines can also be accessed via the presented NR-triggered route. Overall, NfsA and BaNTR1 showed the same reactivity towards all substrates tested, with NfsA always being more active.

Kinetic and mechanistic investigations
This synthesis route for indazoles is crucially dependent on enzymatic activity. The direct two-electron reduction of nitro to nitroso compounds can be achieved by NRs. However, the high selectivity of the reaction compared to spontaneous indazole formation is remarkable and the selectivity is in good agreement with our previous observation that the reduction of nitroso intermediates is poorly performed by BaNTR1 25. The fact that no over-reduction side products 3 and 4 were isolated under the reaction conditions used indicates that the spontaneous cyclization/aromatization of compounds 2 to 6 proceeds rapidly. This is consistent with the finding that nitroso compounds (2) were never detected in our reaction mixture. Although the instability of 2 prohibits kinetic studies, the affinity of NfsA and BaNTR1 for 2 might be low, as suggested by kinetic studies with 1a. The apparent Michaelis-Menten constant (KM,app) values for NAD(P)H of 58 ± 19 µM and 41 ± 9 µM for NfsA and BaNTR1 (Figs. S13 and S15), respectively, are in the typical range for NRs 26,34,35. However, the KM,app values for 1a are very high compared to the KM,app value of bacterial nitroreductases with common substrates, such as nitrofurazone, which are below 1 mM 26,36,37. A KM,app value of 10.8 ± 2.9 mM was determined for BaNTR1 (Fig. S15), while the determination for NfsA was not possible because the correlation between indazole formation and substrate concentration was still linear up to 20 mM (Fig. S17). Therefore, the KM,app value of NfsA towards 1a is more than three orders of magnitude higher than the KM,app value for nitrofurazone (5.5 µM 36). It was not possible to use higher substrate concentrations in the kinetics, because at substrate concentrations above 20 mM, small amounts of the over-reduced product 4a could be detected. These over-reduction products would distort the indazole formation rate. The unusually high KM,app values of NfsA and BaNTR1 for the nitro substrates 1a in combination with the finding that 4a formation could only be observed at higher substrate concentrations and thus higher formation of nitroso intermediates suggest that the affinity for the nitroso compounds is also low. This explains the complete selectivity towards indazole formation via the spontaneous cyclization/aromatization reaction.

Cascade reaction—proof-of-concept
One of the major advantages of enzymatic reactions over chemical reactions is that they typically occur under mild aqueous reaction conditions at neutral pH, which also facilitates the development of enzymatic cascade reactions without an intermediate work-up step. Having demonstrated the versatility and efficiency of NR-triggered indazole formation, we decided to further showcase its synthetic utility by developing an enzymatic cascade for the facile synthesis of indazoles from cheap and readily available building blocks. Thus, we envisioned a sequential enzymatic cascade using imine reductases (IREDs) or reductive aminases (RedAms) as well-established biocatalysts to form secondary amines from a carbonyl and a primary amine compound 38–42. These enzymes could provide the 2-nitrobenzylamines required for the NR-triggered indazole formation from cheap and readily available 2-nitrobenzaldehyde and a primary amine (Fig. 6). Furthermore, one-pot enzymatic cascades including IREDs and NRs have recently been shown to be highly efficient for synthesizing heterocyclic pharmaceutical building blocks 43. To prove this concept, IREDs from Mycobacterium smegmatis (IRED10), Actinomadura rifamycini (IRED15), Streptomyces tsukubaensis (IRED20), Streptomyces viridochromogenes (IRED23) and the RedAm from Aspergillus oryzae (AspRedAm) were investigated for the generation of 1b from 2-nitrobenzaldehyde (12) and methylamine (13) (Fig. 6A).

It was found that all selected enzymes were able to convert 12 and 13 into 1b, with IRED15 and IRED23 showing the highest conversions (Fig. 6B). The reduction of substrate 12 to 2-nitrobenzyl alcohol (15) was observed, which is a known side reaction of the IREDs and AspRedAm 40. For further cascade optimization and upscaling to preparative scale, IRED15 was selected for further experiments. This arose from the fact that the expression of IRED15 at 16.22 µmol/L was much better than the expression of IRED23 at around 0.16 µmol/L. Given the high expression yield, the concentration of IRED15 was increased to 40 µM. This resulted in over 80% conversion after 24 h, and 86% after 48 h (Fig. 6C). When 10 µM NfsA was added to the reaction after 48 h, more than 90% of 6b was formed within 3 h (Fig. 6D). The residual percentages mostly consist of small amounts of unidentified side products that may have arisen from the nitro reduction of 12, 14 and 15.

Cascade reaction—optimization
During the initial upscaling attempts to the 50-mg scale, the first reaction step was observed to occur much more slowly than in analytical-scale reactions, and the NfsA reaction step began only after further BmGDH was added (Fig. 7A). We concluded that BmGDH was inactivated during the initial reaction step, which caused the slower reaction in the upscaled reactions. Therefore, we investigated the influence of temperature, buffer composition, pH and NaCl concentration on the IRED15-catalyzed conversion of 12 and 13 to 1b. Interestingly, increasing the temperature from 25 °C to 30 °C decreased the conversion after 24 h, even though IRED15 was previously used at 30 °C 42, again indicating that the stability of the cofactor regeneration system limits the reaction (Fig. S18). Since BmGDH is unstable at higher pH values 44, the reaction was investigated at pH 7.5 (Fig. S18). However, similar conversions to 1b were achieved, while more side product 15 was formed. Replacing the phosphate-based buffer with tris(hydroxymethyl)aminomethane (Tris) and the cosolvent acetonitrile with dimethyl sulfoxide (DMSO) decreased the reaction conversions (Fig. S19). Although high NaCl concentrations are expected to increase BmGDH stability 44, only an increase in the formation of side product 15 was observed (Fig. S20). To overcome the limitations of the BmGDH-based cofactor regeneration system, we replaced it with a cofactor recycling system based on a mutant formate dehydrogenase (FDH) from Candida boidinii (FDH-M4) 45. Similar conversions were achieved in the IRED15 reaction after 24 h (Fig. S21). However, due to the comparably high concentrations of FDH-M4 required (62 µM), we decided to continue with the BmGDH-based cofactor regeneration system. Nevertheless, the linear progression of the reaction using the FDH-M4-mediated cofactor regeneration indicates that the cofactor regeneration system is rate-limiting. Thus, for the upscaling reaction at a scale of 50 mg, the IRED15 concentration was reduced to 20 µM, and an additional 1.6 µM dose of BmGDH was added after 6 h of reaction time and before the addition of NfsA (after 20 h). Furthermore, to avoid air bubbles that had been observed in the shake-flask, the reactions were split into three 50 mL reaction tubes. Using this optimized system, 87% conversion to 1b was achieved after the first 20-h reaction step, and 85 % conversion to the final product (6b) was achieved after a total reaction time of 24 h with an isolated yield of 68% (30 mg, Figs. 7B, 8).

Cascade reaction—extended substrate scope
To showcase the substrate scope of the cascade reaction, we performed the same reaction with allylamine (16) and propargylamine (17) as the amine substrate instead of 13. While 83% overall conversion (67% isolated yield, 35 mg) was achieved with 16 (Figs. 7C, 8), only 20% conversion (4 % isolated yield, 2 mg) was reached with 17 due to observed protein precipitation (Figs. 7D, 8). In all these cascades, NR-triggered indazole formation was shown to be highly robust, completely converting the available substrates (1a, 1p and 1q) within a 4-h reaction time. This underlines the potential of NR-triggered indazole formation in cascade reactions.

Overall, we demonstrated that an IRED-NR cascade can efficiently be used in a modular synthesis approach for indazoles, in which the nitrogen atoms in the nitrogen-nitrogen bond do not originate from the same molecule. This not only avoids the use of metal catalysts and harsh conditions, but also the use of nitrogen-nitrogen bonded building blocks such as hazardous hydrazine derivatives or azides. Thus, the biocatalytic route reported herein not only expands the existing synthetic toolbox, but also represents an alternative to chemical and electrochemical methods, overcoming sustainability concerns and providing access to indazole derivatives.

Methods
Protein expression
The enzymes BaNTR1 (Bacillus amyloliquefaciens, UniProt accession number: A0AAW3IFH5), NfsA (Escherichia coli, UniProt accession number: P17117), BmoNR (Bacillus mojavensis, UniProt accession number: A0AAP3FXD2), and BpNR (Blautia producta, UniProt accession number: A0A7G5MPE8) were expressed 25. E. coli BL21 (DE3) cells harboring the pET21a plasmid encoding the NR gene were grown in 1 L of 2xYT medium supplemented with 100 µg/mL ampicillin (100 mg/mL stock) at 37 °C. Upon reaching an OD600 of 0.6–0.8, protein expression was induced with 0.3 mM IPTG (1 M stock), and the culture was incubated overnight at 18 °C. Cells were harvested by centrifugation, resuspended, and lysed by sonication. The resulting clarified cell lysate (CFL) was incubated with Ni-NTA agarose beads for 3 h at 4 °C with gentle rotation. The mixture was then loaded onto a gravity flow column, and the bound protein was eluted using appropriate buffers. To replenish any FMN cofactor lost during purification, the eluted enzyme was incubated with 1 mM FMN (10 mM stock) for 3 h at 4 °C with gentle rotation. Finally, the purified enzyme was desalted and buffer-exchanged using PD-10 desalting columns (GE Healthcare).

The expression of FDH-M4, IRED10, IRED15, IRED20, IRED23 and AspRedAm was performed on the basis of a previously described protocol 42,45. The proteins were expressed in E. coli BL21 (DE3) strains containing pET16-, pET22- or pET28-based plasmids with the genes of FDH-M4, IREDs or AspRedAm, respectively. Cultures were grown at 37 °C (180 rpm) in 1 L lysogeny broth (LB) containing 100 µg/mL ampicillin (100 mg/mL stock) or 50 µg/mL kanamycin ampicillin (50 mg/mL stock). Prior to induction at an OD600 of 0.6, the culture was cooled to 20 °C. After induction with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (1 M stock) the culture was further incubated overnight (20 h) at 20 °C. After harvesting, the proteins were lysed by sonication and purified using the His-tag. The elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 250 mM imidazole, pH 8.0) was replaced with a storage buffer (50 mM sodium phosphate with 300 mM sodium chloride, 10 % glycerol, pH 8.0) using PD-10 desalting columns (GE Healthcare, Chicago, IL, USA).

Substrate scope screening
All solutions were prepared in 200 mM sodium phosphate buffer (NaPi) with 300 mM sodium chloride pH 8.0, unless otherwise stated. The total volume of the reactions was 1 mL and 1.5 mL reaction tubes were used. 20 µM NR (NfsA: 650 µM stock; BaNTR1 325 µM stock), 1.6 µM BmGDH (640 µM stock), 50 mM D-glucose (1 M stock) and 0.5 mM NAD+ or NADP+ (10 mM stock) were used. The reaction was started by adding 5 mM substrate 1a-o or 7 from a 10 mM stock solution. Only 1d was added from a 200 mM stock solution in acetonitrile. For solubilization, acetonitrile was added to the reactions to a final concentration of 2.5 % (v/v), and incubated for 24 h at 25 °C and 1000 rpm. Time samples of 100 µL were taken after 0, 0.5, 1, 2, 4 and 24 h. The time samples were quenched by adding a spatula of potassium phosphate (KPO4) and then extracting three times with 100 µl ethyl acetate. For substrates 1b-g and 1i-n, 2 mM 1H-indazole was added to ethyl acetate used as an internal standard. The organic phases were combined and dried over magnesium sulfate before analysis by gas chromatography (GC, see Supplementary information). The conversions of analytical scale reactions are calculated based on the integrals of the GC-FID analysis. Preparative reactions (20 mg scale) for substrates 1a-g, 1i, 1j and 1l-n were performed (see Supplementary Information).

2-Nitrobenzylamine (1a). 0.150 mmol (28 mg) of 1a·HCl were used as a starting material and 0.110 mmol (13 mg, 73 %) of product 6a’ were obtained as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 10.51 (s, 1H), 8.11 (s, 1H), 7.78 (d, J=8.1 Hz, 1H), 7.51 (d, J=8.4 Hz, 1H), 7.40 (t, J=7.7 Hz, 1H), 7.18 (t, J=7.5 Hz, 1H). GC-MS (EI, 70 eV): m/z = 118 (M+, 100), 91 (31), 63 (41), 52 (19). Furthermore, the identity was confirmed by using an authentic standard.

N-Methyl-2-nitrobenzylamine (1b). 0.099 mmol (20 mg) of 1b·HCl were used as a starting material and 0.053 mmol (7 mg, 53 %) of product 6b were obtained as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H), 7.69 (d, J=8.7 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H), 7.29 (d, J=7.5 Hz, 1H), 7.08 (t, J=7.6 Hz, 1H), 4.22 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 149.1, 126.2, 123.8, 122.1, 121.9, 120.1, 117.2, 40.4. GC-MS (EI, 70 eV): m/z = 132 (M+, 100), 104 (24), 63 (24), 51 (18). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 133.0760, found: 133.0758.

N-Ethyl-2-nitrobenzylamine (1c). 0.107 mmol (23 mg) of 1c·HCl were used as a starting material and 0.055 mmol (8 mg, 52 %) of product 6c were obtained as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 7.93 (d, J=1.0 Hz, 1H), 7.74–7.70 (m, 1H), 7.65 (dt, J=8.5, 1.1 Hz, 1H), 7.28 (ddd, J=8.7, 6.6, 1.1 Hz, 1H), 7.08 (ddd, J=8.4, 6.6, 0.9 Hz, 1H), 4.49 (q, J=7.3 Hz, 2H), 1.64 (t, J=7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 148.8, 126.0, 122.0, 121.7, 120.2, 117.4, 48.6, 16.0. GC-MS (EI, 70 eV): m/z = 146 (M+, 40), 118 (100), 91 (20), 63 (29), 51 (15). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 147.0917, found: 147.0915.

N-Propyl-2-nitrobenzylamine (1d). 0.101 mmol (20 mg) of 1d were used as a starting material and 0.091 mmol (15 mg, 90 %) of product 6d were obtained as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J=1.0 Hz, 1H), 7.71 (dq, J=8.8, 0.9 Hz, 1H), 7.65 (dt, J=8.4, 1.1 Hz, 1H), 7.33–7.23 (m, 1H), 7.08 (ddd, J=8.3, 6.6, 0.9 Hz, 1H), 4.38 (t, J=7.1 Hz, 2H), 2.10–1.99 (m, 2H), 0.95 (t, J=7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 148.9, 125.9, 122.8, 121.8, 121.7, 120.2, 117.4, 55.5, 24.1, 11.3. GC-MS (EI, 70 eV): m/z = 160 (M+, 46), 131 (92), 118 (100), 91 (25), 77 (23), 63 (22), 51 (22). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 161.1072, found: 161.1073.

N-Cyclohexyl-2-nitrobenzylamine (1e). 0.068 mmol (21 mg) of 1e·HBr were used as a starting material and 0.045 mmol (9 mg, 66 %) of product 6e were obtained as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 7.95 (d, J=1.0 Hz, 1H), 7.72 (dd, J=8.8, 1.1 Hz, 1H), 7.65 (dt, J=8.5, 1.1 Hz, 1H), 7.33–7.21 (m, 1H), 7.15–6.99 (m, 1H), 4.49–4.33 (m, 1H), 2.34–2.18 (m, 2H), 2.05–1.83 (m, 4H), 1.85–1.71 (m, 1H), 1.59–1.39 (m, 2H), 1.41–1.27 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 148.3, 125.7, 121.5, 120.3, 120.2, 117.5, 63.0, 34.1, 25.6, 25.4. GC-MS (EI, 70 eV): m/z = 200 (M+, 23), 131 (13), 118 (100), 91 (16), 55 (23). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 201.1386, found: 201.1382.

(2-Nitrobenzyl)hydrazine (1f). 0.100 mmol (20 mg) of 1f·HCl were used as a starting material and 0.065 mmol (9 mg, 65 %) of product 6f were obtained as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 7.99 (s, 1H), 7.63 (t, J=9.2 Hz, 2H), 7.34 (dd, J=8.8, 6.7 Hz, 1H), 7.12 (t, J=7.6 Hz, 1H), 4.28 (s, 4H). 13C NMR (101 MHz, CDCl3) δ 145.4, 127.3, 123.2, 122.4, 120.5, 120.5, 116.3. GC-MS (EI, 70 eV): m/z = 133 (M+, 87), 118 (14), 104 (100), 78 (34), 63 (46), 51 (68). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 134.0713, found: 134.0713.

1-(2-Nitrophenyl)ethan-1-amine (1g). 0.100 mmol (20 mg) of 1g·HCl were used as a starting material and 0.053 mmol (7 mg, 53 %) of product 6g’ were obtained as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J=8.1 Hz, 1H), 7.49 (d, J=8.4 Hz, 1H), 7.41 (t, J=7.6 Hz, 1H), 7.17 (t, J=7.5 Hz, 1H), 2.65 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 142.7, 140.8, 127.9, 122.3, 121.0, 120.5, 110.4, 11.8. GC-MS (EI, 70 eV): m/z = 132 (M+, 100), 104 (20), 77 (21), 63 (30), 51 (39). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 133.0760, found: 133.0759.

5-Bromo-2-nitrobenzylamine (1i). 0.100 mmol (27 mg) of 1i·HCl were used as a starting material and 0.055 mmol (11 mg, 55 %) of product 6i’ were obtained as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.92 (dd, J=1.7, 0.8 Hz, 1H), 7.48 (dd, J=8.8, 1.8 Hz, 1H), 7.41 (dd, J=8.7, 0.9 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 138.8, 134.2, 130.2, 125.0, 123.5, 114.3, 111.4. GC-MS (EI, 70 eV): m/z = 198 (78), 196 (M+, 83), 117 (69), 90 (100), 63 (65). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 196.9705, found: 196.9709.

5-Chloro-2-nitrobenzylamine (1j). 0.100 mmol (22 mg) of 1j·HCl were used as a starting material and 0.075 mmol (11 mg, 75 %) of product 6j’ were obtained as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.75 (d, J=1.9 Hz, 1H), 7.45 (d, J=8.7 Hz, 1H), 7.36 (dd, J=8.9, 1.9 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 138.5, 134.5, 127.8, 126.8, 121.5, 120.3, 111.0. GC-MS (EI, 70 eV): m/z = 152 (M+, 100), 125 (35), 90 (35), 63 (43). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 153.0214, found: 153.0214.

4-Chloro-2-nitrobenzylamine (1l). 0.100 mmol (22 mg) of 1l·HCl were used as a starting material and 0.077 mmol (12 mg, 77 %) of product 6l’ were obtained as a yellowish solid. 1H NMR (400 MHz, CD3OD) δ 8.04 (d, J=1.7 Hz, 1H), 7.74 (dd, J=8.6, 0.9 Hz, 1H), 7.55 (dd, J=1.8, 0.9 Hz, 1H), 7.12 (dd, J=8.6, 1.7 Hz, 1H). 13C NMR (101 MHz, CD3OD) δ 140.7, 133.7, 132.7, 121.8, 121.6, 121.5, 109.5. GC-MS (EI, 70 eV): m/z = 152 (M+, 100), 125 (33), 90 (38), 63 (47). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 153.0214, found: 153.0214.

4-Methoxy-2-nitrobenzylamine (1m). 0.100 mmol (22 mg) of 1m·HCl were used as a starting material and 0.079 mmol (12 mg, 79 %) of product 6m’ were obtained as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 7.99 (s, 1H), 7.61 (d, J=8.9 Hz, 1H), 6.89–6.80 (m, 2H), 3.87 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 159.9, 141.5, 134.8, 121.7, 118.0, 113.5, 90.7, 55.6. GC-MS (EI, 70 eV): m/z = 148 (M+, 100), 133 (84), 105 (47), 78 (18). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 149.0709, found: 149.0709.

3-Chloro-2-nitrobenzylamine (1n). 0.100 mmol (22 mg) of 1n·HCl were used as a starting material and 0.058 mmol (9 mg, 58 %) of product 6n’ were obtained as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 8.20 (s, 1H), 7.69 (dd, J=8.0, 0.8 Hz, 1H), 7.40 (dd, J=7.5, 0.8 Hz, 1H), 7.13 (dd, J=7.8, 7.8 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 138.3, 135.8, 126.2, 124.8, 122.0, 119.7, 115.9. GC-MS (EI, 70 eV): m/z = 152 (M+, 100), 125 (17), 117 (20), 90 (27), 63 (17). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 153.0214, found: 153.0214.

Kinetics
For the kinetic studies of BaNTR1 and NfsA towards 1a, a similar biocatalytic setting was used as for the substrate scope screening. The enzyme concentration and reaction time were reduced to 1 µM and 0.4 µM and 45 min and 30 min for BaNTR1 and NfsA, respectively. Also, no acetonitrile was added and the reaction volume was reduced to 0.2 mL. The concentrations of 1a between 0.5 mM and 25 mM and 2.5 mM to 20 mM were investigated for BaNTR1 and NfsA, respectively. No cofactor regeneration system was used for the kinetic studies of NADH for BaNTR1 and NADPH for NfsA. The concentration of the enzyme was lowered to 0.2 µM and the reaction time was set to 10 min. NADH/NADPH concentrations between 0 and 500 µM and 1 mM of 1a were used. Kinetics parameters were calculated using OriginPro 2020 (OriginLab Corporation, Northampton, MA, USA) with a nonlinear Michaelis-Menten function and an orthogonal distance regression.

IRED and RedAm screening
In order to find a suitable enzyme for the formation of 1b from 12 and 13, AspRedAm and the IREDs 10, 15, 20, and 23 were investigated. In a 1.5 mL reaction tube, 0.5 mL reaction mixture consisting of 5 mM 12 (200 mM stock in acetonitrile), 20 mM 13 (200 mM aqueous stock, pH 8), 0.5 mM NADP+ (10 mM stock), 50 mM D-glucose (1 M stock), 1.6 µM BmGDH, 20 µM IRED/AspRedAm were prepared in 50 mM sodium phosphate buffer (pH 8.0) with 300 mM NaCl and 10 % (v/v) glycerol. The reaction was started by adding the IREDs or AspRedAm and incubated for 24 h at 25 °C and 1000 rpm in a shaker (ThermoMixer C, Eppendorf SE, Hamburg, Germany). 100 µL of the reaction mixture was added to another reaction tube containing 200 µL ethyl acetate and 50 µL saturated potassium phosphate solution (KPO4). The reaction was vortexed, centrifuged and the organic phase dried over magnesium sulfate prior to gas chromatographic analysis (see Supplementary Information).

Cascade reaction
The analytical-scale cascade reaction of IRED15 and NfsA was performed in a sequential manner. First, a reaction of 0.5 mL was prepared as in the IRED screening described above, but using 40 µM IRED15. After 4, 24, and 48 h, samples of 100 µL were taken and analyzed as described above. After analyzing the 48 h-time samples, 10 µM NfsA was added and incubation was continued for a further 3 h. A sample of 100 µL was analyzed using the method described above.

The cascade reaction of IRED15 and NfsA was scaled up to the preparative scale (50 mg). For this purpose, 3.31 mL 1 M D-glucose solution, 3.31 mL 10 mM NADP+ solution, 333 µL of 1 M 12 (in acetonitrile, 0.331 mmol, 50 mg), 6.62 mL amines 13, 16, or 17 (200 mM aqueous stock at pH 8.0), 918 µL acetonitrile and 43.6 mL reaction buffer (50 mM sodium phosphate buffer, 300 mM NaCl, 10 % glycerol, pH 8.0) were added to a 200 mL Erlenmeyer flask. After mixing, the reaction of about 60 mL was divided into three equal portions and incubated in 50 mL reaction tubes. The pH was checked before the reaction started. Then 6.62 mL (3×2.21 mL) IRED15 (400 µM stock, 20 µM final conc.) was added. 495 µL (3×165 µL) of BmGDH solution (228 µM stock, 1.6 µM) was added at 0, 6 and 20 h. After 20 h 945 µL NfsA (700 µM stock, 10 µM final conc.) was added. The reaction was incubated at 25 °C and 180 rpm (New Brunswick Innova 42, Eppendorf SE, Hamburg, Germany) for overall 24 h. Time samples were taken after 4, 20, and 24 h. Each reaction tube was extracted five times with 10 mL of methyl tert-butyl ether (MTBE). The combined organic phases were evaporated in the presence of silica and purified by medium-performance liquid chromatography (MPLC). The MPLC separation was performed using a Büchi MPLC system (C-815 Flash, Flawil, Switzerland) equipped with a Flashpure EcoFlex silica column (4 g, 50 µm irregular). The mobile phase consisted of n-heptane and MTBE. Gradient elution began with 5% MTBE, which was maintained for 5 min, followed by a gradual increase in MTBE concentration to 50% over 30 min. The flow rate was set to 20 mL/min. Detection was performed using a UV detector at 254 nm. The fractions containing the desired product were combined and dried with a rotary evaporator.

The cascade reaction described above already utilizes the optimized reaction conditions. In comparison, the initial cascade was carried out in a 200-mL shaking flask, with a reaction time of 48 h for the first step and 24 h for the second step. During the first reaction step, 40 µM IRED15 was used. The second step was supposed to start with the addition of 10 µM NfsA, but the final product 6b only formed after the addition of another 1.6 µM BmGDH.

Methylamine (13). 30 mg (0.224 mmol, 68 %) of product 6b were obtained as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 7.89 (d, J=0.9 Hz, 1H), 7.70 (dq, J=8.7, 0.9 Hz, 1H), 7.64 (dt, J=8.4, 1.1 Hz, 1H), 7.28 (ddd, J=8.7, 6.6, 1.1 Hz, 1H), 7.08 (ddd, J=8.3, 6.7, 0.9 Hz, 1H), 4.22 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 149.1, 126.2, 123.8, 122.1, 121.9, 120.1, 117.2, 40.4. GC-MS (EI, 70 eV): m/z = 132 (M+, 100), 104 (24), 63 (24), 51 (18). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 133.0760, found: 133.0758.

Allylamine (16). 35 mg (0.222 mmol, 67 %) of product 6p were obtained as a yellowish oil. 1H NMR (400 MHz, CDCl3) δ 7.93 (d, J=1.1 Hz, 1H), 7.72 (dq, J=8.7, 1.0 Hz, 1H), 7.65 (dt, J=8.5, 1.1 Hz, 1H), 7.32–7.24 (m, 1H), 7.08 (ddd, J=8.4, 6.6, 0.9 Hz, 1H), 6.20–6.06 (m, 1H), 5.38–5.26 (m, 2H), 5.04 (dt, J=6.1, 1.4 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 149.0, 132.4, 126.1, 122.7, 122.1, 121.8, 120.2, 119.6, 117.6, 56.2. GC-MS (EI, 70 eV): m/z = 158 (M+, 77), 131 (100), 118 (29), 104 (47), 77 (59), 73 (30), 63 (66), 51 (40). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 159.0917, found: 159.0914.

Propargylamine (17). 2 mg (0.012 mmol, 4 %) of product 6q were obtained as a yellowish oil. 1H NMR (400 MHz, CDCl3) δ 8.19 (d, J=1.0 Hz, 1H), 7.70 (dq, J=8.8, 1.0 Hz, 1H), 7.67 (dt, J=8.4, 1.1 Hz, 1H), 7.30 (ddd, J=8.8, 6.6, 1.1 Hz, 1H), 7.09 (ddd, J=8.4, 6.6, 0.9 Hz, 1H), 5.26 (dd, J=2.6, 0.4 Hz, 2H), 2.61 (t, J=2.6 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 149.1, 126.7, 122.8, 122.2, 121.9 120.4, 117.4, 76.1, 75.9, 43.1. GC-MS (EI, 70 eV): m/z = 156 (M+, 48), 129 (31), 102 (34), 76 (30), 73 (100), 63 (64), 57 (27), 51 (29). HRMS (ESI) m/z for C8H8N2 [M+H]+: expected: 157.0760, found: 157.0757.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript submitted by Schmidt and co-workers describes the development of a biocatalytic method for transforming\northo-nitrobenzylamine derivatives into indazoles, using nitroreductase-catalysed reduction of the substrates' nitro group into\na nitroso group as the key step. Extension of the method to ortho-nitrophenethylamine, and hence to cinnoline formation, and\ncoupling of the reaction to an upstream IRED-catalysed reductive amination that forms nitrobenzylamines in situ from\nnitrobenzaldehydes and primary amines are also demonstrated. As pointed out by the authors in the introduction to their\narticle, indazoles are frequent structural motifs in pharmaceutical drugs, but current synthesis methods often require\nproblematic reagents. The new method presented in this manuscript (and, in particular, the two-step cascade) is of\nconsiderable interest as it uses simple starting materials and forms the target compounds cleanly and in high analytical\nyields.\nThe reported research has been carried out competently, and with only a few minor exceptions the presented data firmly\nsupport the authors' conclusions. The manuscript is well structured and written, and the experimental procedures – again\nwith few minor exceptions – are described in sufficient detail to allow them to be reproduced by others in the field. The\nreported reactions are conceptually and synthetically interesting, and the authors have characterised and optimised them\nadequately. The demonstrated substrate scope is a bit narrow, in that most N-substituents are simple alkyl groups, the only\nC-alpha substituents are methyl and carboxyl, and additional substituents on the aromatic ring have not been explored at all.\nHowever, this is acceptable for a first proof-of-concept publication on this novel biocatalytic synthesis method.\nOverall, this submission describes innovative, high-quality research that will appeal to a broad readership and is therefore\nsuitable for publication in Nature Communications. My only minor concerns are related to product quantification, as well as\nterminology and formatting issues, and I trust that they can be dispelled with a moderate additional effort. Hence, my\nrecommendation is Minor Revision. Suggestions for improving the manuscript are provided below:\n(1) Product quantification: The authors state in the captions of Figures 3 and 4 that the reported conversion values are based\non GC areas. However, the way in which the conversions were calculated is not made entirely clear: Are they based on raw\nintegrals, integrals corrected by response factors, or integrals corrected by calibration curves based on an internal standard?\nWhat is the role of the internal standards used in the analyses (toluene for GC-MS, 1a for GC-FID)? If the conversions are\nbased on raw (uncorrected) integrals, I consider this acceptable for GC-FID analysis of the target reaction, as substrate and\nproduct (nitrobenzylamine and indazole) are likely to have a similar FID response. However, I consider it problematic for the\ncascade, as the responses of the additional compounds involved there (aldehyde, alcohol, imine) could differ substantially\nfrom those of nitrobenzylamine and indazole.\nMoreover, as the target reaction proceeds through several (mostly non-enzymatic) steps and via highly reactive\nintermediates that might undergo side reactions, it would be desirable to perform quantification based on an internal\nstandard, so that recoveries (mass balances) can be reported in addition to conversions. This would also help clarify\nwhether the moderate isolated yields at complete conversion are due to the small scale of the preparative experiments or\ndue to side reactions. All that said, I have to commend the authors for carrying out preparative biotransformations for all\nreported substrates.\n(2) Terminology: On page 4, the authors refer to TON = molesproduct × molesNR-1 as the \"total turnover number\". However, the\ntotal turnover number is usually abbreviated TTN and is calculated as the molar quantity of product relative to catalyst\n*over the entire lifetime of the catalyst* (see https://doi.org/10.1021/cs3005264 and https://doi.org/10.1002/cite.202200177 for\ndiscussions of TON and TTN). As the reactions under question reach complete conversion within a few hours, at which time\nthe enzymes are likely still active, the reported number is an underestimation of the maximum (\"total\") number of turnovers\nthat can be achieved under these conditions, and should be referred to as \"turnover number\".\nWhat the authors refer to as \"by-products\" (e.g., alcohol 15 in the cascade process) are actually side products, as they\nreduce the yield of the desired compounds (for a discussion of these terms, see: https://doi.org/10.1021/op300317g). Please\nrevise.\nOn several occasions in the main article and the SI, the authors use the wording \"were dried\" to refer to the removal of\nsolvent by rotary evaporation. This is misleading, as the term \"drying\" usually refers to the removal of residual water from an\norganic solution by drying agents such as anhydrous salts. I suggest to use \"were evaporated\" or \"were concentrated\"\ninstead.\n(3) Formatting: In Figure 4, I suggest to use superscript rather than subscript numbers to distinguish between the two\nvariable residues (R1 and R2), as a subscript R2 can be msiread as \"two times R\". In Figure S2, the colours mentioned in\nthe caption do not always agree with those used in the graph and the legend.\nPlease ensure correct and consistent formatting (lowercase 3 in CDCl3, italic J and δ) in all NMR listings, including the\nreference comparisons, and report 13C chemical shifts with one decimal place. Please ensure consistent formatting of\njournal titles in the SI references.\n(4) p. 4, Figure 2 caption: The wording \"Error bars represent the standard deviation of measurements made in triplicate.\" suggests that only the analysis was performed three times; however, I assume that each experimental condition (setup of the\nbiotransformation and analysis) was replicated. Please use a wording that is clear in this regard, for instance \"Error bars\nrepresent the standard deviation of triplicate experiments.\" For consistency, please add such a statement to all other graphs\nshowing replicate experiments as well.\n(5) p. 7/8: \"However, it was found that the products partially evaporated upon prolonged drying in vacuum.\" With boiling\npoints >200 °C, this problem should have been manageable with careful evaporation. Nevertheless, calculating corrected\nyields from the solvent-containing NMR samples is an acceptable approach. It would be informative, though, to report the\nactual weights of the samples along with their corrected weights in the SI.\n(6) p. 9, discussion of kinetic experiments, and Figures S12–S15: The y-axis of the Michaelis–Menten plots is drawn in units\nof GC area. The plots would be more informative if the y-axes were drawn in units of apparent activity (1/s for apparent kcat,\nor U/mg for apparent specific activity). Also, the vastly different value ranges for Figures S12 and S14 vs Figures S13 and\nS15 seem to indicate that the data points represent vastly different levels of product concentration (and, hence, conversion).\nThe validity of a kinetic analysis based on a non-continuous assay (here: GC) strongly depends on the level of conversion in\nthe individual samples. Please ensure that all samples taken into consideration are representative of initial reaction rates\n(ideally <15% conversion) and clearly report the range of conversion values observed in these samples.\n(7) p. 11: \"This arose from the fact that the expression of IRED15 at 16.22 μmol/L was much better...\" I assume this refers to\nµmol of pure protein obtained per litre of culture medium. The reported 16.22 µmol/L would then correspond to roughly 5 g of\npure protein per litre of culture, which seems like a very high expression level. Are the numbers correct?\n(8) p. 13: \"Using this optimized system, 87 % conversion to 1b was achieved after the first 20-hour reaction step, and 85 %\nconversion to the final product (6b) was achieved after a total reaction time of 24 hours with an isolated yield of 68 % (30 mg,\nFigure 7B and 8).\" I suggest to move this sentence to the end of the previous section.\n(9) Methods section and SI: Please specify the concentrations of stock solutions used for all experimental procedures (for\ninstance, in the way you do for the paragraph on \"Investigation of the IRED15/BmGDH system\" in the SI). Also, please\nspecify the potassium phosphate species that is added during work-up, for instance by giving the formula in parentheses:\n\"potassium phosphate (K3PO4)\".\n(10) p. 15/16: For all substrates that were used in the form of hydrohalides, please indicate the type of salt with the\ncompound abbreviation (e.g., 1a·HCl, 1e·HBr). Otherwise, the specified weights would appear not to correspond to the\nspecified molar quantities.\nInstead of repeating the statement about how NMR analysis was performed (\"The proton nuclear magnetic resonance (1H-\nNMR) analysis was performed...\") for every compound, better use the space for providing listings of the NMR, GC-MS and\nHRMS data.\n(11) p. 16, also Figure 4: The reported yield of compound 6d seems to be incorrect. 8 mg of product correspond to 0.050\nmmol, a yield of 49%.\n(12) SI, p. 3: \"using a 400 MHz NMR\" is jargon; better write \"using a 400 MHz NMR spectrometer\". \"The spectra were related\nto CDCl3...\" It is actually the signal of residual CHCl3 in the deuterated solvent that is used for referencing. Also, it is good\npractice to explicitly state the chemical shift value of the signal that the spectra were referenced to. Hence, I suggest: \"The\nspectra were reference to the solvent signal (δ = 7.26 ppm)...\".\n\"No 13C-NMRs could be measured due to the low amounts of products obtained.\" With several mg of the compounds\navailable, a few hours of pulsing would have provided good-quality 13C NMR spectra. Since all biotransformation products\nare known compounds, reporting carbon spectra is not critically important, but if the samples are still available and in good\ncondition, I suggest to acquire and add the 13C spectra.\n(13) SI, p. 3/4; incomplete analysis conditions: For HRMS analysis, please report the ionisation method (I assume ESI) and\nconditions. For GC-FID and GC-MS analysis, please report the carrier gas and the flow rate or pressure (depending on\nwhether the instrument was operated in constant pressure or constant flow mode). For GC-MS analysis, please also report\nthe ionisation mode (I assume EI, 70 eV) and the m/z scan range.\n(14) SI, NMR spectra: The NMR data are provided in list form, not table form. Please change the Figure captions\naccordingly.\n(15) Typos: Figure 5 caption: Rection -> Reaction; Figure 7 caption: \"starting materials 12 and 13\" -> \"starting materials 12\nand 13, 16 or 17\"; p. 13: \"available substrates (1a, 1i and 1j)\" -> \"available substrates (1b, 1i and 1j)\"; p. 15: product 6a ->\nproduct 6a'; p. 17: 918 mL acetonitrile -> 918 µL acetonitrile; SI, p. 2: intervalls -> intervals; SI, p. 3: \"The sample were\ndiluted\" -> \"The samples were diluted\".", "reviewer_2": "Schmidt and co-workers report an enzymatic strategy for constructing indazole motifs using nitroreductases (NRs), including\na coupled NR–imine reductase cascade that delivers 2H-indazoles directly from inexpensive 2-nitrobenzaldehydes.\nAlthough numerous synthetic routes to indazoles have been estabished, biocatalytic indazole construction, even N–N bond\nformation remains scarcely explored. Accordingly, this study appears to establish the first enzymatic platform for indazole\nconstruction and meaningfully broadens the scope of enzyme-mediated heterocycle synthesis while offering a potentially\nmore sustainable alternative to existing chemical methods. The effort devoted to integrating IREDs with NRs to access 2H-\nindazoles from aldehyde precursors further underscores the synthetic utility of the approach.\nWhen introducing the state-of-the-art, the authors already summarize a variety of organocatalytic and transition-metal-\ncatalyzed strategies. For completeness and to better guide readers from the synthetic community, it would be helpful to\ndiscuss the Davis–Beirut indazole synthesis. In addition, some organophosphorus-catalyzed indazole formations also need\nto be mentioned (e.g., J. Am. Chem. Soc. 2017, 139, 6839–6842; Chem. Eur. J. 2018, 24, 9090–9100).\nRegarding the nitro reduction pathway, the manuscript refers the reduction of nitro groups to amines as “incomplete\nreduction”. Please specify why this process is incomplete. Because nitroso species are typically more reactive than nitro\ncompounds, a lot of enzymatic reductions can lead to a complete reduction to hydroxylamines or amines. BaNTR1 and NfsA\nare two of the exceptions. More evidence needs to be provided for the statement “However, the reaction catalyzed by NRs\nusually leads to xxx”. Relatedly, do you believe indazole formation occurs within the enzyme active site, or after diffusion of a\nnitroso intermediate into solution? This information could be helpful for readers to design transformations.\nMoreover, the authors demonstrate that without enzyme engineering, the transformation works well with purified enzymes.\nHave the lysate or whole-cell formats for any of the biocatalytic transformation been evaluated? These observations would\ninform practical adoption and industrial applications.\nThe substrate scope focusing on side-chain variation is informative. To further demonstrate robustness, please consider\nincluding representative ring-substituted examples. Such data would help potential users gauge the method’s tolerance to\nelectronic and steric variations.\nThe authors also showcased the substrate scope of the nitroreductase-catalyzed indazole formation, different substituents\non the side chain were studied. Have the authors tried substituents on the phenyl ring? Those examples could be a good\ndemonstration for a wider audience that this transformation is robust towards different substituents.\nThe authors also provide careful kinetic analysis, including Michaelis-Menten experiments to obtain the KM for both\nNAD(P)H and 1a. The authors mention the KM value for 1a with BaNTR1 is very high. Please specify comparing to which\nvalue, this KM (10.8   2.9 mM) is very high? Can you also comment on why higher substrate concentration leads to over-\nreduction products?\nFinally, the NR–IRED cascade nicely demonstrates synthetic utility. Cofactor regeneration could be a costly step. Were\nother regeneration methods tried, such as sodium dithionite, to replace this system?\nSome general edits:\n1. Please review the References section. All citations need to be in a consistent format. For example, Ref. 9 and 15 need to\nbe fixed.\n2. For all quantification data, such as conversions, KM value etc., it will be helpful to include the calculation methods in the\nSI. Please add calibration curves and necessary date in the SI.\n3. For all compounds not previously reported, 1H NMR, 13C NMR and HRMS are all needed. Please add.", "reviewer_3": "This study reports the enzymatic synthesis of indazoles via nitroreductase (NR)-triggered cyclization of 2-nitrobenzylamine\nderivatives. The authors used NRs to selectively reduce substrates to reactive nitroso intermediates that spontaneously\ncyclize to form indazoles with high conversions (>99% for most substrates), producing either 1H- or 2H-indazole tautomers\ndepending on substitution patterns. The substrate scope were investigated, and a proof-of-concept cascade combining an\nimine reductase with NfsA enables one-pot synthesis of 2H-indazoles from inexpensive 2-nitrobenzaldehyde and primary\namines (up to 85% conversion, 68% isolated yield). The work expands biocatalytic strategies for N-N bond formation and\nprovides a new route to indazole scaffolds. However, this reviewer finds the paper not reach Nature Communications in this\nstage for the following reasons:\n1. Although the proposed nitroso intermediate is mechanistically plausible, its formation has not been directly detected,\nwhich weakens the mechanistic interpretation. Please provide solid evidence—such as low-temperature NMR, controlled\nreduction kinetics (e.g., limited BmGDH/NADPH addition), or the use of an N-substituted secondary amine substrate that\nprevents over-reduction—to trap or visualize the nitroso/hydroxylamine intermediate and strengthen the proposed\nmechanism.\n2. Less than 10 examples were demonstrated with only substitutions on the nitrogen or adjacent carbon. The diversity of the\nsubstrate scope is not high. For six membered rings, the scope seems to be too narrow. The extension of the nitroreductase-\ntriggered cyclization to six-membered ring systems (e.g., cinnolines) is conceptually promising but remains underdeveloped.\nPlease optimize the oxidation conditions (e.g., addition of a mild oxidant or use of an enzymatic oxidation system) to achieve\nfull aromatization to cinnoline and expand the substrate scope for six-membered heterocycle formation.\n3. Yields are moderate for some cases (6b, 6c, 6e, 6f), and high for 6d, even the conversions were the same. Could further\nengineering of the enzyme expand the scope of the reactions to include more diverse substrates? Though the reaction\nseems quite efficient now, the authors should at least attempt enzyme engineering.\n4. The two enzymatic steps were performed sequentially, not simultaneously, which should be emphasized.\n5.Please cite the recent multienzyme stereodivergent cascade for tetrahydroquinoline synthesis (Angew. Chem. Int. Ed.\n2024, 63, e202411561) to better contextualize your cascade strategy within the current literature.\n6. Three replicates were mentioned, but no error bars or statistical analyses are provided. Please add mean ± SD (or SEM)\nto all bar/line charts and clearly state the sample size (n) and statistical methods used in the figure captions.\n7. Please report the total reaction volume (in x mL) for each experiment in the main text.\n8. Supply ¹³C NMR data for all new compounds (6f). Ensure consistent formatting for NMR solvent descriptions throughout the\nSupporting Information (e.g., “400 MHz, CDCl₃”)."}, "author_response": "We are grateful to the reviewers for their enthusiastic feedback and helpful suggestions.\nWe have uploaded a revised version of the paper that addresses the reviewers' feedback\nand suggestions for improving the manuscript. The specific changes made are outlined\nin the reviewer response file below (author responses are in blue) and highlighted in\nyellow in the revised manuscript and supporting information. In the reviewer response\nfile below, we also highlighted the changes made to the manuscript and supporting\ninformation in yellow. These changes can also be found in the two documents.\nReviewer #1 (Remarks to the Author):\n[...]\nWe are very grateful for the reviewer’s compliments and detailed suggestions, which\nhave further improved the quality of the manuscript. We greatly appreciate that the\nreviewer thoroughly read the main manuscript and supplementary information and\nidentified several important aspects that we have carefully addressed during the\nrevision.\n(1) Product quantification: [...]\nWe are grateful for the reviewers question and we have recapitulated our data. The\nconversions were actually based on the substrate and product integrals. For\nclarification, a sentence on this topic has been added to the 'Methods' and the\n'Supporting Methods' section.\n[...]\n(2) Terminology: [...]\nWe thank the reviewer for these valuable clarifications regarding terminology. We\nappreciate the distinction between turnover number (TON) and total turnover number\n(TTN) and agree that our original wording may lead to misunderstanding. As suggested,\nwe revised the manuscript to refer to the reported values as “turnover numbers,”\nthereby avoiding any implication that these represent the maximum turnovers\nachievable over the full catalyst lifetime.\n[...]\n(3) Formatting: [...]\nWe thank the reviewer for the helpful suggestions regarding formatting and figure\npresentation. We revised Figure 4 and used superscript indices for R1, R2 and R3 to avoid\nany potential misinterpretation. We also appreciate the reviewer’s note concerning\nFigure S2; we carefully checked the colors in the graph, legend, and caption to ensure\nfull consistency.\n[...]\n(4) p. 4, Figure 2 caption: [...]\nWe thank the reviewer for this helpful remark regarding the wording in the caption of\nFigure 2. As suggested, we revised the caption to read “Error bars represent the\nstandard deviation of triplicate experiments,” which more accurately reflects that each\nbiotransformation setup, including both reaction and analysis, was independently\nreplicated. We have carefully checked the entire manuscript and Supporting\nInformation accordingly.\n[...]\n(5) p. 7/8: [...]\nWe agree with the reviewer that we could have carefully evaporated the solvent for most\ncompounds, as we did for the substrates added during this revision. However, when we\nstarted the upscaling reactions, we found that product 6b was evaporating in high\nvacuum. We therefore had to repeat this upscaling experiment. This is why we\nproceeded with greater caution in subsequent upscaling reactions, trying not to\nevaporate the solvent extensively to avoid product loss. Consequently, more solvent\nremained in some products. The actual weights are now noted in the supplementary\ninformation Table S1.\n(6) p. 9, discussion of kinetic experiments, and Figures S12–S15: [...]\nWe thank the reviewer for the comments regarding the kinetic analysis. We have\nconverted the y-axes of all Michaelis–Menten plots to apparent activity units (k , ) to\ncat app\nmake the data more informative and comparable (Figures S12-15). Additionally, based\non a calibration curve (Figure S16), we confirmed that conversions in all kinetic samples\nremained below 6 %, even at the lowest substrate concentrations used. The vast\ndiKerence in area is due to the fact that the K values of NAD(P)H are in the low µM\nM,app\nrange. When we performed the NAD(P)H kinetics, we stopped the reaction at low\nconversions (always below 6 % relative to NAD(P)H), resulting in a low µM concentration\nof the formed product. Meanwhile, the K values for 1a were much higher, enabling\nM,app\nhigher product concentrations (even in the mM range) without exceeding 6 %\nconversion relative to 1a. For clarity, the conversions for the kinetics towards 1a do not\nhave to be relative to NAD(P)H because a cofactor regeneration system was used in the\nlatter experiments, which avoids NAD(P)H depletion. The kinetics towards NAD(P)H\nwere not analyzed using the cofactor regeneration system to avoid influencing the\nreaction speed at low NADPH concentrations through competitive binding or slow\nregeneration.\n(7) p. 11: [...]\nYes, the numbers are correct. Based on the MW of IRED15 of about 31.4 kg/mol\n(corresponding to 31.4 mg/µmol) the amount of protein per liter of culture medium is\nabout 500 mg (16.22 µmol/L* 31.4 mg/µmol). This corresponds to the expression level\nwe obtain from the culture.\n(8) p. 13: [...]\nWe thank the reviewer for this helpful suggestion. We agree that the sentence in\nquestion fits better at the end of the previous section. We adjusted the manuscript\naccordingly.\n(9) Methods section and SI: [...]\nWe thank the reviewer for this constructive comment. We have revised both the\nMethods section and the Supporting Information to specify the concentrations of all\nstock solutions used. In addition, we have clarified the potassium phosphate species\nby adding the chemical formula (K PO ) wherever applicable.\n3 4\n(10) p. 15/16: [...]\nWe thank the reviewer for these helpful suggestions. We have indicated the type of\nhydrohalide salt for all relevant substrates by adding the corresponding notation,\nensuring that the mentioned weights and molar quantities are fully consistent.\nFurthermore, we have removed the repeated NMR-analysis statements and added\nNMR, GC-MS, and HRMS data listings instead.\n(11) p. 16, also Figure 4: [...]\nWe are grateful for spotting this mistake. The mg yield mentioned in paragraph 6d is\nactually incorrect due to a copy-paste error from the paragraph of 6c. While addressing\ncomment 5, we also found that the weight of product 6d (which was measured to be 16\nmg) was not corrected by the remaining solvent as the solvent peaks were comparably\nlow. This has now been corrected, resulting in 15 mg of 6d corresponding to 0.091 mmol\nand a yield of 90 %. We have carefully checked all the other yields reported in the\nmanuscript.\n(12) SI, p. 3: [...]\nWe thank the reviewer very much for the comments on the NMR analysis description.\nThe NMR methods are now described in a separate paragraph in the SI and follow the\nsuggestions. We also followed the advice to measure the 13C NMRs using elongated\npulsing times, and we were able to acquire the 13C spectra that we added to the SI and\nthe main paper (list only).\n(13) SI, p. 3/4; incomplete analysis conditions: [...]\nIndeed, the HRMS ionization mode is ESI. This information was added to the HRMS\nsection in the SI and each HRMS experiment shown in the main paper. Details on the\nGC-FID and GC-MS methods were also added:\n(14) SI, NMR spectra: [...]\nWe agree with the reviewer's comment. We adjusted the figure captions accordingly.\n(15) Typos: [...]\nWe thank the reviewer for carefully reading the manuscript and noting these typos. All\nlisted errors have been corrected in the manuscript and Supporting Information.\nReviewer #2 (Remarks to the Author):\n[...]\nWe thank the reviewer for the valuable comments and suggestions, which we carefully\nconsidered during our manuscript revision. These comments have further improved the\nquality of our manuscript.\nWhen introducing the state-of-the-art, the authors already summarize a variety of\norganocatalytic and transition-metal-catalyzed strategies. For completeness and to\nbetter guide readers from the synthetic community, it would be helpful to discuss the\nDavis–Beirut indazole synthesis. In addition, some organophosphorus-catalyzed\nindazole formations also need to be mentioned (e.g., J. Am. Chem. Soc. 2017, 139,\n6839–6842; Chem. Eur. J. 2018, 24, 9090–9100).\nWe appreciate the reviewer’s comment, which has strengthened the background and\naccessibility of the manuscript for the synthetic community. We have expanded the\nintroduction to include a brief discussion of the Davis–Beirut indazole synthesis as well\nas relevant organophosphorus-catalyzed approaches. We also included a very recent\nexample of Lewis acid-catalyzed 2H-indazole formation. These additions help to\nprovide a more complete overview of current synthetic strategies and better\ncontextualize our biocatalytic method within the broader landscape of indazole\nsynthesis.\nRegarding the nitro reduction pathway, the manuscript refers the reduction of nitro\ngroups to amines as “incomplete reduction”. Please specify why this process is\nincomplete. Because nitroso species are typically more reactive than nitro compounds,\na lot of enzymatic reductions can lead to a complete reduction to hydroxylamines or\namines. BaNTR1 and NfsA are two of the exceptions.\nWe thank the reviewer for this useful comment, and we agree that the current phrasing\nmight be misleading. By \"incomplete reduction,\" we meant that the 6e- nitro reduction\nperformed by nitroreductases is not a concerted reaction, and the 2e- (nitroso) or 4e-\n(hydroxylamine) reduction intermediates can sometimes be observed (e.g., Molecules\n23(2), 211 (2018); J. Biol. Chem. 289, 15203–15214 (2014); Nat. Commun. 14, 5442\n(2023)). This means that the nitroso intermediate exists and can undergo chemical\nreactions when they occur quickly enough, even though it is typically described as a\nmore reactive nitroreductase substrate. The point was clarified by rephrasing the\naccording paragraph:\nMore evidence needs to be provided for the statement “However, the reaction catalyzed\nby NRs usually leads to xxx”.\nWe acknowledge the reviewer's point that NR reactions do not always lead to nitroso- or\nhydroxylamine-type intermediates. We rewrote the entire sentence as described above\nto weaken the statement.\nRelatedly, do you believe indazole formation occurs within the enzyme active site, or\nafter diKusion of a nitroso intermediate into solution? This information could be helpful\nfor readers to design transformations.\nUnfortunately, the nitroso intermediate is too reactive to be detected experimentally. It\nis also known to cyclize spontaneously in an aqueous methanolic solution\n(Tetrahedron, 54(1998):3197-3206). Therefore, the cyclization does not have to take\nplace in the protein. NRs work according to a ping-pong bi-bi mechanism (J Biol Chem.\nthe NR before a further reduction step can occur. However, cyclization could still occur\nbefore the intermediate is released into the solution. Therefore, we cannot exclude the\npossibility that the enzyme is involved in the cyclization; however, the enzyme is most\nlikely not required.\nMoreover, the authors demonstrate that without enzyme engineering, the\ntransformation works well with purified enzymes. Have the lysate or whole-cell formats\nfor any of the biocatalytic transformation been evaluated? These observations would\ninform practical adoption and industrial applications.\nWe agree that this would be a useful experiment for the industrial application of the\nreaction. Therefore, we conducted lysate and whole cell experiments. The results are\npromising and demonstrate the potential application of this reaction in resting cells and\nlysate. We added a sentence to the main paper mentioning the applicability of the\nconcept in lysate and whole cells. The experiments are depicted in Figures S21 and S22.\nThe substrate scope focusing on side-chain variation is informative. To further\ndemonstrate robustness, please consider including representative ring-substituted\nexamples. Such data would help potential users gauge the method’s tolerance to\nelectronic and steric variations. The authors also showcased the substrate scope of the\nnitroreductase-catalyzed indazole formation, diKerent substituents on the side chain\nwere studied. Have the authors tried substituents on the phenyl ring? Those examples\ncould be a good demonstration for a wider audience that this transformation is robust\ntowards diKerent substituents.\nWe thank the reviewer for this valuable suggestion. We agree that including ring-\nsubstituted examples would further demonstrate the robustness and versatility of the\nmethod. We conducted experiments with halogen- and methoxy-substituents in the\northo, meta, and para positions relative to the nitro group (substrates 1i-o, Figure 4).\nWhile the enzymes converted most of the substrates very well, ortho-substitution\nseems to lead to reduced activity due to steric hindrance. BaNTR1 appears to tolerate\northo-substitutions better than NfsA. Substrate 1k was depleted by the enzymes;\nhowever, cyclization did not occur eKiciently, possibly due to the strong electron-\ndonating eKect of the para-methoxy group, which reduces the electrophilicity of the\nnitroso group.\nThe authors also provide careful kinetic analysis, including Michaelis-Menten\nexperiments to obtain the KM for both NAD(P)H and 1a. The authors mention the KM\nvalue for 1a with BaNTR1 is very high. Please specify comparing to which value, this KM\n(10.8 ± 2.9 mM) is very high? Can you also comment on why higher substrate concentration leads to over-\nreduction products?\nTypical NR substrates, such as nitrofurazone, have K values below 1 mM (e.g. 5.5 µM\nM\nfor NfsA), whereas the K values of NfsA and BaNTR1 are higher. The example of\nM\nnitrofurazone was mentioned later in the manuscript. To avoid confusion, the paragraph\nwas reorganized and additional citations were added.\nFinally, the NR–IRED cascade nicely demonstrates synthetic utility. Cofactor regeneration could be a costly step. Were\nother regeneration methods tried, such as sodium dithionite, to replace this system?\nWe are grateful for the acknowledgment of the synthetic utility of the designed cascade.\nYes, other cofactor regeneration systems were investigated. We successfully\nregenerated the cofactor using a variant of formate dehydrogenase (FDH) from Candida\nboidinii (FDH-M4), which was designed for NADPH regeneration (Figure S20). However,\ndue to the fact that about 100 times more FDH is required for similar performance\ncompared to GDH, we continued with the GDH-glucose system to establish the\ncascade reaction. We agree that non-enzymatic regeneration methods or industrial\nGDHs that are more stable at pH 8 or higher could be used to optimize the system from\nan economic point of view. However, since this paper describes the first proof of\nconcept of this cascade and cofactor regeneration is not its focus, we believe that\nexploring this further is beyond its scope and could be done by industrial users facing\nthese issues in all biocatalytic reductions.\nSome general edits:\n1. Please review the References section. All citations need to be in a consistent format.\nFor example, Ref. 9 and 15 need to be fixed.\nWe have carefully checked the references, and we believe they align with the Nature\nCommunications guidelines.\n2. For all quantification data, such as conversions, KM value etc., it will be helpful to\ninclude the calculation methods in the SI. Please add calibration curves and necessary\ndate in the SI.\nWe thank the reviewer for this helpful suggestion. We have added the calculation\nmethods for all quantification data to the appropriate places. Additionally, we have\nincluded the standard curve used for kinetics (Figures S12-16) and the GC response\nfactor in the SI (Figure S25).\n3. For all compounds not previously reported, 1H NMR, 13C NMR and HRMS are all\nneeded. Please add.\nAll of the compounds except 6f and 6q have already been described in the literature.\nNevertheless, we added the1H NMR, HRMS and 13C NMR spectra for all of the\ncompounds that we isolated, except for 1H-indazole and cinnoline, for which\ncommercial standards were available.\nReviewer #3 (Remarks to the Author):\n[...]\nWe thank the reviewer for this insightful comment. We agree that the mechanistic\ninterpretation could be further strengthened by experiments proving that the nitroso\nintermediate is cyclizing. For this we conducted an experiment where we indirectly\nmeasured the turnover of the cofactor regeneration system. We used a pH probe\ncoupled titration to neutralize the proton released from gluconic acid (hydrolyzed\ngluconolactone), an equimolar by-product formed during cofactor recycling. We also\nconsidered that protons are released during the reaction because the amine group of\nthe substrate is mostly protonated at pH 8.0, but the product, 1H-indazole, does not\nreact as a base at this pH value (pK of 1.22; T. K. Adler and A. Albert, J. Chem. Soc.,\na\n1960, 1794). To determine this, we measured the pK value of 1a and found it to be 8.63.\na\nExperimentally, we found that the addition of 1.62 eqivalents of NaOH (relative to the\nsubstrate) were needed to keep the pH stable (at 73% conversion (GC-FID)). From the\ntheoretic calculations we expected the addition of 1.32 eq. of NaOH for one turnover of\nthe cofactor regeneration system. Considering that flavin-dependent enzymes typically\nexhibit a side reaction in which reduced flavin cofactors react with O to form H O ,\n2 2 2\na\nreaction that we also detected, this result clearly indicates that only one 2e- reduction\nstep occurs. Therefore, we are confident that cyclization occurs from the nitroso\nintermediate. We added a statement to the main paper and included the titration\nexperiment in the supplementary information (SI Methods and Figure S24).\n[...]\n2. Less than 10 examples were demonstrated with only substitutions on the nitrogen or\nadjacent carbon. The diversity of the substrate scope is not high. For six membered\nrings, the scope seems to be too narrow. The extension of the nitroreductase-triggered\ncyclization to six-membered ring systems (e.g., cinnolines) is conceptually promising\nbut remains underdeveloped. Please optimize the oxidation conditions (e.g., addition of\na mild oxidant or use of an enzymatic oxidation system) to achieve full aromatization to\ncinnoline and expand the substrate scope for six-membered heterocycle formation.\nWe thank the reviewer for this thoughtful perspective on the substrate scope. We agree\nthat the substrate diversity could be increased. Therefore, we included a series of ring-\nsubstituted substrates. We conducted experiments with halogen- and methoxy- in the\northo, meta, and para positions relative to the nitro group (substrates 1i-o). While the\nenzymes converted most of the substrates very well, ortho-substitution seemed to lead\nto reduced activity due to steric hindrance. BaNTR1 appears to tolerate\northo-substitutions better than NfsA. Substrate 1k was depleted by the enzymes;\nhowever, cyclization did not occur eKiciently, possibly due to the strong electron-\ndonating eKect of the para-methoxy group, which reduces the electrophilicity of the\nnitroso group.\n[...]\n3. Yields are moderate for some cases (6b, 6c, 6e, 6f), and high for 6d, even the\nconversions were the same. Could further engineering of the enzyme expand the scope\nof the reactions to include more diverse substrates? Though the reaction seems quite\neKicient now, the authors should at least attempt enzyme engineering.\nWe thank the reviewer for this thoughtful comment. We agree that the isolated yields\nvary despite comparable conversions. Although we cannot provide a definitive\nexplanation, we note that 6d was the only substrate that was not used as a hydrohalide\nsalt and was therefore employed as a neat liquid. In contrast, the hydrohalide salts\nused for the other substrates may have contained small amounts of moisture, which\ncould contribute to lower isolated yields despite full conversion. Additionally, the\npurifications were performed on a small scale, where material losses during workup\nand chromatography (e.g., transfer losses, adsorption on silica, extraction variability)\ncan have a disproportionate impact on the final yield. Furthermore, we recognized while\naddressing another reviewers comments that the weight of product 6d (which was\nmeasured to be 16 mg) was not corrected by the remaining solvent as the solvent peaks\nwere comparably low. The corrected yield was found to be 90 % (15 mg) instead of 96 %.\nRegarding enzyme engineering, we agree that it could broaden the substrate scope,\ne.g., toward 1o. However, given that this substrate was the only one tested that was less\nwell accepted by the NRs, we believe that the substrate scope is suKiciently broad also\nusing the native enzymes. Moreover, given the high conversions achieved for nearly all\nsubstrates, together with the now significantly increased substrate scope which\nshowcases that the chosen NRs are already very eKicient biocatalysts, and the focus of\nthis study on demonstrating the feasibility of the biocatalytic concept, we believe that\nsystematic engineering eKorts would exceed the scope of this study.\n4. The two enzymatic steps were performed sequentially, not simultaneously, which\nshould be emphasized.\nWe thank the reviewer for this helpful remark. In the revised manuscript, we have\nclarified that the two enzymatic steps were performed sequentially by adding\n\"sequential\" where it was missing. For example, we added it to the description of the\ncascade design, the caption of Figure 6, and the conclusion.\n5.Please cite the recent multienzyme stereodivergent cascade for tetrahydroquinoline\nsynthesis (Angew. Chem. Int. Ed. 2024, 63, e202411561) to better contextualize your\ncascade strategy within the current literature.\nWe thank the reviewer for pointing out the recent relevant work. We have now cited the\naforementioned paper, in the description of the cascade design section (see below).\nThis citation provides a better context for our cascade strategy within the current\nliterature on enzymatic cascade reactions for heterocyclic pharmaceutical building\nblocks.\n6. Three replicates were mentioned, but no error bars or statistical analyses are\nprovided. Please add mean ± SD (or SEM) to all bar/line charts and clearly state the\nsample size (n) and statistical methods used in the figure captions.\nWe thank the reviewer for this important point. Mean ± SD and the corresponding\nsample size have been added to the figures in which replicate experiments were\nperformed (e.g. Figure 2 and S12-16). Statistical annotations were included where\napplicable.\n7. Please report the total reaction volume (in x mL) for each experiment in the main text.\nWe have added the total reaction volume for each experiment to the figure captions and\nthe method sections to ensure full clarity and reproducibility.\n8. Supply ¹³C NMR data for all new compounds (6f). Ensure consistent formatting for\nNMR solvent descriptions throughout the Supporting Information (e.g., “400 MHz, CDCl₃”).\nWe thank the reviewer for this comment. We have added the missing 13C NMR data for\ncompound 6f (and all other compounds isolated) and ensured consistent formatting of\nall NMR solvent descriptions throughout the Supporting Information.\nWe are confident that these changes made in response to the reviewer comments and\nsuggestions have strengthened the manuscript considerably. Once again we thank you\nfor your constructive comments and hope that you are able to accept our manuscript for\npublication in Nature Communications in light of the above points."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In their revised manuscript, Schmidt and co-workers have thoroughly addressed the minor concerns voiced in my original reviewer report, as well as the criticism raised by the other two reviewers. The authors have carried out several additional experiments, the most significant of which are an extension of the substrate scope to ring-substituted derivatives and a titration experiment that supports their original mechanistic proposal. In addition, many small improvements were implemented, ranging from determination of GC response factors to an increased level of detail in figure captions and experimental procedures, and the correction of typos and minor terminological issues.\nAll these revisions have further strengthened the manuscript, and I recommend its publication in Nature Communications after a last round of minor corrections, which can be implemented without further peer review. Suggestions for these final improvements are provided below:\n(1) Terminology: Please ensure consistent spelling of the term \"side product\" (with a space, not a hyphen).\n(2) Listings of GC-MS data: It is not common practice to report expected and observed masses in unit-resolution GC-MS analysis. Instead, provide a list of all significant mass fragments including their relative abundance, with identification of the parent ion (M+); e.g., for 6a': GC-MS (EI, 70 eV): m/z = 118 (M+, 100), 91 (31), 63 (40), 52 (19).\n(3) GC-MS analysis conditions: The flow rate of 13.8 mL/min appears unusually high. I assume it is the total flow (including split flow, and septum purge flow, if applicable) that is reported here, but in order to allow other researchers to reproduce the GC separations, the column flow rate (the part of total carrier gas flow that is directed through the column) should be reported.", "reviewer_2": "The authors have thoroughly addressed all of my comments and concerns, and I have no further objections.", "reviewer_3": "The authors have comprehensively addressed all the core issues raised in the initial review. The inclusion of additional experimental data, an expanded substrate scope, and enhanced data presentation has substantially improved the scientific rigor, completeness, and overall persuasiveness of the manuscript. My primary concerns have been satisfactorily resolved.\nSpecifically, the newly conducted pH-stat titration experiments provide solid quantitative evidence for the proposed \"nitroso-intermediate-mediated cyclization\" mechanism. Furthermore, the addition of seven ring-substituted substrates significantly broadens the synthetic utility of the method and allows for a more in-depth discussion of substituent effects. The authors have also meticulously updated the experimental section with essential details, including error bars and reaction volumes, provided the missing NMR data in a consistent format, and updated the relevant citations. This work represents the first example of nitroreductase-triggered indazole synthesis and demonstrates clear originality. Given its mild reaction conditions, operational simplicity, broad substrate tolerance, and successful integration into a practical enzymatic cascade, I recommend the manuscript for publication."}, "author_response": "We are grateful for the enthusiastic feedback from the reviewers and are delighted that\nall three recommend our manuscript for publication. We have uploaded a revised\nversion of the paper that addresses reviewer 1's final (minor) comments.\nThe specific changes made are outlined in the reviewer response file below (author\nresponses are in blue) and highlighted in yellow in the revised manuscript. We used the\ntrack changes function of Word for changes made to the manuscript based on editorial\nrequests.\nReviewer #1 (Remarks to the Author):\n[...]\n(1) Terminology: Please ensure consistent spelling of the term \"side product\" (with a\nspace, not a hyphen).\nWe thank the reviewer for these helpful comments. The spelling of the term “side\nproduct” has been made consistent throughout the manuscript.\n(2) Listings of GC-MS data: It is not common practice to report expected and observed\nmasses in unit-resolution GC-MS analysis. Instead, provide a list of all significant mass\nfragments including their relative abundance, with identification of the parent ion (M+);\ne.g., for 6a': GC-MS (EI, 70 eV): m/z = 118 (M+, 100), 91 (31), 63 (40), 52 (19).\nThe GC–MS data listings were revised to report significant mass fragments with their\nrelative abundances and identification of the parent ion (M⁺).\n(3) GC-MS analysis conditions: The flow rate of 13.8 mL/min appears unusually high. I\nassume it is the total flow (including split flow, and septum purge flow, if applicable) that\nis reported here, but in order to allow other researchers to reproduce the GC\nseparations, the column flow rate (the part of total carrier gas flow that is directed\nthrough the column) should be reported.\nThe GC–MS analysis conditions were corrected by replacing the total flow rate with the\ncolumn flow rate (1.81 mL/min), which has now been reported to ensure reproducibility\nof the GC separations."}]

Decision letter