Stereoselective cyano translocation reaction enabled by photoenzymatic catalysis

Domain: neuroscience
Published: 2026-01-29
Editorial outcome: major_revision

Abstract
Functional-group translocation constitutes a powerful strategy in organic synthesis, allowing precise modification of molecular architectures and functional group positions. Nevertheless, methodologies capable of delivering precise stereocontrol during such migrations remain exceedingly scarce. Herein, we present a photoenzymatic strategy for cyano translocation that achieves precise stereocontrol. The use of stereo-complementary enzymes grants access to both enantiomers, while mechanistic studies and molecular dynamics simulations delineate the origin of stereoselectivity. This work expands the paradigm of transformations that can be rendered asymmetric by photoenzymatic catalysis and provides a new strategy to tackle persistent challenges in organic synthesis.

Introduction
Functional groups (FGs) govern the properties and functions of organic molecules, making their introduction, removal, or rearrangement fundamental in organic chemistry1,2. Functional-group translocation enables precise modulation of molecular architecture and FG positioning, with profound effects on biological activity and physicochemical properties, thereby thereby expanding the chemical space for drug discovery and materials design3. In recent years, significant progress has been made in the development of radical-mediated migratory processes for key functional groups (FGs), such as aryl, alkynyl, acyloxy, hydroxyl, and cyano groups (CN), in which chemoselectivity and regioselectivity are generally well controlled4–11. Nevertheless, stereocontrol in these reactions has typically relied on chiral substrates, and the direct achievement of precise stereochemical control remains an unresolved challenge (Fig. 1a)12.

Biocatalysis has garnered significant attention in synthesis for its mild, environmentally friendly conditions and inherent stereocontrol13–22. Recently, the incorporation of classical reactive intermediates into enzyme active sites has enabled the development of new-to-nature reactions, particularly those involving highly reactive intermediates such as carbenes and radicals 23–28, thereby broadening the synthetic potential of enzymatic catalysis. Flavin-dependent enzymes represent one of the most prevalent classes of enzymes in nature, orchestrating a broad spectrum of biological redox transformations29. These enzymes facilitate processes including substrate dehydrogenation, monooxygenation, cyclization, and intricate radical-mediated reactions, thereby playing central roles in energy metabolism, signal transduction, and the biosynthesis of diverse natural products30. Flavins exhibit exceptional versatility due to their ability to adopt multiple redox states, including oxidized, single-electron reduced, and two-electron reduced forms31–37. Recent developments have harnessed photoexcitation to enhance their redox capabilities, enabling radical-mediated coupling reactions with precise stereochemical control38–47. Flavin-dependent photoenzymatic catalysis typically proceeds through radical addition to alkenes38,39. More recently, the reaction paradigm has been extended to radical additions to nitro compounds41 and oximes48, as well as radical-mediated 1,5-hydrogen atom transfer (1,5-HAT) reactions (Fig. 1b)49. Nevertheless, alternative pathways within this enzymatic class remain largely unexplored.

We aimed to exploit flavin-dependent enzymes to catalyze stereoselective functional group translocation reactions. Alkyl nitriles are highly valuable synthetic intermediates, as they can be readily transformed into carboxylic acids, amines, and numerous other functional molecules50–52. Consequently, the development of enantioenriched alkyl nitriles is of considerable significance in both academia and industry.

Results
The excited state of flavin hydroquinone (FMN*) possesses a reduction potential of approximately −2.26 V vs SCE53, which enables the reduction of alkyl iodides (E ≈ −2.19 V vs SCE)54. This redox compatibility prompted us to select 5-iodo-2-methyl-2-phenylpentanenitrile 1a as a model substrate to evaluate the feasibility of the CN translocation reaction. Inspired by chemical precedents, our design involves the generation of a neutral alkyl radical within the enzyme active site, which enables addition to the C≡N bond followed by β-fragmentation of the resulting iminyl radical to effect CN migration7–9. The ensuing radical is subsequently quenched via enzyme-catalyzed stereoselective hydrogen atom transfer (HAT), thereby providing access to chiral alkyl nitriles with high stereocontrol (Fig. 1c).

In this work, we selected CN translocation as a representative transformation. Initially, we screened a panel of flavin-dependent enzymes and found that several displayed measurable catalytic activities (Table 1). Among them, old yellow enzyme 1 from Saccharomyces pastorianus (OYE1) catalyzed the formation of the product in 30% yield with 43:57 (S:R) stereoselectivity, whereas old yellow enzyme 3 from Saccharomyces cerevisiae (OYE3) afforded a 27% yield with 45:55 e.r. The optimal result was achieved with Caulobacter segnis arene reductase from Caulobacter segnis (CsER), providing the S-configured product in 69% yield and 98:2 e.r. In contrast, enoate reductase from Gluconobacter oxydans (GluER) afforded the opposite stereoisomer in 57% yield with 1:99 e.r. Control experiments were then performed, as shown in Table 1, entry 5-8. No product was detected in the absence of the enzyme, GDH, or light, indicating that the transformation relies on the excited FMNhq. When the enzymes were replaced with FMN alone, only trace amounts of product were formed without stereoselectivity, highlighting the indispensable role of enzyme scaffold in orchestrating both reactivity and stereocontrol. While our manuscript was under revision, Yang reported a similar cyano migration reaction catalyzed by an NADPH-dependent imine reductase55.

With the optimal reaction conditions in hand, we explored the substrate scope for this photoenzymatic CN translocation reaction. As shown in Fig. 2, substrates bearing either electron-rich (S)-2b-2e or electron-deficient substituents (S)-2f-2g on the phenyl ring were tolerated, delivering products with good reactivity and stereoselectivity. The disubstituted aromatic substrate 1e was also efficiently converted into (S)-2e in 53% yield with 90:10 e.r. In addition to the benzyl-position methyl group, the ethyl-substituted substrate 1h was evaluated, which afforded the corresponding product (S)-2h in 65% yield with 97:3 e.r. Furthermore, substrates bearing larger alkyl groups at benzyl position (R=i-Pr (1i) and Cy (1j)) were not well tolerated. Sterically demanding naphthyl substrate 1k was accommodated in the reaction, affording product (S)-2k in 24% yield with 98:2 e.r. Compounds bearing heterocyclic substituents were well tolerated, affording the pyridine-substituted product (S)-2l in 65% yield with 99:1 e.r. and the thiophene-substituted product (S)-2m in 63% yield with 97:3 e.r. Benzyl-substituted substrates were further examined to expand the substrate scope. The dicyano-substituted benzyl derivative (1n) reacted with moderate yield and decreased stereoselectivity (Table S5). We further employed 1n as a substrate to screen and evolve flavin-dependent enzymes, and identified OYE3-Y196F as the improved mutant, affording (R)-2n in 70% yield and 85:15 e.r. (Table S6). We hypothesize that the reduced stereoselectivity of these substrates may result from accelerated radical quenching facilitated by electron-withdrawing cyano groups. Consistent with this hypothesis, introducing electron-donating groups on the phenyl ring enhanced stereoselectivity ((R)-2o, (R)-2p and (R)-2q), whereas electron-withdrawing groups further diminished it ((R)-2t: 85% yield, 70:30 e.r.), underscoring the critical role of electronic effects in governing stereocontrol. Benzyl ester-substituted substrate 1u also underwent the reaction, providing the desired products (R)-2u in good yields with moderate stereoselectivity. Finally, we examined the reversed-selectivity reactions catalyzed by GluER, identified in the initial screening, and found that its substrate scope was highly limited ((R)-2a: 57% yield, 1:99 e.r.; (R)-2f: 35% yield, 14:86 e.r.). Finally, the scaled-up reaction (0.1 mmol of 1d) was implemented, affording the corresponding product in 50% yield and 99:1 e.r.

To gain insight into the mechanism of this transformation, we carried out a series of control experiments. Radical trapping experiments employing TEMPO significantly suppressed the product yield (Fig. 3a) and enabled the detection of the corresponding TEMPO-substrate adduct (Section 5.2 in SI). This result suggests that the reaction proceeds via a radical pathway. Furthermore, the reaction maintained a yield of 32% even in the presence of TEMPO. This result likely stems from the generation of the alkyl radical intermediate within the confined enzyme pocket, where intramolecular CN trapping is more readily achieved than intermolecular quenching by TEMPO. UV–visible spectroscopy revealed that FMN alone exhibits no absorption in the visible region, whereas the addition of the substrate induces the formation of a distinct Electron Donor-Acceptor (EDA) complex, enhancing the system’s absorption under visible light (Fig. 3b). Experiments were conducted of alkene substrates (1w and 1v) with different carbon-chain lengths. As shown in Fig. 3c, deiodinated products from direct hydrogen atom transfer (HAT) were detected, whereas no cyano migration products were observed (Section 5.5 in SI). To further probe the mechanistic basis of this observation, density functional theory (DFT) calculations were performed on cluster models composed of FMN and the corresponding deiodinated radical intermediates (Fig. S15–S17). The results reveal that the 1,4-cyano translocation pathway of 1a, proceeding via a five-membered cyclic radical intermediate, is energetically favored, in good agreement with the experimental observations. Isotopic labeling experiments were carried out to probe the reaction quenching pathway. No deuterium incorporation was observed in the product using a deuterated buffer. By contrast, in situ generation of deuterated flavin semiquinone (FMNsq) from deuterated glucose led to 56% deuterium incorporation (Fig. 3d), supporting that quenching occurs via FMNsq.

To elucidate the origin of stereoselectivity in this new-to-nature enzymatic reaction at the molecular level, molecular dynamics (MD) simulations were conducted. As the final HAT process determined the stereoselectivity of products, we focused on the stage that the β-fragmentation of the iminyl radical has achieved, with the cofactor under the FMNsq state (Fig. 3e). The RMSD profiles of all binding components and the key distance between the HAT involved atoms, i.e., H5 of FMNsq and C7 of 2a', became converged within 200 ns. However, the dihedral formed by the coplanar C3, C8, and C7 of 2a' and the H5 of FMNsq (∠C3-C8-C7-H5), an indicator of the hydrogen atom incorporation via a re or si face, showed a highly biased distribution (Figs. S4 and S5), which agrees well with the experimentally determined e.r values. The representative binding conformations demonstrated consistent binding features. The cyano group of 2a' anchored to a polar environment built mainly by the H173/H172 and N176/N175 side chains in CsER/GluER in almost all cases. Relative to the pro-(R) binding form, the benzyl group of pro-(S) 2a' formed additional parallel π-π stacking interactions with Y66 (Fig. S8), providing extra binding stability. In binding with GluER, two π-π stacking interactions were observed for both the pro-(R) (with W66 and W342) and pro-(S) (with W66 and F269) 2a'. However, the distances of the pro-(S) 2a' involved π-π stacking are relatively longer than that of the pro-(R) 2a' (Fig. 3e and Fig. S10), indicating a decreased binding strength. For the dicyano-substituted intermediate 2n', a distinct binding feature was discovered in its binding with OYE3 (Y196F), i.e., the H5 atom is almost always coplanar with the radical plane of 2n' (defined by atoms C3, C7, and C8, Fig. S7), leading to a concentrated distribution of dihedral angles around 180° and –180° and a decreased conformational preference (Fig. S6). In comparison with the pro-(S) 2n', the pro-(R) binding form is more favorable by forming a sandwich framework with Y82 and Y375 through π-π stacking interactions, resulting in a dihedral distribution which is in accordance with the stereoselectivity of OYE3 (Y196F) (Figs. S6 and S7). Notably, with the cyano group of 2a'/2n' stably anchored to the polar residues His and/or Asn through hydrogen bond interactions in all binding systems, the prochiral conversion can be primarily attributed to the rotation of the C7‒C8 bond. Together, MD simulations shed light on the mechanism of stereoselective cyano translocation.

Methods
Flavin-dependent enzyme catalyzed cyano translocation reaction

960 μL Enzyme solution (0.025–0.03 μmol pure enzyme, 1 mol%, tris buffer, pH = 8.0) contain NADP+ (7 μL, 0.1 μmol, 15 mM stock in buffer, 3 mol%), GDH (100 μL, about 3 U, 30 U/mL stock in buffer), glucose (100 μmol, 33.3 equiv) was added to a 10 mL Schlenk tube containing a magnetic stir bar. 1 (3 μmol, 1.0 equiv) in DMSO (40 μL) was following added into the system. The mixture was then degassed by freeze-pump-thaw for three times under N2 atmosphere. The reaction was stirred for 12 h at room temperature. Then the reaction was extracted by ethyl acetate for three times (1 mL × 3). The enantioselectivity was determined by GC or HPLC.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Functional group migration offers a powerful approach for precise molecular editing and regiocontrol, yet strategies that achieve exact stereochemical control remain rare. Professor Jian Xu and co-workers have introduced a photoenzymatic cyano migration that enables precise stereocontrol. By employing stereocomplementary enzymes, both enantiomers of the product can be accessed—a selectivity rationalized through mechanistic studies and molecular dynamics simulations. This work significantly broadens the scope of asymmetric transformations achievable via photoenzymatic catalysis and presents a compelling strategy to address long-standing challenges in organic synthesis. Overall, this reviewer finds the MS highly exciting and looks forward to future advances from the authors.\n1. Regarding the substrate scope, electron-withdrawing substituents lead to diminished yield and enantioselectivity. The manuscript merely suggests accelerated radical quenching as a possible cause, which does not sufficiently account for the significantly low yields. A mass balance analysis is recommended to clarify the fate of the substrates.\n2. In the radical trapping experiment, the reaction yield remains at 32% despite the addition of 4 equivalents of TEMPO. Any discussions would be helpful.\n3. Additional review could be cited, such as Chin. J. Chem. 2024, DOI: 10.1002/cjoc.202400656\n4. The Supporting Information lacks detailed procedures for the substrate synthesis and enzymatic reactions.\n5. For racemic product 2o in Table S65 (line 990), the high-resolution mass spectrometry (HRMS) deviation exceeds 0.001 Da. Re-measurement is suggested.\n6. Several NMR spectra in the SI are misinterpreted. For example, in the spectrum of racemic 2g (S92), the aromatic region is incorrectly integrated to 2.77H, and an extra hydrogen is accounted for in the aliphatic region. Similarly, in the spectrum of 2h (S93), a water peak appears to have been misassigned as a product signal. A thorough re-examination of all spectral assignments is suggested.\n7. Minor errors were noted in the SI: inconsistent font type at S3 line 44, \"standard\" misspelled in the table on S17, and inconsistent abbreviation of \"eq\" in the first reaction scheme on S21. A full proofreading is recommended to ensure consistency and accuracy throughout.", "reviewer_2": "The manuscript by Wang and Xu describes a photoenzymatic 1,5-cyano transfer reaction catalyzed by flavin-dependent ene-reductases. While these types of cyano transfer reactions are well known in the photoredox literature, they have not been realized within a photoenzyme. The ERED platform is well established for photoenzymatic reactivity and can control the delivery of a hydrogen atom to prochiral benzylic radicals with excellent enantioselectivity, making it a sensible catalyst for the reported reaction. The yields and enantioselectivities are good with a range of substates. Mechanistic studies confirm that radical initiation occurs via excitation of charge transfer complex. Overall, I would describe this work as novel but not particularly surprising as it relies on many well-established features from the ERED photoenzymatic literature.\nAdditional Comments:\n-TOC graphic should use beta rather than B.\n- Is there a kinetic resolution of the racemic starting material?\n- Does the method tolerate pyridine as an arene?\n- Are larger alkyl groups (beyond methyl) tolerated by the enzyme?", "reviewer_3": "This work introduces a photoenzymatic strategy for stereocontrolled translocation reactions, with cyano translocation taken as a model system. It combines experimental data with computational analysis aimed at rationalizing the stereoselectivity. At this stage, however, the computational component does not appear sufficiently strong, as it is not entirely clear whether the sampling depth and methodological rigor of the analysis are adequate to support firm mechanistic conclusions. Nevertheless, if the results can be maintained and strengthened after addressing the following comments, the study may reach the level required for publication. I would like to see the revised version of the manuscript.\n1) The simulations need to be extended and at least a second replica needs to be performed for each systems.\n2) Rather than showing distances from a single representative configuration (such as those between the substrate and the polar atoms of the active site, or between the benzyl ring and hydrophobic active-site residues), the authors should present the distribution of each distance over the course of the MD simulation, separated for the pro-S and pro-R basins.\n3) One of the main conclusions drawn from the simulations is the following:\n“Notably, the benzyl group formed additional parallel (with W100) and T-shaped (with F269) π-π stacking interactions in the pro-(S) binding form in CsER and the pro-(R) binding form in GluER, respectively, providing extra binding stability. In contrast, the pro-(R) binding form in CsER and the pro-(S) binding form in GluER showed much more exposure of the 2a• hydrophobic group to polar water molecules, indicative of an energetically unfavorable binding mode.”\nThis could be an interesting observation but should be corroborated by specific analyses.\na) Concerning the distances between the benzyl group and hydrophobic residues, the authors should also consider the side chains of residues that could for pi-pi interactions in the “wrong” pro-chiral basin as for example:\n-distance between the benzyl group and Y336 or Y66 in CsER (pro-R)\n-distance between the benzyl group and W66 and W342 in GluER (pro-S)\nb) Concerning the comment on the larger exposure to water molecules of hydrophobic groups, this should be shown with appropriate analyses\n4) Concerning the OYE3 enzyme, the authors claim: “the dihedral distribution and structure characteristics in the binding complex of OYE3(Y196F), FMNsq, and 2i• were in accordance with the reduced stereoselectivity for the dicyano-substituted compounds (Fig. S6).”\nI might agree with this conclusion, but not for the reasons provided by the authors. In fact, for OYE3-Y196F the negative values do not appear to be pro-S but rather achiral, as they lie too close to –180° to define a clear prochiral preference. Similarly, for the positive values the average dihedral angle may also be not too far from 180°, which again implies an unclear chiral assignment. The authors may therefore wish to reconsider and revise their interpretation.\nMinor points:\nWhich software was used for optimizations at the DFT level?\nWhy was the e Berendsen barostat used?\nWhich thermostat was used?\nWhich software (or in-house script?) was used for the clustering analysis?\nWhich atoms were chosen for the fit in the RMSD calculations for each of the three components?\nThe y label in Figure S4, panel b, should be distance, not RMSD.\nSome data on the clustering analysis should be reported to better understand the choice of the representative structure.\n“Substrate scope of for the photoenzymatic enantioselective CN translocation reaction”. “for” needs to be deleted.\nWhat do the authors mean with “solute” in the sentence “with a weak constraint of 10 kcal∙mol-1∙Å -2 imposed on the solute “?", "reviewer_4": "Xu et al reported an interesting cyano translocation reaction with high stereoselectivity under the photoenzymatic catalysis with ene reductases. But it seems this reaction only works with three carbons, limiting its synthetic applications. It did not work with four carbon atoms, possibly due the stability of six-membered cyclic radical intermediate. It is not clear whether it works with other numbers of carbon atoms, that may be dependent on the ability of forming the cyclic intermediate and the stability of the intermediate. The computational analysis may address this point.\nTable 2, CsER or GluER is noted in the footnote, but it is not noted which one under each product. It should be noted under each product for clarity."}, "author_response": "Dear Reviewers:\nThank you very much for your careful reviewing and important suggestions for this submission.\nYour comments are all valuable and very helpful for revising and improving our paper. We have\nstudied these comments carefully and have made suitable corrections which we hope meet with\napproval. Revised portions are marked in yellow in the text. We listed all the response to the\ncomments point by point as following.\nReviewer #1 (Remarks to the Author):\nReviewer’s general comments:\nFunctional group migration offers a powerful approach for precise molecular editing and\nregiocontrol, yet strategies that achieve exact stereochemical control remain rare. Professor Jian Xu\nand co-workers have introduced a photoenzymatic cyano migration that enables precise\nstereocontrol. By employing stereocomplementary enzymes, both enantiomers of the product can\nbe accessed—a selectivity rationalized through mechanistic studies and molecular dynamics\nsimulations. This work significantly broadens the scope of asymmetric transformations achievable\nvia photoenzymatic catalysis and presents a compelling strategy to address long-standing challenges\nin organic synthesis. Overall, this reviewer finds the MS highly exciting and looks forward to future\nadvances from the authors.\nOur response:\nWe greatly appreciate the reviewer’s positive recommendation and carefully revised the\nmanuscript and supporting information according to the reviewer’s valuable suggestion.\nCommnet 1:\nRegarding the substrate scope, electron-withdrawing substituents lead to diminished yield and\nenantioselectivity. The manuscript merely suggests accelerated radical quenching as a possible\ncause, which does not sufficiently account for the significantly low yields. A mass balance analysis\nis recommended to clarify the fate of the substrates.\nReply 1:\nSubstrates containing electron-deficient substituents (1f) on the aryl ring exhibited markedly\nlower yields, accompanied by substantial amounts of unreacted starting material. Similarly,\nsubstrates featuring geminal dicyano groups (1n) at the benzylic position also showed incomplete\nconversion. Please see detail in Supplementary Information (Page S37, Section 5.4).\nCommnet 2:\nIn the radical trapping experiment, the reaction yield remains at 32% despite the addition of 4\nequivalents of TEMPO. Any discussions would be helpful.\nReply 2:\nWe sincerely appreciate your valuable feedback and have incorporated the following discussion\ninto revised manuscript: “Furthermore, the reaction maintained a yield of 32% even in the presence\nof TEMPO. This result likely stems from the generation of the alkyl radical intermediate within\nthe confined enzyme pocket, where intramolecular CN trapping is more readily achieved than\nintermolecular quenching by TEMPO.”\nCommnet 3:\nAdditional review could be cited, such as Chin. J. Chem. 2024, DOI: 10.1002/cjoc.202400656\nReply 3:\nThank you very much for your comment. We have cited the important reference in ref. 22.\nCommnet 4:\nThe Supporting Information lacks detailed procedures for the substrate synthesis and enzymatic\nreactions.\nReply 4:\nThank you very much for your important comment.\nWe have detailed the detailed procedures for the substrate synthesis and enzymatic reactions.\nPlease see the Section 4 “General Procedures” in Supplementary Information (Page S21-S26,\nSection 4.1-4.6).\nCommnet 5:\nFor racemic product 2o in Table S65 (line 990), the high-resolution mass spectrometry (HRMS)\ndeviation exceeds 0.001 Da. Re-measurement is suggested.\nReply 5:\nThank you very much for your important comment.\nWe have re-measured the high-resolution mass spectrometry of 2t, we have added this results in\nSupplementary Information (Page S89, Section 6.4).\nHRMS (ESI) m/z: calcd for C H FN ([M+H]+): 203.0985, found: 203.0978.\n12 12 2\nCommnet 6:\nSeveral NMR spectra in the SI are misinterpreted. For example, in the spectrum of racemic 2g\n(S92), the aromatic region is incorrectly integrated to 2.77H, and an extra hydrogen is accounted for\nin the aliphatic region. Similarly, in the spectrum of 2h (S93), a water peak appears to have been\nmisassigned as a product signal. A thorough re-examination of all spectral assignments is suggested.\nReply 6:\nWe sincerely appreciate your valuable feedback regarding the NMR spectral assignments in the\nSupplementary Information. We have thoroughly re-examined all spectra and made the necessary\ncorrections. We have remeasured the 1H NMR spectra of 2g (Page S123, Section 7) and 2k (Page\nS125, Section 7) to unambiguously confirm their structural assignments. The spectra were carefully\nintegrated, with attention paid to distinguishing residual solvent peaks (residual water and\nchloroform) from product resonances.\nCommnet 7:\nMinor errors were noted in the SI: inconsistent font type at S3 line 44, \"standard\" misspelled in\nthe table on S17, and inconsistent abbreviation of \"eq\" in the first reaction scheme on S21. A full\nproofreading is recommended to ensure consistency and accuracy throughout.\nReply 7:\nThank you very much for your careful checking. We have revised the mistakes.\nReviewer #2 (Remarks to the Author):\nReviewer’s general comments:\nThe manuscript by Wang and Xu describes a photoenzymatic 1,5-cyano transfer reaction\ncatalyzed by flavin-dependent ene-reductases. While these types of cyano transfer reactions are well\nknown in the photoredox literature, they have not been realized within a photoenzyme. The ERED\nplatform is well established for photoenzymatic reactivity and can control the delivery of a hydrogen\natom to prochiral benzylic radicals with excellent enantioselectivity, making it a sensible catalyst\nfor the reported reaction. The yields and enantioselectivities are good with a range of substates.\nMechanistic studies confirm that radical initiation occurs via excitation of charge transfer complex.\nOverall, I would describe this work as novel but not particularly surprising as it relies on many well-\nestablished features from the ERED photoenzymatic literature.\nOur response:\nWe sincerely thank the reviewer for the insightful and thorough feedback. We have carefully\nconsidered the comments and revised the manuscript accordingly, which we believe has improved\nthe overall clarity and rigor of our study.\nCommnet 1:\nTOC graphic should use beta rather than B.\nReply 1:\nThank you very much for your careful checking. We have revised the TOC graphic and\nreplaced\"B\" with \"β\".\nCommnet 2:\nIs there a kinetic resolution of the racemic starting material?\nReply 2:\nYour comment is certainly very insightful.\nIn Table 2, most products were obtained in the yields exceeding 50% while maintaining good\nstereoselectivity, allowing us to preliminarily rule out a kinetic resolution pathway. To further clarify\nthe reaction mechanism, we conducted additional control experiments in Supplementary\nInformation (Page S32-S36, Section 5.3).\n(1) After the reaction was terminated, we examined the stereochemical purity of the remaining\nstarting material and found it to be racemic, providing definitive evidence against a kinetic-\nresolution pathway.\nEnantiomeric excess was established by HPLC analysis using a Chiralcel OD-H column, HPLC\nconditions: 254 nm, n-hexane/isopropanol = 99:1, flow rate 1.0 mL/min; Retention time: t(major)\nr\n= 12.658 min, t(minor) = 15.686 min.\nr\nHPLC trace of rac-1a:\nPeak RT/min Area Area/%\n1 12.528 3444 50.087\n2 15.742 3432 49.913\nHPLC trace of 1a after cyano translocation reaction:\nPeak RT/min Area Area/%\n1 12.658 970 50.059\n2 15.686 968 49.941\n(2) Furthermore, we separated the two enantiomers of 1a (1a-1 and 1a-2) by preparative HPLC\nand used each enantiomer as the starting material in independent reactions. Both·1a-1 and 1a-2\nconsistently yielded (S)-2a, demonstrating a stereo-convergent transformation.\nHPLC Conditions: Chiralcel OD-H column, 254 nm, n-hexane/isopropanol = 99:1, flow rate 1.0\nmL/min; t(1a-1) = 9.828 min, t(1a-2) = 11.638 min.\nr r\nHPLC trace of rac-1a:\nPeak RT/min Area Area/%\n1 9.828 484 50.01\n2 11.638 484 49.99\nHPLC trace of 1a-1:\nHPLC trace of 1a-2:\nAccording to General procedure VI with 1a-1 or 1a-2, The ee values and yield of 2a were\ndetermined by GC. Reactions of either single-enantiomer 1a-1 or 1a-2 consistently afforded (S)-2a,\ndemonstrating a stereoconvergent transformation.\nEnantiomeric excess was established by GC analysis using a CP column, the temperature setting\nwas programmed as follows: T : 110 oC; dT/dt: 0.5 oC/min, T : 180 oC.\n0 1\nGC trace of rac-2a:\nPeak RT/min Area Area/%\n1 37.561 16126 49.956\n2 38.017 16154 50.044\nGC trace of (S)-2a with the reaction of 1a-1:\nPeak RT/min Area Area/%\n1 37.507 19652 96.640\n2 37.997 682 3.360\nGC trace of (S)-2a with the reaction of 1a-2:\nPeak RT/min Area Area/%\n1 37.504 17860 96.820\n2 38.015 587 3.180\nIn conclusion, the experimental results presented above demonstrated that the cyano group\ntransfer reaction of the racemic starting material under enzyme catalysis does not proceed via a\nkinetic resolution pathway, but rather followed a stereoconvergent process.\nCommnet 3:\nDoes the method tolerate pyridine as an arene?\nReply 3:\nThank you very much for your insightful comment. We evaluated pyridine-substituted\nsubstrate 1l, which afforded the corresponding product (S)-2l in 65% yield with >99:1 e.r. (Page\nS65, Section 6.1). Additionally, the thiophene- substituted substrate 1m were also examined within\nthe substrate scope, yielding product (S)-2m in 63% yield with 97:3 e.r. (Page S66, Section 6.1). We\nhave added these results into Table 2.\n(S)-5-(pyridin-3-yl)hexanenitrile (S)-2l\nEnantiomeric excess was established by GC analysis using a CP column, >99: 1 e.r.; The\ntemperature setting was programmed as follows: T : 120 oC; dT/dt: 0.5 oC/min, T : 160 oC;\n0 1\nRetention time: t(major) = 47.611 min, t(minor) = 48.563 min.\nr r\nGC trace of rac-2l:\nPeak RT/min Area Area/%\n1 47.521 97165 50.086\n2 48.232 96833 49.914\nGC trace of (S)-2l:\nPeak RT/min Area Area/%\n1 47.611 21404 99.918\n2 48.563 18 0.082\n(S)-5-(thiophen-2-yl)hexanenitrile (S)-2m\nEnantiomeric excess was established by GC analysis using a CP column, 97:3 e.r.; The temperature\nsetting was programmed as follows: T : 130 oC; dT/dt: 0.5 oC/min, T : 170 oC; Retention time:\n0 1\nt(major) = 20.001 min, t(minor) = 20.348 min.\nr r\nGC trace of rac-2m:\nPeak RT/min Area Area/%\n1 19.974 16596 50.013\n2 20.309 16587 49.987\nGC trace of (S)-2m:\nPeak RT/min Area Area/%\n1 20.001 29043 96.627\n2 20.348 1024 3.373\nCommnet 4:\nAre larger alkyl groups (beyond methyl) tolerated by the enzyme?\nReply 4:\nThank you for this valuable suggestion. In addition to the methyl substitution at benzyl position,\nthe ethyl-substituted substrate 1h was evaluated, affording the corresponding product (S)-2h in 65%\nyield with 97:3 e.r. (Page S63, Section 6.1). Furthermore, substrates bearing larger alkyl groups at\nbenzyl position (R = i-Pr (1i) and Cy (1j)) were not well tolerated. These results have been included\nin Table 2.\n(S)-5-phenylheptanenitrile (S)-2h\nEnantiomeric excess was established by GC analysis using a CP column, 97: 3 e.r.; The temperature\nsetting was programmed as follows: T : 110 oC; dT/dt: 0.5 oC/min, T : 180 oC; Retention time:\n0 1\nt(major) = 21.751 min, t(minor) = 22.060 min.\nr r\nGC trace of rac-2h:\nPeak RT/min Area Area/%\n1 21.758 14370 49.958\n2 22.059 14394 50.042\nGC trace of (S)-2h:\nPeak RT/min Area Area/%\n1 21.751 62947 97.104\n2 22.060 1878 2.896\nReviewer #3 (Remarks to the Author):\nReviewer’s general comments:\nThis work introduces a photoenzymatic strategy for stereocontrolled translocation reactions, with\ncyano translocation taken as a model system. It combines experimental data with computational\nanalysis aimed at rationalizing the stereoselectivity. At this stage, however, the computational\ncomponent does not appear sufficiently strong, as it is not entirely clear whether the sampling depth\nand methodological rigor of the analysis are adequate to support firm mechanistic conclusions.\nNevertheless, if the results can be maintained and strengthened after addressing the following\ncomments, the study may reach the level required for publication. I would like to see the revised\nversion of the manuscript.\nOur response:\nWe are truly grateful for the reviewer’s insightful and comprehensive comments. Your\nconstructive feedback has been invaluable, and we have thoroughly revised the manuscript to\nincorporate your suggestions. We believe these improvements have strengthened the clarity and\naccuracy of our study.\nCommnet 1:\nThe simulations need to be extended and at least a second replica needs to be performed for\neach systems.\nReply 1:\nThank you for this valuable suggestion. According to the suggestion, the simulation time has\nbeen extended to 200 ns and three replicas of MD simulations have been performed for each system.\nPlease find the data in Fig. S4−S6 (Page S42-S44, Section 5.7).\nFig. S4. MD simulations of the binding complex of CsER, FMN , and 2a•. (a-c) The root mean\nsq\nsquare deviation (RMSD) profiles of the binding components. (d-f) The profile of distance between\natoms of H5 of FMN and C7 of 2a• through MD simulation. (g-i) The distribution of dihedral angle\nsq\n∠C3-C8-C7-H5, with the positive and negative values indicating the pro-(R) and pro-(S) binding\nforms of 2a•, respectively.\nFig. S5. MD simulations of the binding complex of GluER, FMN , and 2a•. (a-c) The root mean\nsq\nsquare deviation (RMSD) profiles of the binding components. The RMSD fluctuation of 2a•\ncorresponded to the oscillation of the benzyl group. (d-f) The profile of distance between atoms of\nH5 of FMN and C7 of 2a• through MD simulation. (g-i) The distribution of dihedral angle ∠C3-\nsq\nC8-C7-H5, with the positive and negative values indicating the pro-(R) and pro-(S) binding forms\nof 2a•, respectively.\nFig. S6. MD simulations of the binding complex of OYE3, FMN , and 2n•. (a-c) The root mean\nsq\nsquare deviation (RMSD) profiles of the binding components. The RMSD fluctuation of 2n•\ncorresponded to the oscillation of the benzyl group. (d-f) The profile of distance between atoms of\nH5 of FMN and C7 of 2n• through MD simulation. (g-i) The distribution of dihedral angle ∠C3-\nsq\nC8-C7-H5, with the positive and negative values indicating the pro-(R) and pro-(S) binding forms\nof 2n•, respectively.\nCommnet 2:\nRather than showing distances from a single representative configuration (such as those between\nthe substrate and the polar atoms of the active site, or between the benzyl ring and hydrophobic\nactive-site residues), the authors should present the distribution of each distance over the course of\nthe MD simulation, separated for the pro-S and pro-R basins.\nReply 2:\nThank you for your valuable suggestions; they are highly instrumental in improving the accuracy\nand reliability of the MD results. Accordingly, the distribution of the mentioned key distances over\nthe course of the MD simulations have been provided in Fig. S8−S10, with the distances\ncorresponding to distinct (pro-(S) and pro-(R)) conformations shown in different colors (Page S46-\nS48, Section 5.7).\nFig. S8. Profiles of distances between the binding 2a• and CsER key residues. a‒c Distance between\nthe atom N1 of 2a• and the atom HE2 of residue H173; d‒f Distance between the centroids of the\nbenzyl groups of 2a• and residue Y66; g‒i Distance between the centroids of the benzyl groups of\n2a• and residue Y336. The distances corresponding to the pro-(R) and pro-(S) configured binding\nconformations of 2a• were represented as black and orange dots, respectively. The values were\ncomputed based on three independent MD simulations.\nFig. S9. Profiles of distances between the binding 2a• and GluER key residues. a‒c Distance\nbetween the atom N1 of 2a• and the atom HE2 of residue H172; d‒f Distance between the centroid\nof the benzyl group of 2a• and the centroid of the indolyl group of residue W66; g‒i Distance\nbetween the centroids of the benzyl groups of 2a• and residue F269; j‒l Distance between the\ncentroid of the benzyl group of 2a• and the centroid of the indolyl group of residue W342. The\ndistances corresponding to the pro-(R) and pro-(S) configured binding conformations of 2a• were\nrepresented as black and orange dots, respectively. The values were computed based on three\nindependent MD simulations.\nFig. S10. Profiles of distances between the binding 2n• and OYE3-Y169F key residues. a‒c\nDistance between the atom N1 of 2n• and the atom HE2 of residue H191; d‒f Distance between the\natom N1 of 2n• and the atom HD21 of residue N194; g‒i Distance between the centroids of the\nbenzyl groups of 2n• and residue Y82; j‒l Distance between the centroids of the benzyl groups of\n2n• and residue Y375. The distances corresponding to the pro-(R) and pro-(S) configured binding\nconformations of 2n• were represented as black and orange dots, respectively. The values were\ncomputed based on three independent MD simulations.\nCommnet 3:\nOne of the main conclusions drawn from the simulations is the following:\n“Notably, the benzyl group formed additional parallel (with W100) and T-shaped (with F269) π-\nπ stacking interactions in the pro-(S) binding form in CsER and the pro-(R) binding form in GluER,\nrespectively, providing extra binding stability. In contrast, the pro-(R) binding form in CsER and\nthe pro-(S) binding form in GluER showed much more exposure of the 2a• hydrophobic group to\npolar water molecules, indicative of an energetically unfavorable binding mode.”\nThis could be an interesting observation but should be corroborated by specific analyses.a)\nConcerning the distances between the benzyl group and hydrophobic residues, the authors should\nalso consider the side chains of residues that could for pi-pi interactions in the “wrong” pro-chiral\nbasin as for example:\n-distance between the benzyl group and Y336 or Y66 in CsER (pro-R)\n-distance between the benzyl group and W66 and W342 in GluER (pro-S)\nReply 3a:\nThank you for your valuable suggestions. Analyses on the mentioned distances including the ones\nbetween the aromatic sidechains of Y66/Y336 and the benzyl group of the reaction intermediate 2a•\nin CsER and these between the sidechains of W66/F269/W342 and the benzyl group of 2a• in GluER\nhave been performed. In addition, the distances between the sidechains of Y82/Y375 and the benzyl\ngroup of 2a• in OYE3 were analyzed as well. The corresponding results are provided in Fig. S8−S10,\nin which the distances corresponding to the pro-(R)/pro-(S) configured structures were shown in\ndifferent colors. Combining with the dihedral data (Fig. S4−S6) and visual inspection on the\nstructural conformations, we find that the chiral conversion is closely related to the rotation of the\nC7‒C8 bond. Based on the results of three time-extended MD replicas for each binding system, we\nhave revised the above descriptions of MD simulations as “Relative to the pro-(R) binding form, the\nbenzyl group of pro-(S) 2a• formed additional parallel π-π stacking interactions with Y66 (Fig. S8),\nproviding extra binding stability. In binding with GluER, two π-π stacking interactions were\nobserved for both the pro-(R) (with W66 and W342) and pro-(S) (with W66 and F269) 2a•. However,\nthe distances of the pro-(S) 2a• involved π-π stacking are relatively longer than that of the pro-(R)\n2a• (Fig. 2e and Fig. S9), indicating a decreased binding strength”. Please find the revision on Page\n10, highlighted in yellow.\nb) Concerning the comment on the larger exposure to water molecules of hydrophobic groups,\nthis should be shown with appropriate analyses\nReply 3b:\nAccording to the suggestions, the solvent accessible surface area (SASA) that describes the extent\nof exposure of concerned residue or compound in the binding complex has been calculated for the\nintermediate 2a•/2n• based on the three time-extended MD replicas. However, the results shown in\nFig. S11 indicate that the pro-(R) and pro-(S) configured 2a•/2n• in CsER, GluER, and OYE3 only\ndemonstrated marginal difference in SASA value, with no consistent changing tendency observed\n(Page S49, Section 5.7). The result indicates that solvent exposure may not play a fundamental role\nin determining the binding conformation of 2a•/2n•. Therefore, we have deleted the discussion\ncontents about solvent exposure.\nFig. S11. The solvent accessible surface area (SASA) of 2a• and 2n• in the binding complexes. The\nSASA values corresponding to the pro-(R) and pro-(S) binding states of 2a•/2n• were represented as\ngreen and orange columns, respectively. The values were computed based on three independent MD\nsimulations.\nCommnet 4:\nConcerning the OYE3 enzyme, the authors claim: “the dihedral distribution and structure\ncharacteristics in the binding complex of OYE3(Y196F), FMNsq, and 2n• were in accordance with\nthe reduced stereoselectivity for the dicyano-substituted compounds (Fig. S6).”\nI might agree with this conclusion, but not for the reasons provided by the authors. In fact,\nfor OYE3-Y196F the negative values do not appear to be pro-S but rather achiral, as they lie too close\nto –180° to define a clear prochiral preference. Similarly, for the positive values the average dihedral\nangle may also be not too far from 180°, which again implies an unclear chiral assignment. The\nauthors may therefore wish to reconsider and revise their interpretation.\nReply 4:\nThank you for your valuable suggestion, which is instrumental in enabling us to present our\nexperimental findings with enhanced precision. We fully agree with your perspective. Following\nyour suggestion, we have revised the interpretation as “ For the dicyano-substituted inetermediate\n2n•, a distinct binding feature was discovered in its binding with OYE3 (Y196F), i.e. the H5 atom\nis almost always coplanar with the radical plane of 2n• (defined by atoms C3, C7, and C8, Fig. S7),\nleading to a concentrated distribution of dihedral angles around 180° and –180° and a decreased\nconformational preference (Fig. S6). In comparison with the pro-(S) 2n•, the pro-(R) binding form\nis more favorable by forming a sandwich framework with Y82 and Y375 through π-π stacking\ninteractions, resulting in a dihedral distribution which is in accordance with the stereoselectivity of\nOYE3 (Y196F) (Fig. S6 and S7). Notably, with the cyano group of 2a•/2n• stably anchored to the\npolar residues His and/or Asn through hydrogen bond interactions in all binding systems, the\nprochiral conversion can be primarily attributed to the rotation of the C7‒C8 bond.”. Please find the\nrevision on Page 10 in yellow.\nMinor points:\nCommnet 5:\nWhich software was used for optimizations at the DFT level?\nReply 5:\nThe DFT calculations were performed by using the Gaussian 16 software. This information has\nbeen added to the text with the software properly cited.\nCommnet 6:\nWhy was the Berendsen barostat used?\nReply 6:\nAll MD simulations were performed by using the Amber 24 software in which two kinds of\nbarastats, i.e. Berendsen and MonteCarlo barostat. Though Berendsen barostat does not strictly\nsample from the isothermal-isobaric ensemble and typically yields volume fluctuations that are too\nlow, it is simple and effective for equilibrating systems quickly. Therefore, the Berendsen barostat\nwhich is the default pressure regulation algorithm in Amber software was used.\nCommnet 7:\nWhich thermostat was used?\nReply 7:\nIn all MD simulations, the Langevin thermostat was used for temperature regulation. This\ninformation has been added to the method part (Page S40-S41, Section 5.7).\nCommnet 8:\nWhich software (or in-house script?) was used for the clustering analysis?\nReply 8:\nFollowing the official recommended guide (https://amberhub.chpc.utah.edu/clustering-a-protein-\ntrajectory/), the clustering analysis was performed by applying the K-means clustering algorithm.\nCommnet 9:\nWhich atoms were chosen for the fit in the RMSD calculations for each of the three components?\nReply 9:\nAll atoms of CsER/GluER/OYE3, FMN , and 2a•/2n• were used for the RMSD calculations.\nsq\nCommnet 10:\nThe y label in Figure S4, panel b, should be distance, not RMSD.\nReply:\nWe are sorry for the mistake. The label has been revised.\nCommnet 11:\nSome data on the clustering analysis should be reported to better understand the choice of the\nrepresentative structure.\nReply 11:\nThank you for your valuable suggestion. Tables that summarize the clustering result have been\nprovided (Table S7−S9). Structure comparison demonstrated that the representative structures of\ntop 4 clusters are of considerable conformational similarity. Therefore, the representative structure\nof cluster 0 (the centroid frame) was finally chosen (Page S53, Section 5.7).\nCommnet 12:\n“Substrate scope of for the photoenzymatic enantioselective CN translocation reaction”. “for”\nneeds to be deleted.\nReply 12:\nAccording to the suggestion, the word “for” has been deleted (Table 2 title). Please find the\nrevision on Page 8.\nCommnet 13:\nWhat do the authors mean with “solute” in the sentence “with a weak constraint of 10 kcal∙mol-\n1∙Å -2 imposed on the solute “?\nReply 13:\nSolute means the components of binding complexes, including CsER/GluER/ OYE3, FMNsq,\nand 2a•/2n•. To improve the descriptive accuracy, we have revised the sentence to be “Then, the\nsystem was gently annealed from 0 to 300 K under the NVT ensemble for 500 ps, with a weak\nconstraint of 10 kcal∙mol-1∙Å-2 imposed on CsER/GluER/ OYE3, FMN , and 2a•/2n•”.\nsq\nReviewer #4 (Remarks to the Author):\nReviewer’s general comments:\nXu et al reported an interesting cyano translocation reaction with high stereoselectivity under the\nphotoenzymatic catalysis with ene reductases. But it seems this reaction only works with three\ncarbons, limiting its synthetic applications. It did not work with four carbon atoms, possibly due the\nstability of six-membered cyclic radical intermediate. It is not clear whether it works with other\nnumbers of carbon atoms, that may be dependent on the ability of forming the cyclic intermediate\nand the stability of the intermediate. The computational analysis may address this point.\nOur response:\nThank you for your valuable comment.\nTo examine the feasibility of four-, six-, and seven-membered cyclic radical intermediates in the\nreaction, we conducted experiments using substrates 1w, 1v, and 1x. As shown in the figure below,\nonly products resulting from direct HAT after deiodination were observed, and no products arising\nfrom CN migration were detected. We have added this content to the Supplementary Information\n(Page S38, Section 5.5).\nTo elucidate why the stereoselective cyano translocation reaction favors substrate with 1a (five-\nmembered cyclic radical intermediates), DFT calculations were performed on the cluster models\ncomposed of FMN and the deiodinated radical intermediates derived from 1w, 1a, and 1v,\nsq\ncorresponding to the potential formation of four-, five-, and six-membered cyclic radical\nintermediates. Two plausible reaction pathways were investigated: the formation of cyclic\nintermediates and direct quenching through HAT. The results demonstrate that only the intermediate\nderived from 1a favors the cyclic intermediate pathway with an energy barrier of 4.21 kcal/mol,\nwhich is 3.31 kcal/mol less than the HAT pathway (Fig. S12). For the intermediate derived from 1w\nand 1v, the cyclic intermediate pathway shows obviously higher energy barriers than the\ncorresponding HAT pathway (Fig. S13 and S14). The DFT results agree well with our experimental\nfindings and explain the reaction preference for the substrates which are able to form five-membered\ncyclic intermediate. This result has been included in the Supplementary Information (Page S50-S52,\nSection 5.7). Additionally, we have added the following sentence in the main text: “DFT calculations\nfurther corroborate that the 1,4-CN translocation pathway is energetically preferred.”.\nFig. S12. DFT calculated energy profile and the structures of stationary points of the reaction\nprocess of the deiodinated radical intermediates derived from 1a. TS-1 and Int-2 are the transition\nstate and reaction intermediate corresponding to the cyclic intermediate reaction pathway; TS-1ʹ and\nInt-2ʹ are the transition state and reaction product corresponding to the direct HAT reaction pathway.\nEnergies are in kcal/mol.\nFig. S13. DFT calculated energy profile and the structures of stationary points of the reaction\nprocess of the deiodinated radical intermediates derived from 1w.TS-1 and Int-2 are the transition\nstate and reaction intermediate corresponding to the cyclic intermediate reaction pathway; TS-1ʹ and\nInt-2ʹ are the transition state and reaction product corresponding to the direct HAT reaction pathway.\nEnergies are in kcal/mol.\nFig. S14. DFT calculated energy profile and the structures of stationary points of the reaction\nprocess of the deiodinated radical intermediates derived from 1v. TS-1 and Int-2 are the transition\nstate and reaction intermediate corresponding to the cyclic intermediate reaction pathway; TS-1ʹ and\nInt-2ʹ are the transition state and reaction product corresponding to the direct HAT reaction pathway.\nEnergies are in kcal/mol.\nComments 2:\nTable 2, CsER or GluER is noted in the footnote, but it is not noted which one under each product.\nIt should be noted under each product for clarity.\nReply 2:\nWe thank the reviewer for this helpful suggestion. To avoid any potential confusion, we have\nprovided a more detailed description in the footnote and noted under each product for clarity in\nTable 2.\nTable 2. Substrate scope of the photoenzymatic enantioselective CN translocation reactiona,c"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have well-addressed my comments. I fully support the acceptance of the revised MS. Congratulations!", "reviewer_2": "The authors have addressed my concerns. This work is now suitable for publication. I suggest adding a reference to the recent JACS paper by Yang and coworkers that do a similar nitrile transfer reaction.", "reviewer_3": "The MD simulation section of the manuscript is clearly improved. However, a few minor points still need to be addressed.\nFirst, the y-axis scaling of the RMSD, distance plots, and similar time-dependent data should be reduced so that the fluctuations can be more easily appreciated. Although the resulting profiles may appear less converged, which is understandable for systems of this complexity, it is nevertheless important that the reader can clearly visualize the underlying variation in the data.\nRegarding my original point 3 (reported again below), I asked the authors to present distributions of the relevant distances, not only their time-dependent traces. The authors should therefore include, in addition to the time-dependence, the corresponding normalized distance distributions, separately for the pro-R and pro-S conformational basins.\nOriginal point 3:\n“Rather than showing distances from a single representative configuration (such as those between the substrate and polar atoms of the active site, or between the benzyl ring and hydrophobic active-site residues), the authors should present the distribution of each distance over the course of the MD simulation, separated for the pro-S and pro-R basins.”\nFinally, I have a concern regarding the DFT calculations. If I am not mistaken, the QM models include a radical species. If this is correct, the authors must explicitly state whether restricted or unrestricted DFT was used. Without this information, it is not possible to properly assess the reliability of the computed energies and structures.", "reviewer_4": "The revisions have generally answered the questions.\nFor the reply to the first question of reviewer 4, since they are valuable data, it should be briefly discussed in the main text."}, "author_response": "Dear Reviewers:\nWe sincerely thank you for your careful review and valuable suggestions on our manuscript. Your\ncomments have been immensely helpful in improving the quality of our work. We have thoroughly\nconsidered all your feedback and have implemented appropriate revisions accordingly. All changes\nhave been highlighted in yellow in the revised manuscript for your convenience. We provide our\npoint-by-point responses to each of your comments as following.\nReviewer #1 (Remarks to the Author):\nThe authors have well-addressed my comments. I fully support the acceptance of the revised MS.\nCongratulations!\nOur response:\nThank you for your positive feedback and for your time and effort in reviewing the manuscript.\nWe are delighted to hear that you fully support its acceptance. We sincerely appreciate your valuable\ncomments throughout the review process, which have significantly contributed to improving the\nquality of our work.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed my concerns. This work is now suitable for publication. I suggest\nadding a reference to the recent JACS paper by Yang and coworkers that do a similar nitrile transfer\nreaction.\nOur response:\nThank you for your positive evaluation and for recommending our manuscript for publication.\nWe greatly appreciate the constructive feedback provided throughout the review process. We have\nadded Yang’s paper as ref. 55 and a brief discussion in the revised manuscript as “While our\nmanuscript was under revision, Yang reported a similar cyano migration reaction catalyzed by an\nNADPH-dependent imine reductase.55.”.\nReviewer #3 (Remarks to the Author):\nThe MD simulation section of the manuscript is clearly improved. However, a few minor points\nstill need to be addressed.\nCommnet 1:\nFirst, the y-axis scaling of the RMSD, distance plots, and similar time-dependent data should be\nreduced so that the fluctuations can be more easily appreciated. Although the resulting profiles may\nappear less converged, which is understandable for systems of this complexity, it is nevertheless\nimportant that the reader can clearly visualize the underlying variation in the data.\nOur response:\nThank you for your positive comment on our revisions. We are also highly appreciative of your\nvaluable suggestion regarding the y-axis scaling of our figures.\nWe agree that while the inherent complexity of systems may cause the resulting profiles to appear\nless converged, clearly visualizing the dynamic behavior of the system is essential for scientific\ncommunication. Therefore, as suggested, we have reduced the scaling of the y-axis for all the time-\ndependent data including RMSD plots and distance plots. This modification allows for a clearer\nvisualization of the underlying dynamic fluctuations in the data. Please find the revised data in Fig.\nS4−S6 and Fig. S8−S13. Thank you for your positive comment on our revisions. We are also highly\nappreciative of your valuable suggestion regarding the y-axis scaling of our figures.\nCommnet 2:\nRegarding my original point 3 (reported again below), I asked the authors to present distributions\nof the relevant distances, not only their time-dependent traces. The authors should therefore include,\nin addition to the time-dependence, the corresponding normalized distance distributions, separately\nfor the pro-R and pro-S conformational basins.\nOriginal point 3: “Rather than showing distances from a single representative configuration (such\nas those between the substrate and polar atoms of the active site, or between the benzyl ring and\nhydrophobic active-site residues), the authors should present the distribution of each distance over\nthe course of the MD simulation, separated for the pro-S and pro-R basins.”\nOur response:\nWe appreciate you bringing our oversight regarding the presentation of distance analysis to our\nattention once again. We apologize for not having fully provided the requested distance distribution\nanalysis in the previous revision.\nFollowing your explicit instructions, we have now performed a detailed normalized distribution\nanalysis for all relevant distances, separately for the pro-S and pro-R conformational basins. Please\nfind the corresponding data in Fig. S9, S11, and S13, in which the x-axis represents the distance and\nthe y-axis represents the probability density. [Reference: Unveiling Interactions of a Peptide-Bound\nMonolayer-Protected Metal Nanocluster with a Lipid Bilayer. J. Phys. Chem. Lett. 2025, 16, 3351-\n3358.]\nAs you rightly emphasized, providing this distribution analysis in addition to the time traces\nallows for a more intuitive and quantitative identification of the intermolecular distances that are\nmost crucial for discriminating between the pro-(S) and pro-(R) conformational basins.\nThank you again for this valuable and professional guidance, which significantly enhances the\ndepth and rigor of our molecular dynamics analysis.\nCommnet 3:\nFinally, I have a concern regarding the DFT calculations. If I am not mistaken, the QM models\ninclude a radical species. If this is correct, the authors must explicitly state whether restricted or\nunrestricted DFT was used. Without this information, it is not possible to properly assess the\nreliability of the computed energies and structures.\nOur response:\nThank you for raising this crucial technical concern. You are correct that our QM cluster models\ninvolve radical species, specifically the intermediate radical and the FMN semiquinone (FMN ).\nsq\nTherefore, for the DFT calculations on these open-shell systems, we employed the open-shell\n(unrestricted) singlet DFT method. This unrestricted treatment method is the appropriate choice to\nensure the reliability of the computed energies and optimized structures for such species. As per\nyour suggestion, we have now explicitly updated both the computational methods section and the\nrelevant figure legends to include this specification, thereby enhancing the transparency and rigor\nof our computational details. Please refer to Page S53-S55 in the Supplementary Information for\nthe updated text.\nReviewer #4 (Remarks to the Author):\nThe revisions have generally answered the questions.\nFor the reply to the first question of reviewer 4, since they are valuable data, it should be briefly\ndiscussed in the main text.\nOur response:\nWe sincerely thank the reviewer for this valuable suggestion. Accordingly, Figure 2c has been\nrevised to include the experimental results obtained with substrates of different chain lengths, and\nthe corresponding discussion has been added to the main text. Please see “Experiments were\nconducted of alkene substrates (1w and 1v) with different carbon-chain lengths. As shown in Fig.\n2c, deiodinated products from direct hydrogen atom transfer (HAT) were detected, whereas no\ncyano migration products were observed (Section 5.5 in SI). To further probe the mechanistic basis\nof this observation, density functional theory (DFT) calculations were performed on cluster models\ncomposed of FMN and the corresponding deiodinated radical intermediates. The results reveal that\nsq\nthe 1,4-cyano translocation pathway of 1a, proceeding via a five-membered cyclic radical\nintermediate, is energetically favored, in good agreement with the experimental observations.”"}, {"round": 3, "reviewer_comments": {"reviewer_3": "The analyses are now satisfactory. However, the scale of the y-axis for the time-dependent properties and the scale of the x-axis of the corresponding distributions should be the same for a given property in the three MD simulations."}, "author_response": "Dear Reviewers:\nWe would like to express our sincere gratitude for your thorough review and the constructive\ncomments. Your insightful suggestions have been immensely helpful in improving the quality of\nour work. We have carefully addressed your points raised and have incorporated revisions into the\nmanuscript. We provide a detailed point-by-point response to your comments as following.\nREVIEWERS' COMMENTS\nReviewer #3 (Remarks to the Author):\nReviewer’s comments:\nThe analyses are now satisfactory. However, the scale of the y-axis for the time-dependent\nproperties and the scale of the x-axis of the corresponding distributions should be the same for a\ngiven property in the three MD simulations.\nResponse:\nThank you for your positive feedback on our revisions. We also greatly appreciate your\nvaluable suggestion concerning the axis scaling in our figures–specifically the y-axis for time-\ndependent properties and the x-axis for the corresponding distributions.\nWe fully agree with this point and have adjusted the scales accordingly. Please refer to the\nrevised Fig. S4−S6 and S8−S13 in the updated Supplementary Information."}]

Decision letter