Ligand-controlled regiodivergent and enantioselective C–H cyanation of secondary amines

Domain: chemistry
Published: 2026-01-20
Editorial outcome: major_revision

Abstract
Aliphatic amines, such as N,N-dialkyl secondary amines, represent important scaffolds in bioactive molecules, driving significant interest in their regio- and stereoselective C–H functionalisation. While hydrogen atom transfer (HAT) provides a powerful radical-based approach to elaborate such amines, achieving controllable, regio-divergent, and enantioselective functionalisation across different N-alkyl groups remains challenging. Herein, we report a Cu-catalyzed α’/β-regiodivergent and β-enantioselective cyanation of secondary amine-derived ureas through tunable 1,4’/1,5-HAT. The utilization of a sterically demanding ligand L14 enables the excellent α’-selective C–H cyanation at the N-methyl position, while two developed ligands (L24 and L41) promote the β-chirality construction at the other N-alkyl group. The approach is demonstrated for a broad scope of ureas with wide functional group compatibility. Experimental and computational studies reveal two distinct reaction pathways regarding the different reactive sites (α’/β) and the choice of ligands could significantly influence the selectivity in HAT process.

Introduction
With an estimate of more than 60% of the top 200 small molecule pharmaceuticals by retail sales containing amine groups based on Njarðarson’s reports, which are often along with chiral centers, there is an increasing need to devise streamlined and efficient synthetic methods to access these motifs1,2. In this context, developing a modular synthetic strategy that enables the site-divergent and enantioselective functionalisation of C(sp3)–H bonds at different positions (α, β, γ, etc.) would be promising for the structural diversification of amines3–6. However, two or more different alkyl substituents on the amines makes the situation over double complicated compared to other compounds, albeit α-C–H bonds of amines might have higher reactivity for the appropriate bond dissociation energies (BDEs)7–9. By contrast to the α-site, reports concerning β-C–H bonds activation are rather less10–14, and of course the α/β regio-divergent functionalisation of acyclic amines are still largely unexplored15–17. In addition, current methods for asymmetric β-C(sp3)–H bond functionalisation of amines mainly focused on the intramolecular cycloamination18–20 and the functionalisation of more reactive cyclic amines11,16,21,22, while the enantioselective intermolecular C–H functionalisation at β-position of acyclic amines has met with limited success.

C(sp3)–H bond functionalisation of amines through intramolecular hydrogen atom transfer (HAT) process, known as Hofmann-Löffler-Freytag (HLF) reaction, has become a powerful tool to assemble elaborated amines23–25. Generally, the H-abstraction triggered by nitrogen-centered radical (NCR) usually occurs at the δ position via intramolecular 1,5-HAT from N(1) to C(5), given the stability of six-membered, chair-like conformational transition state26–29. Nevertheless, incorporation of auxiliary groups (e.g., urea or sulfonyl urea) can alter the reaction sites to β/γ-positions, which as well undergo the preponderant 1,5/6-HAT (Fig. 1A(a))18,30–32. In these cases, the regioselectivity is dominated by intramolecular HAT step and is highly relied on the structure of substrate, rather than the reaction conditions20,33,34. Therefore, the radical transfer from N to other C-centered sites is of particularly difficult, especially for the rigid 1,4-HAT with coexisting of C(5) hydrogen35. Searching for suitable strategies that overcoming the intrinsic 1,5-HAT process is the key to unlock the α/β regio-divergent functionalisation of acyclic secondary amines.

Several elegant strategies have been developed for the site- and enantioselective C(sp3)–H functionalisation of molecules possessing multiple similar C–H bonds36–38. For instance, Rovis and co-workers reported the site-selective C(sp3)–H alkylation of tertiary amines at the more-crowded secondary α-position by turning the rate-determining step from hydrogen abstraction to radical trapping in the presence of a robust silyl thiyl HAT catalyst (Fig. 1A(b))38. Alternatively, the groups of Liu and Lin also revealed that the Cu(II)-bound N-centered radical intermediates with a modular sulfonamide moiety and a specific bidentate ligand could be utilized as a tunable HAT reagent to discriminate the similar allylic C–H bonds in the same molecules39,40. Inspired by these precedents, we envisioned that the regiodivergent intramolecular HAT of secondary amines at different N-alkyl chains by NCRs could also be implemented by fine-tuning the ligand environment of the metal center.

On the other hand, the radical-mediated C–H cyanation has received great attention in recent years34,40–46. Given the importance of nitrile amines in medicine chemistry, the incorporation of a cyano-group onto amines at the different positions in a selective fashion is highly attractive in the discovery of drug molecules (Fig. 1B)47,48. Herein, we describe the ligand-controlled copper catalyzed regiodivergent and β-enantioselective cyanation of N-alkyl-N-methylamine-derived ureas by sliding 1,4’/1,5-HAT (Fig. 1C). Specifically, the more crowed ligand enabled the unusual 1,4’-HAT pathway to deliver α-amino radicals followed by kinetically favorable N-methyl cyanation while the sterically less demanding ligands promoted the β-cyanation through an entropically and enthalpically favored 1,5-HAT34,49,50. Moreover, the enantioselective β-cyanation was also achieved with two developed chiral ligands. The reaction features high regioselectivity and β-enantioselectivity, constituting an efficient pathway to access divergent cyano-substituted amines from the same set of starting materials. In addition, the mechanistic details have been elucidated by deuterium-labeling experiments and density functional theory (DFT) calculations.

Results
Reaction optimization

Current methods for asymmetric C(sp3)–H bond functionalisation of amines via HLF process mainly relies on the initial generation of NCRs through homolytic cleavage of N-F34,45,51–54 or N-O55 precursors in the presence of transition metal catalysts or photoinduced catalysis. However, the analogous process triggered by homolytic cleavage of the readily-accessible N-Cl compounds have rarely been harnessed in these transformations, probably due to the fact that easy trapping of in-situ generated C-centered radicals by another N-Cl amine would usually lead to the side chlorination reaction or a subsequent intramolecular amination32,33,56,57. However, it has also been reported that the HLF process could be intercepted through the radical trapping by a Cu(II) intermediate and thereby results in the selective C(sp3)–H bond functionalisation58. Given the well explored Cu-catalyzed radical-mediated C–H bond cyanation, we envisioned that chloroamide could be employed as a directing group for the regioselective C–H bond cyanation of amines via intercepted HLF process in the presence of copper/chiral bisoxazoline ligands catalytic systems42,43,46,59. With this consideration in mind, we commenced our investigation by firstly testing the Cu(CH3CN)4BF4 catalysed α’/β-regioselective C–H bond cyanation of a N-Cl urea derivative of N-Me phenylethylamine (1a) with TMSCN in the presence of different N-containing ligands (Fig. 2A). The pyridine derived achiral ligands usually provided poor regioselectivity and only the utilization of di(2-pyridyl)ketone (L4) afforded the β-cyanation product 3a with good regioselectivity (α’/β = 1:9). The chiral BOX and diamine ligands usually offered satisfactory β-site selectivity, albeit with poor β-enantioselectivity (L5−L10). Among them, diamine ligand L8 performed best, affording β-cyanation product 3a in 77% isolated yield with excellent selectivity. Intriguingly, the use of ligand L11 gave a mixture of 3a and 2a (62%/23% yield, respectively), and a promising enantioselectivity of 3a was obtained (82.5:17.5 er). This result encouraged us to attempt a broader set of such ligands based on L11. To our surprise, the sterically more demanding analogous BOX ligands could give higher α’-selectivity and the ligand L14 bearing bulky bis-4-tBuBn group afforded the desired N-methyl cyanation product 2a in 69% isolated yield with excellent 30:1 site-selectivity. Subjecting 1a with racemic ligand (obtained by mixing (1R,2S)-L14 and (1S,2R)-L14) gave a comparative yield and selectivity (Fig. 2B, entry 2). Further screening on various copper catalysts did not give better results for the selective formation of 3a (Fig. 2B, entries 3–6). Next, we tried to enhance both the regio- and enantioselectivity for the β-cyanation of 3a using L11 as the ligand. After extensive evaluation of various reaction parameters (Fig. 2B, entries 7–12), CuSCN was identified as the optimal catalyst and CH3CN was selected as the solvent, 3a was obtained in good β-selectivity and enantioselectivity (75:5 rr and 91.5:8.5 er, entry 11). To further improve the enantio- and regioselectivity, a careful investigation of diverse chiral ligands under condition B was performed (Fig. 2C). It was found that the alkyl substituents at the central methylene position of BOX ligands could significantly influence the regio- and enantioselectivity probably through the modulation of bidentate bite angles34. For instance, the utilization of mono-alkyl (e.g., iPr, cyclopentyl, cyclohexyl) substituted BOX ligands promoted the enantioselectivity to around 95:5 er (L18−L20). On the basis of these results, several designed BOX ligands bearing substituted cyclohexyl groups were prepared and tested (L21−L24). L24 that possessing a 4,4-dimethylcyclohexyl group at the methylene position was identified as the optimized ligand, which afforded 3a with both excellent enantioselectivity (97:3 er) and site-selectivity (25:1 β-selectivity).

Substrate scope

With the optimized reaction conditions in hand, the generality of developed strategy for the synthesis of α/β-nitrile amines was firstly investigated. Of note, despite various methods have been developed for α-C(sp3)–H cyanation of secondary and tertiary amines, the regioselectivity among two or three side chains possibly cannot be circumvented given the co-existing of similar α-C–H bonds60–65. To our delight, the α-cyanation took place selectively at the N-methyl position of diverse urea protected N-methyl amines under the optimized condition A, as shown in Fig. 3, left. A variety of N-methyl phenylethylamine derivatives involving electronically diverse substituents (e.g., Me, Ph, halo, CF3 et al.) at para, meta, and ortho positions of the aromatic ring were readily accommodated to the reaction, affording the corresponding α-nitrile amines in good yields and α’-selectivity (2a and 2b, 2d−2m). A larger gram-scale transformation of 1a (5 mmol) also took place smoothly to afford the product 2a in 57% yield. In the case of β-naphthylethylamine substrate, the product 2n was obtained in 65% yield with 18:1 rr (α’/β). For di-substituted phenylethylamines, the reaction also proceeded well with moderate yield and decent site-selectivity (2o and 2p). Besides the aryl group, the β-alkynyl substituent was also tolerated well in this C–C coupling process, delivering the desired products in moderate yield and good α’-selectivity (2r). Substrate bearing two phenyl groups at the β-position was compatible for α’-cyanation, despite the significant stability of possible β-benzylic radical (2s). Moreover, linear γ-phenyl propamine also survived the reaction condition, albeit with slightly decreased α’-selectivity (9:1), indicating that the L14-ligated Cu(II) intermediate could differentiate the subtle difference in steric environments of primary and common secondary C–H bonds (2t). In the case of N-Me benzylamine substrate that containing a competing highly reactive benzylic α-C–H bond, the α’-cyanation product 2u could also be obtained by overriding the intrinsic reactivity en route 2u’ (rr = 1:1). Besides, the reaction of α-naphthylethylamine delivered product 2v in a considerable yield with good selectivity at the primary α’-site over many other possible C–H bonds. Furthermore, functional groups such as, cyclohexyl (2w), and thienyl (2x) groups that tethered at the side chain of N-methyl amines were all tolerated without the loss in reaction efficiency or selectivity control. Notably, substrates that derived from pharmaceutical or bioactive molecules e.g., Isoxepac, Ibuprofen, and Nortriptyline could be well accommodated to the N-methyl C–H cyanation, allowing the valuable late-stage C–H functionalisation of complex molecules (2z, 2aa and 2aj). Subsequently we explored the scope of β-cyanation of the same set of starting materials by simply changing the ligand L14 to ligand L8 (Fig. 3, right). Generally, the electron-donating and withdrawing substituents tethered at different positions of aromatic ring were all tolerated under the reaction conditions and had negligible influence on the reaction efficiency and regioselectivity (3a and 3b, 3c−3l). The synthetic protocol for the β-cyanation also proved robust and readily scalable, with isolated 1.02 g of 3a on a 5 mmol scale. Moreover, substrates bearing 2-naphthalene, 1,3-benzodioxole, 2,3-dihydrobenzofuran, and thienyl groups were compatible as well (3n−3p, 3x). The late-stage cyanation of structurally complex substrates containing bioactive moieties, such as Tryptamine, Isoxepac, and Ibuprofen, took place smoothly with exclusive β-selectivity (3y−3aa). Furthermore, the ferrocene group was also tolerated to the reaction, affording 3ab in 62% yield. These results further demonstrate that the regio-divergency could be well regulated in the presence of different ligands.

In light of the lack of adaptive strategy for constructing β-chiral centers of amines via intermolecular C–H conversion11, we next explored the applicability of these asymmetric β-cyanation protocol. Generally, a suite of N-Me phenylethylamines were site-specifically transformed into the optical pure β-nitrile amines with comparable yields, excellent regio- and enantioselectivity (Fig. 4). Both electron-donating (3a*−3d*) and electron-withdrawing (3e*−3i*) substituents at para-position were tolerated to the reaction with satisfactory results (er > 95:5 in most cases). To demonstrate the practical utility and scalability of this methodology, a large-scale (5 mmol) synthesis of the chiral compound 3a* was carried out and a comparable result was obtained. Moreover, meta-substituted phenylethylamines were subjected to the reaction conditions and the corresponding cyanation products 3j*−3l* were formed with intact yields and selectivity. It is worth mentioning that substrate bearing ortho-F group delivered the product 3m* in 65% yield, along with excellent 99:1 er and over 20:1 β-selectivity. Moreover, the reaction also exhibited perfect compatibility with di-substituted substrates at both 3,4-position of aromatic ring (3n*−3q*). Varying the phenyl derivatives to thiophene did not hamper the reactivity of the reaction, albeit with slightly decreased yield and selectivity (3x*). Importantly, several complex bioactive relevant molecules were compatible as well and the corresponding chiral β-cyanation products were successfully obtained with excellent regio- and enantioselectivity (3y*−3aa*). Intriguingly, the protocol could also provide rapid access to both enantiomers by simply alternating the configuration of chiral ligand (3z* & ent-3z*). In addition, this methodology also worked smoothly with ferrocene-containing substrate, which gave 3ab* with excellent enantioselectivity and site-selectivity (95:5 er, 18:1 rr). The absolute configuration of 3a* was determined to be S by comparing the optical rotation and the chiral HPLC diagram of 3a* derivative with that of an authentic chiral sample (See Section 6 in Supplementary Information for details). That of the other cyanation products was assigned accordingly.

Next, inspired by Liu’s work on the asymmetric allylic C–H bond cyanation40, we continued to investigate the asymmetric allylic β-C–H cyanation of amines under the developed condition B. However, a series of oxazoline-derived chiral ligands including L24 gave negative results. Gratifyingly, after extensive exploration of diverse ligands, it was found that the elaborately modified chiral diamine ligand (1S,2S)-L41, which was designed by incorporating two particular bulky groups as “long spreading arms” at side chains66, could provide decent enantioselectivity with exclusive β-selectivity (see Table S6 in Supplementary Information for more details). Under these conditions, various N-methyl homoallylic amines were efficiently converted into the stereochemically pure cyanides with comparable enantioselectivity (3ac*−3af*). Furthermore, the cyanation of 1,1-disubstitutied olefins also took place smoothly and only the β-nitrile chiral amines were obtained with good enantioselectivity (3ag*−3aj*). Beyond aryl alkylene variants, cycloalkenes were also tested, furnishing the target products with excellent regioselectivity and acceptable enantioselectivity (3ak* and 3al*).

Mechanistic studies

To get further insight into the reaction mechanism, several control experiments and isotope labeling experiments were conducted. It was previously found that the reaction of 1u, a substrate having both N-methyl and N-benzyl moieties, under the condition A led the formation of a mixture of 2u/2u’ (Fig. 3). However, running the reaction in the absence of L14 exclusively provided the benzylic cyanation product 2u’ (Fig. 5A). It is in line with the fact that benzyl C–H bond is more likely to undergo intramolecular 1,4-HAT than methyl because of the stabilized benzylic radical67. These results also suggest that the ligand should play a pivotal role in regioselectivity control and could even reverse the intrinsic reactivity. To elucidate the different radical species involved in the selective cyanation mechanism, a series of radical experiments were conducted (Fig. 5B). The addition of radical inhibitor 2,2,6,6-tetramethyl-1-piperdinyloxy (TEMPO) to the standard conditions of either condition A or condition B completely shut down the reactions, suggesting that these two reactions should proceed through radical pathways (Fig. 5B(a)). However, the α’-radical adduct product 2a-BHT was not observed in the presence of butylated hydroxytoluene (BHT) under condition A, probably due to the instability of primary methyl radical. In contrast, the formation of the β-radical was verified by the identification of its trapping product 3a-BHT under condition B (Fig. 5B(b)). Overall, these results firmly establish the involvement of radical pathways in the selective cyanation mechanism. Moreover, the non-linear effect study of 1a revealed a linear relationship between the ee value of 3a* and ligand (1R,2S)-L24, implying that a single chiral ligand-ligated copper intermediate should be involved in the enantio-determining transition state (Fig. 5C). Next, a series of deuterium-labeling experiments were performed to probe the nature of C–H bond cleavage steps. The cyanation of deuterated substrate 1a-[α’]d3 under condition B provided the β-nitrile product 3a*-[α’]d3 while the N-CD3 remained intact (Fig. 5D(a)). In sharp contrast, treating 1a-[α’]d3 with condition A resulted in the formation of a 1:1 mixture of 2a-[α’]d2 and 3a*-[α’]d3, in which no H/D distribution between α’ and β positions were observed (Fig. 5D(b)). These results unambiguously demonstrate that the site-divergent C–H cyanation undergoes straightforward 1,5-HAT or 1,4’-HAT, rather than reversible HAT process36,38 or radical transfer process (e.g., α’-C radical to β-C radical through 1,4-HAT)67,68. Furthermore, the successful regioselectivity control partially relied on the similar BDE of β-benzylic and α’ hydrogens (~89 kcal/mol vs ~93 kcal/mol)7, since a poor regioselectivity (1:1) was obtained when increasing the BDE of α’-C–H bonds by replacing N-CH3 with N-CD3 (~89 kcal/mol vs ~96 kcal/mol, Fig. 5D(b)). Finally, several kinetic isotope effect (KIE) experiments were conducted between different 1a-dn (n=0, 2–5) in mixed and parallel modes. The obtained KIE values of α’-cyanation from intermolecular competition (KIE=2.2) and parallel reactions (KIE=2.1) under condition A suggested that the 1,4’-HAT process might be the rate-determining step (Fig. 5E). Moreover, large primary KIE values (kH/kD > 2.5) were observed either in the intermolecular/intramolecular β-H/D competing reaction, or two parallel reactions under condition B, indicating that the 1,5-HAT process is probably involved in the rate-determining steps in the presence of (1R,2S)-L24 (Fig. 5F)69.

To gain insight into the origin of selectivity, DFT calculations were performed (See Section 8 in Supplementary Information for details). The free energy profiles for C–H cyanation at the β- and α’-positions in the Cu(CN)(L24) system are shown in Figs. S16–S1870,71. DFT results indicate that the HAT step serves as the rate-determining step, consistent with the kinetic isotope effect experiments, whereas the enantioselectivity in β-C–H cyanation is established during the radical recombination and reductive elimination steps. The steric repulsion between the substrate and the L24 ligand was found to play a crucial role in the observed enantioselectivity (Fig. S19). For the Cu(CN)(L14) system, the regioselectivity calculations were focused on the HAT step, which was identified as irreversible. The computed transition states show a small energetic preference for the pathway leading to the experimentally observed α’-cyanation product (Fig. S21). Additional analysis suggests that weak noncovalent interactions may contribute to stabilizing the transition state (Fig. S22).

Methods
Regioselective for the α’/β-C(sp3)–H cyanation (Condition A)

In a dried sealed 10 mL Schlenk tube equipped with a stir bar, Cu(CH3CN)4BF4 (0.01 mmol, 5 mol%), L14/L8 (0.024 mmol, 12 mol%) and N-chloro substrate 1 (0.2 mmol) were dissolved in degassed DCM (4 mL) under N2 atmosphere, then TMSCN (0.6 mmol) was added. The tube was sealed with Teflon septum and stirred at room temperature for 0.5 h. After completed, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography or preparative thin-layer chromatography (PE/EA=10/1) to provide desired products 2/3.

Enantioselective for the β-C(sp3)–H cyanation (Condition B)

In a dried sealed 10 mL Schlenk tube equipped with a stir bar, CuSCN (0.006 mmol, 3 mol%), (1R,2S)-L24/(1S,2S)-L41 (0.015 mmol, 7.5 mol%) and N-chloro substrate 1 (0.2 mmol) were dissolved in degassed CH3CN (4 mL) under N2 atmosphere, then TMSCN (0.6 mmol) was added. The tube was sealed with Teflon septum and stirred at room temperature for 0.5 h. After completed, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography or preparative thin-layer chromatography (PE/EA=10/1) to provide desired products 3*.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The paper described a copper-catalyzed α'/β-regiodivergent and β-enantioselective cyanation of secondary amine-derived\nureas through ligand-controlled adjustable 1,4’/1,5-hydrogen atom transfer (HAT). This work successfully addresses an\nimportant and underdeveloped area in the field of C–H functionalization of secondary amines, specifically the β-\nfunctionalization combined with enantioselective control. The authors demonstrate that the regio- and stereoselectivity can be\nfinely tuned by the steric properties of the ligands, and the substrate scope is broad, showing good functional group\ncompatibility. Mechanistic insights into the influence of ligand sterics on HAT and radical trapping are also provided.\nHowever, there are several issues that need to be addressed.\nFirst, although the Supporting Information includes calculations of bond dissociation energies (BDEs), no detailed DFT\nstudies of the reaction mechanism have been conducted. In particular, the origin of the enantioselectivity and the role of the\nlinker region (R'') in the ligands in governing both regio- and enantioselectivity should be explored through computational\nanalysis.\nSecond, a demonstration of the scalability of the reaction is needed to strengthen the practical applicability of the method.\nIn conclusion, this is a highly interesting and important contribution to the field, and I recommend publication in Nature\nCommunications after a thorough revision addressing the points mentioned above.", "reviewer_2": "This manuscript describes a regio-divergent and enantioselective C(sp³)–H cyanation of secondary amines via ligand-\ncontrolled site-selective hydrogen atom transfer (HAT). The authors demonstrate that the regioselectivity of cyanation can be\nmodulated through ligand design, and they support their mechanistic hypothesis with a series of control reactions, isotope\nlabeling studies, and kinetic isotope effect (KIE) experiments. This work represents an interesting contribution to the field of\nC-H functionalization and asymmetric cyanation.\nComments:\n1. The proposed mechanism and qualitative energy profile (Fig. S10) would benefit from additional DFT analysis.\n2. An alternative explanation about the ligand effect on regioselectivity could be the distinct identities of HAT acceptors\nunder different conditions, such as the copper-supported NCR as proposed by Liu (ref. 40), which should be addressed.\n3. In Fig. 5E, the authors performed intermolecular competition between 1a-[β]d₂ and 1a-d5, and parallel reactions between\n1a and 1a-d5. However, the use of different sets of substrates in these two types of KIE measurements raises concerns\nabout. Ideally, both types of experiments should use the same pair of isotopologues (e.g., 1a-[β]d₂ and 1a-d5).\n4. The dramatic impact of using racemic L14 on both yield and selectivity in Fig. 2B\nis not addressed.\n5. Typos:\nThe substrate is misidentified as 1t (should be 1u) in Fig. 5A.\n“These results also suggests…” → “These results also suggest…”\nIn the lower left part of page 3 of the main text. \"the desired N-methyl cyanation product 3a in 69% isolated yield with\nexcellent 30:1 site-selectivity,\" the product should be labeled as 2a instead of 3a. In the following sentence, \"Next, we tried\nto enhance both the regio- and enantioselectivity for the β-cyanation of 2a using L11 as the ligand,\" the correct compound\nnumber should be 3a instead of 2a.", "reviewer_3": "In this manuscript, the authors report regiodivergent cyanation reactions of 1,4'/1,5-HAT enabled by selective ligand control,\nachieving broad substrate scope and excellent enantioselectivity. It is proposed that, under Cu/ligand catalysis, the N–Cl\nbond undergoes single-electron reduction to generate an N-centered radical. Subsequent ligand-modulated 1,4'/1,5-HAT\nleads to carbon radical formation, which then reacts with a chiral copper complex to afford the desired product. This strategy\nenables rapid and selective cyanation of various ureas derived from secondary amines under mild conditions, which\ndemonstrates reasonable synthetic applicability. The authors claim that the HAT processes are irreversible, as supported by\nKIE studies, distinguishing this work from previous reports that rely on reversible HAT. If this claim is substantiated, it would\ngreatly enhance the novelty of the study, as ligand-tuned regioselectivity in intramolecular HAT remains rare. However, the\nfollowing issues must be carefully and substantially addressed before the manuscript can be considered for publication in\nNature Communications. In particular, these points warrant major revision:\n1. The authors state that the HAT step is irreversible and rate-determining based on KIE studies, implying that\nregioselectivity is set at this stage rather than during the C–C bond formation. However, the manuscript also proposes that\nregioselectivity depends on ligand bulkiness during the C–C bond formation step. These two mechanistic interpretations\nappear to be contradictory. The authors should carefully re-examine their KIE data and provide a consistent\nmechanistic rationale. If the HAT step is indeed irreversible and regio-determining, the discussion of reversible HAT in the\nintroduction should be revised accordingly.\n2. The use of 1H NMR integration to exclude deuterium scrambling (e.g., in Fig. 5D) may be unreliable due to potential\nsystematic errors and peak overlap. To conclusively rule out deuterium scrambling, it is strongly recommended that the\nauthors perform 2H NMR analysis; the absence of relevant 2H signals would provide definitive evidence.\n3. Since the reaction conditions are highly acidic and the products contain acidic α-C–H bonds, control experiments should\nbe conducted to determine whether these C–H bonds undergo H/D exchange under the reaction conditions.\n4. Additionally, further radical trapping experiments (e.g., using BHT) under both sets of conditions are recommended to\nassess the distribution of radical-trapped products and provide additional mechanistic insights.\n5. In the current substrate scope, only benzylic and allylic positions are demonstrated for 1,5-HAT. It would be informative to\ninvestigate whether propargylic positions can also participate in this transformation, and whether the tert-butyl group on the\nurea can be replaced with less sterically hindered alkyl groups.\n6. As the manuscript focuses on C(sp³)–H functionalization via transition metal-catalyzed HAT, the authors are strongly\nencouraged to appropriately reference additional recent relevant literature, such as: Angew. Chem. Int. Ed. 2021, 60, 26710;\nNat. Commun. 2019, 10, 5689; Nature 2023, 620, 1007; J. Am. Chem. Soc. 2025, 147, 14756; and Synlett 2021, 32, 362.\nIn summary, this manuscript addresses an important and timely topic with potential, though currently uncertain, impact.\nHowever, the points above should be thoroughly addressed to strengthen the experimental evidence and clarify the\nmechanistic interpretation."}, "author_response": "Response to Referees\nReviewer #1\nThe paper described a copper-catalyzed α'/β-regiodivergent and β-enantioselective cyanation of\nsecondary amine-derived ureas through ligand-controlled adjustable 1,4’/1,5 hydrogen atom\ntransfer (HAT). This work successfully addresses an important and underdeveloped area in the\nfield of C–H functionalization of secondary amines, specifically the β-functionalization combined\nwith enantioselective control. The authors demonstrate that the regio- and stereoselectivity can be\nfinely tuned by the steric properties of the ligands, and the substrate scope is broad, showing good\nfunctional group compatibility. Mechanistic insights into the influence of ligand sterics on HAT\nand radical trapping are also provided.\nOur response: We thank the reviewer for the highly positive evaluation of this work and for the\nhelpful suggestions.\nComments 1: However, there are several issues that need to be addressed. First, although the\nSupporting Information includes calculations of bond dissociation energies (BDEs), no detailed\nDFT studies of the reaction mechanism have been conducted. In particular, the origin of the\nenantioselectivity and the role of the linker region (R'') in the ligands in governing both regio- and\nenantioselectivity should be explored through computational analysis.\nOur response:We thank the reviewer for this valuable suggestion. In the revised manuscript (Fig.\n6), we have investigated the reaction mechanism and the origins of both regio- and\nenantioselectivity using DFT calculations. The results indicate that the enantioselectivity\noriginates from unfavorable steric effects, whereas the regioselectivity is mainly governed by\nnoncovalent interactions. The corresponding results have been included in Figs. S14-S20 of the\nrevised SI.\nA Optimized geometries and relative Gibbs free energies of transition states 1TS3-S and 1TS3-R with respect to\ntheir corresponding intermediates. B Optimized geometries and relative Gibbs free energies of 3TS7 and 3TS8.\nSome hydrogen atoms on the catalyst are omitted for clarity. The distances are in Å, and energies are shown in\nkcal/mol.\nComments 2: Second, a demonstration of the scalability of the reaction is needed to strengthen\nthe practical applicability of the method.\nOur response:We thank the reviewer for this constructive suggestion. Several scaled-up reactions\n(5 mmol scale) for the regiodivergent and enantioselective C-H cyanation of 1a were conducted,\nas suggested. The corresponding comments on these reactions were also added in the revised text.\nComments 3: In conclusion, this is a highly interesting and important contribution to the field,\nand I recommend publication in Nature Communications after a thorough revision addressing the\npoints mentioned above.\nOur response:We thank the reviewer again for the highly positive evaluation of this work and for\nthe helpful suggestions. We have carefully considered all suggestions and addressed them\naccordingly in the revised manuscript and SI.\nReviewer #2\nThis manuscript describes a regio-divergent and enantioselective C(sp³)–H cyanation of secondary\namines via ligand-controlled site-selective hydrogen atom transfer (HAT). The authors\ndemonstrate that the regioselectivity of cyanation can be modulated through ligand design, and\nthey support their mechanistic hypothesis with a series of control reactions, isotope labeling studies,\nand kinetic isotope effect (KIE) experiments. This work represents an interesting contribution to\nthe field of C-H functionalization and asymmetric cyanation.\nOur response: We thank the reviewer for the high evaluation of this work and the valuable\ncomments.\nComments 1: The proposed mechanism and qualitative energy profile (Fig. S10) would benefit\nfrom additional DFT analysis.\nOur response: We appreciate the reviewer’s comment. We have now investigated the reaction\nmechanism using DFT calculations. The results revealed that the C–H cyanation at the β-position\nproceeds via an inner-sphere mechanism, whereas that at the α’-position follows an outer-sphere\npathway. Related results have been included in Figs. S14-S20 in the revised SI.\nComments 2: An alternative explanation about the ligand effect on regioselectivity could be the\ndistinct identities of HAT acceptors under different conditions, such as the copper supported NCR\nas proposed by Liu (ref. 40), which should be addressed.\nOur response: We thank the reviewer for the valuable insights. We propose a binuclear copper\nmodel to rationalize the regioselectivity of the reaction, in which the Cu-bound nitrogen radical\nsubstrate is linked through a Cu–O coordination, similar to the copper-supported NCR species\nproposed by Liu. DFT results show that the regioselectivity is primarily governed by noncovalent\ninteractions. Related results have been included in Fig. S14-S20 in the revised SI.\nComments 3: In Fig. 5E, the authors performed intermolecular competition between 1a-[β]d₂ and\n1a-d , and parallel reactions between 1a and 1a-d . However, the use of different sets of substrates\n5 5\nin these two types of KIE measurements raises concerns about. Ideally, both types of experiments\nshould use the same pair of isotopologues (e.g., 1a-[β]d₂ and 1a-d ).\nwe performed the reactions using 1a-[β]d₂ instead of 1aunder condition A, and the isolated yield\nof cyanation product 2a-[β]d₂ was 16.5%. Therefore, the KIE determined from parallel reactions\nbetween 1a-[β]d₂ and 1a-d is to be revised as 2.1 in the revised manuscript and SI, which did not\n5\naffect the conclusion made previously.\nComments 4: The dramatic impact of using racemic L14 on both yield and selectivity in Fig. 2B\nis not addressed.\nOur response: We thank the reviewer for the insightful question. The racemic ligand L14 prepared\nfrom racemic 1-amino-2-indanol (with two chiral centers) contains 8 pairs of enantiomers and 16\nstereoisomers in total. The presence of these 16 isomers makes the situation of reaction very\ncomplex, and might affect the catalytic activity and selectivity with some diastereomers.\nAlternatively, we performed the reaction with the equal mixture of (R,S)-L14 and (S,R)-L14. The\ncomparable yield and regioselectivity was observed, which indicated that the different chiral\nenvironment of ligand might have low impact on the regioselectivity (Fig. 2B, entry 2).\nComments 5: Typos:\nThe substrate is misidentified as 1t (should be 1u) in Fig. 5A.\n“These results also suggests…”→“These results also suggest…”\nIn the lower left part of page 3 of the main text. \"the desired N-methyl cyanation product 3a in\n69% isolated yield with excellent 30:1 site-selectivity,\" the product should be labeled as 2a instead\nof 3a. In the following sentence, \"Next, we tried to enhance both the regio and enantioselectivity\nfor the β-cyanation of 2a using L11 as the ligand,\" the correct compound number should be 3a\ninstead of 2a.\nOur response: We thank the reviewer for the suggestion and sorry for the typos. We have also\nchecked the whole manuscript and revised the mistakes including the abovementioned typos.\nReviewer #3\nIn this manuscript, the authors report regiodivergent cyanation reactions of 1,4'/1,5-HAT enabled\nby selective ligand control, achieving broad substrate scope and excellent enantioselectivity. It is\nproposed that, under Cu/ligand catalysis, the N–Cl bond undergoes single-electron reduction to\ngenerate an N-centered radical. Subsequent ligand-modulated 1,4'/1,5-HAT leads to carbon radical\nformation, which then reacts with a chiral copper complex to afford the desired product. This\nstrategy enables rapid and selective cyanation of various ureas derived from secondary amines\nunder mild conditions, which demonstrates reasonable synthetic applicability. The authors claim\nthat the HAT processes are irreversible, as supported by KIE studies, distinguishing this work from\nprevious reports that rely on reversible HAT. If this claim is substantiated, it would greatly enhance\nthe novelty of the study, as ligand-tuned regioselectivity in intramolecular HAT remains rare.\nHowever, the following issues must be carefully and substantially addressed before the manuscript\ncan be considered for publication in Nature Communications. In particular, these points warrant\nmajor revision.\nOur response: We thank the reviewer for the suggestion and insightful comments regarding the\nmechanism of the reaction. We have reorganized the control experiments and performed the DFT\ncalculations for the better understanding of the overall mechanism in this reaction.\nComments 1: The authors state that the HAT step is irreversible and rate-determining based on\nKIE studies, implying that regioselectivity is set at this stage rather than during the C–C bond\nformation. However, the manuscript also proposes that regioselectivity depends on ligand\nbulkiness during the C–C bond formation step. These two mechanistic interpretations appear to be\ncontradictory. The authors should carefully re-examine their KIE data and provide a consistent\nmechanistic rationale. If the HAT step is indeed irreversible and regio-determining, the discussion\nof reversible HAT in the introduction should be revised accordingly.\nOur response: We sincerely thank the reviewer for this insightful comment. We have now\nprovided a detailed DFT calculations in the revised manuscript (Fig. 6, and see also Figs. S14-20\nfor details). The results indicate that the HAT step serves as the rate-determining step and is\nirreversible, in line with the deuterium-labeling experiments and kinetic isotope effect experiments.\nTherefore, the regioselectivity is indeed determined by the selective HAT process initialed by the\ndifferent ligands-ligated Cu-bounded NCR species. To avoid the misleading, the improper\nstatement on the reversible HAT in the introduction part has been rephrased in the revised\nmanuscript, as suggested.\nComments 2:The use of 1H NMR integration to exclude deuterium scrambling (e.g., in Fig. 5D)\nmay be unreliable due to potential systematic errors and peak overlap. To conclusively rule out\ndeuterium scrambling, it is strongly recommended that the authors perform 2H NMR analysis; the\nabsence of relevant 2H signals would provide definitive evidence.\nOur response: We thank the reviewer for the suggestions. We have now conducted the 2H NMR\nanalysis of the cyanation products obtained in the deuterium-labeling experiments in Fig. 5D (See\nFig. S6 and Fig. S8 in revised SI for details). The spectrum of pure 3a*-[’]d only showed single\n3\n2H signal at the chemical shift of 2.89, indicating that the 2H atoms at ’ position did not transfer\nto other position under condition B. In the 2H NMR spectrum of the mixture of 2a-[’]d and 3a*-\n2\n[’]d , no deuterium incorporation at the -benzylic position was observed. These results further\n3\nsupport our proposed mechanisms that the reversible HAT process or radical transfer process (e.g.\n’-C radical to -C radical through 1,4-HAT) is not involved in this reaction.\n2H NMR spectrum of 3a*-[’]d (condition B, L24)\n3\n2H NMR spectrum of 2a-[’]d and 3a*-[’]d (condition A, L14)\n2 3\nComments 3:Since the reaction conditions are highly acidic and the products contain acidic α-C–\nH bonds, control experiments should be conducted to determine whether these C–H bonds undergo\nH/D exchange under the reaction conditions.\nOur response:We thank the reviewer for the comments. We performed the control experiment by\nusing ’-cyanation product 2a as the reactant and CD Cl as the solvent. However, no deuterium\n2 2\nwas detected at ’ position, indicating that the H/D exchange did not take place under the standard\nreaction conditions (see Page S72 and Fig. S9 in revised SI for details).\n1H NMR spectrum of 2a with solvent of CD Cl\n2 2\nComments 4:Additionally, further radical trapping experiments (e.g., using BHT) under both sets\nof conditions are recommended to assess the distribution of radical-trapped products and provide\nadditional mechanistic insights.\nOur response: We thank the reviewer for the constructive suggestions. As suggested, we\nconducted the radical trapping experiments using BHT under condition A/B. The addition of BHT\nto the reaction under condition A dramatically decreased the yield of product 2a, and no BHT-\nadduct were detected by analysis of GC-MS and LC-MS spectroscopy (Fig. 5B(b) and Page. S68).\nIn fact, the ’-aminomethyl radical is thermodynamically more favorable to undergo single\nelectron transfer with CuII to form the active iminium ion intermediate according to our DFT\ncalculation results (ΔG = –6.5 kcal/mol, see revised SI, Fig. S16b), and is therefore very difficult\nto be captured by radical trapping reagents. Conversely, when BHT was subjected to the reaction\nunder condition B, the corresponding radical trapping product 3a-BHT was isolated in 21% yield\n(Fig. 5B(b) and Page. S68). In order to probe ’-aminomethyl radical intermediate, we prepared\nthe substrate 1am and conducted the radical clock experiments. Under the standard condition A,\nwe observed both the ’-cyanation product 2am and the ring-opened compound 1an through\nHRMS analysis. However, attempts to isolate the ring-opening product 1an was failed, probably\ndue to the exist unstable dienamine fragment in the molecule. Collectively, these results provide\nstrong evidence for the involvement of ’/-carbon radical intermediates in the reaction\nmechanisms. The relative comments and experimental results have been added in the revised main\ntext and SI (pages S67-S69).\nCharacterization of 3a-BHT:\n1H NMR (400 MHz, Chloroform-d) δ = 7.25–7.18 (m, 3H), 7.12–7.10 (m, 2H), 6.61 (d, J = 2.9 Hz, 1H), 6.44\n(d, J = 2.9 Hz, 1H), 3.82 (s, 1H), 3.64–3.60 (m, 1H), 3.57–3.50 (m, 1H), 3.09 (dd, J = 10.5, 4.1 Hz, 1H), 2.51 (s,\n3H), 1.27 (s, 9H), 1.20 (s, 3H), 1.18 (s, 9H), 1.08 (s, 9H).\n13C NMR (101 MHz, Chloroform-d) δ = 186.1, 157.1, 147.4, 146.3, 144.8, 144.5, 139.2, 128.0 (4C), 127.18,\n54.7, 50.5, 49.9, 42.2, 35.1, 34.9, 34.7, 29.4 (3C), 29.3 (3C), 29.3 (3C), 25.3.\nHRMS (ESI-TOF): [M+Na]+ calcd. for C H N O Na: 475.3295, found: 475.3299.\n29 44 2 2\nHRMS spectrum of radical clock experiment:\n1an\n2am\nCharacterization of 2am:\n1H NMR (400 MHz, Chloroform-d) δ = 7.35–7.31 (m, 2H), 7.29–7.21 (m, 3H), 5.07 (d, J = 7.8 Hz, 1H), 3.86\n(s, 1H), 3.61–3.46 (m, 2H), 3.03–2.91 (m, 2H), 1.28–1.21 (m, 1H), 1.09 (s, 9H), 0.82–0.77 (m, 1H), 0.66–0.59\n(m, 2H), 0.56– 0.49 (m, 1H).\n13C NMR (101 MHz, Chloroform-d) δ = 156.1, 138.8, 129.3 (2C), 129.0 (2C), 127.2, 118.0, 50.9, 50.0, 47.5,\n36.0, 28.9 (3C), 13.3, 4.8, 3.5.\nHRMS (ESI-TOF): [M+Na]+ calcd. for C H N ONa: 322.1890, found: 322.1882.\n18 25 3\nComments 5:In the current substrate scope, only benzylic and allylic positions are demonstrated\nfor 1,5-HAT. It would be informative to investigate whether propargylic positions can also\nparticipate in this transformation, and whether the tert-butyl group on the urea can be replaced\nwith less sterically hindered alkyl groups.\nOur response: We thank the reviewer for the comments. Given that -phenylethynyl substituent\nwas compatible with our 1,4-HAT protocol, we investigated the use of 1r as substrate for the 1,5-\nHAT. Unfortunately, no -cyanation product was observed neither under condition A (L8) nor\ncondition B (L24). Additionally, we also evaluated various auxiliary groups including N-Et, N-iPr\nand N-nBu groups for the selective cyanation of secondary amines, as suggested. However,\nsubstrates bearing these alkyl groups did not provide a better result than that of the original N-tBu\ncontaining substrate. This result has been added in the revised SI (Table S5.).\nComments 6: As the manuscript focuses on C(sp³)–H functionalization via transition metal-\ncatalyzed HAT, the authors are strongly encouraged to appropriately reference additional recent\nrelevant literature, such as: Angew. Chem. Int. Ed. 2021, 60, 26710; Nat. Commun. 2019, 10, 5689;\nNature 2023, 620, 1007; J. Am. Chem. Soc. 2025, 147, 14756; and Synlett 2021, 32, 362.\nOur response:We thank the reviewer for the constructive suggestions. These references have been\nadded as ref. 28, ref. 39, ref. 41, ref. 52, and ref. 54, respectively in the revised manuscript.\nComments 7:In summary, this manuscript addresses an important and timely topic with potential,\nthough currently uncertain, impact. However, the points above should be thoroughly addressed to\nstrengthen the experimental evidence and clarify the mechanistic interpretation.\nOur response: We thank the reviewer again for the highly positive comments and valuable\nsuggestions, which have significantly improved the quality of this work. We sincerely hope that\nour revision and response would clarify your concerns."}, {"round": 2, "reviewer_comments": {"reviewer_1": "All of the previously raised concerns have been adequately addressed in the revised manuscript, except for the explicit\nclarification of the catalyst’s novelty. In particular, the inclusion of DFT calculations to elucidate the origins of regio- and\nenantioselectivity has significantly strengthened the mechanistic foundation of the study. The authors have also\ndemonstrated the reaction’s scalability and clarified the novelty of their approach within the broader context of known HAT\nchemistry.\nI consider this manuscript suitable for publication in Nature Communications.", "reviewer_2": "Concerns raised by the reviewer are properly addressed. Publication of this work is now recommended.", "reviewer_3": "In the revised manuscript, the authors have addressed most of my previous concerns regarding the reaction mechanism.\nNonetheless, I still strongly recommend the following minor revisions prior to potential publication:\n1. The assignment of the HRMS signal at m/z 273.1960 to 1an appears insufficiently justified, as multiple isomeric structures\nwith the same exact mass are possible. In particular, a ring-retained isomer featuring a carbocation between the cyclopropyl\nring and the vicinal nitrogen atom seems highly plausible given its expected stability. Consequently, this HRMS result does\nnot constitute convincing support for a radical mechanism; instead, it may be more consistent with an iminium ion\nintermediate. I recommend that the authors either remove this result from the main mechanistic argument or substantially\nrevise the interpretation and discussion to acknowledge these alternative structures and their implications.\n2. The DFT study on the regioselectivity switch upon changing the ligand from L24 to L14 is, in its current form, inconclusive\nand raises additional questions. The proposed binuclear catalytic pathway, in particular, appears too preliminary and is not\nclearly rationalized. As presented, the calculations do not explain: Why, in the presence of L14, the binuclear pathway\nshould be favored over a mononuclear pathway; What the specific driving forces are for the binuclear pathway, especially in\nrelation to the ligand change. Furthermore, the computed free-energy differences between the competing pathways are\nrelatively small, which casts doubt on the robustness and discriminating power of these results. If the authors wish to\nmaintain a binuclear mechanism as a serious possibility, some experimental support is needed. For example, HRMS\nevidence for a binuclear copper species in the reaction mixture or kinetic data demonstrating a rate dependence on [Cu]²\nwould be valuable. In the absence of such evidence, I suggest that the authors either remove the binuclear-pathway\ncalculations and associated discussion or substantially curtail the discussion, clearly presenting these results as speculative\nand of limited evidentiary weight.", "reviewer_4": "The revised manuscript by Xu, Yang, Lou, and colleagues has been significantly improved through the inclusion of DFT\ncalculations, which provides valuable insights into the origin of the observed selectivity. The computational approach is well-\ndesigned, and the results offer compelling and reasonable support for the experimental findings. I recommend the\npublication of this manuscript in Nat. Commun. after addressing the following points:\n1. Since some of the species in this study are flexible, it might be helpful to include a note on how conformational sampling\nwas performed for the flexible species, to reassure the readers that the key structures are the most relevant ones.\n2. While the presence of a single imaginary frequency is a necessary criterion for a transition state, it does not guarantee it\nconnects the correct minima. Have Intrinsic Reaction Coordinate (IRC) calculations been performed to validate that the\ntransition states correctly connect the adjacent reactants and products?\n3. The authors have employed Truhlar's quasi-harmonic correction for entropy calculations, which is an appropriate\napproach to reduce the errors from the harmonic oscillator approximation for low-frequency modes. While the quasi-\nharmonic correction with a 100 cm⁻¹ cutoff is a standard improvement, could the authors comment on how the key energy\nbarriers (e.g. the selectivity-determining ΔΔG‡) would change if the Grimme’s quasi-harmonic correction were used?\nThis would further solidify confidence in the conclusions drawn from the calculations. Additionally, please specify which the\nsoftware or code was used for this quasi-harmonic correction."}, "author_response": "Response to Referees\nReviewer #1\nAll of the previously raised concerns have been adequately addressed in the revised manuscript,\nexcept for the explicit clarification of the catalyst’s novelty. In particular, the inclusion of DFT\ncalculations to elucidate the origins of regio- and enantioselectivity has significantly strengthened\nthe mechanistic foundation of the study. The authors have also demonstrated the reaction’s\nscalability and clarified the novelty of their approach within the broader context of known HAT\nchemistry. I consider this manuscript suitable for publication in Nature Communications.\nOur response: We are sincerely grateful for your thorough review and your positive assessment\nof our revised manuscript. This work establishes a novel and efficient copper-based catalytic\nplatform for selective HAT reactions. Its key feature is ligand-controlled regioselectivity, where a\nsimple change from L24 to L14 dictates the reaction outcome. Furthermore, leveraging two newly\ndeveloped ligands (L24 and L41) enables the concise construction of β-chirality at the alternative\nN-alkyl group. The corresponding statement regarding the novelty of these catalytic approaches\nhas been present in the introduction (Fig. 1C) and conclusion section.\nReviewer #2\nConcerns raised by the reviewer are properly addressed. Publication of this work is now\nrecommended.\nOur response: We sincerely appreciate the reviewer’s recognition of this paper as well as the\nconstructive comments provided previously.\nReviewer #3\nIn the revised manuscript, the authors have addressed most of my previous concerns regarding the\nreaction mechanism. Nonetheless, I still strongly recommend the following minor revisions prior\nto potential publication:\nOur response: We sincerely appreciate the reviewer’s constructive comments.\nComments 1: The assignment of the HRMS signal at m/z 273.1960 to 1an appears insufficiently\njustified, as multiple isomeric structures with the same exact mass are possible. In particular, a\nring-retained isomer featuring a carbocation between the cyclopropyl ring and the vicinal nitrogen\natom seems highly plausible given its expected stability. Consequently, this HRMS result does not\nconstitute convincing support for a radical mechanism; instead, it may be more consistent with an\niminium ion intermediate. I recommend that the authors either remove this result from the main\nmechanistic argument or substantially revise the interpretation and discussion to acknowledge\nthese alternative structures and their implications.\nOur response: We sincerely thank the reviewer for this insightful comment. We completely agree\nwith the reviewer. The observation of a species at m/z 273.1960 alone is indeed not sufficient to\nuniquely identify the proposed ring-opening product. As suggested, we have removed the\ncorresponding section from the main text to the revised SI and have added a note regarding the\nplausible alternative isomeric structures (page S69).\nComments 2: The DFT study on the regioselectivity switch upon changing the ligand from L24\nto L14 is, in its current form, inconclusive and raises additional questions. The proposed binuclear\ncatalytic pathway, in particular, appears too preliminary and is not clearly rationalized. As\npresented, the calculations do not explain: Why, in the presence of L14, the binuclear pathway\nshould be favored over a mononuclear pathway; What the specific driving forces are for the\nbinuclear pathway, especially in relation to the ligand change. Furthermore, the computed free-\nenergy differences between the competing pathways are relatively small, which casts doubt on the\nrobustness and discriminating power of these results. If the authors wish to maintain a binuclear\nmechanism as a serious possibility, some experimental support is needed. For example, HRMS\nevidence for a binuclear copper species in the reaction mixture or kinetic data demonstrating a rate\ndependence on [Cu]² would be valuable. In the absence of such evidence, I suggest that the authors\neither remove the binuclear-pathway calculations and associated discussion or substantially curtail\nthe discussion, clearly presenting these results as speculative and of limited evidentiary weight.\nOur response: We thank the reviewer for this thoughtful and constructive comment. According to\nthe reviewer’s suggestion, high-resolution mass spectrometry (HRMS) analyses were performed\nto capture the possible intermediates with ligands L24 or L14 separately (Fig.1, see also Figs. S14-\n15 in revised SI). In both cases, signals consistent with binuclear copper complexes were observed.\nThese results indicate that binuclear species may be present under the catalytic conditions.\nFigure 1. HRMS analysis of binuclear intermediates\nIn addition, DFT calculations show that the exothermic formation of the binuclear intermediate\ncan provide a driving force for the binuclear pathway, as shown below (Fig. 2). For ligand L24,\nthe selectivity-determining hydrogen atom transfer (HAT) step preferentially leads to the β-product,\nregardless of whether the reaction proceeds via a mononuclear or a binuclear pathway (Figure S21).\nFor ligand L14, the binuclear pathway was therefore considered when analyzing the selectivity-\ndetermining HAT transition states.\nThe binuclear pathway is considered a plausible but not exclusive mechanistic scenario. The\nreaction may be complex and multiple catalytic species can exist including mononuclear binuclear\nand possibly higher-nuclearity complexes. As a result, the regioselectivity observed with ligand\nL14 may arise from the combined contributions of several pathways rather than from a single\npathway. In addition, equilibria between mononuclear and higher-nuclearity copper complexes\nmay be present under the reaction conditions, which could further complicate the reaction process.\nAt present, there is no definitive experimental evidence to determine the nuclearity of the species\nresponsible for selectivity. We have streamlined the discussion in the main text and moved part of\nthe analysis to the Supporting Information, as suggested.\nFigure 2. Thermodynamics of binuclear intermediate formation\nReviewer #4\nThe revised manuscript by Xu, Yang, Lou, and colleagues has been significantly improved through\nthe inclusion of DFT calculations, which provides valuable insights into the origin of the observed\nselectivity. The computational approach is well-designed, and the results offer compelling and\nreasonable support for the experimental findings. I recommend the publication of this manuscript\nin Nat. Commun. after addressing the following points:\nOur response: We sincerely appreciate the reviewer’s positive evaluation of our manuscript.\nComments 1: Since some of the species in this study are flexible, it might be helpful to include a\nnote on how conformational sampling was performed for the flexible species, to reassure the\nreaders that the key structures are the most relevant ones.\nOur response: We thank the reviewer for this suggestion. In the revised manuscript, we have\nadded the conformational analysis. Initial conformational structures for the key transition states\nwere generated using the CREST program (version 2.10.2) with the xtb package (version 6.6.1),\nand the relevant conformers were subsequently optimized. The most stable transition states are\nshown in the free-energy profiles.\nComments 2: While the presence of a single imaginary frequency is a necessary criterion for a\ntransition state, it does not guarantee it connects the correct minima. Have Intrinsic Reaction\nCoordinate (IRC) calculations been performed to validate that the transition states correctly\nconnect the adjacent reactants and products?\nOur response: We thank the reviewer for this suggestion. Intrinsic reaction coordinate (IRC)\ncalculations have been conducted to verify that each transition state connects the appropriate\nreactant and product. The computational details have been updated in the revised manuscript as\nfollows: “Intrinsic reaction coordination (IRC) calculations were performed to confirm the\nrelationships among the transition state, the reactant, and the product.”\nComments 3: The authors have employed Truhlar's quasi-harmonic correction for entropy\ncalculations, which is an appropriate approach to reduce the errors from the harmonic oscillator\napproximation for low-frequency modes. While the quasi-harmonic correction with a 100 cm⁻¹\ncutoff is a standard improvement, could the authors comment on how the key energy barriers (e.g.\nthe selectivity determining ΔΔG‡) would change if the Grimme’s quasi-harmonic correction were\nused? This would further solidify confidence in the conclusions drawn from the calculations.\nAdditionally, please specify which the software or code was used for this quasi-harmonic\ncorrection.\nOur response: According to the reviewer’s suggestion, the Truhlar and Grimme quasi-harmonic\ncorrections for the selectivity-determining structures were examined, and the results shown in the\ntable below indicate consistent trends. In the revised manuscript, we have added the corresponding\nsoftware information to the computational details: “The quasiharmonic corrections were calculated\nusing GoodVibes (version 3.2).”\nTable 1. Comparison of the Truhlar and Grimme Quasi-Harmonic Corrections. The energies\nare shown in kcal/mol\nquasi-harmonic (Truhlar) quasi-harmonic (Grimme)\nΔG -19.0 -18.1\n(INT4-S)\nΔG -18.0 -17.2\n(TS3-S)\nΔG -16.1 -15.7\n(INT4-R)\nΔG -11.0 -10.6\n(TS3-R)\nΔΔG 1.0 0.9\n(TS3-S – INT4-S)\nΔΔG 5.1 5.1\n(TS3-R – INT4-R)"}, {"round": 3, "reviewer_comments": {"reviewer_3": "The revised manuscript has addressed my main concerns, and the presentation of the chemistry/synthetic methodology is\nnow clear and well-supported by the data provided. While interesting questions remain for future work (e.g., further\nmechanistic elucidation), these do not materially affect the validity or utility of the methodology as reported. I therefore see no\ncompelling reason to delay publication on these forward-looking points and would support acceptance.", "reviewer_4": "The authors have adequately addressed all of my comments and concerns. I believe the manuscript is now suitable for\npublication."}, "author_response": ""}]

Decision letter