Kinetic resolution of amino acids by phosphine oxide catalyzed enantioselective esterification

Domain: chemistry
Published: 2026-04-13
Editorial outcome: major_revision

Abstract
Chiral amino acids are essential building blocks in asymmetric synthesis and drug discovery, yet their efficient preparation from racemic mixtures remains challenging. Here we show that a rationally designed phosphine oxide catalyst derived from L-pyroglutaminol enables the highly efficient kinetic resolution of racemic amino acids under mild conditions. Using L-pyroglutaminol as the esterification reagent, this catalytic system delivers a broad range of chiral esters and recovered amino acids with excellent stereoselectivities (s > 1057). Mechanistic studies suggest that the superior stereocontrol arises from a cooperative double hydrogen-bonding interaction between the catalyst and the pyroglutaminol core. This work provides a practical and scalable approach to enantioenriched amino acids, highlighting the potential of dual chiral cooperative catalysis in asymmetric synthesis.

Introduction
Chiral amino acids find applications as versatile building blocks in the synthesis of functional molecules, as a source of chiral information in asymmetric synthesis and as tools to expand and explore the function of native biological machinery.1-8 Therefore, chiral amino acids represent a class of valuable and indispensable compounds whose stereoselective synthesis is a major objective within synthetic chemists and synthetic biologists.9-10 An array of effective synthetic strategies for the construction of chiral amino acids have been developed, such as asymmetric hydrogenation and nucleophilic addition of imines11-18, enantioselective carbene insertion into N-H bonds of amines or amides19-23, stereoselective photobiocatalytic cross-coupling24-27, stereocontrolled 1,3-nitrogen migration of carboxylic acids28, and asymmetric hydrolysis of amino acid esters29. However, identifying a highly enantioselective chiral catalyst for a specific reaction is not always an easy task. An alternative well-established strategy relies on the catalytic kinetic resolution (KR) of a racemic mixture (Fig. 1a).30-32 The KR stands out as one of the most practical and straightforward strategies for obtaining enantioenriched molecules and recovering the starting materials, effectively allowing for access to both enantiomers from a single enantiomer of catalyst. Numerous highly efficient catalytic KR processes have been developed that reliably deliver enantiopure compounds, including chiral alcohols33-37, monohydrosilanes38, organoperoxides39, alkenes40-41, sulfonyl ketones42, amines43, imines44, sulfoximines45-46, aldehydes47, phosphindane oxides48-49, and heterocyclic compounds50-53. Despite this significant progress in the field, the catalytic KR of amino acids remains a challenging task and has been rarely explored.54-58

On the other hand, we noticed that the intermediate chlorophosphonium salt (Ph3PCl+/Cl-) generated from triphenylphosphine oxide (Ph3PO) and oxalyl chloride ((COCl)2) can efficiently activate carboxylate group. In 1977, Masaki and Fukui59 reported that the industrial byproduct Ph3PO could be easily converted into Ph3PCl+/Cl-, which was subsequently applied in amide and ester synthesis via stoichiometric activation of carboxylic acids, dehydration of amides to nitriles, Appel reaction and others60-62. In 2021, Ni and co-workers reported an efficient triphenylphosphine oxide catalyzed amidation and esterification for rapid synthesis of a series of dipeptides, amides and esters (Fig. 1b).63 Through analysis of an improved amidation and esterification reaction where catalytic Ph3PO was deoxygenated to the Ph3PCl+/Cl- by (COCl)2, we anticipated that chiral phosphine oxides generating chiral chlorophosphonium salts would be a promising strategy for the kinetic resolution of a broad variety of racemic amino acids. Noticeably, pyroglutaminol, a cost-effective chiral feedstock with both enantiomeric forms readily available, was commonly employed as a starting material in asymmetric synthesis owing to its distinctive features, including a predefined chiral center, five-membered lactam ring, and hydroxyl group.64-68 Conceivably, the lactam moiety in pyroglutaminol offers H-bonding donor and acceptor simultaneously, which could potentially bind to the substrate through double H-bonding. Subsequently, we designed a L-pyroglutaminol-derived organocatalyst featuring both H-bonding site and activator site, which can be prepared from L-pyroglutaminol and potassium diphenylphosphide. Herein, we disclose the development and application of the L-pyroglutaminol-derived phosphine oxide organocatalyst. We report the KR of racemic amino acids with L-pyroglutaminol as esterification reagent via a phosphine oxide catalysis under mild conditions, which provides a wide range of chiral amino acids and esters in good yields with excellent diastereoselectivities and enantioselectivities (s > 1057) (Fig. 1c).

Results
Optimization studies of kinetic resolution of amino acid.
To test the feasibility of our design, the kinetic resolution of racemic amino acid 1a was explored as a model substrate using 20 mol% of the chiral L-pyroglutaminol-derived phosphine oxide catalyst 3a and 1.5 equiv of oxalyl chloride and L-pyroglutaminol as the esterification reagent in chloroform provided the ester (S,S)-4a with 98:2 dr and the recovered (R)-1a with >99% ee, with excellent selectivity factor (s > 259) (Table 1, Entry 1). The absolute configuration of the recovered starting material 1a was unambiguously determined by the optical rotation analysis (Dextrorotation) and that of other recovered starting materials by analogy. Noticeably, the reaction was completed within 10 minutes. The study of variation of the chiral phosphine oxide catalysts indicated that only the catalyst 3e gave decent kinetic resolution performances (s = 20), while other catalysts all provided poor results (Table 1, Entries 2-5). When 3a was replaced with the other enantiomer 3f, the stereoselectivities decreased considerably (Table 1, Entry 6). Thereafter, a series of solvents were evaluated (Table 1, Entries 7-10). The reaction could also be conducted in toluene (PhMe), tetrahydrofuran (THF) or acetonitrile (MeCN), but the stereoselectivities would drop dramatically. The reaction in 1,2-dichloroethane (DCE) could provide good kinetic resolution performance (s = 68). Further reduction of catalyst loading to 10 mol%, a slightly lower selectivity factor (107) was observed (Table 1, Entry 11). In the absence of catalyst 3a, none of the desired product formed in 10 min, indicating that the catalyst was essential (Table 1, Entry 12). The racemic ester was obtained in the presence of triphenylphosphine oxide. The reactions of various alcohols and catalysts were also evaluated under the optimized conditions, while all provided poor results (please see Supplementary Information for details).

Kinetic resolution of amino acids. We next investigated the scope of this method for catalytic kinetic resolution of natural amino acids (Fig. 2). An array of racemic amino acids 1a-1d bearing α-alkyl groups, including methyl, isopropyl, isobutyl, and sec-butyl, serves as effective substrates, yielding both ester products and remaining amino acids in good yields and excellent enantioselectivities (s = 52-422). The amino acids 1e and 1f, whose side-chain contains an amide, were well tolerated with a higher level of enantiocontrol (s = 531 and 1057). Substrates 1g and 1h bearing either benzyl or 3-indolyl group also proved to be effective components, exhibiting enantioselectivities of 1057 and 66, respectively. Methionine (Met) 1i was also a suitable component (s = 149). Notably, good yields and excellent enantiodiscrimination (s = 99-1057) were observed for diverse side-chain protected amino acids (Tyr, Asp, Ser, Glu, Cys, Thr, Lys) 1j-1p. The electronic effects of side chain substituents demonstrated no significant correlation with enantioselectivity.

Second, the non-natural amino acids were also fairly broad in scope (Fig. 3). Good yields and excellent enantioselectivities (s = 353-669) were observed for diverse α-alkyl moieties that vary in steric demand from ethyl to cyclohexyl (5a-5e). Variations in the steric hindrance of the side chain resulted in only minor changes to enantioselectivity. The same catalyst can facilitate the enantioselective KR of α,α-dialkyl substituted amino acid 5f, yielding the corresponding enantioenriched amino acid and ester. The amino acids 5g-5j, whose side-chain contains aryl group (phenyl, pyridyl, naphthyl), also proved to be effective components (s = 39-747). The amino acids 5k and 5l, which contain alkene and urea moieties in their side-chains, exhibited good tolerance and demonstrated excellent enantiocontrol (s = 811 and 1057). The side-chain protected amino acids 5m and 5n also reacted smoothly to give both esters and remaining amino acids in good yields and excellent enantioselectivities (s = 259 and 711). An array of racemic amino acids 5o-5t bearing electronically varied α-benzyl groups, including ortho-, meta-, and para- substituted compounds, serves as effective substrates, providing the corresponding products with good yield and excellent enantiocontrol (s = 33-1057). The electronic effects of side chain substituents demonstrated no significant correlation with enantioselectivity. The α,α-dialkyl substituted amino acids 5u and 5v also reacted smoothly to give both esters and remaining amino acids in good yields and excellent enantioselectivities (s = 79 and 70). The steric effect of α,α-dialkyl substituted amino acids has a significant impact on the enantioselectivity. Specifically, the steric hindrance substrates exhibited a moderate reduction in enantioselectivity control (5u vs 5k and 5v vs 1g).

Gram-scale reaction, control experiments and nonlinear effect experiment. The practicality and scalability of this protocol were successfully demonstrated by performing the reaction for 1a at 5 mmol scale under the optimal conditions to give comparable result (s > 259) without any loss of efficiency (Fig. 4a). Aiming at better understanding the reaction mechanism, control experiments were carried out under the optimal conditions (Fig. 4b-4e). N-Protected pyroglutaminol 2b, N-protected catalyst 3g and L-prolinol 2c were employed to intentionally mask the hydrogen-bonding sites. Not surprisingly, the corresponding esters (4q, 4a and 4r) were obtained in markedly reduced diastereoselectivities. These results suggested that lactams in both pyroglutaminol and catalyst had sizeable influence on the observed diastereoselectivity. The KR of 1a was carried out using catalyst 3f and D-pyroglutaminol (R)-2a provided the enantiomer (R,R)-4a with 99:1 dr and the recovered (S)-1a with >99% ee, with excellent selectivity factor (s > 529) (Fig. 4e). These results demonstrated that the chiral environments of both the catalyst and pyroglutaminol synergistically govern the stereoselectivity. Moreover, this statement was also confirmed through 1H NMR spectroscopic studies on the mixture of (S)-2a with 3a with a molar ratio of 1:1, in which upfield shifts of 0.27 ppm and downfield shifts of 0.11 ppm were induced for the N-H signals in 2a and 3a respectively (see SI for spectra). Additional diversely substituted amino acids were also evaluated under the optimal conditions (Fig. 4f). Unfortunately, the corresponding products were not obtained. This outcome may be attributed to interference from alternative reactive sites, leading to the formation of an undefined byproducts.

In addition, we carried out a nonlinear effect experiment for elucidating the nature of the interaction. We observed a small but positive nonlinear effect (Fig. 4g, left, please see Supplementary Information and Supplementary Data for details), which indicates that more than one molecule of 3a was likely to be involved in the transition state of the enantiodifferentiating step. To gain deeper mechanistic insights and reconcile the observed positive nonlinear effect (Fig. 4g, left), we conducted a complementary nonlinear effect experiment by varying the enantiomeric purity of the chiral esterification reagent, L-pyroglutaminol 2a, while employing enantiopure catalyst 3a. Intriguingly, a distinct trend emerged: the enantiopurity of the recovered amino acid (R)-1a increased nonlinearly from 53% ee (when 2a was racemic) to 99% ee (when 2a was enantiopure) (Fig. 4g, right, please see Supplementary Information and Supplementary Data for details). This nonlinear correlation underscores two critical points. First, the catalyst 3a itself possesses an intrinsic enantioselectivity (baseline of 53% ee), which forms the primary chiral framework for substrate discrimination. Second, the matched chiral environment of (S)-2a acts in a cooperative manner to dramatically amplify and perfect this stereocontrol. This interpretation is strongly corroborated by our earlier observations: the drastic drop in selectivity when the mismatched catalyst enantiomer 3f was used with (S)-2a (Table 1, entry 6), and the full restoration of high selectivity when both components were switched to the matched 3f/(R)-2a pair (Fig. 4e), which unambiguously established the necessity of chiral matching between the catalyst and the esterification reagent. When the catalyst’s ee decreases, the mismatched catalyst enantiomer 3f present in the mixture can engage in detrimental interactions with the ever-present, enantiopure (S)-2a. These unfavorable interactions presumably disrupt the finely tuned double H-bonding network that is optimal for the matched 3a/(S)-2a pair, thereby depressing the overall selectivity in a disproportionate, nonlinear manner. This explanation elegantly unifies all experimental observations: it preserves the 1:1 catalyst-substrate interaction mode depicted in the DFT calculated transition state, which represents the optimal productive pathway for the matched pair, while attributing the nonlinear phenomenon to inhibitory interactions from mismatched catalyst components. It conclusively highlights that the supreme stereocontrol (s > 1057) originates from a synergistic, dual-chiral induction where the phosphine oxide catalyst provides the principal stereodifferentiating element, and the chiral pyroglutaminol reagent serves as an essential co-operative partner that refines and maximizes fidelity through specific, match-dependent non-covalent interactions.

Density functional theory calculations and proposed reaction mechanism. On the basis of the above experimental results, in order to gain greater insights into the origin of stereoselectivity of the KR, density functional theory (DFT) was employed to study 3a-catalyzed esterification of amino acid 1a and L-pyroglutaminol 2a. Both steps of the KR reaction mechanism were as follows (Fig. 5): nucleophilic attack of chlorophosphonium CP with amino acid 1a formed acyl phosphonium salt (step 1), and nucleophilic addition of L-pyroglutaminol 2a with acyl phosphonium salt (step 2). According to our previous study,62 triphenylphosphine oxide and oxalyl chloride rapidly formed chlorophosphonium. Step 1 commences via H-bonding between chlorophosphonium CP, amino acid 1a, and L-pyroglutaminol 2a, producing complex RC. The carboxyl’s oxygen atom acts as the nucleophilic site, allowing nucleophilic attack of 1a to chlorophosphonium CP via transition state TS1 with the energy barrier of 5.1 kcal/mol. In intermediate IM1, double H-bondings were formed spontaneously between L-pyroglutaminol 2a and acyl phosphonium salt through the perfectly matched lactam moieties. In step 2, using the oxygen atom in the hydroxyl group as the nucleophilic site, nucleophilic addition of L-pyroglutaminol 2a to acyl phosphonium salt occurred along the Si face of amino acid 1a via transition state TS2 with an energy barrier of 19.1 kcal/mol. Finally, product complex PC was formed, releasing catalyst 3a, (S,S)-4a and hydrogen chloride. Comparison of the relative free energy of TS1 (-1.4 kcal/mol) and TS2 (6.0 kcal/mol) revealed that the latter was the rate-determining step.

The free energy and geometric property of the transition states were explored in detail in order to comprehensively understand the stereoselectivity of the reaction (Fig. 6). As shown in Figure 6a,b, the relative free energy of (Si,S)-TS2 (ΔG = 6.0 kcal/mol, leading to product (S,S)-4a) was 9.0 kcal/mol (ΔΔG⧧) lower than that of (Si,R)-TS2 (ΔG = 15.0 kcal/mol, leading to product (R,S)-4a), which indicated that (S,S)-4a was the dominant product and showed good agreement with the experimental results (Table 1, Entry 1). When L-pyroglutaminol 2a attacked amino acid 1a from the Re face (Figure 6c,d), the relative free energy of (Re,S)-TS2 (ΔG = 21.2 kcal/mol, leading to product (S,S)-4a) was 3.2 kcal/mol lower than that of (Re,R)-TS2 (ΔG = 24.4 kcal/mol, leading to product (R,S)-4a), which was consistent with the experimental results ((S,S)-4a as the dominant product). Notably, the relative free energies of transition states (Re,S)-TS2 and (Re,R)-TS2 were much higher than those of (Si,S)-TS2 and (Si,R)-TS2, leading to the Si face attack being the dominant attack pathway. As shown in Figure 6e, the theoretical chemical study indicates that, to identify the correct transition state, a comprehensive comparison of the Si and Re face attacks was required. Importantly, the relative free energy of transition state (Si,S)-TS2 was the lowest, corresponding to the key selective step and the major isomer (S,S)-4a.

Based on the related literature23, the control experiments and DFT calculations, a possible KR mechanism was proposed (Fig. 7). Initially, phosphine oxide catalyst 3a reacted with oxalyl chloride to generate catalyst chlorophosphonium A. The in situ generated intermediate A subsequently acts as an activator for the amino acid, leading to the formation of an acyl phosphonium salt B. The key intermediates A and B were successfully characterized by high-resolution mass spectrometry (see Supplementary Information for details). Finally, L-pyroglutaminol 2a underwent a nucleophilic addition to generate the ester 4a and phosphine oxide catalyst 3a to complete the catalytic cycle. Among the amino acids, the reaction rate of the S-enantiomer was faster, and (R)-1a remained. Presumably, double H-bondings were formed spontaneously between L-pyroglutaminol 2a and phosphine oxide catalyst 3a through the perfectly matched lactam moieties. The developed catalyst demonstrated excellent stereocontrol and catalytic activity in the KR of racemic amino acids, which presumably benefited from an intimate double H-bonding interaction.

Methods
General procedure
In a 15 mL oven-dried Schlenk tube, racemic amino acids (0.5 mmol, 1.0 equiv.), L-pyroglutaminol (0.3 mmol, 0.6 equiv.) and catalyst (0.1 mmol, 20 mol %) were well mixed in chloroform (1.0 mL). Then oxalyl chloride (0.75 mmol, 1.5 equiv.) and triethylamine (0.75 mmol, 1.5 equiv.) were added in sequence at ambient temperature under argon atmosphere. The resulting mixture was stirred at room temperature for 10 min. Subseqently, the mixture was partitioned between EtOAc (70 mL) and H2O (30 mL) at room temperature. The organic layer was washed with saturated brine (30 mL×2), dried over Na2SO4, and concentrated in vacuo. The resulting residue was dissolved in dichloromethane (10 mL) and 1M aqueous solution NaOH (3 mL). The organic layer and the aqueous layer were separated. The organic layer was washed with saturated brine (10 mL×2), dried over Na2SO4, and concentrated in vacuo. The resulting residue was purified via silica gel colunm chromatography to yield esters. The aqueous layer was washed with dichloromethane (10 mL×2). The organic layer was discarded. The aqueous layer was made acidic with excess 1M aqueous solution HCl (to pH ~5) and was extracted with dichloromethane (10 mL×2). The combined organic layer was washed with brine (20 mL×2), dried over Na2SO4, then filtered and evaporated to afford the recovered amino acids.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Ren and co-workers report a highly efficient kinetic resolution (KR) of racemic amino acids with L-pyroglutaminol as an\nesterification reagent. The process is catalyzed by a phosphine oxide organocatalyst under mild conditions and provides a\nwide range of chiral esters and recovered amino acids with excellent stereoselectivities. The intimate double H-bonding\ninteraction between the pyroglutaminol core and the catalyst is proposed based on control experiments and DFT\ncalculations.\nAmino acids are undoubtedly an important class of compounds in chemistry and biology. Although many methods have\nbeen developed to make enantioenriched amino acids, kinetic resolution methods have been less developed. This work\nprovides a practical and general solution to the problem. The newly designed phosphine oxide catalyst is a highlight of the\nwork. The efficient resolution of a series of unnatural amino acids shows the importance of the work.\nI suggest accepting the paper after the following issues being addressed:\n1. In Fig 2, actually there are only examples of amino acids with trisubstituted stereocenters. So, the representative reaction\nequation should be revised.\n2. This reviewer is interested in access to amino acids bearing tetrasubstituted stereocenters. But there is only one example.\nIt will be very nice to see more examples.\n3. A control experiment using a compound removing the carbonyl oxygen of (S)-2a should be conducted to the importance of\nthe carbonyl group.", "reviewer_2": "The manuscript by Ren and co-workers describes a phosphine oxide organocatalyst derived from L-pyroglutaminol for the\nkinetic resolution (KR) of racemic amino acids via enantioselective esterification. Using N-protected racemic amino acids\nand L-pyroglutaminol as substrates, the authors achieved highly enantioselective synthesis of over 30 natural and non-\nnatural amino acids, demonstrated scalability to 5 mmol, and provided supporting evidence for a double hydrogen-bonding\ninteraction in the catalytic mechanism through control experiments and DFT calculations. However, in terms of conceptual\nnovelty and originality, I do not believe this work meets the standards of Nature Communications for the following reasons:\n1) Numerous strategies for stereoselective synthesis of chiral amino acids have been reported, including catalytic\nasymmetric hydrolysis of amino acid esters that not mentioned in the manuscript. These approaches enable\nstereoconvergent synthesis of chiral amino acids. In comparison, the KR strategy described here does not demonstrate clear\nsuperiority in terms of concept, mechanism, or application, particularly given the inherent limitation of maximum 50%\ntheoretical yield for KR processes.\n2) The authors employ optically pure L-pyroglutaminol as a template substrate but fail to address how its chiral center\ninfluences stereoselectivity. For instance, in Table 1, the use of the enantiomeric catalyst 3f resulted in reduced\nstereoselectivity (53:47 dr) rather than producing the opposite enantiomer of the product. This raises critical questions: Does\nthe chirality of L-pyroglutaminol play a decisive role in stereocontrol? If so, does this diminish the significance of the catalyst\nitself in governing enantioselectivity? Further mechanistic studies (e.g., using racemic or D-pyroglutaminol) are necessary to\ndisentangle the contributions of the substrate and catalyst to stereoselectivity.\n3) The assertion that this work represents the \"first KR of amino acids\" overlooks prior enzymatic KR strategies for amino\nacids. Such omissions weaken the manuscript’s scholarly context. Moreover, the claim that \"only CO, CO₂, and HCl are\ngenerated as waste\" is misleading, as it neglects the stoichiometric use of triethylamine, which generates ammonium salts\nas byproducts.\nOverall, though the work is technically sound and represents a valid contribution to the field of organocatalytic KR, the\nelements of innovation and broad appeal required for Nature Communications are insufficiently demonstrated. The study\nwould be more appropriately suited for a specialized journal focusing on asymmetric catalysis or synthetic methodology.", "reviewer_3": "Ren and co-workers' manuscript reports a kinetic resolution of racemic amino acids using a newly developed L-\npyroglutaminol-derived phosphine oxide organocatalyst. The methodology is broadly applicable and delivers good\nstereoselectivities. To investigate the operative mechanism and stereocontrol, the authors used control experiments and\nDFT computations. While this work presents an interesting new synthetic methodology, especially for non-natural amino\nacids, the reviewer questions whether it currently meets the broad appeal required for publication in Nature\nCommunications. I recommend publication only after the authors fully address the major issues outlined below.\n1. The role of oxalyl chloride and its reaction/interaction with the phosphine oxide catalyst is crucial for the overall\nmechanism and selectivity. A more detailed mechanistic proposal, supported, for example, by spectroscopic evidence (low-\nT NMR?) for intermediates A and B (Figure 6), would significantly strengthen the manuscript.\n2. The authors should compute the full reaction profile, rather than limiting their analysis to only the selectivity-determining\ntransition states.\n3. The authors state in the Supporting Information that they used “dispersion-corrected” DFT. However, B3LYP is not\ndispersion-corrected by default. The authors should clarify whether a dispersion correction was applied and, if so,\nspecify which one.\n4. The manuscript could benefit from a more explicit discussion of the methodology's limitations. For example, how do\nelectron-rich vs. electron-poor substrates or sterically hindered systems perform? Are there known classes of amino acids\nwhere the method fails?\n5. The authors should include a short comparison with existing KR methods for amino acids (e.g., enzymatic or transition-metal-catalyzed routes), which would help contextualize this organocatalytic method's practical advantages.\n6. While the manuscript is generally well-written, some grammatical errors and awkward phrasing should be corrected (e.g.,\n\"reaction completed in less than 10 min\" → \"the reaction was completed within 10 minutes\")."}, "author_response": "Response: We really appreciate the reviewer’s positive comments on our work.\n1) In Fig 2, actually there are only examples of amino acids with trisubstituted\nstereocenters. So, the representative reaction equation should be revised.\nResponse: Per the reviewer’s suggestion, the representative reaction equation has\nbeen revised in the revised manuscript.\n2) This reviewer is interested in access to amino acids bearing tetrasubstituted\nstereocenters. But there is only one example. It will be very nice to see more examples.\nResponse: Per the reviewer’s suggestion, more amino acids bearing tetrasubstituted\nstereocenters were tested and these results have been included in the revised\nmanuscript and supporting information.\n3) A control experiment using a compound removing the carbonyl oxygen of (S)-2a\nshould be conducted to the importance of the carbonyl group.\nResponse: Per the reviewer’s suggestion, L-prolinol 2c was tested. Not surprisingly,\nthe corresponding ester 4r was obtained in markedly reduced diastereoselectivity.\nThese results have been included in the revised manuscript and supporting\ninformation.\nReviewer 2:\nComments:\nThe manuscript by Ren and co-workers describes a phosphine oxide organocatalyst\nderived from L-pyroglutaminol for the kinetic resolution (KR) of racemic amino acids\nvia enantioselective esterification. Using N-protected racemic amino acids and\nL-pyroglutaminol as substrates, the authors achieved highly enantioselective synthesis\nof over 30 natural and non-natural amino acids, demonstrated scalability to 5 mmol,\nand provided supporting evidence for a double hydrogen-bonding interaction in the\ncatalytic mechanism through control experiments and DFT calculations. However, in\nterms of conceptual novelty and originality, I do not believe this work meets the\nstandards of Nature Communications for the following reasons.\nResponse: We really appreciate the reviewer’s positive comments on our work.\n1) Numerous strategies for stereoselective synthesis of chiral amino acids have been\nreported, including catalytic asymmetric hydrolysis of amino acid esters that not\nmentioned in the manuscript. These approaches enable stereoconvergent synthesis of\nchiral amino acids. In comparison, the KR strategy described here does not\ndemonstrate clear superiority in terms of concept, mechanism, or application,\nparticularly given the inherent limitation of maximum 50% theoretical yield for KR\nprocesses.\nResponse: Per the reviewer’s suggestion, the asymmetric hydrolysis of amino acid\nesters and the corresponding reference have been added in the revised manuscript.\nWhile numerous strategies for stereoselective synthesis of chiral amino acids achieve\ntheoretical 100% yield, they often exhibit limited scope for sterically hindered or\nnon-natural amino acids. In contrast, our KR strategy leverages a novel double\nH-bonding mechanism between the phosphine oxide catalyst and pyroglutaminol,\nenabling precise stereocontrol (s > 1057) unmatched by enzymatic KR. In addition,\nour KR strategy accommodates challenging substrates, including α,α-dialkyl\nsubstituted amino acids and side-chain-functionalized variants, which are often\nproblematic for enzymatic systems due to steric constraints or sensitivity.\nThe inherent 50% yield limitation of KR is mitigated by three key advantages. (1)\nSimultaneous access to both enantiopure products: The recovered amino acids (up\nto >99% ee) and the chiral esters (up to >99:1 dr) are both high-value building blocks\n(for peptidomimetics or asymmetric catalysis). (2) Unprecedented efficiency:\nReactions were completed within 10 minutes (vs. hours/days for enzymatic systems),\nwith exceptional selectivity (s > 1057). (3) Scalability and robustness: Demonstrated\nat gram-scale without sensitivity to ambient conditions (unlike biocatalysts, which\nrequire strict pH/temperature control).\nIn summary, our KR strategy offers a complementary approach to stereoselective\nmethods by combining rapidity, excellent selectivities of amino acids with operational\nsimplicity and scalability—addressing gaps in efficiency and scope for structurally\ndiverse substrates. Therefore, this work should be of wide interest to the community\nof organic chemistry. We have revised the manuscript to more explicitly position our\nwork within the broader landscape of amino acid synthesis strategies.\n2) The authors employ optically pure L-pyroglutaminol as a template substrate but\nfail to address how its chiral center influences stereoselectivity. For instance, in Table\n1, the use of the enantiomeric catalyst 3f resulted in reduced stereoselectivity (53:47\ndr) rather than producing the opposite enantiomer of the product. This raises critical\nquestions: Does the chirality of L-pyroglutaminol play a decisive role in stereocontrol?\nIf so, does this diminish the significance of the catalyst itself in governing\nenantioselectivity? Further mechanistic studies (e.g., using racemic or\nD-pyroglutaminol) are necessary to disentangle the contributions of the substrate and\ncatalyst to stereoselectivity.\nResponse: Per the reviewer’s suggestion, the KR of 1a was carried out using catalyst\n3f and D-pyroglutaminol (R)-2a provided the enantiomer (R,R)-4a with 99:1 dr and\nthe recovered (S)-1a with >99% ee, with excellent selectivity factor (s > 529) (Fig.\n4e). These results demonstrated that the chiral environments of both the catalyst and\npyroglutaminol synergistically govern the stereoselectivity. The corresponding results\nand details have been included in the revised manuscript and supporting information.\n3) The assertion that this work represents the \"first KR of amino acids\" overlooks\nprior enzymatic KR strategies for amino acids. Such omissions weaken the\nmanuscript’s scholarly context. Moreover, the claim that \"only CO, CO₂, and HCl are\ngenerated as waste\" is misleading, as it neglects the stoichiometric use of\ntriethylamine, which generates ammonium salts as byproducts.\nResponse: Per the reviewer’s suggestion, the corresponding changes have been made\nin the revised manuscript.\n4) Overall, though the work is technically sound and represents a valid contribution\nto the field of organocatalytic KR, the elements of innovation and broad appeal\nrequired for Nature Communications are insufficiently demonstrated. The study would\nbe more appropriately suited for a specialized journal focusing on asymmetric\ncatalysis or synthetic methodology.\nResponse: We really appreciate the reviewer’s positive comments on our work. We\nrespectfully contend that our work demonstrates conceptual innovation, mechanistic\nnovelty, and broad interdisciplinary appeal that align with Nature Communications'\nscope. Below, we highlight three key aspects underscoring its significance.\n(1) Our study introduces the first L-pyroglutaminol-derived phosphine oxide\norganocatalyst, which leverages a unique double H-bonding interaction to achieve\nunprecedented stereocontrol (s > 1057). The catalyst’s design-integrating a chiral\nlactam H-bonding motif with a phosphine oxide activator-enables simultaneous\nsubstrate recognition and activation, a strategy unreported in amino acid KR.\n(2) Our method achieves excellent selectivity (s > 1057) and ultrafast resolution (10\nmin) for amino acid KR. In addition, our method accommodates broad substrate\ndiversity-including natural/non-natural amino acids, sterically demanding α,α-dialkyl\nsubstituted amino acids.\n(3) The work bridges organic synthesis, catalyst design, and chemical biology.\nGram-scale synthesis delivers both enantiopure amino acids (e.g., drug intermediates)\nand chiral pyroglutaminol esters (e.g., peptidomimetic precursors). DFT studies reveal\nH-bonding dominated enantiocontrol, offering a blueprint for designing lactam-based\norganocatalysts—a nascent area with untapped potential. Enantioenriched non-natural\namino acids are pivotal in peptide therapeutics and biocatalysis, and our method\nprovides rapid, general access to these motifs.\nWhile specialized methodology journals focus on catalytic efficiency, our work\nadvances fundamental questions in stereoselective catalysis (H-bonding networks in\nenantioselection) and addresses practical limitations in biocatalysis (enzyme\ninstability and narrow scope). This dual impact aligns with Nature Communications’\nemphasis on \"scientific advances that represent a significant breakthrough in their\nfield\".\nReviewer 3:\nComments:\nRen and co-workers' manuscript reports a kinetic resolution of racemic amino acids\nusing a newly developed L-pyroglutaminol-derived phosphine oxide organocatalyst.\nThe methodology is broadly applicable and delivers good stereoselectivities. To\ninvestigate the operative mechanism and stereocontrol, the authors used control\nexperiments and DFT computations. While this work presents an interesting new\nsynthetic methodology, especially for non-natural amino acids, the reviewer questions\nwhether it currently meets the broad appeal required for publication in Nature\nCommunications. I recommend publication only after the authors fully address the\nmajor issues outlined below.\nResponse: We really appreciate the reviewer’s positive comments on our work.\n1) The role of oxalyl chloride and its reaction/interaction with the phosphine oxide\ncatalyst is crucial for the overall mechanism and selectivity. A more detailed\nmechanistic proposal, supported, for example, by spectroscopic evidence (low-T\nNMR?) for intermediates A and B (Figure 6), would significantly strengthen the\nmanuscript.\nResponse: Per the reviewer’s suggestion, the intermediates A and B in Figure 6 were\nsuccessfully characterized by high-resolution mass spectrometry, which tentatively\nsupports the proposed reaction mechanism. The corresponding results and details\nhave been included in the revised manuscript and supporting information.\n2) The authors should compute the full reaction profile, rather than limiting their\nanalysis to only the selectivity-determining transition states.\nResponse: Per the reviewer’s suggestion, relative energy profiles for the reaction\npathway have been added. A detailed discussion has been included in the revised\nmanuscript and supporting information.\n3) The authors state in the Supporting Information that they used\n“dispersion-corrected” DFT. However, B3LYP is not dispersion-corrected by default.\nThe authors should clarify whether a dispersion correction was applied and, if so,\nspecify which one.\nResponse: Actually, the dispersion correction was applied. The Grimme's DFT-D3\nmethod was applied to correct the van der Waals interactions for all molecules. The\ncorresponding changes have been made in the revised supporting information.\n4) The manuscript could benefit from a more explicit discussion of the methodology's\nlimitations. For example, how do electron-rich vs. electron-poor substrates or\nsterically hindered systems perform? Are there known classes of amino acids where\nthe method fails?\nResponse: Per the reviewer’s suggestion, a more explicit discussion and unsuccessful\ncases have been added in the revised manuscript.\n5) The authors should include a short comparison with existing KR methods for\namino acids (e.g., enzymatic or transition-metal-catalyzed routes), which would help\ncontextualize this organocatalytic method's practical advantages.\nResponse: Per the reviewer’s suggestion, a short comparison with existing KR\nmethods has been included in the revised manuscript. Compared to existing methods,\nthis organocatalytic approach offers distinct advantages in efficiency, scope, and\npracticality. Enzymatic KR (e.g., using hydrolases/deaminases) typically achieves\nmoderate selectivity (s ≈ 20-200) but suffers from narrow substrate scope (limited\nto natural amino acids), sensitivity to pH/temperature, and high enzyme costs.53-57 In\ncontrast, our method achieves unprecedented selectivity (s up to 1057) within 10\nminutes under mild conditions, accommodates broad substrate diversity-including\nnatural/non-natural amino acids, sterically demanding α,α-dialkyl substituted amino\nacids. While enzymatic KR remains viable for aqueous biotransformations, this\norganocatalytic system bridges critical gaps in efficiency, versatility, and scalability,\nproviding a practical, sustainable alternative for synthesizing enantiopure amino acid\nbuilding blocks.\n6) While the manuscript is generally well-written, some grammatical errors and\nawkward phrasing should be corrected (e.g., \"reaction completed in less than 10 min\"\n→ \"the reaction was completed within 10 minutes\").\nResponse: Per the reviewer’s suggestion, the error has been thoroughly checked and\ncorrected in the revised manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have made nice revision and conducted necessary addtiional experiments to address all the concerns from the\nreviewers, and I suggest accepting the paper.", "reviewer_2": "After thorough evaluation of the authors' point-by-point response and the revised manuscript with supporting materials, I\nappreciate their substantial efforts in addressing reviewers' concerns. While I still maintain that the core concept of this work\n(i.e. phosphine oxide catalysis for stereoselective amino acid synthesis) lacks transformative novelty given the extensive\nprior literature in both organocatalytic KR process and amino acid derivatization, the authors' rigorous engagement with all\nspecific technical queries raised by myself and other 2 reviewers is recognized. Nevertheless, several critical issues still\nrequire resolution:\nAlthough the authors supplemented experiments using (R)-2a (Fig. 4e) demonstrate stereochemical inversion, they remain\ninadequate to conclusively elucidate the origin of stereocontrol. More critically, the proposed double hydrogen-bonding\ninteraction—central to the catalytic mechanism—cannot be definitively proven through substrate/catalyst analogue studies\nalone. To resolve these mechanistic ambiguities, nonlinear effect studies are strongly recommended to unravel in-depth\nchiral-induction model, complemented by NMR titration to characterize hydrogen-bonding thermodynamics under reaction-\nlike conditions. Moreover, regarding methodological transparency, section 2.2 of the Supporting Information (\"Optimization\nstudies\") requires significant expansion to enhance methodological transparency. Specifically: Rationale for initial catalyst\ndesign choices must be clarified. Was the racemic phosphine oxide catalyst evaluated during preliminary screening? If not,\njustification for its exclusion should be provided. The solvent screening table referenced in the main text must be reproduced\nin full within the SI, including all tested solvents and their corresponding selectivity factors (s). Finally, the current transition-\nstate models (Figures 5–7) remain suboptimally presented, impeding mechanistic comprehension and necessitating\nadditional optimization for clarity.", "reviewer_3": "The authors have addressed all of my concerns. I have only one very minor correction. As stated in their rebuttal, the authors\nused Grimme's DFT-D3 method, but the manuscript still reports: “B3LYP/6-311G(d,p)/SMD(CHCl3)”. This should be\ncorrected to: “B3LYP-D3/6-311G(d,p)/SMD(CHCl3)”."}, "author_response": "Response: We really appreciate the reviewer’s positive comments on our work.\nReviewer 2:\nComments:\nAfter thorough evaluation of the authors' point-by-point response and the revised\nmanuscript with supporting materials, I appreciate their substantial efforts in\naddressing reviewers' concerns. While I still maintain that the core concept of this work\n(i.e. phosphine oxide catalysis for stereoselective amino acid synthesis) lacks\ntransformative novelty given the extensive prior literature in both organocatalytic KR\nprocess and amino acid derivatization, the authors' rigorous engagement with all\nspecific technical queries raised by myself and other 2 reviewers is recognized.\nNevertheless, several critical issues still require resolution.\nResponse: We really appreciate the reviewer’s positive comments on our work.\n1) Although the authors supplemented experiments using (R)-2a (Fig. 4e) demonstrate\nstereochemical inversion, they remain inadequate to conclusively elucidate the origin\nof stereocontrol. More critically, the proposed double hydrogen-bonding\ninteraction—central to the catalytic mechanism—cannot be definitively proven\nthrough substrate/catalyst analogue studies alone. To resolve these mechanistic\nambiguities, nonlinear effect studies are strongly recommended to unravel in-depth\nchiral-induction model, complemented by NMR titration to characterize\nhydrogen-bonding thermodynamics under reaction-like conditions.\nResponse: Per the reviewer’s suggestion, 1H NMR spectroscopic studies on the\nmixture of (S)-2a with 3a with a molar ratio of 1:1 were tested and these results have\nbeen included in the revised manuscript and supporting information.\n2) Moreover, regarding methodological transparency, section 2.2 of the Supporting\nInformation (\"Optimization studies\") requires significant expansion to enhance\nmethodological transparency. Specifically: Rationale for initial catalyst design\nchoices must be clarified. Was the racemic phosphine oxide catalyst evaluated during\npreliminary screening? If not, justification for its exclusion should be provided. The\nsolvent screening table referenced in the main text must be reproduced in full within\nthe SI, including all tested solvents and their corresponding selectivity factors (s).\nResponse: Per the reviewer’s suggestion, the corresponding changes have been made\nin the revised supporting information.\n3) Finally, the current transition-state models (Figures 5–7) remain suboptimally\npresented, impeding mechanistic comprehension and necessitating additional\noptimization for clarity.\nResponse: Thank you for your valuable comment on the presentation of the\ntransition-state (TS) models in Figures 5-7; we fully agree that a clear and optimized\nTS model is crucial for facilitating mechanistic comprehension and appreciate your\ninsight that helps improve this work. As detailed in the manuscript, the current TS\nmodels are rigorously derived from a combination of experimental results and density\nfunctional theory (DFT) calculations: control experiments (Figure 4b-4e) confirmed\nthat the double H-bonding interaction between the lactam moieties of\nL-pyroglutaminol and the phosphine oxide catalyst is essential for stereocontrol\n(masking H-bonding sites significantly reduced diastereoselectivity), while DFT\ncalculations at the B3LYP-D3/6-311G(d,p)/SMD(CHCl ) level yielded relative free\n3\nenergies of key TSs (e.g., (Si,S)-TS2 with ΔG = 6.0 kcal/mol) that align with\nexperimental selectivity (s > 1057) and product configuration ((S,S)-4a as the\ndominant isomer), verifying the models’ rationality. Therefore, the transition-state\nmodels in Figures 5-7 are still being kept and remains unchanged in the revised\nmanuscript.\nReviewer 3:\nComments:\nThe authors have addressed all of my concerns.\nResponse: We really appreciate the reviewer’s positive comments on our work.\n1) I have only one very minor correction. As stated in their rebuttal, the authors\nused Grimme's DFT-D3 method, but the manuscript still reports:\n“B3LYP/6-311G(d,p)/SMD(CHCl3)”. This should be corrected to:\n“B3LYP-D3/6-311G(d,p)/SMD(CHCl3)”.\nResponse: Per the reviewer’s suggestion, the corresponding change has been made in\nthe revised manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The reviewer appreciates the authors' efforts to improve the manuscript, and the revised version is certainly an improvement\nover the previous one. However, my key concern regarding the interaction mode between the catalyst and the substrate has\nnot been fully addressed.\nAlthough the authors have supplemented the manuscript with a 1H NMR titration experiment at a 1:1 molar ratio of catalyst\nto substrate, the previously suggested nonlinear effect study has not been implemented. While the added NMR data do\nindicate an interaction between the catalyst and the substrate, they do not provide further insight into the specific binding\nmode. In this context, a nonlinear effect experiment remains highly important and informative for elucidating the nature of the\ninteraction. Therefore, I strongly recommend that this experiment be carried out before the manuscript can be considered for\nacceptance."}, "author_response": "Response: We really appreciate the reviewer’s positive comments on our work.\n1) Although the authors have supplemented the manuscript with a 1H NMR titration\nexperiment at a 1:1 molar ratio of catalyst to substrate, the previously suggested\nnonlinear effect study has not been implemented. While the added NMR data do\nindicate an interaction between the catalyst and the substrate, they do not provide\nfurther insight into the specific binding mode. In this context, a nonlinear effect\nexperiment remains highly important and informative for elucidating the nature of the\ninteraction. Therefore, I strongly recommend that this experiment be carried out\nbefore the manuscript can be considered for acceptance.\nResponse: Per the reviewer’s suggestion, we carried out a nonlinear effect experiment\nfor elucidating the nature of the interaction. We observed a small but positive\nnonlinear effect, which indicates that more than one molecule of 3a was likely to be\ninvolved in the transition state of the enantiodifferentiating step. These results have\nbeen included in the revised manuscript and supporting information."}, {"round": 4, "reviewer_comments": {"reviewer_2": "As noted previously, the detailed reaction mechanism and interaction model require further experimental validation.\nThrough supplementary nonlinear effect experiments, a positive nonlinear effect was observed, which may suggest the\nexistence of a non-1:1 catalyst-substrate interaction mode. However, the authors did not provide further discussion or\nexplanation for this experimental phenomenon, especially since it was inconsistent with the transition-state model proposed in\nFigs. 5–7. Therefore, the current version of the manuscript, in my assessment, falls short of the completeness and rigor\nexpected for a publishable contribution."}, "author_response": "Response: We sincerely thank the reviewer for this profound and insightful comment,\nwhich prompted us to perform additional experiments that have led to a more nuanced\nand robust understanding of our system. To directly address the concern regarding the\npositive nonlinear effect and its implication for the reaction mechanism, we conducted\na decisive control experiment: we examined the kinetic resolution\nusing enantiopure catalyst 3a while systematically varying the enantiomeric purity of\nthe chiral reagent (S)-2a. The results are unequivocal. The enantiomeric excess of the\nrecovered amino acid (R)-1a increased nonlinearly from 53% ee (when 2a was\nracemic) to 99% ee (when 2a was enantiopure). This nonlinear relationship is critical\nfor three reasons. First, the baseline selectivity of 53% ee with racemic 2a proves that\nthe catalyst 3a itself possesses significant intrinsic enantioselectivity, affirming its role\nas the primary chiral director. Second, the nonlinear enhancement demonstrates that\nthe chiral 2a acts as a co-factor, providing a complementary chiral environment\nthat nonlinearly boosts the catalyst's inherent selectivity to exceptional levels. Third,\nand most importantly, the stark contrast between this nonlinear “substrate nonlinear\neffect” and the positive nonlinear “catalyst nonlinear effect” (Fig. 4g) allows us to\npinpoint the origin of the nonlinearity. This analysis is fully consistent with, and\nreinforced by, key data already present in the manuscript: (i) the drastic loss of\nselectivity when the mismatched catalyst 3f was used with (S)-2a (Table 1, entry 6),\nand (ii) the restoration of high selectivity (s > 529) when the chiralities of both\ncomponents were switched to the matched 3f/(R)-2a pair (Fig. 4e). These results had\nalready established the foundational principle of chiral matching for optimal\nperformance. Building on this, we now posit that the observed nonlinear relationship\nin the original catalyst experiment arises because, when the catalyst's ee is lowered,\nthe introduced mismatched catalyst enantiomer 3f interacts detrimentally—and in a\nnonlinear, inhibitory fashion—with the ever-present enantiopure (S)-2a. This effect\ndisproportionately suppresses the selectivity achieved by the optimal matched pair\n3a/(S)-2a. Therefore, the nonlinear effect reports on the system-level behavior when\nnon-productive interactions are introduced, while the DFT model (Figs. 5-7) correctly\ndepicts the structure and energetics of the productive pathway involving the matched\n1:1 complex. The new data and the unified interpretation do not contradict but rather\nenrich the mechanistic picture, solidifying the conclusion that the extreme selectivity\narises from a synergistic dual-chiral induction. We have incorporated this\ncomprehensive discussion and the new data into the revised manuscript and\nsupporting information, which we believe now fully addresses the reviewer's concern\nand meets the expected standards of completeness and rigor."}, {"round": 5, "reviewer_comments": {"reviewer_2": "Thanks for the authors' diligent work in addressing the points raised in my previous review. The additional control\nexperiments and the nuanced interpretation offered have satisfactorily resolved my concerns about the reaction mechanism.\nThe authors' explanation for the origin of the nonlinear effect is compelling and significantly strengthens the manuscript.\nI believe the revised manuscript, with these supplements, now meets the high standards required for publication.\nI support its acceptance in its present form."}, "author_response": ""}]

Decision letter