Asymmetric synthesis of stereogenic-at-sulfur compounds via biocatalytic oxidation with Unspecific Peroxygenases

Asymmetric synthesis of stereogenic-at-sulfur compounds via biocatalytic oxidation with Unspecific Peroxygenases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Asymmetric synthesis of stereogenic-at-sulfur compounds via biocatalytic oxidation with Unspecific Peroxygenases Gideon Grogan, Jiacheng Li, Benjamin Melling, Katy Cornish, Nicholas Mulholland, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7185896/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Stereogenic-at-sulfur compounds are vitally important biologically active small molecules, with many drugs featuring chiral sulfur(IV) atoms. Methods for the asymmetric synthesis of sulfoxide centres are well established, but methods that produce enantiomerically enriched sulfoximines and sulfinimines are far less well developed, with no known biocatalytic methods based on oxygenation. In this study, we demonstrate that Unspecific Peroxygenases (UPOs) catalyze the biocatalytic oxygenation of sulfilimines and sulfenimines to form enantiomerically enriched sulfoximines and sulfinimines respectively, on preparative scale. In the sulfenimine series, sulfoximines are generated in up to 99% ee via a kinetic resolution approach. In the sulfilimine series, the selective, stereodivergent synthesis of either of the enantiomeric sulfinimine products (both up to 99% ee ) can be achieved, with different UPOs affording products with opposite enantioselectivity. Both series represent novel applications of UPO technology to an ever-growing list of selective, practical and industrially relevant biotransformations. Physical sciences/Chemistry/Catalysis/Biocatalysis Physical sciences/Chemistry/Chemical biology/Biocatalysis Figures Figure 1 Introduction Enantioenriched stereogenic-at-sulfur(IV) compounds have myriad important roles, spanning medicinal chemistry, 1 agrochemistry 2 and asymmetric synthesis/catalysis (Fig. 1 a). 3 For example, the blockbuster gastro-intestinal drug Esomeprazole - prepared as its ( S )-enantiomer 1a – has improved metabolic stability and inhibits gastric acid production more effectively than Omeprazole, the racemic variant that preceded it. 4 Both enantiomers of tert -butanesulfinamide 1b (often referred to as Ellman’s sulfonamide) are available commercially, and are highly effective chiral auxiliaries used in the asymmetric synthesis of amine derivatives. 5 Enantiopure sulfur(IV) compounds are also important in asymmetric organocatalysis ( e.g. 1c ). 3 In terms of biological applications, sulfoximines are arguably the most important class of sulfur(IV) compound, featuring in several commercial products, such as the insecticide Sulfoxaflor 1d . 2 They are also of major current interest in medicinal chemistry ( e.g. anti-cancer drug 1e ). 6 Sulfoximines often exhibit good pharmacokinetic properties, such as high metabolic stability and solubility, rendering them useful isosteres of sulfones and sulfonamides. 1 , 6 The additional nitrogen site opens the avenue for H-bonding interactions, and enables easy manipulation of the physiochemical properties through N -functionalisation reactions. Increased interest in chiral sulfur(IV) compounds has helped to propagate a significant upsurge in the development of methods for their synthesis. This includes catalytic asymmetric methods, typically based on kinetic resolution and/or organocatalysis. 7 A notable recent approach, developed by Tian, Xie, Guo and coworkers and summarised in Fig. 1 b, 8 is their efficient, enantioselective method for the catalytic asymmetric synthesis of chiral sulfonamides 2a and sulfinate esters 2b , enabled by bifunctional 4-arylpyridine N -oxide organocatalysts. Biocatalytic methods to prepare stereogenic-at-sulfur(IV) are far less well-developed in comparison, with methods based on oxygenation limited to the conversion of sulfides to form sulfoxides. 9 A range of oxygenase enzymes, including hemoproteins such as cytochromes P450 and flavoprotein oxygenases, such as Baeyer-Villiger Monooxygenases (BVMOs), has been shown to catalyze this transformation, with one example reported to work on a kg scale for the production of a pharmaceutical intermediate by AstraZeneca. 10 Among hemoprotein biocatalysts, the conversion of simple sulfides to sulfoxides has also been reported for Unspecific Peroxygenases UPOs, 11 , 12 which display advantages over P450s and BVMOs in that they are nicotinamide cofactor independent and require only the addition of hydrogen peroxide to promote their oxygenation reactions. The UPO from Agrocybe aegerita ( Aae UPO) had previously been shown to convert substrates such as phenyl methyl sulfide into its ( R )-sulfoxide product with 80% ee. 13 UPOs have been classified into two broad subclasses, Class I and Class II, based on characteristics including sequence, structure and molecular weight. Class I UPOs are smaller, with MW of around 29 kDa and Class II UPOs are larger enzymes, with MWs of around 44 kDa. In previous work, we have shown that a Class I UPO, an engineered ‘artificial peroxygenase’ 14 (artUPO), which is related to the enzyme from Marasmius rotula , 15 delivers sulfoxide products in the complementary enantiomeric series to the Class II Aae UPO. 16 This diversity clearly has great potential for exploitation with respect to the oxidation of more complex substrates, such as the S(IV) oxidation reactions that are the subject of this study. In this manuscript, we describe novel methods for the biocatalytic oxygenation of two distinct families of sulfur precursors (Fig. 1 d and e). These methods allow the enantioselective synthesis of sulfoximines (Fig. 1 d) and sulfinimines (Fig. 1 e) on preparative scale, using two easy-to-handle UPO enzymes. The sulfoximine-forming series relies on a UPO-mediated sulfilimine kinetic resolution, while in the sulfinimine-forming series (Fig. 1 e), a remarkable enantiodivergence is revealed, whereby different UPOs are able to selectively deliver either enantiomer ( ( S )- or ( R )-7 ) of the sulfilimine product, via UPO catalysed sulfenimine oxygenation. Both approaches enable the biocatalytic formation of important stereogenic-at-sulfur(IV) compounds to be prepared in high ee on preparative scale. These approaches all represent completely novel biocatalytic transformations, and further expand the range and utility of UPOs to perform useful and selective preparative biocatalytic oxygenation reactions. Results and Discussion Given their importance in medicinal chemistry, we started by exploring the potential of UPOs to generate enantioenriched sulfoximines. Various powerful chemical methods for the asymmetric synthesis of sulfoximines and their derivatives have been developed in recent years; these include methods based on the chromatographic separation and resolutions of racemic sulfoximines, 17 the oxidative imination of sulfoxides, 18 electrophilic addition reactions to S -nucleophiles 17c,19 and nucleophilic addition reactions to S -electrophiles. 17c,20 An alternative approach for sulfoximine synthesis is via the oxidation of a sulfilimine ( 4 → 5 ). Methods for the oxidation of sulfilimines (also known as sulfimides) to form racemic sulfoximines have been known for decades, typically using simple peroxide based chemical oxidants. 21 However, to the best of our knowledge, all published syntheses of enantioenriched sulfoximines from sulfilimines start from enantioenriched sulfilimine precursors; as sulfilimines are themselves chiral and configurationally stable, stereochemical information in the precursor is retained following oxidation. 7a Methods for the preparation of enantiomerically enriched sulfilimines are known, but are rare, with notable exceptions being a biocatalytic (cytochrome P450 BM3 ) approach from Arnold and co-workers, 22 and a recently reported organocatalytic method by Zhang, Zhang and coworkers. 23 In recognition of the well-established ability of UPOs to catalyse enantioselective oxygenation reactions, we questioned whether they might be capable of forming enantioenriched sulfoximines via the kinetic resolution of easy-to-prepare racemic sulfilimines. To the best of our knowledge, no asymmetric sulfilimine to sulfoximine transformations are known that start from racemic starting materials. Furthermore, we also know of no biocatalytic methods (neither racemic nor asymmetric) for the oxidation of sulfilimines, using UPOs or indeed any other enzyme class. In view of their stability and ease of synthesis, N -cyano sulfilimines of the type rac-4 were selected for this study, with a view towards developing biocatalytic kinetic resolution transformations of the type summarised in Table 1 (box, rac-4 → ( S )-4 and ( S )-5 ). We started by exploring the kinetic resolution of the simple N -cyano phenyl methyl sulfilimine rac-4a , which was prepared via a straightforward oxidative procedure from phenyl methyl sulfide. 21c All other racemic sulfilimine precursors used throughout this study were made using similar methods (see Supplementary Information Section 2 for full preparative details and characterisation data). Our aim was to develop a kinetic resolution of racemic sulfilimine starting material rac-4a and to isolate the sulfoximine product 5a , along with unreacted sulfilimine 4a , both in enantioenriched form. The biotransformation of rac-4a was therefore tested conducted on a preparative (0.3 mmol) scale using artUPO and the conditions summarised in Table 1 ; the use of 0.6 equivalents of H 2 O 2 was chosen to facilitate approximately 50% conversion of rac-4 , as needed for effective kinetic resolution. Encouragingly, under these conditions kinetic resolution was achieved, with enantioenriched sulfoximine ( S ) - 5a and sulfilimine ( S )-4 isolated in 22% and 35% yields, and 74% and 32% ee respectively following column chromatography. The absolute stereochemistry of the major enantiomer ( S ) - 5a was confirmed by comparison to literature optical rotation data; 21b therefore, the absolute stereochemistry of the major enantiomer of the unreacted sulfilimine is logically assumed to be ( S )-4 . Note that while products ( S ) - 5a and sulfilimine ( S )-4 both have ( S )-stereochemical assignments, this merely reflects the change in Cahn–Ingold–Prelog priorities, and they have opposite sense of absolute stereochemistry as expected. The assignment of absolute stereochemistry of the other related products in this manuscript was made by analogy, supported by optical rotation and HPLC data (see Supplementary Information, Section 3 and 5). r Aae UPO-PaDa-I-H 24 was also tested as an alternative UPO for this transformation but was not pursued, as it led to much poorer conversion when tested on a selection of sulfilimines under the same conditions (see Supplementary Information, Section 2.6). Having established that the proposed UPO mediated kinetic resolution is viable, attention turned to exploring the scope of the artUPO biotransformation with other sulfilimine substrates (Table 1 A–C). All biotransformations were performed on preparative scale, with the yields quoted referring to purified product following column chromatography. The ee s of the product sulfoximines ( S )-5 were measured using chiral HPLC analysis of the isolated product; the ee s of the enantioenriched sulfilimines (S)-4 were also measured by chiral HPLC, in some cases from the sulfilimines directly, and on others following m -CPBA oxidation to the corresponding sulfoximines when this expedited analysis (see Supplementary Information, General Procedure 2.3). We started by exploring substrates similar to rac-4a with different substituents on the phenyl ring (Table 1 A). Ten substrates of this type were tested, with successful kinetic resolution achieved and enantioenriched sulfoximines ( ( S )-5a – k ) and sulfilimines ( ( S )-4a – k ) isolated in all cases. Electron-rich and -poor substituents were both well tolerated. Substrates featuring a para- trifluoromethyl substituent ( rac-4e and rac-4j ) worked especially well, with very efficient kinetic resolution achieved, with > 90% overall yield and > 95% ee obtained for the isolated sulfoximines ( ( S )-5e , 5j ) and enriched sulfilimines ( ( S )-4e, 4j ). Three pyridine-containing substrates (Table 1 B) were also transformed successfully, with good to high ee s for the sulfoximine products ( ( S )-5k – m ) obtained in this series. It is notable that the protocol can be applied to aza-heterocyclic systems given their prominence in medicinal chemistry, while avoiding the proclivity of pyridines to undergo oxidation to form N- oxides with UPOs. 25 Table 1 C summarises results using racemic sulfilimine starting materials with non-methyl alkyl groups ( rac-4n – u ). The resolution of substrate rac-4s is notable, as this shows that the biotransformation is not contingent on the sulfilimine having an aromatic substituent. As before, the most effective examples in this series were those containing electron-poor aromatic groups, with sulfilimines rac-4t and rac-4u affording products in 98% overall yield, and with ee s of 90% and 99% respectively for sulfoximines ( S )-5t and ( S )-5u . Except for selected cases (those highlighted with a blue background) the biotransformations were all performed on 0.3 mmol scale using the same conditions, without optimisation and on a substrate-by-substrate basis. Of course, for any kinetic resolution, it is unlikely that the same reaction conditions will be optimal across all substrates. To demonstrate how additional optimisation can lead to improved results, and to showcase the scalability of the method, the biotransformations of rac-4b , rac-4e , rac-4j , rac-4t and rac-4u were performed on larger (1.0 mmol) scale, leading to the improved results presented (Table 1 A and 1 C, examples highlighted with a blue background). The increase in scale was useful in facilitating more precise control in reaction conversion compared with the standard method; careful monitoring of conversion and the use of additional H 2 O 2 when needed, proved to be effective in achieving the ≈ 50% conversion needed for optimal kinetic resolution (see Supplementary Information, General Procedure 2.5). The resolution of substrate rac-4j (98% and > 99% ee , s > 200) best illustrates the power of this approach. N -Cyano sulfilimines were chosen as starting materials for this study primarily in view of their synthetic tractability. However, if the unfunctionalised sulfoximine products are required, either directly or for further derivatisation, these can easily be accessed using the method summarised in Table 1 D. 26 Thus, reaction of N -cyano sulfoximine ( S )-5e with trifluoroacetic anhydride followed by potassium carbonate in methanol affords sulfoximine ( S )-9e with no erosion in ee . Furthermore, if the enantiomeric ( R )-sulfoximine is required, this can be obtained via simple oxidation of the enantiomerically enriched ( S )-sulfilimine; for example, the reaction of sulfilimine ( S )-4e with m -CPBA gave sulfoximine ( R )-5e in good yield, and with no erosion in ee . Note that the oxidation of ( S )-4e into ( R )-5e is stereo-retentive, and the change from an ( S )- to ( R )-stereochemical assignment merely reflects the change in Cahn–Ingold–Prelog priorities. The enantiopreference of artUPO for N- cyano sulfilimines was investigated through docking of the favoured ( R )-enantiomer of 4e into our previously obtained structure of artUPO (PDB 7ZNM) 16 using Autodock VINA. 27 The lowest energy pose obtained positions the sulphur lone pair of the pro -( S ) face of ( R )- 4e ideally for receiving oxygen from the oxygenating species Compound I (CpdI) with the aromatic group bound in a hydrophobic pocket formed by L65, V69, I91, I160 and I235 ( Figure S69A ) and the N -cyano group positioned between the side chains of I62, A66 and F167. A similar pose for the unflavoured ( S )- enantiomer of 4e would bring the cyano group into close contact with the side chains of I160, L163 and E164, thus providing a plausible structural explanation for the experimentally observed enantioselectivity. After establishing artUPO as a selective biocatalyst for the kinetic resolution of sulfilimines 4 , attention then turned to the exploration of UPOs for the enantioselective synthesis of sulfinimines. Sulfinimines (also known as N- sulfinylimines) are extremely useful synthetic intermediates owing to their ability to undergo a range of diastereoselective transformations, and hence have been widely used to prepare chiral amine derivatives. 5 , 28 When enantiomerically enriched sulfinimines are used in organic synthesis, most commonly they are prepared via the condensation of an enantiomerically enriched sulfonamide (often Ellman’s sulfonamide auxiliary 1b , or the p -tolyl derivative popularised by Davis and coworkers 29 ) with an aldehyde or ketone. In this work, we explored an alternative approach to access enantiomerically enriched sulfinimines, via the UPO catalysed oxidation of sulfenimines 6 . Representatives of both Class I and Class II UPOs were tested for this transformation, revealing intriguing stereodivergent reaction profiles for the different UPO classes. First, Class II r Aae UPO-PaDa-I-H was challenged with a selection of seven different phenyl sulfenimines with different S -alkyl/aryl substituents (to form ( R )-7a – 13a , Table 2 , Box 1). The reactions were performed on preparative scale (0.2 mmol of 6 ) in pH 7 KPi buffer with acetonitrile as co-solvent and using 1 equivalent of H 2 O 2 (added slowly over 10 h) as the stoichiometric oxidant. Conversion was measured by comparing the amounts of 6 and product in the 1 H NMR spectra of the crude reaction mixture, and ee was measured using chiral HPLC (see Supplementary Information, Section 5). In all but one case, some conversion into the expected enantiomerically enriched sulfinimine was observed and the depicted ( R )-enantiomer was formed in excess; the assignment of absolute stereochemistry made by comparison to literature optical rotation data for ( R )-12a 30 (corroborated by several other substrates in the series featured later in Table 2 B, see Supplementary Information Section 3). The most successful example was the oxygenation of the i -Pr-substituted sulfenimine, which was converted into sulfinimine ( R )-12a with 89% conversion and in 98% ee . The only substrate in this series that did not undergo oxygenation was the tert- butyl substituted sulfenimine, where no conversion into ( R )-13a was observed. This result was not wholly surprising, given that r Aae UPO-PaDa-I-H tends to perform poorly in the biotransformations of more sterically demanding substrates. 31 In contrast, artUPO, the same Class I UPO employed in the first half of this manuscript, typically performs better than r Aae UPO-PaDa-I-H with bulky substrates, owing to its more accessible active site. Thus, four sulfenimines were tested using artUPO in the same way (Table 2 , Box 2). All four substrates were converted well, included the bulky tert- butyl substituted sulfenimine, with full conversion into ( S )-13a observed. For the smaller substrates, the products ( R )-7a and rac-9a were formed with little or no ee respectively; again, this was not wholly surprising, as the ability to transform bulkier substrates using artUPO is often offset against reduced enantioselectivity. However, for the bulkier substrates, enantioselectivity was much higher, with products ( S )-12a and ( S )-13a formed in 80% and 92% ee respectively. Remarkably, the opposite ( S )-enantiomer was formed in excess in this series, thus offering complementary enantioselectivity to that of r Aae UPO-PaDa-I-H. The assignment of absolute stereochemistry was made by comparison to literature optical rotation data for ( S )-13a , 32 and corroborated in several cases in the series in Table 2 C (see Supplementary Information Section 3). The complementary enantioselectivity accords with previous preliminary observations made for r Aae UPO-PaDa-I-H and artUPO for the oxidation of sulfides. 16 In the case of sulfenimines such as 6 , this complementarity appears to result from different substrate approach trajectories to the oxidising species CpdI, in the active sites of the enzymes, as revealed by docking 6 (where R = i -Pr) into the enzymes, again using Autodock VINA (see Supplementary Information, Figure S69B). 27 Hence in the case of artUPO, the pro -( S ) lone pair is presented to the CpdI oxygen as the phenyl ring rests in the hydrophobic pocket formed by L65, V69, I91, I160 and I235 previously described. As the equivalent pocket in r Aae UPO-PaDa-I-H is restricted by phenylalanine residues including F188, the substrate approaches CpdI through a more available and less constrained tunnel and thus presents the pro -( R )-lone pair to the oxidant. The best performing cases with each enzyme ( i -Pr and t -Bu derivatives, leading to the formation of (R )-12a and (S )-13a ) are arguably amongst the most synthetically useful systems, considering the established utility of i -Pr and t -Bu sulfinimines in asymmetric synthesis. 5 Therefore, we next examined the scope of these biotransformations on other iso -propyl- and tert -butyl-substituted sulfenimines. Results for the enantioselective synthesis of iso -propyl substituted ( R )-sulfinimines 7 using r Aae UPO-PaDa-I-H are summarised in Table 2 B. As in the initial screening, the reactions were performed on preparative scale (0.2 mmol of 6 ) in pH 7 KPi buffer with acetonitrile as co-solvent and using 1 equivalent of H 2 O 2 (added slowly over 10 h) as the stoichiometric oxidant. The yields quoted refer to isolated yields of purified products following column chromatography, and ee was measured by chiral HPLC. All twelve examples proceeded with excellent enantioselectivity (90–99% ee ), across a range of substituted aromatic substrates ( ( R )-12a – i ). Naphthyl ( ( R )-12h , ( R )-12j ), ketimine ( ( R )-12i – k ), cyclic ( ( R )-12k ) and pyridyl ( ( R )-12l ) substrates also worked well. The opposite enantiomeric series is depicted in Table 2 C, in which results for the formation of tert -butyl substituted ( S )-sulfinimines ( S )-13 using artUPO are summarised. Enantioselectivity was again high, with the ( S )-enantiomer formed in > 90% ee in most examples tested. As before, a range of substituted aromatic substrates were tested and all worked well ( ( S )-13a – g ). Naphthyl ( ( S )-13h ), ketimine ( ( S )-13i ), cyclic ( ( S )-13j ) and heterocyclic ( ( S )-13l – m ) substrates were also well tolerated. The poor conversion (10% yield) for adamantyl derivative ( S )-13n appears to show the limits of enzyme with respect to steric bulk of the substrate, although notably the enantioselectivity remained relatively high (80% ee ). In both series, attempts to test simple alkyl substituted sulfenimines ( e.g. 6 where neither R 1 nor R 2 is aromatic) were thwarted by the instability of the requisite starting materials, meaning the biotransformations were not tested. The method is therefore limited to aromatic and cyclic aliphatic sulfenimines systems, that can be prepared and handled easily. To showcase the utility of the method of the enantiomerically enriched sulfinimines accessible using this method, two relatively simple syntheses of secondary amines used on the synthesis of pharmaceuticals are summarised in Table 2 D. Starting from sulfinimine ( R )-12j (prepared in 99% ee using r Aae UPO-PaDa-I-H), DIBAL reduction, followed by hydrolysis delivered a single enantiomer of amine 14 . Alkylation with iodide 15 then delivered hyperparathyroidism drug Cincalcet 16 . Similarly, in the opposite series, sulfinimine ( S )-13j was prepared in 98% ee ; in this case, the reaction was performed on 3 mmol scale and delivered 400 mg of the enantiomerically enriched product, in almost identical yield to the smaller scale version. A highly diastereoselective reduction and hydrolysis followed to deliver amine 18 in 99% ee , with this amine a key precursor to the obstructive hypertrophic cardiomyopathy drug Mavacamten 19 . Conclusions In summary, two novel, preparative biocatalytic approaches for the high yielding and enantioselective oxygenation of stereogenic-at-sulfur(IV) compounds, have been developed and described for the first time. To the best of our knowledge, both approaches had no known biocatalytic variants, using any enzyme, prior to this manuscript. Selective oxidation reactions in organic chemistry remain a challenge, especially where enantioselective transformations are concerned. Whereas cofactor-dependent enzymes present problems with stability, turnover and expense, UPOs offer real potential for scalable asymmetric oxidations using simple procedures. To facilitate their wider uptake, it is crucial that examples of scalable reactions on useful molecules are investigated and presented. With the transformations to form stereogenic-at-sulfur(IV)functionalities presented in this report, we add another highly promising application of UPO technology to a growing list of reactions that have significant potential for scale-up in an industrial context. Online content Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data availability are available at [insert link at publication]. Declarations Data availability All data, including experimental procedures, compound characterization data and HPLC data, are available within the article and its Supplementary Information. Acknowledgements We thank the U.K. Engineering and Physical Sciences Research Council (EPSRC, EP/X014886/1, J.L. and K.A.S.C.) for funding, and the EPSRC and Syngenta for the award of a PhD studentship to B.M. (project 2602946). Author contributions W.P.U and G.G. designed the study. J. L., B. M. and K.A.S.C. performed the experiments and interpreted the results. J. C. led on all aspects relating to enzyme production. N. M. provided industrial advice and additional guidance (to B. M.). W.P.U and G.G. prepared the manuscript and for publication, supported by J. L. and B. M. Competing interests The authors declare no competing interests. Additional information Supplementary Information. The online version contains supplementary information available at xxxxxxxxxxxxxxxx References (a) Bentley, R. Role of sulfur chirality in the chemical processes of biology. Chem. Soc. Rev . 34 , 609–624 (2005); (b) Mäder, P. & Kattner, L. Sulfoximines as rising stars in modern drug discovery? Current status and perspective on an emerging functional group in medicinal chemistry. J. Med. Chem . 63 , 14243–14275 (2020); (c) Lücking, U. Neglected sulfur(VI) pharmacophores in drug discovery: exploration of novel chemical space by the interplay of drug design and method development. Org. Chem. Front . 6 , 1319–1324 (2019); (d) Han, Y. et al. Application of sulfoximines in medicinal chemistry from 2013 to 2020. Eur. J. Med. Chem . 209 , 112885 (2021); (e) Trost, B. M., Ryan, M. C., Rao, M. & Markovic, T. Z. Construction of enantioenriched [3.1.0] bicycles via a ruthenium-catalyzed asymmetric redox bicycloisomerization reaction. J. Am. Chem. Soc . 136 , 17422–17425 (2014). (f) Trost, B. M. & Rao, M. Development of chiral sulfoxide ligands for asymmetric catalysis. Angew. Chem. Int. Ed . 54 , 5026–5043 (2015). T. C. Sparks et al. Sulfoxaflor and the sulfoximine insecticides: Chemistry, mode of action and basis for efficacy on resistant insects. Pestic. Biochem. Physiol . 107 (2013) 1–7. (a) Han, Z., Krishnamurthy, D., Grover, P., Fang, Q. K. & Senanayake, C. H. Properly designed modular asymmetric synthesis for enantiopure sulfinamide auxiliaries from N-sulfonyl- 1,2,3-oxathiazolidine-2-oxide agents. J. Am. Chem. Soc. 124 , 7880–7881 (2002); (b) Sedelmeier, J., Hammerer, T. & Bolm, C. C1-Symmetric oxazolinyl sulfoximines as ligands in copper-catalyzed asymmetric Mukaiyama aldol reactions. Org. Lett . 10 , 917–920 (2008); (c) Xu, H., Zuend, S. J., Woll, M. G., Tao, Y. & Jacobsen, E. N. Asymmetric cooperative catalysis of strong Brønsted acid-promoted reactions using chiral ureas. Science 327 , 986–990 (2010); M. J. Kendall. Review article: esomeprazole – the first proton pump inhibitor to be developed as an isomer. Aliment Pharmacol. Ther. 17 (Suppl. 1): 1–4 (2003). (a) Liu, G., Cogan, D. A. & Ellman, J. A. Catalytic asymmetric synthesis of tert-butanesulfinamide. Application to the asymmetric synthesis of amines. J. Am. Chem. Soc . 119 , 9913–9914 (1997); (b) Cogan, D. A., Liu, G., Kim, K., Backes, B. J. & Ellman, J. A. Catalytic asymmetric oxidation of tert-butyl disulfide. Synthesis of tert-butanesulfinamides, tert-butyl sulfoxides, and tert-butanesulfinimines. J. Am. Chem. Soc . 120 , 8011–8019 (1998); (c) Robak, M. T., Herbage, M. A. & Ellman, J. A. Synthesis and applications of tert-butanesulfinamide. Chem. Rev. 110 , 3600–3740 (2010); (d) Sun, G., Liu, H., Dong, B., Zhang, Y., Zhao, Z. & Gao, B. Modular synthesis of bis-α-chiral amines using Ellman sulfinamide for consecutive S-to-C chirality induction/transfer. Sci. Adv . 11 , eadv2010 (2025). Mäder, P., Kattner, L. Sulfoximines as Rising Stars in Modern Drug Discovery? Current Status and Perspective on an Emerging Functional Group in Medicinal Chemistry. J. Med. Chem . 63 , 14243–14275 (2020). (a) Zhang, X., Wang, F. & Tan, C.-H. Asymmetric synthesis of (IV) and S(VI) stereogenic centers. JACS Au 3 , 700–714 (2023); (b) Wojaczyńska, E. & Wojaczyński, J. Modern stereoselective synthesis of chiral sulfinyl compounds. Chem. Rev . 120 , 4578–4611 (2020); (c) Evans, J. W., Fierman, M. B., Miller, S. J. & Ellman, J. A. Catalytic enantioselective synthesis of sulfinate esters through the dynamic resolution of tert-butanesulfinyl chloride. J. Am. Chem. Soc . 126 , 8134–8135 (2004); (d) Shibata, N. et al. Cinchona alkaloid/sulfinyl chloride combinations: enantioselective sulfinylating agents of alcohols. J. Am. Chem. Soc . 127 , 1374–1375 (2005); (e) Zhang, X., Ang, E. C. X., Yang, Z., Kee, C. W. & Tan, C.-H. Synthesis of chiral sulfinate esters by asymmetric condensation. Nature 604 , 298–303 (2022); (f) Huang, S. et al. Organocatalytic asymmetric deoxygenation of sulfones to access chiral sulfinyl compounds. Nat. Chem . 15 , 185–193 (2023); (g) Suleman, M., Huang, T. Zhou, T., Chen, Z. & Shi, B. Recent Advances in Asymmetric Synthesis of Chiral-at-Sulfur Sulfoximines. ACS Catal. 15 , 5511–5530 (2025). Wei, T., Wang, H.-L., Tian, Y., Xie, M.-S. & Guo, H.-M. Enantioselective construction of stereogenic-at-sulfur(IV) centres via catalytic acyl transfer sulfinylation. Nat. Chem. 16 , 1310–1311 (2024). Grogan, G. 3.5.2 Oxidation at Sulfur. In Biocatalysis in Organic Synthesis 3 , 1st edition ed.; Science of Synthesis; Georg Thieme Verlag KG, 2015; DOI: 10.1055/sos-SD-216-00175. Goundry, W. R. F., Adams, B., Benson, H., Demeritt, J., McKown, S., Mulholland, K., Robertson, A., Siedlecki, P., Tomlin, P. & Vare, K. Development and Scale-up of a Biocatalytic Process To Form a Chiral Sulfoxide. Org. Process Res. Dev . 21 , 107–113 (2017). Wang, Y., Lan, D., Durrani, R. & Hollmann, F. Peroxygenases en route to becoming dream catalysts. What are the opportunities and challenges? Curr. Opin. Chem. Biol. 37 , 1–9 (2017). Monterrey, D. T., Menés-Rubio, A., Keser, M., Gonzalex-Perez, D. & Alcalde, M. Unspecific peroxygenases: The pot of gold at the end of the oxyfunctionalization rainbow? Curr. Opin. Green. Suss. Chem. 41 , 100786 (2023). Bassanini, I., Ferrandi, E. E., Vanoni, M., Ottolina, G., Riva, S., Crotti, M., Brenna, E. & Monti, D. Peroxygenase-Catalyzed Enantioselective Sulfoxidations. Eur. J. Org. Chem. 2017 , 7186–7189 (2017). Vind, J., Kiemer, L. & Amourgi, E. 2016 WO 2016207373A1. Gröbe, G., Ullrich, R., Pecyna, M. J., Kapturska, D., Friedrich, S., Hofrichter, M. & Scheibner, K. High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula. AMB Express 31 , 1, (2011). Robinson, W. X. Q., Mielke, T., Melling, B., Cuetos, A., Parkin, A., Unsworth, W. P., Cartwright, J., & Grogan, G. Comparing the catalytic and structural characteristics of a ‘short’ unspecific peroxygenase (UPO) expressed in P. pastoris and E. coli. ChemBioChem 24 , e202200558 (2023). (a) Kleymann, G. & Gege, C. Preparation of enantiomers of substituted thiazoles as antiviral compounds. WO Patent 2019/068817 A1 (2019); (b) Brandt, J. & Gais, H.-J. An efficient resolution of (±)-S-methyl-Sphenylsulfoximine with (+)-10-camphorsulfonic acid by the method of half-quantities. Tetrahedron: Asymmetry 8 , 909–912 (1997); (c) Tang, Y. & Miller, S. J. Catalytic Enantioselective Synthesis of Pyridyl Sulfoximines. J. Am. Chem. Soc. 143 , 9230–9235 (2021); (d) Zhang, X., Wang, F. & Tan, C.-H. Asymmetric synthesis of (IV) and S(VI) stereogenic centers. JACS Au 3 , 700–714 (2023); (e) Fan, F.-X., Tang, S.-X., Dang, Y. & Wang, F. Iron-catalysed stereoselective NH transfer enables dynamic kinetic resolution of sulfoxides. Nat. Commun. 16 , 1471 (2025). (a) García-Cárceles, J. et al. 2-(fluoromethoxy)-4′-(s-methanesulfonimidoyl)-1,1′-biphenyl (UCM-1306), an orallybioavailable positive allosteric modulator of the human dopamineD1 receptor for Parkinson’s disease. J. Med. Chem . 65 ,12256–12272 (2022); (b) Zhang, X., Ang, E. C. X., Yang, Z., Kee, C. W. & Tan, C.-H. Synthesis of chiral sulfinate esters. by asymmetric condensation. Nature 604 , 298–303 (2022). (a) Aota, Y., Kano, T. & Maruoka, K. Asymmetric synthesis of chiral sulfoximines via the S-arylation of sulfinamides. J. Am. Chem. Soc. 141 , 19263–19268 (2019); (b) Aota, Y., Kano, T. & Maruoka, K. Asymmetric synthesis of chiral sulfoximines through the S-alkylation of sulfinamides. Angew. Chem. Int. Ed. 58 , 17661–17665 (2019); (c) Maeda, Y. et al. Practical asymmetric synthesis of chiral sulfoximines via sulfur-selective alkylation. J. Org. Chem. 87 , 3652–3660 (2022). (a) Mendonça Matos, P., Lewis, W., Argent, S. P., Moore, J. C. & Stockman, R. A. General method for the asymmetric synthesis of N–H sulfoximines via C–S bond formation. Org. Lett. 22 , 2776–2780 (2020); (b) Greed, S. et al. Synthesis of highly enantioenriched sulfonimidoyl fluorides and sulfonimidamides by stereospecific sulfur–fluorine exchange (SuFEx) reaction. Chem. Eur. J. 26 , 12533–12538 (2020); (c) Greed, S., Symes, O. & Bull, J. A. Stereospecific reaction of sulfonimidoyl fluorides with Grignard reagents for the synthesis of enantioenriched sulfoximines. Chem. Commun. 58 , 5387–5390 (2022); (d) Yang, G.-F. et al. Synthesis of chiral sulfonimidoyl chloride via desymmetrizing enantioselective hydrolysis. J. Am. Chem. Soc. 145 , 5439–5446 (2023); (e) Teng, S., Shultz, Z. P., Shan, C., Wojtas, L. & Lopchuk, J. M. Asymmetric synthesis of sulfoximines, sulfonimidoyl fluorides and sulfonimidamides enabled by an enantiopure bifunctional S(VI) reagent. Nat. Chem. 16 , 183–192 (2024) (a) Johnson, C. R. & Kirchoff, R. A. Oxidation of N-(p-tolylsulfonyl)sulfilimines to N-(p-tolylsulfonyl)sulfoximines with alkaline hydrogen peroxide. J. Org. Chem. 44 , 2280 (1979); (b) Mancheño, O. G. & Bolm, C. Synthesis of N-(1H)-Tetrazole Sulfoximines. Org. Lett. 9 , 2951–2954 (2007); (c) Mancheño, O. G., Bistri, O. & Bolm, C. Iodinane- and Metal-Free Synthesis of N-Cyano Sulfilimines: Novel and Easy Access of NH-Sulfoximines. Org. Lett. 9 , 3809–3811 (2007). Farwell, C. C., McIntosh, J. A., Hyster, T. K., Wang, Z. J. & Arnold, F. H. Enantioselective Imidation of Sulfides via Enzyme-Catalyzed Intermolecular Nitrogen-Atom Transfer. J. Am. Chem. Soc. 136 , 8766–8771 (2014). Wang, F. et al. Synthesis of chiral sulfilimines by organocatalytic enantioselective sulfur alkylation of sulfonamides. Sci. Adv. 10 , eadq2768 (2024). Bonfield, H. E., Mercer, K., Diaz-Rodriguez, A., Cook, G. C., McKay, B. S. J., Slade, P., Taylor, G. M., Ooi, W. X., Williams, J. D., Roberts, J. P. M. et al. The Right Light: De Novo Design of a Robust Modular Photochemical Reactor for Optimum Batch and Flow Chemistry. ChemPhotoChem 4 , 45-51 (2020). Pogrányi, B., Mielke, T. F., Díaz-Rodriguez, A., Cartwright, J., Unsworth, W. P. & Grogan G. Preparative-Scale Biocatalytic Oxygenation of N-Heterocycles with a Lyophilized Peroxygenase Catalyst . Angew. Chem., Int. Ed . 62 , e202214759 (2023). Dong, S., Frings, M., Cheng, H., Wen, J., Zhang, D., Raabe, G. & Bolm, C. Organocatalytic Kinetic Resolution of Sulfoximines J. Am. Chem. Soc . 138 , 2166−2169 (2016). Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. , 31 , 455-461 (2010). Shi, Y., Yuan, Y., Li, J., Yang, J. & Zhang, J. Catalytic Asymmetric Synthesis of Sulfinamides via Cu-Catalyzed Asymmetric Addition of Aryl Boroxines to Sulfinylamines. J. Am. Chem. Soc . 146 , 17580–17586 (2024) Davis, F. A., Zhou, P. & Chen, B,-C. Asymmetric synthesis of amino acids using sulfinimines. Chem. Soc. Rev. 27 , 13–18 (1998); (b) Philip, R. M., Treesa, G. S. S., Saranya, S. & Anilkumar, G. Applications of aryl-sulfinamides in the synthesis of N-heterocycles. RSC Adv. 11 20591–20600 (2021). Vazquez-Chavez, J., Luna-Morales, S., Cruz-Aguilar, D. A., Díaz-Salazar, H., Vallejo Narváez, W. E., Silva-Gutiérrez, R. S., Hernández-Ortega, S., Rocha-Rinza, T. & Hernández-Rodríguez, M . The effect of chiral N-substituents with methyl or trifluoromethyl groups on the catalytic performance of mono- and bifunctional thioureas. Org. Biomol. Chem . 17 , 10045–10051 (2019). Melling, B., Mielke, T., Whitwood, A. C., O’Riordan, T. J. C., Mulholland, N., Cartwright, J., Unsworth, W. P. & Grogan, G. Complementary specificity of unspecific peroxygenases enables access to diverse products from terpene oxygenation. Chem Catalysis , 4 , 100889 (2024). Petrone, D. A., Yoon, H., Weinstabl, H., Lautens, M. Additive Effects in the Palladium-Catalyzed Carboiodination of Chiral N-Allyl Carboxamides. Angew. Chem. Int. Ed. 53 , 7908–7912 (2014). Methods General procedures for used for preparative biosynthetic reactions are as follows. More details, can be found in the Supplementary Information. General procedure for artUPO kinetic resolution of sulfilimines (4) at 0.3 mmol scale (Table 1) Liquid artUPO secretate (1.0 mL, 0.8 U/mL) was added to KPi Buffer (24.0 mL, pH 7.00) at RT and stirred for five min, after which, a solution of sulfilimine (0.300 mmol) in MeCN (6.00 mL) was added. The reaction was initiated by the slow continuous addition of a H 2 O 2 solution (0.180 mmol in 2 mL H 2 O) over 4 h followed by stirring overnight. The reaction was extracted with diethyl ether (3 x 30 mL), and the combined organic phase washed with saturated brine (40 mL), dried over MgSO 4 , filtered and the solvent removed in vacuo. For preparative reactions, the purified products were isolated following flash column chromatography on silica gel. General procedure for r AaeUPO biotransformations with i -Pr N -sulfenylimines (Table 2B) To a round bottom flask containing a magnetic stirring bar was added r Aae UPO-PaDa-I-H (1.3 mL, 57 U/mL) and KPi buffer (10 mL, 0.1 mmol/mL, pH = 7, 10 mL). The solution was diluted by the addition of deionised water (2.7 mL), followed by addition of the appropriate i -Pr N -sulfenylimines (0.2 mmol, 1.0 equiv.,) in MeCN (4 mL). Next, H 2 O 2 solution (2 mL, 0.1 mmol/ml, 1.0 equiv.) was added over a 10 h period, using a syringe pump. After the H 2 O 2 addition was complete, the reaction was then stirred at room temperature for a further 6 h. The reaction mixture was then extracted with ethyl acetate (3 × 20 mL). The combined organic phases were then washed with brine (20 mL), dried over anhydrous MgSO 4 and concentrated in vacuo to give the crude product mixture, which was purified by flash column chromatography on silica to provide the corresponding N -sulfinyl imine product ( R )-12 . General procedure for the artUPO biotransformations with t -Bu N -sulfenylimines (Table 2C) To a round bottom flask containing a magnetic stirring bar was added liquid artUPO secretate (1.0 mL, 0.8 U/mL) and KPi buffer (100 mM, pH = 7, 10 mL). The solution was diluted by the addition of deionised water (3 mL), followed by addition of the appropriate t -Bu N -sulfenylimine 6 (0.2 mmol, 1.0 equiv., final concentration 10 mM) in MeCN (4 mL). Next, 2 mL of a 100 mM H 2 O 2 solution (prepared from 22 µL 30% H 2 O 2 in 2 mL deionised water) was added over a 10 h period, using a syringe pump. After the H 2 O 2 addition was complete, the reaction was then stirred at room temperature for a further 6 h. The reaction mixture was then extracted with ethyl acetate (3 × 20 mL). The combined organic phases were then washed with brine (20 mL), dried over anhydrous MgSO 4 and concentrated in vacuo to give the crude product mixture, which was purified by flash column chromatography on silica gel (to provide the corresponding N -sulfinyl imine product ( S )-13 . Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files Grogan2025SI.pdf Supplementary Information Tables.docx Cite Share Download PDF Status: Published Journal Publication published 12 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7185896","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":503518445,"identity":"c5838c95-5fa1-4527-b19a-8bab4377e5ac","order_by":0,"name":"Gideon Grogan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDklEQVRIiWNgGAWjYDACZgYDJF4FEPNAmAbYVGPRcoYYLShyjG1EaJF3Z9744OMOBrvt7e0XPxfOOyxn3nOA8cMPhsPGuLQYHmYrNpx5hiF5zpkzxdIztx02ljnbwCzZw3DYDKeWZh4zad42hmQJiZwEad5thxNn8DMwSDMwHLbBo8X8N1RL8m/eOWAtzL/xaZFn5jFjBmqxk5BIPybN2wDUwtvABrIFp8MMmNmKJWe2SSRI8Jxhs+Y5lm4swXOwzbLHIB2n9+X7D2/88LHNxl6Cvf3xbZ4aazkJnuTDN35UWBs24LLlAJiSSGxg4AHFRDMQMzbgjUh5qFn2DAzsD4B0HW6lo2AUjIJRMGIBAFi8TVhQbQ31AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1383-7056","institution":"University of York","correspondingAuthor":true,"prefix":"","firstName":"Gideon","middleName":"","lastName":"Grogan","suffix":""},{"id":503518446,"identity":"3601ff6d-bc9d-40f2-8d72-4aebfb4ba737","order_by":1,"name":"Jiacheng Li","email":"","orcid":"","institution":"University of York","correspondingAuthor":false,"prefix":"","firstName":"Jiacheng","middleName":"","lastName":"Li","suffix":""},{"id":503518447,"identity":"a610db37-26d6-4367-8356-240788095807","order_by":2,"name":"Benjamin Melling","email":"","orcid":"","institution":"University of York","correspondingAuthor":false,"prefix":"","firstName":"Benjamin","middleName":"","lastName":"Melling","suffix":""},{"id":503518448,"identity":"ac90cf1f-89c6-4925-ba87-f4eeefc6acbf","order_by":3,"name":"Katy Cornish","email":"","orcid":"","institution":"University of York","correspondingAuthor":false,"prefix":"","firstName":"Katy","middleName":"","lastName":"Cornish","suffix":""},{"id":503518449,"identity":"6a6e1c9f-612e-42c5-a144-9277f98b34e2","order_by":4,"name":"Nicholas Mulholland","email":"","orcid":"","institution":"Syngenta","correspondingAuthor":false,"prefix":"","firstName":"Nicholas","middleName":"","lastName":"Mulholland","suffix":""},{"id":503518450,"identity":"6b6533bd-92eb-4518-bcc4-7af13abe279f","order_by":5,"name":"Jared Cartwright","email":"","orcid":"","institution":"University of York","correspondingAuthor":false,"prefix":"","firstName":"Jared","middleName":"","lastName":"Cartwright","suffix":""},{"id":503518451,"identity":"13ddec42-4573-4730-8b79-41ec5f0e20e1","order_by":6,"name":"William Unsworth","email":"","orcid":"https://orcid.org/0000-0002-9169-5156","institution":"University of York","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"","lastName":"Unsworth","suffix":""}],"badges":[],"createdAt":"2025-07-22 10:33:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7185896/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7185896/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-67405-0","type":"published","date":"2025-12-12T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89624888,"identity":"e3d64aa9-db29-4aef-b249-e214110a9c2e","added_by":"auto","created_at":"2025-08-22 05:37:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":123685,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStereogenic-at-sulfur(IV) compounds and methods for their synthesis.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Examples of stereogenic-at-sulfur(IV) compounds used as pharmaceuticals, agrochemicals and in asymmetric synthesis/catalysis. \u003cstrong\u003eb\u003c/strong\u003e Tian, Xie, Guo and coworkers:\u003csup\u003e8\u003c/sup\u003e enantioselective synthesis of sulfonamides and sulfinate esters using bifunctional 4-arylpyridine \u003cem\u003eN\u003c/em\u003e-oxide organocatalysts. \u003cstrong\u003ec\u003c/strong\u003e Unsworth, Grogan and co-workers:\u003csup\u003e16 \u003c/sup\u003esulfide to sulfoxide oxygenation using a UPO. \u003cstrong\u003ed\u003c/strong\u003e \u003cstrong\u003eThis work\u003c/strong\u003e: enantioselective sulfoximine synthesis \u003cem\u003evia\u003c/em\u003e the UPO catalysed oxygenation of sulfilimines, using a kinetic resolution approach. \u003cstrong\u003ee\u003c/strong\u003e \u003cstrong\u003eThis work\u003c/strong\u003e: stereodivergent, enantioselective synthesis of both sulfinimine enantiomers \u003cem\u003evia \u003c/em\u003ethe UPO catalysed oxygenation of sulfenimines.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7185896/v1/6c370aad0e90f9f20b8bb828.png"},{"id":100661965,"identity":"30b49fc4-9d1c-4a90-b386-7950f9cd4f67","added_by":"auto","created_at":"2026-01-20 08:53:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1187493,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7185896/v1/9d861553-6ee7-4a9c-9889-931455bf25b0.pdf"},{"id":89624895,"identity":"9f1f335f-d8eb-4280-beaa-5a6d4897a63e","added_by":"auto","created_at":"2025-08-22 05:37:32","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16452056,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"Grogan2025SI.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7185896/v1/c73579b0735fb6f5af499395.pdf"},{"id":89624896,"identity":"f213e41c-dcad-49d6-8471-2d6135c08b8c","added_by":"auto","created_at":"2025-08-22 05:37:32","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5635157,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7185896/v1/24f8736e529732f4012baa23.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Asymmetric synthesis of stereogenic-at-sulfur compounds via biocatalytic oxidation with Unspecific Peroxygenases","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEnantioenriched stereogenic-at-sulfur(IV) compounds have myriad important roles, spanning medicinal chemistry,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e agrochemistry\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and asymmetric synthesis/catalysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e For example, the blockbuster gastro-intestinal drug Esomeprazole - prepared as its (\u003cem\u003eS\u003c/em\u003e)-enantiomer \u003cb\u003e1a\u003c/b\u003e \u0026ndash; has improved metabolic stability and inhibits gastric acid production more effectively than Omeprazole, the racemic variant that preceded it.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Both enantiomers of \u003cem\u003etert\u003c/em\u003e-butanesulfinamide \u003cb\u003e1b\u003c/b\u003e (often referred to as Ellman\u0026rsquo;s sulfonamide) are available commercially, and are highly effective chiral auxiliaries used in the asymmetric synthesis of amine derivatives.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Enantiopure sulfur(IV) compounds are also important in asymmetric organocatalysis (\u003cem\u003ee.g.\u003c/em\u003e \u003cb\u003e1c\u003c/b\u003e).\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e In terms of biological applications, sulfoximines are arguably the most important class of sulfur(IV) compound, featuring in several commercial products, such as the insecticide Sulfoxaflor \u003cb\u003e1d\u003c/b\u003e.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e They are also of major current interest in medicinal chemistry (\u003cem\u003ee.g.\u003c/em\u003e anti-cancer drug \u003cb\u003e1e\u003c/b\u003e).\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Sulfoximines often exhibit good pharmacokinetic properties, such as high metabolic stability and solubility, rendering them useful isosteres of sulfones and sulfonamides.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e The additional nitrogen site opens the avenue for H-bonding interactions, and enables easy manipulation of the physiochemical properties through \u003cem\u003eN\u003c/em\u003e-functionalisation reactions.\u003c/p\u003e\u003cp\u003eIncreased interest in chiral sulfur(IV) compounds has helped to propagate a significant upsurge in the development of methods for their synthesis. This includes catalytic asymmetric methods, typically based on kinetic resolution and/or organocatalysis.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e A notable recent approach, developed by Tian, Xie, Guo and coworkers and summarised in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb,\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e is their efficient, enantioselective method for the catalytic asymmetric synthesis of chiral sulfonamides \u003cb\u003e2a\u003c/b\u003e and sulfinate esters \u003cb\u003e2b\u003c/b\u003e, enabled by bifunctional 4-arylpyridine \u003cem\u003eN\u003c/em\u003e-oxide organocatalysts.\u003c/p\u003e\u003cp\u003eBiocatalytic methods to prepare stereogenic-at-sulfur(IV) are far less well-developed in comparison, with methods based on oxygenation limited to the conversion of sulfides to form sulfoxides.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e A range of oxygenase enzymes, including hemoproteins such as cytochromes P450 and flavoprotein oxygenases, such as Baeyer-Villiger Monooxygenases (BVMOs), has been shown to catalyze this transformation, with one example reported to work on a kg scale for the production of a pharmaceutical intermediate by AstraZeneca.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Among hemoprotein biocatalysts, the conversion of simple sulfides to sulfoxides has also been reported for Unspecific Peroxygenases UPOs,\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e which display advantages over P450s and BVMOs in that they are nicotinamide cofactor independent and require only the addition of hydrogen peroxide to promote their oxygenation reactions. The UPO from \u003cem\u003eAgrocybe aegerita\u003c/em\u003e (\u003cem\u003eAae\u003c/em\u003eUPO) had previously been shown to convert substrates such as phenyl methyl sulfide into its (\u003cem\u003eR\u003c/em\u003e)-sulfoxide product with 80% \u003cem\u003eee.\u003c/em\u003e\u003csup\u003e13\u003c/sup\u003e UPOs have been classified into two broad subclasses, Class I and Class II, based on characteristics including sequence, structure and molecular weight. Class I UPOs are smaller, with MW of around 29 kDa and Class II UPOs are larger enzymes, with MWs of around 44 kDa. In previous work, we have shown that a Class I UPO, an engineered \u0026lsquo;artificial peroxygenase\u0026rsquo;\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e (artUPO), which is related to the enzyme from \u003cem\u003eMarasmius rotula\u003c/em\u003e,\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e delivers sulfoxide products in the complementary enantiomeric series to the Class II \u003cem\u003eAae\u003c/em\u003eUPO.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e This diversity clearly has great potential for exploitation with respect to the oxidation of more complex substrates, such as the S(IV) oxidation reactions that are the subject of this study.\u003c/p\u003e\u003cp\u003eIn this manuscript, we describe novel methods for the biocatalytic oxygenation of two distinct families of sulfur precursors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and e). These methods allow the enantioselective synthesis of sulfoximines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) and sulfinimines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) on preparative scale, using two easy-to-handle UPO enzymes. The sulfoximine-forming series relies on a UPO-mediated sulfilimine kinetic resolution, while in the sulfinimine-forming series (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), a remarkable enantiodivergence is revealed, whereby different UPOs are able to selectively deliver either enantiomer (\u003cb\u003e(\u003c/b\u003e\u003cb\u003eS\u003c/b\u003e\u003cb\u003e)-\u003c/b\u003e or \u003cb\u003e(\u003c/b\u003e\u003cb\u003eR\u003c/b\u003e\u003cb\u003e)-7\u003c/b\u003e) of the sulfilimine product, \u003cem\u003evia\u003c/em\u003e UPO catalysed sulfenimine oxygenation. Both approaches enable the biocatalytic formation of important stereogenic-at-sulfur(IV) compounds to be prepared in high \u003cem\u003eee\u003c/em\u003e on preparative scale. These approaches all represent completely novel biocatalytic transformations, and further expand the range and utility of UPOs to perform useful and selective preparative biocatalytic oxygenation reactions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eGiven their importance in medicinal chemistry, we started by exploring the potential of UPOs to generate enantioenriched sulfoximines. Various powerful chemical methods for the asymmetric synthesis of sulfoximines and their derivatives have been developed in recent years; these include methods based on the chromatographic separation and resolutions of racemic sulfoximines,\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e the oxidative imination of sulfoxides,\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e electrophilic addition reactions to \u003cem\u003eS\u003c/em\u003e-nucleophiles \u003csup\u003e17c,19\u003c/sup\u003e and nucleophilic addition reactions to \u003cem\u003eS\u003c/em\u003e-electrophiles.\u003csup\u003e17c,20\u003c/sup\u003e An alternative approach for sulfoximine synthesis is \u003cem\u003evia\u003c/em\u003e the oxidation of a sulfilimine (\u003cstrong\u003e4\u003c/strong\u003e \u0026rarr; \u003cstrong\u003e5\u003c/strong\u003e). Methods for the oxidation of sulfilimines (also known as sulfimides) to form racemic sulfoximines have been known for decades, typically using simple peroxide based chemical oxidants.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e However, to the best of our knowledge, all published syntheses of enantioenriched sulfoximines from sulfilimines start from enantioenriched sulfilimine precursors; as sulfilimines are themselves chiral and configurationally stable, stereochemical information in the precursor is retained following oxidation.\u003csup\u003e7a\u003c/sup\u003e Methods for the preparation of enantiomerically enriched sulfilimines are known, but are rare, with notable exceptions being a biocatalytic (cytochrome P450\u003csub\u003eBM3\u003c/sub\u003e) approach from Arnold and co-workers,\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and a recently reported organocatalytic method by Zhang, Zhang and coworkers.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eIn recognition of the well-established ability of UPOs to catalyse enantioselective oxygenation reactions, we questioned whether they might be capable of forming enantioenriched sulfoximines \u003cem\u003evia\u003c/em\u003e the kinetic resolution of easy-to-prepare racemic sulfilimines. To the best of our knowledge, no asymmetric sulfilimine to sulfoximine transformations are known that start from racemic starting materials. Furthermore, we also know of no biocatalytic methods (neither racemic nor asymmetric) for the oxidation of sulfilimines, using UPOs or indeed any other enzyme class.\u003c/p\u003e\n\u003cp\u003eIn view of their stability and ease of synthesis, \u003cem\u003eN\u003c/em\u003e-cyano sulfilimines of the type \u003cstrong\u003erac-4\u003c/strong\u003e were selected for this study, with a view towards developing biocatalytic kinetic resolution transformations of the type summarised in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e (box, \u003cstrong\u003erac-4\u003c/strong\u003e \u0026rarr; \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-4\u003c/strong\u003e and \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-5\u003c/strong\u003e). We started by exploring the kinetic resolution of the simple \u003cem\u003eN\u003c/em\u003e-cyano phenyl methyl sulfilimine \u003cstrong\u003erac-4a\u003c/strong\u003e, which was prepared \u003cem\u003evia\u003c/em\u003e a straightforward oxidative procedure from phenyl methyl sulfide.\u003csup\u003e21c\u003c/sup\u003e All other racemic sulfilimine precursors used throughout this study were made using similar methods (see Supplementary Information Section 2 for full preparative details and characterisation data). Our aim was to develop a kinetic resolution of racemic sulfilimine starting material \u003cstrong\u003erac-4a\u003c/strong\u003e and to isolate the sulfoximine product \u003cstrong\u003e5a\u003c/strong\u003e, along with unreacted sulfilimine \u003cstrong\u003e4a\u003c/strong\u003e, both in enantioenriched form.\u003c/p\u003e\n\u003cp\u003eThe biotransformation of \u003cstrong\u003erac-4a\u003c/strong\u003e was therefore tested conducted on a preparative (0.3 mmol) scale using artUPO and the conditions summarised in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; the use of 0.6 equivalents of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was chosen to facilitate approximately 50% conversion of \u003cstrong\u003erac-4\u003c/strong\u003e, as needed for effective kinetic resolution. Encouragingly, under these conditions kinetic resolution was achieved, with enantioenriched sulfoximine \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003e5a\u003c/strong\u003e and sulfilimine \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-4\u003c/strong\u003e isolated in 22% and 35% yields, and 74% and 32% \u003cem\u003eee\u003c/em\u003e respectively following column chromatography. The absolute stereochemistry of the major enantiomer \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003e5a\u003c/strong\u003e was confirmed by comparison to literature optical rotation data;\u003csup\u003e21b\u003c/sup\u003e therefore, the absolute stereochemistry of the major enantiomer of the unreacted sulfilimine is logically assumed to be \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-4\u003c/strong\u003e. Note that while products \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003e5a\u003c/strong\u003e and sulfilimine \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-4\u003c/strong\u003e both have (\u003cem\u003eS\u003c/em\u003e)-stereochemical assignments, this merely reflects the change in Cahn\u0026ndash;Ingold\u0026ndash;Prelog priorities, and they have opposite sense of absolute stereochemistry as expected. The assignment of absolute stereochemistry of the other related products in this manuscript was made by analogy, supported by optical rotation and HPLC data (see Supplementary Information, Section 3 and 5). r\u003cem\u003eAae\u003c/em\u003eUPO-PaDa-I-H\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e was also tested as an alternative UPO for this transformation but was not pursued, as it led to much poorer conversion when tested on a selection of sulfilimines under the same conditions (see Supplementary Information, Section 2.6).\u003c/p\u003e\n\u003cp\u003eHaving established that the proposed UPO mediated kinetic resolution is viable, attention turned to exploring the scope of the artUPO biotransformation with other sulfilimine substrates (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;C). All biotransformations were performed on preparative scale, with the yields quoted referring to purified product following column chromatography. The \u003cem\u003eee\u003c/em\u003es of the product sulfoximines \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-5\u003c/strong\u003e were measured using chiral HPLC analysis of the isolated product; the \u003cem\u003eee\u003c/em\u003es of the enantioenriched sulfilimines \u003cstrong\u003e(S)-4\u003c/strong\u003e were also measured by chiral HPLC, in some cases from the sulfilimines directly, and on others following \u003cem\u003em\u003c/em\u003e-CPBA oxidation to the corresponding sulfoximines when this expedited analysis (see Supplementary Information, General Procedure 2.3).\u003c/p\u003e\n\u003cp\u003eWe started by exploring substrates similar to \u003cstrong\u003erac-4a\u003c/strong\u003e with different substituents on the phenyl ring (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Ten substrates of this type were tested, with successful kinetic resolution achieved and enantioenriched sulfoximines (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-5a\u003c/strong\u003e\u0026ndash;\u003cstrong\u003ek\u003c/strong\u003e) and sulfilimines (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-4a\u003c/strong\u003e\u0026ndash;\u003cstrong\u003ek\u003c/strong\u003e) isolated in all cases. Electron-rich and -poor substituents were both well tolerated. Substrates featuring a \u003cem\u003epara-\u003c/em\u003etrifluoromethyl substituent (\u003cstrong\u003erac-4e\u003c/strong\u003e and \u003cstrong\u003erac-4j\u003c/strong\u003e) worked especially well, with very efficient kinetic resolution achieved, with \u0026gt;\u0026thinsp;90% overall yield and \u0026gt;\u0026thinsp;95% \u003cem\u003eee\u003c/em\u003e obtained for the isolated sulfoximines (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-5e\u003c/strong\u003e, \u003cstrong\u003e5j\u003c/strong\u003e) and enriched sulfilimines (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-4e, 4j\u003c/strong\u003e). Three pyridine-containing substrates (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB) were also transformed successfully, with good to high \u003cem\u003eee\u003c/em\u003es for the sulfoximine products (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-5k\u003c/strong\u003e\u0026ndash;\u003cstrong\u003em\u003c/strong\u003e) obtained in this series. It is notable that the protocol can be applied to aza-heterocyclic systems given their prominence in medicinal chemistry, while avoiding the proclivity of pyridines to undergo oxidation to form \u003cem\u003eN-\u003c/em\u003eoxides with UPOs.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC summarises results using racemic sulfilimine starting materials with non-methyl alkyl groups (\u003cstrong\u003erac-4n\u003c/strong\u003e\u0026ndash;\u003cstrong\u003eu\u003c/strong\u003e). The resolution of substrate \u003cstrong\u003erac-4s\u003c/strong\u003e is notable, as this shows that the biotransformation is not contingent on the sulfilimine having an aromatic substituent. As before, the most effective examples in this series were those containing electron-poor aromatic groups, with sulfilimines \u003cstrong\u003erac-4t\u003c/strong\u003e and \u003cstrong\u003erac-4u\u003c/strong\u003e affording products in 98% overall yield, and with \u003cem\u003eee\u003c/em\u003es of 90% and 99% respectively for sulfoximines \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-5t\u003c/strong\u003e and \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-5u\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eExcept for selected cases (those highlighted with a blue background) the biotransformations were all performed on 0.3 mmol scale using the same conditions, without optimisation and on a substrate-by-substrate basis. Of course, for any kinetic resolution, it is unlikely that the same reaction conditions will be optimal across all substrates. To demonstrate how additional optimisation can lead to improved results, and to showcase the scalability of the method, the biotransformations of \u003cstrong\u003erac-4b\u003c/strong\u003e, \u003cstrong\u003erac-4e\u003c/strong\u003e, \u003cstrong\u003erac-4j\u003c/strong\u003e, \u003cstrong\u003erac-4t\u003c/strong\u003e and \u003cstrong\u003erac-4u\u003c/strong\u003e were performed on larger (1.0 mmol) scale, leading to the improved results presented (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC, examples highlighted with a blue background). The increase in scale was useful in facilitating more precise control in reaction conversion compared with the standard method; careful monitoring of conversion and the use of additional H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e when needed, proved to be effective in achieving the \u0026asymp;\u0026thinsp;50% conversion needed for optimal kinetic resolution (see Supplementary Information, General Procedure 2.5). The resolution of substrate \u003cstrong\u003erac-4j\u003c/strong\u003e (98% and \u0026gt;\u0026thinsp;99% \u003cem\u003eee\u003c/em\u003e, s\u0026thinsp;\u0026gt;\u0026thinsp;200) best illustrates the power of this approach.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eN\u003c/em\u003e-Cyano sulfilimines were chosen as starting materials for this study primarily in view of their synthetic tractability. However, if the unfunctionalised sulfoximine products are required, either directly or for further derivatisation, these can easily be accessed using the method summarised in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Thus, reaction of \u003cem\u003eN\u003c/em\u003e-cyano sulfoximine \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-5e\u003c/strong\u003e with trifluoroacetic anhydride followed by potassium carbonate in methanol affords sulfoximine \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-9e\u003c/strong\u003e with no erosion in \u003cem\u003eee\u003c/em\u003e. Furthermore, if the enantiomeric (\u003cem\u003eR\u003c/em\u003e)-sulfoximine is required, this can be obtained \u003cem\u003evia\u003c/em\u003e simple oxidation of the enantiomerically enriched (\u003cem\u003eS\u003c/em\u003e)-sulfilimine; for example, the reaction of sulfilimine \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-4e\u003c/strong\u003e with \u003cem\u003em\u003c/em\u003e-CPBA gave sulfoximine \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-5e\u003c/strong\u003e in good yield, and with no erosion in \u003cem\u003eee\u003c/em\u003e. Note that the oxidation of \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-4e\u003c/strong\u003e into \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-5e\u003c/strong\u003e is stereo-retentive, and the change from an (\u003cem\u003eS\u003c/em\u003e)- to (\u003cem\u003eR\u003c/em\u003e)-stereochemical assignment merely reflects the change in Cahn\u0026ndash;Ingold\u0026ndash;Prelog priorities.\u003c/p\u003e\n\u003cp\u003eThe enantiopreference of artUPO for \u003cem\u003eN-\u003c/em\u003ecyano sulfilimines was investigated through docking of the favoured (\u003cem\u003eR\u003c/em\u003e)-enantiomer of \u003cstrong\u003e4e\u003c/strong\u003e into our previously obtained structure of artUPO (PDB 7ZNM)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e using Autodock VINA.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e The lowest energy pose obtained positions the sulphur lone pair of the \u003cem\u003epro\u003c/em\u003e-(\u003cem\u003eS\u003c/em\u003e) face of (\u003cem\u003eR\u003c/em\u003e)-\u003cstrong\u003e4e\u003c/strong\u003e ideally for receiving oxygen from the oxygenating species Compound I (CpdI) with the aromatic group bound in a hydrophobic pocket formed by L65, V69, I91, I160 and I235 (\u003cstrong\u003eFigure S69A\u003c/strong\u003e) and the \u003cem\u003eN\u003c/em\u003e-cyano group positioned between the side chains of I62, A66 and F167. A similar pose for the unflavoured (\u003cem\u003eS\u003c/em\u003e)- enantiomer of \u003cstrong\u003e4e\u003c/strong\u003e would bring the cyano group into close contact with the side chains of I160, L163 and E164, thus providing a plausible structural explanation for the experimentally observed enantioselectivity.\u003c/p\u003e\n\u003cp\u003eAfter establishing artUPO as a selective biocatalyst for the kinetic resolution of sulfilimines \u003cstrong\u003e4\u003c/strong\u003e, attention then turned to the exploration of UPOs for the enantioselective synthesis of sulfinimines. Sulfinimines (also known as \u003cem\u003eN-\u003c/em\u003esulfinylimines) are extremely useful synthetic intermediates owing to their ability to undergo a range of diastereoselective transformations, and hence have been widely used to prepare chiral amine derivatives.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e When enantiomerically enriched sulfinimines are used in organic synthesis, most commonly they are prepared \u003cem\u003evia\u003c/em\u003e the condensation of an enantiomerically enriched sulfonamide (often Ellman\u0026rsquo;s sulfonamide auxiliary \u003cstrong\u003e1b\u003c/strong\u003e, or the \u003cem\u003ep\u003c/em\u003e-tolyl derivative popularised by Davis and coworkers\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e) with an aldehyde or ketone. In this work, we explored an alternative approach to access enantiomerically enriched sulfinimines, \u003cem\u003evia\u003c/em\u003e the UPO catalysed oxidation of sulfenimines \u003cstrong\u003e6\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eRepresentatives of both Class I and Class II UPOs were tested for this transformation, revealing intriguing stereodivergent reaction profiles for the different UPO classes. First, Class II r\u003cem\u003eAae\u003c/em\u003eUPO-PaDa-I-H was challenged with a selection of seven different phenyl sulfenimines with different \u003cem\u003eS\u003c/em\u003e-alkyl/aryl substituents (to form \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-7a\u003c/strong\u003e\u0026ndash;\u003cstrong\u003e13a\u003c/strong\u003e, Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Box 1). The reactions were performed on preparative scale (0.2 mmol of \u003cstrong\u003e6\u003c/strong\u003e) in pH 7 KPi buffer with acetonitrile as co-solvent and using 1 equivalent of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (added slowly over 10 h) as the stoichiometric oxidant. Conversion was measured by comparing the amounts of \u003cstrong\u003e6\u003c/strong\u003e and product in the \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectra of the crude reaction mixture, and \u003cem\u003eee\u003c/em\u003e was measured using chiral HPLC (see Supplementary Information, Section 5). In all but one case, some conversion into the expected enantiomerically enriched sulfinimine was observed and the depicted (\u003cem\u003eR\u003c/em\u003e)-enantiomer was formed in excess; the assignment of absolute stereochemistry made by comparison to literature optical rotation data for \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-12a\u003c/strong\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e (corroborated by several other substrates in the series featured later in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, see Supplementary Information Section 3). The most successful example was the oxygenation of the \u003cem\u003ei\u003c/em\u003e-Pr-substituted sulfenimine, which was converted into sulfinimine \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e(\u003c/span\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-12a\u003c/strong\u003e with 89% conversion and in 98% \u003cem\u003eee\u003c/em\u003e. The only substrate in this series that did not undergo oxygenation was the \u003cem\u003etert-\u003c/em\u003ebutyl substituted sulfenimine, where no conversion into \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-13a\u003c/strong\u003e was observed. This result was not wholly surprising, given that r\u003cem\u003eAae\u003c/em\u003eUPO-PaDa-I-H tends to perform poorly in the biotransformations of more sterically demanding substrates.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eIn contrast, artUPO, the same Class I UPO employed in the first half of this manuscript, typically performs better than r\u003cem\u003eAae\u003c/em\u003eUPO-PaDa-I-H with bulky substrates, owing to its more accessible active site. Thus, four sulfenimines were tested using artUPO in the same way (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Box 2). All four substrates were converted well, included the bulky \u003cem\u003etert-\u003c/em\u003ebutyl substituted sulfenimine, with full conversion into \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-13a\u003c/strong\u003e observed. For the smaller substrates, the products \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-7a\u003c/strong\u003e and \u003cstrong\u003erac-9a\u003c/strong\u003e were formed with little or no \u003cem\u003eee\u003c/em\u003e respectively; again, this was not wholly surprising, as the ability to transform bulkier substrates using artUPO is often offset against reduced enantioselectivity. However, for the bulkier substrates, enantioselectivity was much higher, with products \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-12a\u003c/strong\u003e and \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-13a\u003c/strong\u003e formed in 80% and 92% \u003cem\u003eee\u003c/em\u003e respectively. Remarkably, the opposite (\u003cem\u003eS\u003c/em\u003e)-enantiomer was formed in excess in this series, thus offering complementary enantioselectivity to that of r\u003cem\u003eAae\u003c/em\u003eUPO-PaDa-I-H. The assignment of absolute stereochemistry was made by comparison to literature optical rotation data for \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-13a\u003c/strong\u003e,\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and corroborated in several cases in the series in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC (see Supplementary Information Section 3). The complementary enantioselectivity accords with previous preliminary observations made for r\u003cem\u003eAae\u003c/em\u003eUPO-PaDa-I-H and artUPO for the oxidation of sulfides.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e In the case of sulfenimines such as \u003cstrong\u003e6\u003c/strong\u003e, this complementarity appears to result from different substrate approach trajectories to the oxidising species CpdI, in the active sites of the enzymes, as revealed by docking \u003cstrong\u003e6\u003c/strong\u003e (where R\u0026thinsp;=\u0026thinsp;\u003cem\u003ei\u003c/em\u003e-Pr) into the enzymes, again using Autodock VINA (see Supplementary Information, Figure S69B).\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Hence in the case of artUPO, the \u003cem\u003epro\u003c/em\u003e-(\u003cem\u003eS\u003c/em\u003e) lone pair is presented to the CpdI oxygen as the phenyl ring rests in the hydrophobic pocket formed by L65, V69, I91, I160 and I235 previously described. As the equivalent pocket in r\u003cem\u003eAae\u003c/em\u003eUPO-PaDa-I-H is restricted by phenylalanine residues including F188, the substrate approaches CpdI through a more available and less constrained tunnel and thus presents the \u003cem\u003epro\u003c/em\u003e-(\u003cem\u003eR\u003c/em\u003e)-lone pair to the oxidant.\u003c/p\u003e\n\u003cp\u003eThe best performing cases with each enzyme (\u003cem\u003ei\u003c/em\u003e-Pr and \u003cem\u003et\u003c/em\u003e-Bu derivatives, leading to the formation of \u003cstrong\u003e(R\u003c/strong\u003e\u003cstrong\u003e)-12a\u003c/strong\u003e and \u003cstrong\u003e(S\u003c/strong\u003e\u003cstrong\u003e)-13a\u003c/strong\u003e) are arguably amongst the most synthetically useful systems, considering the established utility of \u003cem\u003ei\u003c/em\u003e-Pr and \u003cem\u003et\u003c/em\u003e-Bu sulfinimines in asymmetric synthesis.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Therefore, we next examined the scope of these biotransformations on other \u003cem\u003eiso\u003c/em\u003e-propyl- and \u003cem\u003etert\u003c/em\u003e-butyl-substituted sulfenimines. Results for the enantioselective synthesis of \u003cem\u003eiso\u003c/em\u003e-propyl substituted (\u003cem\u003eR\u003c/em\u003e)-sulfinimines \u003cstrong\u003e7\u003c/strong\u003e using r\u003cem\u003eAae\u003c/em\u003eUPO-PaDa-I-H are summarised in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB. As in the initial screening, the reactions were performed on preparative scale (0.2 mmol of \u003cstrong\u003e6\u003c/strong\u003e) in pH 7 KPi buffer with acetonitrile as co-solvent and using 1 equivalent of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (added slowly over 10 h) as the stoichiometric oxidant. The yields quoted refer to isolated yields of purified products following column chromatography, and \u003cem\u003eee\u003c/em\u003e was measured by chiral HPLC. All twelve examples proceeded with excellent enantioselectivity (90\u0026ndash;99% \u003cem\u003eee\u003c/em\u003e), across a range of substituted aromatic substrates (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-12a\u003c/strong\u003e\u0026ndash;\u003cstrong\u003ei\u003c/strong\u003e). Naphthyl (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-12h\u003c/strong\u003e, \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-12j\u003c/strong\u003e), ketimine (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-12i\u003c/strong\u003e\u0026ndash;\u003cstrong\u003ek\u003c/strong\u003e), cyclic (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-12k\u003c/strong\u003e) and pyridyl (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-12l\u003c/strong\u003e) substrates also worked well.\u003c/p\u003e\n\u003cp\u003eThe opposite enantiomeric series is depicted in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC, in which results for the formation of \u003cem\u003etert\u003c/em\u003e-butyl substituted (\u003cem\u003eS\u003c/em\u003e)-sulfinimines \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-13\u003c/strong\u003e using artUPO are summarised. Enantioselectivity was again high, with the (\u003cem\u003eS\u003c/em\u003e)-enantiomer formed in \u0026gt;\u0026thinsp;90% \u003cem\u003eee\u003c/em\u003e in most examples tested. As before, a range of substituted aromatic substrates were tested and all worked well (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-13a\u003c/strong\u003e\u0026ndash;\u003cstrong\u003eg\u003c/strong\u003e). Naphthyl (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-13h\u003c/strong\u003e), ketimine (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-13i\u003c/strong\u003e), cyclic (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-13j\u003c/strong\u003e) and heterocyclic (\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-13l\u003c/strong\u003e\u0026ndash;\u003cstrong\u003em\u003c/strong\u003e) substrates were also well tolerated. The poor conversion (10% yield) for adamantyl derivative \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-13n\u003c/strong\u003e appears to show the limits of enzyme with respect to steric bulk of the substrate, although notably the enantioselectivity remained relatively high (80% \u003cem\u003eee\u003c/em\u003e). In both series, attempts to test simple alkyl substituted sulfenimines (\u003cem\u003ee.g.\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e where neither R\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e nor R\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e is aromatic) were thwarted by the instability of the requisite starting materials, meaning the biotransformations were not tested. The method is therefore limited to aromatic and cyclic aliphatic sulfenimines systems, that can be prepared and handled easily.\u003c/p\u003e\n\u003cp\u003eTo showcase the utility of the method of the enantiomerically enriched sulfinimines accessible using this method, two relatively simple syntheses of secondary amines used on the synthesis of pharmaceuticals are summarised in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD. Starting from sulfinimine \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003e)-12j\u003c/strong\u003e (prepared in 99% \u003cem\u003eee\u003c/em\u003e using r\u003cem\u003eAae\u003c/em\u003eUPO-PaDa-I-H), DIBAL reduction, followed by hydrolysis delivered a single enantiomer of amine \u003cstrong\u003e14\u003c/strong\u003e. Alkylation with iodide \u003cstrong\u003e15\u003c/strong\u003e then delivered hyperparathyroidism drug Cincalcet \u003cstrong\u003e16\u003c/strong\u003e. Similarly, in the opposite series, sulfinimine \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e)-13j\u003c/strong\u003e was prepared in 98% \u003cem\u003eee\u003c/em\u003e; in this case, the reaction was performed on 3 mmol scale and delivered 400 mg of the enantiomerically enriched product, in almost identical yield to the smaller scale version. A highly diastereoselective reduction and hydrolysis followed to deliver amine \u003cstrong\u003e18\u003c/strong\u003e in 99% \u003cem\u003eee\u003c/em\u003e, with this amine a key precursor to the obstructive hypertrophic cardiomyopathy drug Mavacamten \u003cstrong\u003e19\u003c/strong\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, two novel, preparative biocatalytic approaches for the high yielding and enantioselective oxygenation of stereogenic-at-sulfur(IV) compounds, have been developed and described for the first time. To the best of our knowledge, both approaches had no known biocatalytic variants, using any enzyme, prior to this manuscript. Selective oxidation reactions in organic chemistry remain a challenge, especially where enantioselective transformations are concerned. Whereas cofactor-dependent enzymes present problems with stability, turnover and expense, UPOs offer real potential for scalable asymmetric oxidations using simple procedures. To facilitate their wider uptake, it is crucial that examples of scalable reactions on useful molecules are investigated and presented. With the transformations to form stereogenic-at-sulfur(IV)functionalities presented in this report, we add another highly promising application of UPO technology to a growing list of reactions that have significant potential for scale-up in an industrial context.\u003c/p\u003e"},{"header":"Online content","content":"\u003cp\u003eAny methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data availability are available at [insert link at publication].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data, including experimental procedures, compound characterization data and HPLC data, are available within the article and its Supplementary Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the U.K. Engineering and Physical Sciences Research Council (EPSRC, EP/X014886/1, J.L. and K.A.S.C.) for funding, and the EPSRC and Syngenta for the award of a PhD studentship to B.M. (project 2602946).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.P.U and G.G. designed the study. J. L., B. M. and K.A.S.C. performed the experiments and interpreted the results. J. C. led on all aspects relating to enzyme production. N. M. provided industrial advice and additional guidance (to B. M.). W.P.U and G.G. prepared the manuscript and for publication, supported by J. L. and B. M.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Information. The online version contains supplementary information available at xxxxxxxxxxxxxxxx\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e(a) Bentley, R. Role of sulfur chirality in the chemical processes of biology. \u003cem\u003eChem. Soc. Rev\u003c/em\u003e. \u003cstrong\u003e34\u003c/strong\u003e, 609\u0026ndash;624 (2005); (b) M\u0026auml;der, P. \u0026amp; Kattner, L. Sulfoximines as rising stars in modern drug discovery? Current status and perspective on an emerging functional group in medicinal chemistry. \u003cem\u003eJ. Med. Chem\u003c/em\u003e. \u003cstrong\u003e63\u003c/strong\u003e, 14243\u0026ndash;14275 (2020); (c) L\u0026uuml;cking, U. Neglected sulfur(VI) pharmacophores in drug discovery: exploration of novel chemical space by the interplay of drug design and method development. \u003cem\u003eOrg. Chem. Front\u003c/em\u003e. \u003cstrong\u003e6\u003c/strong\u003e, 1319\u0026ndash;1324 (2019); (d) Han, Y. et al. Application of sulfoximines in medicinal chemistry from 2013 to 2020. \u003cem\u003eEur. J. Med. Chem\u003c/em\u003e. \u003cstrong\u003e209\u003c/strong\u003e, 112885 (2021); (e) Trost, B. M., Ryan, M. C., Rao, M. \u0026amp; Markovic, T. Z. Construction of enantioenriched [3.1.0] bicycles via a ruthenium-catalyzed asymmetric redox bicycloisomerization reaction. \u003cem\u003eJ. Am. Chem. Soc\u003c/em\u003e. \u003cstrong\u003e136\u003c/strong\u003e, 17422\u0026ndash;17425 (2014). (f) Trost, B. M. \u0026amp; Rao, M. Development of chiral sulfoxide ligands for asymmetric catalysis. \u003cem\u003eAngew. Chem. Int. Ed\u003c/em\u003e. \u003cstrong\u003e54\u003c/strong\u003e, 5026\u0026ndash;5043 (2015).\u003c/li\u003e\n\u003cli\u003eT. C. Sparks et al. Sulfoxaflor and the sulfoximine insecticides: Chemistry, mode of action and basis for efficacy on resistant insects. \u003cem\u003ePestic. Biochem. Physiol\u003c/em\u003e. \u003cstrong\u003e107\u003c/strong\u003e (2013) 1\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003e(a) Han, Z., Krishnamurthy, D., Grover, P., Fang, Q. K. \u0026amp; Senanayake, C. H. Properly designed modular asymmetric synthesis for enantiopure sulfinamide auxiliaries from N-sulfonyl- 1,2,3-oxathiazolidine-2-oxide agents. \u003cem\u003eJ. Am. Chem. Soc. \u003c/em\u003e\u003cstrong\u003e124\u003c/strong\u003e, 7880\u0026ndash;7881 (2002); (b) Sedelmeier, J., Hammerer, T. \u0026amp; Bolm, C. C1-Symmetric oxazolinyl sulfoximines as ligands in copper-catalyzed asymmetric Mukaiyama aldol reactions. \u003cem\u003eOrg. Lett\u003c/em\u003e. \u003cstrong\u003e10\u003c/strong\u003e, 917\u0026ndash;920 (2008); (c) Xu, H., Zuend, S. J., Woll, M. G., Tao, Y. \u0026amp; Jacobsen, E. N. Asymmetric cooperative catalysis of strong Br\u0026oslash;nsted acid-promoted reactions using chiral ureas. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e327\u003c/strong\u003e, 986\u0026ndash;990 (2010);\u003c/li\u003e\n\u003cli\u003eM. J. Kendall. Review article: esomeprazole \u0026ndash; the first proton pump inhibitor to be developed as an isomer. \u003cem\u003eAliment Pharmacol. Ther. \u003c/em\u003e\u003cstrong\u003e17\u003c/strong\u003e (Suppl. 1): 1\u0026ndash;4 (2003).\u003c/li\u003e\n\u003cli\u003e(a) Liu, G., Cogan, D. A. \u0026amp; Ellman, J. A. Catalytic asymmetric synthesis of tert-butanesulfinamide. Application to the asymmetric synthesis of amines. \u003cem\u003eJ. Am. Chem. Soc\u003c/em\u003e. \u003cstrong\u003e119\u003c/strong\u003e, 9913\u0026ndash;9914 (1997); (b) Cogan, D. A., Liu, G., Kim, K., Backes, B. J. \u0026amp; Ellman, J. A. Catalytic asymmetric oxidation of tert-butyl disulfide. Synthesis of tert-butanesulfinamides, tert-butyl sulfoxides, and tert-butanesulfinimines. \u003cem\u003eJ. Am. Chem. Soc\u003c/em\u003e. \u003cstrong\u003e120\u003c/strong\u003e, 8011\u0026ndash;8019 (1998); (c) Robak, M. T., Herbage, M. A. \u0026amp; Ellman, J. A. Synthesis and applications of tert-butanesulfinamide. \u003cem\u003eChem. Rev.\u003c/em\u003e \u003cstrong\u003e110\u003c/strong\u003e, 3600\u0026ndash;3740 (2010); (d) Sun, G., Liu, H., Dong, B., Zhang, Y., Zhao, Z. \u0026amp; Gao, B. Modular synthesis of bis-\u0026alpha;-chiral amines using Ellman sulfinamide for consecutive S-to-C chirality induction/transfer. \u003cem\u003eSci. Adv\u003c/em\u003e. \u003cstrong\u003e11\u003c/strong\u003e, eadv2010 (2025). \u003c/li\u003e\n\u003cli\u003eM\u0026auml;der, P., Kattner, L. Sulfoximines as Rising Stars in Modern Drug Discovery? Current Status and Perspective on an Emerging Functional Group in Medicinal Chemistry. \u003cem\u003eJ. Med. Chem\u003c/em\u003e. \u003cstrong\u003e63\u003c/strong\u003e, 14243\u0026ndash;14275 (2020).\u003c/li\u003e\n\u003cli\u003e(a) Zhang, X., Wang, F. \u0026amp; Tan, C.-H. Asymmetric synthesis of (IV) and S(VI) stereogenic centers. \u003cem\u003eJACS Au\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 700\u0026ndash;714 (2023); (b) Wojaczyńska, E. \u0026amp; Wojaczyński, J. Modern stereoselective synthesis of chiral sulfinyl compounds. \u003cem\u003eChem. Rev\u003c/em\u003e. \u003cstrong\u003e120\u003c/strong\u003e, 4578\u0026ndash;4611 (2020); (c) Evans, J. W., Fierman, M. B., Miller, S. J. \u0026amp; Ellman, J. A. Catalytic enantioselective synthesis of sulfinate esters through the dynamic resolution of tert-butanesulfinyl chloride. \u003cem\u003eJ. Am. Chem. Soc\u003c/em\u003e. \u003cstrong\u003e126\u003c/strong\u003e, 8134\u0026ndash;8135 (2004); (d) Shibata, N. et al. Cinchona alkaloid/sulfinyl chloride combinations: enantioselective sulfinylating agents of alcohols. \u003cem\u003eJ. Am. Chem. Soc\u003c/em\u003e. \u003cstrong\u003e127\u003c/strong\u003e, 1374\u0026ndash;1375 (2005); (e) Zhang, X., Ang, E. C. X., Yang, Z., Kee, C. W. \u0026amp; Tan, C.-H. Synthesis of chiral sulfinate esters by asymmetric condensation. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e604\u003c/strong\u003e, 298\u0026ndash;303 (2022); (f) Huang, S. et al. Organocatalytic asymmetric deoxygenation of sulfones to access chiral sulfinyl compounds. \u003cem\u003eNat. Chem\u003c/em\u003e. \u003cstrong\u003e15\u003c/strong\u003e, 185\u0026ndash;193 (2023); (g) Suleman, M., Huang, T. Zhou, T., Chen, Z. \u0026amp; Shi, B. Recent Advances in Asymmetric Synthesis of Chiral-at-Sulfur Sulfoximines. \u003cem\u003eACS Catal. \u003c/em\u003e\u003cstrong\u003e15\u003c/strong\u003e, 5511\u0026ndash;5530 (2025).\u003c/li\u003e\n\u003cli\u003eWei, T., Wang, H.-L., Tian, Y., Xie, M.-S. \u0026amp; Guo, H.-M. Enantioselective construction of stereogenic-at-sulfur(IV) centres via catalytic acyl transfer sulfinylation. \u003cem\u003eNat. Chem. \u003c/em\u003e\u003cstrong\u003e16\u003c/strong\u003e, 1310\u0026ndash;1311 (2024). \u003c/li\u003e\n\u003cli\u003eGrogan, G. 3.5.2 Oxidation at Sulfur. In \u003cem\u003eBiocatalysis in Organic Synthesis 3\u003c/em\u003e, 1st edition ed.; Science of Synthesis; Georg Thieme Verlag KG, 2015; DOI: 10.1055/sos-SD-216-00175.\u003c/li\u003e\n\u003cli\u003eGoundry, W. R. F., Adams, B., Benson, H., Demeritt, J., McKown, S., Mulholland, K., Robertson, A., Siedlecki, P., Tomlin, P. \u0026amp; Vare, K. Development and Scale-up of a Biocatalytic Process To Form a Chiral Sulfoxide. \u003cem\u003eOrg. Process Res. Dev\u003c/em\u003e. \u003cstrong\u003e21\u003c/strong\u003e, 107\u0026ndash;113 (2017). \u003c/li\u003e\n\u003cli\u003eWang, Y., Lan, D., Durrani, R. \u0026amp; Hollmann, F. Peroxygenases en route to becoming dream catalysts. What are the opportunities and challenges? \u003cem\u003eCurr. Opin. Chem. Biol. \u003c/em\u003e\u003cstrong\u003e37\u003c/strong\u003e, 1\u0026ndash;9 (2017). \u003c/li\u003e\n\u003cli\u003eMonterrey, D. T., Men\u0026eacute;s-Rubio, A., Keser, M., Gonzalex-Perez, D. \u0026amp; Alcalde, M. Unspecific peroxygenases: The pot of gold at the end of the oxyfunctionalization rainbow? \u003cem\u003eCurr. Opin. Green. Suss. Chem. \u003c/em\u003e\u003cstrong\u003e41\u003c/strong\u003e, 100786 (2023).\u003c/li\u003e\n\u003cli\u003eBassanini, I., Ferrandi, E. E., Vanoni, M., Ottolina, G., Riva, S., Crotti, M., Brenna, E. \u0026amp; Monti, D. Peroxygenase-Catalyzed Enantioselective Sulfoxidations. \u003cem\u003eEur. J. Org. Chem. \u003c/em\u003e\u003cstrong\u003e2017\u003c/strong\u003e, 7186\u0026ndash;7189 (2017). \u003c/li\u003e\n\u003cli\u003eVind, J., Kiemer, L. \u0026amp; Amourgi, E. 2016 WO 2016207373A1.\u003c/li\u003e\n\u003cli\u003eGr\u0026ouml;be, G., Ullrich, R., Pecyna, M. J., Kapturska, D., Friedrich, S., Hofrichter, M. \u0026amp; Scheibner, K. High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula. \u003cem\u003eAMB Express\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 1, (2011).\u003c/li\u003e\n\u003cli\u003eRobinson, W. X. Q., Mielke, T., Melling, B., Cuetos, A., Parkin, A., Unsworth, W. P., Cartwright, J., \u0026amp; Grogan, G. Comparing the catalytic and structural characteristics of a \u0026lsquo;short\u0026rsquo; unspecific peroxygenase (UPO) expressed in P. pastoris and E. coli. \u003cem\u003eChemBioChem\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, e202200558 (2023).\u003c/li\u003e\n\u003cli\u003e(a) Kleymann, G. \u0026amp; Gege, C. Preparation of enantiomers of substituted thiazoles as antiviral compounds. WO Patent 2019/068817 A1 (2019); (b) Brandt, J. \u0026amp; Gais, H.-J. An efficient resolution of (\u0026plusmn;)-S-methyl-Sphenylsulfoximine with (+)-10-camphorsulfonic acid by the method of half-quantities. \u003cem\u003eTetrahedron: Asymmetry\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 909\u0026ndash;912 (1997); (c) Tang, Y. \u0026amp; Miller, S. J. Catalytic Enantioselective Synthesis of Pyridyl Sulfoximines. \u003cem\u003eJ. Am. Chem. Soc. \u003c/em\u003e\u003cstrong\u003e143\u003c/strong\u003e, 9230\u0026ndash;9235 (2021); (d) Zhang, X., Wang, F. \u0026amp; Tan, C.-H. Asymmetric synthesis of (IV) and S(VI) stereogenic centers. \u003cem\u003eJACS Au\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 700\u0026ndash;714 (2023); (e) Fan, F.-X., Tang, S.-X., Dang, Y. \u0026amp; Wang, F. Iron-catalysed stereoselective NH transfer enables dynamic kinetic resolution of sulfoxides. \u003cem\u003eNat. Commun. \u003c/em\u003e\u003cstrong\u003e16\u003c/strong\u003e, 1471 (2025).\u003c/li\u003e\n\u003cli\u003e(a) Garc\u0026iacute;a-C\u0026aacute;rceles, J. et al. 2-(fluoromethoxy)-4\u0026prime;-(s-methanesulfonimidoyl)-1,1\u0026prime;-biphenyl (UCM-1306), an orallybioavailable positive allosteric modulator of the human dopamineD1 receptor for Parkinson\u0026rsquo;s disease. \u003cem\u003eJ. Med. Chem\u003c/em\u003e. \u003cstrong\u003e65\u003c/strong\u003e,12256\u0026ndash;12272 (2022); (b) Zhang, X., Ang, E. C. X., Yang, Z., Kee, C. W. \u0026amp; Tan, C.-H. Synthesis of chiral sulfinate esters. by asymmetric condensation. \u003cem\u003eNature \u003c/em\u003e\u003cstrong\u003e604\u003c/strong\u003e, 298\u0026ndash;303 (2022).\u003c/li\u003e\n\u003cli\u003e(a) Aota, Y., Kano, T. \u0026amp; Maruoka, K. Asymmetric synthesis of chiral sulfoximines via the S-arylation of sulfinamides. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e141\u003c/strong\u003e, 19263\u0026ndash;19268 (2019); (b) Aota, Y., Kano, T. \u0026amp; Maruoka, K. Asymmetric synthesis of chiral sulfoximines through the S-alkylation of sulfinamides. \u003cem\u003eAngew.\u003c/em\u003e \u003cem\u003eChem. Int. Ed. \u003c/em\u003e\u003cstrong\u003e58\u003c/strong\u003e, 17661\u0026ndash;17665 (2019); (c) Maeda, Y. et al. Practical asymmetric synthesis of chiral sulfoximines via sulfur-selective alkylation. \u003cem\u003eJ. Org. Chem. \u003c/em\u003e\u003cstrong\u003e87\u003c/strong\u003e, 3652\u0026ndash;3660 (2022).\u003c/li\u003e\n\u003cli\u003e(a) Mendon\u0026ccedil;a Matos, P., Lewis, W., Argent, S. P., Moore, J. C. \u0026amp; Stockman, R. A. General method for the asymmetric synthesis of N\u0026ndash;H sulfoximines via C\u0026ndash;S bond formation. \u003cem\u003eOrg. Lett. \u003c/em\u003e\u003cstrong\u003e22\u003c/strong\u003e, 2776\u0026ndash;2780 (2020); (b) Greed, S. et al. Synthesis of highly enantioenriched sulfonimidoyl fluorides and sulfonimidamides by stereospecific sulfur\u0026ndash;fluorine exchange (SuFEx) reaction. \u003cem\u003eChem. Eur. J. \u003c/em\u003e\u003cstrong\u003e26\u003c/strong\u003e, 12533\u0026ndash;12538 (2020); (c) Greed, S., Symes, O. \u0026amp; Bull, J. A. Stereospecific reaction of sulfonimidoyl fluorides with Grignard reagents for the synthesis of enantioenriched sulfoximines. \u003cem\u003eChem. Commun. \u003c/em\u003e\u003cstrong\u003e58\u003c/strong\u003e, 5387\u0026ndash;5390 (2022); (d) Yang, G.-F. et al. Synthesis of chiral sulfonimidoyl chloride via desymmetrizing enantioselective hydrolysis. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e145\u003c/strong\u003e, 5439\u0026ndash;5446 (2023); (e) Teng, S., Shultz, Z. P., Shan, C., Wojtas, L. \u0026amp; Lopchuk, J. M. Asymmetric synthesis of sulfoximines, sulfonimidoyl fluorides and sulfonimidamides enabled by an enantiopure bifunctional S(VI) reagent. \u003cem\u003eNat. Chem. \u003c/em\u003e\u003cstrong\u003e16\u003c/strong\u003e, 183\u0026ndash;192 (2024)\u003c/li\u003e\n\u003cli\u003e(a) Johnson, C. R. \u0026amp; Kirchoff, R. A. Oxidation of N-(p-tolylsulfonyl)sulfilimines to N-(p-tolylsulfonyl)sulfoximines with alkaline hydrogen peroxide. \u003cem\u003eJ. Org. Chem. \u003c/em\u003e\u003cstrong\u003e44\u003c/strong\u003e, 2280 (1979); (b) Manche\u0026ntilde;o, O. G. \u0026amp; Bolm, C. Synthesis of N-(1H)-Tetrazole Sulfoximines. \u003cem\u003eOrg. Lett. \u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 2951\u0026ndash;2954 (2007); (c) Manche\u0026ntilde;o, O. G., Bistri, O. \u0026amp; Bolm, C. Iodinane- and Metal-Free Synthesis of N-Cyano Sulfilimines:\u0026thinsp; Novel and Easy Access of NH-Sulfoximines. \u003cem\u003eOrg. Lett. \u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 3809\u0026ndash;3811 (2007).\u003c/li\u003e\n\u003cli\u003eFarwell, C. C., McIntosh, J. A., Hyster, T. K., Wang, Z. J. \u0026amp; Arnold, F. H. Enantioselective Imidation of Sulfides via Enzyme-Catalyzed Intermolecular Nitrogen-Atom Transfer. \u003cem\u003eJ. Am. Chem. Soc. \u003c/em\u003e\u003cstrong\u003e136\u003c/strong\u003e, 8766\u0026ndash;8771 (2014).\u003c/li\u003e\n\u003cli\u003eWang, F. et al. Synthesis of chiral sulfilimines by organocatalytic enantioselective sulfur alkylation of sulfonamides. \u003cem\u003eSci. Adv. \u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, eadq2768 (2024). \u003c/li\u003e\n\u003cli\u003eBonfield, H. E., Mercer, K., Diaz-Rodriguez, A., Cook, G. C., McKay, B. S. J., Slade, P., Taylor, G. M., Ooi, W. X., Williams, J. D., Roberts, J. P. M. et al. The Right Light: De Novo Design of a Robust Modular Photochemical Reactor for Optimum Batch and Flow Chemistry. \u003cem\u003eChemPhotoChem \u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 45-51 (2020).\u003c/li\u003e\n\u003cli\u003ePogr\u0026aacute;nyi, B., Mielke, T. F., D\u0026iacute;az-Rodriguez, A., Cartwright, J., Unsworth, W. P. \u0026amp; Grogan G. Preparative-Scale Biocatalytic Oxygenation of N-Heterocycles with a Lyophilized Peroxygenase Catalyst\u003cem\u003e. Angew. Chem., Int. Ed\u003c/em\u003e. \u003cstrong\u003e62\u003c/strong\u003e, e202214759 (2023).\u003c/li\u003e\n\u003cli\u003eDong, S., Frings, M., Cheng, H., Wen, J., Zhang, D., Raabe, G. \u0026amp; Bolm, C. Organocatalytic Kinetic Resolution of Sulfoximines \u003cem\u003eJ. Am. Chem. Soc\u003c/em\u003e. \u003cstrong\u003e138\u003c/strong\u003e, 2166\u0026minus;2169 (2016).\u003c/li\u003e\n\u003cli\u003eTrott, O. \u0026amp; Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. \u003cem\u003eJ. Comput. Chem.\u003c/em\u003e, \u003cstrong\u003e31\u003c/strong\u003e, 455-461 (2010).\u003c/li\u003e\n\u003cli\u003eShi, Y., Yuan, Y., Li, J., Yang, J. \u0026amp; Zhang, J. Catalytic Asymmetric Synthesis of Sulfinamides via Cu-Catalyzed Asymmetric Addition of Aryl Boroxines to Sulfinylamines. \u003cem\u003eJ. Am. Chem. Soc\u003c/em\u003e. \u003cstrong\u003e146\u003c/strong\u003e, 17580\u0026ndash;17586 (2024)\u003c/li\u003e\n\u003cli\u003eDavis, F. A., Zhou, P. \u0026amp; Chen, B,-C. Asymmetric synthesis of amino acids using sulfinimines. \u003cem\u003eChem. Soc. Rev. \u003c/em\u003e\u003cstrong\u003e27\u003c/strong\u003e, 13\u0026ndash;18 (1998); (b) Philip, R. M., Treesa, G. S. S., Saranya, S. \u0026amp; Anilkumar, G. Applications of aryl-sulfinamides in the synthesis of N-heterocycles. \u003cem\u003eRSC Adv. \u003c/em\u003e\u003cstrong\u003e11 \u003c/strong\u003e20591\u0026ndash;20600 (2021).\u003c/li\u003e\n\u003cli\u003eVazquez-Chavez, J., Luna-Morales, S., Cruz-Aguilar, D. A., D\u0026iacute;az-Salazar, H., Vallejo Narv\u0026aacute;ez, W. E., Silva-Guti\u0026eacute;rrez, R. S., Hern\u0026aacute;ndez-Ortega, S., Rocha-Rinza, T. \u0026amp; Hern\u0026aacute;ndez-Rodr\u0026iacute;guez, M\u003cem\u003e. \u003c/em\u003eThe effect of chiral N-substituents with methyl or trifluoromethyl groups on the catalytic performance of mono- and bifunctional thioureas.\u003cem\u003e Org. Biomol. Chem\u003c/em\u003e. \u003cstrong\u003e17\u003c/strong\u003e, 10045\u0026ndash;10051 (2019).\u003c/li\u003e\n\u003cli\u003eMelling, B., Mielke, T., Whitwood, A. C., O\u0026rsquo;Riordan, T. J. C., Mulholland, N., Cartwright, J., Unsworth, W. P. \u0026amp; Grogan, G. Complementary specificity of unspecific peroxygenases enables access to diverse products from terpene oxygenation. \u003cem\u003eChem Catalysis\u003c/em\u003e, \u003cstrong\u003e4\u003c/strong\u003e, 100889 (2024).\u003c/li\u003e\n\u003cli\u003ePetrone, D. A., Yoon, H., Weinstabl, H., Lautens, M. Additive Effects in the Palladium-Catalyzed Carboiodination of Chiral N-Allyl Carboxamides. Angew. Chem. Int. Ed. \u003cstrong\u003e53\u003c/strong\u003e, 7908\u0026ndash;7912 (2014).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Methods","content":"\u003cp\u003eGeneral procedures for used for preparative biosynthetic reactions are as follows. More details, can be found in the Supplementary Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral procedure for artUPO kinetic resolution of sulfilimines (4) at 0.3 mmol scale (Table 1)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiquid artUPO secretate\u0026nbsp;(1.0 mL, 0.8 U/mL)\u0026nbsp;was added to KPi Buffer (24.0 mL, pH 7.00) at RT and stirred for five min, after which, a solution of sulfilimine (0.300 mmol) in MeCN (6.00 mL) was added. The reaction was initiated by the slow continuous addition of a H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution (0.180 mmol in 2 mL H\u003csub\u003e2\u003c/sub\u003eO) over 4 h followed by stirring overnight. The reaction was extracted with diethyl ether (3 x 30 mL), and the combined organic phase washed with saturated brine (40 mL), dried over MgSO\u003csub\u003e4\u003c/sub\u003e, filtered and the solvent removed in vacuo. For preparative reactions, the purified products were isolated following flash column chromatography on silica gel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral procedure for \u003cem\u003er\u003c/em\u003eAaeUPO biotransformations with\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ei\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-Pr \u003cem\u003eN\u003c/em\u003e-sulfenylimines (Table 2B)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo a round bottom flask containing a magnetic stirring bar was added r\u003cem\u003eAae\u003c/em\u003eUPO-PaDa-I-H (1.3 mL, 57 U/mL) and KPi buffer (10 mL, 0.1 mmol/mL, pH = 7, 10 mL). The solution was diluted by the addition of deionised water (2.7 mL), followed by addition of the appropriate\u0026nbsp;\u003cem\u003ei\u003c/em\u003e-Pr \u003cem\u003eN\u003c/em\u003e-sulfenylimines (0.2 mmol, 1.0 equiv.,) in MeCN (4 mL). Next, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution (2 mL, 0.1 mmol/ml, 1.0 equiv.) was added over a 10 h period, using a syringe pump. After the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e addition was complete, the reaction was then stirred at room temperature for a further 6 h. The reaction mixture was then extracted with ethyl acetate (3 \u0026times; 20 mL). The combined organic phases were then washed with brine (20 mL), dried over anhydrous MgSO\u003csub\u003e4\u003c/sub\u003e and concentrated \u003cem\u003ein\u003c/em\u003e \u003cem\u003evacuo\u003c/em\u003e to give the crude product mixture, which was purified by flash column chromatography on silica to provide the corresponding \u003cem\u003eN\u003c/em\u003e-sulfinyl imine product \u003cstrong\u003e(\u003cem\u003eR\u003c/em\u003e)-12\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral procedure for the artUPO biotransformations with\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003et\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-Bu \u003cem\u003eN\u003c/em\u003e-sulfenylimines (Table 2C)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo a round bottom flask containing a magnetic stirring bar was added\u0026nbsp;liquid artUPO secretate\u0026nbsp;(1.0 mL, 0.8 U/mL) and KPi buffer (100 mM, pH = 7, 10 mL). The solution was diluted by the addition of deionised water (3 mL), followed by addition of the appropriate\u0026nbsp;\u003cem\u003et\u003c/em\u003e-Bu \u003cem\u003eN\u003c/em\u003e-sulfenylimine \u003cstrong\u003e6\u003c/strong\u003e (0.2 mmol, 1.0 equiv., final concentration 10 mM) in MeCN (4 mL). Next, 2 mL of a 100 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution (prepared from 22 \u0026micro;L 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in 2 mL deionised water) was added over a 10 h period, using a syringe pump. After the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e addition was complete, the reaction was then stirred at room temperature for a further 6 h. The reaction mixture was then extracted with ethyl acetate (3 \u0026times; 20 mL). The combined organic phases were then washed with brine (20 mL), dried over anhydrous MgSO\u003csub\u003e4\u003c/sub\u003e and concentrated \u003cem\u003ein\u003c/em\u003e \u003cem\u003evacuo\u003c/em\u003e to give the crude product mixture, which was purified by flash column chromatography on silica gel (to provide the corresponding \u003cem\u003eN\u003c/em\u003e-sulfinyl imine product \u003cstrong\u003e(\u003cem\u003eS\u003c/em\u003e)-13\u003c/strong\u003e.\u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7185896/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7185896/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStereogenic-at-sulfur compounds are vitally important biologically active small molecules, with many drugs featuring chiral sulfur(IV) atoms. Methods for the asymmetric synthesis of sulfoxide centres are well established, but methods that produce enantiomerically enriched sulfoximines and sulfinimines are far less well developed, with no known biocatalytic methods based on oxygenation. In this study, we demonstrate that Unspecific Peroxygenases (UPOs) catalyze the biocatalytic oxygenation of sulfilimines and sulfenimines to form enantiomerically enriched sulfoximines and sulfinimines respectively, on preparative scale. In the sulfenimine series, sulfoximines are generated in up to 99% \u003cem\u003eee via\u003c/em\u003e a kinetic resolution approach. In the sulfilimine series, the selective, stereodivergent synthesis of either of the enantiomeric sulfinimine products (both up to 99% \u003cem\u003eee\u003c/em\u003e) can be achieved, with different UPOs affording products with opposite enantioselectivity. Both series represent novel applications of UPO technology to an ever-growing list of selective, practical and industrially relevant biotransformations.\u003c/p\u003e","manuscriptTitle":"Asymmetric synthesis of stereogenic-at-sulfur compounds via biocatalytic oxidation with Unspecific Peroxygenases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 05:37:27","doi":"10.21203/rs.3.rs-7185896/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"39b55900-1044-401b-92ed-d84c09d4a05d","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53492476,"name":"Physical sciences/Chemistry/Catalysis/Biocatalysis"},{"id":53492477,"name":"Physical sciences/Chemistry/Chemical biology/Biocatalysis"}],"tags":[],"updatedAt":"2026-01-20T08:30:10+00:00","versionOfRecord":{"articleIdentity":"rs-7185896","link":"https://doi.org/10.1038/s41467-025-67405-0","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-12 05:00:00","publishedOnDateReadable":"December 12th, 2025"},"versionCreatedAt":"2025-08-22 05:37:27","video":"","vorDoi":"10.1038/s41467-025-67405-0","vorDoiUrl":"https://doi.org/10.1038/s41467-025-67405-0","workflowStages":[]},"version":"v1","identity":"rs-7185896","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7185896","identity":"rs-7185896","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}