The sulfoximine nitrogenalso provides an additional vector for the inclusion of other functionality.Here, we report improved conditions for rhodium catalyzed synthesisof sulfoximine (and sulfilimine) carbamates, especially for previouslylow-yielding carbamates containing π-functionality. Notablywe report the preparation of propargyl sulfoximine carbamates to providean alkyne as a potential click handle. Using Rh Allyl carbamate for examplegave a 40% yield with methyl p-tolyl sulfoxide.14 In particular, we envisaged that the directincorporation of an alkyne as a click handle at the same time as constructingthe sulfoximine motif would provide a useful process. Given the prevalenceof sulfoxides and sulfones in biologically active compounds,22 as well as sulfoximines themselves,1 we envisaged that the additional vector affordedby a sulfoximine derived from these other S(IV) and S(VI) derivativescould be of value in labeling and provide a handle for further conjugation,for example attachment of a fluorophore or other derivatization ofthe alkyne. On this basis we investigated the use of propargyl carbamateas a substrate for the preparation of sulfoximines to install an alkynemotif suitable for click chemistry 4 in CH2Cl2 afforded the N-propargylcarbamatesulfoximine with a low 24% yield along with an oxidized sulfone sideproduct (2Cl2 to toluene provided a further improved yield of 2a and reduced the formation of side product 3a (entry 4), while also providing a more attractive solvent for development.Changing the rhodium catalyst to Rh2(O2CCF3)4 was unsuccessful, whereas using Rh2(Oct)4 afforded higher yields with Rh2(esp)2 achieving an 87% yield (entries 5–7).23We initially investigated thetransfer of 2-propynyl carbamateto methyl phenylsulfoxide in situ (90%)and isolated yields (85%) of 2a which were well correlatedwith the prediction. No difference was observed by comparing the in situ yield after 16 and 24 h, which indicated that thereaction time could be reduced to 16 h for efficiency purposes. Overall,the optimal yield was achieved by increasing the equivalents of theoxidant and carbamate and importantly reducing the catalyst loading,as well as the reaction time and temperature.Next a design-of-experiments (DoE) optimization wasundertakento further optimize and improve the reaction conditions accountingfor the interplay of conditions. The equivalents of the oxidant andcarbamate, the catalyst loading and the reaction temperature wereidentified as major factors to influence yield. Identical equivalentsof oxidant and carbamate were used to group these as a single variableso as to reduce the number of variables in the DOE study and improvemodel accuracy. Other factors such as the concentration (0.5 M) andthe reaction time (24 h) were set to fixed values. Additionally, theDoE custom design examined second-order interactions of these parameters.The correlation between the predicted yield and the parameters wasvisualized by a three-dimensional response surface of the predictedyield against two major factors , and the dome-shaped surface showed a saddlepoint at 87% 2. These para- and meta-substituted arylmethylsulfoxide derivatives bearing electron-donatingand electron-withdrawing substituents (2a–2g), including halogen and ketone functionality. Enantioenriched(S)-2b was obtained with excellent ee, showing the complete retention of stereochemical informationwith enantioenriched substrates. Scaling the reaction to 3 mmol scaledid not affect the yield significantly (2e). It was noticeablethat low yields were observed for ortho-substitutedexamples 2h and 2i; the corresponding ortho-chloro-derivative (not shown) gave only trace amountsof the sulfoximine product and recovered starting material highlightingthe steric and electronic demands on the nucleophilicity at sulfur.In these lower yielding examples, the mass balance was recovered startingmaterial. Good yields were witnessed for alkyl-aryl substrates andtheir derivatives 2j–m, includingthe cyclopropyl derivative (2n), a structural featurethat has been observed in recent clinical candidates. The reducedyield with isopropyl (2o) was again indicative of stericdemands which was also apparent with diphenyl and dibenzyl substrates(2p and 2q). Other dialkyl substrates (2r and 2s) were investigated, and each was successfulincluding tBu derivative 2s. Cyclicsulfoxide 2t gave an excellent yield. A moderate yieldwas achieved with the 2-pyridyl substrate (2u). Thismethod also showed tolerance of other interesting functional groups,including terminal alcohol (2v) and terminal alkene (2w) where vinyl sulfoximine could be applied as a possibleprobe structure in chemical biology.17 Usingprotected methionine sulfoxide gave the propargylic carbamate derivativeof methionine sulfoximine , MSO itself beingan inhibitor for the biosynthesis of glutathione.With the optimized reaction conditions the scopeof the reactionwas then investigated on a slightly larger 0.5 mmol scale 1. Good t14 we compared these across a rangeof carbamate types . In our previous report, the yields dropped significantly to 60%and 54% for benzyl (Cbz) and phenyl carbamates.14 However, the yields remained at 95% for benzyl carbamate 6 and 90% for phenyl carbamate 7 using the newconditions. In contrast to the disappointing results obtained withallyl and alkynyl carbamates under the previous conditions, allyl 8 and TMS protected propargyl 9 were obtainedin 82% and 93% yields.To make a direct comparison with our previousconditions see 1,14 we cte types 2. For ca21 Applying our propargyl carbamatetransfer to methyl p-tolyl sulfides gave sulfilimine 12 with a 32% yield, where direct oxidation to the correspondingsulfoxide represented the major side product. Moderate to good yieldswere observed when transferring tert-butyl carbamateto sulfides, affording the products 13, 14, and 15 with different aryl-substituents. Interestingly,switching to Cbz carbamate gave sulfilimine 16 in excellentyield. Additionally, the transfer of tert-butyl carbamateto a sulfenamide was successful, affording the unusual sulfinamidineproduct 17 with a 38% yield.24The developed conditions for carbamate transfer were also appliedto sulfide substrates to afford sulfilimines 3. Previo2a and benzylazide cleanly formed triazole 18. Similarly,biotin azide was suitable to prepare the corresponding triazole (19) as may be valuable in chemical biology.Finally, the potential utility of propargylcarbamate-sulfoximinewas demonstrated in CuAAC click reactions with alkyl azides 4. Using 2(esp)2 catalyst and toluene solvent gave improved toleranceof carbamates with unsaturated functionality. This method may furtherenhance the possibilities for the use of sulfoximines, to exploitthat additional vector and directly install a broader range of carbamateswith improved yields. The cycloaddition chemistry from the alkynesmay provide an alternative way to incorporate small sulfoximine motifs,or allow labeling via other sulfur derivatives. It also provides thefirst example of the formation of sulfilimine carbamates without therequirement for a preactivated nitrogen source.In conclusion, improved conditions for the preparationof sulfoximinecarbamates are reported where the use of more reactive Rh2Cl2) or used directly from commercial sources  without drying. Rh2(esp)2 (96%) was purchased from Sigma-Aldrich and PhI(OAc)2 (98%) was purchased from Fluorochem, and used directly withoutfurther treatment. Flash column chromatography was performed using230–400 mesh silica with the indicated solvent system accordingto standard techniques. Analytical thin-layer chromatography (TLC)was performed on precoated, glassbacked silica gel plates. Visualizationof the developed chromatogram was performed by UV absorbance (254nm) or aqueous potassium permanganate stains. Infrared spectra  were recorded in reciprocal centimeters (cm–1). Nuclear magnetic resonance spectra were recordedon 400 MHz spectrometers. Chemical shifts for 1H NMR spectraare recorded in parts per million from tetramethylsilane with theresidual protic solvent resonance as the internal standard . Data are reported as follows: chemicalshift . 13C NMR spectra were recordedwith complete proton decoupling. Chemical shifts are reported in partsper million from tetramethylsilane with the solvent resonance as theinternal standard 2SO: δ = 39.5 ppm, 13CD3OD: δ = 49.0 ppm). J values are reported in Hz. Assignments of 1H/13C spectra were made by the analysis of δ/J values, and COSY, HSQC, and HMBC experiments as appropriate. Meltingpoints are uncorrected. Heating blocks were used for reactions aboveroom temperature.All nonaqueousreactions were run under an inert atmosphere (argon) with flame-driedor oven-dried glassware using standard techniques. Anhydrous solventswere obtained by filtration through drying columns -1b, 1n, 1p–s, and 1w), carbamates ,and sulfides (10a–c) were used assupplied, and others were prepared from the corresponding commerciallyavailable sulfides or thiols as previously reported.25 Sulfenamide 11 was prepared as previouslyreported.26 Benzyl azide was prepared aspreviously reported.27 Azide-PEG3-biotinconjugate was purchased from Sigma-Aldrich.Reagents: Commercial reagents were used assupplied or purified by standard techniques where necessary. Commerciallyavailable sulfoxides  was addeddropwise to a stirred solution of propargyl alcohol  and NaOCN  in anhydrous Et2O (100 mL) at 30 °C. The reaction mixture was stirredat 30 °C overnight. The resulting mixture was diluted with Et2O (100 mL) and filtered. The filtrate was concentrated underreduced pressure. Purification by flash column chromatography  afforded prop-2-yn-1-ylcarbamate as a white solid . Rf = 0.12 (30% Et2O in pentane); mp = 48–51°C; IR (film)/cm–1 3425, 3307, 3280, 3211,2952, 1675, 1647, 1607, 1405, 1328, 1062, 865, 702, 634, 563; 1H NMR  δ 4.93 , 4.69 , 2.50 ; 13C NMR  δ 155.8 (C=O), 77.9(CCH), 74.8 (CCH), 52.7 (CH2). Analytical data (NMR and IR) in agreement with those reportedin the literature.29CF3CO2H  was added dropwiseto a stirred solution of 3-trimethylsilyl-2-propyn-1-ol  and NaOCN  in anhydrousEt2O (40 mL) at 30 °C. The reaction mixture was stirredat 30 °C overnight. The resulting mixture was diluted with Et2O (40 mL) and filtered. The filtrate was concentrated underreduced pressure. Purification by flash column chromatography  afforded 3-(trimethylsilyl)prop-2-yn-1-ylcarbamate as a white solid . Rf = 0.33 (30% EtOAc in pentane); mp = 53–57 °C;IR (film)/cm–1 3451, 3343, 3292, 3185, 2964, 2181,1705 (C=O), 1606, 1388, 1320, 1067, 1011, 838, 764, 703; 1H NMR  δ 4.84 , 4.69 , 0.19 3); 13C NMR  δ 155.8 (C=O), 99.2 (CCSi), 92.1(CCSi), 53.5 (C(CH3)3), −0.3 (C(CH3)3); HRMS (ESI-TOF) m/z: Calcd. forC7H14NO2Si [M + H]+: 172.0788,found: 172.0789.CF2  was added to a suspensionof the sulfoxide or sulfide , carbamate , MgO , and Rh2(esp)2  in toluene (1.0 mL) at rt.The resulting mixture was heated to 30 °C and stirred for 16h. The reaction mixture was diluted with CH2Cl2 (1 mL), filtered through Celite, and concentrated under reducedpressure. The resulting residue was purified by flash column chromatography(SiO2) to afford the corresponding sulfoximine or sulfiliminecarbamate.PhI(OAc)1a  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  afforded sulfoximinepropargyl carbamate 2a  as a pale-yellowgum. Rf = 0.17 (40% EtOAc in hexane);IR (film)/cm–1 3261, 3014, 2925, 2120, 1668 (C=O),1444, 1368, 1223, 1084, 977, 867, 783, 742, 683, 559, 504; 1H NMR  δ 8.02–7.99 , 7.71–7.69 , 7.64–7.60, 4.70–4.65 , 4.65–4.60 , 3.34, 2.43 ; 13C{1H} NMR  δ 158.0 (C=O), 138.0 (Ar–Cq), 134.1(Ar–C), 129.7 (2 × Ar–C), 127.4 (2 × Ar–C),77.9 (CCH), 74.6 (CCH), 53.4 (CH2), 44.5 (SCH3); HRMS (ESI-TOF) m/z: Calcd. for C11H12NO3S [M + H]+: 238.0538, found: 238.0533.Prepared using the General Procedure usingsulfoxide 1b  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  afforded sulfoximinepropargyl carbamate 2b  as a pale-yellowgum. Rf = 0.22 (40% EtOAc in hexane);IR (film)/cm–1 3260, 3016, 2924, 2120, 1668 (C=O),1593, 1368, 1224, 1086, 978, 866, 812, 631, 512, 493; 1H NMR  δ 7.88 , 7.42–7.40 , 4.70–4.66 , 4.65–4.61 , 3.32 , 2.47 , 2.43 ; 13C{1H} NMR δ 158.1 (C=O), 145.3 (Ar–Cq), 134.8 (Ar–Cq), 130.4 (2 × Ar–C),127.4 (2 × Ar–C), 78.0 (CCH), 74.6 (CCH), 53.4 (CH2), 44.6 (SCH3), 21.6(Ar–CH3); HRMS (ESI-TOF) m/z: Calcd. for C12H14NO3S [M + H]+: 252.0694, found: 252.0682.Prepared using the General Procedure usingsulfoxide (S)-1b  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  afforded sulfoximinepropargyl carbamate (S)-2b  as a pale-yellow gum. Rf = 0.22 (40% EtOAc in hexane); Spectroscopicdata same as those for 2b above. [α]21D = +52 . HPLCconditions: Chiralpak IB column, 90:10 n-hexane:iPrOH, flow rate: 1 mL min–1, 35 °C,UV detection wavelength: 250 nm, ((S)-2b) retention time: 38 min. ((rac)-2b) retention times: 38 and42 min.Preparedusing General Procedure using sulfoxide 1c and prop-2-yn-1-yl carbamate . Purificationby flash column chromatography afforded sulfoximine propargyl carbamate 2c  as a white solid. Rf = 0.08 (30%EtOAc in hexane); mp = 84–85 °C; IR (film)/cm–1 3265, 3019, 2930, 2848, 2125, 1670 (C=O), 1595, 1498, 1312,1238, 1088, 984, 867; 1H NMR  δ 7.93–7.90 , 7.08–7.04, 4.70–4.66 , 4.65–4.60 , 3.90, 3.32 , 2.43 ; 13C{1H} NMR δ 164.1 (Ar–Cq), 158.1 (C=O), 129.6 (2 × Ar–C), 128.8(Ar–Cq), 115.0 (2 × Ar–C), 78.0 (CCH),74.6 (CCH), 55.8 (OCH3), 53.3 (CH2), 44.9 (SCH3); HRMS (ESI-TOF) m/z: Calcd. for C12H14NO4S [M + H]+: 268.0644, found: 268.0640.Prepared usingGeneral Procedure using sulfoxide 1d and prop-2-yn-1-yl carbamate . Purificationby flash column chromatography  afforded sulfoximine propargyl carbamate 2d  as a white gum. Rf =0.10 (40% Et2O in pentane); IR (film)/cm–1 3283, 3083, 3016, 2120, 1672 (C=O), 1573, 1366, 1217, 1081,977, 866, 783, 684, 462; 1H NMR  δ 7.95–7.93 , 7.61–7.58, 4.70–4.66 , 4.65–4.60 , 3.34, 2.44 ; 13C{1H} NMR  δ 157.8 (C=O), 141.1 (Ar–Cq), 136.4(Ar–Cq), 130.1 (2 × Ar–C), 128.9 (2× Ar–C), 77.8 (CCH), 74.8 (CCH), 53.5 (CH2), 44.5 (SCH3); HRMS (ESI-TOF) m/z: Calcd. for C11H11NO3S35Cl [M + H]+: 272.0148, found:272.0151.Prepared usingGeneral Procedure using sulfoxide 1e  and prop-2-yn-1-yl carbamate .Purification by flash column chromatography  afforded sulfoximine propargyl carbamate 2e  as a colorless gum. Rf = 0.12 (40% Et2O in pentane); IR (film)/cm–1 3263, 3083, 3012, 2923, 2120, 1668 (C=O),1567, 1367, 1224, 1085, 1065, 978, 867, 780, 676, 565, 508; 1H NMR  δ 7.85–7.82 , 7.75–7.72 ,4.65–4.60 , 4.60–4.55, 3.31 , 2.42; 13C{1H} NMR  δ 157.7 (C=O), 136.8(Ar–Cq), 132.9 (2 × Ar–C), 129.4 (Ar–Cq), 128.8 (2 × Ar–C), 77.7 (CCH),74.7 (CCH), 53.4 (CH2), 44.3 (SCH3); HRMS (ESI-TOF) m/z: Calcd.for C11H11NO3S79Br [M+ H]+: 315.9643, found: 315.9650. Three mmol scale synthesisof 2e: PhI(OAc)2  was added in three-portions over 5 min to a stirring suspensionof sulfoxide 1e , prop-2-yn-1-yl carbamate, MgO and Rh2(esp)2 in toluene (6 mL) at rt. The resulting mixture was heated to 30 °Cand stirred for 16 h. The reaction mixture was diluted with CH2Cl2 (5 mL), filtered through Celite and concentratedunder reduced pressure. Purification by flash column chromatography afforded 2e as a colorless gum .Prepared usingGeneral Procedure using sulfoxide 1f and prop-2-yn-1-yl carbamate . Purificationby flash column chromatography afforded sulfoximine propargyl carbamate 2f  as a colorless gum. Rf = 0.10 (40%EtOAc in hexane); IR (film)/cm–1 3272, 3032, 2926,1682 (C=O), 1663 (C=O), 1367, 1251, 1229, 968, 920,863, 789, 621, 500; 1H NMR δ 8.17–8.15 , 8.12–8.10, 4.69–4.64 , 4.64–4.60 , 3.35, 2.68 , 2.43 ; 13C{1H} NMR δ 196.4 (C=O), 157.8 (C=O),141.9 (Ar–Cq), 141.1 (Ar–Cq),129.4 (2 × Ar–C), 127.9 (2 × Ar–C), 77.7 (CCH), 74.8 (CCH), 53.6 (CH2),44.2 (SCH3), 26.9 (COCH3);HRMS (ESI-TOF) m/z: Calcd. for C13H14NO4S [M + H]+: 280.0644,found: 280.0638.Prepared usingGeneral Procedure using sulfoxide 1g  and prop-2-yn-1-yl carbamate .Purification by flash column chromatography  afforded sulfoximine propargyl carbamate 2g  as a colorless gum. Rf = 0.13 (50% Et2O in hexane); IR (film)/cm–1 3273, 3079, 3019, 2930, 2125, 1670 (C=O), 1365, 1230, 1103,977, 872, 775, 671; 1H NMR δ 8.12–8.09 , 7.91–7.88 , 7.80–7.77 , 7.49–7.45, 4.65–4.61 , 4.60–4.55 , 3.32 , 2.42 ; 13C{1H} NMR  δ157.6 (C=O), 139.7 (Ar–Cq), 137.1 (Ar–C),131.1 (Ar–C), 130.2 (Ar–C), 125.8 (Ar–C), 123.5(Ar–Cq), 77.7 (CCH), 74.8 (CCH), 53.4 (CH2), 44.3 (SCH3); HRMS(ESI-TOF) m/z: Calcd. for C11H11NO3S79Br [M + H]+: 315.9643, found: 315.9649.Prepared usingGeneral Procedure using sulfoxide 1h and prop-2-yn-1-yl carbamate . Purificationby flash column chromatography afforded sulfoximine propargyl carbamate 2h  as a colorless gum. Rf = 0.22 (40%EtOAc in hexane); IR (film)/cm–1 3273, 3019, 2937,2125, 1670 (C=O), 1476, 1230, 1126, 1074, 977, 865, 760; 1H NMR  δ 8.09 , 7.71 , 7.46–7.38, 7.31–7.26 , 4.66,4.59 , 3.47 , 2.42. 13C{1H} NMR  δ 159.7 (C=O), 157.3(Ar–Cq), 136.6 (Ar–C), 131.0 (Ar–C),125.5 (Ar–Cq), 125.1 (Ar–C), 117.4 (Ar–C),77.8 (CCH), 74.7 (CCH), 53.5 (OCH2), 43.5 (SCH3). HRMS (ESI-TOF) m/z: Calcd. for C11H11NO3SF [M + H]+: 256.0444, found: 256.0452.Prepared usingGeneral Procedure using sulfoxide 1i  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  afforded sulfoximinepropargyl carbamate 2i  as a colorlessgum. Rf = 0.27 (40% EtOAc in hexane);IR (film)/cm–1 3265, 3019, 2937, 2125, 1670 (C=O),1446, 1223, 1118, 977, 865, 753; 1H NMR  δ 8.11–8.09 , 7.58–7.54, 7.46–7.42 , 7.39–7.37, 4.68–4.64 , 4.64–4.59 , 3.34 , 2.71 , 2.41 ; 13C{1H} NMR δ 157.8 (C=O), 137.2 (Ar–Cq), 136.0 (Ar–Cq), 134.0 (Ar–C), 133.3(Ar–C), 129.6 (Ar–C), 127.1 (Ar–C), 78.0 (CCH), 74.6 (CCH), 53.4 (OCH2), 43.3 (SCH3), 20.5 (Ar–CH3); HRMS(ESI-TOF) m/z: Calcd. for C12H14NO3S [M + H]+: 252.0694,found: 252.0699.Prepared using General Procedure usingsulfoxide 1j  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  afforded sulfoximine propargyl carbamate 2j  as a white solid. Rf = 0.25 (CH2Cl2:Et2O:hexane= 20:30:50); mp = 100–102 °C; IR (film)/cm–1 3250, 3064, 2132, 1655 (C=O), 1520, 1446, 1267, 1074, 992,880, 790, 738; 1H NMR  δ7.64–7.59 , 7.46–7.42 , 7.32–7.27 ,7.22–7.18 , 6.96–6.94 , 4.77 , 4.73 , 4.72 , 4.65 , 2.45 ; 13C{1H} NMR  δ 158.3 (C=O), 134.4 (Ar–Cq), 134.0 (Ar–Cq), 131.1 (2 × Ar–C),129.2 (Ar–C), 129.0 (2 × Ar–C), 128.6 (2 ×Ar–C), 128.5 (2 × Ar–C), 126.6 (Ar–C), 78.0(CCH), 74.6 (CCH), 62.0 (SCH2), 53.4 (OCH2); HRMS (ESI-TOF) m/z: Calcd. for C17H16NO3S [M + H]+: 314.0851, found: 314.0851.Prepared using General Procedure usingsulfoxide 1k  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  afforded sulfoximine propargyl carbamate 2k  as a white solid. Rf = 0.29 (CH2Cl2:Et2O:hexane= 20:15:65); mp = 79–81 °C; IR (film)/cm–1 3310, 3034, 2982, 2182, 1670 (C=O), 1595, 1498, 1372, 1215,1118, 984, 775, 626, 492; 1H NMR  δ 7.86 ,7.42–7.39 , 7.28–7.21 , 7.11–7.09 ,4.69–4.65 , 4.64–4.60, 3.71 , 3.59 , 3.10 , 2.97, 2.43 ; 13C{1H} NMR  δ 158.1 (C=O),145.3 (Ar–Cq), 136.6 (Ar–Cq),133.0 (Ar–Cq), 130.3 (2 × Ar–C), 128.8(2 × Ar–C), 128.3 (2 × Ar–C), 128.0 (2 ×Ar–C), 127.0 (Ar–Cq), 78.0 (CCH), 74.6 (CCH), 57.2 (SCH2), 53.4 (OCH2), 28.3 (PhCH2), 21.6 (Ar–CH3); HRMS (ESI-TOF) m/z: Calcd. forC19H20NO3S [M + H]+: 342.1164,found: 342.1168.Prepared using General Procedure usingsulfoxide 1l  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  afforded sulfoximine propargyl carbamate 2l  as a colorless gum. Rf = 0.3 (CH2Cl2:Et2O:hexane= 30:20:50); IR (film)/cm–1 3340, 3263, 2937, 2125,1677 (C=O), 1521, 1431, 1208, 1074, 969, 738. 1HNMR  δ 7.80 , 7.39 , 4.66–4.61 , 4.60–4.56 , 3.49–3.42, 3.41–3.34  2.45 , 2.40 , 1.25 ; 13C{1H} NMR  δ 158.0 (C=O), 145.1 (Ar–Cq), 132.2 (Ar–Cq), 130.2 (2 × Ar–C),128.0 (2 × Ar–C), 77.9 (CCH), 74.4 (CCH), 53.1 (OCH2), 50.6 (SCH2), 21.5(Ar–CH3), 6.8 (CH3); HRMS (ESI-TOF) m/z: Calcd. for C13H16NO3S [M + H]+: 266.0851, found: 266.0848.Prepared using General Procedure usingsulfoxide 1m and prop-2-yn-1-yl carbamate . Purificationby flash column chromatography afforded sulfoximine propargyl carbamate 2m  as a white solid. Rf = 0.22 (20%EtOAc in hexane); mp = 83–84 °C; IR (film)/cm–1 3271, 3014, 2944, 2122, 1671 (C=O), 1592, 1432, 1368, 1234,1084, 975, 923, 881, 780, 633, 522; 1H NMR  δ 7.93–7.91 ,7.45–7.42 , 5.20 , 4.87 , 4.76–4.72 , 4.72–4.68 , 2.49 , 2.48 ; 13C{1H} NMR  δ 157.7 (C=O), 146.6 (Ar–Cq), 130.3 (2 × Ar–C), 129.5 (Ar–Cq), 129.4 (2 × Ar–C), 77.7 (CCH), 75.0(CCH), 58.5 (SCH2), 53.8 (OCH2), 21.7 (Ar–CH3); HRMS (ESI-TOF) m/z: Calcd. for C12H13NO3S35Cl [M + H]+: 286.0305, found: 286.0298.Prepared usingGeneral Procedure using sulfoxide 1n and prop-2-yn-1-yl carbamate . Purificationby flash column chromatography  afforded sulfoximine propargyl carbamate 2n  as a colorless gum. Rf = 0.12 (50% Et2O in hexane); IR (film)/cm–1 3265, 3056, 2945, 2117, 1677 (C=O), 1446, 1245, 1088, 977,880, 783; 1H NMR  δ 7.93–7.91, 7.68–7.64 ,7.61–7.56 , 4.63–4.58 , 4.58–4.53 , 2.68–2.62 , 2.39 , 1.68–1.59 , 1.28–1.19 , 1.05–0.94 ; 13C{1H} NMR  δ 157.7 (C=O), 138.3(Ar–Cq), 133.6 (Ar–C), 129.5 (2 × Ar–C),127.4 (2 × Ar–C), 77.9 (CCH), 74.5 (CCH), 53.2 (OCH2), 33.4 (SCH), 6.8 (CH2), 5.2 (CH2); HRMS (ESI-TOF) m/z: Calcd. for C13H14NO3S [M + H]+: 264.0694, found: 264.0695.Prepared usingGeneral Procedure using sulfoxide 1o  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  afforded sulfoximine propargyl carbamate 2o  as a pale-yellow gum; Rf = 0.43 (CH2Cl2:Et2O:hexane= 30:30:40); IR (film)/cm–1 3265, 2937, 2125, 1677(C=O), 1431, 1372, 1238, 1088, 977, 872, 723, 641; 1H NMR  δ 7.77 , 7.40–7.38 , 4.66–4.62 , 4.61–4.57 , 3.54 , 2.46 , 2.40 , 1.41 , 1.24; 13C{1H} NMR δ 158.3 (C=O), 145.1 (Ar–Cq), 130.8(Ar–Cq), 130.2 (2 × Ar–C), 129.0 (2× Ar–C), 78.2 (CCH), 74.4 (CCH), 56.4 (SCH), 53.3 (CH2), 21.6 (Ar–CH3), 15.6 (CHCH3), 15.0 (CHCH3); HRMS (ESI-TOF) m/z: Calcd. for C14H18NO3S [M + H]+: 280.1007, found: 280.0999.Prepared using General Procedure usingsulfoxide 1p  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  affordedsulfoximine propargyl carbamate 2p  asa colorless gum. Rf = 0.13 (40% Et2O in pentane). IR (film)/cm–1 3271, 3084,3056, 2124, 1667 (C=O), 1443, 1368, 1226, 1135, 1074, 982,962, 877, 679, 575, 554. 1H NMR  δ 8.03–8.00 , 7.62–7.58, 7.55–7.51 ,4.65 , 2.41 . 13C{1H} NMR δ 157.8 (C=O), 139.1 (2 ×Ar–Cq), 133.5 (2 × Ar–C), 129.5 (4 ×Ar–C), 127.7 (4 × Ar–C), 77.9 (CCH), 74.6 (CCH), 53.5 (CH2). HRMS (ESI-TOF) m/z: Calcd. for C16H14NO3S [M + H]+: 300.0694; found: 300.0700.Prepared using General Procedure usingsulfoxide 1q  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  afforded sulfoximine propargyl carbamate 2q  as a white solid. Rf = 0.28 (CH2Cl2:Et2O:hexane= 20:30:50); mp = 124–126 °C; IR (film)/cm–1 3289, 3058, 2983, 2933, 2118, 1651 (C=O), 1491, 1428, 1377,1271, 1230, 1152, 1096, 1069, 964, 918, 873, 694, 600, 480; 1H NMR  δ 7.44–7.38 , 4.71 , 4.59 , 4.53 , 2.49 ; 13C{1H} NMR  δ 158.4 (C=O), 131.3 (4 × Ar–C),129.6 (2 × Ar–C), 129.1 (4 × Ar–C), 126.2(2 × Ar–Cq), 78.3 (CCH), 74.5(CCH), 56.5 (2 × SCH2), 53.5 (OCH2); HRMS (ESI-TOF) m/z: Calcd.for C18H18NO3S [M + H]+: 328.1007, found: 328.1012.Prepared using General Procedure usingsulfoxide 1r  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  afforded sulfoximine propargyl carbamate 2r  as a colorless gum. Rf = 0.24 (CH2Cl2:Et2O:hexane= 20:30:50); IR (film)/cm–1 3250, 2960, 2878, 2125,1662 (C=O), 1461, 1230, 1074, 977, 865, 783, 671; 1H NMR  δ 4.67 , 3.42–3.26 , 2.44 ,1.90–1.74 , 1.53–1.44 , 0.97 ; 13C{1H} NMR  δ 158.3 (C=O), 78.1 (CCH),74.5 (CCH), 53.3 (OCH2), 51.1 (2 ×SCH2), 24.0 (2 × SCH2CH2), 21.6 (2 × CH2CH3), 13.5 (2 × CH3); HRMS (ESI-TOF) m/z: Calcd. for C12H22NO3S [M + H]+: 260.1320, found: 260.1317.Prepared using General Procedure usingsulfoxide 1s  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  afforded sulfoximinepropargyl carbamate 2s  as a colorlessoil. Rf = 0.15 (40% EtOAc in hexane);IR (film)/cm–1 3265, 2915, 2125, 1670 (C=O),1461, 1260, 992, 872, 783, 723; 1H NMR  δ 4.73–4.69 ,4.86–4.64 , 3.28 , 2.45 , 1.51 3); 13C{1H} NMR δ 159.2 (C=O), 78.3 (CCH), 74.5 (CCH), 60.5 (C(CH3)3), 53.3 (OCH2), 32.1 (SCH3), 22.9 (C(CH3)3);HRMS (ESI-TOF) m/z: Calcd. for C9H16NO3S [M + H]+: 218.0851,found: 218.0854.Prepared using General Procedure usingsulfoxide 1t  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  afforded sulfoximinepropargyl carbamate 2t  as a pale-red gum.Rf = 0.16 (50% EtOAc in hexane). IR (film)/cm–1 3264, 3029, 2957, 2121, 1654 (C=O), 1369,1225, 1115, 992, 871, 782, 689, 553. 1H NMR  δ 4.69–4.67 , 4.42–4.33, 4.30–4.22 , 2.47–2.46 , 2.44–2.34. 13C{1H} NMR  δ 158.4 (C=O), 77.8 (CCH), 74.8 (CCH), 62.9 (2 × SCH2),53.5 (OCH2), 9.4 (CH2). HRMS (ESI-TOF) m/z: Calcd. for C7H10NO3S [M + H]+: 188.0381, found: 188.0375.Prepared using General Procedure usingsulfoxide 1u and prop-2-yn-1-yl carbamate . Purificationby flash column chromatography afforded sulfoximine propargyl carbamate 2u  as a pale-red gum. Rf = 0.21 (60%EtOAc in hexane); IR (film)/cm–1 3265, 3019, 2930,2125, 1670 (C=O), 1431, 1223, 1111, 984, 872, 760; 1H NMR  δ 8.74 , 8.29 , 8.02 , 7.59 , 4.63 , 4.56 , 3.47 , 2.40 ; 13C{1H} NMR  δ 157.9 (C=O),156.0 (Ar–Cq), 150.1 (Ar–C), 138.3 (Ar–C),127.6 (Ar–C), 123.5 (Ar–C), 77.8 (CCH), 74.7 (CCH), 53.4 (OCH2), 39.8 (SCH3); HRMS (ESI-TOF) m/z: Calcd.for C10H11N2O3S [M + H]+: 239.0490, found: 239.0498.Prepared usingGeneral Procedure using sulfoxide 1v and prop-2-yn-1-yl carbamate . Purificationby flash column chromatography afforded sulfoximine propargyl carbamate 2v  as a colorless gum. Rf = 0.24 (60%EtOAc in hexane); IR (film)/cm–1 3429, 3273, 3064,2937, 2125, 1670 (C=O), 1446, 1372, 1223, 1081, 969, 872, 783,686; 1H NMR  δ 7.99–7.97, 7.75–7.71 ,7.66–7.62 , 4.65 , 4.60 , 4.19–4.11, 4.08–4.00 , 3.68–3.62 , 3.52–3.46, 3.41–3.37 ; 13C{1H} NMR  δ157.7 (C=O), 136.7 (Ar–Cq), 134.4 (Ar–C),129.9 (2 × Ar–C), 127.9 (2 × Ar–C), 77.7 (CCH), 74.8 , 58.9 (SCH2), 55.8 (SCH2CH2), 53.6 (OCH2); HRMS (ESI-TOF) m/z: Calcd.for C12H13NO4NaS [M + Na]+: 290.0463, found: 290.0451.Prepared usingGeneral Procedure using sulfoxide 1w  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  affordedsulfoximine propargyl carbamate 2w  asa colorless gum. Rf = 0.12 (50% Et2O in hexane). IR (film)/cm–1 3263, 3055,2121, 1673 (C=O), 1443, 1368, 1230, 1082, 972, 873, 744, 686,635, 536, 494. 1H NMR  δ7.98–7.96 , 7.68–7.65 , 7.61–7.57 ,6.75 , 6.54 , 6.21 , 4.70–4.65, 4.65–4.61 , 2.43 . 13C{1H} NMR  δ157.7 (C=O), 137.0 (Ar–Cq), 136.5 (SCH),134.0 (Ar–C), 129.6 (2 × Ar–C), 129.5 (HCCH2), 127.9 (2 × Ar–C), 77.9 (CCH), 74.7 (CCH), 53.5 (OCH2). HRMS (ESI-TOF) m/z: Calcd. forC12H12NO3S [M + H]+: 250.0538,found: 250.0533.Prepared using General Procedure usingsulfoxide 1x  and prop-2-yn-1-ylcarbamate . Purification by flash columnchromatography  afforded sulfoximinepropargyl carbamate 2x  as a colorlessgum. Rf = 0.22 (35% EtOAc in hexane);IR (film)/cm–1 3355, 3280, 2982, 2125, 1707 (C=O),1670 (C=O), 1513, 1372, 1238, 1148, 1044, 992, 872, 783, 634,497; 1H NMR  δ 5.30–5.28, 4.65–4.64 , 4.24–4.21, 3.55–3.31 , 3.26 , 2.45–2.44, 2.43–2.35 , 2.21–2.08 ,1.45 3), 1.41 3); 13C{1H} NMR  δ 169.9 (C=O), 158.1 (C=O), 155.4 (C=O),83.2 (C(CH3)3), 80.3 (C(CH3)3), 77.9 (CCH), 74.7 (CCH), 53.3 (OCH2), 52.3 (CHCO2), 50.6 (SCH2), 39.3 , 28.2 (C(CH3)3),27.8 (C(CH3)3), 25.7 ; HRMS (ESI-TOF) m/z: Calcd. for C18H30N2O7SNa [M + Na]+: 441.1671, found: 441.1668.Prepared using General Procedure usingsulfoxide 1b  and tert-butylcarbamate. Purification by flash column chromatography afforded N-Boc sulfoximine 4  as a white solid.Rf = 0.5 (50% EtOAc in hexane); mp = 108–112°C; IR (film)/cm–1 3027, 2974, 2109, 1662 (C=O),1364, 1267, 1148, 984, 865, 790; 1H NMR  δ 7.84 , 7.37 ,3.21 , 2.44 , 1.37 3); 13C{1H} NMR  δ 157.6 (C=O),144.7 (Ar–Cq), 135.6 (Ar–Cq),130.2 (2 × Ar–C), 127.3 (2 × Ar–C), 80.4 (C(CH3)3), 44.8 (SCH3),27.9 (C(CH3)3), 21.5 (Ar–CH3). Analytical data (NMR and IR) in agreement with those reportedin the literature.14Prepared using General Procedure using sulfoxide 1b  and methyl carbamate . Purification by flash column chromatography  afforded N-CO2Me sulfoximine 5  as a colorlessoil. Rf = 0.26 (50% EtOAc in hexane);mp = 92–96 °C; IR (film)/cm–1 3019,2930, 2117, 1670 (C=O), 1431, 1223, 1088, 984, 872, 790; 1H NMR  δ 7.87 , 7.40 , 3.67 , 3.30 , 2.46 ; 13C{1H} NMR  δ 159.4 (C=O), 145.1 (Ar–Cq), 135.1 (Ar–Cq), 130.3 (2 × Ar–C),127.4 (2 × Ar–C), 53.1 (CO2CH3), 44.6 (SCH3), 21.6 (Ar–CH3). Analytical data (NMR and IR) in agreement with those reportedin the literature.14Prepared using General Procedure using sulfoxide 1b  and benzyl carbamate . Purification by flash column chromatography  afforded N-Cbz sulfoximine 6  as a white solid. Rf = 0.4 (50% EtOAc in hexane); mp = 90–94 °C; IR(film)/cm–1 3027, 2922, 2110, 1655 (C=O),1379, 1215, 1088, 969, 895 775; 1H NMR  δ 7.82 , 7.35 ,7.29–7.22 , 5.09 , 5.01 , 3.25 , 2.43 ; 13C{1H} NMR  δ 158.4 (C=O), 144.9(Ar–Cq), 136.1 (Ar–Cq), 134.9(Ar–Cq), 130.1 (2 × Ar–C), 128.1 (2× Ar–C), 128.0 (2 × Ar–C), 127.7 (Ar–C),127.2 (2 × Ar–C), 67.5 (OCH2), 44.5 (SCH3), 21.4 (Ar–CH3). Analytical data (NMR andIR) in agreement with those reported in the literature.14Prepared using General Procedure using sulfoxide 1b  and phenyl carbamate . Purification by flash column chromatography  afforded N-CO2Ph sulfoximine 7  as a white solid.Rf = 0.5 (50% EtOAc in hexane); mp = 110–116°C; IR (film)/cm–1 3042, 2072, 1670 (C=O),1491, 1260, 1185, 977, 880, 716; 1H NMR  δ 7.92 , 7.43–7.41 , 7.34–7.30, 7.18–7.11 ,3.38 , 2.47 ; 13C{1H} NMR δ 157.2 (C=O), 151.3 (Ar–Cq), 145.2(Ar–Cq), 134.6 (Ar–Cq), 130.3(2 × Ar–C), 129.0 (2 × Ar–C), 127.2 (2 ×Ar–C), 125.1 (Ar–C), 121.5 (2 × Ar–C), 44.3(SCH3), 21.5 (Ar–CH3). Analytical data(NMR and IR) in agreement with those reported in the literature.14Prepared using General Procedure using sulfoxide 1b  and allyl carbamate . Purification by flash column chromatography  afforded sulfoximine allyl carbamate 8  as a colorless oil. Rf = 0.13 (30% EtOAc in hexane); IR (film)/cm–1 3019, 2930, 2110, 1670 (C=O), 1446, 1357, 1223, 1088, 977,872, 790; 1H NMR  δ 7.84, 7.38 , 5.92–5.82, 5.28–5.22 , 5.17–5.13 , 4.56–4.51 , 4.51–4.46, 3.27 , 2.43; 13C{1H} NMR δ 158.5 (C=O), 145.0 (Ar–Cq), 135.0 (Ar–Cq), 132.4 (CHCH2), 130.2 (2 × Ar–C), 127.3 (2 × Ar–C),117.7 (CHCH2), 66.5 (OCH2),44.5 (SCH3), 21.5 (Ar–CH3). Analyticaldata (NMR and IR) in agreement with those reported in the literature.14Prepared using General Procedure using sulfoxide 1b  and TMS-protected propargyl carbamate . Purification by flash column chromatography  afforded sulfoximine propargyl carbamate 9  as a colorless gum. Rf = 0.38 (30% EtOAc in hexane); IR (film)/cm–1 3027, 2960, 2117, 1670 (C=O), 1372, 1230, 1088, 1029, 977,842, 760;1H NMR  δ 7.84–7.82, 7.38–7.35 ,4.64–4.59 , 4.58–4.54, 3.27 , 2.42, 0.11 3); 13C{1H} NMR  δ 158.0 (C=O), 145.1 (Ar–Cq), 134.6(Ar–Cq), 130.2 (2 × Ar–C), 127.2 (2× Ar–C), 99.3 (CCSi), 91.5 (CCSi), 54.1 (OCH2), 44.4 (SCH3), 21.5(Ar–CH3), −0.5 ((CH3)3). HRMS (ESI-TOF) m/z: Calcd. forC15H22NO3SSi [M + H]+:324.1090, found: 324.1081.Preparedusing General Procedure using sulfoxide 10a  and 2-propynyl carbamate. Purification by flash column chromatography afforded sulfilimine propargyl carbamate 12  as a colorless gum. Rf = 0.36 (EtOAc); IR (film)/cm–1 3235, 3019,2922, 2117, 1759, 1625 (C=O), 1245, 1074, 954, 813, 671; 1H NMR  δ 7.68 , 7.34 , 4.67 , 4.63 , 2.82 , 2.42 , 2.40 ; 13C{1H} NMR δ 163.6 (C=O), 143.3 (Ar–Cq), 132.7 (Ar–Cq), 130.6 (2 × Ar–C),126.4 (2 × Ar–C), 79.1 (CCH), 73.9 (CCH), 53.2 (OCH2), 36.1 (SCH3), 21.4(Ar–CH3). HRMS (ESI-TOF) m/z: Calcd. for C12H14NO2S [M + H]+: 236.0745, found: 236.0755.Prepared using General Procedure usingsulfide 10a  and tert-butyl carbamate . Purification byflash column chromatography  afforded N-Boc sulfilimine 13  as a whitesolid. Rf = 0.3 (EtOAc); mp = 152–155°C; IR (film)/cm–1 3011, 2975, 2925, 1625,1361, 1275, 1163, 1079, 985, 835, 817, 786, 753; 1H NMR δ 7.64 , 7.31 , 2.76 , 2.39, 1.44 3); 13C{1H} NMR  δ 164.4 (C=O), 142.8 (Ar–Cq), 133.6(Ar–Cq), 130.5 (2 × Ar–C), 126.1 (2× Ar–C), 78.9 (C(CH3)3), 35.8 (SCH3), 28.4 (C(CH3)3), 21.4 (Ar–CH3). Analyticaldata (NMR and IR) in agreement with those reported in the literature.14Prepared using General Procedure usingsulfide 10b and tert-butyl carbamate . Purification by flash column chromatography  afforded N-Boc sulfilimine 14  as a white solid. Rf =0.15 (EtOAc); mp = 146–149 °C; IR (film)/cm–1 3086, 3012, 1625 (C=O), 1498, 1260, 1156, 984, 835; 1H NMR  δ 7.72 , 7.02 , 3.86 , 2.78 , 1.46 3); 13C{1H} NMR  δ 164.5 (C=O), 162.7 (Ar–Cq), 128.2 (2 × Ar–C), 127.5 (Ar–Cq),115.3 (2 × Ar–C), 78.8 (C(CH3)3), 55.6 (OCH3), 35.9 (SCH3), 28.4(C(CH3)3). HRMS (ESI-TOF) m/z: Calcd. for C13H20NO3S [M + H]+: 270.1164, found: 270.1162.Prepared usingGeneral Procedure using sulfide 10c and tert-butyl carbamate . Purification by flash column chromatography  afforded N-Boc sulfilimine 15  as a white solid. Rf = 0.18 (EtOAc); mp = 142–144 °C; IR (film)/cm–1 3012, 1625 (C=O), 1476, 1282, 1163, 835; 1H NMR δ 7.72 , 7.52 , 2.80 , 1.463); 13C{1H} NMR  δ 164.4 (C=O), 138.5(Ar–Cq), 135.6 (Ar–Cq), 130.2(2 × Ar–C), 127.4 (2 × Ar–C), 79.3 (C(CH3)3), 35.8 (SCH3),28.3 (C(CH3)3). HRMS (ESI-TOF) m/z: Calcd. for C12H17NO2S35Cl [M + H]+: 274.0669, found:274.0662.Prepared usingGeneral Procedure using sulfide 10a  and benzyl carbamate. Purification by flash column chromatography afforded N-Cbz sulfilimine 16  as a colorless oil. Rf = 0.19 (EtOAc); IR (film)/cm–1 3027, 2922,2095, 1625 (C=O), 1446, 1252, 1074, 969, 813, 746; 1H NMR  δ 7.67 , 7.40–7.38 , 7.34–7.24 ,5.13 ,5.07 ,2.80 , 2.41 ; 13C{1H} NMR δ 164.5 (C=O), 143.1 (Ar–Cq), 137.3(Ar–Cq), 133.1 (Ar–Cq), 130.6(2 × Ar–C), 128.2 (2 × Ar–C), 128.1 (2 ×Ar–C), 127.6 (Ar–C), 126.3 (2 × Ar–C), 67.5(OCH2), 36.1 (SCH3), 21.4 (Ar–CH3). HRMS (ESI-TOF) m/z: Calcd.for C16H18NO2S [M + H]+: 288.1058, found: 288.1059.Prepared using General Procedure usingsulfide 11 and tert-butyl carbamate . Purification by flash column chromatography afforded sulfinamidine 17  as a colorless oil. Rf = 0.26 (30% EtOAc in pentane); IR (film)/cm–1 2972, 2936, 2853, 2116, 1630 (C=O), 1273, 1247, 1161, 1029,848, 751, 688, 548; 1H NMR δ 7.91–7.83 , 7.53–7.47, 3.18 , 2.95 , 1.77–1.55 , 1.53 3), 1.53–1.50 ; 13C{1H} NMR  δ 165.0 (C=O),134.6 (Ar–Cq), 131.2 (Ar–C), 128.9 (2 ×Ar–C), 128.1 (2 × Ar–C), 78.7 (C(CH3)3), 48.2 (2 × NCH2), 28.4(C(CH3)3), 26.1 (2 × NCH2CH2), 23.5 (NCH2CH2CH2); HRMS (ESI-TOF) m/z: Calcd. for C16H25N2O2S [M + H]+: 309.1637, found: 309.1630.Prepared using General Procedure using1-(phenylsulphenyl)piperidylamide 4 anhydrous  was added to sulfoximine 2a , benzyl azide , and sodium ascorbate  in tert-butyl alcohol (1.0 mL) and H2O (0.5 mL) at rt. The resulting mixture was stirred at rt for 24h, then quenched with saturated aqueous NH4Cl solution(1.0 mL), and extracted with CH2Cl2 (3 ×5 mL). The combined organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Purificationby flash column chromatography afforded triazole 18  as a white solid.Rf = 0.52 (5% EtOH in EtOAc); mp = 131–134°C; IR (film)/cm–1 3129, 3089, 3062, 2957,2923, 2106, 1660 (C=O), 1443, 1338, 1249, 1224, 1084, 945,859, 720, 683; 1H NMR  δ7.92–7.89 , 7.65–7.61 , 7.54–7.50 ,7.48 , 7.35–7.30 ,7.23–7.20 , 5.47 , 5.43 , 5.14 , 5.09 , 3.25 ; 13C{1H} NMR δ 158.3 (C=O), 143.3 (Ar–Cq), 137.8(CCH), 134.4 (Ar–Cq), 133.9 (Ar–C),129.5 (2 × Ar–C), 128.9 (2 × Ar–C), 128.5(Ar–C), 127.9 (2 × Ar–C), 127.1 (2 × Ar–C),123.4 (CCH), 59.2 (OCH2), 53.9 (NCH2), 44.3 (SCH3); HRMS (ESI-TOF) m/z: Calcd. for C18H19N4O3S [M + H]+: 371.1178, found: 371.1183.CuSO2a , biotin-PEG3-azide ,and sodium ascorbate  were added toa microwave vial and sealed. The vial was degassed and backfilledwith argon three times. CuSO4 anhydrous  was dissolved in a mixture of tBuOH (750 μL)and H2O (375 μL), degassed, and backfilled with argonthree times. CuSO4  was added to the reaction vialand stirred at rt for 24 h. Purification by flash column chromatography affordedthe biotin-PEG3-triazole 19 as a white gum . Rf = 0.18 (5% MeOH in CH2Cl2); IR (film)/cm–1 3295, 2922, 2867,1698 (C=O), 1669 (C=O), 1541, 1449, 1253, 1119, 1090,977, 893, 740, 687, 546, 512; 1H NMR  δ 7.99 , 7.70 ,7.62 , 6.84, 6.40 , 5.52 , 5.19 , 4.54 , 4.35 , 3.88 , 3.59–3.53 , 3.41 , 3.35 , 2.21, 1.67 , 1.43; 13C{1H} NMR  δ 173.4 (C=O), 158.6 (C=O),138.0 (Ar–Cq), 134.1 (Ar–C), 129.8 (2 ×Ar–C), 127.4 (2 × Ar–C), 77.2 (SCH), 70.5 (OCH2), 70.4 (OCH2), 70.3 (OCH2), 70.0 (OCH2), 69.9 (OCH2), 69.3 (OCH2), 61.8 (CONHCH), 60.3 (CONHCH), 59.3 (OCH2), 55.5 (NCH2), 50.5 (NCH2), 44.5 (SCH3), 39.1 (CH2), 35.9 (CH2), 28.2 (2 ×CH2), 25.6 (CH2); HRMS (ESI-TOF) m/z: Calcd. for C29H44N7O8S2 [M + H]+: 682.2693,found: 682.2711. Note: Broadening of the 1H and 13C NMR signals of the triazole ring  occurred toan extent that these were not visible. Similarly the 13C NMR signal for the biotin urea carbonyl (HN-CO-NH) is not observed.Sulfoximine


An elevated amount of free fatty acids (FFA) is a milk quality concern that can contribute to off-flavor, rancidity, reduced foaming ability, and inhibited fermentation that affects cheese coagulation. Free fatty acid concentrations >1.2 mmol/100 g of fat are considered high, although there are various thresholds and units used to quantify levels. The FFA result from milk fat breakdown through spontaneous, bacterial, or induced lipolysis. The amount of bulk tank milk FFA can vary between farms as well as within farms daily, and this mini-review aimed to identify those risk factors at the farm level associated with elevated FFA. A search of the literature identified 5 current sources selected for this review based on relevance. Cows that were milked by automated milking systems (AMS) are suggested to produce milk that is higher in FFA compared with conventional parlors. Factors associated with AMS contributing to spontaneous lipolysis include higher milking frequencies, reduced milking intervals, and low milk yields at each robot visit. Automated milking systems also have characteristics of quarterly milking and high milk lines that can increase vacuum fluctuations and air admission contributing to induced lipolysis. Both AMS and conventional systems with poor tank cooling or without precooling mechanisms can be at risk for higher bulk tank FFA. Bacterial lipolysis can occur when milk temperatures fluctuate and rise, or when there is insufficient milking system cleaning and sanitization. Feed factors such as high saturated fatty acid diets can increase the likelihood of spontaneous lipolysis. We concluded that the major factors associated with increased levels of FFA are non-parlor milking systems, increased air admission, the absence of additional cooling, temperature fluctuations in the bulk tank, and rations high in saturated fatty acids. Future research further investigating these factors can help to minimize FFA and ensure milk quality. FFA) concentration should also be considered a milk quality attribute   . Free faTAG) , a catabTAG) .There is inconsistency in the literature with respect to a suggested sensory threshold for FFA because fatty acid composition in milk varies, and various analytical techniques are used to quantify their levels . Wiking LPL) and influenced by the physiological state of the cow and her nutrition  increases milk fat yields, but it can also increase FFA . SaturatLactating ration ingredients can also influence FFA, and it is thought that diets higher in UFA can be protective . In an eAMS). An association between milking system and higher levels of FFA has been reported in the literature, with tie-stalls and AMS having, on average, higher FFA levels compared with parlors  implemented on a farm include a plate cooling exchanger to efficiently lower the milk temperature . Wiking Maintaining bulk tank function within regulatory requirements and routine system checks are important to prevent elevated FFA concentrations . In AMS,Temperature fluctuations in the bulk tank can add stress to the milk fat globule membrane and risks higher FFA concentrations , 2019. TMinimizing and responding to any bulk tank alarms has also been associated with reduced FFA concentration . Farms wThe range and number of farm factors contributing to elevated FFA concentrations could explain why the increase in FFA is greatest within the first 24 h of milking . It is e


Rehabilitation engineering is the use of engineering sciences and principles to provide technological solutions to improve the quality of life for people experiencing disabilities. Although rehabilitation engineering is a relatively new field of study compared with traditional engineering disciplines, earliest forms of practice date back thousands of years , 2. ThusAccording to World Health Organization (WHO), 1 in 6 people worldwide currently experience a significant disability, 1 in 3 people are living with a health condition that can benefit from rehabilitation, and these numbers are expected to increase with growing aging populations and people living with chronic disease . As rehaLife changing breakthroughs have been made in rehabilitation engineering, resulting in a great variety of devices, tools, and systems that meet a wide range of needs for individuals experiencing functional deficits in mobility, communication, hearing, vision, or cognition . The achFor rehabilitation engineering, much of the activities are driven by the needs of people experiencing disabilities. Rehabilitation engineers, instead of working in isolation, should work closely with patients and clinicians, viewing them as part of the team to facilitate better outcomes. It is worth noting that the current development in rehabilitation engineering has not fully met the priority needs of patients or consumers. There exists a discrepancy between areas of research of the most cited rehabilitation engineers and consumer priorities . A pressIn a recent review of NIDILRR's RERC activities, it was reported that 70% of the research and development staff of RERCs are in engineering fields, 23% in clinical fields, and only 7% come from basic science fields . AlthougRehabilitation engineering also faces a grand challenge in education and training. Given the demands of the field to produce positive outcomes for patients, there is an unmet need to establish professional qualification programs in rehabilitation engineering. Initiatives to address the challenge can include establishment of common core curriculum and specialist curriculum , development of textbooks and other teaching and learning materials, summer schools, continued education, and online courses, etc. Of particular note, rehabilitation technology is to serve our patients, so there is an urgent need to develop practical technology design and implementation techniques. These may well require cross-disciplinary training of engineers by clinicians and similar training of clinicians by engineers. In addition, a systematic examination has revealed gender and geographical disparities in rehabilitation engineering . There iFrontiers in Rehabilitation Sciences (As a nascent but fast-growing field of endeavor, it can be anticipated that rehabilitation engineering will remain intensely active and vibrant. The field offers nearly unlimited potential for continued assistance of day-to-day activities related to independent living, education, employment, and recreation of people experiencing disabilities. The journey promises to be exciting and hopeful, but challenging. In line with the mission of Sciences , the “Re


Considerable progress has been made in saving the lives of children younger than 5 years. Nevertheless, these advances have failed to help all children thrive, particularly children with disabilities. We describe the increasing prevalence of disability among children and adolescents. We evaluate the current situation regarding children with disabilities and rehabilitation in the context of health systems, particularly those in low- and middle-income countries. Within the newborn health agenda, congenital anomalies often require early intervention and rehabilitation. We provide Argentina as an example of a country where rehabilitation for congenital anomalies is integrated into the health system. We argue that congenital anomalies that require rehabilitation have the potential to strengthen rehabilitation systems and policies by: strengthening coordination between primary care and rehabilitation; identifying and understanding pathways that allow families to engage with services; providing human resources for rehabilitation; and building systems and resources that support assistive technology and rehabilitation. We propose ways for countries to prioritize and integrate early identification, referral and care for children with congenital anomalies to strengthen health systems for all. We identify opportunities to expand policy and planning and to design service delivery and workforce strategies through World Health Organization guidelines and frameworks for rehabilitation. We argue that the global health community must act to ensure that rehabilitation services to support functioning from birth are well established, accepted and integrated within health systems, and that disability is prioritized within child health. These steps would strengthen health systems, ensure functioning from birth and make rehabilitation accessible to all. There are at least three complementary reasons why countries should urgently prioritize rehabilitation services to promote functioning from birth. First, while remarkable gains in child survival have been made over the past three decades, this progress has exposed a growing inequity. The likelihood of a child having a disability before their fifth birthday was 10 times higher than the likelihood of dying (377 versus 38 per 1000 live births) in 2019,,Second, affordable, timely and accessible rehabilitation and assistive technology improves health and well-being, functioning and participation,Third, providing access to appropriate care, including rehabilitation, upholds the rights of people with disabilities. Global political support to address inequity in child health is enshrined in the Convention on the Rights of the Child,,,Despite a large and growing need, provision of rehabilitation and assistive technology is affected by systemic exclusion from global health financing, leadership, and programming relative to investments in other conditions.,The need to strengthen early intervention and rehabilitation is recognized within the survive and thrive newborn health agendaIn 2020, WHO published updated guidance for screening and reporting congenital anomalies.Among the 15 major external congenital anomalies, three external anomalies are classified as incompatible with life: anencephaly, craniorachischisis and iniencephaly. Three conditions – exomphalos , gastroschisis and hypospadias – require surgical correction only. The remaining nine conditions all benefit considerably from early intervention, rehabilitation and provision of assistive technology .,,Systems and policies to support rehabilitation in children with congenital anomalies do not have to be at odds with providing health care for all. Moreover, they may provide an entry point to make rehabilitation accessible to all. Clubfoot and limb reduction are good examples of congenital anomalies that are easily recognizable and detected at birth, and require early intervention to support functioning. Rehabilitation and assistive technology can result in substantial, sometimes complete, improvement of function for children with clubfootStrengthening the provision of rehabilitation services for such conditions within primary health-care clinics and centres builds capacity that will be useful for all individuals who may benefit from rehabilitation services. The systems and resources supporting assistive technology for these two conditions are similar for most birth anomalies, injuries and childhood disabilities. Therefore, the shared challenges and benefits of addressing clubfoot and limb reduction can be extrapolated to numerous childhood conditions to build appropriate models of care in low- and middle-income countries.,Argentina is an example of a country that has prioritized systems for integrating early identification, referral and care for children with congenital anomalies. The National Network of Congenital Anomalies of Argentina started in November 2009 in four provinces in Argentina as a hospital-based registry.We foresee several ways for countries to prioritize and integrate early identification and referral of and care for children with congenital anomalies, and thus to strengthen health systems for all.Identifying children with congenital anomalies within the primary care system and referring them for care would strengthen coordination between primary care and rehabilitation. This approach would also address policy and leadership gaps for rehabilitation at health ministries and in health systems governance.,At the community level, stigma, negative attitudes of caregivers and health-care providers, and a lack of knowledge and skills among health workers are barriers to early intervention.Strengthening multidisciplinary coordination for congenital anomalies can improve the health system functioning for all. When rehabilitation is prioritized from birth, as is indicated for clubfoot and limb reduction, for example, these multidisciplinary connections are enhanced, as rehabilitation services are coordinated across several care teams. For clubfoot, the Ponseti method may require a surgeon to perform an outpatient Achilles tenotomy or to address recurrence;Strengthening systems and resources that support rehabilitation and assistive technology at the policy level will assist policy-makers in implementing services for care and rehabilitation. Currently, the absence of appropriate assistive technology supply chains, political will and global investment limits rehabilitation efforts; only an estimated 5–15% of people who need assistive technology can access it in low- and middle-income countries.Opportunities exist to expand planning for rehabilitation through WHO guidelines and frameworks, which shape governments’ health expenditure, development assistance and targets. For example, in the absence of adequate government-led investment in rehabilitation in low- and middle-income countries, WHO’s Rehabilitation 2030For referral systems to function, they require national data, formalized roles, supervision and leadership, and targets. In Argentina, reporting into centrally managed open source medical records systems allows patients to be tracked and trends in treatment to be monitored over time. Adequate record-keeping is essential to identify children who are being lost to follow-up. While success of treatment can be determined through analysis of documented clinical findings, complex paperwork systems or the lack of a recording system make central analysis and identification of individual clinic results difficult. Rehabilitation practitioners may be motivated to record data that are seen as immediately beneficial, and identification of simple processes to collect these data is needed. Data are also vital for monitoring service availability and quality. Integrating the Rehabilitation 2030 service delivery indicatorsGaps remain in the health-policy and systems-research agenda to prioritize functioning from birth, and these gaps present opportunities for future research. More widespread and nuanced evidence of systems approaches to managing common congenital and childhood causes of impairment in low- and middle-income countries is essential to inform national rehabilitation plans that guide efforts to make these services accessible from birth. Valid, reliable and timely data are essential for priority-setting, planning and delivery of high-quality rehabilitation services to all. For example, accurate national birth prevalence data and population estimates of congenital anomalies are needed for strategic planning and proportionate resource allocation for rehabilitation and assistive technology services. These data require enhanced country-level information systems to support complete and accurate reporting of newborn and child health indicators on functioning and impairments. These enhancements, in turn, will link rehabilitation to broader improvements in national health systems and health information systems. Likewise, reporting on and monitoring rehabilitation-specific professionals in national human resources information systems is vital to inform workforce development and distribution. Global evidence-based guidelines for the treatment and rehabilitation of children with congenital anomalies are also needed to strengthen the quality of service provision. Data on coverage and access to rehabilitation services for children with congenital anomalies, as well as other social determinants of health and equity measures, need to be included at local levels of health systems to ensure performance management for equity and to inform rehabilitation and assistive technology provision. Integrating these data with routine health information systems and ensuring the data are made accessible and actionable for national and subnational health leadership is essential to strengthen rehabilitation services from birth.Rehabilitation needs a focus to move beyond rhetoric to strategy and harness political will and resources. A strategy that prioritizes identifying and supporting babies with congenital anomalies can strengthen rehabilitation and assistive technology delivery for children and adolescents with other conditions. A rights-based approach is vital. This approach requires a fundamental shift in understanding that the functioning of an individual is central to population health and to society, and equal in benefit to preventing disability and death. By prioritizing disability in child health, governments and global agencies can support the health system infrastructure required to enhance rehabilitation and disability-inclusive services for all.


Psychiatry has long been battling with a recruitment crisis in the UK which is also reflected across much of Europe. Covid-19 has brought about widespread changes to our ways of working, as well as driving technological developments, which provides potential opportunities for the profession to draw people into the speciality. Covid-19 has brought interest in digital psychiatry from the peripheries to the mainstream. Mental health professionals are currently using sophisticated technologies such as Virtual Reality, Artificial Intelligence and Natural Language Processing in the diagnosis and treatment of mental health disorders. Highlighting the ways in which our profession is at the cutting edge of innovation to junior doctors offers a fruitful avenue to improve recruitment into the speciality. Many outpatient clinics have made the move to online service delivery during the pandemic to varying degrees. For many clinicians this has allowed more flexible and efficient ways of working. Psychiatry is better placed than most other medical disciplines to retain online patient contact in future clinical practice, post pandemic and may provide an attractive proposition for future psychiatrists. This talk will review some of the ways in which developments in digital psychiatry have been used to help generate interest for recruitment into the discipline as well as evaluating the benefits and challenges of the shift to telepsychiatry during Covid-19 and will offer some suggestions what the profession can learn from this to help future recruitment.No significant relationships.


Elevated androgen receptor (AR) expression is a hallmark of castration-resistant prostate cancer (CRPC) and contributes to the restoration of AR signaling under the conditions of androgen deprivation. However, whether overexpressed AR alone with the stimulation of castrate levels of androgens can be sufficient to induce the reprogramming of AR signaling for the adaptation of prostate cancer (PCa) cells remains unclear. In this study, we used a PCa model with inducible overexpression of AR to examine the acute effects of AR overexpression on its cistrome and transcriptome. Our results show that overexpression of AR alone in conjunction with lower androgen levels can rapidly redistribute AR chromatin binding and activates a distinct transcription program that is enriched for DNA damage repair pathways. Moreover, using a recently developed bioinformatic tool, we predicted the involvement of EZH2 in this AR reprogramming and subsequently identified a subset of AR/EZH2 co-targeting genes, which are overexpressed in CRPC and associated with worse patient outcomes. Mechanistically, we found that AR-EZH2 interaction is impaired by the pre-castration level of androgens but can be recovered by the post-castration level of androgens. Overall, our study provides new molecular insights into AR signaling reprogramming with the engagement of specific epigenetic factors. AR gene expression that can happen rapidly through a negative-feedback mechanism or by AR gene amplification or alterations of AR regulatory elements  plays a pivotal role in prostate cancer (PCa) initiation and progression . AR tranelements –7. Whilegression , it is sEZH2, a key component of polycomb complex (PRC2), functions as a lysine methyltransferase to methylate histone 3 lysine 27 (H3K27)  and is oAR. Treating with the combination of doxycycline and different doses of androgens, this system can provide ARlow/DHThigh or ARhigh/DHTlow status to mimic the levels of the receptor and ligand under pre- and post-castration conditions. Our data demonstrated that induced overexpression of AR can rapidly alter the AR chromatin binding, which results in specific activation of a subset of genes that are highly enriched for DNA replication and damage repair pathways. Using a recently developed transcription factor predication algorism, we have revealed the involvement of EZH2 in this reprogramming of AR and identified a 68-gene signature to predict activities of AR/EZH2 co-regulated gene transcription in CRPC. Our data further revealed that the expression of these genes is significantly increased in CRPC tumor samples in comparison with primary PCa samples and is associated with poor clinical outcomes. Moreover, we also found that AR-EZH2 interaction is impaired by high-dose androgen stimulation but maintained by low-dose androgens. Together, our data provide a global view of AR signaling reprogramming in CRPC driven by increased AR expression and decreased ligand levels and suggest the engagement of EZH2 for this activity of AR.In this study, we have generated a lentiviral stable cell line with doxycycline-inducible overexpression of LNCaP and C4-2 cell lines were purchased from ATCC, authenticated every six months using short tandem repeat (STR) profiling, and frequently tested for mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza). LNCaP cells were cultured in RPMI with 10% FBS  and C4-2 cells were cultured with 2% FBS plus 8% charcoal-stripped FBS (CSS). LNCaP stable cell line overexpressing tetracycline-regulated AR (LNCaP-tet-AR) was generated by lentiviral infection of pLIX_403 tetracycline-inducible lentiviral vector with wild-type full-length AR, using the Gateway Technology with Clonase II . LNCaP-tet-AR cells were cultured in RPMI with 10% tetracycline-free FBS. For androgen stimulation assays, cells were grown to 50-60% confluence in culture medium containing 5% CSS for 3 days (d) and then treated with DHT or inhibitors.For immunoprecipitation assays (IP), cells were lysed in Triton Lysis buffer and treated with protein inhibitor cocktails (Thermo Fisher Scientific). Lysates were then immunoprecipitated with anti-AR  antibody and proteins were eluted by boiling in Laemmli buffer with 5% beta-mercaptoethanol. For immunoblotting, cells were lysed with RIPA buffer containing protease inhibitor cocktail. Anti-BRD4 , anti-EZH2 , anti-AR , and anti-GAPDH  were used as primary antibodies.RNA was isolated from cells with TRIzol reagent (Invitrogen), Quantitative real-time PCR was performed using Fast 1-step Mix (Thermo Fisher Scientific) on QuantStudio 3 PCR machine. All TaqMan primer/probe sets were predesigned and purchased from Thermo Fisher Scientific, and PCR results were normalized to GAPDH.LNCaP-tet-AR cells were pre-cultured with 5% CSS. Cells were then plated into 12-well plates supplemented with/out 0.25 mg/ml doxycycline. After 1-2d, cells were treated with DHT for 24 hours (h) and then treated with DMSO, olaparib , cisplatin , or GSK126  for 0-6d. Cells were trypsinized, collected, and counted by countess II automated cell counter (Invitrogen).For the preparation of ChIP, dispensed cells were formalin-fixed, lysed, and sonicated to break the chromatin into 200–300 bp fragments (500-800 bp fragments for ChIP-qPCR assays), followed by immunoprecipitation with ChIP grade antibodies: anti-AR , anti-EZH2 , or Rabbit/Mouse IgG (Millipore). Primer sequences for ChIP-qPCR (using SYBR green) are: BRCA1-ARE forward, 5’-AATGGGGATGACAAGACAGG-3’; BRCA1-ARE reverse, 5’-AGGGGTGGACCCTACATTATC-3’; BRCA2-ARE forward, 5’-TGTAAGCAGATTTGTTGAATATTTG-3’; BRCA2-ARE reverse,5’-ATCCAGGAGGCATTGCATAA-3’; BRIP1-ARE forward, 5’- TCAGCACCACATCGCACT-3’; BRIP1-ARE reverse, 5’-CACACTGAGAGATTCTGGTACG-3’; MCM2-ARE forward, 5’-GTACAGTGGCACGCAGCTC-3’; MCM2-ARE reverse, 5’-AGGGCGGAGCTTTGTGTAT-3’.q-value = 0.05, fix-bimodal and extend size set as 200 using the narrow peak caller. The bedGraph files containing signal per million reads produced from MACS were converted into bigwig files using UCSC tools (version 372). The R package ChIPpeakAnno (version 3.26.4) was used to analyze peak intervals and determine the overlapped regions. Venn diagrams were generated using VennDiagram (version 1.7.3) R package. The signals associated with genomic regions were visualized by using computeMatrix and plotHeatmap tools from deepTools (version 3.0.2). computeMatrix with reference-point mode was used to calculate scores for each genomic region, and plotHeatmap was used to create a heatmap for scores associated with genomic regions. MACS-generated peaks were ranked by their score ) and the top 5,000 peaks were imported to SeqPos in Galaxy/Cistrome (ChIP-seq libraries were constructed using the SMARTer ThruPLEX DNA-Seq Prep Kit (Takara Bio USA). Next-generation sequencing  was performed using Illumina NextSeq 2000 Genome Analyzer. ChIP-sequencing reads were mapped to the hg19 human genome using BWA (version 0.7.5a) with aln and samse sub-commands (-l 32 -q 5 -k 2) . SamtoolCistrome  for motiCistrome  to integP value  . RNA-Seq library was prepared using TruSeq Stranded RNA LT Kit (Illumina). Sequencing was performed on NextSeq 2000 Illumina Genome Analyzer. The single-end reads were processed by FastQC (version 0.11.6)  and aligP value  with msin 0.2.1)  was utilARhigh/DHTlow and ARlow/DHThigh gene signatures is as follows: (i) The genes for both conditions were filtered by fold-change > 1.5, FDR < 0.05 and direct targets of peaks were generated by ChIP-seq; (ii) these two filtered gene sets were overlapped and the unique genes for each group were selected; (iii) For each unique gene sets, the genes that have fold-change difference of greater than 1.5 to other groups, were selected. The public RNA-seq data of siEZH2 in LNCaP-ABL cells (GSE39452) (The process to identify the SE39452)  were retARlow/DHThigh and ARhigh/DHTlow were defined as the fold-change > 1.5 with FDR < 0.05 in comparison with vehicle. The genes were ranked by fold-change and FDR and fed to Causaul Inference Engine (CIE)   to identt-test by comparing treatment versus vehicle control or otherwise as indicated. P-value < 0.05 was considered to be statistically significant . Box plots of the signature score and gene expression were compared using the Wilcoxon test for comparison between the two conditions. All statistical analyses and visualization were performed with R (version 4.1.1) unless otherwise specified.Data in bar graphs represent mean ± SD of at least 3 biological repeats. Statistical analysis was performed using Student’s ARlow/DHThigh and ARhigh/DHTlow, which mimic the AR status under pre- and post-castration conditions, respectively. Cells can proliferate under both conditions but ARhigh/DHTlow cells grew slightly slower (ARhigh/DHTlow condition) with our previously published AR ChIP-seq in parental LNCaP cells stimulated with 10nM DHT (ARlow/DHThigh condition) . As shown in ARhigh/DHTlow cells, indicating a reprogramming of AR cistrome despite that the genomic distribution of AR binding sites was similar under two conditions  overexpressing tetracycline-regulated AR. As shown in  in CRPC –26.ARhigh/DHTlow cells. A comprehensive RNA-seq analysis was performed in LNCaP-tet-AR cells under both ARlow/DHThigh and ARhigh/DHTlow conditions, and DHT-upregulated and downregulated genes were identified  with hallmark gene set was conducted to provide a comprehensive view of AR transcriptomes under different conditions. As predicted, the androgen response pathway was enriched under all four conditions  and MTORC1 signaling. In addition, AR also uniquely repressed several pathways including apoptosis, hypoxia, p53, and myogenesis in ARhigh/DHTlow cells. By directly comparing androgen-regulated genes in ARlow/DHThigh versus ARhigh/DHTlow cells, we identified 579 ARlow/DHThigh-unique genes, 1,242 common genes, and 1,213 ARhigh/DHTlow-unique genes. Remarkably, the ARhigh/DHTlow-unique genes were highly enriched for DNA replication and damage repair functions (We next sought to determine the change of AR transcriptome in is genes . More imPC cells . Other AARhigh/DHTlow unique AR binding sites (2801) were also associated with AR-activated genes, suggesting that these AR bindings are transcriptionally active (ARlow/DHThigh and ARhigh/DHTlow conditions. While the ARlow/DHThigh signature (58-gene) had a similar expression pattern as the previously reported classic AR target signature (10-gene) (ARhigh/DHTlow signature (27-gene) was clearly regulated in a different pattern  , we foun10-gene) , the ARgnature 2-gene wasBRCA1, BRCA2, BRIP1 (BRCA1 interacting helicase 1), and MCM2 (subunit of DNA helicase protein), which all harbor unique AR binding sites in ARhigh/DHTlow cells (KLK3 and NKX3.1) which are similarly induced under both ARlow/DHThigh and ARhigh/DHTlow conditions  but negatively associated with ARlow/DHThigh signature  with poor clinical outcomes in response to ARSi treatments , we found that 71 genes were possibly co-targeted by EZH2 and AR in reatment , 35. MorARlow/DHThigh condition but recovered under ARhigh/DHTlow condition, suggesting that EZH2 may enhance AR transcription program preferentially in CRPC but not in primary PCa. Interestingly, AR-BRD4 interaction also showed a similar pattern as EZH2. Consistent with the protein-protein interaction results, EZH2 chromatin binding at those gained AR binding sites was markedly higher in ARhigh/DHTlow cells than in ARlow/DHThigh cells  and post-castration (ARhigh/DHTlow) AR status and then examined whether the combination of AR overexpression with low levels of ligands may significantly alter AR transcription program. Our study clearly indicates that PCa cells switching from ARlow/DHThigh to ARhigh/DHTlow status can acutely and dramatically alter AR chromatin binding and transcription outputs. More importantly, we fully characterized this AR reprogramming and revealed that one critical outcome is the distinct activation of genes mediating DNA replication and damage repair pathways, which may allow CRPC cells to become resistant to genotoxic stress. Interestingly, our previous studies indicated that the combination of AR overexpression and high-dose androgens can transcriptionally suppress DNA replication genes, which provides a molecular basis for the high-dose testosterone treatment in CRPC  can be found below: Gene Expression Omnibus, series number: GSE211641.ML, DH, and CC designed the study. ML, MQL, LT, BK, SZ, SG, SP, JM, KZ, and DH performed experiments and analyzed the results. ML, DH, and CC wrote the manuscript. All authors contributed to the article and approved the submitted version.This work is supported by grants from NIH (R01 CA211350 to CC and U54 CA156734 to JM and CC), DOD . CC and DH were supported by Proposal Development Award from University of Massachusetts Boston. ML and SZ were supported by the graduate fellowship from the Integrative Biosciences/Computational Sciences Programs at the University of Massachusetts Boston. LT and BK were supported by University of Massachusetts Boston/Dana-Farber Cancer Institute Partnership Summer Program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Androgen-dependent nuclear accumulation of pathogenic AR protein causes degeneration of lower motor neurons, leading to progressive muscle weakness and atrophy. While the successful induction of SBMA-like pathology has been achieved in mouse models, mechanisms underlying motor neuron vulnerability remain unclear. In the present study, we performed a transcriptome-based screening for genes expressed exclusively in motor neurons and dysregulated in the spinal cord of SBMA mice. We found upregulation of Mid1 encoding a microtubule-associated RNA binding protein which facilitates the translation of CAG-expanded mRNAs. Based on the finding that lower motor neurons begin expressing Mid1 during embryonic stages, we developed an organotypic slice culture system of the spinal cord obtained from SBMA mouse fetuses to study the pathogenic role of Mid1 in SBMA motor neurons. Impairment of axonal regeneration arose in the spinal cord culture in SBMA mice in an androgen-dependent manner, but not in mice with non-CAG-expanded AR, and was either exacerbated or ameliorated by Mid1 overexpression or knockdown, respectively. Hence, an early Mid1 expression confers vulnerability to motor neurons, at least by inducing axonogenesis defects, in SBMA.Spinal and bulbar muscular atrophy (SBMA) is an adult-onset hereditary neurodegenerative disease caused by the expansions of CAG repeats in the  SBMA is accompanied by the degeneration of lower motor neurons in the spinal cord and specific brainstem motor nuclei and atrophy of skeletal muscle resulting in progressive motor impairment . PatientAR) gene .Androgen-dependent nuclear accumulation of mutant AR protein causes neurological symptoms , typicalcpg15/neuritin [Androgen-dependent regulation of axonogenesis in motor neurons has been extensively studied so far . Androgeneuritin , 12.dynactin1 and impairs retrograde axonal transport driven by dynein [Several mechanisms have been proposed for how pathogenic AR disrupts axonal transport. The aggregates of pathogenic nuclear AR cause toxicity, which downregulates the gene expression of y dynein . Non-aggy dynein . Hence, y dynein . Pathogey dynein , 18. Midy dynein , which py dynein , 21. They dynein . HoweverMid1, a development-related gene, specifically expressed in the spinal motor neurons and upregulated in the spinal cord of SBMA mice. Using spinal cord slice cultures from the embryos of SBMA mice, we found that overexpression of Mid1 increased the pathogenic AR protein levels, thereby inducing the androgen-dependent impairment of axonogenesis. Our findings indicate a mechanism for the Mid1-associated axonal vulnerability in SBMA motor neurons.Here, we performed a transcriptome-based screening of genes and identified AR-24Q and pCR3.1-AR-97Q, which drive expression of full-length human AR containing 24 and 97-expanded CAGs, respectively, under the control of a cytomegalovirus enhancer and a chicken β-actin promoter as previously described [Mid1 cDNA was amplified from the cDNA obtained from mouse spinal cord on embryonic day 14.5 (E14.5) and was cloned into the NotI/EcoRI site of pcDNA3.1-mycHisC to create pcDNA3.1-Mid1-mycHisC. Infusion system (Invitrogen) was used for cloning Mid1 cDNA together with the sequence encoding the self-cleaving peptide 2 A into pLenti-GFP(C), resulting in pLenti-GFP-2A-Mid1. The plasmids for shRNA expression were obtained by inserting the target sequences into the AgeI/EcoRI site of pLKO.1 puro (Addgene). The target sequences of shRNA for mouse Mid1 and human AR were GACTTGCGTTACTTGTGAA [The plasmids pCR3.1-escribed  were useCTTGTGAA , respect2. The plasmids were transfected using OPTI-MEM (Gibco) and Lipofectamine 2000 (Invitrogen). Ten hours after lipofection, the culture medium was changed to DMEM containing 2% FBS and 50 nM dihydrotestosterone (DHT). In the case of cycloheximide (CHX) treatment experiments, the culture medium was changed to DMEM containing 2% FBS and 80 μg/mL CHX, 24 h after lipofection.Mouse NSC34 motor neuron-like cells  were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). In addition, cells were maintained in a 37 °C incubator with a humidified atmosphere of 95% air/5% COProtein isolation was performed using Cellytics MT Cell Lysis Reagent (Sigma-Aldrich). We separated equal amounts of protein on 5–20% SDS-PAGE gels (FUJIFILM Corp.) and transferred them to Hybond-P membranes . The following primary antibodies were used with indicated dilutions: rabbit anti-AR pAb , mouse anti-Gapdh mAb , rabbit anti-MID1 pAb , rabbit anti-PP2Ac pAb , rabbit anti-GFP pAb . The density of each band was quantified using ImageJ software . For RNA pull-down assay, cell lysate containing ~300 μg of total protein derived from HEK293T cells overexpressed with Mid1 was incubated for 1 h with 3 μg of in vitro-transcribed RNA biotinylated with Biotin-16-UTP (Sigma-Aldrich). It was followed by incubation with Dynabeads M-280 streptavidin (Invitrogen) for 1 h at 4 °C and washed with wash buffer three times.https://www.r-project.org). The resultant data was subjected to the differential gene expression analysis using an integrated web application for Differential Expression and Pathway analysis (iDEP) [http://spinalcordatlas.org).Previously published microarray data on Gene Expression Omnibus, GSE39865 , was nors (iDEP) . Cell tys (iDEP)   and RNeasy Mini Kit (QIAGEN). The extracted RNA was then reverse-transcribed into first-strand cDNA using ImProm-II Reverse Transcription system (Promega). Quantitative PCR was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO), and the amplified products were detected with the iCycler system (Bio-Rad Laboratories). The following primers were used for PCR: Mid1 and AR-24Q or AR-97Q and anti-His-tag mAb-Magnetic beads (MBL) (or nonspecific Mouse IgG2a (isotype control)-Magnetic beads (MBL) antibody) for 1 h at 4 °C and washed with Cellytics MT for three times, followed by RNA purification using TRIzol (Invitrogen) and RNeasy Mini Kit (QIAGEN) and examined by RT-PCR using rTaq DNA polymerase (TaKaRa) and the AR forward/reverse primers mentioned above.RNA immunoprecipitation (RIP) was performed using the total cell lysates from HEK293T cells co-expressing His-tagged Cultured spinal cord slices were fixed with 4% PFA, immersed in 20% sucrose, embedded in OCT compound, and cryo-sectioned (16 μm thickness). Sections were incubated overnight at 4 °C with the following primary antibodies with the indicated dilution: rabbit anti-MID1 pAb ; mouse anti-Islet1&2 mAb ; rabbit anti-AR  mAb; mouse anti-non-phosphorylated NF-H mAb ; rabbit anti-Pnpt1 . After PBS washes, the sections were incubated for 1 h at room temperature with the following secondary antibodies: Donkey anti-Rabbit IgG-Alexa488 and Donkey anti-Mouse IgG-Alexa546 . After incubation, the sections were counterstained with DAPI and mounted with a mounting solution. Images were acquired using CoolSNAP CCD camera (Photometrics) assembled on BX60 upright microscope (Olympus).GFP(C), pLenti-GFP-2A-Mid1, pLKO.1-contol shRNA , pLKO.1-Mid1 shRNA, or pLKO.1-human AR shRNA together with pLP1, pLP2, and pVSV-G (Invitrogen) using Lipofectamine 2000 (Invitrogen). The lentiviral-containing supernatants were collected 48 h after transfection and were concentrated by ultracentrifugation using Lenti-X Concentrator (Takara Bio USA). The viral titers were measured using Lenti-X qRT-PCR Titration Kit . The final concentration of lentiviral particles in the culture medium was ~5.0 × 107 IU/mL.Lentiviral particles were produced in HEK293T cells by transfection of pLenti-β-actin promoter were generated in our previous study [AR forward/reverse, 5'-CTTCTGGCGTGTGACCGGCG-3'/ 5'-TGAGCTTGGCTGAATCTTCC-3'; Sry forward/reverse, 5'-TGGGACTGGTGACAATTGTC-3'/5'-GAGTACAGGTGTGCAGCTCT-3'. Mice were fed with a standard diet and maintained on a 12 h light/dark cycle. Pregnant mice were obtained by crossing female AR-97Q transgenic mice with wild-type C57BL/6 J male mice.AR-97Q transgenic mice expressing full-length AR containing 97-expanded CAGs under the control of a cytomegalovirus enhancer and a chicken us study . The mic2, 40% O2) to allow for further gel solidification (5–10 min). An enriched medium (1 ml) was added to the gel and spread over the entire dish surface. Lentivirus  or DHT  was directly added to the dish at the start of culture (day 0). Images of cultured spinal cord slices were acquired using ORCA-flash 4.0 v3 camera (Hamamatsu Photonics) assembled on an IX71 inverted microscope (Olympus).Cross-sectional slices of the spinal cord (250–300 μm thickness) were prepared as previously described for embryonic cerebral wall , 29 and t-test to compare two groups and analysis of variance (ANOVA) with Tukey’s test for multiple comparisons using Prism 8 Software (GraphPad). We considered p < 0.05 to indicate statistical significance. The data were checked for normality using Shapiro-Wilk test and for equality of variances using F-test. In the microarray data analysis, the false discovery rate (FDR) < 0.05 was considered to be statistically significant.We analyzed the data with unpaired two-sided To identify genes involved in the degeneration of motor neurons, we reanalyzed the transcriptomes from the spinal cord of AR-97Q mice at different ages: 7–9, 10–12, and 13–15 weeks  and combMid1, which encodes a microtubule-associated RNA binding protein [Mid1 was restricted to the spinal motor neurons according to the single-cell transcriptional atlas -derived motor neurons from SBMA patients [Pnpt1, which had the second-largest fold change, using immunofluorescence, but the expression pattern of it was not motor neuron-specific in the spinal cord  in the cultured AR-97Q spinal cord slices treated with DHT , which strongly stains the cell bodies and weakly stains the axons of the motor neurons . We founMid1 could exacerbate the androgen-dependent impairment of axonogenesis in the spinal cord slices from AR-97Q mice, Mid1 overexpression would further impair the axonogenesis defects. To test this possibility, we introduced Mid1 to the spinal cord slice culture by lentivirus-mediated transduction (Fig. Mid1 resulted in increased pathogenic AR protein levels in a similar manner to that observed in the cellular model (Figs. Mid1 overexpression alone without DHT treatment resulted in only moderate impairment of axonogenesis, while Mid1 overexpression showed severe impairment of axonogenesis compared with that caused by DHT alone (Fig. Mid1 exacerbates the androgen-dependent impairment of axonogenesis in the spinal cord slice culture from AR-97Q mice, especially under androgen exposure.If ion Fig. . Overexpel Figs. b, 2a. Siel Figs. , we usedel Figs.  and thusone Fig. , which wone Fig. . These rMid1 knockdown rescues the axonogenesis defects. We first confirmed that knockdown of Mid1 did not affect axonogenesis in the spinal cord slice culture from wild-type mice (Supplementary Fig. Mid1 knockdown decreased the pathogenic AR protein levels (Fig. Mid1 knockdown increased the protein levels of PP2Ac, a ubiquitination target of Mid1 involved in the translational regulatory complex of mTOR [Mid1 knockdown ameliorated the axonogenesis defects (Fig. Mid1 is responsible for the androgen-dependent impairment of axonogenesis in the spinal cord slice culture from AR-97Q mice. In addition, knockdown of the human AR transgene rescued the impairment of axonogenesis regardless of co-silencing of Mid1 (Fig. We further examined whether els Fig. . Further of mTOR  (Supplemcts Fig. , suggestid1 Fig. . OverallAR mRNA and increases the protein levels of pathogenic AR in both cellular and organotypic slice culture models of SBMA. The transcriptional upregulation of Mid1 by pathogenic AR induced the translational upregulation of pathogenic AR in the spinal cord of early symptomatic SBMA mice and accelerated the motor neuron pathology in SBMA. Interestingly, AR has been shown to repress the transcription of Mid1, forming a negative feedback loop leading to suppression of AR protein levels [Mid1 transcription. The bi-directional feedbacks between pathogenic AR and Mid1 could contribute to the vulnerability of motor neurons in SBMA.The present study demonstrates that Mid1, a microtubule-associated RNA binding protein exclusively expressed in the motor neurons of the spinal cord, interacts with CAG-repeat expanded n levels . Hence, Although SBMA is an adult-onset disease, studies in the primary culture of embryonic motor neurons from the mouse model of SBMA have suggested that both transcriptional and translational dysregulation by pathogenic AR impair motor neuron homeostasis as an early dysregulation during development , 40. OurMid1 (Supplementary Fig. Mid1 and AR suggest that the impairment of axonogenesis by Mid1 depends on the pathogenic AR (Fig. Our study indicates that the impairment of axonal growth by pathogenic AR partly depends on the translational upregulation of pathogenic AR by Mid1. It has also been reported that axonal growth could be directly regulated by Mid1-PP2A system , 42. Sti AR Fig. . AnotherOur study demonstrated that the embryonic spinal cord slice culture is a useful model for elucidating the axonal vulnerability in SBMA, which might reflect the early dysregulation of motor neurons in vivo. In the organotypic slice culture, axonogenesis is supported by the surrounding extracellular matrix and glial cells , in addiAll animal experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and with the approval of the Nagoya University Animal Experiment Committee.Reproducibility checklistOriginal Data FileSupplemental Data