In this study, we identified C7/C8-cyclitols and C7N-aminocyclitols as potential inhibitors of Streptomyces coelicolor (Sco) GlgEI-V279S based on the docking scores. The protein and the ligand  were prepared, the states were generated at pH 7.0 ± 2.0, and the ligands were docked into the active sites of the receptor via Glide™. The synthetic route to these targets was similar to our previously reported route used to obtain 4-⍺-glucoside of valienamine (AGV), except the protecting group for target 12 was a p-bromobenzyl (PBB) ether to preserve the alkene upon deprotection. While compounds 11–13 did not inhibit Sco GlgEI-V279S at the concentrations evaluated, an X-ray crystal structure of the Sco GlgE1-V279S/13 complex was solved to a resolution of 2.73 Å. This structure allowed assessment differences and commonality with our previously reported inhibitors and was useful for identifying enzyme–compound interactions that may be important for future inhibitor development. The Asp 394 nucleophile formed a bidentate hydrogen bond interaction with the exocyclic oxygen atoms (C(3)-OH and C(7)-OH) similar to the observed interactions with the Sco GlgEI-V279S in a complex with AGV (PDB:7MGY). In addition, the data suggest replacing the cyclohexyl group with more isosteric and hydrogen bond–donating groups to increase binding interactions in the + 1 binding site.C For example, derivatives of valienol (streptol)  (4) are also known inhibitors of α-glucosidases, 5) has important applications in agriculture due to its insecticidal, fungicidal, and fungistatic activities. The antifungal activity of 5 is attributed to its ability to inhibit trehalase (Treh) in the fungi (6) is a common core for validamycins A, C, D, E, and F and is also known inhibitor of Treh of various origins (7), an essential unit in many commercial glucosidase inhibitors such as 6 and acarbose, is a potent inhibitor itself. Other closely related aminosugars such as trehazolin (8) (9)   or some osidases . Closelytins (3)  and adiphe fungi . (+)-Val origins . Valienaolin (8)  and kirk (8) (9)  share si (8) (9)  and mann (8) (9) . The lat (8) (9) .7/C8-cyclitols and C7N-aminocyclitols. Bennett et al. have synthesized 1 and 2, among other covalent inhibitors, using a linear synthetic route from 2,3,4,6-tetra-O-benzyl glucopyranose  and acarbose (10 (10′) to obtain the targets 11, 12, and 13 (Streptomyces coelicolor (Sco) GlgEI V279S.Although most of these covalent and non-covalent inhibitors are naturally occurring, various synthetic approaches have been adopted to chemically synthesize the Cpyranose . The leapyranose . Compouns groups  using sis groups . Oligosaacarbose  have beeacarbose . Birch racarbose . Recentlacarbose . In thisbose (10  or 10′) , and 13 , which wSco GlgEI is a homolog of Mycobacteria tuberculosis (Mtb) GlgE. In Mtb, GlgE is part of a biosynthetic pathway that generates α-glucans from trehalose and is a genetically validated anti-TB target (Mtb to form a capsule correlated with increased virulence (Mtb GlgE and Sco GlgEI are similar to glycoside hydrolases (GH), but have no hydrolase activity on α-glucan, and are present in over 10% of sequenced genomes from bacteria and archaea, see Sco GlgEI-V279S, which possesses the same active site topology and active site residues as Mtb GlgE; however, Sco GlgEI-V279S is more stable and is highly amenable to structural studies  which could later be converted into a labile leaving group via a Buchwald–Hartwig amination  relative to 19B′ (H-1′ = 5.41 ppm) was similar to the upfield shift of 19A (H-1′ = 5.30 ppm) relative to 19B (H-1′ = 5.44 ppm), which is indicative of the desired isomer.The protected thiomaltosides mediates   under reflux conditions to afford 21 and 21′ after Zemplén deacetylation  formed using phosphorous tribromide in 55% yield  and XP GScore  .13 lacks any apparent inhibition of Sco GlgEI-V279S at concentrations as high as 1 mM. To assess any difference or commonality with our previously tested inhibitors and identify any additional enzyme–compound interactions that may be important for future drug development, we analyzed the X-ray crystal structure of the Sco GlgEI-V279S/13 complex. In particular, it is important to assess the conformation of the carbocycle in the −1 site as the computational docking model of compound 13 differs from the carbocycle conformation in the previously published Sco GlgEI-V279S complex with a 4-α-glucoside of valienamine (AGV). The Sco GlgEI-V279S/13 complex structure was resolved to a resolution of 2.73 Å, and the 2Fo-Fc composite omit map illustrated the presence of compound 13 in the M1P binding site of Sco GlgEI-V279S -OH and C(7)-OH functional groups are in an axial conformational and form a bidentate hydrogen-bonded interaction with the side chain oxygen atoms of the Asp 394 nucleophile . A similwith AGV   difference between Asp 394 in the Sco GlgEI-V279S/13 and Sco GlgEI-V279S/AGV complexes  were weakened by addition of the cyclohexyl moiety due to steric hindrance. This also promotes bond rotation in the Asp 394 side chain, which weakens an otherwise strong bidentate hydrogen-bonded interaction between Asp 394 and hydroxyls 2 and 7 of compound 13. Unfortunately, these minor disruptions in binding lack compensating interactions between the +1 subsite and the cyclohexyl moiety of 13, which appears to form only transitory interactions with side chains of the +1 subside. However, it is still possible to use compound 13 as the lead molecule in future GlgE inhibitor design studies. Taken together, the structure of the Sco GlgEI-V279S/13 complex strongly supports changing the stereo centers at carbocycle positions 2 and 3 to allow for equatorial hydroxyl groups at these positions. However, this would eliminate one of the hydrogen bonds to Asp 394 and lower conformational energy of the molecule and the addition of three other hydrogen bonds with Arg 392 and Asp 480 as seen in GlgE structures with bound maltose or maltose analogs  under UV light or by charring (5% H2SO4-MeOH), and the purification was performed by column chromatography on a silica gel (230–400 mesh) using the solvent system specified. Solvents were used without purification for chromatography. 1H NMR was recorded on the Bruker Avance III 600 MHz spectrometer using CDCl3 and D2O as solvents with residual CDCl3 and D2O as references. 13C were recorded on the Bruker Avance III 600 MHz spectrometer using CHCl3 and HDO as internal references. High resolution mass spectrometry was carried out using the TOF MS-ES + instrument. Low resolution mass spectrometry was carried out on ESquire-LC-MS.All chemicals and solvents were purchased from Fisher Scientific, Acros Organics, Alfa Aesar, or Sigma-Aldrich. Solvents were dried by using a solvent purification system by passing through activated alumina and copper catalyst columns. All reactions were carried out at room temperature under a nitrogen atmosphere using a nitrogen balloon, unless mentioned otherwise. Reactions were monitored by TLC  in dry N,N-dimethylformamide (50 ml) was cooled to 0°C. The solution was treated dropwise with a suspension of sodium hydride   in dry N,N-dimethylformamide. p-Bromobenzyl bromide  was added dropwise over 15 min, and the solution was stirred at room temperature for 16 h. The reaction was poured over ice and extracted with ethyl acetate (50 ml). The combined organic layers were washed with brine. The resulting organic phase was dried (anhydrous Na2SO4) and filtered, and the filtrate was concentrated under reduced pressure to obtain a product. The product was purified by silica gel flash column chromatography. The product fractions were combined, concentrated, and dried in vacuum to afford a yellow oily product 15′: yield 85% (2.50 g); silica gel TLC Rf = 0.61 (25% ethyl acetate:hexane). 1H NMR  δ 7.52–7.35 , 7.33–6.89 , 5.51 , 4.80 , 4.76–4.66 , 4.63 , 4.58 , 4.52–4.42 , 4.35–4.29 , 4.23 , 4.00 , 3.84 , 3.81–3.74 , 3.70 , 3.55 , 3.51 , 3.48 , 3.46–3.42 , 3.39 , 3.32 , 2.93 , and 2.35 . 13C NMR  δ 136.93 (Ar), 136.61 (Ar), 132.65 (Ar), 131.64 (Ar), 131.56 (Ar), 131.54 (Ar), 131.52 (Ar), 131.51 (Ar), 131.46 (Ar), 131.43 (Ar), 130.73 (Ar), 129.79 (Ar), 129.73 (Ar), 129.65 (Ar), 129.20 (Ar), 129.18 (Ar), 128.98 (Ar), 128.60 (Ar), 127.65 (Ar), 122.12 (Ar), 121.83 (Ar), 120.72 (Ar), 120.68 (Ar), 120.52 (Ar), 97.34 (C1′), 87.67 (C1), 86.95 (C5′), 81.91 (C4′), 81.11 (C4), 79.59 (C2), 78.91 (C2′), 77.70 (C5′), 76.82 (C3′), 74.74 (C3), 74.38 (OCH2Ph), 72.90 (OCH2Ph), 71.12 (OCH2Ph), 69.30 (C6′), 68.36 (C6), 64.85 (OCH2Ph), and 21.20 (-CH3). Mass spectrum (HRMS), m/z = 1654.8255 (M+Na)+; C68H63Br7O10S requires 1654.8335 (M+Na)+.A solution of deacetylated N-bromosuccinimide  was added to a solution of 15′  in 9:1 acetone:water (70 ml) and stirred at room temperature for 45 min. The solvent was evaporated at room temperature until turbid. A solution of the residue in ethyl acetate (100 ml) was washed successively with satd. aq. NaHCO3, (3 X 50 ml) and water (3 X 50 ml). The solution was dried with anhydrous Na2SO4 and evaporated. The product was purified by silica gel flash column chromatography. The product fractions were combined, concentrated, and dried in vacuum to afford as yellow oil 16′: yield 80% (2.00 g); silica gel TLC Rf = 0.19 (30% ethyl acetate:hexane). 1H NMR  δ 7.50—7.33 , 7.17—6.90 , 5.54 , 5.27 , 4.89–4.82 , 4.81 , 4.78–4.73 , 4.68 , 4.63 , 4.55 , 4.47 , 4.42 , 4.35 , 4.30–4.23 , 4.18–4.11 , 4.05 , 3.98 , 3.96 , 3.81 , 3.70 , 3.60–3.52 , 3.43 , and 3.38–3.32 . 13C NMR  δ 137.69, 137.49, 137.39, 137.35, 136.98, 136.94, 136.89, 136.72, 136.66, 136.64, 136.62, 136.42, 131.71, 131.68, 131.67, 131.66, 131.62, 131.61, 131.59, 131.57, 131.56, 131.53, 131.51, 131.49, 131.49, 131.47, 131.46, 131.42, 131.41, 131.40, 129.69, 129.68, 129.67, 129.65, 129.62, 129.60, 129.36, 129.33, 129.32, 129.31, 129.30, 129.28, 129.15, 129.14, 129.13, 129.09, 129.07, 129.06, 129.05, 129.05, 129.04, 129.04, 129.03, 129.02, 129.01, 127.92, 127.80, 122.06, 121.85, 121.83, 121.77, 121.75, 121.71, 121.63, 121.55, 121.54, 121.50, 121.07, 121.06, 97.41, 96.94, 96.84, 90.56, 84.38, 82.82, 81.72, 81.32, 80.16, 79.45, 79.28, 77.56, 77.34, 77.13, 76.92, 74.58, 74.24, 74.20, 73.68, 73.43, 73.21, 73.13, 73.07, 72.75, 72.74, 72.69, 72.58, 72.22, 71.06, 70.96, 69.70, 69.34, 69.22, 68.06, and 60.50. Mass spectrum (HRMS), m/z = 1548.8017 (M+Na)+; C61H57Br7O11 requires 1548.8057 (M+Na)+.n-Butyl lithium (2.22 M) in hexanes  was added dropwise to a suspension of methyltriphenylphosphonium bromide  in tetrahydrofuran (50 ml) at −20°C. The solution was stirred at −20°C for 15 min and raised to ambient temperature over 1 h. The solution was cooled to −20°C and compound 16′  dissolved in 50 ml of tetrahydrofuran was added dropwise. The solution was stirred at −20°C for 15 min and allowed to warm to ambient temperature and stirred for an additional 12 h. The solution was diluted with acetone (6 ml) and stirred for 30 min. Diethyl ether (40 ml) was added to precipitate triphenylphosphine oxide. The latter was removed by filtration using a Celite™ 545 filter aid. The filtrate was washed successively with saturated aq. NaHCO3 and brine (50 ml X 3). The solution was dried with anhydrous Na2SO4 and filtered, and the filtrate concentrated under reduced pressure to obtain the crude product. The product was purified by silica gel flash column chromatography on a silica gel to give product as a yellow oil 17′: yield 65% (1.30 g); Rf = 0.63 (30% ethyl acetate:hexane). 1H NMR  δ 7.50–7.33 , 7.22 –7.06 , 6.97 , 5.88 , 5.27 , 5.00 , 4.80 , 4.74 , 4.72 , 4.70 , 4.68 , 4.66 , 4.64 , 4.62 , 4.54 , 4.49 , 4.45 , 4.41 , 4.39 , 4.37 , 4.34 , 4.23 , 4.20 , 4.14 , 4.10 , 4.01 , 3.90 , 3.73 , and 3.60–3.45 . 13C NMR  δ 137.42 (Ar), 137.05 (Ar), 137.04 (Ar), 136.91 (Ar), 136.81 (Ar), 136.66 (Ar), 135.06 (Ar), 131.61 (Ar), 131.61 (Ar), 131.60 (Ar), 131.59 (Ar), 131.58 (Ar), 131.55 (Ar), 131.50 (Ar), 131.49 (Ar), 129.65 (Ar), 129.49 (Ar), 129.37 (Ar), 129.28 (Ar), 129.27 (Ar), 129.16 (Ar), 121.57 (Ar), 121.52 (Ar), 119.33 (=CH2), 98.43 (C1′), 81.66 (C3), 81.39 (C2′), 80.62 (C3), 79.83 (C2′), 78.96 (C4′), 77.78 (-OCH2Ph), 77.08 (-OCH2Ph), 76.87 (-OCH2Ph), 74.55 (-OCH2Ph), 74.22 (-OCH2Ph), 73.50 (-OCH2Ph), 72.71 (-OCH2Ph), 72.54 (-OCH2Ph), 72.52 (-OCH2Ph), 71.29 (-OCH2Ph), 71.19 (-OCH2Ph), 70.85 (C5′), 69.77 (C6′), and 68.28 (C6). Mass spectrum (HRMS), m/z=1545.8219 (M+Na)+; C62H59Br7O10 requires 1545.8451 (M+Na)+.17′  was dissolved by gentle warming in anhydrous toluene (3.0 ml). To this solution was added dry dimethyl sulfoxide (3.0 ml). To the clear solution was added anhydrous pyridine , trifluoracetic acid , and N,N′-dicyclohexylcarbodiimide  in that order. The reaction was left to stir at room temperature for 18 h. After completion of reaction, which was monitored by TLC, toluene (10 ml) was added. The resulting crystalline dicyclohexylurea was removed by filtration and washed with benzene. The combined filtrates and washings were extracted with water (20 ml X 3) to remove dimethyl sulfoxide. The organic layer was dried over anhydrous Na2SO4, evaporated under reduced pressure, and subjected to flash column chromatography on a silica gel with 1:6 ethyl acetate–hexane to give product as colorless viscous liquid 18′: yield 88% (1.1 g); silica gel TLC Rf = 0.72 (30% ethyl acetate:hexane). 1H NMR  δ 7.51–7.31 , 7.17–6.96 , 5.91 , 5.35–5.27 , 4.90–4.84 , 4.82–4.75 , 4.71 , 4.68 , 4.55 , 4.55–4.54 , 4.53 , 4.46 , 4.38 , 4.36 , 4.33 , 4.31 , 4.28 , 4.25 , 4.23 , 4.20 , 4.15 , 4.12 , 4.08 , 3.95–3.89 , 3.85 , 3.78 , 3.65–3.55 , 3.46 , and 3.37 . 13C NMR  δ 203.97 (-C=O), 137.47 (Ar), 137.15 (Ar), 136.91 (Ar), 136.90 (Ar), 136.69 (Ar), 136.55 (Ar), 136.37 (Ar), 134.34 (Ar), 131.61 (Ar), 131.60 (Ar), 131.59 (Ar), 131.58 (Ar), 131.55 (Ar), 131.53 (Ar), 131.52 (Ar), 131.51 (Ar), 131.51 (Ar), 131.49 (Ar), 131.47 (Ar), 131.46 (Ar), 129.67 (Ar), 129.64 (Ar), 129.63 (Ar), 129.55 (Ar), 129.35 (Ar), 129.34 (Ar), 129.34 (Ar), 129.32 (Ar), 129.30 (Ar), 129.30 (Ar), 129.23 (Ar), 129.02 (Ar), 129.01 (Ar), 129.00 (Ar), 129.00 (Ar), 128.99 (Ar), 121.81 (Ar), 121.77 (Ar), 121.75 (Ar), 121.73 (Ar), 121.69 (Ar), 121.57(Ar), 119.47 (=CH2), 99.46 (C-1′), 81.40 (C3), 81.07 (C2′), 80.70 , 79.82 (C4′), 79.07 (OCH2Ph), 77.50 (OCH2Ph), 77.40 (OCH2Ph), 77.19 (OCH2Ph), 76.97 (OCH2Ph), 74.63 (OCH2Ph), 74.38 (OCH2Ph), 73.99 (OCH2Ph), 73.83 (OCH2Ph), 72.64 (OCH2Ph), 72.51 (OCH2Ph), 72.31 (OCH2Ph), 70.96 (C5′), 69.95 (C6′), and 67.98 (C6). Mass spectrum (HRMS), m/z=1543.7928 (M+Na)+; C62H57Br7O10 requires 1543.8105 (M+Na)+.Compound 18′  in tetrahydrofuran (15 ml) was added of vinylmagnesium bromide (0.7 M) in tetrahydrofuran  dropwise. The reaction mixture was stirred for 1 h at the same temperature. The reaction mixture was warmed to room temperature. Diethyl ether (30 ml) and aq. NH4Cl (30 ml) were added to the reaction mixture. The organic layer was separated, washed with brine (50 ml X 2), and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure and purification was performed by flash column chromatography on a silica gel with 1:8 ethyl acetate:hexane to afford product as colorless viscous liquid 19A′: yield 93% (0.97 g); silica gel TLC Rf = 0.65 (30% ethyl acetate:hexane). 1H NMR  δ 7.49–7.31 , 7.15–7.06 , 6.09 , 5.65 , 5.53 , 5.26 , 4.80 , 4.67 , 4.54 , 4.42 , 4.37 , 4.33 , 4.28 , 4.20 , 4.15 , 3.95 , 3.89–3.82 , 3.79 , 3.63 , 3.57–3.52 , 3.49 , and 3.33–3.22 . 13C NMR  δ 139.19 (Ar), 137.65 (Ar), 137.45 (Ar), 137.35 (Ar), 137.01 (Ar), 136.88 (Ar), 136.71 (Ar), 136.47 (Ar), 135.47 (Ar), 131.57 (Ar), 131.52 (Ar), 131.44 (Ar), 131.42 (Ar), 131.33 (Ar), 129.81 (Ar), 129.70 (Ar), 129.27 (Ar), 129.22 (Ar), 129.16 (Ar), 129.08 (Ar), 128.92 (Ar), 121.92 (Ar), 121.72 (Ar), 121.56 (Ar), 121.49 (Ar), 121.33 (Ar), 121.32 (Ar), 120.06 (Ar), 116.20 (Ar), 95.86 (C1′), 83.64 (C5), 81.49 (C2′), 79.75 , 79.36 (C4′), 77.43 (OCH2Ph), 77.33 (OCH2Ph), 77.03 (OCH2Ph), 76.81 (OCH2Ph), 74.84 (OCH2Ph), 74.54 (OCH2Ph), 74.19 (OCH2Ph), 73.95, 72.74 (OCH2Ph), 72.25 (OCH2Ph), 71.67 (OCH2Ph), 70.56 (C5′), 69.71 (C6′), and 67.68 (C9). Mass spectrum (HRMS), m/z = 1572.5124 (M + Na)+; C64H61Br7O10 requires 1572.5223 (M + Na)+.To a cooled (−78°C) solution of 19B′: yield 23% (0.24 g); silica gel TLC Rf = 0.72 (30% ethyl acetate:hexane). 1H NMR  δ 7.51 –7.30 , 7.19–6.85 6.16 , 5.76 , 5.59 , 5.41 , 5.32 , 5.31–5.25 , 4.79 , 4.66 , 4.47 , 4.45–4.39 , 4.38 , 4.34 , 4.28–4.25 , 4.24 , 4.19–4.11 , 3.97 , 3.87 , 3.78 , 3.64 , 3.57–3.52 , 3.45 , 3.24 , 3.19 , and 2.74 . 13C NMR  δ 139.19 (Ar), 138.14 (Ar), 137.46 (Ar), 137.25 (Ar), 136.89 (Ar), 136.57 (Ar), 131.56 (Ar), 131.47 (Ar), 131.45 (Ar), 131.38 (Ar), 131.27 (Ar), 131.14 (Ar), 129.90 (Ar), 129.41 (Ar), 129.28 (Ar),129.25 (Ar), 129.18 (Ar),128.78 (Ar), 128.60 (Ar), 121.98 (Ar), 121.74 (Ar), 121.52 (Ar), 121.49 (Ar), 121.31 (Ar), 121.20 (Ar), 120.06 (Ar), 116.20 (Ar), 97.45 (C1′), 83.52 (C5), 81.41 (C2′), 79.75 (C4), 79.30 (C4′), 77.43 (C3′), 77.33 (OCH2Ph), 77.03 (OCH2Ph), 76.98 (OCH2Ph), 74.94 (OCH2Ph), 74.78 (OCH2Ph), 74.02 (C5), 73.55 (OCH2Ph), 72.74 (OCH2Ph), 72.25 (OCH2Ph), 71.67 (OCH2Ph), 70.56 (C5′), 68.91 (C9), and 67.89 (C6′). Mass spectrum (ESI-MS), m/z = 1572.4 (M + Na)+; C64H61Br7O10 requires 1572.52 (M + Na)+.Flash column chromatography on a silica gel with 1:6 ethyl acetate–hexane afforded product as colorless liquid 19A′ in dichloromethane was degassed by passing nitrogen gas through it for 20 min. After that, 1st generation Grubbs catalyst (10 mol%) was added to the solution and the reaction was kept under nitrogen atmosphere using nitrogen balloon for 7 days or until the catalyst turned dark brown or black. Then, everything was evaporated under reduced pressure and purified using flash column chromatography on a silica gel. 10′: yield 80% (0.60 g); silica gel TLC Rf = 0.5 (30% ethyl acetate:hexane). 1H NMR  δ 7.41–7.14  7.11–6.92  5.92 , 5.67 , 5.54 , 4.81 , 4.75–4.65 , 4.61 , 4.52–4.46 , 4.40 , 4.35 , 4.27 , 4.17–4.13 , 4.10 , 3.84 , 3.64 , 3.52 , 3.44 , 3.38 , and 3.38–3.36 . 13C NMR  δ 137.89 (Ar), 137.31 (Ar), 137.02 (Ar), 136.90 (Ar), 136.78 (Ar), 136.68 (Ar), 136.50 (Ar), 131.56 (Ar), 131.52 (C6), 131.51 (Ar), 131.49 (Ar), 131.32 (C1), 130.76 (Ar), 130.00 (Ar), 129.66 (Ar), 129.28 (Ar), 129.21 (Ar), 129.11 (Ar), 128.99 (Ar), 128.22 (Ar), 121.85 (Ar), 121.77 (Ar), 121.70 (Ar), 121.60 (Ar), 121.59 (Ar), 121.55 (Ar), 120.99 (Ar), 96.86 (C1′), 81.59 (C3′), 80.68 (C3), 80.03 (C2′), 79.66 (C2), 77.48 (C5), 77.26 (C4′) (OCH2Ph), 77.05 (OCH2Ph), 76.83 (OCH2Ph), 74.85 (OCH2Ph), 74.55 (C4), 74.15 (OCH2Ph), 73.72 (OCH2Ph), 73.34 (C7), 73.01 (OCH2Ph), 72.73 (OCH2Ph), 72.36 (OCH2Ph), 72.13 (OCH2Ph), 70.72 (C5′), and 68.11 (C6′). Mass spectrum (HRMS), m/z = 1544.6995 (M + Na)+; C62H57Br7O10 requires 1544.7132 (M + Na)+.A solution of dialkene 10  in tetrahydrofuran (5 ml) was added 200 µl of (2M sodium bis(trimethylsilyl) amide in tetrahydrofuran) dropwise. The reaction mixture was stirred for 30 min at the same temperature. After that acetyl chloride  was added to the reaction and let it run for 24 h at room temperature. After completion of the reaction, which was monitored by TLC, ethyl acetate (6 ml) was added to the reaction mixture. Washings were given to the organic layer with NaHCO3 solution (20 ml) and brine (20 ml), dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure and purification was performed by flash column chromatography on a silica gel to afford product as colorless viscous liquid 20: yield ; Rf = 0.79 (30% ethyl acetate:hexane). 1H NMR : δ 7.33–7.22 , 7.18–7.16 , 6.24 , 5.96 , 5.42 , 5.1 , 4.87 , 4.76 , 4.66 , 4.53 , 4.45 , 4.38 , 4.2 , 4.11 , 4.06 , 3.63 , 3.52 , and 2.03 . 13C NMR : δ 169.85 (C=O), 139.42 (Ar), 138.80 (Ar), 138.58 (Ar), 138.38 (Ar), 138.21 (Ar), 138.05 (Ar), 137.91 (Ar), 130.95 (Ar), 129.42 (Ar), 128.41 (Ar), 128.38 (Ar), 128.33 (Ar), 128.19 (Ar), 128.08 (Ar), 127.98 (Ar), 127.97 (Ar), 127.71 (Ar), 127.67 (Ar), 127.60 (Ar), 127.52 (Ar), 127.44 (Ar), 127.42 (Ar), 127.06 (Ar), 126.98 (Ar), 98.40 (C1′), 81.75 (C3′), 81.26 (C2′), 81.18 (C3), 80.01 (C2), 79.67 (OCH2Ph), 77.74 (C4′), 77.37 (OCH2Ph), 75.47 (C5), 75.11 (OCH2Ph), 74.74 (OCH2Ph), 73.47 (C4), 73.09 (OCH2Ph), 72.12 (OCH2Ph), 70.91 (C5′), 69.51 (C7), 68.68 (C6′), and 21.90 (CH3). Mass spectrum (HRMS), m/z = 1033.4484 (M + Na)+; C64H66O11 requires 1033.4503 (M + Na)+.To a cooled (0°C) solution of 10′  in tetrahydrofuran (10 ml) was cooled at 0°C. To this, excess (1.50 mmol) potassium bis(trimethylsilyl) amide (1.0 M) was added slowly. The reaction mixture was stirred for 45 min at the same temperature. 1.50 mmol of acetyl chloride was added dropwise to the reaction mixture. The reaction mixture was warmed to room temperature and stirred for 10–12 h. After completion of the reaction, ethyl acetate was added. The organic layer was washed with aq. brine and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure and purification was performed by flash column chromatography on a silica gel to afford product as colorless viscous liquid 20′: yield 80% (0.38 g); silica gel TLC Rf = 0.25 (30% ethyl acetate:hexane). 1H NMR  δ 7.45–7.39 , 7.35 , 7.15–6.96  6.18 , 5.94 , 5.34 , 4.90 , 4.70 , 4.70 , 4.67 , 4.59 , 4.50 , 4.42–4.35 , 4.29 , 4.10 , 4.02 , 3.92 , 3.53 , 3.52–3.47 , 3.43 , and 2.03 . 13C NMR  δ 169.61 (C=O), 138.34 (Ar), 137.55 (Ar), 137.23 (Ar), 137.01 (Ar), 136.99 (Ar), 136.91 (Ar), 136.62 (Ar), 131.57 (Ar), 131.57 (Ar), 131.55 (Ar), 131.55 (Ar), 131.51 (Ar), 131.51 (Ar), 131.37 (Ar), 131.20 (Ar), 130.56 (Ar), 129.55 (Ar), 129.42 (Ar), 129.13 (Ar), 129.13 (Ar), 129.05 (Ar), 129.00 (Ar), 128.27 (Ar), 121.84 (Ar), 121.76 (Ar), 121.63 (Ar), 121.60 (Ar), 121.56 (Ar), 121.47 (Ar), 120.90 (Ar), 98.43 (C1′), 81.48 (C2′), 81.12 (C3), 81.05 (C3′), 80.94 (C2), 80.12 (OCH2Ph), 79.56 (OCH2Ph), 77.02 (OCH2Ph), 76.81 (C5), 74.49 (C4′), 74.27 (OCH2Ph), 72.69 (C4), 72.48 (OCH2Ph), 71.72 (C7), 71.13 (OCH2Ph), 70.88 (C5′), 68.62 (C6′), and 21.82 (CH3). Mass spectrum (HRMS), m/z = 1585.9246 (M + Na)+; C64H59Br7O11 requires 1585.9117 (M + Na)+.A solution of 20  in ethyl acetate (4 ml) was added bis(benzonitrile) palladium (II) chloride , and reaction was left for stirring under refluxing conditions for 36 h. The solvent was evaporated under reduced pressure and purification was performed by flash column chromatography on a silica gel (7% ethyl acetate:hexane) to afford product as colorless viscous liquid. Rf = 0.78 (30% ethyl acetate:hexane). 1H NMR : δ 7.34–7.2 , 7.15 , 5.98 , 5.63 , 4.96 , 4.91 , 4.82 , 4.73 , 4.59 , 4.45 , 4.37 , 4.29 , 4.23 , 3.98 , 3.91 , 3.73 , 3.69 , 3.57 , 3.42 , and 2.13 ; 13C NMR : δ 170.84 (C=O), 140.45 (Ar), 138.88 (Ar), 138.76 (Ar), 138.62 (Ar), 138.16 (Ar), 138.12 (Ar), 138.00 (Ar), 128.49 (Ar), 128.46 (Ar), 128.43 (Ar), 128.23 (Ar), 128.16 (Ar), 127.98 (Ar), 127.93 (Ar), 127.89 (Ar), 127.86 (Ar), 127.82 (Ar), 127.79 (Ar), 127.76 (Ar), 127.74 (Ar), 127.68 (Ar), 127.41 (Ar), 127.01 (Ar), 122.92 (C5′), 97.06 (C1′), 82.27 (C3′), 79.79 (C2), 79.57 (C2′), 77.96 (C4′), 75.65 (OCH2Ph), 75.07 (OCH2Ph), 74.64 (OCH2Ph), 73.74 (OCH2Ph), 73.66 (OCH2Ph), 73.10 (OCH2Ph), 72.45 (OCH2Ph), 72.45 (OCH2Ph), 72.13 (C5′), 70.45 (C7), 68.35 (C6′), and 21.40 (CH3). Mass spectrum (HRMS), m/z = 1033.499 (M + Na)+; C64H66O11 requires 1033.450 (M + Na)+. To this viscous product, in methanol (8 ml), was added a small piece of sodium and the reaction was left to run for 40 min at room temperature under N2 atmosphere. The completion of reaction was monitored by TLC after which the reaction mixture was neutralized to pH = 7 with amberlite resin. Resin was filtered off followed by the evaporation of solvent under reduced pressure, and purification was performed by flash column chromatography on a silica gel (20% ethyl acetate:hexane) to afford product as colorless viscous liquid 21. Yield ; fR = 0.38 (30% ethyl acetate:hexane). 1H NMR : δ 7.35–7.12  5.98 , 5.36 , 4.80 , 4.75 , 4.71–4.64 , 4.60–4.52 , 4.43 , 4.33–4.28 , 4.18 , 3.95 , 3.70 , 3.60 , 3.57 , and 3.45 . 13C NMR : δ 138.70 (Ar), 138.46 (Ar), 138.35 (Ar), 138.23 (Ar), 138.03 (Ar), 137.94 (Ar), 137.32 (Ar), 136.09 (Ar), 128.44 (Ar), 128.40 (Ar), 128.35 (Ar), 128.32 (Ar), 128.28 (Ar), 128.02 (Ar), 127.91 (Ar), 127.86 (Ar), 127.75 (Ar), 127.73 (Ar), 127.70 (Ar), 127.69 (Ar), 127.57 (Ar), 127.52 (Ar), 127.02 (Ar), 97.75 (C1′), 82.00 (C3′), 79.61 , 76.81 (C4), 75.37 (OCH2Ph), 74.89 (OCH2Ph), 74.00 (OCH2Ph), 73.83 (OCH2Ph), 73.49 (OCH2Ph), 71.96 (OCH2Ph), 71.77 (C5′), 71.19 (C7), and 64.79 (C6′). Mass spectrum (HRMS), m/z = 991.418 (M + 23)+; C62H64O10 requires 991.439 (M + 23)+.To a solution of 20′  in ethyl acetate (6 ml) was added bis(benzonitrile) palladium (II) chloride , and the reaction was left for stirring under refluxing conditions for 12 h. After confirming the completion of the reaction, the mixture was filtered to remove the deactivated palladium catalyst. The solvent was evaporated under reduced pressure and the crude was dissolved in methanol. Sodium methoxide was added till the pH reached 9. The reaction mixture was stirred at room temperature for 45 min. The completion of reaction was monitored by TLC after which the reaction mixture was neutralized to pH = 7 with amberlite resin. Resin was filtered off followed by the evaporation of the solvent under reduced pressure and purification was performed by flash column chromatography on a silica gel (20% ethyl acetate:hexane) to afford product as colorless viscous liquid 20′: yield 80% (0.15 g); silica gel TLC Rf = 0.43 (30% ethyl acetate:hexane). 1H NMR  δ 7.48–7.34 , 7.24–7.12 , 7.09–6.88 , 5.99 , 5.27 , 4.94–4.87 , 4.84 , 4.73 , 4.69 , 4.66 , 4.64–4.61 , 4.59 , 4.57 , 4.56–4.53 , 4.52 , 4.48 , 4.43 , 4.40 , 4.38 , 4.36 , 4.09 , 4.01 , 3.93 , 3.87–3.75 , 3.70–3.61 , 3.56 , 3.47 , and 3.32 . 13C NMR  δ 137.34 (Ar), 137.23 (Ar), 136.98 (Ar), 136.70 (Ar), 136.63 (Ar), 131.69 (Ar), 131.60 (Ar), 131.56 (Ar), 131.53 (Ar), 131.51 (Ar), 131.48 (Ar), 129.71 (Ar), 129.64 (Ar), 129.60 (Ar), 129.58 (Ar), 129.56 (Ar), 129.36 (Ar), 129.27 (Ar), 129.26 (Ar), 129.23 (Ar), 129.17 (Ar), 129.12 (Ar), 128.97 (Ar), 128.24 (Ar), 121.74 (Ar), 121.58 (Ar), 121.49 (Ar), 97.25 (C1′), 84.29, 81.76, 81.57 (C2), 81.48 (C4′), 79.63 (C2′), 77.99 (OCH2Ph), 77.25 (OCH2Ph), 76.82 (OCH2Ph), 74.75 (OCH2Ph), 74.41 (OCH2Ph), 74.07 (OCH2Ph), 73.71 (OCH2Ph), 73.02 (OCH2Ph), 72.73 (OCH2Ph), 72.41 (OCH2Ph), 71.20 (C5′), 68.54 (C7), and 68.31 (C6′). Mass spectrum (HRMS), m/z = 1544.6942 (M + Na)+; C62H57Br7O10 requires 1544.7113 (M + Na)+.To a solution of 2 atmosphere, diethyl zinc  15% by wt. in toluene solution was added to a cooled dry toluene (3 ml). The mixture was stirred at −15°C for 10 min and then dimethyl iodide  was added dropwise to the reaction mixture. After 10 min, trifluoroacetic acid , was added dropwise to the cooled solution following which the cooling bath was removed and the reaction mixture was stirred at room temperature for 5 min. To the resultant mixture, a solution of compound 21  in dry toluene (3 ml) was added and the reaction mixture was stirred at room temperature for 15 h. The reaction was quenched by the addition of aq. HCl (10%) and then diluted with ethyl acetate (10 ml). After separation, the organic layer was washed with NaHCO3 solution (20 ml) and brine (20 ml), dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure and purification was performed by flash column chromatography on a silica gel (15% ethyl acetate:hexane) to afford product as colorless viscous liquid 22: yield ; Rf = 0.38 (30% ethyl acetate:hexane); 1H NMR  δ 7.35–7.29 , 7.28–7.21 , 7.16 , 5.13 , 4.79 , 4.61 , 4.52–4.39 , 4.29 , 4.05 , 4.00–3.95 , 3.93 , 3.78 , 3.74 , 3.68 , 3.62 , 3.54 , 2.68 , 2.47 , 1.57 , 1.40–1.34 , 0.93–0.88 , and 0.44 . 13C NMR  δ 138.81 (Ar), 138.65 (Ar), 138.58 (Ar), 138.29 (Ar), 138.19 (Ar), 137.93 (Ar), 128.38 (Ar), 128.36 (Ar), 128.33 (Ar), 128.30 (Ar), 128.02 (Ar), 128.00 (Ar), 127.85 (Ar), 127.78 (Ar), 127.72 (Ar), 127.63 (Ar), 127.47 (Ar), 127.43 (Ar), 127.39, (Ar) 127.32 (Ar), 98.77 (C1′), 81.88 (C2), 80.06 (C2′), 78.92 (C3), 78.01 (C1), 77.83 (OCH2Ph), 76.82 (OCH2Ph), 76.62 (OCH2Ph), 76.10 (OCH2Ph), 75.38 (OCH2Ph), 75.14 (OCH2Ph), 73.53 (OCH2Ph), 73.16 (OCH2Ph), 72.81(C4′), 72.37 (OCH2Ph), 72.26 (OCH2Ph), 70.96 (C5′), 68.48 (C7), 64.40 (C6′), 29.73, 27.16, 24.36, and 9.43. Mass spectrum (HRMS), m/z = 1005.492 (M + 23)+; C63H66O10 requires 1005.455 (M + 23)+.Under N21′  in 2 ml of DMF was added dropwise. The resulting mixture was stirred for 30 min at the same temperature. Potassium benzoate  was added and stirred for 30 min. During this time, excess 1,3,5-trifluorobenzene (1.53 mmol) was added slowly. After 1 h the reaction was quenched by addition of NH4Cl. Following addition of brine (8 ml) organic layer was extracted with diethyl ether (10 ml), dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure and purification was performed by flash column chromatography on a silica gel to afford product as colorless viscous liquid 23: yield 60% (0.09 g); silica gel TLC Rf = 0.68 (30% ethyl acetate:hexane). 1H NMR  δ 8.03 , 7.46–7.33 , 7.16–6.90 , 6.46 , 6.06 , 5.38 , 4.88 , 4.74 , 4.66 , 4.59–4.47 , 4.43 , 4.42–4.30 , 4.31–4.20 , 3.95 , 3.85 , 3.74 , 3.57 , 3.49 , 3.44 , and 3.31 . 13C NMR  δ 139.21 (Ar), 137.18 (Ar), 136.98 (Ar), 136.82 (Ar), 136.68 (Ar), 136.64 (Ar), 136.52 (Ar), 131.58 (Ar), 131.57 (Ar), 131.56 (Ar), 131.54 (Ar), 131.52 (Ar), 131.44 (Ar), 130.39 (Ar), 129.62 (Ar), 129.33 (Ar), 129.30 (Ar), 129.20 (Ar), 129.16 (Ar), 129.00 (Ar), 128.32 (Ar), 127.78 (Ar), 123.21 (Ar), 121.81 (Ar), 121.74 (Ar), 121.70 (Ar), 121.60 (Ar), 121.52 (Ar), 121.45 (Ar), 99.66 (C1), 99.47 (C1), 96.78 (C1′), 81.77 (C2), 79.60 (C3′), 77.67 (C4′), 77.24 (C2′), 77.03 (OCH2Ph), 76.82 (OCH2Ph), 74.51 (OCH2Ph), 74.12 (OCH2Ph), 72.91 (OCH2Ph), 72.74 (OCH2Ph), 72.15, 71.59 (OCH2Ph), 71.35 (OCH2Ph), 71.06 (C5′), 70.53 (C7), and 68.09 (C6′). Mass spectrum (HRMS), m/z = 1654.3391 (M + Na)+; C68H59Br7F2O10 requires 1654.3372 (M + Na)+.Sodium hydride (60% in hexanes) (0.38 mmol) was added to dry DMF in a flask at 0°C. To this mixture, a solution of compound N,N-dimethylformamide (6 ml) into a flask. To this mixture a solution of compound 22  in 2 ml of N,N-dimethylformamide was added dropwise. The resulting mixture was stirred for 30 min. Potassium benzoate  was then added and stirring was continued for 30 min. During which time 1,3,5-trifluorobenzene was added slowly. After 2 h the reaction was quenched by addition of NH4Cl. Following addition of brine (8 ml) organic layer was extracted with diethyl ether (10 ml), dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure and purification was performed by flash column chromatography on a silica gel (7% ethyl acetate:hexane) to afford product as colorless viscous liquid 24: yield 37 ; Rf = 0.8 (30% ethyl acetate:hexane). 1H NMR  δ 7.37–7.29 , 7.28–7.21 , 7.14–7.12 , 6.49 , 6.43 , 5.24 , 4.94 , 4.88–4.78 , 4.72 , 4.67–4.62 , 4.62–4.38 , 4.07–3.99 , 3.98–3.93 , 3.83 , 3.73–3.61 , 3.54 , 3.49 , 2.61 , 1.40–1.34 , 1.09 , 0.93–0.88 , and 0.48 ; 13C NMR  δ 138.95 (Ar), 138.77 (Ar), 138.43 (Ar), 138.26 (Ar), 137.84 (Ar), 128.41 (Ar), 128.38 (Ar), 128.29 (Ar), 128.24 (Ar), 128.20 (Ar), 128.07 (Ar), 128.01 (Ar), 127.88 (Ar), 127.79 (Ar), 127.75 (Ar), 127.72 (Ar), 127.64 (Ar), 127.59 (Ar), 127.55 (Ar), 127.50 (Ar), 127.45 (Ar), 127.39 (Ar), 127.23 (Ar), 99.56 (C1), 98.73 (C1′), 81.79 (C2), 80.52 (C3), 79.87 (C3′), 77.88 (C4), 77.24 (C4′), 77.03 (OCH2Ph), 76.82 (OCH2Ph), 75.60 (OCH2Ph), 75.54 (OCH2Ph), 75.22 (OCH2Ph), 73.95 (OCH2Ph), 73.54 (OCH2Ph), 72.98 (OCH2Ph), 72.74 (OCH2Ph), 72.58, 71.80 (C5′), 70.88 (C7), 68.44 (C6′), 29.72, 28.35, 22.75, 14.15 (CH), and 10.42 (CH2). Mass spectrum (HRMS), m/z = 1095.4851 (M + 23)+; C69H68F2O10 requires 1095.4814 (M + 23)+.After a suspension of sodium hydride  in mineral oil (60%) was washed with hexane (2 X 5 ml), it was transferred in dry 24  in 3 ml of methanol add 5% Palladium on carbon (8 mg). The mixture was stirred for 4 h under hydrogen (1 atm.). The catalyst was filtered away through a plug of Celite® 545 and washed with methanol (5ml). The combined filtrate and washings were concentrated to dryness afford 11 as a viscous syrup . 1H NMR  δ 6.66 , 6.52 , 5.22 , 4.21–4.11 , 3.84–3.65 , 3.56–3.52 , 3.51–3.47 , 3.06 , 1.34–1.30 , 1.28–1.22 , 1.04–0.99 , and 0.49 , 13C NMR : δ 161.90 (Ar), 161.66 (Ar), 161.44 (Ar), 101.48, 99.17 (C1′), 98.97 (C1′), 73.80 , 73.71 (C4′), 72.94 (C5′), 72.85, 71.14 (C7), 70.21 (C6′), 30.36, 21.26, 13.03 (CH2), and 8.77 (CH2). Mass spectrum (HRMS), m/z = 487.1401 (M + 23)+; C20H26F2O10 requires 487.1392 (M + 23)+.Dissolve compound 23  in toluene (4 ml), palladium (II) acetate , XPhos  and potassium phosphate tribasic  were added at room temperature. N-Methylaniline was added dropwise to the mixture, the reaction was heated to 85°C and stirred at the same temperature for 5 days. After the completion of the reaction, the mixture was cooled to room temperature. The organic phase was washed with brine (2X 10 ml) and dried over anhydrous Na2SO4. The solvent was evaporated, and the product was isolated via flash column chromatography on a silica gel (20% ethyl acetate:hexane) to afford product as yellow viscous liquid 25: yield 40% (0.04 g); silica gel TLC Rf = 0.70 (30% ethyl acetate:hexane). 1H NMR  δ 7.36–7.11 , 7.06–6.80 , 6.51–6.35 , 6.01 , 5.46 , 4.84 , 4.76 , 4.72 , 4.64 , 4.58 , 4.47–4.40 , 4.40–4.34 , 4.33 , 4.26 , 4.02–3.91 , 3.81 , 3.75–3.57 , and 3.30–3.17 . 13C NMR  δ 148.87 (Ar), 148.81 (Ar), 148.77 (Ar), 148.54 (Ar), 148.48 (Ar), 129.65 (Ar), 129.43 (Ar), 129.32 (Ar), 129.22 (Ar), 129.17 (Ar), 128.64 (Ar), 128.36 (Ar), 122.41 (Ar), 121.73 (Ar), 121.66 (Ar), 121.54 (Ar), 121.46 (Ar), 121.29 (Ar), 121.17 (Ar), 121.08 (Ar), 121.02 (Ar), 120.81 (Ar), 120.78 (Ar), 120.73 (Ar), 120.49 (Ar), 120.24 (Ar), 119.93 (Ar), 119.80 (Ar), 119.43 (Ar), 99.52 (C1′), 82.01 (C1), 77.25 (C3′), 77.04 (C3), 76.83 (C2), 75.40 (OCH2Ph), 74.77 (OCH2Ph), 73.59 (OCH2Ph), 73.27 (OCH2Ph), 72.79 (OCH2Ph), 72.21 (OCH2Ph), 71.74 (C7), 71.34 (C6′), 40.28, 40.25, 40.21, and 40.19 (CH3). Mass spectrum (HRMS), m/z = 1840.2834 (M + Na)+; C62H57Br7O10 requires 1839.9321 (M + Na)+.To a solution of compound 25  was dissolved in anhydrous dichloromethane (2.00 ml). To this, 7 ml of 5%TFA/DCM solution was dropwise added at room temperature. Stir till the reaction becomes blue-green in color. Evaporate dichloromethane and dissolve compound in minimal water. The compound 12 was purified with reverse phase column chromatography using C18. The product was eluted with 1:1 Methanol/Water. Yield quantitative (2.00 mg); silica gel TLC Rf = 0.30 (10% methanol: dichloromethane). 1H NMR  δ 6.66  6.49 , 6.05 , 5.43 , 5.28 , 4.96–4.90 , 4.33–4.15 , 3.87 , 3.76 , 3.65 , 3.46 , and 3.27 . 13C NMR  δ 143.70 (Ar), 132.87 (Ar), 130.81 (Ar), 129.03 (Ar), 128.06 (Ar), 119.18 (C6), 100.13, 99.36 (C1′), 80.80 , 73.78 (C3′), 73.39 (C4′), 72.60 , 71.91 (C3), 70.06 (C5′), 61.65 (C7), and 61.56 (C6’). Mass spectrum (HRMS), m/z = 473.3106 (M + Na)+; C19H24F2O10 requires 473.3913 (M + Na)+.Compound 18  was dissolved in dichloromethane (1.5 ml) at 0°C. To this solution was added anhydrous phosphorous tribromide . The reaction was left to stir at room temperature for 3 h. After completion of reaction, which was monitored by TLC, brine was added. The combined filtrates and washings were extracted with water (20 ml X 3) and organic layer was dried over anhydrous Na2SO4, evaporated under reduced pressure, and subjected to flash column chromatography on a silica gel with 1:7 ethyl acetate–hexane to give mixture of a and b products as yellowish brown viscous liquids 26a:26b (1:1) total yield 55% (0.15 g); silica gel TLC Rf = 0.78 (30% ethyl acetate:hexane). (26a)1H NMR  δ 7.35–7.24 , 7.20 –7.11 , 6.04 , 5.73 , 4.97 , 4.88 , 4.86–4.80 , 4.80–4.73 , 4.69 , 4.58 , 4.49 , 4.37 , 4.32 , 4.00–3.89 , 3.87 , 3.72–3.65 , 3.57 , and 3.43 . 13C NMR  δ 138.65 (Ar), 138.42 (Ar), 138.39 (Ar), 137.92 (Ar), 137.88 (Ar), 137.64 (Ar), 136.02 (Ar), 128.38 (Ar), 128.36 (Ar), 128.34 (Ar), 128.31 (Ar), 128.26 (Ar), 128.08 (Ar), 127.96 (Ar), 127.85 (Ar), 127.75 (Ar), 127.73 (Ar), 127.67 (Ar), 127.64 (Ar), 127.60 (Ar), 127.55 (Ar), 127.21 (Ar), 126.23 (Ar), 97.40 (C1′), 85.00 (C3′), 84.54 (C2), 82.28 (C2′), 79.16 (C3), 77.82 (OCH2Ph), 77.25 (OCH2Ph), 77.04 (OCH2Ph), 76.83 (OCH2Ph), 75.89 (OCH2Ph), 75.52 (OCH2Ph), 74.97 (OCH2Ph), 74.11 (OCH2Ph), 73.80 (OCH2Ph), 73.60 (C4), 73.53 (C4′), 72.04 (OCH2Ph), 71.13 (C5′), 69.60 (C7), 68.15 (C6′), and 50.75 (C1). Mass spectrum (HRMS), m/z = 1054.3691 (M + Na)+; C62H63O9Br requires 1055.0672. (26b)1H NMR  δ 7.39–7.20 , 7.18–7.12 , 6.11 , 5.69 , 5.06 , 4.92 , 4.83 , 4.71 , 4.66 , 4.60–4.53 , 4.45 , 4.39–4.31 , 4.27 , 4.02–3.94 , 3.89 , 3.68  3.61–3.51 , and 3.43–3.37 . 13C NMR  δ 138.74 (Ar), 138.55 (Ar), 138.48 (Ar), 138.09 (Ar), 137.99 (Ar), 137.89 (Ar), 137.52 (Ar), 128.45 (Ar), 128.36 (Ar), 128.34 (Ar), 128.30 (Ar), 128.27 (Ar), 128.07 (Ar), 128.01 (Ar), 127.89 (Ar), 127.86 (Ar), 127.82 (Ar), 127.73 (Ar), 127.66 (Ar), 127.63 (Ar), 127.60 (Ar), 127.56 (Ar), 127.53 (Ar), 127.19 (Ar), 126.85 (Ar), 125.15 (Ar), 96.58 (C1′), 82.05 (C3), 80.98 (C3′), 79.56 (OCH2Ph), 77.77 (C2′), 77.50 (C2), 77.26 (C4), 77.05 (OCH2Ph), 76.83 (OCH2Ph), 75.52 (OCH2Ph), 75.46 (C4′), 74.94 (OCH2Ph), 73.55 (OCH2Ph), 73.51 (OCH2Ph), 73.46 (OCH2Ph), 71.62 (C4), 71.14 (C5′), 70.09 (C7), 68.22 (C6’), and 48.57 (C1). Mass spectrum (ESI-MS), m/z = 1054.6 (M + Na)+; C62H63O9Br requires 1055.06.Compound 26a and 26b  were dissolved in dichloromethane (1.0 ml) at room temperature. To this solution was added anhydrous cyclohexylamine . The reaction was left to stir at reflux for 5 days. After completion of reaction, which was monitored by TLC, the reaction mixture was cooled to room temperature and brine was added. The combined filtrates and washings were extracted with water (20 ml X 3) and organic layer was dried over anhydrous Na2SO4, evaporated under reduced pressure, and subjected to flash column chromatography on a silica gel with 1:1 ethyl acetate–hexane to give a yellowish brown viscous liquid 27: yield 60% (0.09 g); silica gel TLC Rf = 0.3 (50% ethyl acetate:hexane). 1H NMR  δ 7.38–7.32 , 7.31 –7.16 , 5.92 , 5.22 , 4.81 , 4.76 , 4.71–4.65 , 4.65–4.61 , 4.58 , 4.46 , 4.41 , 4.34 , 4.28 , 4.22 , 3.99–3.89 , 3.74–3.69 , 3.69–3.57 , 3.46 , 2.29 , 1.79 , 1.65–1.58  1.25–1.13 , and 0.92–0.86 . 13C NMR  δ 138.79 (Ar), 138.59 (Ar), 138.52 (Ar), 138.08 (Ar), 138.02 (Ar), 128.51 (Ar), 128.40 (Ar), 128.34 (Ar), 128.33 (Ar), 128.27 (Ar), 128.24 (Ar), 128.21 (Ar), 128.18 (Ar), 128.14 (Ar), 127.99 (Ar), 127.81 (Ar), 127.73 (Ar), 127.64 (Ar), 127.62 (Ar), 127.48 (Ar), 127.40 (Ar), 127.30 (Ar), 98.01 (C1′), 81.98 (C2), 79.89 (C4), 77.89 (C2′), 77.25 (C3′), 77.04 (C4), 76.83 (OCH2Ph), 75.32 (OCH2Ph), 74.78 (OCH2Ph), 73.96 (OCH2Ph), 73.47 (OCH2Ph), 72.05 (OCH2Ph), 71.62 (OCH2Ph), 71.41 (OCH2Ph), 71.17 (C5′), 70.93 (C7), 68.22 (C6’), 52.99 (C1), 49.08 (C-Hex), 31.95 (C-Hex), 29.72 (C-Hex), 29.38 (C-Hex), 26.11 (C-Hex), 24.98 (C-Hex), 24.94 (C-Hex), and 22.72 (C-Hex). Mass spectrum (HRMS), m/z = 1072.2873 (M + Na)+; C68H75NO9 requires 1072.5262.Compounds 27  in tetrahydrofuran (3.0 ml) using a dry ice cooled cold finger apparatus. The solution was treated with sodium in small pieces, until a blue color in the solution persisted. After stirring for 10 min at −78°C , the mixture was treated with NH4Cl (70 mg), stirred at room temperature overnight and evaporated. The residue was extracted with methanol, filtered, and evaporated. The residue (25.0 mg) was redissolved in water and purified using reverse phase C18 column with a gradient of water:methanol to give 13 as a white solid: yield quantitative (32.0 mg). 1H NMR  δ 5.85 , 5.32 , 4.21 , 4.14 , 4.10–4.05 , 3.99–3.95 , 3.93 , 3.79 , 3.69 , 3.59– 3.54 , 3.54–3.50 , 3.34 , 3.11 , 3.05 , 1.97 , 1.77 , 1.58 , and 1.29–1.17 . 13C NMR  δ 138.24 (C5), 119.43 (C6) 97.43 (C1′), 74.43 (C2), 72.81 (C3′), 72.76 (C2′), 71.12 , 69.68 (C5′), 69.30 (C6′), 66.54 (C7), 61.85 (C1), 60.45, 55.24 (CN-Hex), 51.53 (C-Hex), 48.83 (C-Hex), 29.84 (C-Hex), 24.72 (C-Hex), and 24.18 (C-Hex). Mass spectrum (HRMS), m/z = 420.4788 (M + H)+; C19H33NO9 requires 420.4791.Ammonia was condensed into a solution of Sco GlgEI crystal structure was prepared using the Protein Preparation Wizard incorporated in Maestro (v.11.8) and Prime modules (via the OPLS3e force field (via Glide docking in Maestro (v11.8)  .Sco GlgEI-V279S construct was used to transform T7 express E. coli cells. At 37°C, the largescale cultures were grown in LB media with a 264 mM concentration of carbenicillin. When O.D600 nm reached 0.6, the temperature was lowered to 16°C, and 1 mM Isopropyl β-D-1-thiogalactopyranoside was added to cultures. After 16 h of induction, cells were harvested and resuspended in the lysis buffer  phosphine (TECP)), and the resulting cell suspension was incubated on ice with lysozyme and DNase I. After 30 mins, the cell suspension was sonicated and centrifuged for 45 min. The clarified lysate was applied to a 5 ml metal affinity cobalt column, which was pre-equilibrated with lysis buffer. To remove unbound protein, 25 column volumes of lysis buffer were passed through the column and unbound protein was washed away. Finally, Sco GlgEI-V279S was eluted isocratically with elution buffer  and dialyzed against 20 mM Tris pH 7.5, 150 mM NaCl, and 0.3 mM TCEP to get rid of excess salt and imidazole.Protein purification: The protein purification was carried out according to the previously published method . In brie13 were performed separately using a real-time assay and the EnzChek Phosphate Assay Kit. According to the literature, when the phosphate concentration of the reaction mixture is above 25 mM, GlgE catalyzes the reverse reaction of the production of M1P from glucans  into M1P and 2-chloro-4-nitrophenyl glucose12,13. The 2-chloro-4-nitrophenyl glucose is further hydrolyzed into 2-chloro-4-nitrophenyl and glucose by α-glucosidase. The resulting 2-chloro-4-nitrophenyl exhibits an increased absorbance at 410 nm wavelength, and production of 2-chloro-4-nitrophenyl was measured continuously during the reaction. The reactions were carried out in a 96-well format, with a 50 µl reaction volume at 25°C for 40 min, and the velocity was measured at 410 nm wavelength using a Synergy H4 plate reader (Bio Tek). The reaction mixture consisted of 1.5 mM 2-chloro-4-nitrophenyl-D-maltotrioside, 20 mU α-glucosidase, 100 mM sodium phosphate, and 20 mM Tris pH 7.5. Finally, the reaction was initiated by adding 250 nM Sco GlgI-V279S to the reaction mixture. In the reaction, the compound 13 concentrations varied from 0 to 3000 µM. The positive and negative controls were lacking compound 13 and Sco GlgEI-V279S, respectively. The percent enzymatic activity was calculated by using the equation (V/V0) × 100, where V and V0 denote the rates of the inhibited and uninhibited enzyme (positive control), respectively.Inhibition studies: The inhibition studies on compound  glucans . In the 13 concentration. In the reaction, 2-chloro-4-nitrophenyl-α-D-glucopyranoside was used as the substrate, and the production of 2-chloro-4-nitrophenyl was monitored throughout the reaction. The reaction mixture comprises 1.5 mM 2-chloro-4-nitrophenyl-α-D-glucopyranoside, 20 mM Tris pH 7.5, and each concentration of compound 13. The reaction was initiated by adding 20 mU α-glucosidase. The compound 13 concentrations varied from 0 to 3000 μM. The percent enzymatic activity was calculated using the equation mentioned above. Finally, the total inhibition observed for Sco GlgEI-V279S and α-glucosidase was deducted using α-glucosidase inhibition at each concentration, and the actual GlgE inhibition was calculated. The results indicated that a 3000 μM concentration of compound 13 inhibits the activity of GlgE by 50%.The percent inhibition of α-glucosidase was calculated at each compound Sco GlgEI-V279S to a final concentration of 50 nM. All the reactions were carried out in 96 well formats in a triplicate manner, and a Synergy H4 plate reader (Bio Tek) was used to measure the absorbance at 360 nm. The 100 and 1000 µM concentrations of compound 13 were tested against Sco GlgEI-V279S, and neither of those concentrations exhibited inhibition of the Sco GlgE1-V279S activity.The EnzChek phosphate detection assay was performed according to the previously published method. The reaction mixture consisted of 20 mM Tris pH 7.5, 150 mM NaCl, 500 nM MESG, 0.25 U PNP, 0.5 mM glycogen , and 250 µM M1P . The reaSco GlgEI-V279S/13 mixture consisted of 8 mg/ml Sco GlgEI-V279S and 10 mM concentration of compound 13. The crystallization experiments were performed using the hanging drop vapor diffusion method. Each drop consisted of 2 μl of Sco GlgEI V279S/compound mixture and 2 μl of the well solution of 0.2 mM sodium citrate pH 7 and 10% w/v PEG 3,350 , using Phaser (X-ray diffraction experiments: The LS-CAT beamline at Advanced Photon Source of Argonne National Labs, IL, was used to perform X-ray diffraction experiments. The collected data were indexed, integrated, and scaled by using HKL 2000 . The molg Phaser  in PHENIg Phaser . The refg Phaser . Finallyg Phaser  in PHENI


The health-promoting phytochemicals and antioxidant capacity of four organs  of two cultivars (Chuankui–1 and Chuankui–2) of wasabi from two producing areas, Leibo and Guangyuan in Sichuan Province, China, were investigated in this study. The results showed that leaves were rich in pigments, soluble protein, ascorbic acid, and total phenolics and had the highest antioxidant capacity. Soluble sugars were highest in the petioles and were 1.1- to 5-fold higher than those in the other three organs. Glucosinolates and glucosinolate breakdown products (GBPs) were the most abundant in rhizomes, and their maximum values were 271.61 mmol kg-1 DW and 249.78 mmol kg-1 DW, respectively. The rhizomes of Chuankui–1 in Leibo and the leaves of Chuankui–1 in Guangyuan were superior in terms of glucosinolates and GBPs. These findings provide new insights that will aid the use of wasabi cultivars; they also have implications for the environmental characteristics needed to obtain better quality wasabi products. In the future, metabolome and transcriptome can be used to analyze the potential mechanism of differences among typical varieties, origins and parts.Wasabi (Eutrema  The average summer temperatures in the two areas are 13 and 23°C respectively; the average winter temperatures are 0.1 and -6°C respectively; the annual rainfall is 813.6 and 1300 mm respectively. The soil is loose and fertile, with a pH of 5.9 to 7.0, with clean and sufficient irrigation water. The seeds of wasabi were sown in March 2020, and 6 months later, plants were transferred to the field at the four-leaf stage. In March of the following year, apply 750 kg of potassium sulfate compound fertilizer per hectare. At the end of September and in March of the following year, continue to apply potassium sulfate compound fertilizer, 450 kg and 750 kg per hectare respectively. Water was given as needed throughout the growth period. Carbendazim was sprayed to prevent soft rot. Materials were harvested uniformly in May 2022. Four groups  of 60 wasabi plants were harvested at the same time in the morning and quickly transported to the laboratory. Fifteen plants from each group were divided into three replicate groups for experiments. In the laboratory, the surfaces of wasabi plants were washed, dried, sorted into four organs  (Two cultivars of wasabi (Eutrema F − WD)/WF × 100, where WF is the fresh weight, and WD is the dry weight.Samples (20 g) were placed in an oven at 80°C until a constant weight was achieved to determine the dry matter content of each organ. The dry matter content (%) was calculated using the formula (WF − WD)/WF × 100 (The water content (%) was calculated from the dry matter content: 100 – (WWF × 100 .Frozen powder (50 mg) of leaves and other samples (300 mg) were ground and extracted with 10 mL 96.0% ethanol. After centrifugation at 4000 g for 5 min, the supernatant was collected and total chlorophyll content was measured by reading the absorbance at 665 nm and 649 nm with a spectrophotometer .−1 of dry weight (Frozen powder (50 mg) was extracted with 10 mL of a mixture of acetone and petroleum ether  and petroleum ether was used for constant volume. The content of total carotenoid was measured by reading the absorbance at 451 nm with spectrophotometer. Result of chlorophyll and carotenoids content was expressed as g kgy weight .2O, centrifuged for 5 min at 4000 g and 1 mL transferred to a polypropylene tube. Subsequently, Coomassie brilliant blue G250 was combined with 1 mL of supernatant. The absorbance was measured at 595 nm within 20 min after the reaction. Result of soluble protein content was expressed as g kg−1 of dry weight . The amount of ascorbic acid was calculated from absorbance values at 243 nm. Result of ascorbic acid content was expressed as g kgy weight .−1 of dry weight  were boiled in 5 mL water for 10 min. The supernatant was collected and applied to a DEAE-Sephadex A-25 column. The glucosinolates were converted into their desulpho analogues following aryl sulphatase, then the desulphoglucosinolates were eluted, and analyzed by HPLC. Result of glucosinolate content was expressed as mmol kgy weight .−1 of dry weight (Freeze-dried samples (100 mg) were ground in 2 mL of water in the presence of 0.2 μmol of the internal standard benzonitrile. The solution was incubated at 25°C for 2 h, and dichloromethane (5 mL) was added, vortexed, and then centrifuged. The supernatant (1 mL) was transferred and analyzed using an Agilent 7890A Series GC System with an HP5 column (30 m × 0.25 mm × 0.25 μm). Result of GBPs content was expressed as mmol kgy weight .Data were analyzed using two-way analysis of variance. The means were compared through the least significant difference (LSD) test at a significance level of 0.05. Hierarchical clustering analysis and heatmap were generated using Origin 2022. Principal component analysis (PCA)and orthogonal partial least squares-discriminant analysis (OPLS-DA) were performed using SIMCA 14.1 software .The petioles made up most of the weight of the plants (52%), followed by the leaves, rhizomes, and roots ; the sinigrin content in the petioles and roots was lower, and it was only 17% of the content in the rhizomes on average. The sinigrin content in the leaves was highest in C1 grown in Guangyuan, followed by the rhizomes, and it was lowest in the petioles, which was 22% of that in the leaves. In C2, the sinigrin content was higher in the rhizomes and roots of wasabi grown in Leibo, with an average of 167.5 mmol kg-1, which was 3.0- and 3.4-fold higher than that in the petioles and leaves, respectively. The sinigrin content was high in the rhizomes of plants grown in Guangyuan, which was approximately 4.7-fold higher than that in the petioles, which had the lowest sinigrin content. In addition, the sinigrin content was slightly higher in the rhizomes of C2 grown in Leibo than that in Guangyuan, and the sinigrin content was much higher in the roots in C2 grown in Leibo than in C2 grown in Guangyuan . In C2, the content of gluconapin was high in the rhizomes and roots of wasabi grown in Leibo; the highest gluconapin content was observed in the rhizomes of plants grown in Guangyuan , and the content of 4-hydroxyglucobrassicin in the roots was 3.0-fold higher than that in the rhizomes, which had the lowest content of 4-hydroxyglucobrassicin. The overall content of C1 grown in Guangyuan was relatively low, and the trend was opposite. C2, regardless of producing area, has the highest content in the leaves, followed by the rhizomes, and the lowest in the petioles and roots. The overall 4-hydroxyglucobrrassicin content was higher in C2 grown in Leibo than in plants grown in Guangyuan (-1), followed by the leaves. The content of 4-methoxyglucobrassicin in C1 grown in Guangyuan was highest in the leaves (as high as 4.5 mmol kg-1) followed by the roots and rhizomes; the content of 4-methoxyglucobrassicin in C1 was lowest in the petioles (0.4 mmol kg-1). Differences between the organs of C2 grown in the two producing areas were similar; the 4-methoxyglucobrassicin content was highest in the roots and lowest in the petioles ; the content of SIN-ITC was lowest in the petioles (41.1 mmol kg-1), which was 17% of that in the rhizomes. The content of SIN-ITC was low in C1 grown in Guangyuan (average of 34.3 mmol kg-1). The content of SIN-ITC was high in the rhizomes and roots of C2 grown in Leibo (approximately 110 mmol kg-1); the content of SIN-ITC was highest in the rhizomes of C2 grown in Guangyuan ; the content of GNA-CN was highest in the rhizomes of C2 grown in Guangyuan, which was as high as 3.9 mmol kg-1 (The content of 3-butenenitrile (SIN-CN) was highest in the rhizomes of C1 grown in Leibo, which was 4.3-fold higher than that in the leaves, which had the lowest content of SIN-CN. The SIN-CN content of the two cultivars grown in Guangyuan and C2 grown in Leibo was less than 2.0 mmol kg+ level was strongly correlated with the total phenolics content , and FRAP was strongly correlated with the ascorbic acid and total phenolic content . Glucosinolates were significantly and positively correlated with sinigrin, gluconapin, and progoitrin; GBPs were strongly correlated with SIN-ITC and GNA-ITC.Correlation analysis of various phytochemicals provided various new insights  are to 1. A model is considered effective when Q2 > 0.5. Producing areas (Q2 = 0.849) were more distinguishable than cultivars. After the alignment verification of OPLS-DA , models were meaningful, and differential components can be screened based on variable importance plot (VIP) values. According to VIP values (>1) of the OPLS-DA, SIN-ITC, SIN-CN, GNA-ITC, ITCs, and GBPs contributed to variation among producing areas. Differences in C1 (Q2= 0.765) grown in Leibo and Guangyuan were more pronounced than differences in C2 grown in Leibo and Guangyuan (Q2 = 0.209). In addition to the above-mentioned VIP values, CNs and GNA-CN also contributed to differences among groups  are rich in protein and have a balanced profile of amino acids and micronutrients, including bioactive chemicals , mainly exist in the rhizomes and roots of P. ginseng (quinquefolius)  , and Panoginseng ; they haoginseng . Sinigrioginseng . Most GBoginseng .Eutrema species  had the highest content of glucosinolates and GBPs. Producing area had a greater effect on the quality of wasabi than cultivar. In C1, rhizomes in Leibo and leaves in Guangyuan should be used reasonably according to local conditions. As far as rhizomes grown in Guangyuan are concerned, C2 is a better choice.The original contributions presented in the study are included in the article/HD: Investigation, Writing- Original draft preparation. CC: Data curation, Writing- Original draft preparation. PF: Investigation, Writing- Original draft preparation. JM: Data curation. MH: Investigation. ML: Data curation. WL: Investigation. FZ: Data curation. YZ: Funding acquisition, Writing- Reviewing and Editing, and Conceptualization. All authors: review and editing.This work was supported by the Sichuan Science and Technology Program (2021ZHFP0027), Central Guidance on Local Science and Technology Development Fund of Shaanxi Province (2022ZY1-CGZY-07), and Undergraduate Research Interest Cultivation Project of Sichuan Agricultural University (20220038).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.


The androgen receptor (AR) is a nuclear receptor governing gene expression programs required for prostate development and male phenotype maintenance. Advanced prostate cancers display AR hyperactivation and transcriptome expansion, in part, through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR’s domains and cofactors cooperate to bind DNA has remained elusive. Using single particle cryo-electron microscopy, we isolated three conformations of AR bound to DNA showing AR forms a non-obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome, and reinforced by AR’s oncoprotein cofactor, ERG, and by DNA binding motifs. Finally, we present evidence this plastic dimer interface may have been adopted for transactivation at the expense of DNA binding. Our work highlights how fine-tuning AR’s cooperative interactions translate to consequences in development and disease.  et al. reveal a tunable mode of dimerization and define allosteric surfaces important for AR activities and development.Molecular features regulating DNA binding and transactivation activities of the androgen receptor (AR) and the greater steroid receptor family have remained elusive. Using cryo-EM and mass spectrometry, Wasmuth  AR signaling is a tightly controlled and multifaceted process, regulated through an orchestra of intramolecular and external cues. A better understanding of the rules governing AR activation is of great importance, as multiple pathologies are associated with aberrant AR transcriptional output, including prostate cancer and androgen insensitivity syndrome (AIS). That these disorders present with a spectrum of physical and molecular phenotypes  suggestsA type I nuclear receptor (NR) and member of the 3-ketosteroid receptor (3K-SR) subfamily, AR encodes an approximately 100 kilodalton (kDa) protein with an intrinsically disordered N-terminal domain (NTD), a DNA binding domain (DBD), a flexible Hinge, and a ligand binding domain (LBD)  Weikum . AndrogeNuclear AR binds both palindromic and direct repeats of DNA hexamers known as androgen response elements (AREs) to activate its gene expression program, and is further regulated through association with numerous protein cofactors that bind the NTD or LBD through LXXLL and related motifs . AR can Despite decades of work, the structural underpinnings of AR regulation conferred by its domains, auxiliary cofactors, and ARE sequence remain unclear. The prevailing view of NR activation comes primarily through work on the distantly related type II NRs, including HNF-4α and PPARγ-RXR, with crystal structures of these multidomain variants revealing cooperative mechanisms of LBD-mediated dimerization to bind DNA . A similGreater clarity on the determinants of 3K-SR activation could also be instructive for novel pharmacological intervention, particularly in metastatic prostate cancer where patients inevitably develop resistance to current AR-targeted therapies, including the anti-androgen enzalutamide (ENZ)  that areTo visualize how AR and ERG cooperate to bind DNA at the single molecule level, we imaged recombinant AR and ERG in the presence of duplex ARE DNA by atomic force microscopy (AFM). For these studies, the 52 kDa full-length (FL) ERG protein was used , as the We exploited the dramatic solubilizing effect of ERG to visualize how AR is activated using higher resolution structural methods. We reconstituted and trapped an AR complex designed to model a fully primed state that lacked its NTD, and was bound to DHT, palindromic ARE DNA, and ERG. To enrich for and identify productively bound AR complexes, we performed gentle cross-linking during ultracentrifugation  followedThe purified complex was comprised of hetero- and homo-cross-linked species between AR and ERG or with AR, respectively, that migrated around 100 and 150 kDa by SDS-PAGE . Using sThe most striking difference defining these states is plasticity around a common LBD dimer interface that converges at a prominent surface near beta-sheet 1 and helix 5 of the LBD . While aDensity for the two DBDs in all three models are consistent with the head-to-head conformation previously reported in the X-ray structure of the AR DBD dimer bound to non-palindromic ARE repeats . The simTo validate the structural models, we performed cross-linking mass spectrometry (XL-MS) with the lysine cross-linker disuccinimidyl sulfoxide (DSSO) to identify candidate surfaces responsible for interdomain and intermolecular cooperativity . The higWe next investigated how LBD contacts at the dimer and DBD interfaces impact AR’s ability to bind DNA by targeting conserved residues identified by our cryo-EM and XL-MS data , B. To bTo test the model proposed by Thornton and colleagues that ARE repeats within the DNA template drive AR dimerization and transactivation , or whetInterestingly, while ERG is detected in our complexes by XL-MS, SDS-PAGE and immunoblotting , B, we cOne prediction derived from these three conformational states is that the Divorced state, which exhibits the least interdomain connectivity and unassigned electron density , may be We next introduced AR and ERG truncations and point mutations in the reporter system to determine the surfaces responsible for these interactions. FL ERG repressed activity of an AR variant lacking the N-terminus, but exerted no effect on AR V7, a splice isoform lacking the LBD that is expressed in CRPC patients and associated with anti-androgen resistance  Fig. S1, F. In cWe subsequently queried whether the AR LBD mutants were more susceptible to ERG regulation, given that ERG cross-links to AR were detected exclusively along the length of the LBD , and becBecause the LBDs and LXXLL cofactor interacting surfaces of 3K-SRs are highly conserved , M Edga, we querPten  bound DNA nearly five-fold tighter than WT AR, while the fusion with the longer linker (AR-AR27Linker) bound DNA almost three-fold weaker   .Trans factors serve to reinforce these interactions, as we have shown for the protein cofactor ERG and the nature of the ARE DNA consensus site. Overall, our data suggest that ERG cooperative interactions more readily influence partly primed AR through interactions with the LBD by potentially inducing a more compact state, providing proof-of-principle evidence for how overexpression of an AR cofactor can confer ENZ resistance (sistance . Conversin vivo (in vivo (70%), even in the presence of oncoprotein cofactors  was required to solubilize and stabilize distinct populations of DNA-bound dimeric AR, it is possible that this approach limited overall resolution of our cryo-EM reconstructions and that alternative native conformations may exist.sawyersc@mskcc.org).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Charles Sawyers  cells (Novagen), grown at 37°C, with expression induced by addition of 0.1mM isopropyl-β-D-thiogalactoside (IPTG) and overnight shaking at 16°C.E. coli (Genscript) and subsequently cloned into pRSF-Duet1 (Novagen). The ETS domain of human ERG isoform 2 (residues Q272-E388) was cloned into pRSF-Duet1. The AR DBD (residues D548-E651) was cloned into pET-Duet1 (Novagen). All AR mutants were cloned by HiFi assembly (NEB). For AR Hinge variants, residues of unknown function proximal to the LBD (652-671) were excised, as this region is distal from the bipartite nuclear localization sequence previously implicated in DNA binding and acetylation , P>0.05. Data presented as mean +/− standard deviation from n=4 experiments.Unlabeled and 5’ fluorescein-labeled duplex DNAs were purchased from IDT and had the following sequences, with ARE sites in bold and ETS sites in italics: ARE/Scr: 5’ TACCTAGCGTGGCCeviously , equimolProtein-DNA complexes were prepared as described in previous section as native Main  or Grafi35 DNA were applied on glow discharged 400 mesh copper grids with carbon support (Electron Microscopy Sciences) and stained with Nano-W (Nanoprobes). Datasets were collected at 120 kV on a Tecnai T12 microscope (Thermofisher Scientific) and consisted of 264 micrographs and 107,445 particles (Fraction 9) and 365 micrographs and 61,034 particles (Fraction 14). Data processing, including particle autopicking and 2D classification were performed in cisTEM (3 microliters of the indicated Grafix fraction with AREn cisTEM .35 DNA (Fraction 11) was applied to glow discharged R1.2/1.3 holey carbon grids (Quantifoil) at 4°C and plunge frozen in liquid ethane using a Vitrobot Mark IV (Thermofisher Scientific). Data were collected at 300 kV on a Titan Krios (Thermofisher Scientific) with energy filter using a K3 Summit Detector in counting mode. 17,798 images were recorded at 1.069Å per pixel with a nominal magnification of 81,000. A total dose of 61.27 e−/Å was fractionated over 50 frames, with a defocus range of −0.8 μm to −2.5 μm. Frames were motion-corrected using MotionCor2  using HRP-anti-rabbit IgG. For detection of protein from mammalian cells, total protein was extracted by MPER lysis (Thermofisher Scientific), quantitated by BCA assay (Pierce), and 10 μg lysate resolved by 4-12% SDS-PAGE. The following antibodies were used: androgen receptor antibody , ERG , B Actin  and Cyclophilin B .m/z 200); data-dependent higher-energy collisional dissociation (HCD) spectra were acquired at the same time in the linear trap using the “top speed” option . Other MS scan parameters included: mass window for precursor ion selection, 0.7; charge states, 2 – 5; dynamic exclusion, 15 sec (± 10 ppm); intensity to trigger MS2, 50,000. Mascot  was used to search the spectra against a combination of the SwissProt database [SwissProt 2019_10 . Cysteine carbamidomethylation was set as a fixed modification and methionine oxidation was considered as a variable modification. Trypsin was specified as the proteolytic enzyme, with two missed cleavages allowed. The Mascot cross-linking feature for DSSO was used for the corresponding sample searches. The results were exported in xiView-CSV format and as a FASTA file containing the identified peptides sequences for import into xiView for data visualization. Cross-linked lysines were depicted in 2D using xiNET cross-link viewer and rendered in 3D as solid-colored spheres mapped onto the Entrenched model using PyMol.DSSO cross-linked complexes were separated by SDS-PAGE (12% Bis-Tris) and stained with Coomassie blue. The bands of interest (~100 kDa) were manually excised, individually reduced in situ with TCEP and alkylated in the dark with iodoacetamide prior to treatment with trypsin . Each digest was analyzed by capillary HPLC-electrospray ionization tandem mass spectrometry on a Thermo Scientific Orbitrap Fusion Lumos mass spectrometer. On-line HPLC separation was accomplished with an RSLC NANO HPLC system (Thermo Scientific/Dionex): column, PicoFrit  packed to 15 cm with C18 adsorbent . Precursor ions were acquired in the orbitrap in centroid mode at 120,000 resolution  as N-terminal VP16 fusions using HiFi assembly (NEB). The human ERG ETS domain, and human VP16-AR WT and mutants were cloned into pCDNA3.1 using HiFi assembly. All other constructs were described previously . For len−/−;sgERG organoids previously generated by electroporation of Cas9-sgRNA ribonucleoprotein complexes against ERG .HEK293T cells were cultured in DMEM with high glucose, 10% fetal bovine serum, and penicillin streptomycin. Mouse prostate organoids were isolated and cultured in Matrigel (Corning) using standard methods . Pten−/−inst ERG  were traThe ARE repeat reporter  has been previously described . The hal−/−;ERG organoids were trypsinized to single cells and plated at a density of 84 cells per 25 μl dome of Matrigel, during which time EGF was removed from the media. Cells were treated with either no ligand (0 nM DHT), with DHT (1 nM), or with enzalutamide (10 μM) for 11 days before quantifying establishment, refreshing media every 2-3 days. Percent formation was derived from number of established organoids divided by the total number of cells plated, multiplied by 100. Data from an average of 8 replicate wells were analyzed using two-way ANOVA, with ****P<0.0001; n.s. (not significant), P>0.05. Data presented as mean +/− standard deviation from n=3 experiments.PtenRNA was extracted from organoids using an RNeasy Kit (Qiagen) followed by on-column DNase treatment. cDNA was generated with the High Capacity cDNA Reverse Transcription Kit (Thermofisher Scientific). qPCR was performed with QuantiFast SYBR Green PCR master mix (Qiagen). Primers used for mouse Actb (QT00095242), Ar (PPM05196F), Krt8 (PPM04776F), Ndrg1 (QT00119266), Nkx3-1 (PPM05232A), Plau (QT00103159), Serpine1 (QT00154756), Tp63 (PPM03458A), and Tmprss2 (QT00156093) were purchased from Qiagen. Data were analyzed using the ΔCt method and statistical significance calculated using two-way ANOVA, with ****P<0.0001; n.s. (not significant), P>0.05. Data presented as mean +/− standard deviation from n=3 experiments.Details regarding statistical analyses, including test types, sample size, and definition of statistical significance may be found in Method Details under respective assays.1Figures S1-13, Table S1 (Separate PDF)3Video S1 – Plasticity of the AR dimer interface is apparent in the transition of Entrenched to Divorced states, Related to 4Table S2 – DSSO cross-links identified in a ΔNTD AR, ERG, ARE DNA complex, Related to


Background: Since the beginning of the SARS-CoV-2 epidemic in Poland, 6,128,006 people have been diagnosed, of which 116,798 died. Patients who recovered from COVID-19 and require rehabilitation due to varied impairments should be provided an opportunity to participate in an individualized, complex rehabilitation program starting from acute care and being continued in the post-acute and long-term rehabilitation phase. It is recommended to offer out-patient and in-hospital rehabilitation procedures depending on the type and persistence of symptoms and dysfunctions. The aim of this paper is to present the qualification process of post-COVID19 patients for an in-hospital complex rehabilitation program developed on the basis of pulmonary physical therapy. Methods: The presented qualification program was developed on the basis of clinical experience of over 2000 patients participating in the pilot program of in-hospital rehabilitation launched in September 2020 and based on the Regulation of the Polish Minister of Health of 13 July 2020. Results: The proposed model of patients’ qualification rests on well-known and validated tools for functional assessment: exercise tolerance assessment, dyspnea intensity assessment, functional fitness assessment, assessment of arterial blood saturation, lung ventilation function assessment, assessment of long-lasting COVID-19 symptoms, and patient’s basic mental health condition. Conclusions: The proposed qualification model for the post-COVID rehabilitation program allows us to introduce adequate qualifications followed by much needed assessment of the health effects. According to WHO data at the beginning of August 2022, there have been 589,680,368 confirmed cases of COVID-19, including 6,436,519 deaths [Scientific research published so far indicates that COVID-19 survivors may experience persistent symptoms affecting various organ systems after the acute phase of infection involving not only the respiratory system, but also cardiovascular, musculoskeletal, gastrointestinal, immunological, endocrine, and neurological systems with varied intensity and duration. Histopathological studies have shown direct and indirect damage to many organs caused by COVID-19. A recently published paper indicates more than 50 health effects observed are fatigue, headache, attention disorder, hair loss, and dyspnea as the five most common manifestations ,7. VarieAccording to WHO rehabilitation is a set of interventions designed to optimize functioning and reduce disability in individuals with health conditions in interaction with their environment and consist of different types of services, such as physical therapy, occupational therapy, or speech therapy, and can be conducted as inpatient and outpatient treatment . The verThe aim of this paper is to present the qualification process of adult post-COVID-19 patients for an in-hospital uniquely defined complex rehabilitation program developed on the basis of pulmonary physical therapy. During the pilot program, the qualification program was modified and adapted to the existing functional disorders in the ranges described.The presented qualification program was developed on the basis of clinical experience of over 2500 patients participating in the pilot program in the field of hospital rehabilitation after COVID-19 disease, launched in September 2020. The aim of the pilot program is not only to assess the effects of the rehabilitation model used and to improve the limited exercise capacity associated with impaired ventilation and dyspnea in people after coronavirus infection, but also to identify the persistent additional symptoms and the resulting rehabilitation needs related to neurological and psychiatric disorders and symptoms from the musculoskeletal system.The research hypothesis adopted for the rehabilitation program of adult post-COVID-19 patients assumed that the use of different rehabilitation models based on the existing pulmonary rehabilitation programs and depending on the individually obtained results in the qualification process is the right procedure for this group of patients.The program is financed under the funds provided for in the financial plan of the National Health Fund. The cost of treatment is 200 PLN per person per day. The pilot program is aimed at COVID-19 survivors who received a referral from a health insurance physician . The diagnosis of COVID-19 was based on a clinical picture of the infection with well-defined symptoms and positive nasopharyngeal swab polymerase chain reaction test for SARS-CoV-2, serologic anti-SARS-CoV-2 antibody test, and/or typical findings on chest computed tomography or x-ray. Patients were referred for rehabilitation based on physicians’ clinical judgment of whom may benefit from exercises to improve functional status. There was no separate protocol for the referrals, however, the patients must have presented post-COVID symptoms after 12 weeks from recovery.The duration of therapeutic rehabilitation is a maximum of 21 days. The conditions for the provision of services, including those relating to medical personnel, equipment, and medical apparatus, meet the requirements set out in Annex 2 to the Regulation of the Minister of Health of 13 July 2020. The pilot project is carried out within the existing Pulmonary Rehabilitation Department of the SP ZOZ Specialist Hospital of the Ministry of the Interior and Administration in Glucholazy, Poland. The program is still ongoing.The rehabilitation program is complex, yet based on currently recommended models of complex respiratory physiotherapy with adequate qualification rules including individualized treatment and rehabilitation program followed by an assessment of the effects in patients who recovered from COVID-19 with complications. Existing clinical observations allowed for modifications of the applied rehabilitation program based on the adequate qualification of the patient and appropriate selection of rehabilitation model ,21,22.A brief characterization of the patients’ group in the pilot program, including gender, required care due to COVID-19 infection, and qualification for individual rehabilitation models (A–E), can be found below .The proposed model of patients’ qualification rests on well-tested and validated tools for functional assessment which allow the physiotherapist to adequately program physical exercises being the core of the therapy. The proposed battery of tests includes: exercise tolerance assessment , dyspnea intensity assessment , functional fitness assessment (Fullerton Test), assessment of arterial blood saturation by pulse oximetry, lung ventilation function assessment via body plethysmography, assessment of long-lasting COVID-19 symptoms (authors’ questionnaire) and patient’s mental health condition .Exercise tolerance is evaluated with the use of a 6-min Walk Test (6MWT), also known as a walk test or a corridor test. It allows global examination of the respiratory system, cardiovascular system, and neuromuscular system functions through the assessment of an aerobic capacity and endurance. Designed for geriatric and cardiopulmonary patients, but widely used in varied adult and pediatric populations. Can be conducted in any place, both indoors and outdoors, with a straight distance of 30 m on a hard, flat surface. Walking distance should be marked with cones for better orientation of the patient and include clear indications for every 3 m. The equipment required to conduct the test includes a stopwatch, pulse oximeter, blood pressure monitor, oxygen supply, and a questionnaire for subjective assessment of the level of dyspnea and tiredness . Before the test, the patient should be asked to rest in the sitting position for 10 min. Intensive physical effort is prohibited 2 h prior. The test should also be conducted on an empty stomach or after a light meal. Blood pressure, pulse, saturation, and dyspnea level should be checked directly before the beginning of the test. The patients are instructed to walk at their own speed and not to run; it is allowed to change their pace, accelerate, or slow down depending on their abilities, they can slow down or stop if they feel tired or short of breath. The goal of the 6MWT is to cover the longest possible distance rather than achieve the shortest time.2 × kg−1 × min−1. MET is determined based on the covered distance, calculated into walk speed, with the following equation: distance × 10/1000, e.g., 500 m = 5.0 km/h.After the test, energy expenditure expressed in metabolic equivalent units (MET) is calculated. One MET equals average energy expenditure at rest (sitting position) and is equivalent to oxygen use at rest in sitting position by a person weighing 70 kg, i.e., 3.5 mL OThe previously existing formula for calculating MET based on the 6MWT was intended for people with severe limitations in exercise capacity, excluding patients who cover a distance of more than 300 m (the results obtained were biased). Modified formula based on the estimation developed on COPD patients (with various exercise impairments) can be used for all patients who require a quick, simple, and effective assessment of their exercise capacity, including those who can cover more than 300 m in the 6MWT . The forThe figure below helps to read MET values: walk speed in km/h on the horizontal axis, and MET values on the vertical axis .Dyspnea intensity is assessed with the use of the subjective 10-point Modified Borg Dyspnea Scale (MBS). MBS is a safe and simple method, used in hospital conditions. ‘0’ at the top of the scale represents no perceived exertion while ‘10’ at the bottom represents the most intensive exertion ever experienced or possible to imagine. To determine the intensity, the patient selects the value which best describes the exertion they experience at the moment .Functional fitness is assessed with the use of one of the components of the Fullerton test known also as the Senior Fitness Test (SFT). It is a safe and simple test comprised of 6 components measuring the strength of the upper and the lower body, aerobic endurance, motor coordination, and balance. For qualification purposes, the ‘30-s Chair Stand’ test was used.The patient sits on the chair, back to a wall, with feet flat on the floor and arms folded across their chest. On the ‘Start’ command, the patient stands up to assume an upright position and sits back on the chair, stands up, and sits back down again. The number of cycle repetitions within 30 s represents the test result. The ‘30-s Chair Stand’ test assesses muscle endurance in the lower body part used for getting up, walking, climbing stairs, and maintaining body balance.Based on exercise tolerance and energy expenditure expressed in MET in the 6MWT, dyspnea assessment (10-pointMBS) and functional fitness assessment with the use of Fullerton test (‘30-s Chair Stand’ test) patients are qualified for adequate rehabilitation models A, B, C, D, or E . The resArterial blood oxygen saturation is another parameter assessed in the process of qualifying patients for the rehabilitation program. The level of hemoglobin saturation with oxygen in arterial blood is tested with the pulse oximetry method (spectrophotometry transmission) during the 6MWT. Saturation norm is approx. 95–99%.Another parameter assessed in the qualification process is the Total Lung Capacity (TLC) index measured in body plethysmography, also known as body plethysmography. It is a lung function test used for the assessment of the total amount of air in the lungs as a certain amount of air remains in the lungs that cannot be exhaled. This is called residual volume (RV) and cannot be measured during spirometry. Plethysmography also allows us to determine the resistance to which air meets going through the respiratory tract. Obturation degree can be measured indirectly in this way. The lowest limit for TLC is set at the 5 percentile in reference population and equals the predicted value. The percentile is calculated with the function of cumulated probability for normal distribution and is equivalent to a given deviation from the predicted value. The result expressed in percentiles shows what percentage of the healthy population  scores lower than the patient tested . The principles for interpreting the results presented are based on the Recommendations of the Polish Society of Lung Diseases. The limits of the norm are determined arbitrarily on the basis of the probability theory and allow a certain margin of error, therefore the borderline results are always interpreted with great caution.In case of saturation below 92% and/or TLC < 5 percentile, the rehabilitation model is lowered by one group.Given the deficits which occur long after the infection, complex therapy focused on restoring the function of skeletal muscles, as well as physical and mental aspects is recommended. Apart from symptoms from the respiratory system, COVID-19 convalescents frequently experience weakened muscle strength, coordination and balance disorders, memory and concentration disorders, anxiety, and depression symptoms. The qualification for rehabilitation procedure, therefore, includes the results of the authors’ questionnaire on the subjective presence of additional post-COVID symptoms, Hospital Anxiety and Depression Scale (HADS), and record of the assessment of PCFS syndrome  . TreatmeThe proposed post-COVID-19 rehabilitation program includes five different models of therapeutic activities  based on the individual exercise capacity of the patient. The models differ mainly in the intensity of exercise, which is determined for each patient also based on additional COVID-related symptoms and comorbidities. The specific abilities and needs of the patient should be taken into account, which are analyzed both on the basis of the results obtained in screening tests, anamnesis, and previous medical history.Models A, B, and C consist of physical efficiency training on a cycle ergometer, walking training, resistance training, general fitness and circuit training, breathing exercises, procedures used to directly remove bronchial secretions, inhalations, and relaxation techniques. While the elements of the rehabilitation program for each of the models are the same, the difference is related to the intensity of training, starting from 80% of the submaximal heart rate for model A to 60% of the submaximal heart rate for model C. Models D and E consist of breathing exercises, general fitness exercises, circuit training, procedures used to directly remove bronchial secretions, inhalations, and relaxation techniques. The elements included in the rehabilitation program for both models are the same, the difference results from the intensity of the training sessions: heart rate increase during exercises by 20–30% in relation to the heart rate at rest in model D while in model E exercises are conducted in a sitting position on a chair with a heart rate increase during exercises by 20–30% in relation to the heart rate at rest.Particularly important in the program is the use of new technologies, including breathing exercises with biofeedback, in order to improve respiratory dysfunction; relaxation in virtual reality (VR) conditions; interactive exercises combining cognitive exercises and physical exercises in VR focused on improving concentration, perception, memory, understanding and efficiency of communication, balance and coordination, strengthening the muscles of the lower and upper limbs, improving movement control, improving reaction time. Thanks to VR systems, patients can use sports facilities, such as a volleyball court, a climbing wall, a tennis court, and an obstacle course in hospital conditions.The rehabilitation program also uses a high-intensity electromagnetic field to stimulate diaphragm contractility, intercostal muscles, and respiratory auxiliary muscles; stochastic resonance to normalize muscle tone, improve static and dynamic balance and coordination, stimulate deep sensory perception, and restore gait dysfunction; microcirculation therapy and vascular therapy.The individual elements for each model in the presented rehabilitation program have been described and published in a separate article .The proposed activities are carried out both individually and in groups depending on the patient’s current condition, postoperative complications, observed symptoms, their scope, and severity with a special focus on disorders of the musculoskeletal and nervous systems, as well as disorders of mental. It should be remembered that during the rehabilitation process, the patient’s health should be assessed regularly not only in order to assess the progress, but also for possible deterioration and undesirable effects.Patients who recovered from COVID-19 and require rehabilitation due to varied health impairments should be provided an opportunity to participate in an individualized, complex rehabilitation program starting from acute and early post-acute care and being continued in the post-acute and long-term rehabilitation phase. It is recommended to offer out-patient and in-hospital rehabilitation procedures depending on the type and persistence of symptoms and dysfunctions ,27. DataTaking into account both the previous experience related to physiotherapy in respiratory system diseases as well as the occurring and described post-COVID-19 symptoms lasting more than 12 weeks and requiring rehabilitation in hospital conditions among survivors, a simple, point-coded qualification model based on the available, verified and described in the literature on the subject tests has been developed. The basic assumption was a balance between a holistic approach to the highly unspecific symptoms, yet underlying functional impairments of patients, the availability of therapeutic services in healthcare facilities, capacity of sharing different therapeutic approaches within the multidisciplinary team, and the selection of a set of diagnostic tools that would be understandable and known to physiotherapists. The novelty in the proposed qualification is the scoring system. It is based on the results of the described tests, enabling the appropriate assignment of the patient to the specific post-COVID rehabilitation models, differing in the intensity of physical exertion and specific treatments adapted to the symptoms from the individual ranges mentioned above . The calPhysical exercises treated and dosed like medicine are the basis of physical therapy. Adjusting the effort to the individual abilities of the patient enables an appropriate and planned qualification process based on the original, modified determination of energy expenditure expressed in MET and an easy and quick assessment of indicators of other elements of the functional assessment, enabling self-assessment of physiotherapeutic methods. Published papers point out that early rehabilitation for COVID-19 hospitalized patients was associated with lower in-hospital mortality even with a rather low dosage of exercises . Lack ofThe presented process of qualification of adult post-COVID-19 patients to the rehabilitation program consisting of 5 different therapeutic models, has some limitations. First of all, conducting a full functional examination in accordance with the described qualification and then continuing a comprehensive rehabilitation program requires housing facilities, administrative support, and a large, multidisciplinary team of health care workers. The size of the center does not determine the possibility of introducing the presented qualification, but it can significantly affect the effectiveness and the number of patients who can participate in the rehabilitation program at the same time. Modifications for the smaller centers and outpatient care facilities, as well as further development of therapeutic activities using new technologies, should be considered. The second significant limitation of the proposed qualification is the lack of a comprehensive assessment of the mental state conducted by specialized clinicians. Mental health may affect both the course of the underlying disease and the effects of the rehabilitation process.The methodology presented in the paper is associated with new perspectives regarding the health care for adult post-COVID-19 patients. As it is universal in terms of the assessment of varied needs related to in-/outpatient rehabilitation and subsequently, further therapeutic decisions, thus can be introduced outside hospital-based rehabilitation units. It allows a functional assessment of the patient’s post-COVID-19 fitness, so it can fulfill more than just the role of qualification for rehabilitation models.Additionally, the presented qualification indicates the need for the usage of a modified formula for calculating MET based on the 6MWT in order to qualify homogeneous groups of patients after COVID-19 with limited exercise capacity and load them appropriately with physical effort. A cardiac stress test is time-consuming and costly in comparison, equipment-related requirements may make it impossible to perform in smaller therapeutic centers.The proposed qualification model, confirmed by the previous theoretical knowledge and clinical experience, is, therefore, an attempt to present a transparent, uniform functional assessment of the patient before starting the rehabilitation process. Such a qualification enables appropriate planning and adaptation of therapeutic activities to the needs of patients, as well as the subsequent broad assessment of therapeutic effects after the end of the program.The wide application of the unified qualification model also enables a more universal functional assessment of post-COVID-19 patients, which will allow further better and more directed healthcare. Structured clinical observation over a longer period of time may also give a better understanding of the long COVID-19 symptoms.However, for holistic patient care, consideration should be given to introducing regular mental status assessments.