Macrocyclic Nonapeptides Incorporating Uncharacterized Amino Acids with Inhibitory Effects on Th17 Differentiation

Open AccessCCS ChemistryRESEARCH ARTICLE1 Feb 2021Macrocyclic Nonapeptides Incorporating Uncharacterized Amino Acids with Inhibitory Effects on Th17 Differentiation Kai-Long Ji, Yao-Yue Fan, Hio-Ha Kuok, Qun-Fang Liu, Ting Li and Jian-Min Yue Kai-Long Ji State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203 , Yao-Yue Fan State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203 , Hio-Ha Kuok State Key Laboratory of Quality Research in Chinese Medicines, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macau 999078 , Qun-Fang Liu State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203 , Ting Li State Key Laboratory of Quality Research in Chinese Medicines, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macau 999078 and Jian-Min Yue *Corresponding author: E-mail Address: [email protected] State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203 https://doi.org/10.31635/ccschem.020.202000265 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Four unprecedented macrocyclic nonapeptides, orberryamides A–D ( 1– 4), were isolated from Glycosmis pentaphylla (orangeberry) and structurally characterized by obtaining solid data from numerous analytical measurements. Compound 1 incorporated a new amino acid residue, named orangeberrine (Orgb), compounds 2 and 3 integrated the tryptophan, an essential amino rare, nonproteinogenic amino acid residues (Kyn and Dioia, respectively), and compound 4 existed as two major conformational isomers in solution at ambient temperature. The biosynthetic pathways proposed for compounds 1– 4 are of considerable biological significance for the modification and metabolism of tryptophan (Trp) and/or Trp containing proteins in nature. Besides, compounds 1– 4 suppressed Th17 differentiation significantly, and the effects of 1– 3 was achieved through targeting the ligand-binding domain (LBD) of the retinoic acid-related orphan receptor gamma t (RORγt). Download figure Download PowerPoint Introduction Cyclopeptides that originated from natural resources have been attracting enormous attention from the communities of chemists and biologists owing to the complex and intriguing chemical structures and their broad spectrum of biological activities.1–3 A subset of immune CD4 T cells, Th17 cells, and their signature cytokine IL-17 orchestrate the pathology of many common human autoimmune diseases, such as psoriasis, rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis.4 Retinoic-acid-related orphan receptor gamma t (RORγt) acts as a critical transcription factor for the induction of IL-17 transcription and the manifestation of the Th17-dependent autoimmune diseases.5 In the presence of IL-6, transforming growth factor-β (TGF-β) induces the differentiation of naive CD4+ T cells into Th17 cells by activating RORγt.6 RORγt is composed of modular protein structures with DNA- and ligand-binding domains (DBDs and LBDs), and the RORγt LBD provides binding sites for the antagonists, which is becoming an attractive target for drug development to treat autoimmune diseases.7 The development of novel RORγt antagonists, thus, remains a high priority. The plant, Glycosmis pentaphylla (orangeberry), is mainly distributed in the Southeast Asian countries,8 and has been used as folk medicine by Dai minority people in China for the treatment of bronchitis, ulcer, and traumatic injuries.9 Previous chemical investigations on this plant afforded several carbazoles, acridone, and quinolone alkaloids with cytotoxic,10,11 antibacterial,12,13 and antimutagenic14 activities. The important medicinal indication and the structurally diverse chemical constituents of this plant have thus attracted our research interests. As part of our ongoing projects for the discovery of structurally attractive and immunologically active agents from medicinal plants,15–18 four unprecedented macrocyclic nonapeptides, orberryamides A–D ( 1– 4) (Figure 1) were isolated from the titled plant mainly via semi-preparative high performance liquid chromatography (HPLC). Their full structures were determined by a comprehensive analysis of nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), X-ray crystallography, and electronic circular dichroism (ECD) data, as well as Marfey's method of enantiomeric analysis. The results showed that these compounds shared a rare sequence of eight amino acid residues, and each of them incorporated different ones in their sequences to furnish four unique 27-membered macrocyclic nonapeptides. It is noteworthy that compound 1 possessed a new amino acid residue, named orangeberrine (Orgb), compounds 2 and 3 integrated the rare nonproteinogenic amino acid residues of kynurenine (Kyn) and dioxindolyalanine (Dioia), respectively, and compound 4 had two major conformers in solution at room temperature (RT). A plausible biosynthetic origin of compounds 1– 4 was proposed; compounds 1– 3 were believed to be derived from 4 via enzyme catalytic oxidation cascades. These compounds 1–4 are of particular interest to us due to their inherent connections with the modification and metabolism of tryptophan, an essential amino acid that serves as a precursor for neurotransmitters (serotonin and tryptamine) and the hormone melatonin, which plays a vital role in the regulation of the sleep cycle. Additionally, we found that compounds 1– 4 could exhibit significant inhibition of Th17 differentiation, and the mode of action of compounds 1– 3 could be accomplished through targeting of the LBD of RORγt. Herein, we present the isolation, structural elucidation, biosynthetic consideration, and biological evaluation of compounds 1– 4. Figure 1 | Structures of compounds 1–4. Download figure Download PowerPoint Experimental Method General experimental procedures Melting points of compounds 1– 4 were obtained (uncorrected) with an SGW X-4 melting point apparatus (Drawell Scientific Instrument Co., Ltd., Shanghai, China). Optical rotations were measured on an Autopol VI polarimeter (Rudolph Research Analytical, Shanghai, China). UV spectra were acquired on a Shimadzu UV-2550 spectrophotometer (Shanghai, China). ECD spectra were collected on a JASCO J-810 spectrometer (JASCO, Shanghai, China). Infrared (IR) spectra were acquired on a Thermo IS5 infrared spectrometer with KBr disks (Thermo Fisher Scientific, Shanghai, China). The one-dimensional (1D) and two-dimensional (2D) NMR spectra were recorded in DMSO-d6 solvents on Brucker AVANCE III 400, AVANCE III 500 (Beijing, China), and/or Ascend 600 NMR instruments (Bruker, Shanghai, China). Liquid chromatography–mass spectrometry (LC-MS) and tandem MS (MS/MS) data were acquired on an Agilent G6520 Q-TOF mass spectrometer (Shanghai, China). ESI-MS and HRESI-MS data were obtained on a Waters Micromass Q-TQF Ultima Global mass spectrometer (Shanghai, China). Analytical high-performance liquid chromatography (HPLC) was performed using Agilent 1100 series (Shanghai, China), and semipreparative HPLC was conducted using a Waters 1525 system (Shanghai, China) with a Waters 2489 UV detector and a Fortis H2o column (250 × 10 mm, S-5 μm). Silica gel (300–400 mesh, Qingdao Maring Chemical Co. Ltd.), MCI gel (CHP20P, 75–150 μm, Mitsubishi Chemical Industries Ltd.), and Sephadex LH-20 gel (40–70 μm, Amersham BioSciences, Beijing China) were used for column chromatography (CC). All solvents used for CC were of analytical grade (Shanghai Chemical Reagents Ltd., China), and solvents used for HPLC were of HPLC grade (J&K Scientific Ltd., Beijing, China). Plant material The roots of G. pentaphylla were collected from Mengla County of Yunnan Province, China, in November 2015 and identified by Professor You-Kai Xu (Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences). A voucher specimen (accession no. Orberry-2015-YN-1Y) has been deposited in Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Extraction and isolation Dried powder of the roots of G. pentaphylla (5.0 kg) was extracted three times with 95% EtOH at RT to give a crude extract (400 g), which was dissolved in water and then partitioned with EtOAc. The EtOAc-soluble fraction (∼ 100 g) was subjected to MCI gel ion exchange column (MeOH/H2O, 1∶1 to 9∶1) to produce three major fractions F1–F3. Fraction F2 (36 g) was fractionated over a silica gel column and eluted with gradient mixtures of petroleum ether/EtOAc (from 20∶1 to 1∶2) to give seven subfractions F2a–F2g. Then the fraction F2g (5.0 g) was separated by a Sephadex LH-20 gel column (EtOH), which afforded six fractions F2g1–F2g6. Further purification of the fraction F2g2 by semipreparative reverse phase (RP)-HPLC (mobile phase: 58% acetonitrile in water; flow rate: 3 mL/min; and UV detection wavelength: 210 nm), yielding the following compounds at the specified retention times: compounds 1 (5.0 mg, tR = 15.5 min), 2 (8.0 mg, tR = 18.0 min), 3 (1.5 mg, tR = 20 min), and 4 (50.0 mg, tR = 16.5 min). Other characterization outcomes are as follows: Orberryamide A (1) Colorless crystal; mp 198–200 °C; [α]18D –47.9 (c 0.12, MeOH); UV (MeOH) λmax (log ɛ) 245 (4.02), 286 (3.76) nm; CD (MeOH) λmax (Δɛ) 200 (–32.89) nm; IR (KBr) νmax 3318, 3069, 2961, 2932, 2873, 1652, 1527, 1454, 1387, 1370, 1262, 1160, 1107, 1029 cm–1; 1H and 13C NMR data, see Table 1; (+)-ESI-MS m/z 1033.6 [M + Na]+; (+)-HRESI-MS m/z 1033.6061 [M + Na]+ (calcd for C51H82N10O11Na, 1033.6062). Table 1 | 1H and 13C NMR Spectroscopic Data for Compounds 1 and 2 Position 1a Position 2a δH (J in Hz) δC δH (J in Hz) δC Orgb1 NH 7.54, d (5.7) Kyn1 NH 7.48, d (6.2) 1 170.2 1 170.2 2 4.74, m 48.7 2 4.83, m 49.0 3 2.91, m (2H) 37.1 3 3.43, m 39.2b 4 169.6 3.13, dd (15.8, 8.6) 5 125.8 4 199.9 6 147.8 5 116.3 7 6.84, dd (8.0, 1.2) 115.3 6 151.4 8 6.92, td (8.0, 1.3) 124.6 7 6.75, dd (8.5, 1.2) 117.1 9 6.65, td (8.0, 1.2) 118.3 8 7.25, ddd (8.5, 6.9, 1.5) 134.6 10 7.80, dd (8.0, 1.3) 121.7 9 6.53, ddd (8.3, 6.9, 1.2) 114.5 NH2 9.80, br s 10 7.62, dd (8.3, 1.5) 131.0 9.45, br s NH2 7.29, br s (2H) Leu2 NH 8.07, d (6.4) Leu2 NH 8.32, d (6.0) 11 172.1 11 172.4 12 4.05, m 53.3 12 3.96, m 53.3 13 1.42, m (2H) 40.4 13 1.38, m (2H) 40.2 14 1.56, m 24.3 14 1.54, m 24.4 15 0.85, d (6.4)b 21.2 15 0.86, d (6.7) 20.8 16 0.81, d (6.4) 22.7 16 0.78, d (6.7)b 22.8 Leu3 NH 8.37, br sb Leu3 NH 8.52, br s 17 172.0b 17 172.7 18 3.70, m 55.0 18 3.63, m 56.0 19 1.83, m 38.6 19 1.89, m 38.7 1.58, m 1.59, m 20 1.53, m 24.5b 20 1.50, m 24.5 21 0.87, d (6.4) 22.6 21 0.87, d (6.5) 22.5 22 0.85 d (6.4)b 21.3 22 0.85, d (6.5) 21.4 Val4 NH 8.37, br sb Val4 NH 8.45, br s 23 172.8 23 172.5 24 3.57, m 60.9 24 3.54, m 60.9 25 1.94, m 29.1 25 1.92, m 29.0b 26 0.95, d (6.6) 18.9 26 0.95, d (6.7) 18.8 27 0.92, d (6.6) 19.7 27 0.92, d (6.7)b 19.5 Val5 NH 7.37, br s Val5 NH 7.32, br s 28 172.0b 28 172.8 29 4.26, m 57.5 29 4.27, m 57.3 30 1.86, m 29.0 30 1.85, m 29.0b 31 0.90, d (6.7) 19.2 31 0.92, d (6.7)b 19.3 32 0.79, d (6.7) 18.1 32 0.78, d (6.7)b 18.0 Gly6 NH 7.58, t (6.0) Gly6 NH 7.56, t (6.0) 33 168.7 33 168.5 34 3.98, m 42.8 34 3.98, m 42.7 3.42, m 3.38, dd (16.5, 6.0) Leu7 NH 7.14, d (9.5) Leu7 NH 7.07, d (9.3) 35 172.3 35 172.0 36 4.21, m 50.0 36 4.20, td (9.3, 4.7) 49.7 37 1.63, m 39.7 37 1.57, m 39.4 1.36, m 1.02, m 38 1.33, m 24.0 38 1.28, m 24.3 39 0.60, d (6.4) 20.6 39 0.48, d (6.6) 20.7 40 0.58, d (6.4) 22.7 40 0.38, d (6.6) 22.1 Leu8 NH 7.73, d (5.6) Leu8 NH 7.92, br s 41 172.2 41 171.7 42 3.95, m 53.9 42 3.95, m 54.0 43 1.71, m 38.7 43 1.72, m 39.2b 1.60, m 1.62, m 44 1.67, m 24.5b 44 1.77, m 24.5 45 0.89, d (6.6) 22.4 45 0.97, d (6.4) 22.4 46 0.88, d (6.6) 21.7 46 0.88, d (6.4) 21.8 Pro9 47 173.9 Pro9 47 174.5 48 4.02, m 61.8 48 3.99, m 62.2 49 2.28, m 29.2 49 2.29, m 29.0b 1.81, m 1.83, m 50 1.97, m (2H) 24.6 50 1.98, m (2H) 24.7 51 3.79, dt (9.4, 6.7) 47.0 51 3.79, dt (9.1, 6.7) 46.9 3.44, m 3.40, m aRecorded at 600 MHz (1H) and 125 MHz (13C) in DMSO-d6. bOverlapping signals. Orberryamide B (2) Pale yellow crystal; mp 259–261 °C; [α]19D –16.4 (c 0.07, MeOH); UV (MeOH) λmax (log ɛ) 227 (4.36), 260 (3.78) nm; CD (MeOH) λmax (Δɛ) 198 (–53.12) nm; IR (KBr) νmax 3453, 3333, 3073, 2959, 2931, 2873, 1659, 1631, 1518, 1469, 1451, 1390, 1369, 1316, 1267, 1212, 1163 cm–1; 1H and 13C NMR data, see Table 1; (+)-ESI-MS m/z 1017.6 [M + Na]+; (+)-HRESI-MS m/z 1017.6114 [M + Na]+ (calcd for C51H82N10O10Na, 1017.6113). Orberryamide C (3) White, amorphous powder; [α]18D –78.1 (c 0.16, MeOH); UV (MeOH) λmax (log ɛ) 250 (3.70) nm; CD (MeOH) λmax (Δɛ) 198 (–26.74), 212 (+14.70), 240 (–15.14), 265 (+1.06) nm; IR (KBr) νmax 3310, 2959, 2926, 2873, 2854, 1715, 1650, 1523, 1472, 1387, 1372, 1335, 1260, 1205, 1184, 1099, 1027 cm–1; 1H and 13C NMR data, see Table 2; (+)-ESI-MS m/z 1045.6 [M + Na]+; (+)-HRESI-MS m/z 1045.6067 [M + Na]+ (calcd for C52H82N10O11Na, 1045.6062). Table 2 | 1H and 13C NMR Spectroscopic Data for Compound 3a Position δH (J in Hz) δC Position δH (J in Hz) δC Dioia1 NH 7.18, br s Val5 NH 7.48, br s 1 171.3 28 172.2 2 5.27, m 46.4 29 4.07, m 58.6 3 2.39, m 38.3 30 1.91, m 28.9 1.86, m 31 0.88, d (6.4)b 19.3 4 73.4 32 0.78, d (6.4)b 17.9 4a 141.0 Gly6 NH 7.44, br s 5 179.0 33 169.4 NH-6 10.23, br s 34 3.98, m 42.8 7 6.88, d (7.4) 110.5 3.48, m 7a 132.4 Leu7 NH 7.42, br s 8 7.00, t (7.4) 122.3 35 172.6 9 7.24, t (7.4) 129.3 36 4.17, m 51.1 10 7.30, d (7.4) 123.6 37 1.43, m (2H) 40.4 Leu2 NH 7.98, br s 38 1.46, m 23.8 11 171.4b 39 0.53, d (5.9) 20.9b 12 3.96, m 53.2 40 0.45, d (5.9) 22.3 13 1.57, m (2H) 39.2b Leu8 NH 7.56, br s 14 1.60, m 24.4b 41 172.4 15 0.82, d (6.4)b 21.1 42 4.09, m 53.3 16 0.88, d (6.4)b 23.1 43 1.88, m 38.4 Leu3 NH 7.93, br s 1.54, m 17 171.7b 44 1.68, m 24.8 18 3.92, m 53.0 45 0.92 d (6.6) 22.9 19 1.50, m (2H) 39.2b 46 0.84, d (6.6) 20.9b 20 1.64, m 24.4b Pro9 47 173.5 21 0.82, d (6.3)b 23.0 48 4.20, m 61.6 22 0.81, d (6.3) 21.3 49 2.23, m 29.5 Val4 NH 7.38, br s 1.84, m 23 171.6b 50 2.00, m (2H) 24.5 24 3.84, m 59.3 51 3.80, m 47.4 25 1.97, m 28.9 3.69, m 26 0.88, d (6.4)b 18.5 27 0.78, d (6.4)b 19.6 aRecorded at 600 MHz (1H) and 125 MHz (13C) in DMSO-d6. bOverlapping signals. Orberryamide D (4) White, amorphous powder; [α]18D –92.5 (c 0.08, MeOH); UV (MeOH) λmax (log ɛ) 220 (4.62), 283 (4.58) nm; CD (MeOH) λmax (Δɛ) 200 (–38.08) nm; IR (KBr) νmax 3313, 3057, 2961, 2936, 2875, 1652, 1529, 1470, 1448, 1387, 1372, 1340, 1282, 1252, 1166, 1097, 1029 cm–1; 1H and 13C NMR data, see Table 3; (+)-ESI-MS m/z 1013.6 [M + Na]+; (+)-HRESI-MS m/z 1013.6166 [M + Na]+ (calcd for C52H82N10O9Na, 1013.6164). Table 3 | 1H and 13C NMR Spectroscopic Data for Compound 4 Position 4aa 4ba δH (J in Hz) δC δH (J in Hz) δC Trp1 NH 7.38, br s 7.36, br s 1 171.3 171.5 2 4.97, q (8.0) 51.1 4.45, m 53.5b 3 3.06, m 26.5 3.11, m (2H) 27.4 2.93, dd (15.0, 8.0) 4 109.6 108.5 4a 127.7 126.9 5 7.11, d (2.2) 123.1 7.15, d (2.2) 124.2 NH-6 10.81, d (2.2) 11.00, d (2.2) 7 7.31, d (7.8)b 111.2 7.31, d (7.8)b 111.4 7a 135.9 136.1 8 7.04, t (7.8) 120.7 7.08, t (7.8) 121.3 9 6.95, t (7.8)b 118.0 6.95, t (7.8)b 118.4 10 7.42, d (7.8)b 117.2 7.42, d (7.8)b 118.2 Leu2 NH 7.38, br s 7.36, br s 11 171.8 171.9 12 4.48, ovb 50.2 4.48, ovb 50.3 13 1.48, m (2H) 41.4 1.36, m (2H) 41.5 14 1.51, ovb 24.5 1.51, ovb 24.4b 15 0.86, ovb 21.3b 0.92, ovb 21.8c 16 0.86, ovb 22.8 0.86, ovb 23.2d Leu3 NH 8.08, d (7.4) 8.27, br s 17 171.0 170.9 18 3.92, m 53.4 3.71, m 54.9 19 1.33, m (2H) 40.2b 1.81, m (2H) 40.2b 20 1.24, m 24.0 1.56, m 23.9 21 0.74, d (6.2) 22.5 0.92, ovb 23.0b,d 22 0.60, d (6.2) 21.1 0.86, ovb 21.3b,e Val4 NH 8.36, br s 7.86, br s 23 172.2 172.4 24 3.64, m 60.4 4.00, m 58.7 25 1.95, m 29.3 2.16, m 29.8 26 0.92, ovb 18.9 0.92, ovb 18.3 27 0.86, ovb 19.0 0.86, ovb 19.3 Val5 NH 7.40, br s 7.70, d (8.7) 28 172.0 172.4 29 4.21, m 57.4 4.19, m 58.9 30 2.01, m 29.1 2.06, m 30.6 31 0.92, ovb 19.5 0.92, ovb 19.4 32 0.86, ovb 18.0 0.86, ovb 18.3 Gly6 NH 7.64, t (5.4) 8.31, br s 33 168.6 169.1 34 4.05, m 42.7 3.88, m 43.3 3.45, dd (16.5, 5.4) 3.68, m Leu7 NH 7.57, d (9.3) 7.83, br s 35 172.1 173.0 36 4.26, m 50.4 4.19, m 51.9 37 1.46, m 39.3 1.58, m 39.7 1.39, m 1.53, m 38 1.43, m 24.3 1.42, m 24.2 39 0.65, d (6.1) 20.7 0.83, d (6.2) 21.2b,e 40 0.53, d (6.1) 22.6 0.76, d (6.2) 23.0b,d Leu8 NH 8.26, br s 7.78, br s 41 170.2 170.8 42 4.03, m 53.5b 4.07, m 53.5b 43 1.65, ov (2H)b 38.8 1.65, ov (2H)b 38.7 44 1.70, ovb 24.6 1.70, ovb 24.4b 45 0.97, d (6.2) 22.7 0.92, ovb 23.1d 46 0.87, d (6.2) 21.2b 0.86, ovb 21.6c Pro9 47 174.6 174.5 48 4.11, m 62.2 3.37, m 59.9 49 2.24, m 28.8 1.61, m (2H) 28.5 1.79, m 50 1.88, m (2H) 24.8 1.28, m 21.4 1.14, m 51 3.57, m 47.0 3.13, m 45.8 3.29, dt (9.8, 6.8) 3.09, m aRecorded at 400 MHz (1H) and 125 MHz (13C) in DMSO-d6, and coupling constants were not averaged ( 4a and 4b: trans- and cis-oriented amide bond of Pro9, respectively). bOverlapping signals. c,d,eNMR values with same letters can be interchanged. Marfey's analysis of compounds 2 and 4 Each of the peptide samples (1 mg) was treated with 1 mL of 6 M HCl at 80 °C in a sealed vial for 5 h and then dried under vacuum. The residue was treated with 1 M NaHCO3 (40 μL) and Marfey's reagent, L-FDAA (1% solution in acetone, 100 μL) at 40 °C for 1 h. After the reaction was neutralized with 1 M HCl (40 μL), the mixture was diluted with acetonitrile (200 μL), filtered with 4.5 μm filter, and kept at 4 °C for HPLC analysis. Authentic Amino acids were also derivatized with L-FDAA in the same way. The L-FDAA derivatives of both the peptide hydrolysates and authentic amino acids were analyzed by LC-MS using an Agilent G6520 HPLC-DAD-ESIMS apparatus (0.5 mL/min, 45 min linear gradient elution from 30% to 50% MeCN/H2O with 0.1% formic acid) equipped with a Waters X-Bridge C18 column (5 μm, 250 × 4.6 mm, column temperature at 30 °C). The absolute configurations of the chiral amino acids in the cyclic peptides were determined by comparing the retention times and the mass data with those of authentic samples. X-Ray crystallographic analysis of compounds 1 and 2 The crystals of 1 and 2 were obtained from the recrystallization of each compound in isopropanol. X-Ray analyses were carried out on a Bruker D8 Venture diffractometer with Ga Kα radiation (λ = 1.34139 Å) for 1 and a Bruker APEX-II CCD diffractometer with Cu Kα radiation (λ = 1.54178 Å) for 2. The acquisition parameters for 1 and 2 were provided in the Supporting Information Tables S3 and S4, respectively, and crystallographic data for compounds 1 (Deposition no. CCDC 1935025) and 2 (Deposition no. CCDC 1935024) have been deposited at the Cambridge Crystallographic Data Center. Copies of the data are available free of charge at: www.ccdc.cam.ac.uk/conts/retrieving.html. Bioassays Reagents Antimouse CD3ɛ, CD28, anti-IL-4, recombinant mouse IL-6 and IL-23, recombinant human TGF-β1, PE antimouse IL-17A, PerCP/Cy5.5 antimouse CD4, monensin solution, brefeldin A solution, and red blood cell lysis buffer were obtained from Biolegend (Beijing, China). CytofixTM Fixation buffer and stain buffer were obtained from BD Bioscience (Shanghai, China). CD4+ T cell isolation kit was purchased from Miltenyi Biotec (Beijing, China). RPMI-1640, DMEM, trypsin, and fetal bovine serum (FBS) were obtained from GIBCO (Shanghai, China). Animals C57BL/6 mice (6–8 weeks) were provided by the Chinese University of Hong Kong. All mice were kept under specific pathogen-free conditions in the animal care facility at Macau University of Science and Technology. Animal care and experiments were conducted in accordance with the Laboratory Animal Research Committee Guidelines of Macau University of Science and Technology. Recombinant protein preparation LBD of human RORγt gene (Genbank accession number NP_005051, aa 259-518) containing 6 × His tag in the C terminal was constructed and cloned into pET21b. The plasmids of RORγt LBD was transformed into the Escherichia coli strain BL21 (DE3) cells for overexpression. The transformed cells were grown at 37 °C in Luria-Bertani broth containing 50 μg/mL ampicillin and the protein expression of RORγt LBD was induced by 0.2 mM isopropyl β-d-thiogalactopyranoside (IPTG) by shaking overnight at 18 °C. The E. coli cells were harvested by centrifugation at 4000 rpm for 20 min and resuspended in precooled nickel–nitrilotriacetic acid (Ni-NTA) buffer A [50 mM Tris-HCl 8.5, 300 mM NaCl, 10 mM imidazole, 5% Glycerol, 1 mM β-mercaptoethanol (β-ME), 1 mM PMSF]. Resuspended cells were passed through a cryogenic overpressure cell breaker at 1200 psi. Both the supernatant and the precipitate were obtained by centrifugation at 14,000 rpm for 1 h at 4 °C. The supernatant was then applied to Ni-NTA affinity column (Qiagen, Shanghai, China), which was previously equilibrated with Ni-NTA buffer A. The target protein was eluted with Ni-NTA buffer B (50 mM Tris-HCl 8.5, 300 mM NaCl, 250 mM imidazole, 5% Glycerol, 1 mM β-ME, 1 mM PMSF). The RORγt-containing fractions were pooled, dialyzed in a buffer containing 300 mM NaCl, 50 mM Tris-HCl 8.5, 5% Glycerol, 1 mM dithiothreitol (DTT). The protein was purified further by anion-exchange chromatography on a QHP column (GE Healthcare, Shanghai, China) with a NaCl gradient. The soluble purified protein obtained was stored at –80 °C until further use. T cell subsets differentiation Naive CD4+ T cells were sorted by magnetic bead (Miltenyi Biotec) from spleen of C57BL/6 mice according to the manual, and then CD4+ T cells (5 × 105 cells/well) were incubated with 2 μg/mL of plate-bound antimouse CD3ɛ. To determine the effects of compounds on Th17 differentiation, cells were cultured for 3 days in the presence of 5 μg/mL anti-CD28, 50 ng/mL IL-6, 1 ng/mL TGF-β1, 10 μg/mL anti-IL-4, and 5 ng/mL IL-23 with or without the indicated compounds. The cells were restimulated with 50 ng/mL PMA, 1 μg/mL ionomycin, and 1 μg/mL Brefeldin A for 5 h after 3 days. For Th1 and Th2 differentiation, cells were restimulated with 50 ng/mL PMA, 1 μg/mL ionomycin for 1 h followed incubation of 2 μM Monensin for additional 4 h before intracellular staining. To analyze T cell subpopulations, the cells were stained with the indicated fluorophore-conjugated monoclonal antibodies. PerCP/Cy5.5 anti-CD4 was applied to indicate the surface marker of T lymphocytes. After the permeabilization of cell, PE anti-IL-17A was used to detect the presence of the cytokine. The expression level of IL-17A was determined by BD FACSAriaTM III (BD Bioscience) and analyzed by FlowJo software ( https://www.flowjo.com/). Binding affinity assay During the Biolayer interferometry experiment, the steady-state analysis was performed by detecting each probe in the buffer (0.1% DMSO). The probe-protein binding results were obtained based on fitting steady-state results. The binding kinetics of indicated compounds on RORγt LBD were conducted using Octet RED96 instrument (Pall ForteBio, Shanghai, China). Briefly, the recombinant human RORγt LBD protein was biotinylated using EZ-Link™ NHS-LC-LC-Biotin (ThermoFisher, Shanghai, China), according to the manufacturer's instructions. To immobilize the biotinylated protein onto biosensors, the streptavidin biosensors (Pall ForteBio) were incubated in the biotinylated protein at a final concentration of 6 ng/µL in PBS at 4 °C overnight. 200 µM of indicated compounds and digoxin were prepared and serially diluted at 1∶2 and incubated with biotinylated protein. The acquired data were analyzed using the custom ForteBio software Data Acquisition 9.0 and Data Analysis 9.0 (Shanghai, China). Results and Discussion Orberryamide A ( 1) had a molecular formula of C51H82N10O11, and incorporates 16 double-bond equivalents (DBEs), as determined by HRESI-MS ion peak at m/z 1033.6061 [M + Na]+ (calcd 1033.6062) and NMR analysis (Table 1). The 1H NMR, HSQC, and TOCSY spectra of 1 showed typical signals for eight amido protons (–NH–, ranging from δH 7.14 to 8.37), an amino group (–NH2, δH 9.45 and 9.80), four coupling aromatic protons (δH 6.65, 6.84, 6.92, and 7.80), and 12 doublet methyls. Analysis of the 13C NMR (Table 1), distortionless enhancement by polarization transfer (DEPT), and heteronuclear single quantum coherence spectroscopy (HSQC) spectra revealed the presence of 10 carbonyls (ranging from δC 168.7 to 173.9), a disubstituted phenyl group, as well as 12 methyls, 9 methylenes, and 14 methines. Further interpretation of the 1H and 13C NMR data (Table 1), and also, the total correlated spectroscopy (TOCSY) analysis (Figure 2a and Supporting Information Figure S1) revealed eight amino acid residues of four leucines (Leu), two valines (Val), a glycine (Gly), and a proline (Pro), with one structural fragment, C10H10N2O3, left unidentified as its NMR characteristics did not match any of the known amino acids. Then this key fragment was established by detailed examination of the 1D (Table 1) and 2D (Figure 2a) NMR spectra, in which the TOCSY correlation sequence of H-7/H-8/H-9/H-10 and the key heteronuclear multiple bond correlations (HMBCs) of H-7 and H-9/C-5 and H-8 and H-10/C-6, along with the chemical shifts of C-5 to C-10, and the identified amino group suggested the presence of a 2-aminophenol moiety. Also, the TOCSY correlations of –NH–/H-2/H2-3 and the HMBC correlations of –NH–/C-1, and H2-3/C-1 and C-4 revealed highly oxygenated C-1 to C-4 subunits. The linkage between C-4 and C-5 was definitely fixed to form an ester functionality by the chemical shifts of C-4 (δC 169.6) and C-5 (δC 125.8), revealing a new amino acid residue that was named orangeberrine (Orgb). The aforementioned functionalities accounted for 15 out of 16 DBEs and the remaining one, thus, required compound 1 to be a cyclic nonapeptide. Figure 2 | Selected two-dimensional nuclear magnetic resonance (2D NMR) correlations (a) and X-ray structure (b) of compound 1. Download figure Download PowerPoint Comprehensive analysis of its 2D NMR spectra (Figure 2a) revealed an unprecedented cyclo-[Orgb1-Leu2-Leu3-Val4-Val5-Gly6-Leu7-Leu8-Pro9] nonapeptide scaffold for compound 1. The sequence of nine amino acid residues from Orgb1 to Pro9 was first furnished by the key HMBC correlation networks (Orgb1-NH/C-11, Leu2-NH/C-17, Val5-NH/C-33, H2-34 and Gly6-NH/C-35, and H-36, and Leu7-NH/C-41), and the mutual ROESY cross peaks (Orgb1-NH/Leu2-NH, Leu2-NH/H-18, Leu3-NH/H-24, Val4-NH/H-29, Val5-NH/H2-34, Gly6-NH/Leu7-NH, Leu7-NH/Leu8-NH, and Leu8-NH/H-48). The two "loose ends" of Orgb1 and Pro9 residues were then fixed by the rotating frame nuclear Overhauser effect spectroscopy (ROESY) correlation of H-2/H2-51, which also indicated a trans-Pro9 amide bond. This assignment was supported by its ESI-MS/MS fragmentations ( Supporting Information Figure S13). For the determination of the stereo chemistry of compound 1, high quality crystals were obtained in isopropanol at RT after an extensive screening of solvents and conditions, which allowed a successful performance of X-ray crystallography study. Although the Flack parameter [0.26(8)]19 acquired for 1 was unsatisfactory, its Hooft parameter [0.18(9)]20 was acceptable. Thus, the absolute configuration of 1 (2S, 12S, 18S, 24S, 29S, 36S, 42S, 48S) (Figure 2b) was established as depicted by refinement of Hooft parameter, which is a reliable factor in determining the absolute configuration of natural products.21,22 We noted that compound 1 was an unprecedented macrocyclic nonapeptide integrated with a new amino acid, orangeberrine (Orgb). Orberryamide B ( 2) was isolated as pale crystals and possessed a molecular formula of C51H82N10O10 as established by the HRESI-MS ion peak at m/z 1017.6114 [M + Na]+ (calcd 1017.6113) and NMR data (Table 1), which is 16 mass units less than that of 1. Comprehensive analysis of its 1D (Table 1) and 2D (Figure 3a) NMR spectra revealed that