Macrocyclic Nonapeptides Incorporating Uncharacterized Amino Acids with Inhibitory Effects on Th17 Differentiation
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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 Ji State Key Laboratory of Drug Research, Shanghai Institute Materia Medica, Chinese Academy Sciences, 201203 Google Scholar More articles by this author , Fan Kuok Quality Research in Medicines, Macau for Applied Medicine Health, University Science Technology, 999078 Liu *Corresponding author: E-mail Address: [email protected] 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) structurally characterized obtaining solid data numerous analytical measurements. Compound 1 incorporated a new amino acid residue, named orangeberrine (Orgb), compounds 2 3 integrated the tryptophan, an essential rare, nonproteinogenic residues (Kyn Dioia, respectively), compound 4 existed as two major conformational isomers solution at ambient temperature. The biosynthetic pathways proposed are considerable biological significance modification metabolism tryptophan (Trp) and/or Trp containing proteins nature. Besides, suppressed differentiation significantly, effects was achieved through targeting ligand-binding domain (LBD) retinoic acid-related orphan receptor gamma t (RORγt). Download figure PowerPoint Introduction Cyclopeptides that originated natural resources have been attracting enormous attention communities chemists biologists owing complex intriguing chemical structures their broad spectrum activities.1–3 A subset immune CD4 T cells, signature cytokine IL-17 orchestrate pathology many common human autoimmune diseases, such psoriasis, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis.4 Retinoic-acid-related (RORγt) acts critical transcription factor induction manifestation Th17-dependent diseases.5 In presence IL-6, transforming growth factor-β (TGF-β) induces naive CD4+ cells into activating RORγt.6 RORγt is composed modular protein DNA- domains (DBDs LBDs), LBD provides binding sites antagonists, which becoming attractive target drug development treat diseases.7 novel thus, remains high priority. plant, (orangeberry), mainly distributed Southeast Asian countries,8 has used folk medicine Dai minority people China treatment bronchitis, ulcer, traumatic injuries.9 Previous investigations plant afforded several carbazoles, acridone, quinolone alkaloids cytotoxic,10,11 antibacterial,12,13 antimutagenic14 activities. important medicinal indication diverse constituents thus attracted our research interests. As part ongoing projects discovery immunologically active agents plants,15–18 four 4) (Figure 1) titled via semi-preparative performance liquid chromatography (HPLC). Their full determined comprehensive analysis nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), X-ray crystallography, electronic circular dichroism (ECD) data, well Marfey’s method enantiomeric analysis. results showed these shared rare sequence eight residues, each them different ones sequences furnish unique 27-membered nonapeptides. It noteworthy possessed kynurenine (Kyn) dioxindolyalanine (Dioia), respectively, had conformers room temperature (RT). plausible origin proposed; believed be derived enzyme catalytic oxidation cascades. These 1–4 particular interest us due inherent connections serves precursor neurotransmitters (serotonin tryptamine) hormone melatonin, plays vital role regulation sleep cycle. Additionally, we found could exhibit significant inhibition differentiation, mode action accomplished RORγt. Herein, present isolation, structural elucidation, consideration, evaluation 4. Figure | Structures 1–4. Experimental Method General experimental procedures Melting points obtained (uncorrected) SGW X-4 melting point apparatus (Drawell Scientific Instrument Co., Ltd., Shanghai, China). Optical rotations measured Autopol VI polarimeter (Rudolph Analytical, UV spectra acquired Shimadzu UV-2550 spectrophotometer (Shanghai, ECD collected JASCO J-810 spectrometer (JASCO, Infrared (IR) Thermo IS5 infrared KBr disks (Thermo Fisher Scientific, one-dimensional (1D) two-dimensional (2D) NMR recorded DMSO-d6 solvents Brucker AVANCE III 400, 500 (Beijing, China), Ascend 600 instruments (Bruker, Liquid chromatography–mass (LC-MS) tandem MS (MS/MS) Agilent G6520 Q-TOF ESI-MS HRESI-MS Waters Micromass Q-TQF Ultima Global Analytical high-performance (HPLC) performed using 1100 series semipreparative HPLC conducted 1525 system China) 2489 detector Fortis H2o column (250 × 10 mm, S-5 μm). Silica gel (300–400 mesh, Qingdao Maring Chemical Co. Ltd.), MCI (CHP20P, 75–150 μm, Mitsubishi Industries Sephadex LH-20 (40–70 Amersham BioSciences, Beijing (CC). All CC grade (Shanghai Reagents (J&K Beijing, Plant material roots G. Mengla County Yunnan Province, China, November 2015 identified Professor You-Kai Xu (Xishuangbanna Tropical Botanical Garden, Sciences). voucher specimen (accession no. Orberry-2015-YN-1Y) deposited Sciences. Extraction isolation Dried powder (5.0 kg) extracted three times 95% EtOH RT give crude extract (400 g), dissolved water then partitioned EtOAc. EtOAc-soluble fraction (∼ 100 g) subjected ion exchange (MeOH/H2O, 1∶1 9∶1) produce fractions F1–F3. Fraction F2 (36 fractionated over silica eluted gradient mixtures petroleum ether/EtOAc (from 20∶1 1∶2) seven subfractions F2a–F2g. Then F2g separated (EtOH), six F2g1–F2g6. Further purification F2g2 reverse phase (RP)-HPLC (mobile phase: 58% acetonitrile water; flow rate: mL/min; detection wavelength: 210 nm), yielding following specified retention times: mg, tR = 15.5 min), (8.0 18.0 (1.5 20 (50.0 16.5 min). Other characterization outcomes follows: Orberryamide (1) Colorless crystal; mp 198–200 °C; [α]18D –47.9 (c 0.12, MeOH); (MeOH) λmax (log ɛ) 245 (4.02), 286 (3.76) nm; CD (Δɛ) 200 (–32.89) IR (KBr) νmax 3318, 3069, 2961, 2932, 2873, 1652, 1527, 1454, 1387, 1370, 1262, 1160, 1107, 1029 cm–1; 1H 13C see Table 1; (+)-ESI-MS m/z 1033.6 [M + Na]+; (+)-HRESI-MS 1033.6061 Na]+ (calcd C51H82N10O11Na, 1033.6062). Spectroscopic Data Compounds Position 1a 2a δH (J Hz) δC Orgb1 NH 7.54, d (5.7) Kyn1 7.48, (6.2) 170.2 4.74, m 48.7 4.83, 49.0 2.91, (2H) 37.1 3.43, 39.2b 169.6 3.13, dd (15.8, 8.6) 5 125.8 199.9 6 147.8 116.3 7 6.84, (8.0, 1.2) 115.3 151.4 8 6.92, td 1.3) 124.6 6.75, (8.5, 117.1 9 6.65, 118.3 7.25, ddd 6.9, 1.5) 134.6 7.80, 121.7 6.53, (8.3, 114.5 NH2 9.80, br s 7.62, 131.0 9.45, 7.29, Leu2 8.07, (6.4) 8.32, (6.0) 11 172.1 172.4 12 4.05, 53.3 3.96, 13 1.42, 40.4 1.38, 40.2 14 1.56, 24.3 1.54, 24.4 15 0.85, (6.4)b 21.2 0.86, (6.7) 20.8 16 0.81, 22.7 0.78, (6.7)b 22.8 Leu3 8.37, sb 8.52, 17 172.0b 172.7 18 3.70, 55.0 3.63, 56.0 19 1.83, 38.6 1.89, 38.7 1.58, 1.59, 1.53, 24.5b 1.50, 24.5 21 0.87, 22.6 (6.5) 22.5 22 0.85 21.3 21.4 Val4 8.45, 23 172.8 172.5 24 3.57, 60.9 3.54, 25 1.94, 29.1 1.92, 29.0b 26 0.95, (6.6) 18.9 18.8 27 0.92, 19.7 19.5 Val5 7.37, 7.32, 28 29 4.26, 57.5 4.27, 57.3 30 1.86, 29.0 1.85, 31 0.90, 19.2 19.3 32 0.79, 18.1 Gly6 7.58, 7.56, 33 168.7 168.5 34 3.98, 42.8 42.7 3.42, 3.38, (16.5, 6.0) Leu7 7.14, (9.5) 7.07, (9.3) 35 172.3 172.0 36 4.21, 50.0 4.20, (9.3, 4.7) 49.7 37 1.63, 39.7 1.57, 39.4 1.36, 1.02, 38 1.33, 24.0 1.28, 39 0.60, 20.6 0.48, 20.7 40 0.58, 0.38, 22.1 Leu8 7.73, (5.6) 7.92, 41 172.2 171.7 42 3.95, 53.9 54.0 43 1.71, 1.72, 1.60, 1.62, 44 1.67, 1.77, 45 0.89, 22.4 0.97, 46 0.88, 21.7 21.8 Pro9 47 173.9 174.5 48 4.02, 61.8 3.99, 62.2 49 2.28, 29.2 2.29, 1.81, 50 1.97, 24.6 1.98, 24.7 51 3.79, dt (9.4, 6.7) 47.0 (9.1, 46.9 3.44, 3.40, aRecorded MHz (1H) 125 (13C) DMSO-d6. bOverlapping signals. B (2) Pale yellow 259–261 [α]19D –16.4 0.07, 227 (4.36), 260 (3.78) 198 (–53.12) 3453, 3333, 3073, 2959, 2931, 1659, 1631, 1518, 1469, 1451, 1390, 1369, 1316, 1267, 1212, 1163 1017.6 1017.6114 C51H82N10O10Na, 1017.6113). C (3) White, amorphous powder; –78.1 0.16, 250 (3.70) (–26.74), 212 (+14.70), 240 (–15.14), 265 (+1.06) 3310, 2926, 2854, 1715, 1650, 1523, 1472, 1372, 1335, 1260, 1205, 1184, 1099, 1027 2; 1045.6 1045.6067 C52H82N10O11Na, 1045.6062). 3a Dioia1 7.18, 171.3 5.27, 46.4 4.07, 58.6 2.39, 38.3 1.91, 28.9 73.4 17.9 4a 141.0 7.44, 179.0 169.4 NH-6 10.23, 6.88, (7.4) 110.5 3.48, 7a 132.4 7.42, 7.00, 122.3 172.6 7.24, 129.3 4.17, 51.1 7.30, 123.6 1.43, 7.98, 1.46, 23.8 171.4b 0.53, (5.9) 20.9b 53.2 0.45, 22.3 24.4b 0.82, 21.1 4.09, 23.1 1.88, 38.4 7.93, 171.7b 1.68, 24.8 3.92, 53.0 0.92 22.9 0.84, 1.64, 173.5 (6.3)b 23.0 61.6 (6.3) 2.23, 29.5 7.38, 1.84, 171.6b 2.00, 3.84, 59.3 3.80, 47.4 3.69, 18.5 19.6 D (4) –92.5 0.08, 220 (4.62), 283 (4.58) (–38.08) 3313, 3057, 2936, 2875, 1529, 1470, 1448, 1340, 1282, 1252, 1166, 1097, 3; 1013.6 1013.6166 C52H82N10O9Na, 1013.6164). 4aa 4ba Trp1 7.36, 171.5 4.97, q (8.0) 4.45, 53.5b 3.06, 26.5 3.11, 27.4 2.93, (15.0, 8.0) 109.6 108.5 127.7 126.9 7.11, (2.2) 123.1 7.15, 124.2 10.81, 11.00, 7.31, (7.8)b 111.2 111.4 135.9 136.1 7.04, (7.8) 120.7 7.08, 121.3 6.95, 118.0 118.4 117.2 118.2 171.8 171.9 4.48, ovb 50.2 50.3 1.48, 41.4 41.5 1.51, 21.3b 21.8c 23.2d 8.08, 8.27, 171.0 170.9 53.4 3.71, 54.9 40.2b 1.24, 23.9 0.74, 23.0b,d 21.3b,e 8.36, 7.86, 3.64, 60.4 4.00, 58.7 1.95, 29.3 2.16, 29.8 18.3 19.0 7.40, 7.70, (8.7) 57.4 4.19, 58.9 2.01, 2.06, 30.6 19.4 7.64, (5.4) 8.31, 168.6 169.1 3.88, 43.3 3.45, 5.4) 3.68, 7.57, 7.83, 173.0 50.4 51.9 39.3 1.39, 24.2 0.65, (6.1) 0.83, 21.2b,e 0.76, 8.26, 7.78, 170.8 4.03, 1.65, ov (2H)b 38.8 1.70, 23.1d 21.2b 21.6c 174.6 4.11, 3.37, 59.9 2.24, 28.8 1.61, 28.5 1.79, 1.14, 45.8 3.29, (9.8, 6.8) 3.09, 400 DMSO-d6, coupling constants not averaged 4b: trans- cis-oriented amide bond Pro9, respectively). c,d,eNMR values same letters can interchanged. Each peptide samples (1 mg) treated mL M HCl 80 °C sealed vial h dried under vacuum. residue NaHCO3 (40 μL) reagent, L-FDAA (1% acetone, h. After reaction neutralized μL), mixture diluted (200 filtered 4.5 μm filter, kept Authentic acids also derivatized way. derivatives both hydrolysates authentic analyzed LC-MS HPLC-DAD-ESIMS (0.5 mL/min, min linear elution 30% 50% MeCN/H2O 0.1% formic acid) equipped X-Bridge C18 (5 4.6 °C). absolute configurations chiral cyclic peptides comparing those samples. X-Ray crystallographic crystals recrystallization isopropanol. analyses carried out Bruker D8 Venture diffractometer Ga Kα radiation (λ 1.34139 Å) APEX-II CCD Cu 1.54178 2. acquisition parameters provided Supporting Information Tables S3 S4, (Deposition CCDC 1935025) 1935024) Cambridge Crystallographic Center. Copies available free charge at: www.ccdc.cam.ac.uk/conts/retrieving.html. Bioassays Antimouse CD3ɛ, CD28, anti-IL-4, recombinant mouse IL-6 IL-23, TGF-β1, PE antimouse IL-17A, PerCP/Cy5.5 CD4, monensin solution, brefeldin red blood cell lysis buffer Biolegend CytofixTM Fixation stain BD Bioscience kit purchased Miltenyi Biotec RPMI-1640, DMEM, trypsin, fetal bovine serum (FBS) GIBCO Animals C57BL/6 mice (6–8 weeks) Hong Kong. specific pathogen-free conditions animal care facility Technology. Animal experiments accordance Committee Guidelines Recombinant preparation gene (Genbank accession number NP_005051, aa 259-518) His tag terminal constructed cloned pET21b. plasmids transformed Escherichia coli strain BL21 (DE3) overexpression. grown Luria-Bertani broth μg/mL ampicillin expression induced 0.2 mM isopropyl β-d-thiogalactopyranoside (IPTG) shaking overnight °C. E. harvested centrifugation 4000 rpm resuspended precooled nickel–nitrilotriacetic (Ni-NTA) [50 Tris-HCl 8.5, 300 NaCl, imidazole, 5% Glycerol, β-mercaptoethanol (β-ME), PMSF]. Resuspended passed cryogenic overpressure breaker 1200 psi. Both supernatant precipitate 14,000 applied Ni-NTA affinity (Qiagen, previously equilibrated A. (50 β-ME, PMSF). RORγt-containing pooled, dialyzed dithiothreitol (DTT). purified further anion-exchange QHP (GE Healthcare, NaCl gradient. soluble stored –80 until use. subsets Naive sorted bead (Miltenyi Biotec) spleen according manual, 105 cells/well) incubated plate-bound CD3ɛ. To determine cultured days anti-CD28, ng/mL IL-23 or without indicated compounds. restimulated PMA, ionomycin, Brefeldin after days. For Th1 Th2 ionomycin followed incubation μM Monensin additional before intracellular staining. analyze subpopulations, stained fluorophore-conjugated monoclonal antibodies. anti-CD4 indicate surface marker lymphocytes. permeabilization cell, anti-IL-17A detect cytokine. level IL-17A FACSAriaTM (BD Bioscience) FlowJo software https://www.flowjo.com/). Binding assay During Biolayer interferometry experiment, steady-state detecting probe (0.1% DMSO). probe-protein based fitting results. kinetics Octet RED96 instrument (Pall ForteBio, Briefly, biotinylated EZ-Link™ NHS-LC-LC-Biotin (ThermoFisher, manufacturer’s instructions. immobilize onto biosensors, streptavidin biosensors ForteBio) final concentration ng/µL PBS overnight. µM digoxin prepared serially 1∶2 protein. custom ForteBio Acquisition 9.0 Analysis Results Discussion molecular formula C51H82N10O11, incorporates double-bond equivalents (DBEs), peak 1033.6062) (Table 1). NMR, HSQC, TOCSY typical signals amido protons (–NH–, ranging 7.14 8.37), group (–NH2, 9.45 9.80), aromatic (δH 7.80), doublet methyls. 1), distortionless enhancement polarization transfer (DEPT), heteronuclear single quantum coherence spectroscopy (HSQC) revealed carbonyls (ranging 173.9), disubstituted phenyl group, methyls, methylenes, methines. interpretation also, total correlated (TOCSY) S1) leucines (Leu), valines (Val), glycine (Gly), proline (Pro), one fragment, C10H10N2O3, left unidentified its characteristics did match any known acids. key fragment established detailed examination 1D 2D 2a) spectra, correlation H-7/H-8/H-9/H-10 correlations (HMBCs) H-7 H-9/C-5 H-8 H-10/C-6, along shifts C-5 C-10, suggested 2-aminophenol moiety. Also, –NH–/H-2/H2-3 HMBC –NH–/C-1, H2-3/C-1 C-4 highly oxygenated C-1 subunits. linkage between definitely fixed form ester functionality (δC 169.6) 125.8), revealing (Orgb). aforementioned functionalities accounted DBEs remaining one, required nonapeptide. Selected (2D NMR) (a) structure (b) 1. Comprehensive cyclo-[Orgb1-Leu2-Leu3-Val4-Val5-Gly6-Leu7-Leu8-Pro9] nonapeptide scaffold nine first furnished networks (Orgb1-NH/C-11, Leu2-NH/C-17, Val5-NH/C-33, H2-34 Gly6-NH/C-35, H-36, Leu7-NH/C-41), 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, Leu8-NH/H-48). “loose ends” rotating frame Overhauser effect (ROESY) H-2/H2-51, trans-Pro9 bond. This assignment supported ESI-MS/MS fragmentations S13). determination stereo chemistry 1, quality isopropanol extensive screening conditions, allowed successful crystallography study. Although Flack parameter [0.26(8)]19 unsatisfactory, Hooft [0.18(9)]20 acceptable. Thus, configuration (2S, 12S, 18S, 24S, 29S, 36S, 42S, 48S) 2b) depicted refinement parameter, reliable determining products.21,22 We noted acid, 2) pale
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ژورنال
عنوان ژورنال: CCS Chemistry
سال: 2021
ISSN: ['2096-5745']
DOI: https://doi.org/10.31635/ccschem.020.202000265