Supplementary Information Cycloexpansamines A and B: Spiroindolinone alkaloids from a marine isolate of Penicillium sp. (SF-5292) Running Head: Spiroindolinone alkaloids from a marine isolate of Penicillium sp. Chiwook Lee,1,2 Jae Hak Sohn,2 Jae-Hyuk Jang,3 Jong Seog Ahn,3 Hyuncheol Oh,1 Jonas 4 Baltrusaitis , In Hyun Hwang5, and James B. Gloer5 1 College of Pharmacy, Wonkwang University, Iksan, Korea; 2College of Medical and Life Sciences, Silla University, Busan, Korea; 3Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongwon, Korea; 4PhotoCatalytic Synthesis Group, University of Twente, Enschede, The Netherlands, and 5Department of Chemistry, University of Iowa, Iowa City, IA, USA Correspondence. Professor Hyuncheol Oh, College of Pharmacy, Wonkwang University, Iksan 570-749, Korea. E-mail: hoh@wonkwang.ac.kr List of Supplementary Information Experimental Section Physical Properties of Compounds 1 and 2 Figure S1. Experimental ECD spectrum (top) and TDDFT-calculated ECD spectrum (bottom) with energy-minimized model of 1. Figure S2. 1H NMR spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5) Figure S3. 13 C NMR spectrum of cycloexpansamine A (1, 100 MHz, pyridine-d5) Figure S4. HMQC spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5) Figure S5. HMBC spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5) Figure S6. COSY spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5) Figure S7. NOESY spectrum of cycloexpansamine A (1, 600 MHz, pyridine-d5) Figure S8. 1H NMR spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5) Figure S9. 13 C NMR spectrum of cycloexpansamine B (2, 100 MHz, pyridine-d5) Figure S10. HSQC spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5) Figure S11. HMBC spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5) Figure S12. COSY spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5) Figure S13. NOESY spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5) Figure S14. Expanded NOESY spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5) Figure S15. IR spectrum of cycloexpansamine A (1) Figure S16. IR spectrum of cycloexpansamine B (2) 2 Experimental Section General experimental procedures. Optical rotations were recorded on a Perkin Elmer 341 digital polarimeter. UV data for compound 1 were obtained in MeOH using a Varian Cary III UV/vis spectrophotometer and ECD data were recorded in MeOH with an Olis Cary-17 spectrophotometer (1-cm cell). UV data for compound 2 were obtained in MeOH using a Beckman Coulter UV/vis spectrophotometer. IR spectra were obtained using a Varian 640-IR spectrometer. NMR spectra (1D and 2D) were recorded in pyridine-d5 or CDCl3 using a JEOL JNM ECP-400 spectrometer (400 MHz for 1H and 100 MHz for spectrometer (600 MHz for 1H and 150 MHz for relative to tetramethylsilane (H/C = 0). All 13 13 C) or JEOL JNM ECA-600 C), and chemical shifts were referenced 13 C NMR multiplicities were determined by analysis of DEPT and/or HMQC data, and are consistent with the position assignments. HMQC/HSQC and HMBC experiments were optimized for 1JCH = 140 Hz and nJCH = 8 Hz, respectively. ESIMS data were obtained using a Q-TOF micro LC-MS/MS instrument (Waters, USA) at Korea University, Seoul, Korea. Solvents for extractions and flash column chromatography were reagent grade and used without further purification. Solvents used for HPLC were analytical grade. Flash column chromatography was carried out using YMC octadecyl-functionalized silica gel (C18). HPLC separations were performed on a Shiseido Capcell Pak C18 column (10 250 mm; 5 m particle size; 2 ml/min) or Agilent prep-C18 column (21.2 150 mm; 5 m particle size; 5 ml/min). Compounds were detected by UV absorption at 210 nm. Energy minimization and ECD calculations. Initial structures were obtained from a Spartan’10 conformation search using MMFF,1 then further fully optimized using the 6-31G(d) basis set2,3 combined with the M06-2X functional4 followed by frequency calculations using the 3 same level of theory. Absence of the imaginary frequencies confirmed that all conformers were minima on the potential energy surface. No symmetry constraints were used during the optimization. Solvent methanol and the PCM solvation model5 were used in Gaussian 09 Rev. B0.1.6 Time-dependent density functional (TDDFT) calculations were performed to calculate ECD spectra at the optimized geometry using the same basis set and functional. A total of 20 excited states were calculated, and only singlet excited states were considered. For TDDFT calculations, B3LYP/aug-cc-pVDZ combined with COSMO solvation were used as implemented in NWChem 6.3.7 This combination of density functional and basis set has been shown to provide good ECD simulations.8 To visualize the ECD spectra, SpecDis version 1.62 software by Dr. Torsten Bruhn was used.9 1 Shao, Y. et al. Advances in methods and algorithms in a modern quantum chemistry program package. Phys. Chem. Chem. Phys. 8, 3172-3191 (2006). 2 Ditchfield, R., Hehre, W. J. & Pople, J. A. Self‐Consistent Molecular‐Orbital Methods. IX. An Extended Gaussian‐Type Basis for Molecular‐Orbital Studies of Organic Molecules. J. Chem. Phys. 54, 724-728 (1971). 3 Hehre, W. J., Ditchfield, R. & Pople, J. A. Self-Consistent Molecular Orbital Methods. XII. Further Extensions of Gaussian—Type Basis Sets for Use in Molecular Orbital Studies of Organic Molecules J. Chem. Phys. 56, 2257-2261 (1972). 4 Zhao, Y. & Truhlar, D. The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals Theor. Chem. Acc. 120, 215-241 (2008). 5 Mennucci, B.; Tomasi, J. Continuum solvation models: A new approach to the problem of solute’s charge distribution and cavity boundaries. J. Chem. Phys. 1997, 106, 5151-5158. 4 6 Frisch, M. J. et al. Gaussian 09, revision B.01. Gaussian, Inc., Wallingdorf, CT, 2009. 7 Valiev, M. et al. NWChem: A comprehensive and scalable open-source solution for large scale molecular simulations. Comput. Phys. Commun. 181, 1477-1489 (2010). 8 Autschbach, J. Time-dependent density functional theory for calculating origin- independent rotation and rotatory strength tensors. ChemPhysChem 12, 32249 optical 3235 (2011). Bruhn, T.; A.Schaumloffel; Y.Hemberger; Bringmann, G. SpecDis, Version 1.62, University of Wuerzburg, Germany, 2013. PTP1B assay procedures. PTP1B (human, recombinant) was purchased from BIOMOL Research Laboratories, Inc. The enzyme activity was measured in a reaction mixture containing 2 mM p-nitrophenyl phosphate (pNPP) in 50 mM citrate, pH 6.0, 0.1 M NaCl, 1 mM EDTA and 1mM dithiothreitol (DTT). The reaction mixture was placed in a 30 oC incubator for 30 min, and the reaction was terminated by the addition of 10 N NaOH. The amount of produced pnitrophenol was estimated by measuring the increase in absorbance at 405 nm. The nonenzymatic hydrolysis of 2 mM pNPP was corrected by measuring the increase in absorbance at 405 nm obtained in the absence of PTP1B enzyme. A known PTP1B inhibitor, ursolic acid (IC50 = 3.08 M) was used as a positive control in the assay.10,11 10 Na, M. et al. Inhibition of protein tyrosine phosphatase 1B by ursane-type triterpenes isolated from Symplocos paniculata. Planta Med. 72, 261-263 (2006). 11 Zhang, W. et al. Ursolic acid and its derivative inhibit protein tyrosine phosphatase 1B, enhancing insulin receptor phosphorylation and stimulating glucose uptake. Biochem 1505-1512 (2006). 5 Biophy Acta 1760, Physical Properties of Compounds 1 and 2 Cycloexpansamine A (1): yellow solid; []25D + 33 (c 1.39, MeOH); UV (MeOH) λmax (log ε) 263 (3.78), 348 (3.26), and 365 (3.37) nm; CD (MeOH) λmax (Δε) 227 (+23), 242 (−11), 267 (+21), 305 (−0.16), 322 (+0.74), and 360 (−2.8) nm; IR (CHCl3) max 3234, 2954, 2916, 1716, 1660, 1650, 1608, 1459, 1372 cm-1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 450.2380 [M + H]+ (calcd for C26H32N3O4, 450.2393). Cycloexpansamine B (2): yellow solid; []25D + 20 (c 0.15, MeOH); UV (MeOH) max (log ) 263 (4.12), 362 (3.83); IR (CHCl3) max 3284, 2955, 2921, 1715, 1650, 1603, 1546, 1459, 1367, 755 cm-1; 1H and 13 C NMR data, see Table 2; HRESIMS m/z 454.2349 [M + H]+ (calcd for C25H32N3O5, 454.2342). 6 30 20 10 0 190 240 290 340 390 440 190 240 290 340 390 440 -10 -20 20 10 0 -10 -20 Figure S1. Experimental ECD spectrum (top) and TDDFT-calculated ECD spectrum (bottom) with energy-minimized model of 1. 7 Figure S2. 1H NMR spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5) Figure S3. 13 C NMR spectrum of cycloexpansamine A (1, 100 MHz, pyridine-d5) 9 JF34(3)-4-3-PYR-C-4.jdf JF34(3)-4-3-HMQC-4.jdf Y : parts per Million : 13C 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 (Millions) 1.0 3.0 5.0 JF34(3)-4-3-PYR-H-8.jdf 9.0 8.0 X : parts per Million : 1H 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 1.0 (Billions) Figure S4. HMQC spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5) 10 2.0 3.0 JF34(3)-4-3-PYR-C-4.jdf Y : parts per Million : 13C 210.0200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 (Millions) 0 JF34(3)-4-3-PYR-H-8.jdf JF34(3)-4-3-PYR-BC-3.jdf 9.0 8.0 X : parts per Million : 1H 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 (Billions) Figure S5. HMBC spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5) 11 1.0 (Millions) 0 3.0 6.0 JF34(3)-4-3-PYR-H-8.jdf JF34(3)-4-3-PYR-H-8.jdf Y : parts per Million : 1H 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 JF34(3)-4-3-COSY-PYR-3.jdf 9.0 8.0 X : parts per Million : 1H 7.0 6.0 5.0 4.0 3.0 2.0 1.0 Figure S6. COSY spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5) 12 0 2.0 4.0 6.0 8.0 (Millions) (Millions) 0 3.0 6.0 JF34(3)-4-3-PYR-H-8.jdf JF34(3)-4-3-PYR-H-8.jdf Y : parts per Million : 1H 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 JF34(3)-4-3-NOESY-3.jdf 9.0 8.0 X : parts per Million : 1H 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 0 3.0 6.0 (Millions) Figure S7. NOESY spectrum of cycloexpansamine A (1, 600 MHz, pyridine-d5) 13 9.0 Figure S8. 1H NMR spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5) 14 Figure S9. 13 C NMR spectrum of cycloexpansamine B (2, 100 MHz, pyridine-d5) 15 JF34(3)-4-5-2-C-7.jdf JF34(3)-4-5-2-HSQC-4.jdf 0.6 Y : parts per Million : 13C 170.0160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 abundance 20.0 0 0.2 0.4 JF34(3)-4-5-2-H-4.jdf 8.0 X : parts per Million : 1H 7.0 6.0 5.0 4.0 3.0 2.0 1.0 -10.0 10.0 (thousandths) Figure S10. HSQC spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5) 16 30.0 JF34(3)-4-5-2-C-6.jdf JF34(3)-4-5-2-HMBC-4.jdf Y : parts per Million : 13C 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 abundance 20.0 0 0.2 0.4 0.6 0.8 JF34(3)-4-5-2-H-4.jdf 7.0 X : parts per Million : 1H 6.0 5.0 4.0 3.0 2.0 1.0 -10.0 0 10.0 20.0 30.040.0 (thousandths) Figure S11. HMBC spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5) 17 1.0 abundance 0 JF34(3)-4-5-2-H-4.jdf JF34(3)-4-5-2-H-4.jdf Y : parts per Million : 1H 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 JF34(3)-4-5-2-COSY-4.jdf 9.0 8.0 X : parts per Million : 1H 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 0.2 0.4 abundance Figure S12. COSY spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5) 18 0.6 0.8 1.0 abundance 0 JF34(3)-4-5-2-H-4.jdf JF34(3)-4-5-2-H-4.jdf Y : parts per Million : 1H 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 JF34(3)-4-5-2-NOESY-4.jdf 8.0 X : parts per Million : 1H 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 abundance Figure S13. NOESY spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5) 19 1.0 Figure S14. Expanded NOESY spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5) 20 Figure S15. IR spectrum of cycloexpansamine A (1) 21 Figure S16. IR spectrum of cycloexpansamine B (2) 22