Supporting Information

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1 Supporting Information Three Minor Diterpenoids with Three Carbon Skeletons from Euphorbia peplus Luo-Sheng Wan,, Yin Nian,, Chen-Jun Ye,, Li-Dong Shao, Xing-Rong Peng, Chang-An Geng, Zhi-Li Zuo, Xiao-Nian Li, Jian Yang,, Ming Zhou,*,, and Ming-Hua Qiu.*, State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, 32 Lanhei Road, Kunming 52, P. R. China. Key Laboratory of Animal Models and Human Diease Mechanisms, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 5223, P. R. China. Department of Biological Sciences, Columbia University, New York, NY27, United States. Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 773, United States. S

2 Table of Contents Figure S. Key 2D NMR correlations for pepluacetal ().... S4 Figure S2. Key 2D NMR correlations for pepluanol A (2)... S5 Figure S3. Key 2D NMR correlations for pepluanol B (3)... S Figure S4. Normalized current-voltage (I-II) curves of Kv.3... S7 Experimental section... S8 General experimental procedures... S8 Plant material... S8 Extraction and isolation... S8 Physical constants and spectral data of 3... S9 Bioassays... S9. Oocyte preparation and expression... S9 2. Electrophysiology... S9 3. Data analysis and statistics... S9 Computational methods for ECD a of pepluanol A (2)... S X-ray crystallographic data for Pepluacetal ()... S2 X-ray crystallographic data for Pepluanol A (2)... S3 X-ray crystallographic data for Pepluanol B (3)... S4 X-ray crystallographic data for Pepluacetal () and Pepluanol A B (2 3):... S5 H NMR spectrum of Pepluacetal () in CDCl 3... S 3 C NMR and DEPT spectra of Pepluacetal () in CDCl 3... S7 HSQC spectrum of Pepluacetal () in CDCl 3... S8 HMBC spectrum of Pepluacetal () in CDCl 3... S9 H- H COSY spectrum of Pepluacetal () in CDCl 3... S2 ROESY spectrum of Pepluacetal () in CDCl 3... S2 HR-ESI (+) MS spectrum of Pepluacetal ()... S22 CD spectrum of Pepluacetal () in MeOH... S23 H NMR spectrum of Pepluanol A (2) in CDCl 3... S24 3 C NMR and DEPT spectra of Pepluanol A (2) in CDCl 3... S25 HSQC spectrum of Pepluanol A (2) in CDCl 3... S2 HMBC spectrum of Pepluanol A (2) in CDCl 3... S27 H- H COSY spectrum of Pepluanol A (2) in CDCl 3... S28 ROESY spectrum of Pepluanol A (2) in CDCl 3... S29 HR-ESI (+) MS spectrum of Pepluanol A (2)... S3 CD spectrum of Pepluanol A (2) in MeOH... S3 H NMR spectrum of Pepluanol B (3) in CDCl 3... S32

3 3 C NMR and DEPT spectra of Pepluanol B (3) in CDCl 3... S33 HSQC spectrum of Pepluanol B (3) in CDCl 3... S34 HMBC spectrum of Pepluanol B (3) in CDCl 3... S35 H- H COSY spectrum of Pepluanol B (3) in CDCl 3... S3 ROESY spectrum of Pepluanol B (3) in CDCl 3... S37 HR-EI (+) MS spectrum of Pepluanol B (3)... S38 CD spectrum of Pepluanol B (3) in MeOH... S39

4 Figure S. Key 2D NMR correlations for pepluacetal (). S4

5 Figure S2. Key 2D NMR correlations for pepluanol A (2). S5

6 Figure S3. Key 2D NMR correlations for pepluanol B (3). S

7 Figure S4. Normalized current-voltage (I-II) curves of Kv.3 current in the absence or presence of 3 M, and 2. Currents were evoked from a HP of -8 mv by 2-ms depolarizations ranging from 4 mv to + mv in -mv increments at a 5-s interval. Data points reflect mean ± SEM of three to five determinations. The inhibition ratio for, and 2 are 24.9 ±8.%, and 4. ± 9.%, respectively. S7

8 Experimental section General experimental procedures UV spectra was obtained using Shimadzu UV24PC spectrophotometry. Optical rotations were recorded with a Jasco P-2 polarimeter. H and 3 C NMR spectra were measured on Bruker AV-4 and DRX-5 instruments (Bruker, Zurich, Switzerland) using TMS as internal standard for chemical shifts. Chemical shifts ( ) were expressed in ppm with reference to solvent peak ( H 7.2 and C 77.). ESIMS and HRTOF-ESIMS data were recorded on API QSTAR Pulsar spectrometer. EIMS and HRTOF-EIMS data were taken on an Waters Auto Spec Premierp 77 spectrometer (America, Waters). Infrared spectra were recorded on a Bruker Tensor-27 instrument by using KBr pellets. An agilent series instrument equipped with Agilent ZORBAX SB-C8 column (5 μm, 4. mm 25 mm) was used for high-performance liquid chromatography (HPLC) analysis. Preparative TLC and TLC was performed on precoated TLC plates (2-25 μm thickness, F254 Si gel, Qingdao Marine Chemical, Inc.) with compounds visualized by spraying the dried plates with 5% aqueous H 2 SO 4 followed by heating until dryness. Silica gel (2-3) mesh, Qingdao Marine Chemical, Inc.), Lichroprep RP-8 (4-3 μm, Fuji) and Sephadex LH-2 (2-5 μm, Pharmacia) were used for column chromatography. Methanol, chloroform, ethyl acetate, acetone, petroleum ether and 2-propanol were purchased from Tianjing Chemical Reagents Co. (Tianjing, China). All other materials were of the highest grade available. Plant material The whole plants of Euphorbia peplus were collected from September of 23 on Kunming, P. R. China, and identified by Prof. W-D Liu, Kunming Institute of Botany (KIB), Chinese Academy of Sciences. A voucher specimen has been deposited in the herbarium of KIB (number kep-9-3). Extraction and isolation The air-dried powder of the plant (25 kg) was extracted twice with acetone ( L), heat and reflux for 4 hours, to give kg of crude extract which was suspended in water (2L) and partitioned with petroleum ether (PE) (5 2L). The PE-soluble part (3 kg) was fractioned by a macroporous resin (AB-8) column eluted with MeOH/H 2 O (5% %) to give four fractions F F4. Fraction F2 (43 g) was extensively subjected to silica gel CC, eluted with PE/EtOAc (: :), to give 8 subfactions subf subf8. subf (4. g) were further subjected to RP-8 CC and eluted with MeOH-H 2 O (2:8 to :) to obtain 3 fractions (subf- subf-3). SubF-2 (3 mg) was further purified by semi-preparative HPLC (Acetonile:Water = 75:25, 3 ml/min, wavelength at 2 nm) to yield 2 (5.2 mg, t R =.2 min). SubF-32 (5 mg) was further purified by semi-preparative HPLC (Acetonile:Water = 78:22, 3 ml/min, wavelength at 2 nm) to yield (2.3 mg, t R = 9.7 min). subf3 (23 g) were separated by RP-8 CC and eluted with MeOH-H 2 O (2:8 to :) to afford 44 factions (subf3- subf3-44). subf3-27 (2 mg) was chromatographed on LH2 (% MeOH) to five fractions (subf3-27a 27e). subf3-27a ( mg) was purified by preparative TLC (PE:i-Propanol, 3:) to S8

9 yield 3 (.5 mg, R f =.4). Physical constants and spectral data of 3 Pepluacetal (): Colorless prismatic crystals; [α] 25 D (.2, MeOH); UV (MeOH) λ max (log ε) 23 (3.7), 237 (3.8) nm; CD (MeOH): λ( ε) = 23 ( 4.7), 247 (4.5), 287 ( 3.), 32 (3.9); IR (KBr) υ max 293, 87, 2, 4, 379, 9, 953 cm - ; H and 3 C NMR data (see Table ); (+)HRESIMS m/z [M + Na] (calcd for C 22 H 32 O 4 Na, ). Pepluanol A (2):. Colorless prismatic crystals; [α] 25 D +8.3 (.27, MeOH); UV (MeOH) λ max (log ε) 2 (3.8), 233 (3.9) nm; CD (MeOH): λ( ε) = 2 ( 4.4), 24 (4.9), 32 ( 4.3); IR (KBr) υ max 3453, 2932, 77, 24, 45, 437, 37, 34, 24, 28, 947 cm - ; H and 3 C NMR data (see Table ); (+)HRESIMS m/z [M + Na] (calcd for C 2 H 2 O 3 Na, ) Pepluanol B (3): Colorless needle crystals; [α] 25 D (., MeOH); UV (MeOH) λ max (log ε) 2 (3.9), 242 (3.9), 33 (3.) nm; CD (MeOH): λ( ε) = 95 ( 4.8), 28 (4.5), 25 ( 4.2), 35 (4.3), 342 ( 3.8); IR (KBr) υ max 3444, 29, 77, 29, 45, 375, 32, 3, 72, 7 cm - ; H and 3 C NMR data (see Table ); (+)HREIMS m/z [M] (calcd for C 2 H 2 O 3, ). Bioassays. Oocyte preparation and expression Oocytes were obtained from adult Xenopus laevis by a surgical procedure approved by Kunming Institute of Zoology. Preparation of mrna and its expression in oocytes were performed following ref (PMC282575). 2. Electrophysiology All experiments were performed at 2-22 o C. Whole-oocyte recordings were conducted by two-electrode voltage clamp. Electrodes were filled with KCl (3mM) and had resistances of.3- MΩ. The bath solution used to record currents contained NaCl (9 mm), KCl (2. mm), MgCl 2 (mm),hepes (5 mm),cacl 2 (.8 mm). PH was adjusted to 7.4 with NaOH and the solution was filtered. Kv.3 currents were evoked from a holding potential of -8 mv by 2-ms depolarizations ranging from -4 mv to + mv in -mv increments at a 5-s interval. All currents were sampled at -2 khz and filtered at 5- khz. 3. Data analysis and statistics Data acquisition and analysis were carried out using pclamp (Molecular Devices Corporation, California, United States of America). Data fitting and statistical analyses were performed using PRISM 5. (GraphPadSoftware Inc., San Diego, CA). IC 5 value and Hill coefficient were determined by fitting the data points to a Hill equation with the form of Y = I Min + (I Max - I Min )/[+(LogIC 5 -C) Hillslope]. Where IC 5 is the concentration at which half-maximal currents were inhibited, C is the concentration of compounds, I Min is the minimum inhibition ratio, I Max is the maximum inhibition ratio, and Hillslope is the Hill coefficient. All the data were presented as mean ± S9

10 SEM. Computational methods for ECD of pepluanol A (2) The 3D structure of 2 were obtained from X-ray data, and the ECD calculations were performed on the Gausssian9 program using a TD-DFT-B3LYP/-3++G (2d, p) level of theory (in methanol). Standard orientation of compound 2: Center Number Atomic Number Atomic Type Coordinates (Angstroms) X Y Z S

11 S

12 X-ray crystallographic data for Pepluacetal () Empirical formula Formula weight Temperature Wavelength Crystal system, space group Unit cell dimensions C 22 H 32 O (2) K.5478 A Monoclinic, C 2 a = 8.29(5) A alpha = 9 deg. b = 8.772(3) A beta = 3.232() deg. c = 2.993(4) A gamma = 9 deg. Volume Z, Calculated density Absorption coefficient F() () A^3 4,.8 Mg/m^3.34 mm^- 784 Crystal size Theta range for data collection Limiting indices Reflections collected / unique Completeness to theta = 9.3 Absorption correction Max. and min. transmission Refinement method Data / restraints / parameters Goodness-of-fit on F^2 Final R indices [I>2sigma(I)] R indices (all data) Absolute structure parameter Largest diff. peak and hole.98 x.39 x.8 mm 3.49 to 9.3 deg. -2<=h<=2, -9<=k<=8, -5<=l<= / 37 [R(int) =.394] 93.3 % Semi-empirical from equivalents.95 and.575 Full-matrix least-squares on F^2 37 / 7 / R =.42, wr2 =.278 R =.43, wr2 =.282.4(9).33 and e.a^-3 View of the hydrogen-bonded motif of Hydrogen-bonds are shown as dashed lines. S2

13 X-ray crystallographic data for Pepluanol A (2) Empirical formula C 4 H 54 O 7 [2(C 2 H 2 O 3 ) H 2 O] Formula weight Temperature Wavelength Crystal system, space group Unit cell dimensions 4.83 (2) K.5478 A Tetragonal, I 4 a = 9.753(3) A alpha = 9 deg. b = 9.753(3) A beta = 9 deg. c = 9.3(7) A gamma = 9 deg. Volume 352.(4) A^3 Z, Calculated density Absorption coefficient F() Crystal size Theta range for data collection Limiting indices Reflections collected / unique Completeness to theta = 8.97 Absorption correction Max. and min. transmission Refinement method Data / restraints / parameters Goodness-of-fit on F^2 Final R indices [I>2sigma(I)] R indices (all data) Absolute structure parameter Extinction coefficient Largest diff. peak and hole 4,.28 Mg/m^3.53 mm^- 4.3 x.3 x.34 mm 3. to 8.97 deg. -22<=h<=22, -7<=k<=23, -8<=l<= 5232 / 244 [R(int) =.59] 9.2 % Semi-empirical from equivalents.884 and.473 Full-matrix least-squares on F^2 244 / / R =.759, wr2 =.225 R =.79, wr2 =.22.4(5).4(7).4 and e.a^-3 View of the hydrogen-bonded motif of 2 Hydrogen-bonds are shown as dashed lines. S3

14 X-ray crystallographic data for Pepluanol B (3) Empirical formula Formula weight Temperature Wavelength Crystal system, space group C 2 H 2 O (2) K.5478 A Orthorhombic, P Unit cell dimensions a = 9.79(2) A alpha = 9 deg. b =.38(2) A beta = 9 deg. c = 7.528(3) A gamma = 9 deg. Volume Z, Calculated density Absorption coefficient F() 72.7() A^3 4,.29 Mg/m^3.32 mm^- 8 Crystal size Theta range for data collection Limiting indices Reflections collected / unique Completeness to theta = 9.8 Absorption correction Max. and min. transmission Refinement method Data / restraints / parameters Goodness-of-fit on F^2 Final R indices [I>2sigma(I)] R indices (all data) Absolute structure parameter Extinction coefficient Largest diff. peak and hole.45 x.3 x.23 mm 5.4 to 9.8 deg. -<=h<=, -<=k<=2, -4<=l<=2 998 / 2935 [R(int) =.322] 9. % Semi-empirical from equivalents.883 and.742 Full-matrix least-squares on F^ / / R =.29, wr2 =.77 R =.29, wr2 =.77.(7).8(5).99 and -.45 e.a^-3 View of the hydrogen-bonded motif of 3 Hydrogen-bonds are shown as dashed lines. S4

15 X-ray crystallographic data for Pepluacetal () and Pepluanol A B (2 3): The crystallographic data for (deposition NO. CCDC 44389), 2 (deposition NO. CCDC 44345), and 3 (deposition NO. CCDC 44344) have been deposited in the Cambridge Crystallographic Data Centre. Copies of the data can be obtained free of charge from the Cambridge Crystallographic Data Centre, 2 Union Road, Cambridge CB2EZ, UK (Fax: ; or deposit@ccdc.cam.ac.uk). S5

16 H NMR spectrum of Pepluacetal () in CDCl 3 S

17 3 C NMR and DEPT spectra of Pepluacetal () in CDCl 3 S7

18 HSQC spectrum of Pepluacetal () in CDCl 3 S8

19 HMBC spectrum of Pepluacetal () in CDCl 3 S9

20 H- H COSY spectrum of Pepluacetal () in CDCl 3 S2

21 ROESY spectrum of Pepluacetal () in CDCl 3 S2

22 HR-ESI (+) MS spectrum of Pepluacetal () S22

23 CD spectrum of Pepluacetal () in MeOH File: CD compound -mm(95-4)595.dsx ProBinaryX Attributes : - Time Stamp :Fri Sep 7:2: File ID : {A3F8D5-35-4e2a CE2C535F} - Is CFR Compliant : false - Original unaltered data Remarks: - HV (CDDC channel): v - Time per point: s - Description: Sample - Concentration:.24mg/mL MeOH - Pathlength: mm Settings: - Time-per-point: s (25us x 4) - Wavelength: 95nm - 4nm - Step Size: nm - Bandwidth: 2nm S23

24 H NMR spectrum of Pepluanol A (2) in CDCl 3 S24

25 3 C NMR and DEPT spectra of Pepluanol A (2) in CDCl 3 S25

26 HSQC spectrum of Pepluanol A (2) in CDCl 3 S2

27 HMBC spectrum of Pepluanol A (2) in CDCl 3 S27

28 H- H COSY spectrum of Pepluanol A (2) in CDCl 3 S28

29 ROESY spectrum of Pepluanol A (2) in CDCl 3 S29

30 HR-ESI (+) MS spectrum of Pepluanol A (2) S3

31 CD spectrum of Pepluanol A (2) in MeOH File: CD compound 2-mm(95-4)5244.dsx ProBinaryX Attributes : - Time Stamp :Sat Oct 24 9:49: File ID : {E97DEE-C45F-4d5-B7E-24B838823CBA} - Is CFR Compliant : false - Original unaltered data Remarks: - HV (CDDC channel): v - Time per point: s - Description: Sample - Concentration:.225mg/mL MeOH - Pathlength: mm Settings: - Time-per-point: s (25us x 4) - Wavelength: 95nm - 4nm - Step Size: nm - Bandwidth: 2nm S3

32 H NMR spectrum of Pepluanol B (3) in CDCl 3 S32

33 3 C NMR and DEPT spectra of Pepluanol B (3) in CDCl 3 S33

34 HSQC spectrum of Pepluanol B (3) in CDCl 3 S34

35 HMBC spectrum of Pepluanol B (3) in CDCl 3 S35

36 H- H COSY spectrum of Pepluanol B (3) in CDCl 3 S3

37 ROESY spectrum of Pepluanol B (3) in CDCl 3 S37

38 HR-EI (+) MS spectrum of Pepluanol B (3) S38

39 CD spectrum of Pepluanol B (3) in MeOH File: CD compound 3-mm(95-)524.dsx ProBinaryX Attributes : - Time Stamp :Sat Oct 24 9:7: File ID : {C37B-BE3-485d-B2E8-AE8592FEF78} - Is CFR Compliant : false - Original unaltered data Remarks: - HV (CDDC channel): v - Time per point: s - Description: Sample - Concentration:.2mg/mL MeOH - Pathlength: mm Settings: - Time-per-point: s (25us x 4) - Wavelength: 95nm - nm - Step Size: nm - Bandwidth: 2nm S39