Supporting Information. Jasisoquinolines A and B, Architecturally New Isoquinolines, from. a Marine Sponge Jaspis sp.

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1 Supporting Information Jasisoquinolines A and B, Architecturally ew Isoquinolines, from a Marine Sponge Jaspis sp. Yasufumi Imae, Kentaro Takada, Shuhei Murayama, Shigeru kada, Yuji Ise, and Shigeki Matsunaga Table of Contents I. Experimental Procedures... 3 General Procedures... 3 Animal Material... 3 Extraction and Isolation... 3 Chemical Degradation of Jasisoquinoline A (1)... 4 Preparation of (R)-MTPA Ester of 3-Methyl-1-decanol Cathepsin B Inhibitory Assay... 5 II. Tables... 6 Table S1. 1 and 13 C MR data for jasisoquinoline A (1) in CD3D Table S2. 1 and 13 C MR data for jasisoquinoline A (1) in DMS-d Table S3. 1 and 13 C MR data for jasisoquinoline B (2) in CD3D III. Figures... 9 Figure S1. 1 MR spectrum for jasisoquinoline A (1) in CD3D Figure S2. CSY spectrum for jasisoquinoline A (1) in CD3D Figure S3. TCSY spectrum for jasisoquinoline A (1) in CD3D Figure S4. SQC spectrum for jasisoquinoline A (1) in CD3D Figure S5. MBC spectrum for jasisoquinoline A (1) in CD3D Figure S6. 1 spectrum for jasisoquinoline A (1) in DMS-d Figure S7. SQC spectrum for jasisoquinoline A (1) in DMS-d Figure S8. MBC spectrum for jasisoquinoline A (1) in DMS-d Figure S9. RESY spectrum for jasisoquinoline A (1) in DMS-d Figure S10. 1 MR spectrum for jasisoquinoline B (2) in CD3D Figure S11. CSY spectrum for jasisoquinoline B (2) in CD3D Figure S12. SQC spectrum for jasisoquinoline B (2) in CD3D Figure S13. MBC spectrum for jasisoquinoline B (2) in CD3D Figure S14. 1 MR spectrum for schulzeine A (3) in CD3D S1-

2 Figure S15. 1 MR spectrum for schulzeine C (4) in CD3D Figure S16. 1 MR spectrum for MTPA ester Figure S17. Tandem FABMS fragment ions for Figure S18. Tandem FABMS analysis of 5 (m/z 0-772) Figure S19. Tandem FABMS analysis of 5 (m/z ) Figure S20. Tandem FABMS analysis of 5 (m/z ) Figure S21. Tandem FABMS analysis of 5 (m/z ) Figure S22. Tandem FABMS analysis of 5 (m/z ) Figure S23. CD spectra for schulzeines S2-

3 I. Experimental Procedures General Procedures MR spectra were recorded on a JEL delta 600 MR spectrometer at 600 Mz. 1 and 13 C MR chemical shifts were referenced to the solvent cross peak on SQC spectra at δ 3.31 and δ for CD 3 D and at δ 2.50 and δ for DMS-d 6, respectively. ESIMS spectra were measured on a JEL JMS-T100LC time-of-flight mass spectrometer. FABMS spectra were measured on a JEL JMS-700T MStation. Fluorescence for enzyme inhibition assay was detected with a Molecular Devices SPECTRA MAX fluorescence spectrometer. UV spectra were measured in Me using a Shimadzu BioSpec-1600 spectrophotometer. ptical rotations were measured on a JASC DIP-1000 degital polarimeter. CD spectra were recorded in Me on a JASC J-720 spectrapolarimeter. Animal Material The sponge sample was collected by dredging at a depth of 150 m at shima-shinsone (28 80 ; E) in The specimen was frozen after collection and kept at -20 C until extraction. Extraction and Isolation The sponge (400 g, wet weight) was extracted with Me (500 ml) and Et (500 ml). The combined extracts were concentrated and partitioned between 2 and CCl 3, and the aqueous layer was further extracted with n-bu. The CCl 3 layer was separated between 90% Me and n-hexane, and the 90% Me layer was partitioned between 60% Me and CCl 3. The n-bu layer and the 60% Me layer were combined and separated by silica gel column chromatography using a stepwise gradient (CCl 3 /Me/ 2 10/0/0 5/5/2). Fractions eluted with CCl 3 /Me/ 2 6/4/1 and 5/5/2 were combined and separated by DS flash chromatography using a stepwise gradient of aqueous Me (0 -S3-

4 100%) and aqueous MeC (70 and 80%). The 40% Me elute was chromatographed by RP-PLC (Cosmosil AR-II; mm) with 50% MeC containing 1.0 M acl 4. Active fractions were further purified by RP-PLC (Develosil C 30 -UG-5; mm) with 50% MeC containing 0.7M acl 4 to afford jasisoquinolines A (2.0 mg), B (0.7 mg), schulzeines A (4.0 mg), and C (1.5 mg). Jasisoquinoline A (1): white powder; [α] 24 D -1.1 (c 0.05, Me); UV (Me) λ max 211 nm (ε 17,000), 278 nm (1760); CD Δε (Me) (242 nm), +(284 nm); 1 and 13 C MR data, see Table S1 and S2; RFABMS m/z (M a) - [C S 3 a 2 (Δ 0.4 mmu)]. Jasisoquinoline B (2): white powder; [α] 24 D +7.6 (c 0.03, Me); UV (Me) λ max 215 nm (ε 16,200), 282 nm (2720); CD Δε (Me) (242 nm), +(284 nm); 1 and 13 C MR data, see Table S3; RFABMS m/z (M a) - [C S 3 a 2 (Δ +2.8 mmu)]. Chemical Degradation of Jasisoquinoline A (1) 1 (1 mg) was diluted with Me (1 ml) and desulfated with p-ts (catalytic amount) at room temperature for 2 h. The reaction mixture was neutralized with 10% ac 3 followed by desalting with Inertsep RP-1. The resulting triol 5 was cleaved by the treatment with 2eq. of ai 4 in 75% Me at room temperature followed by reduction with 50 mg of ab 4. The reaction mixture was quenched with Ac and extracted with C 2 Cl 2 to give diol 6 and primary alcohol 7. The presence of 6 was verified by FABMS analysis (m/z 601 [M + ] + ). Preparation of (R)-MTPA Ester of 3-Methyl-1-decanol mg portion of 7 was treated with (S)-MTPACl in C 2 Cl 2 /pyridine (1:1, 100μL). The reaction mixture was partitioned between 2 and CCl 3, and the organic layer was -S4-

5 purified by RP-PLC (Develosil C 30 -UG-5; mm) with linear gradient of % aqueous Me to give (R)-MTPA ester 8. 8: MR (CD 3 D) δ 7.72, 7.63, 4.22 (1), 3.64 (Me), 1.69 (3), 1.44 (2), (4 11). Cathepsin B Inhibitory Assay Cathepsin B inhibitory assay was carried out according to the modified method of iwasa et al. 1 Stock solution of bovine catheosin B (Sigma Chemical Company; 1unit/mL in 0.05 M MES p 6.0 and 0.1% Brij-35) was diluted by 100 times with the buffer. 50 μl of 25μM substrate solution (Z-Arg-Arg-AMC; Peptide Institute,Inc. in DMS) was added to the mixture of 100 μl cathepsin B solution and 4 μl of test sample solution. The mixture was incubated at 37 C for 30 min. The fluorescence of the reaction mixture was measured with an excitation at 345 nm and an emission at 440 nm. 1 T. iwasa, J. Fujita-Yoshigaki, M. Shirouzu,. Koide, T. Sawada, S. Sakiyama, S. Yokokawa, Cancer Lett. 1993, 69, S5-

6 II. Tables Table S1. 1 and 13 C MR data for jasisoquinoline A (1) in CD 3 D. jasisoquinoline A (1) in CD 3 D 1 13 C MBC 1 b (brs), 3.03 a a, 2.76 (brd, 13.3) a 4a (brs) , 6, 7, 8a (brs) , 6, a (m), 2.28 (m) (m) (m) (m) (quin, 5.9) (m) , (m) (m) c , 26, , 1.41 (m) (m) , 1.18 (m) (m) (m) , (t, 7.0) , (d, 6.4) , 27, 28 1' b 2' 3' 3.51, 3.43 (dt, 6.9, 13.3) ', 5' 4' 2.64 (t, 6.8) ', 5', 6', 10' 5' ' 6.10 (brd, 2.1) ', 8', 10' 7' ' 6.12 (brd, 2.1) ', 9' 9' ' 6.10 (brd, 2.1) ', 8', 9' a Signals overlapped. b Signals were undetectable in CD 3 D. c This signal overlapped with D signal. -S6-

7 Table S2. 1 and 13 C MR data for jasisoquinoline A (1) in DMS-d 6. a jasisoquinoline A (1) in DMS-d C MBC (m), 2.93 (brd, 7.75) , 4a (brs), 2.38 (brs) a 4a (d, 2.4) , 6, 7, 8a (brs) (d, 2.4) , 8, 8a (brs) 8a (m), 1.8 (m) , 8a, 10, 1' (m) (m) (m) (m) (quin, 5.4) (m) , (m) (dt, 8.3, 4.2) (dt, 9.5, 3.6) (m), 1.24 (m) (brs) (m), 1.06 (m) (m) (m) , (t, 7.2) , (d, 6.0) ' ' 8.78 (brd, 2.4) 1', 3' 3' 3.30 b ', 4', 5' 4' 2.55 (t, 7.2) ', 6', 10' 5' ' 6.05 (d, 1.8) ', 7', 8', 9', 10' 7' ' (brs) 8' 6.04 (t, 1.8) ', 9' 9' ' (brs) 10' 6.05 (d, 1.8) ', 6', 7', 8', 9' a Contaminated Ac signals were detected: 1 MR δ 1.88 (2); 13 C MR δ (C1), 21.6 (C2). b This signal overlapped with D signal. -S7-

8 Table S3. 1 and 13 C MR data for jasisoquinoline B (2) in CD 3 D. jasisoquinoline B (2) in CD 3 D , , a a a , ' a 2' 3' 3.49, ' ' ' ' ' ' ' a Signals were undetectable in CD 3 D. a 13 C -S8-

9 III. Figures Figure S1. 1 MR spectrum for jasisoquinoline A (1) in CD 3 D. 8 S 3 a a 3 S a 3 S Jasisoquinoline A (1) -S9-

10 Figure S2. CSY spectrum for jasisoquinoline A (1) in CD 3 D. 8 S 3 a a 3 S a 3 S Jasisoquinoline A (1) -S10-

11 Figure S3. TCSY spectrum for jasisoquinoline A (1) in CD 3 D. 8 S 3 a a 3 S a 3 S Jasisoquinoline A (1) -S11-

12 Figure S4. SQC spectrum for jasisoquinoline A (1) in CD 3 D. 8 S 3 a a 3 S a 3 S Jasisoquinoline A (1) -S12-

13 Figure S5. MBC spectrum for jasisoquinoline A (1) in CD 3 D. 8 S 3 a a 3 S a 3 S Jasisoquinoline A (1) -S13-

14 Figure S6. 1 spectrum for jasisoquinoline A (1) in DMS-d 6. 8 S 3 a a 3 S a 3 S Jasisoquinoline A (1) -S14-

15 Figure S7. SQC spectrum for jasisoquinoline A (1) in DMS-d 6. 8 S 3 a a 3 S a 3 S Jasisoquinoline A (1) -S15-

16 Figure S8. MBC spectrum for jasisoquinoline A (1) in DMS-d 6. 8 S 3 a a 3 S a 3 S Jasisoquinoline A (1) -S16-

17 Figure S9. RESY spectrum for jasisoquinoline A (1) in DMS-d 6 8 S 3 a a 3 S a 3 S Jasisoquinoline A (1) -S17-

18 Figure S10. 1 MR spectrum for jasisoquinoline B (2) in CD 3 D. 8 S 3 a a 3 S a 3 S Jasisoquinoline B (2) -S18-

19 Figure S11. CSY spectrum for jasisoquinoline B (2) in CD 3 D. 8 S 3 a a 3 S a 3 S Jasisoquinoline B (2) -S19-

20 Figure S12. SQC spectrum for jasisoquinoline B (2) in CD 3 D. 8 S 3 a a 3 S a 3 S Jasisoquinoline B (2) -S20-

21 Figure S13. MBC spectrum for jasisoquinoline B (2) in CD 3 D. 8 S 3 a a 3 S a 3 S Jasisoquinoline B (2) -S21-

22 Figure S14. 1 MR spectrum for schulzeine A (3) in CD 3 D. 9 S 3 a a 3 S a 3 S Schulzeine A (3) -S22-

23 Figure S15. 1 MR spectrum for schulzeine C (4) in CD 3 D. 9 S 3 a a 3 S a 3 S Schulzeine C (4) -S23-

24 Figure S16. 1 MR spectrum for MTPA ester 8. -S24-

25 Figure S17. Tandem FABMS fragment ions for (C 2 ) S25-

26 Figure S18. Tandem FABMS analysis of 5 (m/z 0-772). -S26-

27 Figure S19. Tandem FABMS analysis of 5 (m/z ). -S27-

28 Figure S20. Tandem FABMS analysis of 5 (m/z ). -S28-

29 Figure S21. Tandem FABMS analysis of 5 (m/z ). -S29-

30 Figure S22. Tandem FABMS analysis of 5 (m/z ). -S30-

31 Figure S23. CD spectra for schulzeines 9 S 3 a a 3 S a 3 S RESY P-helicity 9 S 3 a a 3 S a 3 S RESY M-helicity -S31-