Biosynthesis of Rishirilide B

Transcription

1 Supporting Informations Biosynthesis of Rishirilide B Philipp Schwarzer 1,#, Juli Wunsch Palasis 1,#, Andreas Bechthold 1, and Thomas Paululat 2, 1 P. Schwarzer, Dr. J. Wunsch Palasis, Prof. Dr. A. Bechthold, Pharmaceutical Biology and Biotechnology, University of Freiburg, Stefan Meier Str. 19, Freiburg i. Br. (Germany); andreas.bechthold@pharmazie.uni freiburg.de 2 Dr. T. Paululat, Organic Chemistry, University of Siegen, Adolf Reichwein Str. 2, Siegen (Germany); thomas.paululat@uni siegen.de # P. Schwarzer and Dr. J. Wunsch Palasis contributed equally to this work Correspondence: andreas.bechthold@pharmazie.uni freiburg.de and thomas.paululat@uni siegen.de

2 Tables and Figures: Table S1: Table S2: Figure S1: Figure S2: Table S3: Figure S3: Figure S4: Figure S5: Table S4: Figure S6: Table S5: Figure S7: Figure S8: Figure S9: Table S6: NMR data of rishirilide B (600/150 MHz, DMSO d6, 35 C) Calculation of enrichment and specific enrichment for rishirilide B from feeding experiment with [1 13 C]acetate Labelling positions of rishirilide B after feeding experiment with [1 13 C]acetate 13 C NMR spectra (150 MHz, DMSO d6, 35 C) of rishirilide B after feeding of [1 13 C]acetate in comparison to rishirilide B at natural abundance Table of NMR data of rishirilide B from feeding experiment with [1,2 13 C2]acetate Labelling positions of rishirilide B after feeding experiment with [1,2 13 C2]acetate 13 C NMR spectra (150 MHz, DMSO d6, 35 C) of rishirilide B after feeding of [1,2 13 C2]acetate in comparison to rishirilide B at natural abundance Inadequate (150 MHz, DMSO d6, 35 C) of rishirilide B after feeding of [1,2 13 C2]acetate Results of feeding experiment with L [methyl 13 C]methionine 13 C NMR spectra (150 MHz, DMSO d6, 35 C) of rishirilide B from feeding experiment with L [methyl 13 C]methionine in comparison to rishirilide B at natural abundance Calculation of enrichment and specific enrichment for rishirilide B from feeding experiment with [2 13 C]acetate Labelling positions of rishirilide B after feeding experiment with [2 13 C]acetate 13 C NMR spectra (150 MHz, DMSO d6, 35 C) of rishirilide B after feeding of [2 13 C]acetate in comparison to rishirilide B at natural abundance Mass spectrum of rishirilide B from feeding experiment with [ 13 C5, 15 N1] L valine Table of NMR data rishirilide B from feeding experiment with [ 13 C5, 15 N1] L valine Figure S10: Labelling positions of rishirilide B after feeding experiment with [ 13 C5, 15 N1] L valine Figure S11: 13 C NMR spectra (150 MHz, DMSO d6, 35 C) of rishirilide B from feeding experiment with [ 13 C5, 15 N1] L valine in comparison to rishirilide B at natural abundance Figure S12: Inadequate (150 MHz, DMSO d6, 35 C) of rishirilide B from feeding experiment with [ 13 C5, 15 N1] L valine Figure S13: Expansion of Inadequate (150 MHz, DMSO d6, 35 C) of rishirilide B from feeding experiment with [ 13 C5, 15 N1] L valine.

3 Table S1: NMR data of rishirilide B (600/150MHz, DMSO-d 6, 35 C) Pos. δ C [ppm] δ H (J Hz) [ppm] COSY a HMBC and bsghmbc( ppm) H, 2-H, 17-H q (6.8) 17-H 3 17-H H, 17-H H, (11-H a ) 4a H, (11-H a ) H, 7-H, 8-H, 10- H d (7.6) 7-H 8-H dd (8.3, 7.6) 6-H, 8-H 6-H, 8-H d (8.3) 7-H 6-H, 9-H 8a H, 8-H, 10-H s (8-H) 8-H 9a H s 6-H, 9-H 10a H, 8-H, 9-H H a : 2.23 dt 11-H b, 12-H a, H b (13.1, 3.9) H b 11-H b : 1.61 ddd 11-H a, 12-H a, 12- (13.3, 12.8, 4.7) H b H a : 1.38 m 11-H a, 11-H b 11-H b, 13-H, 14- H 3, 15-H 3 12-H b : 0.78 m 11-H a, 11-H b, 13- H m 12-H b, 14-H 3, 15- H 3 12-H a, 14-H 3, 15- H d (6.5) 13-H 15-H d (6.5) 13-H 12-H a, 13-H, 14- H H d (6.8) 2-H 2-H 10.2 s br a weak signals in brackets

4 Table S2: Calculation of enrichment and specific enrichment for rishirilide B from feeding experiment with [1-13 C]acetate Pos. δ C [ppm] Intensity reference signal Intensity from feeding experiment Normalized intensities from feeding experiment (factor: 1.318) Enrichment (specific enrichment) [%] (3.8) (4.9) 4a Missing signal (4.6) (8.0) a (5.2) a (4.2) (6.8) 10a c (5.1) a reference signal for normalization a Figure S1: Labelling positions of rishirilide B after feeding experiment with [1-13 C]acetate O H CO H 3 C

5 Figure S2: 13 C NMR spectra ( 150MHz, DMSO-d 6, 35 C) of rishirilide B after feeding of [1-13 C]acetate (red) in comparison to rishirilide B at natural abundance (black)

6 Table S3: Table of NMR data of rishirilide B from feeding experiment with [1,2-13 C 2 ]acetate Pos. δ C [ppm] J CC Inadequate [Hz] C C C C-11 4a C C-10a C C C-8a 8a C C-9a 9a C C-10a 10a C C C Figure S3: Labelling positions of rishirilide B after feeding experiment with [1,2-13 C 2 ]acetate 55Hz 64Hz O 41Hz H 55Hz 64Hz 64Hz 39Hz 51Hz CO H 3 C

7 Figure S4: 13 C NMR spectra ( 150MHz, DMSO-d 6, 35 C) of rishirilide B after feeding of [1,2-13 C 2 ]acetate (red) in comparison to rishirilide B at natural abundance (black) Figure S5: Inadequate (150MHz, DMSO-d 6, 35 C) of rishirilide B after feeding of [1,2-13 C 2 ]acetate

8 Table S4: Results of feeding experiment with L-[methyl- 13 C]methionine Pos. δ C [ppm] Intensity reference signal Intensity from feeding experiment Normalized intensities from feeding experiment (factor: 0.985) Enrichment (specific enrichment) [%] a a a a a Reference Signal for normalization and calculation of enrichment a No labelled position could be observed.

9 Figure S6: 13 C NMR spectra ( 150MHz, DMSO-d 6, 35 C) of rishirilide B from feeding experiment with L-[methyl- 13 C]methioninee (red) in comparison to rishirilide B at natural abundance (black) No enrichment visible

10 Table S5: Calculation of enrichment and specific enrichment for rishirilide B from feeding experiment with [2-13 C]acetate Pos. δ C [ppm] Intensity reference signal Intensity from feeding experiment Normalized intensities from feeding experiment (factor: 0.985) Enrichment (specific enrichment) [%] (3.3) (4.5) a (6.4) (2.7) (1.3) 8a (3.8) 9a a (2.0) (2.4) a (1.4) a Reference Signal for normalization and calculation of enrichment a Figure S7: Labelling positions of rishirilide B after feeding experiment with [2-13 C]acetate O H CO H 3 C

11 Figure S8: 13 C NMR spectra ( 150MHz, DMSO-d 6, 35 C) of rishirilide B after feeding of [2-13 C]acetate (red) in comparison to rishirilide B at natural abundance (black)

12 Figure S9: Mass spectrum of rishirilide B from feeding experiment with [ 13 C 5, 15 N 1 ]-L-valine

13 Table S6: Table of NMR data of rishirilide B from feeding experiment with [ 13 C 5, 15 N 1 ]-L-valine Pos. δ C Enrichment a Multiplicity a Inadequate b [ppm] (strong or weak) [Hz] Weak 40 d Weak 40 d Weak 55 d Weak 37 d - 4a Weak 63 d Weak 64 d Weak 56 d Weak 56 d Weak 55 d - 8a Weak 55 d Weak 62 d - 9a Weak 62 d Weak 63 d - 10a Weak 64 d Weak 37 d Strong 35 d C Strong 35, 35 dt C-12, C-14, C Strong 35 d C Strong 35 d C Weak 51 d No - s - a from 13 C NMR spectrum b from INADEQUATE NMR spectrum J CC a Figure S10: Labelling positions of rishirilide B after feeding experiment with [ 13 C 5, 15 N 1 ]-Lvaline O H H 2 N O CO L-Valine, 98% 13 C, 98% 15 N H 3 C O

14 Figure S11: 13 C NMR spectra (150MHz, DMSO-d 6, 35 C) of rishirilide B from feeding experiment with [ 13 C 5, 15 N 1 ]-L-valine (red)) in comparison to rishirilide B at natural abundance (black) Figure S12: Inadequate (150MHz, DMSO-d 6, 35 C) of rishirilide B from feeding experiment with [ 13 C 5, 15 N 1 ]-L-valine

15 Figure S13: Expansion of Inadequate (150MHz, DMSO-d 6, 35 C) of rishirilide B from feeding experiment with [ 13 C 5, 15 N 1 ]-L-valine