Supporting Information Cylindrofridins A–C, Linear Cylindrocyclophane-Related Alkylresorcinols from the Cyanobacterium Cylindrospermum stagnale Michael Preisitsch,† Timo H. J. Niedermeyer,‡, §, ┴ Stefan E. Heiden,|| Inga Neidhardt,†,# Jana Kumpfmüller,||, ◊ Martina Wurster†, Kirsten Harmrolfs,∇ Christoph Wiesner, Rolf Müller,∇ and Sabine Mundt*,† † Institute of Pharmacy, Department of Pharmaceutical Biology, Ernst-Moritz-Arndt-University, Friedrich- Ludwig-Jahn-Straße 17, 17489 Greifswald, Germany ‡ Interfaculty Institute of Microbiology and Infection Medicine, Eberhard Karls University, Auf der Morgenstelle 28, 72076 Tübingen, Germany § German Centre for Infection Research (DZIF), Partner Site Tübingen, Germany ┴ Cyano Biotech GmbH, Magnusstraße 11, 12489 Berlin, Germany || Institute of Pharmacy, Department of Pharmaceutical Biotechnology, Ernst-Moritz-Arndt-University, Felix- Hausdorff-Straße 3, 17489 Greifswald, Germany # Present Address: Institute of Technology, Department of Pharmacology, Toxicology and Clinical Pharmacy, Technical University of Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany ◊ Present Address: Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Department of Biomolecular Chemistry, Beutenbergerstraße 11a, 07745 Jena, Germany ∇ Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research, and Department of Pharmaceutical Biotechnology, Saarland University, Campus E8.1, 66123 Saarbrücken, Germany Sealife PHARMA GmbH, Technopark 1/Obj.C/EG, 3430 Tulln, Austria Table of Contents Figure S1. Influence of extraction time used for the one-step extraction and enrichment procedure on the determination of the total [7.7]paracyclophane amount in Nostoc sp. CAVN2 biomass. ............................... 1 Figure S2. Influence of extraction procedures on the extract yield obtained from Nostoc sp. CAVN2 biomass.. 2 Figure S3. Influence of extraction procedures on the determination of the total [7.7]paracyclophane amount in 10.0 mg Nostoc sp. CAVN2 biomass. ............................................................................................................. 3 Figure S4. Influence of extraction agents used for the one-step extraction and enrichment procedure on the determination of the total [7.7]paracyclophane amount in Nostoc sp. CAVN2 biomass. ............................... 4 Figure S5. Phylogenetic tree based on the secondary structure alignment of cyanobacterial 16S rRNA gene sequences. ........................................................................................................................................................ 9 Figure S6. Alignment of partial 16S rRNA gene sequences of Anabaena azotica Ley HB686 (A, accession number: AJ488603), Trichormus azollae Kom BAI/1983 (B, accession number: AJ630454), redetermined partial 16S rRNA gene sequence of A. azotica FACHB-118 (C, designated as A. azotica CCY 0405 in this study), and A. azotica FACHB-118 (also known as HB686) (D, accession number: AY422691). .............. 10 1 Figure S7. H NMR spectrum (500 MHz, MeOH-d4) of cylindrofridin A (1). ................................................... 12 Figure S8. COSY spectrum (500 MHz, MeOH-d4) of cylindrofridin A (1). ....................................................... 12 Figure S9. HSQC-DEPT spectrum (500 MHz, MeOH-d4) of cylindrofridin A (1)............................................. 13 Figure S10. HMBC spectrum (500 MHz, MeOH-d4) of cylindrofridin A (1). .................................................... 14 Figure S11. ATR-IR (film) spectrum of cylindrofridin A (1).............................................................................. 15 Figure S12. ECD spectrum of cylindrofridin A (1) ............................................................................................. 15 1 Figure S13. H NMR spectrum (500 MHz, MeOH-d4) of cylindrofridin B (2). ................................................. 16 Figure S14. COSY spectrum (500 MHz, MeOH-d4) of cylindrofridin B (2). ..................................................... 16 Figure S15. HSQC-DEPT spectrum (500 MHz, MeOH-d4) of cylindrofridin B (2). .......................................... 17 Figure S16. HMBC spectrum (500 MHz, MeOH-d4) of cylindrofridin B (2). .................................................... 18 Figure S17. ATR-IR (film) spectrum of cylindrofridin B (2). ............................................................................. 19 Figure S18. ECD spectrum of cylindrofridin B (2). ............................................................................................ 19 1 Figure S19. H NMR spectrum (500 MHz, MeOH-d4) of cylindrofridin C (3). ................................................. 20 Figure S20. COSY spectrum (500 MHz, MeOH-d4) of cylindrofridin C (3). ..................................................... 20 Figure S21. HSQC-DEPT spectrum (500 MHz, MeOH-d4) of cylindrofridin C (3). .......................................... 21 Figure S22. HMBC spectrum (500 MHz, MeOH-d4) of cylindrofridin C (3). .................................................... 22 Figure S23. ATR-IR (film) spectrum of cylindrofridin C (3). ............................................................................. 23 Figure S24. ECD spectrum of cylindrofridin C (3) ............................................................................................. 23 I S1. Physical, spectroscopic, and spectrometric data of cylindrocyclophane A (4). ............................................. 24 S2. Physical, spectroscopic, and spectrometric data of cylindrocyclophane B (5). .............................................. 25 S3. Physical, spectroscopic, and spectrometric data of cylindrocyclophane D (6). ............................................. 27 1 Figure S1a. H NMR spectrum (600 MHz, MeOH-d4) of cylindrocyclophane A (4). ........................................ 24 1 Figure S2a. H NMR spectrum (600 MHz, MeOH-d4) of cylindrocyclophane B (5). ........................................ 25 Figure S2b. ATR-IR (film) spectrum of cylindrocyclophane B (5). ................................................................... 26 1 Figure S3a. H NMR spectrum (600 MHz, MeOH-d4) of cylindrocyclophane D (6). ........................................ 27 Figure S3b. ATR-IR (film) spectrum of cylindrocyclophane D (6). ................................................................... 28 Table S1. GC-MS detection of components occurring in the upper and lower phase after biomass extraction of Nostoc sp. CAVN2 with the biphasic solvent system (n-heptane/EtOAc/EtOH/H2O; 5:2:5:2, v/v/v/v) ........ 5 Table S2. Cyanobacterial strains screened in this study ........................................................................................ 6 Table S3. Redetermined partial phylogenetic marker gene sequences of Cylindrospermum stagnale PCC 7417 ....................................................................................................................................................................... 11 II a ) % ( t n 100 u o m a e n a h p o l c y 50 c a r a p ] 7 . 7 [ l a t o T 0 0.5 1 2 3 6 9 12 18 24 36 48 Extraction time (h) Figure S1. Influence of extraction time used for the one-step extraction and enrichment procedure on the determination of the total [7.7]paracyclophane amount in Nostoc sp. CAVN2 biomass. Values shown are expressed as mean + standard deviation (SD), n = 6‒9. Data were tested for Gaussian distribution using the Kolmogorov-Smirnov test. Statistical analysis with one-way ANOVA followed by Tukey's multiple comparisons test revealed no statistically significant differences. aExtraction and subsequent HPLC-UV analysis at 226 nm were done as described in the method part of the article. Calculation was performed based on peak area integration of compounds eluting in the range 5‒30 min and revealing carbamidocyclophane-like UV spectra.1 The average value of treatments using an extraction time of 0.5 hours was defined as 100% total [7.7]paracyclophane amount. 1 Figure S2. Influence of extraction procedures on the extract yield obtained from Nostoc sp. CAVN2 biomass. The dry biomass was subjected either to the five-step MeOH extraction procedure according to Preisitsch et al.1 or to the one-step extraction and enrichment procedure as described in the manuscript. Values shown are expressed as mean + SD, n = 4‒6. Data were tested for Gaussian distribution using the Kolmogorov-Smirnov test. Statistical analysis was performed using unpaired Mann-Whitney U-test. Two asterisks (**) indicate a significant difference of p < 0.01. aThe experimental set-up and solvent volumes have not been changed for the five-step extraction procedure. However, biomass was reduced to 10.0 mg for better comparison. 2 Figure S3. Influence of extraction procedures on the determination of the total [7.7]paracyclophane amount in 10.0 mg Nostoc sp. CAVN2 biomass. Previously reported extraction procedure for carbamidocyclophane quantification of Nostoc sp. CAVN10 biomass2 was used as reference extraction procedure and compared to the one-step extraction and enrichment procedure. Values shown are expressed as mean + SD, n = 6. Data were tested for Gaussian distribution using the Kolmogorov-Smirnov test. Statistical analysis was performed using the unpaired t-test with Welch's correction. One asterisk (*) indicates a significant difference of p < 0.05. aThe total [7.7]paracyclophane amount was determined by HPLC-UV analysis at 226 nm using a Varian