Department of Chemistry and Biochemistry, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA 95064, USA

Transcription

1 Supplemental Information: Integration of high-content phenotypic screening and untargeted metabolomics analysis for the comprehensive functional annotation of complex natural products libraries. Kenji L. Kurita, Emerson Glassey, Roger G. Linington, * Department of Chemistry and Biochemistry, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA 95064, USA *Corresponding author. rliningt@sfu.ca Current address: Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6 1

2 Supplemental Table of Contents: Data Processing and Analysis: Source and Mass Spectrometer Settings: MassHunter Workstation Qualitative Analysis Settings... S3 Find by Molecular Feature... S3 Export Peak Lists... S3 Compound Activity Mapping: Isotope Pattern Matching... S3 Blank Removal... S3 Saturation Removal... S3 The D Prefractions... S3 Supplemental Figure 1 - Compound Activity Mapping Workflow... S4 Feature Validation... S5 Supplemental Figure 2 - Validation Decision Tree... S5 Supplemental Figure 3 - Extract Cytological Profiling Data... S6 Supplemental Figure 4 - Network View of Cytological Profiling Data... S7 Supplemental Figure 5 - Graphical Representations of Activity and Cluster Scores... S8 Supplemental Figure 6 - Fluostatins Clustered with ENZ... S9 Supplemental Figure 7 - Rosaramicin Clustered with ENZ... S9 Supplemental Figure 8 - Staurosporine and Actinomycin Synthetic Fingerprint Clustering... S10 Supplemental Figure 9 - Quinocinnolinomycin Clustered with ENZ... S11 Quinocinnolinomycin Growth and Isolation... S12 Supplemental Figure 10 - HPLC Trace of Quinocinnolinomycins A-D (1-4)... S12 Supplemental Figure 11 - UV-Vis Spectra of (1-4)... S13 Supplemental Table 1 - Molar Absorptivity for (1-4)... S13 Supplemental Table 2 - Quinocinnolinomycin NMR Data Table (1-4)... S14 Structure Elucidation of Quinocinnolinomycin A -... S15 NMR Spectra for Quinocinnolinomycin A (1)... S16-S21 NMR Spectra for Quinocinnolinomycin B (2)... S22, S23 NMR Spectra for Quinocinnolinomycin C (3)... S24, S26-29 NMR Spectra for Quinocinnolinomycin D (4)... S25, S26 Synthesis of (S) and (R)-MTPA Esters (5, 6) of Quinocinnolinomycin A (1)... S30 NMR Spectra for (S)-MTPA Ester of Quinocinnolinomycin A (5)... S31, S32 NMR Spectra for (R)-MTPA Ester of Quinocinnolinomycin A (6)... S33, S34 Supplemental Figure 12 - MTPA Ester Conformational Analysis... S35 Supplemental Table 3 - Mosher s Ester NMR Data... S35 NMR Spectra for Rosaramicin... S36, 37 NMR Spectra for Fluostatin D and J... S38 2

3 Source and Mass Spectrometer Settings: The mass spectrometer was run with a detector range from 100 to 1700 m/z. The ESI source was operated with a desolvation temperature of 350 C and a drying gas flow rate of 11 L/ min. The fragmentor voltage was held at 135 V. In positive ESI mode, the capillary voltage was ramped from 2500 V at 0 min to 2750 V at 1 min, and to 3000 V at 3 min. In negative ESI mode, the capillary voltage was held at 2750 V. Each sample was run in both positive and negative ESI source modes and in both high resolution (4 GHz) and extended dynamic range (2 GHz) detector modes. Data Processing and Analysis: MassHunter Workstation Qualitative Analysis Settings: Find by Molecular Feature: We used default settings for the find by molecular feature algorithm from MassHunter 5.0 with a minimum peak height cutoff of 700 counts. Export Peak Lists: Peak lists are exported in XML file format and include compound, adduct, and isotope information. Unless otherwise stated, we use adduct information described as m/z features and adduct isotope information for isotope pattern matching. Compound Activity Mapping: Isotope Pattern Matching: Feature alignment between sample runs uses three different criteria to determine if two m/z features are in fact the same: retention time, high accuracy mass, and isotope pattern. Isotope pattern matching was done using the method described by Pluskal, et al. by finding the product of one minus the peak height differences within 20 ppm for each peak between two normalized isotope patterns(28). Unless specified, the tolerances used for m/z feature alignment are 7 ppm, 0.4 minutes, and 0.5 isotope similarity. Blank Removal: In order to correct for retention time shift in HPLC experiments and to remove systematic signals, five blank runs were performed before every plate analyzed. The first two were used to clean and equilibrate the system, while the third through fifth are used for blank removal. Runs of fractionated starch/yeast/peptone (SYP) medium are also subtracted in the same step to remove systematic media contaminants. We align m/z features between blanks/syp (20 ppm, 0.4 minutes, 0.5 isotope difference), condense the blank/media background runs into one m/z feature list, and remove all of those features from the 96-well plate data they preceed. Blanks and SYP are acquired in both high-resolution and high-dynamic range modes, and subtracted from their respective data modes. Saturation Removal: Detector saturation causes a number of errors in data acquisition including loss of mass accuracy and signal ringing of false features around the real saturated feature. During initial data analysis, after exporting peak lists, we identify m/z features which are saturated, and remove all features within 0.4 minutes and 1 mass unit of the saturated feature. Because the features are saturated, we do not use isotope pattern matching, because isotope patterns are not reliable for saturated features. The D prefractions: It was observed that the D or 80% methanol/water prefractions contained many nonspecific masses that were present in most of the D fractions, but absent 3

4 from all other fractions; therefore, any features that were only associated with D prefractions were omitted from network analysis in order to reduce non-specific clustering. These masses were likely due to the degradation of XAD-16 polymer resin used in the fermentation media to sequester organic molecules. Supplemental Figure 1: verview of the Compound Activity Mapping work flow from data acquisition in 4 GHz and 2 GHz modes in postive and negative ionization mode to data integration and vizualization. Acquisition Raw Spectra Data Processing Peak Picking Adduct Matching Remove Blanks, Media, and Saturation Databases Store Compound, Adduct, and Isotope Peak Tables Data Validation Replicate Comparison Biological Screening Data Calculate Compound Activity Score and Cluster Score Remove Inactive Compounds Align Compounds Between Samples Data Integration and Visualization Activity Plots Network Diagrams 4

5 Feature Validation: In order to guarantee the fidelity of the m/z features used by our platform, we perform a variation of technical replicates during data acquisition. The Agilent Time of Flight mass spectrometer can acquire data in two modes: high-resolution (4 GHz) mode, which provides greater m/z resolution and increased sensitivity, but has low tolerance for concentration variances so the detector is more readily saturated, and high dynamic range (2 GHz), which sacrifices m/z resolution for greater detector range, reducing saturation. ur samples are microbial extracts with mixtures of unknown constitution and concentration, but we also require high mass accuracy. We acquire data in both modes and align features between the two modes (7 ppm or 20 ppm, 0.4 minutes, 0.5 isotope difference), storing data from the mode that is not saturated, with preference for high-resolution data (Supplemental Figure 2). Because we are dynamically changing the data we store, we assign a binary qualifier (bq) value to each m/z feature that specifies data origin. The bq value is 5 digit binary number that correspond to: peak present in 2 GHz, peak saturated in 2 GHz, peak present in 4 GHz, peak saturated in 4 GHz, aligned between modes with 7 ppm (assigned 0) or 20 ppm (assigned 1). This is shown visually in Supplemental Figure 2. Supplemental Figure 2: The decision tree for m/z feature alignment and scoring displaying how peaks are compared across mass spectrometry experiments of the same prefraction in the same ionization mode. 5

6 Supplemental Figure 3: Cytological Profiling heatmap output of diluted DMS prefractions. These are the processed data from which the synthetic fingerprints for m/z features and thereby the activity and cluster scores are derived. RLPA-1011A_-5 RLPA-1020A_-5 RLPA-2003B_-5 RLPA-1010B_-5 RLPA-1010F_-5 RLPA-2004C_-5 RLPA-2017B_-5 RLPA-2004E_-20 RLPA-1022E_-400 RLPA-2002C_-10 RLPA-2002D_-10 RLPA-2008B_-5 RLPA-2013C_-5 RLPA-2022C_-5 RLPA-1014E_-10 RLPA-2022E_-5 RLPA-1020F_-5 RLPA-1012E_-40 RLPA-2005A_-5 RLPA-2010D_-320 RLPA-2008A_-5 RLPA-2021A_-5 RLPA-1011E_-1280 RLPA-1011F_-640 RLPA-1012A_-5 RLPA-1014D_-5 RLPA-1011C_-20 RLPA-2002B_-5 RLPA-1014A_-40 RLPA-1020B_-10 RLPA-1020C_-40 RLPA-1020D_-20 RLPA-2002A_10 RLPA-1010D_-200 RLPA-1014B_-20 RLPA-2004A_-160 RLPA-2005C_-40 RLPA-1020E_-5 RLPA-2001C_-5 RLPA-2001D_-5 RLPA-2001F_-5 RLPA-1014C_-5 RLPA-1012F_-5 RLPA-2012E_-5 RLPA-2028D_-5 RLPA-1021E_-5 RLPA-2003D_-5 RLPA-1021A_-5 RLPA-2005B_-5 RLPA-1021D_-5 RLPA-2007B_-5 RLPA-2007C_-5 RLPA-2007D_-5 RLPA-2007A_-5 RLPA-1011B_-5 RLPA-1021B_-10 RLPA-1022F_-40 RLPA-2004B_-10 RLPA-2005F_-800 RLPA-2008D_-1600 RLPA-1021C_-5 RLPA-2021B_-5 RLPA-2020B_-5 RLPA-2003E_-10 RLPA-2003F_-5 RLPA-2004D_-10 RLPA-2008E_-3200 RLPA-2008F_-6400 RLPA-1010C_-5 RLPA-1010E_-5 RLPA-2008C_-5 RLPA-2011E_-5 RLPA-2005D_-10 RLPA-2002E_-5 RLPA-2030F_-5 RLPA-2023C_-5 RLPA-2028E_-5 6 RLPA-2003C_-5 RLPA-2022F_-5 RLPA-2020E_-400 RLPA-2022D_-10 RLPA-2001E_-20 RLPA-1011D_-20 RLPA-2005E_-160 RLPA-2021E_-10 RLPA-2023D_-5 RLPA-2021D_-5 RLPA-2030D_-5 RLPA-2010E_-5 RLPA-2021C_-5 RLPA-2023F_-5 RLPA-2023E_-5 RLPA-2010C_-5 RLPA-2030E_-40 RLPA-2031E_-5 RLPA-2032C_-5 RLPA-2014C_-5

7 Supplemental Figure 4: A network representation of the data displayed as a heatmap in Supplental Figure 3. Prefractions that induce phenotypes with a pearson correlation greater than are connected and colored using Gephi s modularity package. RLPA-2007C RLPA-2007D RLPA-1021A RLPA-1021D RLPA-2007B RLPA-2005B RLPA-2028E RLPA-2023C RLPA-1012A RLPA-1014D RLPA-2021B RLPA-1021C RLPA-2020B RLPA-2004A RLPA-1020B RLPA-1014B RLPA-2005C RLPA-1020C RLPA-1020D RLPA-1022F RLPA-1021B RLPA-2004B RLPA-1021E RLPA-2030E RLPA-1011E RLPA-2001E RLPA-2023E RLPA-2012E RLPA-2005D RLPA-2005F RLPA-2021A RLPA-2013C RLPA-2031E RLPA-2013D RLPA-2006F RLPA-1010F RLPA-2024B RLPA-2002A RLPA-1012F RLPA-2031C RLPA-2026CRLPA-2019D RLPA-1010B RLPA-2020C RLPA-2032B RLPA-2031B RLPA-2016C RLPA-2022E RLPA-2017A RLPA-2009D RLPA-2003CRLPA-2028D RLPA-2002E RLPA-1011F RLPA-2032F RLPA-2026E RLPA-1022B RLPA-2011F RLPA-2008D RLPA-2010D RLPA-2008A RLPA-2030C RLPA-2031D RLPA-1022E RLPA-2016D RLPA-2006D RLPA-2014B RLPA-2015A RLPA-2012C RLPA-2020A RLPA-2032A RLPA-1022D RLPA-2015F RLPA-1022C RLPA-2003F RLPA-2004E RLPA-2007A RLPA-2012D RLPA-2022F RLPA-2016E RLPA-2013E RLPA-2003B RLPA-2003ERLPA-2025C RLPA-2028F RLPA-2011B RLPA-2029E RLPA-2027A RLPA-2015D RLPA-2015C RLPA-2011C RLPA-1014C RLPA-2017B RLPA-2019E RLPA-2015E RLPA-2007E RLPA-2009F RLPA-2032C RLPA-2025D RLPA-2018D RLPA-1012B RLPA-2024F RLPA-2010C RLPA-2008E RLPA-2007F RLPA-2017D RLPA-2024E RLPA-2027D RLPA-2027E RLPA-2004C RLPA-2019F RLPA-1021F RLPA-2019C RLPA-1011A RLPA-2031F RLPA-2029B RLPA-2016F RLPA-2018A RLPA-2006C RLPA-2004D RLPA-2029D RLPA-2031A RLPA-2010B RLPA-2013A RLPA-1014E RLPA-2018E RLPA-2026B RLPA-2010F RLPA-2018C RLPA-2024A RLPA-2024C RLPA-2009E RLPA-2027C RLPA-2016A RLPA-2001B RLPA-2014C RLPA-2021F RLPA-2012F RLPA-2022D RLPA-2001D RLPA-2006E RLPA-2016B RLPA-1012D RLPA-2026F RLPA-2025B RLPA-1014F RLPA-2010A RLPA-2018F RLPA-2015B RLPA-2029A RLPA-2026D RLPA-2008F RLPA-2009B RLPA-2003A RLPA-2019A RLPA-2009C RLPA-2013F RLPA-2023B RLPA-2024D RLPA-2022A RLPA-2017F RLPA-2003D RLPA-2030D RLPA-2011A RLPA-2020D RLPA-2014A RLPA-1010D RLPA-2001C RLPA-2017E RLPA-2032E RLPA-1020A RLPA-2012B RLPA-2025F RLPA-2001F RLPA-2011D RLPA-2025E RLPA-2023A RLPA-2030A RLPA-1020E RLPA-1010A RLPA-2022C RLPA-2025A RLPA-2014D RLPA-2032D RLPA-2006A RLPA-2029C RLPA-2001A RLPA-1020F RLPA-2017C RLPA-2006B RLPA-2012A RLPA-2019B RLPA-1012C RLPA-2030B RLPA-2028B RLPA-2014E RLPA-2026A RLPA-2020F RLPA-2028C RLPA-2020E RLPA-2009A RLPA-2029F RLPA-2027F RLPA-2022B RLPA-2023D RLPA-2028A RLPA-1022A RLPA-2018B RLPA-2008B RLPA-2013B RLPA-2027B RLPA-2023F RLPA-2021D RLPA-2002F RLPA-2005A RLPA-2014F RLPA-1012E RLPA-2030F RLPA-1011B RLPA-1014A RLPA-2008C RLPA-1010C RLPA-2002C RLPA-2002D RLPA-2021C RLPA-2010E RLPA-2002B RLPA-1011C RLPA-2011E RLPA-1010E RLPA-2005E RLPA-1011D RLPA-2021E 7

8 Supplemental Figure 5: Graphs displaying the activity and cluster score values of each m/z feature. (a and c) depict activity and cluster score respectively versus feature count. (b and d) histograms of number of m/z features versus activity and cluster scores respectively. 8

9 Supplemental Figure 6: The cytological profiles of the ENZ compound library clustered with the purified fluostatins C, D, and J. The compound name is followed by the in well μm concentration. Supplemental Figure 7: The cytological profiles of the ENZ compound library with the purified rosaramicin. The compound name is followed by the in well μm concentration. 9

10 Supplemental Figure 8: The cytological profiles of the ENZ compound library with synthetic fingerprints (the predicted cytological profiles of m/z features). The first cluster contains m/z features corresponding to the compounds staurosporine and echinomycin while the second contains m/z features corresponding to actinomycin D. 10

11 Supplemental Figure 9: The cytological profiles of the ENZ compound library clustered with the purified quinocinnolinomycins (1-4) in a dilution series. The compound name is followed by the in well μm concentration. The strong similarity of the cytological fingerprints of the quinocinnolinomycins (1-4) with compounds known to cause endoplasmic reticulum stress (thapsigargin, tunicamycin, lycorine, and brefeldin A) suggest that (1-4) have a similar mechanism of action. 11

12 Quinocinnolinomycin growth and isolation: Bacterial frozen stock was struck out on solid media (DIFC Marine Broth 37.4 g and 18.0 g of agar). Colonies were inoculated into a capped 40 ml culture tube with 7.0 ml of liquid media containing 31.2 g of instant ocean, 10.0 g of Soluble Starch, 4.0 g of yeast extract, and 2.0 g of peptone per liter of water. All liquid media cultures were maintained at r.t. and shaken at 210 r.p.m. After 4 days 6 ml from the small-scale culture was used to inoculate 60 ml of the same media in a 250 ml wide neck Erlenmeyer flask with a 1 cm diameter metal spring coil and milk filter top. After 4 days 45 ml of this medium-scale culture were inoculated into 1 L of media in a 2.8 L wide mouth Fernbach flask containing a large spring coil and then topped with a milk filter. This culture was grown for 5 days. The cells were then filtered using a glass filter, washed with sterile water, transferred to a 1.0 L Erlenmeyer flask, and extracted with 250 ml of 1:1 dichloromethane in methanol. The cell debris was filtered off and the extract solution was evaporated under vacuum. This dried extract was prefractionated using the eluotropic series of methanol water described in the methods section. The 80% methanol fraction was dried under vacuum and resuspended in minimal methanol and centrifuged. The supernatant was purified by HPLC on a (Phenomonex Kinetix 2.6 μm XB-C x 4.6 mm) using a gradient of MeCN:H % formic acid (50% MeCN for 2 min, 50%-65% MeCN over 20 min) at a flow rate of 2 ml min -1. The peaks at minutes 8, 14, 15, 17 and 18 (diazaquinomycin C, and 1-4 respectively)were collected separately and dried under vacuum. Supplemental Figure 10: HPLC-DAD trace of RLPA-2003D with peaks labeled. m/z diazaquinomycin C m/z m/z (1)(2) (3)(4) Min 12

13 Supplemental Figure 11: UV-Vis absorbance spectra for quinocinnolinomcyins A-D (1-4). Abs v nm of QCM A(1) Abs v nm of QCM B(2) Abs v nm QCM C(3) Abs v nm QCM D(4) Supplemental Table 1: Table of extinction coefficients for quinocinnolinomycins A-D (1-4) at 318 and 450 nm in M -1 cm -1. (log ε) 318 (log ε) 450 QCM A (1) QCM B (2) QCM C (3) QCM D (4)

14 Supplemental Table 2: Tabulated NMR data from 1-4. All spectra were acquired in DMS-d6 at 600 MHz and 150 MHz for 1 H and 13 C respectively. The structure of the core of the quinocinnolinomycins and each of the different tails displayed and numbered for clarity. 6 H3C N H 7 Position (1) C (1) H(J in Hz) (2) C (2) H(J in Hz) (3) C (3) H(J in Hz) (4) C (4) H(J in Hz) , s , s , s , s 4a , s , s , s , s a , dd (16.0, 1.4) , dd (16.0, 1.5) , dd (16.0, 1.6) , dd (16.0, 1.6) , dd (16.0, 4.9) , dd (16.0, 4.9) , dd (16.0, 5.0) , dd (16.0, 5.0) , m , m , m , m , m , m , m , m 1.51, m 1.51, m 1.51, m 1.51, m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m , m 1.05, m , m , m , m , dt (13.7, 6.4) , d ( 6.6) , m , m , m 1.08, m , t (7.0, 7.0) , t (7.4, 7.4) , m , d ( 4.9) , s , d (6.2) , d (5.1) , d (5.0) NH 8.17, q (4.7) 8.16, s 8.17, q (5.2) 8.16, q (4.8) H 5.12, d (4.9) 5.13, s 5.12, d (4.7) 14 N N 8 8a (1) 5 4a 4 R H (2) (3) (4)

15 Structure Elucidation Details for quinocinnolinomycin A (1): The molecular formulae C 23 H 33 N 3 3 and C 24 H 35 N 3 3 were determined based on the strong consensus between the [M + H] + and [M + Na] + m/z features for each set of two constitutional isomers. The earliest eluting compound with the molecular formula C23H33N33 was solved by NMR analysis, using a combination of 1 H, 13 C, gcsy, ghsqc, ghmbc, and 1D-TCSY spectra (Fig. 5). Consideration of the 1 H-NMR spectrum indicated the presence of two vinylic and two aromatic signals, an aryl amine, one N-methyl doublet, two methyl doublets, and multiple overlapping resonances in a methylene envelope at ppm (Table S2). Interpretation of the 13 C and ghsqc spectra confirmed the presence of two ketones and four aromatic/quaternary carbons. The planar structure of the tail of the molecule was assigned from either side of the methylene region using gcsy and HMBC correlations (Fig. 5B). 1D-TCSY from the H11 proton to the H21 was used to confirm that the allyl oxymethine was connected to the tail through the methylene region. The remaining C 9 N 3 2 belong to the headgroup. The attachment to the tail was assigned by ghmbc correlations from the vinylic protons at δ 7.01 and 7.22 to the carbons at positions 3 and 4 and ghmbc correlations from the aromatic proton on position 4 at δ 8.11 to the carbon at position 9. ne of the ketones could be placed at position 5 based on ghmbc correlations from the aromatic resonance at δ 8.11 (Fig. 5B). The resonance at δ 5.80 could be assigned to the vinylic proton at position 6 between the two ketones that form the quinone based on ghmbc correlations to the carbons at positions 4a, 5, 7, and 8. The ghmbc correlation from the N-methyl at δ 2.84 to the carbons at positions 6, 7, and 8 placed the relative orientation of the vinylic amine (Fig. 5B). Finally, the incorporation of the remaining atoms (CN 2 ) could only satisfy the requirement for three additional degrees of unsaturation by inclusion of the quaternary carbon at position 8a and two heteroaromatic nitrogens in positions 1 and 2, thus completing the structural assignment (Fig. 5B). The R stereochemistry of quinocinnolinomycin A was determined using Mosher s ester method (Fig. 5C), and this assignment extended to quinocinnolinomycins B-D based on their common biosynthetic origin (fig. S12). 15

16 1 H-NMR of quinocinnolinomycin A (1) at 600 MHz in DMS-d6 H H3C N H N N Chemical Formula: C 23 H 33 N 3 3 Exact Mass:

17 13C-NMR of quinocinnolinomycin A (1) at 150 MHz in DMS-d6 13C-NMR of quinocinnolinomycin A (1) at 150 MHz in DMS-d6 17

18 18 gcsy of quinocinnolinomycin A (1) at 600 MHz in DMS-d6

19 19 HSQC of quinocinnolinomycin A (1) at 600 MHz in DMS-d6

20 20 HMBC of quinocinnolinomycin A (1) at 600 MHz in DMS-d6

21 1D TCSY of quinocinnolinomycin A (1) at 600 MHz in DMS-d6 excited at 4.27 ppm δ (ppm)

22 1 H-NMR of quinocinnolinomycin B (2) at 600 MHz in DMS-d6 H H3C N N N H Chemical Formula: C 23 H 33 N 3 3 Exact Mass:

23 23 13 C-NMR of quinocinnolinomycin B (2) at 150 MHz in DMS-d6

24 1 H-NMR of quinocinnolinomycin C (3) at 600 MHz in DMS-d6 H H3C N N N H Chemical Formula: C 24 H 35 N 3 3 Exact Mass:

25 1 H-NMR of quinocinnolinomycin D (4) at 600 MHz in DMS-d6 H H3C N N N H Chemical Formula: C 24 H 35 N 3 3 Exact Mass:

26 13C-NMR of quinocinnolinomycin C (3) at 150 MHz in DMS-d6 13C-NMR of quinocinnolinomycin D (4) at 150 MHz in DMS-d6 26

27 27 CSY of quinocinnolinomycin C (3) at 600 MHz in DMS-d6

28 28 HSQC of quinocinnolinomycin C (3) at 600 MHz in DMS-d6

29 29 HMBC of quinocinnolinomycin C (3) at 600 MHz in DMS-d6

30 Synthesis of (S) and (R)-MTPA esters (5, 6) of quinocinnolinomycin A (1): In two vials with teflon septa pierced by a blunt needle containing a small stir bar, 1.0 mg of compound 1 was dried under vacuum. The vessels were flushed with argon and ml of dry pyridine added to each. The R and S MTPA-Cl were added in 6-fold excess, one to each vial, and the reaction was run for 1 h at RT. The reaction was quenched by addition of a drop of methanol. The products were run through silica plugs, eluted with dichlormethane, and dried under vacuum. The subsequent products were purified by HPLC by (Eclipse XDB-C18 5 μm 4.6 x 150 mm) reverse phase column on a gradient of using a gradient of MeH:H % formic acid (60%-100% MeH over 16 min) at a flow rate of 2 ml min -1. The S-ester eluted at 11 minutes while the R-ester eluted at 15 minutes. 30

31 1 H-NMR of quinocinnolinomycin A (S)-MTPA ester (5) at 600 MHz in DMS-d6 (S)-MTPA H3C N N N H 31

32 32 CSY of quinocinnolinomycin A (S)-MTPA ester (5) at 600 MHz in DMS-d6

33 1 H-NMR of quinocinnolinomycin A (R)-MTPA ester (6) at 600 MHz in DMS-d6 (R)-MTPA H3C N N N H 33

34 34 CSY of quinocinnolinomycin A (R)-MTPA ester (6) at 600 MHz in DMS-d6

35 Supplemental Figure 12: Quinocinnolinomycin A (1) is displayed with the δ SR values for the modified Mosher s ester method. Shielding from in the phenyl ring in the suggested major comformer displayed below causes the affected protons to be shifted upfield for that particular diastereomer. SR MTPA H 3 C N H MTPA N N H H L 1 L 2 L 1 H H L 2 L 1 L 2 MTPA L APTM L 1 2 H H 3 C Ph F 3 C S H L 1 L 2 H 3 C Ph F 3 C R Supplemental Table 3: Tabulated 1 H NMR data for (1, 5, and 6). All spectra were acquired in DMS-d6 at 600 MHz. H L 1 L 2 Position (1) δ H S-Ester (5) δ H R-Ester (6) δ H δ SR = δ S - δ R a a

36 1 H NMR of rosaramicin at 600 MHz in CDCl3 H H H N Chemical Formula: C 31 H 51 N 9 Exact Mass:

37 37 13C NMR of rosaramicin at 150 MHz in CDCl 3

38 13C NMR of fluostatin D at 150 MHz in CDCl 3 13C NMR of fluostatin J at 150 MHz in CDCl 3 38