Chemical Investigations of Fungal Natural Products for Drug Discovery Danielle H

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Graduate Theses and Dissertations Graduate School

July 2017 Chemical Investigations of Fungal Natural Products for Drug Discovery Danielle H. Demers University of South Florida, [email protected]

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Chemical Investigations of Fungal Natural Products

for Drug Discovery

Danielle H. Demers

A dissertation submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida

Major Professor: Bill J. Baker, Ph.D. Lindsey N. Shaw, Ph.D. James W. Leahy, Ph.D. Edward Turos, Ph.D.

Date of Approval: June 27, 2017

Keywords: screening, epigenetic modification, Phomopsis , Penicillium , ESKAPE, leishmaniasis

Dedication

This work is wholehear tedly dedicated to my incredible family. To say I wish to thank my parents, Chris and Sharon, hardly sounds like enough . In my entire life, t hese two have never doubted me for one second. I would not be the person I am today without their ceaseless support, enc ouragement, sacrifice, and love. Truly, this body of work would not exist were it not for all the phone calls, packages, emails, and visits that made Florida feel a little less far f rom home over the past 6 years. I am so lucky to have them, and the amazing family they have built, in my corn er.

To my brothers, Luke and CJ, and my “older sister” Cyndi, thank you for your love, friendship, and pat ience with me over these years. T o the friends and extended family members that are far too numerous to list here (such is the blessing and the curse of such a beautifully large, crazy clan) , thank you for your continued interest, compassion, and support . And finally , to Kieryn Emilee, I dedica te this to you, and hope you always remember that nothing and no one can keep you from your dreams.

“Keep moving forward”

~ Walt Disney

Acknowledgements

This dissertation is built on the shoulders of giants and the work of an army of scientists.

I must first and foremost thank Dr. Bill Baker for sharing his knowledge, financial support, and

for telling me “no” when I did not want to hear it. I was blessed with amazing experiences,

opportunities, and life lessons during my years here, and none of it would have been possible

without you .

To my committee, past and present Baker L ab members, collaborating teams at USF and

beyond , and my many undergrads : thank you for your help, enthusiasm, and dedication to this work. No scientist works alone and I am so grateful to have met and worked with so many excellent researchers .

To the colleagues who became friends, and the friends who became family: I would not have survived without you. There are so many people who deserve to be acknowledged for their comradery over these past 6 years, and though I cannot possibly list you all, please know that your contributions to my life here at USF do not go unrecognized. Thank y ou for turning Florida into my second home. I cannot wait to see what comes next for all of us.

Table of Contents

List of Tables ...... iii

List of Figures ...... iv

List of Schemes ...... ix

List of Abbreviations and Units ...... x

Abstract ...... xiii

Chapter 1. An Introduction to Marine Natural Product Drug Discovery ...... 1 1.1. Why Natural Products ...... 1 1.2. Sources of Marine Natural Products ...... 2 1. 2.1. Marine M acro - organism s ...... 2 1.2.2. Marine M icro - organisms ...... 3 1.2.3. Marine P lant E ndophytes ...... 4 1.2.4. Collection Locations and Non - marine S ources ...... 5 1.2.4.1. Antarctica ...... 5 1.2.4.2. Florida ...... 7 1.2.4.3. Terrestrial and fresh water environments ...... 8 1.3. Approaches Towards the Isolation of Microbial Natural Products ...... 9 1.3.1. Collection and I solation ...... 9 1.3.2. Culture T echniqu e s ...... 10 1.4. Drug Discovery Targets ...... 11 1.4.1. The ESKAPE P athogens ...... 13 1.4.2 . Leishmania donovani ...... 13 1.4.3. Mycobacterium tuberculosis ...... 13 1.4.4. Clostridium difficile ...... 14 1.4.5. Naegleria fowleri ...... 14 1.4.6. Cancer T argets ...... 15 1.5. Future Directions ...... 15 1.6. References ...... 16

C hapter 2. New Bioactive Meroterpenes from Phomopsis sp...... 23 2 .1 . BB11 - 2 Isolation ...... 23 2 .2 . Initi al Bioactivities and Scale - up ...... 24 2 .3 . Compound Isolation and Structure Elucidation ...... 25 2 .4 . Bioactivities ...... 38 2 .5 . Future Directions ...... 40 2 .6 . References ...... 40

Chapter 3 . The Creation of a Large Fungal Isolate and Extract Library ...... 42 3 .1 . Curating a Fungal Library ...... 42 3 .2 . Screening Protocols, Accomplishments ...... 45 3 .3 . Metabolomic Analysis ...... 47 3 .4 . Training Set Results ...... 50 3 .5 . Future Directions ...... 52 3 .6 . References ...... 52

Chapter 4 . A New Citreohybrid d ione Discovered Via Epige netic Modification ...... 54 4.1 . Citreohybridones and Citreohybriddiones ...... 54 4.2 . KML12 - 14MG - B2a I solation ...... 55 4.3 . Initial Bioactivities, Scale - up, Epigenetic Modification , and Identification ...... 57 4.4 . Compound Isolation and Structure Elucidation ...... 59 4.5 . Future Directions ...... 63 4.6 . References ...... 64

Appendix A: Experime ntal and Supporting Data for Chapter 2 ...... 65

Appen dix B: Experime ntal and Supporting Data for Chapter 3 ...... 97

Appendix C: Experime ntal and Supporting Data for Chapter 4 ...... 103

List of Tables

Table 2.1. 1D and 2D data for phomopsichromin A ( 1 ) in CDCl 3 ...... 2 6

Table 2.2. 1D and 2D data for phomopsichromin B ( 2 ) in CDCl 3 ...... 30

Table 2.3. 1D and 2D data for phomopsichromin C ( 3 ) in CDCl 3 ...... 33

Table 2.4. 1D and 2D data for phomopsichromin D ( 4 ) in DMSO - d 6 ...... 36

Table 2.5. 1D and 2D data for phomopsichromin E ( 5 ) in DMSO - d 6 ...... 38

Table 3.1. Collection details for collection trips from Fall 2011 to Spring 2017...... 44

Table 3.2. Weekly sequence of events for continuous production of fungal isolates and extract libraries...... 46

Table 4.1. Screening results for KML12 - 14MG - B2a...... 57

Table 4.2. 1D and 2D data for citreohybriddione D ( 1 ) in CDCl 3 ...... 61

Table A1 . The forward and reverse primers used for the fungal strain identification of BB11 - 2...... 66

Table A2. Pure compound bioactivities of 1 - 6 ...... 96

iii

List of Figures

Fig. 1.1. The Antarctic continent as seen from space on Google Earth with common collection sites for natural products featured...... 6

Fig. 1.2. The Swiss Polar Institute’s ACE cruise plan, December 2016 - March 2017...... 7

Fig. 1.3. Left panel: Mangroves growing in highly developed intercoastal waterways in Dunedin, FL; Right panel: Mangroves growing in the protected, pristine Everglades National Park, FL...... 8

Fig. 2.1. Chemical structures of new compounds 1 - 5 and known compound 6 ...... 26

Fig. 2.2. NP HPLC chromatogram of fraction D_C (Scheme 2.1) from the control growth treatment of Phomopsis sp...... 27

Fig. 2.3. Important COSY, HMBC, and NOE correlations in phomopsichromin A ( 1 ) ...... 29

Fig. 2.4. Important COSY, HMBC, and NOE correlations in phomopsichromin B ( 2 )...... 31

Fig. 2.5. Methylation reaction of 2 (stereochemistry unknown at the time) to yield 7 with diazomethane...... 32

Fig. 2.6. Important COSY and HMBC corr elations in phomopsichromin C ( 3 )...... 34

Fig. 2.7. Proposed biosynthetic pathway towards the cyclohexane substructure of 1 - 6 from Tanabe and Suzuki, 1974...... 34

Fig. 2.8. Proposed stereocenters in the cyclohexane ring of 3 sent for ECD calculations...... 35

Fig. 2.9. Important COSY, HMBC, and NOE correlations in phomopsichromin D ( 4 )...... 37

Fig. 2.10. Important COSY and HMBC correlations in phomopsichromin E ( 5 )...... 37

Fig. 3.1. a) Representative pie chart from bioassay data, in this case, ESKAPE activity, that shows the activity boosting effects of the epigenetic modification; b) Representative pie chart from bioassay data, in this case, ESKAPE activity, that demonstrates distribution of activity across the three treatment conditions...... 47

Fig. 3.2. An MDS plot of the Bray Curtis Similarity matrix of square root transformed data for 123 extracts (replicates averaged) from the screening program...... 48

Fig. 3.3. An MDS plot of the Bray Curtis Similarity matrix of square root transformed data for 9 randomly selected organism s from those represented in Figure 3.2...... 49

Fig. 3.4. Selectivity of active extra cts...... 51

Fig. 4.1. Some of the previously reported citreohybridones, citreohybriddiones A - C, and the new citreohybriddione D ( 1 )...... 55

Fig. 4.2. Google Maps satellite view of c ollection areas around Keys Marine Lab, Long Key Florida...... 56

Fig. 4.3. The NP MPLC chromatogram of KML12 - 14MG - B2a HDACi...... 59

Fig. 4.4. The NP HPLC chromatogram of KML12 - 14MG - B2a HDACi - F...... 60

Fig. 4.5. Important COSY, HMBC, and NOESY correlations in Citre ohybriddione D ( 1 )...... 62

Fig. 4.6. A 3D depiction of 1 for stereochemical review...... 62

Fig. A1. NCBI nucleotide blast results for BB11 - 2 forward primers...... 66

Fig. A2. NCBI nucleotide blast result s for BB11 - 2 reverse primers...... 67

Fig. A3. NP MPLC chromatogram of BB 11 - 2 Control EtOAc partition...... 68

Fig. A4. A second NP MPLC of BB11 - 2 Control fraction D from the first MPLC separation. Fraction C (bottles 4 & 5) co ntained the phomopsichromins...... 69

Fig. A5. NP MPLC chromatogram of BB11 - 2 HDACi E tOAc partition...... 70

Fig. A6. NP MPLC chromatogram of BB11 - 2_DNMTi EtOAc partition...... 71

Fig. A7. NP HPLC chromatogram (black) and ELS D trace (blue) of BB11 - 2_D_C...... 72

1 Fig. A8. H NMR Spectrum (500 MHz, CDCl 3 ) of Phomopsichromin A ( 1 ) ...... 74

13 Fig. A9. C NMR Spectrum (125 MHz, CDCl 3 ) of Phomopsichromin A ( 1 ) ...... 75

1 13 Fig. A10. H - C gHSQCAD NMR Spectrum (600 MHz, CDCl 3 ) of Phomopsichromin A ( 1 ) ...... 75

1 13 Fig. A11. H - C gHMBCAD NMR Spectrum (500 MHz, CDCl 3 ) of Phomopsichromin A ( 1 ) ...... 76

1 1 Fig. A12. H - H gCOSY NMR Spectrum (500 MHz, CDCl 3 ) of Phomopsichromin A ( 1 ) ...... 76

1 1 Fig. A13. H - H NOESY NMR Spectrum (600 MHz, CDCl 3 ) of Phomopsichromin A ( 1 ) ...... 77

1 Fig. A14. H NMR Spectrum (6 00 MHz, CDCl 3 ) of Phomopsichromin B ( 2 ) ...... 78

13 Fig. A15. C NMR Spectrum (125 MHz, CDCl 3 ) of Phomopsichromin B ( 2 ) ...... 79

1 13 Fig. A16. H - C gHSQCAD NMR Spectrum (600 MHz, CDCl 3 ) of Phomopsichromin B ( 2 ) ...... 79

1 13 Fig. A17. H - C gHMBCAD NMR Spectrum (500 MHz, CDCl 3 ) of Phomopsichromin B ( 2 ) ...... 80

1 1 Fig. A18. H - H gCOSY NMR Spectrum (600 MHz, CDCl 3 ) of Phomopsichromin B ( 2 ) ...... 80

1 1 Fig. A19. H - H NOESY NMR Spectrum (600 MHz, CDCl 3 ) of Phomopsichromin B ( 2 ) ...... 81

1 Fig. A20. H NMR Spectrum (400 MHz, CDCl 3 ) of methylated phomopsichromin B ( 7 ). A new methoxy signal can be seen at δ H 3.92...... 81

13 Fig. A21. C NMR Spectrum (500 MHz, CHCl 3 ) of methylated phomopsichromin B ( 7 ). 24 13 C signals can be seen...... 82

1 Fig. A22. H NMR Spectrum (6 00 MHz, CDCl 3 ) of Phomopsichromin C ( 3 ) ...... 83

13 Fig. A23. C NMR Spectrum (125 MHz, CDCl 3 ) of Phomopsichromin C ( 3 ) ...... 84

1 13 Fig. A24. H - C gHSQCAD NMR Spectrum (600 MHz, CDCl 3 ) of Phomopsichromin C ( 3 ) ...... 84

1 13 Fig. A25. H - C gHMBCAD NMR Spectrum (500 MHz, CDCl 3 ) of Phomopsichromin C ( 3 ) ...... 85

1 1 Fig. A26. H - H gCOSY NMR Spectrum (600 MHz , CDCl 3 ) of Phomopsichromin C ( 3 ) ...... 85

1 Fig. A27. H NMR Spectrum (500 MHz, DMSO - d 6 ) of Phomopsichromin D ( 4 )...... 86

13 Fig. A28. C NMR Spectrum (125 MHz, DMSO - d 6 ) of Phomopsichromin D ( 4 )...... 87

1 13 Fig. A29. H - C gHSQCAD NMR Spectrum (600 MHz, DMSO - d 6 ) of Phomopsichromin D ( 4 )...... 88

1 13 Fig. A30. H - C gHMBCAD NMR Spectrum (500 MHz, DMSO - d 6 ) of Phomopsichromin D ( 4 )...... 89

1 1 Fig. A31. H - H gCOSY NMR Spectrum (500 MHz, DMSO - d 6 ) of Phomopsichromin D ( 4 )...... 90

1 Fig. A32. H NMR Spectrum ( 600 MHz, DMSO - d 6 ) of Phomopsichromin E ( 5 )...... 92

13 Fig. A33. C NMR Spectrum (201 MHz, DMSO - d 6 ) of Phomopsichromin E ( 5 )...... 93

1 13 Fig. A34. H - C gHSQCAD NMR Spectrum (600 MHz, DMSO - d 6 ) of Phomopsichromin E ( 5 )...... 93

1 13 Fig. A35. H - C gHMBCAD NMR Spectrum (600 MHz, DMSO - d 6 ) of Phomopsichromin E ( 5 )...... 94

1 1 Fig. A36. H - H gCOSY NMR Spectrum (600 MHz, DMSO - d 6 ) of Phomopsichromin E ( 5 )...... 94

1 Fig. A37. H NMR Spectrum (6 00 MHz, CDCl 3 ) of LL - Z1272ε ( 6 ) ...... 95

13 Fig. A38. C NMR Spectrum (125 MHz, CDCl 3 ) of LL - Z1272ε ( 6 ) ...... 96

Fig. C1. NCBI nucleotide blast results for KML 12 - 14MG - B2a forward primers...... 103

Fig. C2. NCBI nucleotide blast results for KML 12 - 14MG - B2a reverse primers...... 103

Fig. C3. NP MPLC chromatogram of KML12 - 14 MG - B2a_HDAC EtOAc partition...... 104

Fig. C4. NP HPLC ELSD trace of KML12 - 14MG - B2a_Control_D...... 105

Fig. C5. NP HPLC ELSD trace KML12 - 14MG - B2a_HDAC_F...... 105

Fig. C6. NP HPLC ELSD tra ce of KML12 - 14MG - B2a_DNMT_C...... 106

1 Fig. C7. H NMR Spectrum (500 MHz, CDCl 3 ) of citreohybriddione D...... 107

13 Fig. C8. C NMR Spectrum (200 MHz, CD Cl 3 ) of citreohybriddione D...... 108

1 13 Fig. C9. H - C gHSQCAD NMR Spectrum (800 MHz, CDCl 3 ) of citreohybriddione D...... 109

vii

1 13 Fig. C10. H - C gHMBCAD NMR Spectrum (500 MHz, CDCl 3 ) of citreohybriddione D...... 110

1 1 Fig. C11. H - H gCOSY NMR Spectrum (600 MHz, CDCl 3 ) of citreohybriddione D...... 110

1 1 Fig. C12. H - H NOESY NMR Spectrum (800 MHz, CDCl 3 ) of citreohybriddione D...... 111

viii

List of Schemes

Scheme 1.1. A generic flow chart for natural products isolation ...... 12

Scheme 2.1. Extraction scheme for Phomopsis sp. Control...... 25

List of Abbreviations and Units

1, 2, 3D 1, 2, 3 - dimensional [α] specific rotation = 100α/lc ACE Antarctic Circumnavigation Expedition br d broad doublet (NMR) br t broad triplet (NMR) °C degrees Celsius C carbon C18 octadecyl bonded silica CDCl 3 deuterated chloroform CDI Clostridium difficile infection CDDI C enter for Drug Discovery and I nnovation CH 2 N 2 diazomethane COSY correlation spectroscopy (NMR) d doublet (NMR) DI deionized (water) DMSO dimethyl sulfoxide DMSO - d 6 deuterated dimethyl sulfoxide DNA deoxyribonucleic acid DNMT DNA methyltransferase DNMTi DNA methyltransferase inhibited δ chemical shifts (NMR) ECD electronic circular dichroism ESI electrospray ionization (MS) ESKAPE Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae ELSD evaporating light scattering detector (liquid chromatography) EtO Ac ethyl acetate ε the molar extinction coefficient in UV spectroscopy FL Florida g grams gCOSY gradient correlation spectroscopy (NMR) gHMBCAD gradient heteronuclear multiple bond connectivity adiabatic decoupling (NMR) gHSQCAD gradient heteronuclear single quantum correlation adiabatic decoupling (NMR) H hydrogen H 2 O water HDAC histone deacetylase HDACi histone deacetylase inhibited HMBC heteronuclear multiple bond connectivity (NMR)

HPLC high performance/ pressure liquid chromatography HRESIMS high resolution electrospray ionization mass spectrometry hr hour HTS high throughput screening IC 50 inhibitory concentration 50% IR infrared spectroscopy J coupling constant LC/MS liquid chromatography mass spectrometry LC - QToF - MS liquid chromatog raphy quadrupole time of flight mass spectrometry LD 50 lethal dose λmax maximum absorption wavelength (UV) m multiplet (NMR) MBC 99 minimum bactericidal concentration MDR - TB multi - drug resistant tuberculosis MDS multidimensional scaling MeOH methanol mg milligrams MHz megahertz MIC minimum inhibitory concentration µg micrograms µL microliters µM micromolar mL milliliters MPLC medium pressure liquid chromatography MRSA methicillin resistant Staphylococcus aureus MS mass spectrometry MW molecular weight m/z mass/ charge ratio (MS) NCBI National Center for Biotechnology Information NIH National Institutes of Health NMR nuclear magnetic resonance NOE nuclear overhauser effect (NMR) NOESY nuclear overhauser effect spectroscopy (NMR) NP normal phase (liquid chromatography) NTD neglected tropical disease OSMAC one strain many compounds (fungi) PAM primary amoebic meningoencephalitis PCA principle component analysis PCR polymerase chain reaction PDB po tato dextrose broth PKS polyketide synthase ppm parts per million, chemical shifts (NMR) q quartet (NMR) QToF quadrupole time of flight RP reverse phase (liquid chromatography) rt room temperature

RV research vessel s singlet (NMR) SAR structure activity relationship SCUBA self - contained underwater breathing apparatus SDA sabouraud dextrose agar SDB sabouraud dextrose broth t triplet (NMR) TB tuberculosis TSB tryptic soy broth USF University of South Florida UV ultraviolet WHO World Health Organization XDR - TB extensively drug resistant tuberculosis

xii

Abstract

N atural products have , historically , played an important role in drug discovery.

Nevertheless , drug resistance , pathogen evolution, and global climate change threaten human health and nearly all current anti - infective treatments on the market today . It is undeniable that n ew drug discovery efforts are needed with increasing urgency.

Bolstered by a rich history of discovering treatments i n the world around us, natural products chemists continue to look to the environment with increasing understanding and emerging technologies that allow efficient, effective isolation of new chemical entities. This thesis will describe one such endeavor.

F ocusing on fungal natural products, herein is described the isolation and structure elucidation of new, bioactive natural products. Further, the development and implementation of a large fungal screening program will be discussed, the results of which stan d to advance microbial drug discovery in the Baker lab for years to come.

xiii

Chapter 1 .

An Introduction to Marine Natural Product Drug Discovery

1.1. Why Natural Products

From single celled protozoa to homo sapiens , all living beings are comprised of organic

molecules that make up the cellular machinery essential for life. While some of these compounds

must be acquired from the organisms’ surroundings, many of these metabolites that are necessary for basic cellular function are produced de novo by the cell . These primary metabol ites - sugars, proteins, lipids , etc. may vary s ignificantly form one life f orm to another, but the basic chemical composition of each living thing is the same. While DNA structure and composition surely separates a bacterium from a human being, a n investigation of the chemicals found within a single ce ll from either of those organisms will expose a high level of similarity in the aforementioned primary metabolites . What may differ chemically within this investigation, however, is the myriad of small molecules seemingly unnecessary to primary cellular fu nction.

These “ secondary metabolites ” , or natural products, are generally signaling molecules that allow the organism not only to survive, but to thrive. All of these metabolites - primary and secondary -

allow living organisms to live amongst and interact w ith one another . At a purely chemical level ,

all of life is comp eting on the same playing field, and this is the basis for interaction s between

species.

Given that all life forms have the potential to interact with each other chemically - one

organism produces a molecule that interacts with a receptor in another organism - it therefore

follows that the study of these natural products can be of great benefit to human health. For, what is human disease but an interaction between life forms? What is a drug, but a molecule that interacts with a receptor?

With advances in technologies that allow us to visit and sample from all corners of the globe, we now unders tand that there is immeasurable biological and chemical diversity just waiting to be discovered. This chemical diversity is an incredible source for drug discovery. The creativity of the human mind is nothing compared to the creativity of evolution, and we continue to discover natural products with novel chemical scaffolds never before seen or imagined through synthetic methods. Further, n atural products have consistently played a strong role in the development of new drugs for human health, with a full 65% of all small molecule approved drugs since 1981 being inspired by, derivatives of, or directly, natural products. 1

While the natural products of terrestrial communities have long been stu died for their applicat ions to human health, 1 – 4 marine environments have added their own repertoire of

i mportant compounds to the list over the past few decades . 1,5 – 8 With relatively newly accessible regions of the marine environment (namely, deep water and polar communities) 9 – 11 the biological and chemical diversity of marine environments poses great potential for bioprospecting.

1.2. Sources of Marine Natural Products

1.2.1 . Marine M acro - organisms

The world’s oceans cover m ore than 70% of the planet. The exceptionally varie d environments within these oceans range around the globe from the tropics to the poles. E ven in the harshest of these environments, intense biodiversity can be found. Within these biodiversity

hotspots, marine organisms have adapted unique physical and ch emical attributes to support their

survival and proliferation. Benthic marine macro - organisms - multicellular organisms,

specifically those that are sedentary and lack any physical defense mechanisms - have been of

interest for their chemistry for some time , both in the chemical ecology and drug discovery arena s 12 – 15 .

Many new and novel pharmacophores have been discovered from organisms on the

marine benthos, 1,5 and currently, there are two such compounds that are commercially available

as drugs: ziconotide (Prialt ® ) from the marine cone snail Conus magnus 8 , and trabectedin

(Yondelis ® ) isolated from the tunicate Ecteinascidia turbinate 6,7 . Countless other marine natural products that have not yet made it through the drug development pipeline exhibit early and exciting activit ies in various bioassays 5,12,13,16 , inspiring future marine natural products discovery efforts.

1.2.2 . Marine M icro - organisms

While marine macro - organisms have been found to contain interesting secondary metabolites, 1,5 effo rts towards any drug development with these compounds are often met with the issue of supply. Many benthic marine organisms cannot be cultured in an artificial setting 17 and are often originally collected in some of the most remote and difficult to access reg ions of the worl d. H arvesting the biomass of these organisms needed for complete drug development would be impossible without great ecological impact. Though they could present a source - independent supply, s ynthetic routes to some of these complex scaffold s remain extremely difficult. 18 T herefore, attaining an adequate supply of marine macro - organism derived compounds of interest for the rigor ous drug development pipeline can be an extremely daunting challenge.

In fact , faced with this problem, and utilizing advances in genetic sequencing and microbial isolation techniques, it has been discovered that many natural products initially isolated from marine macro - organisms actually originate from invertebrate associated micro - organism s . 5,13 With attention turning towards the density, d iversity, and biosynthetic capabilities of marine microorganisms, the collection, isolation, and culture of these important and sometimes elusive marine participants has become an interesting area of study . 13,19 – 21

1.2.3 . Marine Plant E ndophytes

The discovery of the potent anti - cancer drug paclitaxel (taxol ®) from the yew tree, Taxus brevifolia , added to the already successful narrative of terrestrial plant natural products as an important source of bioactive compoun ds. 22 However, everything changed after paclitaxel was later isolated from the endophytic fungus Taxomyces andreanae . 23 The study of endophytic micro - organis ms, specifically endophytic fungi, was elevated significantly, and today, endophytic fungi are considered an important source of natural products . 24 – 26

In the marine realm, mangrove plants have emerged as a significant source of endophytic fungi. Found in the harsh fringe marine environments across the globe, mangrove forests are well known for intense biodiversity, as well as their ada ptations to withstand consistently changing temperature and salinity. Endophytic fungi isolated from these environments have been of particular interest , 26 – 29 but interestingly, none of the work reported included the mangrove environments in Florida, US A . After a number of collections in Ev erglades National Park and other mangrove communities in Florida, a pr oof of concept study by Beau et al . demonstrated that, unsurprisingly, these local mangrove environments were an unto uched resource of endophyte biological and chemical diversity . 30 The wor k described herein in chapters 3 and 4

seeks to build on these initial studies, hopefully adding to the case for further investigation ,

appreciation, and protection of these exciting marine environments.

1.2.4 . C ollection L ocations and Non - marine S ources

Perhaps as important as choosing a target organism is choosing a collection location. The

marine environment is a vast and variable biome, and much of it still remains to be explored.

Even the most well s tudied marine environments - the tropics, for example, with their easily

accessible reef systems and temperate climates - are changing and evolving with climate change and anthropomorphic stressors. With these changes, come changes in the diversity of life a nd

chemistry that can be found there. T he same can be said for terrestri al and fresh water

environments, and as scientific tools and instrumentation become more advan ced the study of

natural products from all corners of the globe remains an exciting advent ure.

1.2.4.1 . Antarctica . Niche environments in the low to middle latitude seas of the

world are extremely interesting, for here, currents make possible the diverging and converging of

species across the globe. This means, in some instances, that specie s may have an extremely

large distribution and remain relatively similar. Antarctica, however, is an example of an

environment in which this is not true. Upon the splitting of the continent from Australia and

South America some 30 million years ago, the An tarctic Circumpolar current was formed,

effectively cutting the continent and its surrounding waters off from the rest of the globe. These

freezing, isolated waters are now home to organisms that have evolved through pe riods of

fluctuating glaciation and w ithout influence from the currents and winds from the tropics. Those

organisms living on the benthos in this environment - both macro - organisms and their micro -

organism compatriots - have evolved many chemical interactions with one another as a way to

surviv e. These species often exhibit rich biosynthetic potential, and the study of secondary

metabolites produced in this environment is of great interest to many groups - including ours . 9,31 –

The Baker lab Antarctic collection features organisms collected by SCUBA and t rawling in the areas of Palmer S tation and throughout the Scotia Arc (Figure 1 .1 ) over decades of visits to the continent .

Figure 1.1. The Antarctic continent as seen from space on Google Earth with common collection sites for natural products featured. (Image credit: J. L. Fries) 34

In the winter of 2016 - 17, we participated in the Swiss Polar Institute’s Antarctic

Circumnavigation Expedition (ACE) , sampling, in some places for the first time, via Agassiz

trawl, ROV, and intertidal collection s throughout the sub - Antarctic waters and is lands (Figure

1. 2).

Figure 1. 2. The Swiss Polar Institute’s ACE cruise plan, December 2016 - March 2017. ( Imag e Credit: Swiss Polar Institute)

Samples collected during this expedition include bulk material for chemical investigation as well

as tissue samples preserved for the isolation of associated fungi and bacteria. All macro -

organisms collected will be genetically identified at the Western Australia Museum by Dr.

Nerida Wilson. Illustrating an ideal example of an understudied and int eresting niche

environment, our Antarctic investigations are bolstered by strong collaborations that help to

advance our studies of this wildest of marine frontiers.

1.2.4.2 . Florida . Though we travel to the ends of the earth to collect samples, the

Bake r lab calls the University of South Florida, in Tampa, Florida, home. Fortunately, Florida is

home to a number of unique marine environments that are also ideal collection locations for the

isolation of natural products. Florida supports vast mangrove fore sts in both developed and

protected areas (Figure 1. 3). These mangrove communities, as mentioned above, are rather understudied, and are ideal for the collection of endophytic fungi and bacteria.

Figure 1.3. Left panel: Mangroves growing in highly developed intercoastal waterways in Dunedin, FL; Right panel: Mangroves growing in the protected, pristine Everglades National Park, FL.

Florida’s warm waters also support beautiful coral reef communities in the Ke ys and up the west

coast in to the Gulf of Mexico . With just a short boat ride, SCUBA divers from the lab can collect

a vast array of benthic organisms for chemical and microbi al investigation. Convenience is not to

be taken for granted in the choosing of a site from which to collect material for natural products

research, and sometimes you only have to look as far as your own back yard.

1.2.4.3 . Terrestrial and fresh water environments . Nowhere is this mantra better

proven than in chapter 2 of this manus cript. Sedentary and microbial organisms in all

environments - marine, terrestrial, and fresh water - must produce secondary metabolites with

which they can interact with the world around them. Micro - environments, such as fresh water

8 ponds, inner - city forest s, or coastal estuaries, to name a few, may be home to countless organisms that must respond to incredibly localized stressors that make no two environm ents exactly the same. Chapter 2 will tell the fortuitous story of noticing a new organism (a fresh wate r bryozoan) growing in a neighborhood retention pond that resulted in the isolation of a suite of new, bioactive compounds.

Natural products exist all around us, and nature is the supreme synthetic chemist. Even our own human gut microbiome is currently demanding attention for second ary metabolite production and the potential for therapeutic applications. 35 – 37 New chemical structures are emerging all the time from countless environmental sources, and as threats to human health evolve, it could certainly be argued that natural products research is the way forwards.

1.3 . Approaches Towards the Isolation of Microbial Natural Products

1.3.1 . Co llection and I solation

The majority of marine microbes are still considered “unculturable” , however, much work has gone into shrinking that percentage by investigating new collection, isolation, and c ulture techniques . 20,26,38 The fungal library in the Baker lab at the University of South Florida serves as a comprehensive example of microbial isolates obtained using an evolving repertoire of techniques from a variety of sources. Marine organisms such as sponges, tunicates, and coral s are collected either by SCUBA or trawling, and small tissue samples are surface sterilized before being plated on a variety of solid media plates featuring varying chemical and nutrient compositions. Mangrove and mangrove - associated plant material is sim ilarly c ollected and inoculated. Sediment and water column samples are concentrated, or filtered, before being plated on solid media or suspended in liquid media. Collection and isolation techniques are adaptable to

the source organism or targeted isolates . Microbial isolate culture s can be miniaturized and

standardized, a s will be discussed in chapter 3 , for screening purposes, or scaled up and tailored

for titer improvement of compounds of interest . 30 By kee ping a functional archive of all isolates

(stored at - 80°F) this microbial library can be investigated again and again as new culture

techniques and screening targets emerge.

1.3.2 . Culture T echniques

Thanks to genomic advancements, it is clear that micro - organisms cultured in the lab

routinely produce only a fraction of the secondary metabolites that are coded for in their DNA . 39

This is accomplished by t he regulation of transcription by enzymes activated and deactivated

based on environmental factors . 40 I n filamentous fungi, w e know that most secondary metabolite genes are clustered to allow for the most efficient regulation , 40,41 and these clusters can be activated or deactivated by culture conditions, resulting in vastly different metabolite production .

Once isolated, a micro - organism of interest can be cultured in the lab under any number of easily accessible stressors that can chang e secondary metabolite production. Culture variations can be as simple as changing the shape of the culture vessel, or as complex as the addition of biological material from another microbe or host organism . In this way, a single strain can produce a multi tude of different compounds . 39,42 – 45 While this ‘OSMAC’ (‘One Strain Many

Compounds’) 44 strategy is extremely useful in exploiting the full biosynthetic potential of a micro - organism of interest, it is rather intensive in time and consumables.

Rather than systematically changing culture conditions, the biosynthetic potential of a

micro - organism of interest can also be explored through whole genome sequencing. Many

secondary metabolites are products of known biosynthetic pathways. The ability to ascribe a

product to the genes that code for it allows for the unique ability to analyze a whole genome and

predict the metabolites that can be produced. Culture conditions curated to that biosynthetic

pathway can then be employed to isolate specific compo unds of interest . 46 – 51

Genome mining and the OSMAC approach are both useful techniques for the discovery of the biosynthetic potential of a single organism. If, however, you have a microbial library that you would like to screen, these techniques may not be the most efficient. Epigenetic modification - that is, the use of small molecule enzyme inhibitors to promote the expression and

prevent the silencing or downregulation of secondary metabolite gene clusters 52 – 55 - can be u sed

as a more ubiquitous technique to exploit the biosynthetic potential of a larger numb er of micro - organisms. Chapter 3 will discuss the use of histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors 30 as culture additives to epigenetically ‘tur n on’ secondary

metabolite gene clusters in a library of filamentous fungi for the maximum surveying of

bioactive natural product potential therein.

1.4 . Drug Discovery Targets

Natural products isolation efforts largely follow the same generic scheme (Scheme 1).

Efforts aimed at drug discovery can take place at any of the stages, from extraction to pure

compound isolation. There are pros and cons to each approach, though it is ge nerally accepted

that the earlier you can prioritize your efforts, the better.

Crude Extract

Liquid/Liquid Liquid/Liquid Partition Partition

Fraction Fraction

Purified Purified Purified Fraction Fraction fraction

Pure compound Scheme 1 .1 . A generic flow chart for natural product isolation.

Crude extracts can contain thousands of compounds, however, it is possible to get useful

information from that complex mixture in a high - throughput way. Metabolite profiling of crude extracts can be used for initial dereplication and more advanced matabolo mic analysis can reveal chemical outliers that may be of interest. 56,57 High - throughput bioassays that are tolerant of complex mixtures can be used to discover and prioritize activity early in the investigation process. More s ensitive and selective bioassays that are not tolerant of complex mixtures would require more purified fractions or pure compounds. It is important, therefore, when embarking on a natural products screening program, to coordinate bioassay capabilities to isolation protocols, in addition to other target selecti on criteria. The descriptions that follow comprise the panel of targets tha t will be discussed in Chapter 3 . These targets are of great contemporary rele vance to

12 human health concerns and each feature robust bioassay methodologies that assist in early crud e extract level prioritization.

1.4.1 . The ES KAPE P athogens

With growing antibiotic resistance, and a decrease in antibiotic drug discovery, the

Infectious Disease Society of America issued a ‘call to arms’ in 2009 to the drug discovery community to combat what they called the ESKAPE pathogens: the gram positive En terococcus faecium and Staphylococcus aureus, and gram negative Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae . 58 These clinically relevant, highly drug resistant pathogens represent a continuously growing threat to human health and an important target for drug discovery efforts . 59,60

1.4.2 . Leishmania donovani

A neglected tropical disease (NTD), Leishmaniasis is a parasitic infection caused by an intramacrophage protozoa that is transmitted to humans by the bite of infected sandflies. The visceral form of this disease, most commonly caused by Leishmania donovani , is typically fatal when left untre ated. Upon entering the host, the parasite – in a non - flagel l ated amastigote life stage - invades macrophage cells to travel through the body and reproduce . 61 Recent a dvances in infected macrophage in - vitro culture techniques allow for more clinically relevant a ssays to be performed in a high throughput screening (HTS) context . 62 – 64 These advancements will hopefully aid in the discovery of new treatments for this disease in the face of increasing resistance to existing treatments . 65

1.4.3 . Mycobacterium tuberculosis

Tuberculosis (TB) remains a global health crisis, despite the advances of the whole genome sequencing project that revealed the genome of Mycobacterium tuberculosis . This

13 disease, whose latent form is estimated to infect one third of the world ’ s population, poses many drug development hurdles. M ulti - drug resistant (MDR - TB) and extensively drug resistant (XDR -

TB) strains have emerged despite the curr ent course of treatment typically involving combinatorial therapies aimed directly at preventing resistance. Drug discovery efforts, therefore, must address new mechanisms of action or M. tuberculosis targets. Additiona lly, TB drugs have the burden of needing to be compatible in combinatorial treatments for the immunocompromised, particularly those with HIV/AIDS, among whom incidence of this disease are highest . 66 Natural products based drug discovery against this target have revealed promising results, with many existing treatments coming from natural products. With such a demanding target comes the need to screen a broad swath of chemical space, confirming natural products drug discovery efforts as a promising way forward in the search for treatments of this disease . 67

1.4.4 . Clostridium difficile

The leading cause of healthcare related infection, Clostridium difficile is an easily spread, diarrhea causing bacteria that is considered a threat to human health worldwide. The use of antibiotics which upset the human gut microbiome is the primary cause of C. difficile infection

(CDI), but any immuno compromised individuals are at risk. With increasing incidences of resistance, recurrence, and mortality, the need for discovery of new treatments against this bacteria is imperative. Most challengingly, new d rugs to fight CDI must act without impact on the normal human gut fauna . Many novel treatment avenues have been suggested, among which, the discovery and use of bacterial natural products remain of high interest . 68 – 70

1.4.5 . Naegleria fowleri

Naegleria fowleri is a free living, warm - water loving amoeba that causes the nearly always fatal primary amoebic meningoencephalitis (PAM). Diagnosis of PAM is extremely

difficult, and current treatment options are time sensitive and limited to existing drug

combinations (e.g. Amphotericin B, fluconazole, and miltefosine ). 71,72 As clinicians start to

understand and diagnose PAM better, and the risk of this disease continues to rise with

i ncreasing global temperatures, new drugs that specifically target this amoeba are urgently

needed.

1.4.6 . Cancer T argets

With structures as diverse as their targets, n atural products have long played a role in the treatment of various cancers . 1,22,73 As understanding of the complex physiology of human cells

(both healthy and cancerous) continues to grow, assays directed at testing compounds against

highly specifi c cellular targets continue to emerge. Rather than whole cancer - cell assays, these

target specific assays can help to exclude compounds that are broadly cytotoxic to all cells in

favor of compounds that are active within the specific mechanism of action th at is desired.

Autopalmitoylation dysregulation is implicated in many disease states. In a newly

developed assay, palmitoylation of proteins can be monitored for modulation by compounds of

interest in a HTS manner. This allows compounds to be rapidly scr eened not for their effect on

the whole cell, but rather just on this particular pathway of interest. 74

1.5. Future Directions

As I have attempted to illustrate, there are many different techniques available for natural

products drug discovery efforts. While exploring the biosynthetic potential of a single organism

can be very lucrative, screening efforts are needed in order to identify those “lead” organisms.

W ith a robustly designed screening program, natural product extracts from a multitude of source s

can be screened side by side in high - throughput capable bioassays against a wide variety of

disease targets . The resulting diversity of bioactivity information combined with metabolite

profiling can afford intense prioritization of extracts at a very ear ly stage (Scheme 1 .1 ),

streamlining further chemical investigation to a highly time and cost effective level of efficiency.

Focusing on marine fungi, t he remaining chapters in this dissertation will put forth evidence in

support of one such screening initi ative, its successes, lessons, and some recommendations for

improvements moving forward. The work discussed herein will present a number of new,

bioactive microbial natural products that resulted from this initiative. At the conclusion, I hope to

convince the reader that microbial natural products drug discovery can be an efficient, cost

effective endeavor towards combating some of the most difficult challenges facing human health

today.

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Chapter 2

New Bioactive Meroterpenes from Phomopsis sp.

2.1 . BB11 - 2 Isolation

I n the winter of 2011 small colonies of the fresh water bryozoan Pectinatella magnifica were noticed on a morning run in a retention pond located in north Tampa , Florida. These colonial organisms were collected for chemical analysis. While collecting, one of the colonies was found to be growing around the end of a tree branch that was partially submerged in the water. A small piece of this branch was collected along with the bryozoan and returned to the lab for processing. Once in the lab, the branch was processed for microbial isolation (procedure in

Appendix B). From two SDA plates, 7 fungal isolates were o btained based on morphological features. These organisms were given a name of “BB11” for “Baker’s Backyard 2011” and archived as described in Appendix B.

Following chemical i nvestigation of isolate BB11 - 2, fungal id entification was obtained via Sanger seq uencing of the 18S ribosomal spacer region (Appendix A). An NCBI nucleotide blast returned identification as Diaporthe or Phomopsis sp. ; genera that have been determined to be the same but represent diff erent life cycles of these common, 1 chemically rich 2 – 7 endophytic fungi . For purposes of this dissertation, t he isolate will be referred to as Phomopsis sp.

2.2 . Initial Bioactivities and Scale - up

The BB11 fungal isolates were screened in a number of assays and assay development protocols and their extracts were known within the lab to be highly active with low cytotoxicity.

Isolate BB11 - 2 was chosen for its reproducible bioactivity and tolerance towards multiple media types. As a part of a scale - up optimization study, BB11 - 2 was scaled up on rice media in three growth conditions : control (700g o f rice media + 100mL SDB), HDACi (700g of rice media + a

435µM solution of sodium butyrate in 100mL SDB), and DNMTi (700g of rice media + a

435µM solution of 5 - azacytidine in 100mL SDB). After 21 days of growth, each extract was extracted in 1:3 MeOH/ EtOA c solution overnight, followed by 2 subsequent 24 hour extractions in 100% EtOAc. The extracts for each of the three culture conditions were dried down and subjected to a H 2 O: EtOAc partition. The lipophilic partition s were each separated on NP MPLC

(Schem e 2.1) . The fractions of the control extract were further investigated.

Phomopsis sp. Control Crude Extract 50g

H2O partition EtOAc partition 25g 25g

NP MPLC: (Hexanes  Ethyl Acetate  Methanol)

A B C D E F G H

NP MPLC: (Hexanes  Ethyl Acetate)

A B C D E F

Compounds 1 - 6

Scheme 2.1. Extraction scheme for Phomopsis sp. Control.

2.3. Compound Isolation and Structure Elucidation

The extract of the c ontrol culture condition of Phomopsis sp. was investigated by NMR and bioactivity guided fractionation (Scheme 2.1) and yielded 5 new meroterpenes: phomopsichromins A - E ( 1 - 5 ), and the known compound LL - Z1272ε ( 6 ) 8 (Figure 2.1).

1 2

3 4

5 6

Figure 2.1. Chemical structures of new compounds 1 - 5 and known compound 6 .

In the first MPLC separation of the lipophilic partition of the Control extract, the majority of the mass eluted in one large peak in approximately 1:1 n - hexanes: ethyl acetate. This fraction

(Fraction D) was further purified via NP MPLC on an extended non - polar gradient ( n - hexanes to ethyl acetate). Again, the majority of the mass elu ted in a single peak in approximately 1:1 n -

hexanes: ethyl acetate (Fraction C). Further purification was accomplished on NP HPLC using a

26 cyano column (CN capping of the silica particles) and UV detection. Compounds 1 - 6 (Figure

2.1) were isolated as illustrated in Figure 2.2 .

2 1 5 6 4

Figure 2.2 . NP HPLC chromatogram of fraction D_C (Scheme 2.1) from the control growth treatment of Phomopsis sp. Peaks are annotated with their isolated compounds (Figure 2.1) . Each compound required some further purification on reverse phase (RP) HPLC for structure elucidation and bioassay purposes, but represented the major component of each of the peaks seen. Black is the UV chromatogram at 320nm, and blue is the ELSD trace.

Compounds 1 - 6 all share a se s quiterpene backbone functionalized diff erentially at C - 9 . 1 - 3 share a chromene substructure while compounds 4 - 6 feat ure a ring - opened subunit . The

structures of Phomopsichromins A - E ( 1 - 5 ) were elucidated as described below. NMR spectra,

IR, UV, and optical rotation data for all compounds can be found in Appendix A.

Table 2.1. 1D and 2D data for phomopsichromin A ( 1 ) in CDCl 3 . b a c Pos δ C δ H (m, J (Hz)) HMBC 1 50.4 2.41 (q, 6.6 x 2 , 1 H) 2, 5, 6, 7, 12, 14 2 213.8 3 41.5 2.33 (m, 1 H) 2, 4, 5 2.36 (m, 1 H) 4 30.8 1.64 (m, 1 H) 3, 5, 6, 13 1.85 (m, 1 H) 2, 3, 5, 6 5 36.1 1.97 (m, 1 H) 1, 3, 4, 6, 13 6 43.2 7 30.6 1.41 (m, 1 H) 1, 5, 6, 8, 14 1.48 (m, 1 H) 1, 5, 6, 8, 14 8 34.3 1.52 (m, 1 H) 7, 9, 10 1.77 (m, 1 H) 7, 15, 9, 10 9 79.89 10 125.8 5.46 (d, 10.2 , 1 H) 8, 9, 15, 16, 21 11 116.9 6.76 (d, 10.2 , 1 H) 9, 15, 16, 17, 21 12 7.5 0.91 (d, 6.8 , 3 H) 1, 2, 6 13 14.9 0.89 (d, 6.8 , 3 H) 4, 5, 6 14 15.4 0.59 (s, 3 H) 1, 5, 6, 7 15 27 1.44 (s, 3 H) 8, 9, 10, 11 16 106.8 17 160.6 - OH 11.74 (s, 1 H) 16, 17, 18, 21 18 103.6 19 144.4 20 111.9 6.24 (s, 1 H) 10, 11, 16, 18, 21, 22, 23 21 158.6 22 24 2.55 (s, 3 H) 17, 18, 19, 20, 21, 23 23 175.1 a 1 H NMR recorded at 500 MHz, reported in ppm (multiplicity, J - coupling in Hz, integration); b 13 C NMR recorded at 125 MHz; c recorded from a gHMBCAD experiment at 500 MHz and reported as positions of carbons.

20 1 Phomopsichromin A ( 1 ) , [α] D +1.2 , was isolated as a white powder. The H NMR spectrum of 1 (Table 2.1) notably displayed 3 olefinic protons, peaks for 5 methyl substituents,

13 and a phenol. The C NMR spectrum indicated the presence of a carboxylic acid (δ C 175.18) 28

13 and ketone (δ C 213.87). Remaining C signals were split between the aromatic and alkene

regions and implied a high level of substitution on the aromatic ring (as evidenced by the small

number of olefinic protons in the 1 H NMR spectrum ). The chromene substructure was tentatively

assigned based on 2D NMR experiments and 13 C ppm shifts of carbons 9 and 21. The remaining

terpene scaffold was completed based on HMBC and COSY NMR data (Figure 2.3) . HR ESI MS

of 1 ( m/z 387 .2137 [M + H] + ) resemble d compounds in the literature 8 – 12 but did not match any

exactly, conf irming that 1 was a new compound. Using the NMR data from these related

compounds, the chromene substructure was conf irmed. Relative stereochemical assignmen ts at

methyl - bearing carbons 1 ( R ), 5 ( R ), 6 ( S ), and 9 ( S ) were assigned via 1 and 2D NOE experime nts (Figure 2.3) and comparison to the literature 11 – 13 . ECD will be used to confirm absolute stereochemistry .

Figure 2.3. Important COSY, HMBC, and NOE correlations in phomopsichromin A ( 1 ).

Table 2.2. 1D and 2D data for phomopsichromin B ( 2 ) in CDCl 3 . b a c Pos δ C δ H (m, J (Hz)) HMBC 1 39.4 1.43 (m , 1H) 2, 6, 12, 14 2 73.1 3.85 (q, 2.8 , 1 H) 3, 4, 6, 12 3 33.9 1.55 (m, 1 H) 1, 5 1.8 (m, 1 H) 1, 2, 4, 5 4 25.4 1.27 (m, 1 H) 1.63 (m, 1 H) 3, 5 5 36.5 1.46 (m, 1 H) 6, 7, 14 6 38.2 7 30.9 1.35 (m, 2 H) 5, 6, 8, 14 8 34.4 1.45 (m, 1 H) 6, 7, 9 1.63 (m, 1 H) 6, 7, 9, 10, 15 9 80.1 10 126.2 5.46 (d, 10.2 , 1 H) 8, 9, 15, 16 11 116.6 6.74 (d, 10.2 , 1 H) 9, 16, 17, 21 12 12.3 0.95 (d, 7.1 , 3 H) 1, 2, 6 13 15.6 0.82 (d, 6.6 , 3 H) 4, 5, 6 14 17.3 0.86 (s, 3 H) 7, 6, 5 15 27 .1 1.41 (s, 3 H) 8, 9, 10 16 106.9 17 160.6 - OH 11.80 (s, 1 H) 16, 17, 18 18 103.4 19 144.1 20 111.9 6.22 (s, 1 H) 16, 18, 21, 22 21 158.7 22 24 .4 2.53 (s, 3 H) 18, 19, 20 23 174.7 a 1 H NMR recorded at 600 MHz, reported in ppm (multiplicity, J - coupling in Hz, integration); b 13 C NMR recorded at 125 MHz; c recorded from a gHMBCAD experiment at 500 MHz and reported as positions of carbons.

Phomopsichromin B ( 2 ) was isolated as a white powder, C 23 H 32 O 5 , HR ESI MS m/z

+ 20 389.2322 [M + H] (calculated, 389.2283), [α] D - 1.5. Comparison to the HRMS of 1 indicated

a difference of 2 protons. The appearance of a broad multiplet at δ H 3.85 and the loss of the

ketone signal at δ C 213.87 inferred a reduction a t C - 2. The 2D NMR spectra fur ther supported

this assignment (Figure 2.4). The stereochemistry at position 2 was determined to be S based on

the multiplicity observed in the proton NMR spectrum . The observed J - values of the quartet at

δ H 3.85 most closely matched those expected from the axial orientation of the hydroxyl group.

This orientation could a lso be observed in other related compounds in the literature. 14 The remaining chiral centers were determined to be the same as in 1 . Again, the stereochemistry will be confirmed via ECD.

Figure 2.4. Important COSY, HMBC, and NOE correlations in phomopsichromin B ( 2 ).

Curiously, in multiple 1D NMR analyses of 2 over time, notable shifts in ppm were observed for some peaks (e.g. δ H 6.22, 3.85 , and 2.53; δ C 73.17, 103.46, 158.73, and 174.72).

The assumption was made that there were diastereo iso mers present (further supported by some poorly resolved crystal data), however, various NP and RP HPLC attempts eluted a single peak in all conditions. The beginning, middle, and end of the peak were collected as separate fractions

(a, b, and c) in an attempt to separate the diastereo iso mers . Clear ppm differences were observed between fractions a and c , but they were chromatographically indistinguishable . The decision was made to methylate the carboxylic acid at C - 23 in fractions a and c t o reduce the effects of hydrogen bonding in separation attempts and aid in NMR spectra l evaluation (Figure 2.5) .

Figure 2.5. Methylation reaction of 2 (stereochemistry unknown at the time) to yield 7 with diazomethane.

The resulting methyl derivative s of fractions a and c ( 7 ) were confirmed by 1D NMR (Appendix

A). A ll ppm differences between fractions a and c were lost, indica ting that all previously noted shifts in ppm had been a concentration - based artifact of hydrogen bonding of 2 to itself in solution . 2 was determi ned to be a single diastereo iso mer and remaining chemical and biological analyses were completed (Appendix A) .

20 1 Phomopsichromin C ( 3 ) was isol ated as a white powder, [α] D +1.3. H NMR data resembled 1 and 2 in the chromene region, however, the methyl signals at position 12, 13, and 14 were notably shifted downfield and were all overlapping (Table 2.3). New peaks at δ C 170.87

1 and 21.4, with a new methyl signal in the H spectrum (δ H 2.05) indicated added ester functionalization in the molecule . HRESIMS m/z 431.2434 [M + H] + (calculated, 431.2389) gave a molecular formula of C 25 H 34 O 6 , confirming the addition o f a – CO(O)CH 3 group . The available

2D NMR data confirmed that the new functionalization was at C - 2 (δ C 75.16) (Figure 2.6) .

Table 2.3. 1D and 2D data for phomopsichromin C ( 3 ) in CDCl 3 . b a c Pos δ C δ H (m, J (Hz)) HMBC 1 38.5 1.55 (m, 1 H) 6, 12, 14 2 75.1 4.97 (q, 2.0 , 1 H) 1 3 30.9 1.5 (m, 1 H) 1, 2, 4, 5 1.81 (m, 1 H) 4 25.8 1.29 (m, 1 H) 2, 3, 5, 6, 13 1.5 (m, 1 H) 5 36.2 1.47 (m, 1 H) 3, 4, 6, 14 6 38.3 7 30.7 1.4 (m, 2H) 1, 5, 8, 9, 14 8 34.4 1.4 (m, 1H) 7, 9, 10, 11, 15 1.65 (m, 1 H) 9 80.1 10 126.1 5.45 (d, 10.2 , 1 H) 8, 9, 15, 16, 21 11 116.7 6.74 (d, 10. 0 , 1 H) 9, 15 - 17, 21 12 12.0 0.83 (m , 3 H) 2 13 15.6 0.83 (m, 3 H) 4 14 16.7 0.83 (m , 3 H) 1, 5, 7, 8 15 27.1 1.4 (s, 3 H) 8 - 11 16 106.8 17 160. 6 - OH 11.82 (s, 1 H) 16, 17, 18, 21 18 103.3 19 144.1 20 111.9 6.22 (s, 1 H) 11, 16 - 18, 21 - 23 21 158.7 22 24.4 2.53 (s, 3 H) 16 - 20, 23 23 174.4 24 170.8 25 21.4 2.05 (s, 3 H) 2, 24 a 1 H NMR recorded at 600 MHz, reported in ppm (multiplicity, J - coupling in Hz, integration); b 13 C NMR recorded at 125 MHz; c recorded from a gHMBCAD experiment at 500 MHz and reported as positions of carbons.

Figure 2.6. Important COSY and HMBC correlations in phomopsichromin C ( 3 ).

Due to the overlapping signals of the methyl signals on the cyclohexane ring of 3 , elucidating the stereochemistry at C - 1, 2, 5, and 6 based on NMR data posed a significant challenge. The methyl group at C - 9 was assumed, as in compounds 1 and 2 , to be S based on ch emical shift . The stereochemistry at C - 2 was again determined to be S based on the 1D 1 H multiplicity. Figure 2.7 illustrates the proposed biosynthetic pathway for the cyclohexane substructure of 1 - 5 based on work done by Tanabe and Suzuki in 1974 on a related compound. 13 This pathway indicates that the ketone at C - 2 is a part of the precursor molecule, and therefore all reductions at C - 2 occur post translationally. This suggest s a conserved chirality at the centers at C - 1, C - 5, and C - 6.

Figure 2.7. Proposed biosynthetic pathway towards the cyclohexane substructure of 1 - 6 from Tanabe and Suzuki, 1974.

Based on these assumptions, a compound with proposed s tereochemistry (Figure 2.8) was submitted for ECD.

Fi gure 2.8 . Proposed stereocenters in the cyclohexane ring of 3 sent for ECD calculations.

+ Phomopsichromin D ( 4 , C 23 H 34 O 5 , HRESIMS m/z 391 .2473 [M + H] (calculated, 391.2440),

20 [α] D +0.2) was isolated as a white powder from the last and least lipophilic HPLC fraction of

MPLC fraction D_C (Figure 2.2). 4 was notably distinct from 1 - 3 in that it was quite different in polarity (not soluble in CDCl 3 ) and so all NMR data was obtained in DMSO - d 6 (Table 2.4). The olefin region of the proton NMR spectrum of 4 was the most drastically different from compounds 1 - 3 and there was an additional phenol signal at δ H 10.02, indicating that 4 lacked the completed chromene substructure.

Table 2.4. 1D and 2D data for phomopsichromin D ( 4 ) in DMSO - d 6 . b a c Pos δ C δ H (m, J (Hz)) HMBC 1 39.0 1.35 (m, 1 H) 2, 5, 6, 12, 14 2 70.4 3.62 (q, 2.4 , 1 H) 4, 6, 12 3 34.1 1.4 (m, 1 H) 4 1.6 (m, 1 H) 4 4 25.5 1.13 (m, 1 H) 1, 3, 5, 6, 14 1.53 (m, 1H) 5 36.0 1.4 (m, 1 H) 4 6 38.0 7 36.0 1.17 (m, 1 H) 1, 5, 6, 8, 9, 14 1.23 (m, 1 H) 5, 6, 8, 9, 14 8 32.4 1.69 (m, 1 H) 5, 15 1.75 (m, 1 H) 5, 10, 11, 15 9 134.7 10 121.8 5.12 (br t, 6.9 , 1 H) 8, 11, 15, 16 11 21.5 3.16 (br d, 7.2 , 2 H) 7, 8, 9, 10, 15, 16, 17, 21 12 12.7 0.84 (d, 7.2 , 3 H) 1, 2, 6 13 15 .7 0.74 (d, 6.6 , 3 H) 4 14 17.3 0.75 (s, 3 H) 1, 6, 7 15 16.1 1.70 (s, 3 H) 7, 8, 9, 10, 11, 16 16 112.0 17 159.6 - OH 12.61 (s, 1 H) 16, 17, 18, 21 18 103.6 19 139.8 20 110.4 6.23 (s, 1 H) 11, 16, 17, 18, 21, 22, 23 21 162.8 - OH 10.02 (s, 1 H) 16, 17, 20, 21 22 23.8 2.38 (s, 3 H) 18, 19, 20, 21, 23 23 174.1 a 1 H NMR recorded at 500 MHz, reported in ppm (multiplicity, J - coupling in Hz, integration); b 13 C NMR recorded at 125 MHz; c recorded from a gHMBCAD experiment at 500 MHz and reported as positions of carbons.

2D NMR data (Figure 2.9 ) further supported this, and the “open” carbon skeleton was assigned and confirmed by comparison to literature data on known compound 6 . Stereochemistry on the cyclohexane ring was set as in compounds 1 - 3 . The double bond of the open chain was determined t o be E based on the comparison of the J - value of H - 10 to the literature.

Figure 2.9 . Important COSY, HMBC, and NOE correlations in phomopsichromin D ( 4 ) .

Phomopsichromin E ( 5 ) was isolated as a white powder, C 23 H 32 O 5 ; HRESIMS m/z 389.2363 [M

+ 20 + H] , [α] D - 0.4. By comparing 5 to 4 it was immediately evident that the only difference

between the two was the increased oxidation at C - 2 . Comparison to 1 and 4 , along with the 2D

data for 5 (Table 2.5) , completed the struc ture (Figure 2.10 ).

Figure 2.10 . Important COSY and HMBC correlations in phomopsichromin E ( 5 ).

Table 2.5. 1D and 2D data for phomopsichromin E ( 5 ) in DMSO - d 6 . b a c Pos δ C δ H (m, J (Hz)) HMBC 1 49.2 2.53 (q , 6.7, 1 H ) 2, 5, 6, 12, 14 2 212.6 3 40.7 2.1 (m , 1 H ) 1, 2, 4, 5 2.4 (m , 1 H ) 4 30.3 1.47 (m , 1 H ) 3, 5, 13 1.76 (m , 1 H ) 6 5 35.0 1.99 (m , 1 H ) 3, 4, 6, 13, 14 6 42.7 7 35.4 1.23 (m , 1 H) 5, 6, 8, 14 1.32 (m, 1 H) 1, 6, 8, 9, 14 8 32.0 1.8 (m , 1 H ) 7, 9, 10, 15 1.91 (m , 1 H ) 7, 9, 10, 15 9 134. 2 10 122.2 5.18 (br t, 6.8 , 1 H) 8, 11, 15, 16 11 21.5 3.17 ( d, 7.1 , 2 H) 9, 10, 16, 17, 21 12 7.5 0.76 (d, 6.6 , 3 H) 1, 2, 6, 7 13 14.7 0.81 (d, 6.6 , 3 H) 4, 5, 6, 12 14 15.0 0.46 (s, 3 H) 1, 5, 6 15 16.1 1.73 (s , 3 H ) 8, 9, 10, 16 16 111.9 17 159.6 - OH 12.62 (s, 1 H) 18 103.6 19 139.9 20 110.4 6.23 (s, 1 H) 16, 17, 18, 22 21 162.8 - OH 10.04 (s, 1 H) 16, 17, 20, 21 22 23.7 2.39 (s , 3 H ) 18, 19, 20, 23 174.1 a 1 H NMR recorded at 600 MHz, reported in ppm (multiplicity, J - coupling in Hz, integration); b 13 C NMR recorded at 200 MHz; c recorded from a gHMBCAD experiment at 600 MHz and reported as positions of carbons.

2.4 . Bioactivities

Phomopsichromins A ( 1 ), B ( 2 ), C ( 3 ), and E ( 5 ) all show reasonable activity against the infected macrophage model of the Leishmania donovani parasite at 3, 1.9, 0.67, and 0.80 µM, respectively. Interestingly, neither phomopsichromin D ( 4 ) nor LL - Z1272ε ( 6 ) showed any activity tow ards the parasite or the macrophages.

Phomopsichromin C ( 3 ) was the only compound with any notable activity against any of

the ESKAPE pathogens, with an MIC against MRSA of 58 µM. In a drug wash - out study, 3 was determined to be bactericidal. Impressively, this compound exhibits an MBC 99 of 47 µM, which

is reasonably close to the control gentamicin (20 µM in this assay). However, 3 also displays

notable cytotoxicity towards HepG4 human liver cells, with an LD 50 of 37 µM . Further

supporting its cytotoxicity and lack of specificity, 3 is also active against the macrophage

contained Leishmania donovani as mentioned above.

1 , 2 , and 5 all displayed activity against MRSA at extremely high concentrations (> 500

µM ) and further cytotoxicity testing may be useful. If these compounds continue to be selective against the Leishmania donovani parasite, further biological testing against that, and other parasite targets would be warranted.

While none of the phomopsichrom ins immediately emerge as promising lead compounds

against ESKAPE or Leishmania donovani , with a group of compounds of this size, and

interesting “ natural structure activity relationship (SAR) study” is beginning to take form. It is

notable that 4 remains inactive, despite being nothing more than the ring - open form of 2 .

Meanwhile, 1 and its ring opened form, 5 , display similar bioactivities. While six compounds is hardly enough to constitute a true SAR study, on e can imagine that with the isolation (or synthesis) of more of these analogs, a pharmacophore may well begin to emerge. A fungal source is ideal for such studies as more material of these, and any other discovered compounds, could be obtained quite easily and in high quantities.

2.5 . Future Directions

Our Phomopsis sp. isolate proved to be a producer of new, bioactiv e chemistry, and I

believe, may still have biosynthetic potential remaining to be discovered. All six compounds

discussed herein were i solated from the extract of the control growth condition . T he extracts

from the modified growth conditions remain to be explored. Additionally, NMR data suggests

that there may be more phomopsichromins (or other known or new derivatives) as minor

component s of the investigated fractions that were abandoned due to time constraints. This strain

remains archived in the Baker l ab fungal library, along with the other 6 isolates from the branch

piece . These organisms are all known to produce bioactive extracts and would all be interesting

targets for further chemical analysis and growth culture optimization.

This genera is known to have great biosynthetic potential and despite being such a well - studied organism, environmental strains such as the one investigat ed here continue to be producers of new compounds. I believe that the work discussed her e in stands as another layer of support for environmental microbial isolates as a promising source of new chemistry for drug discovery.

2.6 . References

(1) Gomes, R. R.; Glienke, C.; Videira, S. I. R.; Lombard, L.; Groenewald, J. Z.; Crous, P. W. Diaporthe: A Genus of Endophytic, Saprobic and Plant Pathogenic Fungi. Persoonia Mol. Phylogeny Evol. Fungi 2013 , 31 , 1 – 41 DOI: 10.3767/003158513x666844. (2) Dai, J.; Krohn, K.; Flörke, U.; Gehle, D.; Aust, H. J.; Draeger, S.; Schulz, B.; Rheinheimer, J. Novel Highly Substituted Biraryl Ethers, Phomosines D - G, Isolated from the Endophytic Fungus Phomopsis Sp. from Adenocarpus Foli olosus. European J. Org. Chem. 2005 , 4 (23), 5100 – 5105 DOI: 10.1002/ejoc.200500471. (3) Kobayashi, H.; Meguro, S.; Yoshimoto, T.; Namikoshi, M. Absolute Structure, Biosynthesis, and Anti - Microtubule Activity of Phomopsidin, Isolated from a Marine - Derived Fungus Phomopsis Sp. Tetrahedron 2003 , 59 (4), 455 – 459 DOI: 10.1016/S0040 - 4020(02)01566 - 1. (4) Isaka, M.; Jaturapat, A.; Rukseree, K.; Danwisetkanjana, K.; Tanticharoen, M.;

Thebtaranonth, Y. Phomoxanthones A and B, Novel Xanthone Dimers from the Endophyt ic Fungus Phomopsis Species. J. Nat. Prod. 2001 , 64 (8), 1015 – 1018 DOI: 10.1021/np010006h. (5) Tang, J. W.; Wang, W. G.; Li, A.; Yan, B. C.; Chen, R.; Li, X. N.; Du, X.; Sun, H. D.; Pu, J. X. Polyketides from the Endophytic Fungus Phomopsis Sp. sh917 by U sing the One Strain/many Compounds Strategy. Tetrahedron 2016 , 10 , 2 – 9 DOI: 10.1016/j.tet.2017.02.019. (6) Lin, X.; Huang, Y.; Fang, M.; Wang, J.; Zheng, Z.; Su, W. Cytotoxic and Antimicrobial Metabolites from Marine Lignicolous Fungi, Diaporthe Sp. FEMS Microbiol. Lett. 2005 , 251 (1), 53 – 58 DOI: 10.1016/j.femsle.2005.07.025. (7) Dettrakul, S.; Kittakoop, P.; Isaka, M.; Nopichai, S.; Suyarnsestakorn, C.; Tanticharoen, M.; Thebtaranonth, Y. Antimycobacterial Pimarane Diterpenes from the Fungus Diaporthe Sp . Bioorganic Med. Chem. Lett. 2003 , 13 (7), 1253 – 1255 DOI: 10.1016/S0960 - 894X(03)00111 - 2. (8) Ellestad, G. A.; Evans, R. H.; Kunstmann, M. P. Some New Terpenoid Metabolites from an Unidentified Fusarium Species. Tetrahedron 1969 , 25 (6), 1323 – 1334 DOI: 10 .1016/S0040 - 4020(01)82703 - 4. (9) Singh, S. B.; Zink, D. L.; Bills, G. F.; Jenkins, R. G.; Silverman, K. C.; Lingham, R. B. Cylindrol A: A Novel Inhibitor of Ras Farnesyl - Protein Transferase from Cylindrocarpon Lucidum. Tetrahedron Lett. 1995 , 36 (28), 4935 – 4938 DOI: 10.1016/0040 - 4039(95)00896 - K. (10) Singh, S. B.; Ball, R. G.; Bills, G. F.; Cascales, C.; Gibbs, J. B.; Goetz, M. A.; Hoogsteen, K.; Jenkins, R. G.; Liesch, J. M.; Lingham, R. B.; Silverman, K. C.; Zink, D. L. Chemistry and Biology of Cylindrols: Novel Inhibitors of Ras Farnesyl - Protein Tra nsferase from Cylindrocarpon Lucidum. J. Org. Chem. 1996 , 61 (22), 7727 – 7737 DOI: 10.1021/jo961074p. (11) Ishibashi, M.; Ohizumi, Y.; Cheng, J.; Nakamura, H.; Sasaki, T.; Kobayashi, J. Metachromins A and B, Novel Antineoplastic Sesquiterpenoids from the O kinawan Sponge Hippospongia Cf. Metachromia. J. Org. Chem. 1988 , 53 (12), 2855 – 2858. (12) Takahashi, Y.; Yamada, M.; Kubota, T.; Fromont, J.; Kobayashi, J. Metachromins R -- T, New Sesquiterpenoids from Marine Sponge Spongia Sp. Chem. Pharm. Bull. (Tokyo). 2007 , 55 (12), 1731 – 1733 DOI: 10.1248/cpb.55.1731. (13) Tanabe, M.; Suzuki, K. Detection of C - C Bond Fission during the Biosynthesis of the Fungal Triprenylphenol Ascochlorin Using [ 1,2 - 13C] - Acetate. J.C.S. Chem. Comm. 1974 , No. 1, 445 – 446. (14) Biebe r, L. W.; Messana, I.; Lins, S. C.; Da Silva Filho, A. A.; Chiappeta, A. A.; De Mello, J. F. Meroterpenioid Naphthoquinones from Cordia Corymbosa. Phytochemistry 1990 , 29 (6), 1955 – 1959.

Chapter 3

The Creation of a Large Fungal Isolate and Extract Library

3 .1 . Curating a Fungal Library

The Baker l ab is currently home to over 7 000 microbial isolates. With a focus on

filamentous fungi, this library includes micro - organisms isolated from marine and terrestrial environments in Florida and Antarctica. The isolation techniques employed to build this library

have evolved over time, inspired by the work of colleagues, techniques found in the literature , 1 – 3

and the findings of various pr otocol optimization experiments . Isolation technique s vary

according to source and isolate targets, but can be described by the following general protocol : 1)

tissue surface sterilization with a 10% bleach solution and/or isopropyl alcohol; 2) tissue

subsa mpling into 1 cm cross - sections; 3) plating in triplicate onto solid media plates of variable

com position; 4) careful monitoring of colony growth on solid media plates for a period of 1 - 4

weeks in the lab ; and 5) transferring individual colonies to new isolation plates of similar

comp osition . The last step is repeated until a pure colony is established for each isolate.

Variations in solid media plate composition are employed to target a wide range of fungi

and bacteria, and all tissue samples collecte d are plated ac ross 6 - 10 different media types to

access the microbial diversity tha t can be found living within each target organism . 2 Each plate

type is designed with the following general ingredients in varying concentrations: a nutrient

source, agar, salt, and small molecule antibacterial and antifungal agents in sub - lethal doses to

discourage the growth of fast - growing organisms . Available nutrient mixtures include : Sabaraud

Dextrose Broth (SDB), Potato Dextrose Broth (PDB), Tryptic Soy Broth (TSB), Malt Broth,

Actino Agar, and glycerol. S mall molecule additiv es include : nystatin, cycloheximide, and

chloramphenicol. Solid media types are curated for each collection expedition based on source

and microbial targets.

Collection locations and source organisms represented in the isolate library are varied.

Some exa mples of organisms commonly sampled for microbial iso lation include: Floridian man groves ( Rhizophora mangle, Avicennia germinans , and Laguncularia racemose ) , mangrove associated trees ( Conocarpus erectus , and Coccoloba uvifera ), and benthic marine invertebrates including s ponges, tunicates, and corals. Collection d ate, location, and source organism identification is all carefully reco rded in field notebooks during each expedition .

To accommodate different field conditions, two different collection techniques have been deve loped. The first technique is field plating, in which tissue samples are surface sterilized and directly plated on location on to solid media plates (“field plates”) , which are then transferred back to the lab for monitoring of growth . The second technique is glycerol cryotube preservation, in which tissue samples are surface sterilized, frozen and stored for transit in a 20% glycerol solution in cryotubes before being plated in the lab . This allows for microbia l collections to take place around the world, preserving tissues and micro - organisms until they can be processed in the lab. Table 3.1 illustrates collection details for all collection trips by the Baker l ab between

2011 and 2017.

Table 3.1 . Collectio n details for collection trips from Fall 2011 to Spring 2017. Target Target Collection Season, Year Location Source Micro - Technique Organism(s) Organism(s) Spring, 2012 Everglades National Park , FL MG F, B FP Summer, 2012 Keys Marine Lab , FL MG, MA F, B, A, M FP Summer, R.V. Bellows, Dry Tortugas MA F, B FP 2011, 2013 National Park , FL Inter - coastal Islands, Fall, 2013 MG F FP Dunedin, FL Tampa Bay, St. Petersburg , Spring, 2014 MG F FP FL Summer, 2014 Keys Marine Lab, FL MG, MA F FP Summer, Inter - coastal Islands, 2014 - MG F, B L Dunedin, FL Fall, 2015 R.V. Weatherbird, Gulf of Summer, 2015 MA F, B GC Mexico Spring 2016 Palmer Station, Antarctica MA F, B GC Fall 2016 - R.V. Akademik Troyshnikov, MA F, B GC Spring 2017 ACE, Antarctica Spring, 2017 Palmer Station, Antarctica MA F, B GC MG= mangrove and mangrove associate plant material; MA= benthic marine macro - organisms (ie, sponges, tunicates, corals); F= fungi; B= bacteria; A= actinobacteria; M=myxobacteria; FP= field plating; GC= glycerol cryotube preservation; L= whole tissue samples returned to lab (not frozen) to follow isolation protocol (utilized for sampling sites close to the lab).

After establishing a pure isolate , a ll micro - organisms are archived in a 20% glycerol solution at - 80 °C. Bacterial cells are suspended in the solution, while fungal material is st ored as small cubes of growth cut from SDA isolation plates . Isolates are identified according to the following nomenclature: “Collection Location, Year - Source Organism Number - Isolation Plate

Type - Isolate Number”. In this way, isolates can easily be grouped and retrieved according to any one of the collection o r isolation parameters. Nearly 75% of the existing fungal isolates in this library were subjected to an epigenetics bas ed high throughput screening (HTS) project.

3 .2 . Screening Protocols, Accomplishments

In accordance with two NIH R21 grants (NIH AI103673 and AI103715) a high throughput, epigenetics based fungal screening program was designed. A culture miniaturizatio n and modification protocol was developed to accommodate the use of 20mL scintillation vials and a brown ri ce media. The histone deacetylas e (HDAC) inhibitor sodium butyrate, and the DNA methyltransferase (DNMT) inhibitor 5 - azacytidine were employed at concentration s of 100µ M for epigenetic modification. 3 With a target of screening 500 organisms in 3 growth conditions

(Control, HDACi, and DNMTi) each month f or 12 months, goal - oriented timelines and protocols

(Table 3 .2) were developed and put in place. Screening was completed with full - time staffing by

2 graduate and approximately 20 undergraduate students. Training s and proper protocol regulation s were employed to ensure uniformity in the extracts generated b y the program. A full methodology for the culture , extraction and related proc edure s can be found in Appendix B . By implementing the developed protocols, a library of nearly 10,000 fungal extracts was produced over a time period of 18 months. At the height of productivity, 1,500 extracts were being produced and screened each month.

Table 3.2. Weekly sequence of events for continuous production of fungal isolates and extract libraries. Monday Tuesday Wednesday Thursday Friday

□ Make rice media ≥ □ Harvest, start □ Decant □ Weigh □ Get hit report, 4 boxes (400 vials) extraction of 21 extracts extracts slant & day old samples organize hits □ Make isolation □ Dry extracts □ Solvate plates (types A,B,D □ Inoculate rice under air extr acts in □ Plate extracts & TSA =1L; C & ≥ 4 boxes (400 DMSO for bioassay and SDA ≥ 2L) vials) □ Weigh storage (CDDI) samples □ Create plate □ Make 200mL x 3 □ Wash vials maps □ Wash vials SDB for Eppendorf □ Pre - weigh vials □ Wash vials □ Isolate/ □ Make glycerol describe/ cryotubes □ Wash vials □ Isolate/ archive fungi describe/ □ fill eppendorfs □ Isolate/ archive fungi (SDB, HDAC, describe/ DNMT) archive fungi

□ Wash vials

□ Isolate/ describe/ archive fungi

The resulting extract library was dispersed for screening and archived in DMSO at a concentration of 10mg/mL in 96 - well plate format . Beyond the scope of the original two gran t funded screening targets (the ESKAPE pathogens and Leishmania donovani ), extra cts have been distributed for screening in a number of additional targets, resulting in a body of data that can inform high level prioritization of active extracts. Additional screening is ongoing, but extract data to date includes bioactivities against th e ESKAPE pathogens, Leishmania donovani , J774 macrophage cells, Mycobacterium tuberculosis , Clostridium difficile , and Naegleria fowleri . The

46 library was additionally screened against a number of cell - based cancer targets and in a yeast based multiplex ass ay for anti - helminthics. Some active extracts were investigated by metabolomic analysis.

3 .3 . Metabolomic Analysis

Extracts from a subset of organisms producing bioactivities were subjected to metabolomics analysis to investigate and confirm chemical diversity of the extract library and effectiveness of epigenetic modification techniques. Bioassay results showed desirable distribution of active extracts among the three culture conditions, and in each assay, organisms displaying activity only after modi fication accounted for 15 - 30% of all activity observed (Figure

3 .1).

Active Control Organisms HDAC Modified Only DNMT Total Total Screened Screened a b Figure 3 .1 . a) Representative pie chart from bioassay data, in this case, ESKAPE activity, that shows the activity boosting effects of the epigenetic modification; b) Representative pie chart from bioassay data, in this case, ESKAPE activity, that demonstrates distri bution of activity across the three treatment conditions. This is the overall trend amongst all testing completed to date.

To support and further investigate this diversity of activity, 123 extracts from 41 active organisms were analyzed via LC - Q ToF - MS. This subset of samples included active and non - active extracts (for instance, in the case that an organism only displayed activity after

modification, the control extract was still included in the analysis). It included a wide range of

isolates from diffe rent collections, isolation media , and source organisms. The LC - Q ToF - MS data were summarized into a list of ‘chemical entities’ (HR ESI MS @ retention time) for each sample using Agilent Qualitative Analysis and Mass Profiler Professional software (full

prot ocols in Appendix B) . Each sample, identified by its list of chemical entities, could then be

statistically compared using Multidimensional Scaling (MDS) . Each sample was also identified

by a number of additional factors, including biological activity, cul ture treatment, and isolate

identity . Using Primer 6 software, MDS plots could be generated and annotated with any of these

factors. This analysis allowed for a visual representation of the chemical similarity of the isolates

and extracts to one another, a nd verified the chemical diversity of both the fungal library as well

as the different culture treatments (Figure 3.2 and 3.3).

Figure 3.2. An MDS plot of the Bray Curtis Similarity matrix of square root transformed data for 1 23 extracts (replicates averaged) from the screening program . Chemically unique samples (i.e. sample 93 in the top right quadrant) are clearly visible.

Figure 3.3. An MDS plot of the Bray Curtis Similarity matrix of square root transformed data for 9 randomly sel ected organisms from those represented in Figure 3.2 . Each organism is denoted by an individual color, while each culture treatment is represented by a shape (control = triangle, HDAC = circle, DNMT = square). Full shapes represent extracts with ESKAPE activity, empty shapes represent no activity. Similarity contours highlight cluster analysis across organisms and treatments.

In Figure 3.3 we can see that some organisms share less than 20% similarity with the other analyzed organisms (e.g. KML12 - 14MG - B2a). Additionally, we can see that for some organisms, different culture conditions in duce an extract dissimilarity as high as 60% (e.g.

EG10 - 47C - 1). This data illustrates not only chemical uniqueness between dif ferent fungal isolates - validating our collection and isolation protocols - but also allows organisms in which the epigenetic modification has had a large impact on the metabolite profile to be identified.

Organisms whose modified extracts exhibit unique ch emistry and biological activities would be of high interest for scale up and chemical analysis.

Through the processing for MDS analysis, this data is also ideally prepared for der e plication efforts. W ith a robustly annotated database of known cytotoxins, nuisance compounds could be quickly identified and their extracts de - prioritized.

This data represents an exercise in metabolomics analysis of a small subset of screened extracts, but could be scaled up and performed on the entire extract library for high - level analysis of all available chemistry.

3.4 . Training Set Results

A nother subset of the extract library ( 1305 extracts, 13% of the total library ) was screened fully against Mycobacterium tuberculosis (TB) , the ESAKPE pathogens, Leishmania donovani , Naegleria fowleri , and the J774 murine macrophage cell line . U sing stringent def initions of 'active' to moderate the hit rate, 16% of these 1305 extracts were determined to be active. While this number is quite large, this is a result of the fact that 77% of the hits were hits in only one assay. Within this subset, accounting for all bioactivities, the previously observed trends in each of the individual assays (Figure 3.1) were verified; the three culture treatments were equally effectiv e in producing active extracts, and 39% of active fungi only produced hits in the HDACi and DNMTi c ulture conditions .

To analyze this data in the most meaningful way, s trict activity cut - offs were set for each bioassay. For simplicity, the ESKAPE pathogens’ MICs were rep orted as a single scaled score 4 for all 6 pathogens; an extract was considered ‘active’ in this assay if it had a scaled sc ore ≥ 7.

For highest clinical relevance, only extracts exhibiting an IC50 value < 1 μg mL - 1 in the infected

50 macrophage model of the Leishmania donovani parasite were included. TB activity was included when ≥ 85%, and activity against Naegleria fowleri was defined as inhibition of >33% at either concentration tested (50 and 5 µg/mL). Any cytotoxicity against the J774 macrophage cells up to 20 μg/mL was also included. Results can be seen in Figure 3.4.

Naeglaria Leish IM ESKAPE TB J774 Non-Specific Non-Active

Figure 3.4. Selectivity of active extra cts.

Only 48 active extracts (23% of total active, 4% of total screened) were not specific to a single target organism. 17 extracts (8%, 1%) were active only against N. fowleri , 53 (25%, 4%) against the ESKAPE panel, 37 (17%, 3%) against Mycobacterium tuberculosis , 41 (20%, 3%) against the L. donovani infected macrophage, and 14 (7%, 1%) were cytotoxic only against the J774 macrophages. When t he definition of ‘active’ was relaxed in a secondary hit, (i.e. an extract was only considered ‘selective’ if it had reporte d activity only one of the 4 assays ) 100 extracts, 48% of active extracts, retained their qualification as ‘selective’.

This is a unique set of data featuring extracts of diverse fungal origin screened against a

wide range of eukaryotic and prokaryotic dis ease causing organisms. The specificity of bioactivity in this data set suggests unique underlying chemical profiles and , gratifyingly, provides strong support to our screening program design. With this data, we have demonstrated that vigorous front - end in vestigation (multiple bioassays, metabolomic analyses, dereplication, etc.) of a library of extracts can inform scale - up prioritization in a highly effective way for the greatest chance of isolating new, bioactive natural products. Scale - up efforts are und erway to further validate these methods.

3.5 . Future Directions

With such promising data from the extract library, scale - u ps of bioactive fungi have commenced. A scale - up protocol was developed (Appendix B) and samples prioritized according to data available at the time. With the completed analysis of the training set, newly prioritized organisms have been identified and are available for chemical investigation in future. Extracts that feature specific, potent, and induced bioactivities can be scaled up and subjected to chemical analysis including new dereplication protocols as updated databases become available. With the plet hora of front - end analysis that has been employed and presented here, organisms can now be chosen for chemical investigation with confidence in the chances of discovering new, bioactive secondary metabolites.

3 .6 . References Cited

(1) Pang, K.; Vrijmoed, L. L. P.; Goh, K.; Plaingam, N.; Jones, E. B. G. Fungal Endophytes Associated with Kandelia c andel ( Rhizophoraceae ) in Mai Po Nature Reserve , Hong Kong a. 2008 , 51 , 171 – 178 DOI: 10.1515/BOT.2008.012.

(2) Kjer, J.; Debbab, A.; Aly, A. H.; Proksch, P. Methods for Isolation of Marine - Derived 52

Endophytic Fungi and Their Bioactive Secondary Products. Nat. Protoc. 2010 , 5 (3), 479 – 490 DOI: 10.1038/nprot.2009.233.

(3) Beau, J.; Mahid, N.; Burda, W. N.; Harrington, L.; Shaw, L. N.; Mutka, T.; Kyle, D. E.; Barisic, B.; Van Olphen, A.; Baker, B. J. Epigenetic Tailoring for the Production of Anti - Infective Cytosporones from the Marine Fungus Leucostoma p ersoonii . Mar. Drugs 2012 , 10 (4), 762 – 774 DOI: 10.3390/md10040762.

(4) Fleeman, R.; Lavoi, T. M.; Santos, R. G.; Morales, A.; Nefzi, A.; Welmaker, G. S.; Medina - Franco, J. L.; Giulianotti, M. A.; Houghten, R. A.; Shaw, L. N. Combinatorial Libraries as a Tool for the Discove ry of Novel, Broad - Spectrum Antibacterial Agents Targeting the ESKAPE Pathogens. J. Med. Chem. 2015 , 58 (8), 3340 – 3355 DOI: 10.1021/jm501628s.

Chapter 4

A N ew Citreohybrid d ione Discovered Via Epigenetic Modification

4.1 . Citreohybridones and Citreohybriddiones

The h ybrid s train KO 0031 of Penicillium citreo - viride B. IFO 6200 and 4692 has been reported to be a prolific producer of meroterpenoid secondary metabolites . 1 Many of these hybrid polyketide - terpenoids are known to be feeding deterrents against the crop pest Plutella xylostella . 2 – 5 The previously reported c itreohybridones and citreohybriddiones are two groups of these fungal natural products (Figure 4. 1). Herein, the isolations of a new citreohybriddione

(citreohybriddione D, 1 ) from an environmental Penicillium sp. fungus will be discussed.

Figure 4 .1. S ome of the previously reported citreohybridones, citreohybriddiones A - C, and the new citreohybriddione D ( 1 ).

4.2 . KML12 - 14MG - B2a isolation

A number of collection trip s to the Keys Marine Lab, Long Key, FL have targeted

mangrove endophytes. Traveling by canoe and using field plating techniques ( as described in

Appendix B) samples were collected from areas all around the facility targ eting a broad range of

55 micro - organisms . The Keys Marine Lab is uniquely located with access to a wide variety of man grove environments (F igure 4.2 ).

Figure 4.2 . Go ogle Maps satellite view of c ollection areas around Keys Marine Lab, Long Key Florida. Location markers: Blue = Keys Marine Lab; Red = developed mangrove areas; Green = protected mangrove areas within Long Key State Park; Yellow = exposed mangrove areas; Purple = landfill mangrove areas.

KML12 - 14MG - B2a is a Penicillium sp. isolated from the 2012 Keys Marine Lab trip.

This organism was isolated from the stem tissue of a juvenile Rhizophora mangle tree in an exp osed mangrove community on the w est side of Lo ng Key. This fungus was isolated on a solid water agar plate containing sub - lethal doses of both nystatin and cycloheximide. On SDA this organism has a typical Penicillium morphology, growing radially from the inoculated mycelia with a light green/ white c olor and fluffy sporulating bodies.

4.3 . Initial Bioactivities, Scale - up, Epigenetic Modification , and Identification

Like most of the fungal isolates in the Baker Lab microbial library, KML12 - 14MG - B2a was cultured and screened as a part of the screening program introduced in Chapter 3. This isolate emerged early as an important hit against the ESKAPE pathogens, hitting against both gram positive and gram negative pathogens in the C ontrol an d HDACi growth conditions ( Table

4.1 ). In support of the design of the screening program, the HDACi culture produced a consistently active extract across replicate cultures , while repeated Control cultures produced inconsistent activity. Demonstrating the ability of these organisms to regulate their biosynth etic machin ery and the effectiveness of the HDACi culture conditions, KML12 - 14MG - B2a became a model organism for scale - up and chemical investigation protocols.

Table 4.1. Screening results for KML12 - 14MG - B2a. Sample Information MIC (µg/mL)

Date Loc Extract ID 200 100 50 25 10

1/9/2014 F1 KML12 - 14MG - B2a (HIT) CONTROL EKAP EKAP EKAP EAP EA

1/9/2014 F2 KML12 - 14MG - B2a (HIT) HDAC EKAP EKAP - - -

1/9/2014 F3 KML12 - 14MG - B2a (HIT) DNMT - - - - -

2/3/2014 P2 D7 KML12 - 14MG - B2a (HIT) CONTROL - - - - -

2/3/2014 P2 D8 KML12 - 14MG - B2a (HIT) HDAC ESKAP ESKAP ESKAP EKP EK

2/3/2014 P2 D9 KML12 - 14MG - B2a (HIT) DNMT - - - - -

This organism was screened multiple times as a part of a subset of isolates chosen to verify reproducibility in the screening protocol. Activity is indicated by the first letter of the pathogen the extract showed activity against. E = Enterococcus faecium , S = Staphylococcus aureus , K = Klebsiella pneumoniae , A = Acinetobacter baumannii , P = Pseudomonas aeruginosa.

KML12 - 14MG - B2a was scaled up in control culture conditions on 700g of brown rice

(autoclaved with 1.4L DI water) and inoculated in 100mL of S DB in 2 types of Unicorn brand

mycobags and a 3L fernbach flask. After 21 days of incubation, both types of mycobag (Unicorn

types 3T and 14A) were found to produce sterile, fully optimized growth ( i.e. all rice material

covered with growth) that resulted in extracts of similar mass. The fernbach flask culture proved

significantly less desirable, with fungal growth only on the top of the solid rice due to an

inability to agitate the culture throughout the culture process.

Epigenetically modified cultures on KML12 - 14MG - B2a were then scaled up using the

same protocols (at this time, Unicorn bag types 3T and 14A were used interchangeably

according to supply. Type 3T bags were later purchased in bulk and all further scale - ups done in

those bags). Again, resul ting growth was uncontaminated and represented complete media

coverage . The cultures each resulted in approximately 70g of crude extract after a 3 day

exhaustive extraction (day 1: 50mL MeOH, 750 mL EtOAc; days 2 and 3: 800 mL EtOAc) .

Extracts were filtered over celite and sent for bioassay. Unfortunately, the crude extracts of the scale - ups did not replicate the activity seen in the small scale cultures. A liquid: liquid partition between EtOAc and H 2 O, followed by NP MPLC separation of the EtOAc partition was

performed on each extract, and the resulting fractions sent for bioassay. Again, disappointingly,

the fractions returned inactive. Scale - up optimization efforts were continued with other

organis ms in search of a scale up protocol that could be standardized and used in a scale - up

screening program . Due to interesting NMR data, chemical investigation continued on the

fractions of the HDACi extract of KML12 - 14MG - B2a.

KML12 - 14MG - B2a was identified using sanger sequencing of the 18S ribosomal spacer

region (Appendix C). Agreeing with the previously observed morphology, the isolate was

identified to the genus level as a Penicillium sp.

4.4 . Compound Isolation and Structure Elucidation

The MPLC separation of KML12 - 14MG - B2a HDACi resulted in 10 fraction (Figure

4.3 ). With no bioactivity in any of the fractions, compound elucidation proceeded via NMR guided fractionation. NMR analysis of all 10 fractions identified fraction F as interestin g for further investigation. HPLC separation of F was completed in normal phase ( n - hexanes: ethyl acetate) on a silica column to yield 10 fraction s (Figure 4.4 ). Fraction F - 2 was discovered to contain the new meroterpenoid , citreohybriddione D ( 1 ).

A B C D E F G H I J

Figure 4.3 . The NP MPLC chromatogram of KML12 - 14MG - B2a HDACi.

2 5 4 7

6 8 3 10 9 1

Figure 4.4 . The NP HPLC chromatogram of KML12 - 14MG - B2a HDACi - F.

Fraction F - 2 was further purified on silica to yield compound 1 . The 1 H NMR spectrum of 1 was notable in that it contained 8 methyl signals (Table 4.2) and almost nothing in the olefin

region. Investigation of the 13 C data confirmed the presence of a large number of quaternary carbons, indicating a hig hly functionalized fused ring system such as a polyketide. The

HRESIMS of 503.2640 [M + H] + resemble d the terpenoid skeletons of the citreohybridones and citreohybriddiones, but di d not match any of the previo usly reported compounds . 13 C NMR

signals of carbons 1 - 12 strongly matched citreohybridone D, however carbons 13 - 18 more

closely resembled citreohybriddione B. 1

Table 4.2. 1D and 2D data for citreohybriddione D ( 1 ) in CDCl 3 . b a c Pos δ C δ H (m, J (Hz)) HMBC 1 27.8 1.03 (m, 1 H) 2, 9, 10, 22, 23, 25 2.38 (m, 1 H) 3, 5, 9, 10 2 23.3 1.6 (m, 2 H) 10 3 76.8 4.68 (t, 2.6 , 1H) 1, 5, 26 4 36.9 5 47.7 1.83 (m, 1 H) 4, 6, 10, 23, 24 6 16.9 1.8 (m, 1 H) 4, 8, 10 2.01 (m, 1 H) 7, 10 7 30.8 2.83 (m, 1 H) 5, 6, 8, 22 2.38 (m, 1 H) 5, 9, 10 8 38.6 8, 10, 11, 12, 23 9 53.5 2.21 (m, 1 H) 10 52.2 11 126.4 5.85 (s, 1 H) 8, 9, 10, 13, 21 12 133.0 13 60.8 14 70.4 15 210.6 16 72.1 17 206.7 18 19.8 1.4 (s, 3 H) 15, 16, 17 19 167.3 20 16.4 1.31 (s, 3 H) 12, 13, 14, 17 21 18.8 1.7 (s, 3 H) 11, 12, 13 22 19.5 1.17 (s, 3 H) 7, 8, 9, 14 23 204.4 10.14 (s, 1 H) 1, 10, 22 24 26.5 0.97 (s, 3 H) 2, 3, 4, 5, 25 25 21.3 0.9 (s, 3 H) 3, 4, 5, 24 3 - OAc 170.6 3 - OAc 21.3 2.12 (s, 3 H) 26 16 - OH 2.16 (s, 1 H) 15, 16, 17 19 - OMe 52.0 3.63 (s, 3 H) 19 a 1 H NMR recorded at 500 MHz, reported in ppm (multiplicity, J - coupling in Hz, integration); b 13 C NMR recorded at 200 MHz; c recorded from a gHMBCAD experiment at 500 MHz and reported as positions of carbons.

With conformation from 2D NMR experiments (Figure 4.5), it was determined that 1 is, in fact a new citreohybriddione lacking any lactone or epoxide ring structures. 2D NOESY data confirmed that the absolute stereochemistry matches what has been previously reported for similar compounds (Figure 4.6). 1 61

Figure 4.5 . Important COSY, HMBC, and NOESY correlations in Citreohybriddione D ( 1 ).

NOE correlations between the methoxy at C - 19 (δ H 3.63) and the methyl groups at C - 18 (δ H

1.40) and C - 20 (δ H 1.31) confirmed that the D - ring in 1 is up as in the other citreohybriddiones.

The alcohol on C - 16 of 1 has the same orientation as that in citreohybriddione B . Through - space correlations between C - 22, 23, and 25 confirm that the stereo chemistry of the A and B rings of 1 are as reported in citreohybriddione A.

O OAc H Me OH H H H Me A D Me C B O Me Me Me CO 2 Me H O

Figure 4.6. A 3D depiction of 1 for stereochemical review.

4.5 . Future Directions

Citreohybriddione D ( 1 ) was isolated from the HDACi treatment extract. Initial investigation via HPLC of similar fractions indicated that 1 may have also been present in the

DNMTi extract as well, but appeared absent in the control treatment fractions. However,

HRESIMS investig ation of the ethyl acetate partitions of all three treatment extracts revealed that

1 could be found in all treatments. Nevertheless, HRESIMS and NMR analysis shows that there are notable chemical differences between the control and modified conditions, in dicating that there may be many other new compounds to be found from this Penicillium sp. yet.

The culture optimization of KML12 - 14MG - B2a was not completed due to time restraints.

This organism remains active in the small scale, and warrants further stud y to be able to replicate that activity on a culture scale that allows for chemical investigation of the active compound(s).

The isolation of 1 confirms that this Penicillium sp . contains PKSs (polyketide synthases) that are capable of producing interesting secondary metabolites. Additionally, the small scale screening results confirm that this organism responds favorably to HDACi treatment, indicating that efforts in culture optimization would likely be rewarded with the prod uction of many otherwise silenced natural products.

While there are no reported activities for any of the citreohybriddiones (including 1 ) against human disease, further testing of this new compound is warranted. Due to m ass limitations, 1 was only screened against the ESKAPE pathogens and the Leishmania donovani parasite. Feeding studies and cytotoxicity profiling would be interesting to see how it compares to the rest of the compounds in this class .

4.6 . References

(1) Kosemura, S. Meroterpenoids from Penicillium citreo - v iride B. IFO 4692 and 6200 Hybrid. Tetrahedron 2003 , 59 (27), 5055 – 5072 DOI: 10.1016/S0040 - 4020(03)00739 - 7.

(2) Kosemura, S.; Yamamura, S. Isolation and Biosynthetic Pathway for Citreohybridones from the Hybrid Strain KO 0031 Derived from Penicillium s pecies. Tetrahedron Lett. 1997 , 38 (35), 6221 – 6224 DOI: 10.1016/S0040 - 4039(97)01430 - 5.

(3) Kosemura, S.; Matsunaga, K.; Yamamura, S. Citreohybriddiones A and B and Related Terpenoids, New Metabolites of a Hybrid Strain KO 0031 Derived from Penicillium c itre o - v iride B. IFO 6200 and 4692. Chem. Lett. 1991 , 1811 – 1814.

(4) Kosemura, S.; Matsou, S.; Yamamura, S. Citreohybriddione C, A Meroterpenoid of a Hybrid Strain KO 0031 Derived from Penicillium citreo - v iride B. IFO 6200 and 4692. Phytochemistry 1996 , 43 (6), 1231 – 1234.

(5) Kosemura, S.; Miyata, H.; Yamamura, S.; Albone, K.; Simpson, T. J. Biosynthetic Studies on Citreohybridones, Metabolites of a Hybrid Strain KO 0031 Derived from Penicillium citreo - v iride B. IF0 6200 and 4692. J. Chem. Soc. Perkin Trans 1994 , No. 1, 135 – 139 .

Appendix A: Experimental and Supporting Data for Chapter 2

Fungal Strain Identification MPLC Run Parameters and Chromatograms HPLC Run Parameters and Chromatograms Compound Data

Fungal Strain Identification

The fungal isolate “BB11 - 2” was sequenced using sanger sequencing of the 18S

ribosomal spacer region. The forward and reverse primers used can be found in table A1 . The

PCR product was sent to europhins sequencing company which yielded the sequences that were blasted using NCBI nucl eotide blast for sequence similarity.

Table A1. The forward and reverse primers used for the fungal strain identification of BB11 - 2.

NNNNNNNNNNNNNTTGGTTTCTAGGACCGCCGTAATGATTAAT 18S forward AGGGACAGTCGGGGGCATCAGTATTCAATCGTCAGAG GTGAAATTCTTGGATCGATTGAAGACTAACTACTGCGAAAGCA primers TTTGCCAAGGATGTTTTCATTAATCAGGAACGAAAGT

TAGGGGATCGAAAACGATCAGATACCGTTGTAGTCTTAATCAT AAACTATGCCCACTAGGGATCNGGCGGTGTTATTTCT

NNNNNNNNNNNNNNNCNGNTCNCCCCTTGTGGTGCCCTTCCGT CAATTTCTTTAAGTTTCAGCCTTGCGACCATACTCCC 18S reverse CCCAGAACCCAAAAACTTTACTTTCGTGTAAGGTGCCGAGCGG GTCAAGAAATAACACCGCCCGATCCCTAGTCGGCATA primers GTTTATGGTTAAGACTACAACGGTATCTGATCGTTTTCGATNCC CTAACTTTCGTTCCTGATTNANGANAACATCCTTGG

GAAATGCTTTCCN ANTAATNNGNCTTCNATCAAATCCTCA

Figure A1. NCBI nucleotide blast results for BB11 - 2 forward primers.

Figure A2 . NCBI nucleotide blast resu lts for BB11 - 2 reverse primers.

MPLC Run Parameters and Chromatograms

Figure A3. NP MPLC chromatogram of BB11 - 2 Control EtOAc partition.

Figure A4. A second NP MPLC of BB11 - 2 Control fraction D from the first MPLC separation. Fraction C (bottles 4 & 5) contained the phomopsichromins.

Figure A5. NP MPLC chromatogram of BB11 - 2 HDACi EtOAc partition.

Figure A6 . NP MPLC chromatogram of BB11 - 2_DNMTi EtOAc partition.

HPLC Run Parameters and Chromatograms

Normal phase HPLC separation was completed with a normal phase gradient of hexanes to ethyl acetate over 35 minutes on a semi - preperative Phenominex ® Cyano column (5µm, 100Å, 250 x

10mm) using UV and ELSD detection. Reverse phase HPLC separation was completed with a

RP gradient of water to acetonitrile or methanol on a Phenominex ® C18 column (5µm, 100Å,

250x 4.6mm) using UV and E LSD detection.

Figure A7 . NP HPLC chromatogram (black) and ELSD trace (blue) of BB11 - 2_D_C.

Compound Data

+ Phomopsichromin A ( 1 ): C 23 H 30 O 5; HRESIMS m/z 369.2034 [M + H – H 2 O] ( C 23 H 29 O 4

+ calculated, 369.2066) , m/z 387 .2137 [M + H] ( C 23 H 31 O 5 calculated, 387.2171 ), m/z 409.1954

+ 20 [M + Na] ( C 23 H 30 O 5 Na calculated, 409.1991) ; UV (MeOH) λmax (log ε) 250 (4.97) nm ; [α] D

+1.2 ( c 0.1, MeOH) ; IR (thin film) 3450, 2980, 2360, 1700, 1600, 1575, 1450, 1400, 1300, 1090

- 1 1 cm ; H NMR Data (500 MHz, CDCl 3 ) δ ppm 0.59 (s, 3 H), 0.89 (d, J =6.8 Hz, 3 H), 0.91 (d,

J =6.8 Hz, 3 H), 1.41 (m, 1 H), 1.44 (s, 3 H), 1.48 (m, 1 H), 1.52 (m, 1 H), 1.64 (m, 1 H), 1.77

(m, 1 H), 1.85 (m, 1 H), 1.97 (m, 1 H), 2.33 (m, 1 H) , 2.36 (m, 1 H), 2.41 (q, J =6.6 Hz, 1 H),

2.55 (s, 3 H), 5.46 (d, J =10.2 Hz, 1 H), 6.24 (s, 1 H), 6.76 (d, J =10.2 Hz, 1 H), 11.74 (s, 1 H);

13 C NMR (125 MHz, CDCl 3 ) δ ppm 7.5 (CH 3 , C - 12), 14.9 (CH 3 , C - 13) , 15.4 (CH 3 , C - 14 ) , 30.6

( CH 2 , C - 7 ) , 30.8 ( CH 2 , C - 4 ) , 34.3 ( CH 2 , C - 8 ) , 36.1 ( CH, C - 5 ) , 41. 5 ( CH 2 , C - 3 ) , 43.2 ( C - 6 ) , 50.4

( CH, C - 1 ) , 79.8 ( C - 9 ) , 103.6 ( C - 18 ) , 106.8 ( C - 16 ) , 111.9 ( CH, C - 20 ) , 116.9 ( CH, C - 11 ) , 125.8

( CH, C - 10 ) , 144.4 ( C - 19 ) , 158.6 ( C - 21 ) , 160.6 ( C - 17 ) , 175.1 ( C - 23 ) 213.8 ( C - 2 ).

DHD-1_PROTON_01.esp

1.2 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 1.1 9 9 9 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 . . . 5 5 5 ...... 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 0 0 0 5 5 5 0 0 0 0 0 0 0 0 0 0 0 0 4 4 4 0 0 0 . . . 4 4 4 4 4 . . . . . 4 4 4 4 4 . . . . . 4 4 4 4 4 . . . . . 4 4 4 ...... 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.0 1 1 1

0.9

0.8

0.7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 1 1 1 9 9 9 9 1 1 1 1 1 9 9 1 1 1 1 1 1 1 1 1 1 1 1 1 ...... 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . . . 0 0 0 0 . . . . . 0 0 . . . . . 0.6 . . . . . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0.5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 ...... 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 0.4 4 4 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ...... 1 1 1 . . . 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

0.3 5 5 5 . . . 5 5 5 . . . . . 5 5 5 5 5 . . . . . 5 5 5 5 5 7 7 7 . . . . . 5 5 5 5 5 7 7 7 7 7 . . . 5 5 5 7 7 7 7 7 7 7 7 7 7 8 8 8 7 7 7 5 5 5 8 8 8 8 8 5 5 5 5 5 8 8 8 8 8 5 5 5 5 5 8 8 8 5 5 5 1 1 1 7 7 7 1 1 1 1 1 7 7 7 7 7 1 1 1 1 1 7 7 7 7 7 4 4 4 4 4 1 1 1 1 1 7 7 7 4 4 4 4 4 4 4 4 4 4 7 7 7 4 4 4 4 4 4 7 7 7 7 7 4 4 4 4 4 7 7 7 7 7 4 4 4 4 4 7 7 7 7 7 . . . 4 4 4 4 4 7 7 7 . . . . . 4 4 4 4 4 4 ...... 4 4 4 4 4 ...... 4 4 4 4 4 ...... 4 4 4 4 4 . . . . . 4 4 4 ...... 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 5 5 5 3 3 3 3 3 6 6 6 6 6 5 5 5 5 5 3 3 3 3 3 6 6 6 5 5 5 5 5 3 3 3 3 3 5 5 5 5 5 3 3 3 6 6 6 5 5 5 5 5 5 6 6 6 6 6 5 5 5 5 5 6 6 6 6 6 5 5 5 5 5 6 6 6 6 6 5 5 5 5 5 6 6 6 5 5 5 ...... 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 ...... 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 8 8 8 2 2 2 8 8 8 8 8 2 2 2 2 2 8 8 8 8 8 2 2 2 2 2 8 8 8 8 8 2 2 2 2 2 8 8 8 2 2 2

0.2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 7 7 7 4 4 4 7 7 7 7 7 4 4 4 4 4 3 3 3 7 7 7 7 7 4 4 4 4 4 3 3 3 3 3 7 7 7 4 4 4 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 1 1 . . . 4 4 4 4 4 . . . 1 1 1 1 . . . . . 4 4 4 4 4 . . . . . 1 1 1 1 . . . . . 4 4 4 . . . . . 1 1 1 1 ...... 1 1 ...... 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 6 6 6 6 6 6 6 6 4 4 1 1 1 6 6 6 6 6 1 1 1 4 4 4 4 1 1 1 1 1 6 6 6 6 6 1 1 1 1 1 4 4 4 4 . . . 1 1 1 1 1 1 1 1 1 1 4 4 . . . . . 1 1 1 1 1 1 ...... 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0.1 ...... 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

0 12 11 10 9 8 7 6 5 4 3 Chemical Shift (ppm)

1 Figure A8 . H NMR Spectrum (500 MHz, CDCl 3 ) of Phomopsichromin A ( 1 ) .

DHD-1_CARBON_01.esp 0.060

0.055

0.050

0.045

0.040 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 ...... 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 1 1 1 4 4 4 1 1 1 1 1 4 4 4 4 4 1 1 1 1 1 4 4 4 4 4 1 1 1 1 1 4 4 4 4 4 1 1 1 4 4 4 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

0.035 9 9 9 ...... 5 5 2 2 2 5 5 5 5 2 2 2 2 5 5 5 5 2 2 2 2 5 5 5 5 2 2 2 2 5 5 2 2 2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 1 1 1 1 1 1 1 1 1 1 1 1 1 . . . . . 1 1 1 1 1 ...... 1 1 1 ...... 8 8 8 . . . . . 8 8 8 8 8 8 8 8 0 0 0 8 8 8 8 0 0 0 0 0 8 8 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 7 7 7 0 0 0 0 0 1 1 1 1 7 7 7 7 0 0 0 0 0 1 1 1 1 7 7 7 7 0 0 0 1 1 1 1 7 7 7 7 1 1 8 8 8 8 7 7 7 8 8 8 8 2 2 2 8 8 8 8 2 2 2 2 2 8 8 8 2 2 2 2 2 2 2 2 2 2 2 2 2 4 4 4 . . . 4 4 4 4 4 . . . . 4 4 4 4 4 ...... 4 4 4 4 4 . . . . 4 4 4 4 4 . . . . . 4 4 4 4 4 . . . 4 4 4 4 4 . . . . . 4 4 4 4 4 4 4 4 4 . . . 4 4 4 4 4 4 4 4 4 4 4 7 7 7 4 4 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 ...... 0 0 0 . . . 3 3 3 0 0 0 0 8 8 8 3 3 3 3 3 0 0 0 0

0.030 8 8 8 8 8 3 3 3 3 3 3 3 3 0 0 0 0 8 8 8 8 8 3 3 3 3 3 3 3 3 3 3 0 0 0 8 8 8 8 8 3 3 3 3 3 3 3 3 8 8 8 3 3 3 3 3 3 3 3 6 6 6 9 9 9 6 6 6 6 9 9 9 9 9 . . . 6 6 6 6 9 9 9 9 9 . . . . . 6 6 6 6 9 9 9 9 9 0 0 0 . . . . . 6 6 6 0 0 0 0 0 . . . . . 0 0 0 0 0 0 0 0 0 0 0 0 0 3 3 3 . . . 9 9 9 3 3 3 3 . . . . 9 9 9 9 9 3 3 3 3 . . . . 9 9 9 9 9 4 4 4 3 3 3 3 . . . . 9 9 9 9 9 4 4 4 4 4 3 3 3 . . . 9 9 9 4 4 4 4 4 4 4 4 4 4 4 4 4 8 8 8 4 4 4 . . . 8 8 8 8 8 4 4 4 4 4 . . . . . 8 8 8 8 8 4 4 4 4 4 . . . . . 8 8 8 8 8 4 4 4 4 4 . . . . . 8 8 8 5 5 5 4 4 4 . . . 5 5 5 5 5 5 5 5 5 5 9 9 9 5 5 5 5 5 9 9 9 9 9 5 5 5 0 0 0 9 9 9 9 9 0 0 0 0 9 9 9 9 9 . . . 0 0 0 0 9 9 9 . . . . . 0 0 0 0 ...... 3 3 3 1 1 1 3 3 3 3 3 1 1 1 1 1 3 3 3 3 3 1 1 1 1 1 3 3 3 3 3 1 1 1 1 1 4 4 4 3 3 3 1 1 1 4 4 4 4 4 9 9 9 9 9 4 4 4 4 4 9 9 9 9 9 4 4 4 3 3 3 9 9 9 9 9 9 9 9 3 3 3 3 9 9 9 9 9 9 9 9 3 3 3 3 9 9 9 9 9 3 3 3 3 9 9 9 9 9 3 3 3 9 9 9 ...... 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 . . . . . 6 6 6 . . . . . 6 6 6 6 6 . . . . . 6 6 6 6 6 . . . 6 6 6 6 6 1 1 1 6 6 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 6 6 6 6 6 6 6

0.025 1 1 1 6 6 6 6 6 1 1 1 1 1 6 6 6 6 6 1 1 1 1 1 6 6 6 3 3 3 1 1 1 1 1 3 3 3 3 3 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1 3 3 3 3 3 1 1 1 1 1 3 3 3 3 3 8 8 8 8 8 1 1 1 1 1 3 3 3 8 8 8 8 8 1 1 1 8 8 8 8 8 8 8 8 ...... 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 9 9 9 1 1 1 9 9 9 9 9 1 1 1 1 1 9 9 9 9 9 1 1 1 1 1 0.020 9 9 9 1 1 1 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 ...... 8 8 . . . 8 8 8 8 8 8 8 8 8 8 8 8 8 8 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 ...... 7 7 7 6 6 6 7 7 7 7 7 6 6 6 6 6 7 7 7 7 7 6 6 6 6 6 7 7 7 7 7 6 6 6 6 6 1 1 1 7 7 7 6 6 6 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 5 5 3 3 3 5 5 5 5 5 5 5 5 5 5 5 5 8 8 8 8 8 5 5 1 1 1 8 8 8 8 8 1 1 1 1 1 8 8 8 8 8 1 1 1 1 1 6 6 6 8 8 8 1 1 1 1 1 6 6 6 6 6 1 1 1 6 6 6 6 6 6 6 6 6 6 6 6 6 . . . 4 4 4 . . . . . 2 2 2 4 4 4 4 4 . . . . . 2 2 2 2 2 4 4 4 4 4 . . . . . 7 7 2 2 2 2 2 4 4 4 4 4 . . . . . 7 7 7 7 2 2 2 . . . . . 7 7 7 7 . . . . . 7 7 7 7 . . . 7 7 ......

0.015 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 6 6 6 4 4 4 3 3 3 3 3 6 6 6 6 6 3 3 3 3 3 6 6 6 6 6 3 3 3 3 3 6 6 6 6 6 3 3 3 3 3 3 1 1 6 6 6 3 3 3 3 3 1 1 1 1 3 3 3 3 3 1 1 1 1 3 3 3 3 3 1 1 1 1 3 3 3 1 1 . . . 4 4 4 . . . . . 4 4 4 4 4 . . . . . 4 4 4 4 4 . . . 4 4 4 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 1 1 1 8 8 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 . . . 0 0 0 0 0 . . . . . 0 0 0 . . . . . 4 4 4 . . . 8 8 8 4 4 4 4 4 8 8 8 8 8 4 4 4 4 4 8 8 8 8 8 4 4 4 4 4 8 8 8 8 8 4 4 4 8 8 8 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 2 2 2 1 1 1 1 1 1 1 1 1 1 6 6 6 1 1 1 1 1 6 6 6 6 6 1 1 1 6 6 6 6 6 6 6 6 6 6 5 5 5 1 1 1 6 6 6 5 5 5 5 5 1 1 1 1 1 5 5 5 5 5 1 1 1 1 1 5 5 5 5 5 1 1 1 1 1 5 5 5 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.010 1 1 1

0.005

220 200 180 160 140 120 100 80 60 40 Chemical Shift (ppm)

13 Figure A9. C NMR Spectrum (125 MHz, CDCl 3 ) of Phomopsichromin A ( 1 ) .

100

150

200

F2 Chemical Shift (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

1 13 Figure A10 . H - C gHSQCAD NMR Spectrum (600 MHz, CDCl 3 ) of Phomopsichromin A ( 1 ) .

100

150

200

F2 Chemical Shift (ppm) 9 8 7 6 5 4 3 2 1

1 13 Figure A11 . H - C gHMBCAD NMR Spectrum (500 MHz, CDCl 3 ) of Phomopsichromin A ( 1 ) .

7 F2 Chemical Shift (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

1 1 Figure A12 . H - H gCOSY NMR Spectrum (500 MHz, CDCl 3 ) of Phomopsichromin A ( 1 ) .

7 F2 Chemical Shift (ppm) 5 4 3 2 1

1 1 Figure A13 . H - H NOESY NMR Spectrum (600 MHz, CDCl 3 ) of Phomopsichromin A ( 1 ) .

+ Phomopsichromin B ( 2 ) : C 23 H 32 O 5; HRMS m/z 371.2222 [M + H – H 2 O] ( C 23 H 31 O 4 calculated,

+ + 371.2222) , m/z 389.2322 [M + H] ( C 23 H 33 O 5 calculated, 389.2328 ), m/z 411.2145 [M + Na]

20 ( C 23 H 32 O 5 Na calculated, 411.2147) ; UV (MeOH) λmax (log ε) 250 (4.08 ) nm; [α] D - 1.5 ( c 0.1,

MeOH) ; IR (thin film) 3450, 2930, 2380, 1675, 1625, 1600, 1575, 1460, 1400, 1300, 1090 cm - 1 ;

1 H NMR Data ( 6 00 MHz, CDCl 3 ) δ ppm 0.82 ( d, J =6. 6 Hz, 3 H) , 0.86 (s, 3 H) , 0.95 ( d, J =7.1

Hz, 3 H) , 1.27 (m, 1 H) , 1.35 (m, 2 H) , 1.41 (s, 3 H) , 1.43 (m, 1H), 1.45 (m, 1 H) , 1.46 (m, 1H),

1.55 (m, 1 H) , 1.63 (m , 2 H) , 1.8 (m, 1 H) , 2.53 (s, 3 H) , 3.85 (q, 2.8 , 1 H) , 5.46 ( d, J =10.2 Hz, 1

13 H) , 6.22 (s, 1 H) , 6.74 ( d, J =10.2 Hz, 1 H) , 11.80 (s, 1 H) ; C NMR (125 MHz, CDCl 3 ) δ ppm

12.3 (CH 3 , C - 12), 15.6 (CH 3 , C - 13), 17.3 (CH 3 , C - 14), 24.4 (CH 3 , C - 22), 25.4 (CH 2 , C - 4), 27.0

(CH 3 , C - 15), 30.9 (CH 2 , C - 7), 33.9 (CH 2 , C - 3), 34.4 (CH 2 , C - 8), 36.5 (CH, C - 5), 38.2 (C - 6),

39.4 (CH, C - 1), 73.1 (CH, C - 2), 80.1 (C - 9), 103. 4 (C - 18), 106.9 (C - 16), 111.9 (CH, C - 20), 116.6

(C H, C - 11), 126.2 ( CH, C - 10), 144.1 (C - 19), 158.7 (C - 21), 160.6 (C - 17), 174.7 (C - 23).

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 DHD-2_600_Proton_opt.esp 4 4 4 ...... 3 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1 3 3 3 3 3 1 1 1 1 1 3 3 3 1 1 1 1 1 1 1 1 1 1 1 1 1 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 ...... 6 6 6 . . . . . 6 6 6 6 6 . . . 6 6 6 6 6 6 6 6 6 6 6 6 6 2 2 2 2 2 2 2 2 2 2 2 2 2 8 8 8 2 2 2 2 2 8 8 8 8 8 8 8 8 8 8 8 8 8 ......

1.0 ...... 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0.9

0.8

0.7

0.6 6 6 6 6 6 6 6 6 6 6 6 6 6 2 2 2 6 6 6 6 6 3 3 2 2 2 2 2 6 6 6 3 3 3 3 2 2 2 2 2 3 3 3 3 2 2 2 2 2 3 3 3 3 2 2 2 3 3 9 9 9 9 9 9 9 9 9 9 9 9 9 8 8 8 9 9 9 9 9 8 8 8 8 8 8 8 9 9 9 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 ...... 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.5 0 0

0.4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ...... 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 . . .

0.3 ...... 4 4 4 . . . 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 . . . 7 7 7 . . . . . 7 7 7 7 . . . . . 7 7 7 7 . . . . . 7 7 7 7 . . . 7 7 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7 7 7 7 7 7 7 7 7 7 7 5 5 5 7 7 7 7 5 5 5 5 7 7 7 5 5 5 5 5 5 5 5 1 1 1 2 2 2 5 5 5 1 1 1 1 1 2 2 2 2 1 1 1 1 1 2 2 2 2 1 1 1 1 1 2 2 2 2 1 1 1 2 2 2 5 5 5 1 1 1 5 5 5 5 5 1 1 1 1 1 5 5 5 5 5 1 1 1 1 1 5 5 5 . . . 1 1 1 ...... 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 3 3 4 4 4 3 3 3 3 3 3 3 3 3 3 . . . 3 3 3 . . . . 8 8 8 ...... 8 8 8 8 8 ...... 8 8 8 8 8 ...... 8 8 8 8 8 . . . . 8 8 8 1 1 1 . . . 1 1 1 1 1 1 1 1 5 5 5 1 1 1 1 5 5 5 5 1 1 1 5 5 5 5 . . . 5 5 5 5 . . . . . 6 6 6 6 6 . . . . . 6 6 6 6 6 . . . 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 5 5 7 7 7 5 5 5 5 . . . 5 5 5 5 5 5 5 . . . . . 5 5 5 5 5 5 5 5 . . . . . 5 5 5 5 5 5 5 . . . . . 5 5 5 5 ...... 5 5 5 7 7 7 . . . . . 7 7 7 7 . . . . . 7 7 7 7 . . . 3 3 3 7 7 7 7 3 3 3 3 3 7 7 7 3 3 3 3 3 3 3 3 3 3 3 3 3 ...... 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 9 9 9 6 6 6 6 6 6 6 6 6 9 9 9 9 9 6 6 6 6 6 6 6 6 6 9 9 9 9 9 6 6 6 6 6 6 6 9 9 9 9 9 0.2 6 6 6 9 9 9 2 2 2 2 2 2 2 2 7 7 7 2 2 2 2 2 1 1 7 7 7 7 7 2 2 2 2 2 1 1 1 1 7 7 7 7 7 2 2 2 1 1 1 1 7 7 7 7 7 1 1 1 1 7 7 7 1 1 ...... 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 . . 1 1 1 1 1 1 1 1 1 1 . . . . 1 1 1 1 1 1 1 1 . . . . 1 1 1 1 1 . . . . 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.1

13 12 11 10 9 8 7 6 5 4 3 2 Chemical Shift (ppm)

1 Figure A14 . H NMR Spectrum (6 00 MHz, CDCl 3 ) of Phomopsichromin B ( 2 ) .

5 5 5 0 0 0 5 5 5 5 5 5 5 0 0 0 0 5 5 5 5 5 5 5 5 5 0 0 0 0 5 5 5 5 5 5 5 5 0 0 0 5 5 5 2 2 2 0 0 0 7 7 7 2 2 2 2 0 0 0 0 7 7 7 7 7 2 2 2 2 0 0 0 0 7 7 7 7 7 2 2 2 2 0 0 0 0 7 7 7 7 7 DHD-2_dd500_CARBON_01.esp 2 2 2 0 0 0 7 7 7 ...... 7 7 7 7 7 7 6 6 6 7 7 7 7 7 7 7 7 6 6 6 6 6 7 7 7 7 7 7 7 7 6 6 6 6 6 7 7 7 7 7 7 7 7 6 6 6 6 6 7 7 7 7 7 7 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

0.060

0.055

0.050

0.045

0.040

0.035 4 4 4 4 4 4 4 4 4 4 4 4 4 8 8 8 4 4 4 4 4 8 8 8 8 8 4 4 4 8 8 8 8 8 8 8 8 8 8 8 8 8 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 ...... 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 2 2 2 2 2 2 2 2 2 2 2 2 2 7 7 7 2 2 2 2 2 7 7 7 7 7 2 2 2 7 7 7 7 7 . . . . . 7 7 7 ...... 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 1 1 1 3 3 3 3 1 1 1 1 1 3 3 3 3 1 1 1 1 1 3 3 3 1 1 1 1 1 1 1 1 1 1 1 1 1 5 5 5 1 1 1 1 1 1 1 1 5 5 5 5 5 1 1 1 1 1 5 5 5 5 5 1 1 1 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4

0.030 4 4 4 4 4 4 4 8 8 8 4 4 4 8 8 8 8 8 4 4 4 4 8 8 8 8 8 4 4 4 4 8 8 8 8 8 4 4 4 4 8 8 8 . . . 4 4 4 6 6 6 . . . . 1 1 1 6 6 6 6 6 . . . . 8 8 8 2 2 2 1 1 1 1 1 6 6 6 6 6 . . . . 8 8 8 8 8 2 2 2 2 1 1 1 1 1 6 6 6 6 6 . . . 8 8 8 8 8 2 2 2 2 1 1 1 1 1 5 5 5 8 8 8 2 2 2 . . . 5 5 5 5 5 2 2 2 . . . . 5 5 5 5 5 2 2 2 2 2 . . . . 5 5 5 5 5 2 2 2 2 2 . . . . 5 5 5 2 2 2 2 2 . . . 1 1 1 2 2 2 1 1 1 1 1 1 1 1 1 1 3 3 3 9 9 9 1 1 1 1 1 3 3 3 3 9 9 9 9 9 1 1 1 3 3 3 3 9 9 9 9 9 3 3 3 3 9 9 9 9 9 9 9 9 3 3 3 6 6 6 9 9 9 9 9 9 9 6 6 6 6 6 9 9 9 9 6 6 6 6 6 9 9 9 9 4 4 4 4 4 4 6 6 6 6 6 9 9 9 4 4 4 4 4 4 4 4 4 6 6 6 4 4 4 4 4 4 4 4 4 2 2 2 . . . 4 4 4 4 4 4 4 4 4 4 4 4 2 2 2 2 2 . . . . . 4 4 4 4 4 4 4 4 4 4 2 2 2 2 2 . . . . . 5 5 5 4 4 4 4 2 2 2 2 2 . . . . . 5 5 5 5 5 4 4 4 4 2 2 2 . . . 7 7 7 5 5 5 5 5 4 4 4 . . . 7 7 7 7 7 5 5 5 5 5 . . . . . 7 7 7 7 7 2 2 2 5 5 5 . . . . . 7 7 7 7 7 2 2 2 2 2 . . . . . 9 9 9 ...... 2 2 2 2 2 . . . . . 9 9 9 9 ...... 2 2 2 . . . . . 9 9 9 9 ...... 9 9 9 9 ...... 9 9 9 . . . . . 3 3 3 ...... 3 3 3 3 . . . . . 3 3 3 3 . . . . . 3 3 3 3 ...... 3 3 3 ...... 1 1 1 ...... 2 2 2 1 1 1 1 1 ...... 2 2 2 2 1 1 1 1 1 . . . . 2 2 2 2 1 1 1 1 1 1 2 2 2 6 6 6 1 1 1 1 1 6 6 6 6 6 1 1 1 1 1 4 4 4 5 5 5 6 6 6 6 6 1 1 1 1 1 4 4 4 4 4 5 5 5 5 6 6 6 6 6 1 1 1 4 4 4 4 4 5 5 5 5 6 6 6 3 3 3 4 4 4 4 4 5 5 5 5 6 6 6 3 3 3 3 3 4 4 4 5 5 5 6 6 6 6 6 3 3 3 3 3 6 6 6 6 6 6 6 6 3 3 3 3 3 6 6 6 6 6 6 6 6 6 6 3 3 3 6 6 6 6 6 6 6 6 . . . 6 6 6 6 6 . . . . . 8 8 8 8 8 6 6 6 . . . . . 8 8 8 8 8 0 0 0 1 1 1 . . . 8 8 8 8 8 0 0 0 0 1 1 1 1 1 8 8 8 0 0 0 0 1 1 1 1 1 0 0 0 0 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 2 2 2 1 1 1 1 1 3 3 3 3 3 2 2 2 2 1 1 1 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 3 3 3 3 3 3 2 2 2 2 2 2 2 2 1 1 1 1 1 3 3 3 3 3 2 2 2 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1 3 3 3 3 3 3 3 3 3

0.025 1 1 1 1 1 . . . 3 3 3 3 3 3 3 1 1 1 1 1 . . . . . 3 3 3 3 1 1 1 1 1 . . . . . 3 3 3 1 1 1 . . . . . 1 1 1 1 1 . . . 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 2 2 2 . . . 2 2 2 2 2 . . . . . 2 2 2 2 2 . . . . . 2 2 2 2 2 . . . . . 2 2 2 . . . 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3

0.020 3 3 3 3 3 3 3 3 ...... 7 7 7 . . . . . 7 7 7 7 7 0 0 0 . . . . . 7 7 7 7 7 0 0 0 0 0 . . . 7 7 7 7 7 0 0 0 0 0 7 7 7 0 0 0 0 0 0 0 0 . . . 2 2 2 . . . . . 7 7 7 2 2 2 2 . . . . . 7 7 7 7 7 2 2 2 2 . . . . . 7 7 7 7 7 2 2 2 2 . . . 7 7 7 7 7 2 2 2 7 7 7 4 4 4 4 4 4 4 4 6 6 6 4 4 4 4 4 6 6 6 6 6 4 4 4 4 4 6 6 6 6 6 . . . 4 4 4 6 6 6 ...... 9 9 9 4 4 4 9 9 9 9 4 4 4 4 4 9 9 9 9 4 4 4 4 4 9 9 9 9 4 4 4 4 4 9 9 9 4 4 4 4 4 4 4 4 6 6 6 4 4 4 4 4 6 6 6 6 1 1 1 4 4 4 4 4 . . . 6 6 6 6 1 1 1 1 1 4 4 4 . . . . 6 6 6 6 1 1 1 1 1 8 8 8 . . . . 6 6 6 1 1 1 1 1 8 8 8 8 8 . . . . 1 1 1 8 8 8 8 8 . . . 8 8 8 8 8 8 8 8 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 1 1 1 4 4 4 1 1 1 1 1 4 4 4 4 1 1 1 1 1 4 4 4 4 1 1 1 1 1 6 6 6 4 4 4 4 5 5 5 1 1 1 6 6 6 6 4 4 4 5 5 5 5 5 6 6 6 6 5 5 5 5 5 6 6 6 6 5 5 5 5 5 6 6 6 5 5 5 ...... 1 1 1 . . . . 1 1 1 1 1 . . . . 1 1 1 1 1 . . . 1 1 1 1 1 1 1 1 0 0 0 1 1 1 0 0 0 0 1 1 1 1 1 0 0 0 0 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 3 3 3

0.015 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.010

0.005

0 180 160 140 120 100 80 60 40 Chemical Shift (ppm)

13 Figure A15 . C NMR Spectrum (125 MHz, CDCl 3 ) of Phomopsichromin B ( 2 ) .

100

150

F2 Chemical Shift (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

1 13 Figure A16 . H - C gHSQCAD NMR Spectrum (600 MHz, CDCl 3 ) of Phomopsichromin B ( 2 ) .

100

150

200

F2 Chemical Shift (ppm) 10 8 6 4 2 0

1 13 Figure A17 . H - C gHMBCAD NMR Spectrum (500 MHz, CDCl 3 ) of Phomopsichromin B ( 2 ) .

F2 Chemical Shift (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

1 1 Figure A18 . H - H gCOSY NMR Spectrum (600 MHz, CDCl 3 ) of Phomopsichromin B ( 2 ) .

F2 Chemical Shift (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

1 1 Figure A19. H - H NOESY NMR Spectrum (600 MHz, CDCl 3 ) of Phomopsichromin B ( 2 ) .

1.0 4-2c1a_inova400_methyl_PROTON_01.esp

0.9

0.8

0.7

0.6

0.5 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 ...... 0.4 . . . 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 9 9 9 9 9 9 5 5 5 5 5 9 9 9 9 5 5 5 5 5 9 9 5 5 5 5 5 5 5 5 3 3 3 3 3 3 8 8 8 3 3 3 3 8 8 8 8 8 3 3 3 3 8 8 8 8 8 3 3 8 8 8 8 8 8 8 8 ...... 1 1 1 1 1 1 0 0 0 1 1 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 1 1 0 0 0 0 0 0.3 0 0 0 9 9 9 9 9 6 6 6 6 6 9 9 9 9 9 6 6 6 6 6 9 9 9 9 9 6 6 6 6 6 9 9 9 6 6 6 9 9 9 4 4 4 9 9 9 9 9 4 4 4 4 4 9 9 9 9 9 4 4 4 4 4 9 9 9 9 9 4 4 4 4 4 9 9 9 4 4 4 ...... 1 1 1 2 2 2 1 1 1 1 1 2 2 2 2 2 1 1 1 1 1 2 2 2 2 2 1 1 1 1 1 2 2 2 2 2 1 1 1 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 5 5 5 5 5 5 5 5 5 5 2 2 2 8 8 5 5 5 5 2 2 2 2 2 8 8 8 8 5 5 5 2 2 2 2 2 8 8 8 8 2 2 2 2 2 8 8 8 8 2 2 2 8 8 ...... 9 9 9 9 9 9 9 9 9 9 9 8 8 8 9 9 9 9 8 8 8 8 8 9 9 9 8 8 8 8 8 8 8 8 8 8 8 8 8 ...... 0 0 ...... 0 0 0 0 ...... 0 0 0 0 . . . . . 0 0 0 0

0.2 . . . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 8 8 6 6 6 8 8 8 8 8 6 6 6 6 6 4 4 4 8 8 8 8 8 6 6 6 6 6 4 4 4 4 4 8 8 8 8 8 6 6 6 6 6 4 4 4 4 4 8 8 8 6 6 6 4 4 4 4 4 4 4 4 1 1 1 4 4 4 1 1 1 1 1 4 4 4 4 4 4 4 4 1 1 1 1 1 4 4 4 4 4 4 4 4 4 4 1 1 1 4 4 4 4 4 4 4 4 4 4 4 8 8 8 8 8 8 8 8 . . . 8 8 8 8 8 ...... 8 8 8 8 8 ...... 8 8 8 ...... 2 2 ...... 2 2 2 2 ...... 5 5 5 2 2 2 2 . . . 5 5 5 5 5 2 2 2 2 5 5 5 5 5 2 2 5 5 5 1 1 1 1 1 1 1 1 6 6 6 1 1 1 1 1 5 5 5 6 6 6 6 6 1 1 1 1 1 5 5 5 5 5 5 5 5 7 7 6 6 6 6 6 5 5 5 5 5 5 5 5 5 5 7 7 7 7 6 6 6 5 5 5 5 5 5 5 5 7 7 7 7 7 7 7 5 5 5 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 . . . 7 7 7 7 7 . . . . . 7 7 7 ......

0.1 ...... 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0 13 12 11 10 9 8 7 6 5 4 3 Chemical Shift (ppm) 1 Figure A20. H NMR Spectrum (400 MHz, CDCl 3 ) of methylated phomopsichromin B ( 7 ). A new methoxy signal can be seen at δ H 3.92.

D_C_4-2c1a_methyl_dd500_CARBON_01.esp

0.25

0.20

0.15

0.10 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 7 7 7 7 7 7 7 7 0 0 0 7 7 7 7 7 0 0 0 0 0 7 7 7 3 3 3 0 0 0 0 0 3 3 3 3 3 3 3 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 0 0 0 2 2 2 3 3 3 3 3 3 3 3 3 3 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 1 1 1 2 2 2 2 3 3 3 3 3 3 3 1 1 1 1 1 2 2 2 2 3 3 3 3 1 1 1 1 1 2 2 2 3 3 3 3 1 1 1 1 1 9 9 9 3 3 3 3 3 3 1 1 1 9 9 9 9 9 3 3 3 3 3 9 9 9 9 9 3 3 3 3 3 . . . 9 9 9 9 9 3 3 3 3 3 . . . . . 3 3 3 . . . . . 3 3 3 3 3 1 1 . . . 3 3 3 3 3 . . . 1 1 1 1 3 3 3 3 3 3 3 3 . . . . 1 1 1 1 3 3 3 3 3 3 3 3 . . . . 8 8 8 1 1 1 1 3 3 3 3 3 1 1 1 . . . . 8 8 8 8 8 1 1 3 3 3 3 3 1 1 1 1 1 . . . 8 8 8 8 8 3 3 3 1 1 1 1 1 2 2 2 8 8 8 8 8 1 1 1 1 1 2 2 2 2 2 8 8 8 1 1 1 2 2 2 2 2 4 4 4 2 2 2 2 2 6 6 6 4 4 4 4 2 2 2 6 6 6 6 6 4 4 4 4 6 6 6 6 6 . . . . . 4 4 4 . . . 6 6 6 3 3 3 . . . . . 4 4 4 . . . . . 3 3 3 3 3 . . . . . 4 4 4 4 4 7 7 7 . . . . . 3 3 3 3 3 . . . 2 2 2 4 4 4 4 4 7 7 7 7 7 ...... 3 3 3 3 3 2 2 2 2 2 4 4 4 4 4 7 7 7 7 7 ...... 3 3 3 2 2 2 2 2 5 5 5 4 4 4 7 7 7 7 7 6 6 6 . . . . . 2 2 2 2 2 5 5 5 5 . . . 7 7 7 6 6 6 6 6 2 2 2 ...... 5 5 2 2 2 5 5 5 5 . . . . 6 6 6 6 6 2 2 2 2 ...... 5 5 5 5 5 5 5 5 . . . . 6 6 6 6 6 2 2 2 2 . . . . . 5 5 5 5 5 5 5 . . . . 6 6 6 2 2 2 2 . . . . . 5 5 5 5 9 9 9 . . . 2 2 2 ...... 5 5 9 9 9 9 9 . . . 3 3 3 3 . . . . . 9 9 9 9 9 . . . . . 3 3 3 3 . . . . . 9 9 9 . . . . . 3 3 3 3 ...... 3 3 ...... 4 4 4 ...... 4 4 4 4 4 ...... 4 4 4 4 4 . . . 6 6 6 . . . . 4 4 4 4 4 . . . 6 6 6 6 6 . . 4 4 4 . . . . . 1 1 1 6 6 6 6 6 9 9 9 . . . . . 1 1 1 1 1 6 6 6 6 6 6 6 6 9 9 9 9 9 . . . . . 1 1 1 1 1 6 6 6 6 6 6 6 6 9 9 9 9 9 . . . 9 9 9 5 5 5 1 1 1 1 1 6 6 6 6 6 9 9 9 9 9 9 9 9 9 5 5 5 5 1 1 1 1 1 1 1 6 6 6 6 6 1 1 1 1 1 9 9 9 9 9 9 9 5 5 5 5 1 1 1 1 1 1 1 1 1 9 9 9 9 9 9 9 9 5 5 5 1 1 1 1 1 1 1 1 1 9 9 9 9 9 9 9 9 4 4 4 1 1 1 1 1 1 9 9 9 9 9 9 9 9 9 9 4 4 4 4 4 1 1 9 9 9 9 9 9 9 9 4 4 4 4 4 1 1 1 1 9 9 9 9 9 . . . 4 4 4 4 4 1 1 1 1 9 9 9 . . . . . 4 4 4 5 5 5 1 1 1 1 ...... 5 5 5 5 5 1 1 6 6 ...... 5 5 5 5 5 6 6 6 6 3 3 3 ...... 5 5 5 5 5 6 6 6 6 3 3 3 3 3 . . . . 5 5 5 1 1 1 6 6 6 6 3 3 3 3 3 0 0 0 . . . 1 1 1 1 1 6 6 3 3 3 3 3 0 0 0 0 0 2 2 2 1 1 1 1 1 . . 0 0 0 0 0 2 2 2 2 2 1 1 1 . . . . 0 0 0 2 2 2 2 2 . . . . 2 2 2 2 2 2 2 2 1 1 1 . . . . 2 2 2 2 2 2 2 1 1 1 1 1 . . 2 2 2 2 1 1 1 1 1 7 7 7 2 2 2 2 1 1 1 1 1 7 7 7 7 7 2 2 2 1 1 1 7 7 7 7 7 3 3 3 2 2 2 7 7 7 7 7 3 3 3 3 3 2 2 2 2 2 7 7 7 3 3 3 3 3 3 3 3 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 3 3 3 3 3 3 3 3 6 6 6 6 2 2 2 1 1 1 1 1 3 3 3 3 3 6 6 6 6 1 1 1 1 1 3 3 3 3 6 6 6 6 1 1 1 1 1 . . . 3 3 3 3 6 6 6 1 1 1 1 1 1 . . . . . 3 3 3 3 3 3 3 1 1 1 1 1 . . . . . 3 3 3 3 3 3 3 1 1 1 1 1 3 3 . . . . . 3 3 3 3 3 1 1 1 1 1 1 1 1 3 3 3 3 . . . 3 3 3 3 3 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 1 1 1 1 1 3 3 3 3 1 1 1 1 1 1 1 1 3 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 1 1 1 1 1 3 3 3 3 3 1 1 1 1 1 3 3 3 3 3 2 2 2 1 1 1 1 1 3 3 3 3 3 2 2 2 2 1 1 1 3 3 3 2 2 2 2 1 1 1 2 2 2 2 1 1 1 1 1 2 2 2 1 1 1 1 1 1 1 1 1 1 7 7 1 1 1 7 7 7 7 4 4 4 7 7 7 7 4 4 4 4 4 4 4 4 7 7 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 9 9 9 . . . 9 9 9 9 9 7 7 7 2 2 2 . . . . . 9 9 9 9 9 7 7 7 7 7 7 7 7 2 2 2 2 2 . . . . . 9 9 9 9 9 7 7 7 7 7 7 7 7 7 7 2 2 2 2 2 . . . . . 9 9 9 7 7 7 7 7 7 7 7 7 7 2 2 2 2 2 . . . 7 7 7 5 5 5 7 7 7 7 7 2 2 2 5 5 5 5 5 7 7 7 5 5 5 5 5 5 5 5 5 5

0.05 5 5 5 ...... 8 8 8 ...... 8 8 8 8 8 . . . 8 8 8 8 8 8 8 8 8 8 3 3 3 8 8 8 9 9 9 3 3 3 3 3 9 9 9 9 9 9 9 9 3 3 3 3 3 9 9 9 9 9 9 9 9 9 9 3 3 3 3 3 9 9 9 9 9 9 9 9 9 9 3 3 3 7 7 7 9 9 9 9 9 9 9 9 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 ...... 9 9 9 ...... 9 9 9 9 9 2 2 2 ...... 9 9 9 9 9 2 2 2 2 2 . . . . . 8 8 8 9 9 9 9 9 . . . 2 2 2 2 2 . . . 8 8 8 8 8 9 9 9 . . . . . 2 2 2 2 2 8 8 8 8 8 . . . . . 2 2 2 8 8 8 8 8 . . . . . 8 8 8 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 ...... 2 2 2 2 2 2 2 2 5 5 5 6 6 6 2 2 2 2 2 5 5 5 5 5 4 4 4 4 4 6 6 6 6 6 2 2 2 2 2 5 5 5 5 5 4 4 4 4 4 6 6 6 6 6 5 5 5 7 7 7 4 4 4 4 4 6 6 6 7 7 7 7 7 4 4 4 7 7 7 7 7 7 7 7 7 7 7 7 7 4 4 4 4 4 4 4 4 4 4 4 4 4 7 7 7 4 4 4 7 7 7 7 7 0 0 0 7 7 7 7 7 1 1 1 0 0 0 0 0 7 7 7 7 7 1 1 1 1 1 1 1 1 0 0 0 0 0 7 7 7 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 1 1 1 1 1 5 5 5 1 1 1 1 1 0 0 0 1 1 1 5 5 5 5 5 1 1 1 1 1 5 5 5 5 5 1 1 1 5 5 5 5 5 5 5 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0 180 160 140 120 100 80 60 40 Chemical Shift (ppm) 13 Figure A21 . C NMR Spectrum (500 MHz, CHCl 3 ) of methylated phomopsichromin B ( 7 ). 24 13 C signals can be seen.

+ Phomopsichromin C ( 3 ) : C 25 H 34 O 6 ; HRESIMS m/z 413.2330 [M + H – H 2 O] ( C 25 H 33 O 5

+ calculated, 413.2328) , m/z 431.2434 [M + H] ( C 25 H 35 O 6 calculated, 431.2434 ), m/z 453.2256

+ 20 [M + Na] ( C 25 H 34 O 6 Na calculated, 453.2253) ; UV (MeOH) λmax (log ε) 255 (4.21) nm; [α] D

+1.3 ( c 0.1, MeOH); IR (thin film) 2930, 1740, 1675, 1600, 1580, 1400, 1300, 1350, 1090 cm - 1 ;

1 H NMR Data ( 600 MHz, CDCl 3 ) δ ppm 0.83 (2d, J =6.6 Hz, 6 H ; s, 3 H ) , 1.29 (m, 1 H) , 1.40 -

1.43 (m, 6 H) , 1.47 (m, 1 H) , 1.5 (m, 2 H) , 1.55 (m, 1 H) , 1.65 (m, 1 H) , 1.81 (m, 1 H) , 2.05 (s, 3

H) , 2.53 (s, 3 H) , 4.97 (q, 2.0, 1 H) , 5.45 (d, J =10.2 Hz, 1 H) , 6.22 (s, 1 H) , 6.74 (d, J =10. 0 Hz, 1

13 H) , 11.82 (s, 1 H) ; C NMR (125 MHz, CDCl 3 ) δ ppm 12 .0 (CH 3, C - 12), 15.6 (CH 3, C - 13), 16.7

(CH 3, C - 14), 21.4 (CH 3, C - 25), 24. 4 (CH 3, C - 22), 25.8 (CH 2 , C - 4), 27.1 (CH 3, C - 15), 30.7 (CH 2 ,

C - 7), 30.9 (CH 2 , C - 3), 34.4 (CH 2 , C - 8), 36.2 (CH, C - 5), 38.3 (C, C - 6), 38.5 (CH, C - 1), 75.1

(CH, C - 2) , 80.1 (C, C - 9), 103.3 (C, C - 18), 106.8 (C, C - 16), 111.9 (CH, C - 20), 116.7 (CH, C - 11),

126.1 (CH, C - 10), 144.1 (C, C - 19), 158.7 (C, C - 21), 160.6 (C, C - 17), 170.8 (CO 2 CH 3 , C - 24),

174.4 (C OOH, C - 23).

y t i

s DHD-7_600_1D1H_opt M01(d) n e t 3 3 3 3 3 3 3 3 3 n 8 8 8 8 8 8 8 8 8 8 8 8 I ......

0 0 0 0 0 0 0 0 0 0 0 0 d e z i l a m r o N

0.90

0.85

0.80

0.75 M09(br d) M08(s) 0.70 5 5 5 5 5 5 5 5 5 0 0 0 0 0 0

0 0 0 M14(m) M07(s) 0 0 0 ...... 2 2 2 2 2 2 2 2 2 0.65 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 5 5 5 5 5 5 5 5 5 5 5 5 ...... M15(br s) 2 2 2 2 2 2 2 2 2 2 2 2 0.60 4 4 4 4 4 4 4 4 8 8 8 8 8 8 8 8 ...... 0 0 0 0 0 0 0 0 0.55

0.50

0.45

0.40

0.35 M12(br d)

M10(m) 0.30 M11(m) 0.25 M04(s) M05(d) M13(br d) 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 0 0 0 0 0 0 0 0 ......

0.20 5 5 5 5 5 5 5 5 5 6 6 5 5 5

6 6 6 6 M06(br s) 6 6 6 6 6 6 ...... 1 1 1 1 1 1 1 1

M02(s) 1 1 1 1 1 1 1 1 1 M03(d) 1 1 1 5 5 5 5 5 5 5 5 ...... 6 6 6 6 6 6 6 6 6 6 4 4 4 4 6 6 4 4 4 4 4 4 1 1 1 1 1 1 1 1 4 4 4 4 4 4 4 4 4 4 2 2 2 2 4 4 4 4 4 4 4 4 2 2 2 2 4 4 4 4 7 7 7 7 2 2 7 7 7 7 . . 7 7 7 7 ...... 5 5 5 ...... 5 5 5 3 3 3 . . 5 5

0.15 3 3 3 3 3 3 8 8 4 4 7 7 7 8 8 8 8 9 9 4 4 4 4 7 7 7 8 8 8 8 5 5 5 9 9 9 9 4 4 4 4 7 7 7 8 8 5 5 5 9 9 9 9 4 4 7 7 7 5 5 5 5 5 5 5 9 9 4 4 5 5 5 5 5 5 4 4 4 5 5 . . . . 5 5 5 5 5 5 5 5 4 4 4 5 5 5 . . . . 5 5 5 5 . . . . 4 4 5 5 5 . . 7 7 7 . . . . 5 5 7 7 7 . . . . 6 6 6 7 7 7 6 6 6 . . . 6 6 6 . . . 5 5 5 5 4 4 4 6 6 6 . . . . . 5 5 5 5 4 4 4 7 7 7 . . . 5 5 5 5 4 4 4 7 7 7 . . . 9 9 9 7 7 7 6 6 9 9 9 1 1 6 6 6 . . . . . 9 9 9 1 1 1 1 4 4 6 6 6 ...... 1 1 1 1 . . . 4 4 4 4 ...... 1 1 . . . 4 4 4 4 ...... 4 4 . . 4 4 4 . . . 1 1 4 4 4 . . . 1 1 1 4 4 4 . . 1 1 1 6 6 6 1 1 6 6 6 . . . 2 2 2 6 6 6 . . . 2 2 2 . . . 2 2 2 1 1 1 1 1 1 1 1 1 1 1 . . . 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 6 6 1 1 1 1 1 1 1 6 6 6 . . . 6 6 6 1 1 . . . 1 1 1 . . . 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.10 1 1 1

0.05

0 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Chemical Shift (ppm)

1 Figure A22 . H NMR Spectrum (6 00 MHz, CDCl 3 ) of Phomopsichromin C ( 3 ) .

DHD-7_dd500_Dec8_CARBON_01 0.075

0.070

0.065

0.060

0.055

0.050

0.045

0.040

0.035

0.030

0.025 0 0 0 8 8 8 0 0 0 0 8 8 8 8 8 0 0 0 0 8 8 8 8 8 0 0 0 0 8 8 8 8 8 0 0 0 8 8 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 4 4 7 7 7 4 4 4 4 7 7 7 7 7 4 4 4 4 7 7 7 7 7 4 4 4 4 7 7 7 7 7 4 4 4 7 7 7 2 2 2 ...... 0 0 0 2 2 2 2 2 ...... 0 0 0 0 0 2 2 2 2 2 ...... 0 0 0 0 0 2 2 2 2 2 ...... 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 . . . 1 1 1 1 1 . . . . . 1 1 1 ...... 7 7 7 2 2 2 1 1 1 6 6 6 7 7 7 7 7 2 2 2 2 2 1 1 1 1 6 6 6 6 6 7 7 7 7 7 2 2 2 2 2 1 1 1 1 6 6 6 6 6 7 7 7 7 7 2 2 2 2 2 1 1 1 1 6 6 6 6 6 7 7 7 2 2 2 1 1 1 6 6 6 1 1 1 1 1 1 1 1 2 2 2 1 1 1 1 1 4 4 4 2 2 2 2 2 1 1 1 1 1 4 4 4 4 2 2 2 2 2 1 1 1 4 4 4 4 6 6 6 . . . 2 2 2 2 2 8 8 8 4 4 4 4 6 6 6 6 6 2 2 2 . . . . . 2 2 2 8 8 8 8 8 4 4 4 6 6 6 6 6 2 2 2 2 2 . . . . . 8 8 8 8 8 6 6 6 2 2 2 2 2 0.020 . . . 8 8 8 2 2 2 6 6 6 6 6 6 3 3 6 6 6 6 3 3 3 3 3 3 3 2 2 2 1 1 1 . . . 6 6 6 6 3 3 3 3 3 3 3 3 3 2 2 2 2 1 1 1 1 1 . . . . . 5 5 5 6 6 6 6 6 3 3 3 3 3 3 3 3 3 2 2 2 2 1 1 1 1 1 . . . . . 5 5 5 5 5 6 6 6 6 6 3 3 3 3 3 3 3 2 2 2 2 1 1 1 1 1 . . . . . 5 5 5 5 5 6 6 6 6 6 3 3 3 2 2 2 1 1 1 . . . 5 5 5 5 5 6 6 6 6 6 9 9 9 5 5 5 6 6 6 5 5 5 9 9 9 9 9 4 4 4 5 5 5 5 5 9 9 9 9 9 4 4 4 4 5 5 5 5 5 9 9 9 4 4 4 4 6 6 6 5 5 5 5 5 . . . 5 5 5 1 1 1 4 4 4 4 1 1 1 6 6 6 6 6 5 5 5 . . . . . 5 5 5 5 5 1 1 1 1 1 4 4 4 1 1 1 1 1 6 6 6 6 6 . . . . . 5 5 5 5 5 1 1 1 1 1 1 1 1 1 1 6 6 6 6 6 . . . . . 5 5 5 5 5 1 1 1 1 1 1 1 1 1 1 6 6 6 . . . 5 5 5 1 1 1 1 1 1 . . . 9 9 . . . . . 9 9 9 9 ...... 9 9 9 9 . . . . . 2 2 ...... 5 5 5 9 9 9 9 . . . . . 2 2 2 2 ...... 5 5 5 5 5 1 1 1 1 1 9 9 1 1 1 . . . . . 2 2 2 2 ...... 5 5 5 5 5 1 1 1 1 1 1 1 1 1 1 2 2 . . . . . 5 5 5 1 1 1 1 1 1 1 1 1 1 . . . . . 1 1 1 1 1 1 1 1 . . . 1 1 1 ...... 1 1 1 . . . . 8 8 8 . . 1 1 1 1 1 . . . . 8 8 8 8 8 . . . . 4 4 4 . . . 1 1 1 1 1 . . 8 8 8 8 8 . . . . 4 4 4 4 4 . . . . . 1 1 1 1 1 . . . 8 8 8 8 8 . . . . 4 4 4 4 4 . . . . . 1 1 1 . . . . . 8 8 8 . . 4 4 4 4 4 ...... 4 4 4 0 0 0 0 0 . . . . . 8 8 8 0 0 0 0 0 4 4 4 8 8 8 8 8 0 0 0 0 0 4 4 4 4 8 8 8 8 8 0 0 0 4 4 4 4 1 1 1 7 7 7 8 8 8 8 8 7 7 7 4 4 4 4 1 1 1 1 1 7 7 7 7 7 8 8 8 . . . 7 7 7 7 7 4 4 4 1 1 1 1 1 7 7 7 7 7 . . . . . 7 7 7 7 7 1 1 1 1 1 7 7 7 7 7 . . . . . 7 7 7 7 7 1 1 1 7 7 7 . . . . . 7 7 7 . . . 1 1 1 1 1 1 6 6 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 6 6 6 6 6 6 6 1 1 1 1 1 1 1 1 1 1 1 5 5 5 3 3 3 3 3 6 6 6 6 6 6 6 6 6 1 1 1 1 1 1 1 1 1 5 5 5 5 5 3 3 3 3 3 6 6 6 6 6 6 6 1 1 1 1 1 1 1 5 5 5 5 5 3 3 3 6 6 6 6 6 1 1 5 5 5 5 5 3 3 3 6 6 6 5 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 6 6 6 3 3 3 3 3 3 3 3 3 3 2 2 2 2 8 8 8 6 6 6 6 6 3 3 3 3 3 3 3 3 2 2 2 2 8 8 8 8 8 6 6 6 6 6 3 3 3 3 3 4 4 4 2 2 2 2 2 2 2 8 8 8 8 8 6 6 6 6 6 3 3 3 4 4 4 4 4 2 2 2 2 2 2 2 2 8 8 8 8 8 6 6 6 4 4 4 4 4 2 2 2 2 2 8 8 8 4 4 4 4 4 2 2 2 2 2 2 2 2 4 4 4 1 1 4 4 4 4 4 1 1 1 1 4 4 4 4 4 1 1 1 1 3 3 . . . 4 4 4 4 4 1 1 1 1 3 3 3 3 . . . . . 4 4 4 2 2 2 1 1 1 1 3 3 3 3 . . . . . 2 2 2 2 2 2 2 2 1 1 1 1 7 7 7 3 3 3 3 4 4 4 . . . . . 2 2 2 2 2 2 2 2 2 2 1 1 1 1 7 7 7 7 7 3 3 4 4 4 4 4 . . . 2 2 2 2 2 2 2 2 2 2 1 1 1 1 7 7 7 7 7 4 4 4 4 4 2 2 2 2 2 2 2 2 1 1 7 7 7 7 7 4 4 4 4 4 2 2 2 7 7 7 4 4 4 6 6 6

0.015 6 6 6 6 6 6 6 6 6 6 3 3 3 6 6 6 . . 3 3 3 3 3 . . . . 3 3 3 3 3 . . . . 3 3 3 3 3 . . . . 3 3 3 1 1 1 8 8 8 . . 1 1 1 1 1 1 1 8 8 8 8 8 . . . 1 1 1 1 1 1 1 1 1 8 8 8 8 8 . . . . . 1 1 1 1 1 1 1 1 1 8 8 8 8 8 0 0 0 . . . . . 1 1 1 1 1 1 1 8 8 8 0 0 0 0 0 . . . . . 7 7 7 1 1 1 1 1 9 9 9 4 4 4 0 0 0 0 0 . . . 7 7 7 7 7 1 1 1 1 1 9 9 9 9 9 4 4 4 4 4 0 0 0 0 0 7 7 7 7 7 1 1 1 1 1 9 9 9 9 9 4 4 4 4 4 7 7 7 1 1 1 9 9 9 4 4 4 ...... 0 0 . . . 0 0 0 0 ...... 0 0 0 0 ...... 0 0 0 0 ...... 8 8 8 0 0 ...... 8 8 8 8 8 . . . 0 0 0 8 8 8 8 8 7 7 7 0 0 0 0 0 8 8 8 7 7 7 7 7 0 0 0 0 0 3 3 3 7 7 7 7 7 0 0 0 0 0 3 3 3 3 3 7 7 7 7 7 0 0 0 3 3 3 3 3 7 7 7 3 3 3 3 3 3 3 3 . . . 4 4 4 . . . . . 4 4 4 4 4 . . . . . 4 4 4 4 4 . . . . . 4 4 4 4 4 . . . 4 4 4 . . . 8 8 4 4 4 8 8 8 8 8 . . . . . 8 8 8 8 4 4 4 4 4 8 8 8 8 8 . . . . . 8 8 8 8 4 4 4 4 4 8 8 8 8 8 . . . 8 8 4 4 4 4 4 8 8 8 4 4 4 6 6 6 6 6 6 6 6 1 1 1 6 6 6 6 6 1 1 1 1 1 6 6 6 6 6 1 1 1 1 1 6 6 6 1 1 1 1 1 1 1 1 4 4 4 4 4 6 6 6 4 4 4 4 4 6 6 6 6 6 4 4 4 4 4 6 6 6 6 6 4 4 4 6 6 6 5 5 5 3 3 3 7 7 7 5 5 5 5 5 3 3 3 3 3 7 7 7 7 7 5 5 5 5 5 3 3 3 3 3 7 7 7 7 7 5 5 5 5 5 3 3 3 3 3 7 7 7 7 7 5 5 5 3 3 3 7 7 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 0 0 0 0 0 1 1 1 1 1 0 0 0 0 0 1 1 1 1 1 0 0 0 0 0 1 1 1 1 1 0 0 0 1 1 1 0 0 0 1 1 1 0 0 0 0 0

0.010 1 1 1 1 1 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.005

0 180 160 140 120 100 80 60 40 Chemical Shift (ppm)

13 Figure A23 . C NMR Spectrum (125 MHz, CDCl 3 ) of Phomopsichromin C ( 3 ) .

100

120

140

160

180

F2 Chemical Shift (ppm) 6 5 4 3 2 1

1 13 Figure A24 . H - C gHSQCAD NMR Spectrum (600 MHz, CDCl 3 ) of Phomopsichromin C ( 3 ) .

100

120

140

160

180

200

F2 Chemical Shift (ppm) 10 8 6 4 2 0

1 13 Figure A25 . H - C gHMBCAD NMR Spectrum (500 MHz, CDCl 3 ) of Phomopsichromin C ( 3 ) .

F2 Chemical Shift (ppm) 10 9 8 7 6 5 4 3 2 1

1 1 Figure A26 . H - H gCOSY NMR Spectrum (600 MHz, CDCl 3 ) of Phomopsichromin C ( 3 ) .

+ Phomopsichromin D ( 4 ) : C 23 H 34 O 5 ; HR ESI MS m/z 373.2373 [M + H – H 2 O] ( C 23 H 33 O 4

+ calculated, 373.2379) , m/z 391 .2473 [M + H] ( C 23 H 35 O 5 calculated, 391.2484 ) , m/z 413.2298

+ 20 [M + Na] ( C 23 H 34 O 5 Na calculated, 413.2304) ; UV (MeOH) λmax (log ε) 225 (5.89) nm; [α] D

+0.2 ( c 0.1, MeOH); IR (thin film) 3400, 2930, 1600, 1500, 1450, 1360, 1300, 1180, 1080 cm - 1 ;

1 H NMR (500 MHz, DMSO - d 6 ) δ ppm 0.74 (d, J =6.6 Hz, 3 H) , 0.75 (s , 3 H) , 0.84 (d, J =7.1 Hz,

3 H) , 1.13 ( m , 1 H) , 1.17 (m, 1 H) , 1.23 (m, 1 H) , 1.35 (m, 1 H) , 1.4 (m, 2 H) , 1.53 (m, 1H), 1.6

(m, 1 H) , 1.69 (m, 1 H), 1.70 (s, 3 H) , 1.75 (m, 1 H) , 2.38 (s, 3 H) , 3.16 (br d, J =7.1 Hz, 2 H) ,

3.62 (q, 2.4, 1 H) , 5.12 (br t, J =6.9 Hz, 1 H) , 6.23 (s, 1 H) , 10.02 (s, 1 H) , 12.61 (s, 1 H) ; 13 C

NMR (125 MHz, DMSO - d 6 ) δ ppm 12.7 ( CH 3 , C - 12), 15 .7 ( CH 3 , C - 13) , 16.1 ( CH 3 , C - 15 ) , 17.3

( CH 3 , C - 14 ) , 21.5 ( CH 2 , C - 11 ) , 23.8 ( CH 3 , C - 22 ) , 25.5 ( CH 2 , C - 4 ) , 32.4 ( CH 2 , C - 8), 34.1 ( CH 2 ,

C - 3 ) , 36.0 ( CH , C - 5 ) , 36.1 ( CH , C - 7 ) , 38.0 ( C, C - 6 ) , 39.0 ( CH , C - 1 ) , 70.4 ( CH , C - 2 ) , 103.6 ( C,

C - 18 ) , 110.4 ( CH , C - 20 ) , 112.0 ( C, C - 16 ) , 121.8 ( CH , C - 10 ) , 134.7 ( C, C - 9 ) , 139.8 ( C, C - 19 ) ,

159.6 ( C, C - 17 ) , 162.8 ( C, C - 21 ) , 174.1 ( C, C - 23 ) .

y t i M15(m) s DHD-5_PROTONa_01.esp n e t 5 5 5 5 5 5 M04(m) 5 5 5 n 7 7 7 7 7 7 7 7 7 7 I ......

0 0 0 0 0 0 0 0 0 0 d

e M13(m) z i l

a M06(m) M18(m) m r o 8 8 8 8 8 8 8 8 8 8 8 8 3 3 3 3 3 3 3 3 3 N 3 3 3 ...... M05(m) 2 2 2 2 2 2 2 2 2 2 2 2 M19(m) 0.8 0 0 0 0 0 0 0 0 0 0 0 0 7 7 7 7 7 7 7 7 7 7 7 7 ...... 1 1 1 1 1 1 1 1 1 1 0.7 3 3 3 3 3 3 3 3 0.6 3 3 3 3 7 7 7 7 7 7 7 7 7 7 7 7 ...... 4 4 4 4 4 4 4 4 4 4 4 4 0 0 0 0 0 0 0 0 0 0 0 0 8 8 8 8 8 8 8 8 8 8 8 8 ...... 0 0 0 0 0 0 0 0 0 0.5 0 0 0

M10(m) 0.4 M07(br d) M11(s) 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2

. . . . M17(br d) . . . . M12(s) . . 3 3 6 6 6 6 3 3 3 3 6 6 6 6 3 3 3 3 6 6 6 6 3 3 0 0 0 0 0 0 0 0 0 0 0 0 . .

0.3 ...... 6 6 6 6 6 6 6 6 6 5 5 5 5 5 5 5 5 5 5 5 5 2 2 2 2 2 2 2 2 2 2 2 2 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

M08(m) 1 1 1 1 1 1 . . . 6 6 . . . 6 6 6 6 . . . . . 6 6 6 6 1 1 . . . . 6 6 1 1 1 1 . . . . 1 1 1 1 . . 1 1 ...... 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 M09(br t) 1 1 2 2 1 1 1 1 2 2 2 2 2 2 2 1 1 1 1 2 2 2 2 2 2 2 1 1 2 2 2 2 2 2 2 2 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 ......

0.2 ...... 9 9 9 . . . . . 9 9 9 . . . 9 9 9 9 9 9 2 2 2 2 2 2 2 2 2 4 4 4 2 2 2 0 0 4 4 4 3 3 3 3 3 3 3 0 0 0 0 4 4 4 3 3 3 3 3 3 3 0 0 0 0 4 4 4 3 3 3 3 3 3 3 0 0 3 3 3 3 3 3 3 3 3 2 2 2 2 3 3 3 2 2 2 2 2 2 ...... 1 1 1 . . . 1 1 1 2 2 2 . . . 1 1 1 2 2 2 4 4 1 1 1 2 2 2 4 4 4 4 1 1 1 2 2 2 4 4 4 4 1 1 1 4 4 ...... 4 4 4 4 ...... 4 4 4 4 . . . . . 4 4 4 4 ...... 1 1 1 3 3 1 1 1 . . . . 3 3 3 3 7 7 7 1 1 1 . . . . 3 3 3 3 7 7 7 . . . . 3 3 7 7 7 7 7 7 5 5 5 5 5 5 . . . 5 5 5 . . . 1 1 1 1 1 3 3 . . . 1 1 1 1 1 1 1 3 3 3 3 1 1 1 1 . . . 1 1 1 1 1 1 1 3 3 3 3 1 1 1 1 1 1 3 3 1 1 1 1 7 7 2 2 7 7 7 7 2 2 2 2 7 7 7 7 2 2 2 2 7 7 ...... 1 1 1 . . . . 1 1 1 5 5 1 1 . . 1 1 1 5 5 5 5 1 1 1 1 1 1 1 1 1 5 5 5 5 1 1 1 1 1 1 1 1 5 5 1 1 1 1 1 1 4 4 4 4 4 4 4 4 4 4 . . 4 4 ...... 1 1 1 1 1 1 1 1 1 1 2 2 1 1 2 2 2 2 2 2 2 2 8 8 2 2 8 8 8 8 8 8 8 8 4 4 4 4 8 8 4 4 4 4 . . 5 5 4 4 4 4 . . . .

0.1 5 5 5 5 5 5 5 5 5 . . . . 5 5 5 5 5 5 5 5 5 5 5 . . 5 5 5 5 5 5 5 5 5 5 5 5 0 0 5 5 . . . . 0 0 0 0 . . . . 0 0 0 0 . . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 2 2 2 1 1 1 1 . . 2 2 2 1 1 . . . . 2 2 2 . . . . 2 2 2 2 2 1 1 1 1 . . 2 2 2 2 1 1 1 1 2 2 2 2 1 1 1 1 . . . 2 2 ...... 1 1 1 1 1 1 1 1 1 1 1 1 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 0 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)

1 Figure A27. H NMR Spectrum (500 MHz, DMSO - d 6 ) of Phomopsichromin D ( 4 ) .

y t i

s DHD-5_CARBON_01.esp n e t n I

d e z i l a m r o N

0.7

0.6

M12(s) 0.5 M11(s)

0.4 M09(s) M01(s) M10(s) M16(s) M02(s) 0.3 M13(s) M20(s) M22(s) M15(s) M06(s) M03(s) 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 ...... M19(s) . . 9 9 9 9 9 9 9 9 9 9 0.2 M21(s) M17(s) 9 9 M04(s) 3 3

3 3 3 3 M08(s) 3 3 3 3 M23(s) M18(s) M14(s) 3 3 5 5 5 5 5 5 5 5 5 5 5 5 8 8 8 2 2 8 8 8 3 3 2 2 2 2 8 8 8 3 3 3 3 2 2 2 2 3 3 3 3 2 2 5 5 3 3 7 7 5 5 5 5 7 7 7 7 4 4 4 4 5 5 5 5 7 7 7 7 4 4 4 4 2 2 5 5 7 7 4 4 4 4 2 2 2 2 3 3 3 3 1 1 1 2 2 2 2 3 3 3 3 1 1 1 3 3 4 4 4 4 4 . . 1 1 1 4 4 4 4 4 4 4 . . . . 6 6 1 1 1 1 1 1 4 4 4 4 4 4 4 . . . . 6 6 6 6 3 3 1 1 1 4 4 8 8 4 4 4 . . 6 6 6 6 3 3 3 3 1 1 1 8 8 8 8 6 6 3 3 3 3 1 1 1 8 8 8 8 7 7 3 3 8 8 7 7 7 0 0 9 9 9 0 0 8 8 6 6 6 7 7 7 7 9 9 9 7 7 7 0 0 0 0 9 9 9 0 0 0 8 8 8 8 6 6 6 . . . 7 7 7 7 9 9 9 0 0 0 0 9 9 9 0 0 0 8 8 8 8 6 6 6 8 8 . . . 7 7 . . 9 9 9 0 0 9 9 9 0 0 8 8 6 6 6 8 8 8 8 ...... 7 7 7 7 . . . . 8 8 8 8 ...... 7 7 7 7 . . . . 0 0 0 8 8 . . . . 7 7 7 7 1 1 . . 6 6 6 0 0 0 . . 1 1 1 1 6 6 6 4 4 4 4 . . . . 0 0 0 0 0 0 0 . . . . 1 1 1 1 6 6 6 4 4 4 4 . . . . 0 0 0 0 0 0 0 0 . . . . 1 1 6 6 6 4 4 4 4 . . . . 0 0 0 0 0 0 0 0 ...... 0 0 0 0 ...... 8 8 8 ...... 8 8 8 8 8 8 4 4 4 6 6 6 4 4 . . . 5 5 5 ...... 8 8 8 8 8 4 4 4 6 6 6 4 4 4 4 . . . 1 1 5 5 5 6 6 6 ...... 8 8 8 4 4 4 6 6 6 4 4 4 4 ...... 1 1 1 1 5 5 5 7 7 6 6 6 . . . . . 8 8 8 4 4 . . . . 1 1 1 1 5 5 5 7 7 7 7 6 6 6 2 2 2 2 . . . . 1 1 7 7 7 7 6 6 6 2 2 2 2 . . 7 7 2 2 2 2 3 3 ...... 5 5 5 5 . . . 3 3 3 3 . . . 2 2 ...... 5 5 5 5 . . . 3 3 3 3 . . 2 2 2 2 ...... 4 4 4 4 . . . 3 3 5 5 5 5 . . . 3 3 2 2 2 2 ...... 4 4 4 4 . . . 3 3 3 3 2 2 2 2 4 4 3 3 3 3 2 2 2 2 6 6 6 3 3 2 2 2 2 6 6 6 4 4 4 4 9 9 9 8 8 6 6 6 1 1 1 1 1 4 4 4 4 9 9 9 8 8 8 8 1 1 1 1 . . 1 1 1 4 4 4 4 6 6 9 9 9 8 8 8 8 1 1 1 1 . . . . 1 1 1 1 1 1 1 6 6 6 6 8 8 1 1 . . . . 1 1 1 1 1 1 1 6 6 6 6 1 1 . . 1 1 1 1 6 6 1 1 1 1 1 9 9 9 1 1 1 1 1 1 1 1 1 1 1 0 0 0 3 3 3 0 0 0 0 5 5 5 1 1 1 1 9 9 9 1 1 1 1 1 1 1 1 1 1 0 0 0 3 3 3 0 0 0 0 . . . . 5 5 5 2 2 1 1 1 1 9 9 9 1 1 1 1 0 0 0 3 3 0 0 . . . . 5 5 5 2 2 2 2 1 1 9 9 9 . . . . 2 2 2 2 1 1 1 1

0.1 3 3 3 2 2 1 1 1 1 7 7 5 5 5 3 3 3 1 1 7 7 7 7 5 5 5 3 3 3 7 7 7 7 5 5 5 3 3 3 3 3 3 3 2 2 2 2 7 7 5 5 5 3 3 3 3 2 2 2 2 1 1 1 1 3 3 3 3 2 2 2 2 1 1 1 1 2 2 2 1 1 2 2 2 1 1 1 0 0 3 3 3 2 2 2 1 1 1 1 1 1 1 0 0 0 7 7 7 7 4 4 2 2 2 3 3 3 1 1 1 1 1 1 1 0 0 0 7 7 7 7 4 4 4 4 2 2 2 3 3 3 1 1 1 1 1 1 1 0 0 7 7 7 7 4 4 4 4 2 2 2 4 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 1 1 1 1 1 1 1 1 3 3 3 3 1 1 1 3 3 3 3

220 200 180 160 140 120 100 80 60 40 20 Chemical Shift (ppm)

13 Figure A28. C NMR Spectrum (125 MHz, DMSO - d 6 ) of Phomopsichromin D ( 4 ) .

DHD-5_600_gHSQCAD.fid

100

110

120

130

140

150

160

170

180

190

200

210

220 F2 Chemical Shift (ppm) 11 10 9 8 7 6 5 4 3 2 1

1 13 Figure A29 . H - C gHSQCAD NMR Spectrum (600 MHz, DMSO - d 6 ) of Phomopsichromin D

( 4 ) .

DHD-5_gHMBCAD_01.fid.esp

-10

100

110

120

130

140

150

160

170

180

190

200

210

220

F2 Chemical Shif t (ppm) 11 10 9 8 7 6 5 4 3 2 1

1 13 Figure A30 . H - C gHMBCAD NMR Spectrum (500 MHz, DMSO - d 6 ) of Phomopsichromin D

( 4 ) .

DHD-5_gCOSY_01.fid.esp

F2 Chemical Shif t (ppm) 11 10 9 8 7 6 5 4 3 2 1

1 1 Figure A31 . H - H gCOSY NMR Spectrum (500 MHz, DMSO - d 6 ) of Phomopsichromin D ( 4 ) .

+ Phomopsichromin E ( 5 ) : C 23 H 32 O 5 ; HRESIMS m/z 371.2229 [M + H – H 2 O] ( C 23 H 31 O 4

+ + calculated 371.2222) , 389.2363 [M + H] ( C 23 H 33 O 5 calculated 389.2328 ) , 411.2152 [M + Na]

20 ( C 23 H 32 O 5 Na calculated 411.2147) ; UV (MeOH) λmax (log ε) 220 (4.14) ; [α] D - 0.4 ( c 0.1,

MeOH); IR (thin film) 3390, 2950, 1590, 1450, 1300, 1160, 1090, 1020 cm - 1 ; 1 H NMR Data

( 600 MHz, DMSO - d 6 ) δ ppm 0.46 (s, 3 H) , 0.76 (d, J =6.6 Hz, 3 H) , 0.81 (d, J =6.6 Hz, 3 H) , 1.23

(m , 1 H) , 1.32 (m, 1 H), 1.47 (m, 1 H) , 1.73 (s, 3 H) , 1.76 (m, 1 H) , 1.80 (m, 1 H) , 1.91 (m, 1

H) , 1.99 (s, 1 H) , 2.10 (m, 1 H) , 2.39 (s, 3 H) , 2.40 (m, 1 H) , 2.53 (q , J =6.7 Hz, 1 H) , 3.17 ( d,

J =7.1 Hz, 2 H) , 5.18 (br t, J =6.7 Hz, 1 H) , 6.23 (s, 1 H) , 10.04 (s, 1 H) , 12.62 (s, 1 H); 13 C NMR

(125 MHz, DMSO - d 6 ) δ ppm 7.5 (CH 3, C - 12), 14.7 (CH 3, C - 13), 15.0 (CH 3, C - 14), 16.1 (CH 3, C -

15), 21.5 (CH 2 , C - 11), 23.7 (CH 3, C - 22), 30.3 (CH 2 , C - 4), 32.0 (CH 2 , C - 8), 35.0 (CH, C - 5), 35.4

(CH 2 , C - 7), 40. 7 (CH 2 , C - 3), 42.7 (C, C - 6), 49.2 (CH, C - 1), 103.6 (C, C - 1 8), 110.4 (CH, C - 20),

111.9 (C, C - 16), 122.2 (CH, C - 10), 134.2 (C, C - 9), 139.9 (C, C - 19), 159.6 (C, C - 17), 162.8 (C,

C - 21), 174.1 (COOH, C - 23), 212.6 (C, C - 2).

2.5 DHD-8_1H1D_opt

2.0

1.5

1.0

0.5

0 13 12 11 10 9 8 7 6 5 4 3 2 Chemical Shift (ppm)

1 Figure A32. H NMR Spectrum ( 600 MHz, DMSO - d 6 ) of Phomopsichromin E ( 5 ) .

DHD8-1D-13C_121916

0.15

0.10 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 ...... 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 2 2 4 4 4 2 2 2 2 2 4 4 4 4 4 2 2 2 2 2 4 4 4 4 4 2 2 2 2 2

0.05 4 4 4 4 4 2 2 2 4 4 4 8 8 8 0 0 0 8 8 8 8 8 0 0 0 0 0 8 8 8 8 8 0 0 0 0 0 8 8 8 8 8 0 0 0 7 7 7 7 7 7 7 7 . . . 7 7 7 7 7 ...... 7 7 7 7 7 ...... 7 7 7 ...... 4 4 4 . . . 4 4 4 4 4 6 6 6 4 4 4 4 4 6 6 6 6 6 4 4 4 6 6 6 6 6 6 6 6 6 6 6 6 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 0 0 0 4 4 4 0 0 0 0 0 5 5 5 4 4 4 4 4 0 0 0 0 0 5 5 5 5 5 4 4 4 4 4 2 2 0 0 0 5 5 5 5 5 4 4 4 2 2 2 2 5 5 5 2 2 2 2 4 4 4 2 2 2 2 4 4 4 4 4 2 2 4 4 4 4 4 2 2 2 4 4 4 4 4 9 9 9 2 2 2 2 2 4 4 4 . . . 9 9 9 9 2 2 2 2 2 . . . . . 9 9 9 9 2 2 2 2 2 . . . . . 9 9 9 9 9 9 9 2 2 2 . . . . . 9 9 9 3 3 3 9 9 9 9 9 . . . 3 3 3 3 9 9 9 9 9 3 3 3 3 9 9 9 9 9 . . . . . 3 3 3 9 9 9 ...... 1 1 1 ...... 1 1 1 1 1 1 1 1 . . . . . 1 1 1 1 1 1 1 1 1 1 . . . . . 1 1 1 1 1 1 1 1 1 1 5 5 ...... 1 1 1 1 1 1 1 1 5 5 5 5 . . . . 1 1 1 5 5 5 5 . . . . 5 5 5 5 . . . . 5 5 . . . 5 5 5 0 0 0 8 8 8 8 8 5 5 5 5 5 0 0 0 0 8 8 8 8 8 5 5 5 5 5 0 0 0 0 7 7 7 . . 8 8 8 8 8 5 5 5 0 0 0 7 7 7 7 7 . . . . 8 8 8 7 7 7 7 7 . . . . 5 5 5 7 7 7 7 7 . . . . 5 5 5 5 5 7 7 7 . . 5 5 5 5 5 9 9 9 5 5 5 5 5 9 9 9 9 9 5 5 5 . . . 9 9 9 9 9 . . . . 9 9 9 9 9 . . . . 9 9 9 . . . . 0 0 0 ...... 0 0 0 0 . . . . . 0 0 0 0 . . . . . 0 0 0 . . . 2 2 2 3 3 3 2 2 2 2 2 8 8 8 3 3 3 3 3 2 2 2 2 2 8 8 8 8 3 3 3 3 3 2 2 2 2 2 8 8 8 8 3 3 3 3 3 7 7 2 2 2 8 8 8 8 3 3 3 7 7 7 7 8 8 8 7 7 7 7 3 3 3 7 7 7 7 3 3 3 3 3 7 7 3 3 3 3 3 . . . 4 4 4 4 4 3 3 3 3 3 2 2 2 2 . . . . . 4 4 4 4 4 2 2 2 2 . . . . . 4 4 4 4 4 2 2 2 2 3 3 3 . . . . . 4 4 4 2 2 2 3 3 3 3 3 3 3 3 . . . 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 2 2 2 . . . 3 3 3 3 2 2 2 2 2 . . . . 3 3 3 3 2 2 2 2 2 . . . . 3 3 3 3 2 2 2 2 2 2 2 2 . . . . 3 3 3 2 2 2 2 2 2 2 2 . . . 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 2 2 2 2 2 2 2 3 3 3 3 3 2 2 2 2 2 4 4 4 2 2 2 2 3 3 3 3 3 2 2 2 2 2 4 4 4 4 4 4 4 4 2 2 2 2 3 3 3 3 3 2 2 2 4 4 4 4 4 4 4 4 4 4 2 2 2 2 3 3 3 4 4 4 4 4 4 4 4 4 4 2 2 2 2 2 4 4 4 4 4 4 4 4 4 4 2 2 2 2 2 2 2 2 4 4 4 0 0 0 4 4 4 4 2 2 2 2 2 2 2 2 2 2 0 0 0 0 4 4 4 4 2 2 2 2 2 2 2 2 2 2 0 0 0 0 4 4 2 2 2 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 8 8 8 2 2 2 2 2 1 1 8 8 8 8 8 2 2 2 2 2 7 7 7 6 6 6 1 1 1 1 8 8 8 8 8 2 2 2 2 2 9 9 9 7 7 7 7 7 6 6 6 6 6 1 1 1 1 8 8 8 8 8 2 2 2 9 9 9 9 9 7 7 7 7 7 6 6 6 6 6 1 1 1 1 8 8 8 9 9 9 9 9 7 7 7 7 7 6 6 6 6 6 9 9 9 1 1 1 9 9 9 1 1 1 6 6 6 6 9 9 9 9 9 1 1 1 1 1 1 1 1 1 6 6 6 6 9 9 9 9 9 1 1 1 1 1 1 1 1 1 6 6 6 6 9 9 9 9 9 . . . 1 1 1 1 1 1 1 1 1 . . . 6 6 9 9 9 . . . . . 1 1 1 1 1 1 ...... 6 6 6 ...... 6 6 6 6 6 ...... 6 6 6 6 6 ...... 6 6 6 6 6 ...... 6 6 6 ...... 7 7 7 7 7 7 7 7 . . . 7 7 7 7 7 . . . . . 7 7 7 7 7 . . . . . 7 7 7 1 1 1 . . . . . 1 1 1 1 . . . 1 1 1 1 4 4 4 1 1 1 1 2 2 2 4 4 4 4 4 1 1 1 4 4 2 2 2 2 2 4 4 4 4 4 . . . 2 2 2 2 2 4 4 4 4 2 2 2 2 2 4 4 4 4 4 1 1 1 1 1 . . . . . 2 2 2 2 2 4 4 4 4 2 2 2 1 1 1 1 1 . . . . . 2 2 2 2 2 4 4 1 1 1 1 1 . . . 2 2 2 9 9 9 9 9 1 1 1 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 6 6 6 3 3 3 3 3 7 7 6 6 6 6 6 3 3 3 3 3 2 2 2 1 1 1 7 7 7 7 6 6 6 6 6 3 3 3 3 3 1 1 1 2 2 2 2 2 1 1 1 1 1 7 7 7 7 6 6 6 6 6 3 3 3 1 1 1 1 1 2 2 2 2 2 1 1 1 1 1 7 7 7 7 6 6 6 1 1 1 1 1 2 2 2 2 2 1 1 1 1 1 7 7 1 1 1 1 1 2 2 2 1 1 1 5 5 3 3 3 1 1 1 5 5 5 5 3 3 3 3 3 5 5 5 5 3 3 3 3 3 5 5 5 5 3 3 3 3 3 5 5 3 3 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 4 4 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 4 4 4 4 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 4 4 4 4 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 4 4 4 4 4 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 4 4 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0 220 200 180 160 140 120 100 80 60 Chemical Shift (ppm)

13 Figure A33 . C NMR Spectrum (201 MHz, DMSO - d 6 ) of Phomopsichromin E ( 5 ) .

100

120

F2 Chemical Shift (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

1 13 Figure A34 . H - C gHSQCAD NMR Spectrum (600 MHz, DMSO - d 6 ) of Phomopsichromin E

( 5 ) .

100

150

200

F2 Chemical Shift (ppm) 10 9 8 7 6 5 4 3 2 1

1 13 Figure A35 . H - C gHMBCAD NMR Spectrum (600 MHz, DMSO - d 6 ) of Phomopsichromin E

( 5 ) .

F2 Chemical Shift (ppm) 10 9 8 7 6 5 4 3 2 1

1 1 Figure A36. H - H gCOSY NMR Spectrum (600 MHz, DMSO - d 6 ) of Phomopsichromin E ( 5 ) .

+ LL - Z1272ε ( 6 ): C 23 H 32 O 4 ; HRESIMS m/z 373.2411 [M + H] ( C 23 H 33 O 4 calculated 373.2334) ;

UV (MeOH) λmax (log ε) 224 (4.03) nm ; IR (thin film) 3400, 2950, 2360, 1600, 1460, 1380,

- 1 1 1300, 1080 cm ; H NMR Data ( 6 00 MHz, CDCl 3 ) δ ppm 0.59 (s, 3 H) , 0.89 (d, J =6. 6 Hz, 3 H) ,

0.92 (d, J =6. 6 Hz, 3 H) , 1.39 (m, 1 H) , 1.45 (m, 1 H) , 1.62 (m, 1 H) , 1.85 (m, 1H), 1.85 (s, 3 H) ,

1.91 (m, 1 H) , 2.00 (m, 1 H) , 2.06 (m, 1 H) , 2.34 (m, 1 H), 2.35 (m, 1 H) , 2.47 (q, J =6.5 H z, 1

H) , 2.51 (s, 3 H) , 3.41 (d, J =7.1 Hz, 2 H) , 5.29 (br t, J =6.6 Hz, 1 H) , 6.12 (br s , 1 H) , 6.23 (s, 1

13 H) , 10.09 (s, 1 H) , 12.76 (s, 1 H) ; C NMR (125 MHz, CDCl 3 ) δ ppm 7.5 ( CH 3 , C - 12 ) , 1 5.0

( CH 3 , C - 13 ) , 15.3 ( CH 3 , C - 14 ) , 16.5 ( CH 3 , C - 15 ) , 18 .0 (CH 3 , C - 22), 21.2 ( CH 2 , C - 11 ) , 30.9

( CH 2 , C - 4 ) , 32.6 ( CH 2 , C - 8 ) , 35.6 ( CH 2 , C - 7 ) , 36.1 ( CH, C - 5 ) , 41. 5 ( CH 2 , C - 3 ) , 43.4 (C - 6 ) , 50.4

( CH, C - 1 ) , 110.6 ( CH, C - 20 ) , 111.5 ( C - 16 ) , 113.2 ( C - 18 ) , 121.1 ( CH, C - 10 ) , 139.2 ( C - 9 ) , 142.0

( C - 19 ) , 162.1 ( C - 21 ) , 163.5 ( C - 17 ) , 193.0 ( CH, C - 23 ) , 213.9 ( C - 2 ) .

DHD-4_April16_600_Proton.esp M18(s) M20(br d) M09(m) 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 1 1 1 1 1 1 1 1 . . . 1 1 1 1 1 . . . . . 1 1 1 1 1 . . . . . 1 1 1 . . . . . M12(m) . . . 5 5 5 5 5 5 5 5 0 0 0 5 5 5 5 5 0 0 0 0 0 5 5 5 5 5 0 0 0 0 0 5 5 5 0 0 0 0 0 0 0 0 ...... 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1.0 2 2 2 M11(m)

M13(s) 0.9 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 ...... 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.8

0.7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 . . . 9 9 9 9 9 . . . . . 9 9 9 9 9 . . . . . 9 9 9 ...... 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.6 0 0 0

0.5 M02(s) M04(m) M06(br d) 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 M01(s) 9 9 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ...... 0.4 . . . M03(s) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 1 1 1 1 1 0 0 0 0 1 1 1 1 1 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 6 6 6 6 6 1 1 1 1 1 4 4 4 6 6 6 6 6 1 1 1 4 4 4 4 4 4 4 6 6 6 6 6 4 4 4 4 4 4 4 4 4 6 6 6 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 ...... 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 . . . 3 3 3 . . . . . 3 3 3 3 3 3 3 . . . . . 3 3 3 3 3 3 3 3 3 . . . . . 3 3 3 3 3 3 3 3 3 . . . 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ...... 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 0.3 6 6 6 4 4 4 4 4 4 4 4 5 5 5 4 4 4 4 5 5 5 5 5 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 3 3 3 3 3 3

M05(br t) 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 ...... 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 2 2 2

0.2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ...... 9 9 9 . . . . . 9 9 9 9 9 . . . 9 9 9 9 9 9 9 9 9 9 9 9 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 5 5 5 5 5 5 5 8 8 8 5 5 5 5 5 8 8 8 8 8 5 5 5 5 5 8 8 8 8 8 5 5 5 8 8 8 8 8 5 5 5 8 8 8 5 5 5 5 5 5 5 5 5 5 5 5 5 2 2 2 2 5 5 5 5 5 8 8 8 8 8 5 5 5 5 5 2 2 2 2 8 8 8 8 8 5 5 5 5 5 2 2 2 2 8 8 8 8 8 5 5 5 2 2 8 8 8 9 9 9 9 9 9 9 9 9 9 . . . 9 9 9 9 . . . . . 9 9 . . . . . 0 0 0 . . . . . 4 4 4 0 0 0 0 0 . . . 4 4 4 4 4 3 3 3 0 0 0 0 0 4 4 4 4 4 3 3 3 3 3 0 0 0 . . 4 4 4 4 4 3 3 3 3 3 . . . . 3 3 3 ...... 1 1 2 2 2 . . 1 1 1 1 2 2 2 2 2 . . . 1 1 1 1 2 2 2 2 2 ...... 1 1 1 1 2 2 2 2 2 ...... 1 1 2 2 2 ...... 1 1 1 1 1 ...... 1 1 1 1 1 . . . . 3 3 3 . . . . . 1 1 1 1 1 . . . . 3 3 3 3 3 . . . . . 1 1 1 . . 3 3 3 3 3 . . . 3 3 3 3 3 3 3 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 . . . 1 1 1 . . . . . 1 1 1 1 1 1 1 1 . . . . . 1 1 1 1 1 1 1 1 1 1 . . . 1 1 1 1 1 1 1 1 1 1 1 6 6 5 5 5 6 6 6 6 5 5 5 5 5 6 6 6 6 5 5 5 5 5 6 6 6 6 5 5 5 5 5 6 6 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 0.1 5 5 5

0 13 12 11 10 9 8 7 6 5 4 3 Chemical Shift (ppm)

1 Figure A37 . H NMR Spectrum (6 00 MHz, CDCl 3 ) of LL - Z1272ε ( 6 ) .

5 5 5 0 0 0 5 5 5 5 5 5 5 5 0 0 0 0 0 5 5 5 5 5 5 5 5 5 5 0 0 0 0 0 5 5 5 5 5 5 5 5 0 0 0 5 5 5 2 2 2 0 0 0 7 7 7 2 2 2 2 2 0 0 0 0 0 7 7 7 7 7 2 2 2 2 2 0 0 0 0 0 7 7 7 7 7 2 2 2 2 2 0 0 0 0 0 7 7 7 7 7 DHD-4_dd500_CARBON_01_BEST.esp 2 2 2 0 0 0 7 7 7 ...... 7 7 7 7 7 7 6 6 6 7 7 7 7 7 7 7 7 7 7 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 6 6 6 6 6 7 7 7 7 7 7 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

0.09

0.08

0.07

0.06

0.05

0.04 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1 3 3 3 1 1 1 1 1 7 7 8 8 8 1 1 1 1 1 7 7 7 7 8 8 8 8 1 1 1 1 1 7 7 7 7 4 4 4 0 0 0 0 0 8 8 8 8 1 1 1 7 7 7 7 4 4 4 4 4 0 0 0 0 0 0 0 0 0 8 8 8 8 7 7 4 4 4 4 4 0 0 0 0 0 0 0 0 0 8 8 8 4 4 4 4 4 0 0 0 0 0 0 0 0 0 3 3 3 4 4 4 0 0 0 0 0 3 3 3 3 3 3 3 3 . . . 3 3 3 3 4 4 4 4 4 . . . . . 3 3 3 4 4 4 4 4 5 5 . . . . . 2 2 2 4 4 4 4 4 5 5 5 5 . . . 2 2 2 2 2 4 4 4 5 5 5 5 2 2 2 2 2 2 2 2 5 5 5 5 2 2 2 2 2 2 2 2 2 2 5 5 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 6 6 6 6 6 6 6 6 6 6 6 6 6 0 0 1 1 1 6 6 6 6 6 6 6 6 0 0 0 0 0 0 0 1 1 1 1 1 6 6 6 6 5 5 5 6 6 6 6 0 0 0 0 0 0 0 0 0 1 1 1 1 1 6 6 6 5 5 5 5 5 6 6 0 0 0 0 0

0.03 0 0 0 0 1 1 1 1 1 5 5 5 5 5 . . . . . 3 3 3 0 0 0 5 5 5 . . . . . 3 3 3 3 6 6 6 . . . . . 3 3 3 3 6 6 6 6 6 . . . 3 3 3 3 . . . 4 4 4 6 6 6 6 6 2 2 2 . . 3 3 3 . . . . 4 4 4 4 4 6 6 6 6 6 2 2 2 2 2 ...... 4 4 4 4 4 4 4 6 6 6 2 2 2 2 2 . . . . 9 9 9 . . . 9 9 9 ...... 4 4 4 4 4 4 4 4 4 2 2 2 2 2 . . . . 9 9 9 9 9 . . . . . 9 9 9 9 9 ...... 4 4 4 4 4 4 4 2 2 2 . . 9 9 9 9 9 . . . . . 9 9 9 9 9 ...... 4 4 4 4 9 9 9 9 9 . . . . . 9 9 9 9 9 . . . . . 4 4 9 9 9 ...... 9 9 9 . . . . 3 3 3 ...... 3 3 3 3 3 ...... 3 3 3 3 3 ...... 5 5 5 . . . 3 3 3 3 3 ...... 5 5 5 5 5 . . . . . 1 1 1 3 3 3 ...... 5 5 5 5 5 . . . . . 1 1 1 1 1 . . . . 5 5 5 5 5 . . . . . 1 1 1 1 1 . . 5 5 5 . . . 1 1 1 1 1 3 3 3 1 1 1 3 3 3 3 3 2 2 0 0 0 3 3 3 3 3 6 6 6 2 2 2 2 0 0 0 0 3 3 3 3 3 6 6 6 6 6 2 2 2 2 5 5 5 3 3 1 1 1 0 0 0 0 3 3 3 6 6 6 6 6 2 2 2 2 5 5 5 5 5 5 5 5 3 3 3 3 1 1 1 1 1 0 0 0 0 . . . . . 6 6 6 5 5 5 5 5 5 5 5 5 5 3 3 3 3 1 1 1 1 1 . . . . . 5 5 5 5 5 5 5 5 5 5 5 3 3 1 1 1 . . . . . 5 5 5 5 5 5 5 . . . 5 5 5 5 5 5 5 5 0 0 0 5 5 5 5 5 5 0 0 0 0 0 . . . 5 5 5 5 5 0 0 0 0 0 3 3 . . . . . 0 0 0 5 5 5 5 5 . . . 0 0 0 0 0 3 3 3 3 . . . . . 0 0 0 0 0 5 5 5 5 5 . . . . . 0 0 0 3 3 3 3 . . . . . 0 0 0 0 0 2 2 2 5 5 5 . . . . . 3 3 3 3 . . . 0 0 0 0 0 2 2 2 2 2 . . . . . 4 4 4 3 3 0 0 0 2 2 2 2 2 . . . 4 4 4 4 4 2 2 2 2 2 4 4 4 4 4 1 1 1 3 3 4 4 4 1 1 1 1 3 3 3 3 . . . 9 9 2 2 2 1 1 1 1 3 3 3 3 . . . . . 1 1 1 9 9 9 9 2 2 2 2 2 1 1 1 1 1 1 1 3 3 3 3 . . . . . 1 1 1 1 1 9 9 9 9 2 2 2 2 2 1 1 1 1 1 1 1 1 3 3 . . . . . 1 1 1 1 1 9 9 9 9 2 2 2 2 2 1 1 1 1 1 . . . 1 1 1 1 1 9 9 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 5 5 5 5 5 1 1 1 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 4 4 5 5 5 5 5 3 3 3 3 3 7 7 7 7 7 5 5 5 4 4 4 4 5 5 5 3 3 3 3 3 7 7 7 7 7 2 2 2 4 4 4 4 3 3 3 3 3 7 7 7 7 7 2 2 2 2 2 4 4 3 3 3 7 7 7 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 6 6 6 1 1 1 1 6 6 6 6 6 1 1 1 1 1 7 7 7 1 1 1 1 6 6 6 6 6 1 1 1 1 1 1 1 1 1 7 7 7 7 7 1 1 1 1 4 4 4 6 6 6 6 6 1 1 1 1 1 1 1 1 1 7 7 7 7 7 1 1 1 4 4 4 4 4 6 6 6 1 1 1 1 1 1 1 1 1 7 7 7 7 7 4 4 4 4 4 1 1 . . . . . 1 1 1 7 7 7 4 4 4 4 4 ...... 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 6 6 6 2 2 2 6 6 6 6 6 6 6 6 6 6 1 1 1 9 9 9 9 9 6 6 6 6 6 2 2 2 2 2 1 1 1 1 1 9 9 9 9 9 2 2 2 2 2 1 1 1 1 1 9 9 9 9 9 2 2 2 2 2 1 1 1 9 9 9 2 2 2 9 9 9 9 9 9 9 9 1 1 9 9 9 9 9 1 1 1 1 9 9 9 9 9 1 1 1 1 9 9 9 1 1 1 1 . . . 1 1 . . . . . 9 9 9 . . . . . 9 9 9 9 . . . . . 9 9 9 9 1 1 1 9 9 9 1 1 1 1 1 1 1 1 1 1 5 5 5 1 1 1 1 1 5 5 5 5 5 1 1 1 5 5 5 5 5 5 5 5 5 5 . . . 5 5 5 . . . . 3 3 3 . . . . 3 3 3 3 3 ...... 0 0 3 3 3 3 3 ...... 0 0 0 0 3 3 3 3 3 ...... 0 0 0 0 3 3 3 ...... 0 0 3 3 3 . . . . . 3 3 3 3 3 . . . . . 3 3 3 3 3 . . . 3 3 3 3 3 3 3 3 ......

0.02 ...... 3 3 3 3 1 1 1 3 3 3 3 1 1 1 1 1 3 3 3 3 2 2 2 1 1 1 1 1 3 3 3 2 2 2 2 2 1 1 1 1 1 2 2 2 2 2 1 1 1 3 3 3 2 2 2 2 2 1 1 1 3 3 3 3 3 2 2 2 1 1 1 1 1 3 3 3 3 3 1 1 1 1 1 3 3 3 3 3 1 1 1 1 1 3 3 3 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 6 6 1 1 1 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 1 1 1 6 6 6 6 6 1 1 1 1 1 6 6 6 1 1 1 1 1 1 1 1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.01

0 220 200 180 160 140 120 100 80 60 40 Chemical Shift (ppm)

13 Figure A38 . C NMR Spectrum (125 MHz, CDCl 3 ) of LL - Z1272ε ( 6 ) .

Table A2 . Pure compound bioactivities of 1 - 6 .

Pure Compound Bioactivities

Compound MRSA L. donovani Other IC 50 (µM) IC 50 (µM) 1 518 3 2 515 1.9

MRSA MBC 99 : 47 µM 3 58 0.67 HepG4 LD 50 : 37 µM 4 NA NA 5 515 0.80 6 NA 13

Appendix B : Experimental and Supporting Data for Ch. 3

Microbial Isolation Protocols Screening Protocols LC - QToF - MS Protocols Metabolomic and Statistical Analysis Protocols Scale - up Protocols

Microbial Isolation Protocols

Nutrient media components (SDB, PDB, TSA, Actinomycete Isolation Agar, Malt Extract Agar) and agar were produced by BD ™ Difco ™ and purchased through Fisher Scientific. Glycerol,

nystatin, cycloheximide, an d chloramphenicol were purchased from Sigma Aldrich ® .

Solid media was mixed, heated, and autoclaved according to manufacturer’s instructions and poured to set in Fisherbrand ™ petri dishes.

After collections, f ield plates were incubated at 20 - 25°C (room temperature), 4°C (refrigerated), or 26 - 30°C (heated) as source organism or microbial targets dictated. Generally, bacterial plates were heated and fungal plates were left at room temperature for collections in w arm climates.

Cold water microbial isolation generally took place with refrigeration. Field plates were incubated for 1 - 4 weeks, and disposed of after all colonies were isolated and/or after the whole plate was covered in microbial growth.

Isolated p ure co lonies were grown on SDA (fungi) and TSA (bacteria) for archiving as described above . Descriptions of each organism on this standard media were recorded.

Screening Protocols

Each f ungal isolat e was gr own on SDA from either glycerol stocks or isolation pla tes, and after a

colony was established, was subsampled for screening. 1cm cubes of fungal material and agar

were inoculated in triplicate into 3 sterile Eppendorf ™ tubes containing 1.25mL each : SDB

(control), 100 µM sodium butyrate in SDB (HDACi), and 100µM 5 - aza - cytidine in SDB

(DNMTi). Sodium butyrate and 5 - aza - cytidine were purchased from Sigma Aldrich ® . The

SDB/modifier/ fungal mixture was agitated, and then each poured over 1g rice media in a

Fisherbrand ™ 20mL glass scintillation vial. Rice media was made by autoclaving 1g (1/4 tsp) of

brown rice with 4mL DI water. Once inoculated, rice vials were incubated at 28°C for 21 days.

After 21 days, all contaminated/ non - growing cult ure sets were removed. Cultures we re s pritz ed

with ~500µL d istilled MeOH . The f ungal rice cake was broken apart with a clean spatula. 10mL

d istilled EtOAc was added using a glass 10mL pipette and a llow to extract overnight on bench

top . Extracts were carefully decanted into clean, pre - weig hed scintillation vials after 24 hours.

Extracts were dried under air for 24 hours, resuspended in DMSO at a concentration of

10mg/mL, and plated in 96 - well format on a TECAN Freedom EVO 150 liquid handling automated workstation . Five replicate Corning ™ clear polystyrene 96 - well microplates were prepared with 150µL of extracts/ well for bioassay. Remaining extract material was stored in duplicate Fisherbrand ™ 96 - well DeepWell ™ polypropylene microplates. All extract plates were stored at - 20°C.

LC - QT oF - MS Protocols

Extracts were dried of DMSO and suspended in MeOH at a concentration of 0.1mg/mL. The

resulting solution was filtered over 0.2µm Phenomenex ® RC syringe filters and were injected in

triplicate on the LC - QToF - MS for analysis.

Analysis was completed on an Agilent 6540 LC/QTOF with Agilent Jet - stream Electrospray

Ionization . A Kinetex C18 (5µm, 100 Å, 2.1mm ID, 50mm length) column was used with the

following instrument parameters:

Acquisition Method Report

TOF/Q - TOF Mass Spectrometer Parameters: Component Name MS Q - TOF Component Model G6540A Tune File Autotune.tun MS Abs. threshold 200 MS Rel. threshold (%) 0.010 Can wait for temp. Enable Fast Polarity N/A Ion Source Dual AJS ESI Stop Time (min) No Limit/As Pump Storage Mode Both Ion Mode Dual AJS ESI Acquisition Mode MS1: Scan Rate (spectra/sec) 5.46 Max Range (m/z) 1000 Min Range (m/z) 50 Source Parameters: Sheath Gas Flow 8 Sheath Gas Temp 300 Nebulizer (psig) 40 Gas Flow (l/min) 10 Gas Temp (°C) 300 Ion Polarity Positive Scan Source Parameters: Octopole RF Peak 750 Skimmer1 65 Fragmentor 125 Nozzle Voltage (V) 1000

VCap 3500 Chrom Type : TIC; Label : TIC; Offset : 15; Y - Range : 10000000 Auto Recalibration : Min Height (counts) 1000 Detection Window (ppm) 100 Average Scans 1 Reference Masses : Ref Nebulizer (psig) 0 Use Bottle A Ref Nebulizer True Ref Mass Enabled Enabled Reference Masses : 922.00979800 121.05087300 HiP Sampler Parameters: Compon ent Name HiP Samplet Component Model G1367E Draw Speed 100µL/min Eject Speed 100µL/min Draw Position Offset 0.0 mm Wait Time After Drawing 0.0 s Sample Flush Out Factor 5.0 Vial/Well bottom sensing Yes Injection Mode Injection with needle wash Injection Volume 10.0µL/min Needle Wash Location Flush Port Wash Time 5.0 s Automatic Delay Vol Red No Enable Overlap. Inj. No Valve Switching No Stop Time As pump/ No limit Post Time Mode Off Pump Parameters: Component Name Binary Pump Component Model G1312B Flow 0.600 mL/min Use Solvent Types Yes Low Pressure Limit 0.00 bar High Pressure Limit 600.00 bar Maximum Flow Gradient 100.000 mL/min 2 Auto Stroke Calc. A Yes Auto Stroke Calc. B Yes Compressibility A 50 10e - 6/bar Compressibility B 115 10e - 6/bar Stoptime 9.00 min Posttime 1.00 min

100

Timetable Time Function Parameter 1 0.00 min Change Flow Flow 0.6 mL/min 2 0.00 min Change Solvent Composition Solvent Composition A: 75.0 % B:25.0 % 3 0.50 min Change Solvent Composition Solvent Composition A: 75.0 % B:25.0 % 4 3.00 min Change Solvent Composition Solvent Composition A: 5.0 % B:95 .0 % 5 7.00 min Change Solvent Composition Solvent Composition A: 5.0 % B:95 .0 % 6 8.00 min Change Solvent Composition Solvent Composition A: 75.0 % B:25.0 % 7 9.00 min Change Flow Flow 0.6 mL/min 8 9.00 min Change Solvent Composition Solvent Composition A: 75.0 % B:25.0 % Solvent Composition Channel Solvent 1 Name 1 Used Percent 1 A H 2 O H 2 O+0.1% Formic Acid Yes 75.0 % 2 B ACN ACN+0.1% Formic Acid Yes 25.0 % Column Comp. Parameters: Component Name Column Comp Component Model G1316A Left Temperature Control: Temperature Control Mode Temperature Set Temperature 35.00 °C Enable Analys. Left Temp No Right Temperature Control: Right Temp Cont Mode Combined Enable Analys. Right Temp No Stoptime mode As pump/injector Posttime mode Off

Metabolomic and Statistical Analysis Protocols

Each sample for metabolomics analysis was prepared as above and run in triplicate

according to the run parameters above. Resulting chromatograms were subjected to processing

by Agilent MassHunter Qualitative Analysis. First, a list of compounds was generat ed using the

Find By Molecular Feature tool. A peak height cutoff was set at 100 counts and results were

limited to the largest 5000 compounds. Results were exported to .cef files that were transferred

to Agilent Mass Profiler Professional (MPP). In MPP re plicates were averaged and blanks were

subtracted. The resulting table of samples and chemical entities (mass @ retention time) was

101

exported via Microsoft Excel and opened in Primer 6 for statistical analysis (Clarke, K. R.;

Gorley, R. N. 2006. PRIMER v6: User Manual/Tutorial. Primer - E, Plymouth) . Abundances were

square root normalized and factors such as culture conditions and bioactivities were added to

each sample identity. A Bray - Curtis similarity matrix was constructed from which cluster

(dendogram) an d multidimensional scaling (MDS) plots could be created.

Scale - up Protocols

A scale - up protocol that mirrored the screening culture protocol was developed. The optimized

procedure was enacted for all scale - up level screening. Rice media was prepared in a Type 3T

Unicorn bag according to the following procedure: 300g of brown rice was mixed with 500mL

DI water and a heat sealer was used to seal the bag. Rice was autoclaved on a liquid cycle for 30 minutes at 121°C. Each hit organism chosen for scale - up was grown on SDA from either glycerol stocks or isolation plates, and after a colony was established, was subsampled for screening. 1cm cubes of fungal material and agar were inoculated in triplicate into 3 sterile 50 mL Falcon ™ tubes containing 50mL each: SDB (control), 100 µM sodium butyrate in SDB

(HDACi), and 100µM 5 - aza - cytidine in SDB (DNMTi). Rice bags were cut open in a sterile environment, the fungal/ liquid media mixture poured in, and resealed. Rice was agitated once a

week for a 21 day culture period. Extraction was completed by spritzing the culture with distilled

MeOH to dampen the spores , transferring the material to a large beaker, and extracting overnight

in a 1:3 Me OH: EtoAc mixture, followed by two 24 - hour extr actions in EtOAc. Extracts were

collected, filtered, and dried down for chemical analysis.

102

Appendix C: Experimental and Supporting Data for Ch. 4

Fungal Strain Identification MPLC Run Parameters and Chromatograms HPLC Run Parameters and Chromatograms Compound Data

Fungal Strain Identification

The fungal isolate KML12 - 14MG - B2a was sequenced using sanger sequencing of the 18S ribosomal spacer region. The resulting sequences were blasted using NCBI nucleotide blast for sequence si milarity.

Figure C1 . NCBI nucleotide blast results for KML12 - 14MG - B2a forward p rimer s .

Figure C2 . NCBI nucleotide blast results for KML12 - 14MG - B2a reverse primers.

103

MPLC Run Parameters and Chromatograms

Figure C3 . NP MPLC chromatogram of KML12 - 14MG - B2a_HDAC EtOAc partition. Control and DNMT EtOAc partitions were run with the same parameters.

HPLC Run Parameters and Chromat ograms

Normal phase HPLC s eparation was completed wit h a normal phase gradient of hexanes to e thyl acetate over 35 minutes on a semi - preperative Phenominex ® Luna Silica (2) column (5µm,

100Å , 250 x 10mm ) using UV and ELSD detection.

104

Figure C4. NP HPLC ELSD trace of KML12 - 14MG - B2a_Control_D . This fraction from MPLC was most similar in elution time to fraction KML12 - 14MG - B2a_HDAC_F and was used for comparison.

Figure C5. NP HPLC ELSD trace KML12 - 14MG - B2a_HDAC_F . Citreohybriddione D was isolated from the most abundant peak, fraction 2.

105

Figure C6. NP HPLC ELSD trace of KML12 - 14MG - B2a_DNMT_C. This fraction from MPLC was most similar in elution time to fraction KML12 - 14MG - B2a_HDAC_F and was used for comparison.

Compound Data

+ Citreohybriddione D ( 1 ): C 28 H 38 O 8 ; HRESIMS m/z 443.2431 [M - OAc] ( C 26 H 35 O 6 calculated,

+ + 443.2434) , 503.2640 [M + H] ( C 28 H 39 O 8 calculated, 503.2645 ), 525.2465 [M + Na]

20 1 ( C 28 H 38 O 8 Na calculated, 525.2464) ; [α] D + 0.4 ( c 0.1, MeOH); H NMR Data ( 5 00 MHz,

CDCl 3 ) δ ppm 0.90 (s, 3 H) , 0.97 (s, 3 H) , 1.03 (m, 1 H) , 1.17 (s, 3 H) , 1.31 (s, 3 H) , 1.40 (s, 3

H) , 1.68 (m, 1 H) , 1.70 (m , 3 H) , 1.80 (m, 1 H), 1.83 (m , 1 H) , 2.01 (m, 1 H) , 2.12 (s, 3 H) , 2.16

(s, 1 H) , 2.21 (m, 1 H) , 2.38 (m , 1 H) , 2.83 (m, 1 H) , 3.63 (s, 3 H) , 4.68 (t, J =2.6 Hz, 1 H) , 5.85

13 (s, 1 H) , 10.14 (s, 1 H) ; C NMR (200 MHz, CDCl 3 ) δ ppm 16.4 (CH 3, C - 20 ), 16.9 (CH 2 , C - 6),

18.8 (CH 3 , C - 21), 19.5 (CH 3 , C - 22), 19.8 (CH 3 , C - 18), 21.3 (CH 3 , C - 25), 21.3 (CH 3 , C - 27), 23.3

(CH 2 , C - 2), 26.5 (CH 3 , C - 24), 27.8 (CH 2 , C - 1), 30.8 (CH 2 , C - 7), 36.9 (C, C - 4), 38.6 (C, C - 8),

47.7 (CH, C - 5), 52.0 (CH 3 , C - 28), 52.2 (C, C - 10), 53.5 (CH, C - 9), 60.8 (C, C - 13), 70.4 (C, C -

14), 72.1 ( C, C - 16), 76.8 (CH, C - 3), 133.0 (C, C - 12), 126.4 (CH, C - 11), 167.3 ( C, C - 19), 170.6

(C, C - 26), 204.4 (CH, C - 23), 206.7 (C, C - 17), 210.6 (C, C - 15).

106

1.0 KML12-14MG-B2a_HDAC_F2-1f_dd500_PROTON_01.esp

0.9

0.8 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ...... 2 2 2 2 2 2 2 2 3 3 3 2 2 2 2 2 3 3 3 3 3 2 2 2 2 2 3 3 3 3 3 2 2 2 3 3 3 3 3 0.7 3 3 3 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 ...... 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 4 4 4 4 4 4 4 4 4 4 4 ...... 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 ...... 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.6 1 1 1 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 ...... 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0

0.5 0 0 0 0 0 0 0 0 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 9 9 9 7 7 7 9 9 9 9 9 7 7 7 7 7 9 9 9 9 9 7 7 7 7 7 9 9 9 7 7 7 7 7 7 7 7 ...... 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 9 9 9 9 9 9 9 9 9 9 9 9 9 . . . 9 9 9 9 9 . . . . 9 9 9 ...... 0 0 0 . . . 0 0 0 0 0 . . . . . 0 0 0 0 0 . . . . . 0 0 0 ...... 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.3 0 0 0 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 ...... 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 4 4 4 1 1 1 4 4 4 4 1 1 1 1 4 4 4 4 1 1 1 1 4 4 4 4 1 1 1 1 4 4 4 1 1 1 ...... 0.2 . . . 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 . . . 2 2 2 2 . . . . 2 2 2 2 . . . . 2 2 2 2 . . . . 2 2 2 2 2 0 0 0 2 2 2 2 0 0 0 0 2 2 2 2 0 0 0 0 2 2 2 2 0 0 0 0 2 2 0 0 0 8 8 8 8 8 8 8 8 5 5 5 8 8 8 8 8 5 5 5 5 5 8 8 8 8 8 5 5 5 5 5 5 5 5 0 0 1 1 1 0 0 0 0 1 1 1 1 0 0 0 0 1 1 1 1 0 0 0 0 1 1 1 1 0 0 1 1 1 6 6 6 . . 6 6 6 6 6 . . . . 8 8 8 6 6 6 6 6 . . . . 8 8 8 8 8 6 6 6 6 6 . . . . 8 8 8 8 8 6 6 6 . . 8 8 8 8 8 8 8 8 ...... 8 8 8 8 8 8 8 8 2 2 8 8 8 8 8 2 2 2 2 8 8 8 8 8 2 2 2 2 8 8 8 2 2 2 2 2 2 7 7 7 4 4 4 7 7 7 7 7 8 8 8 4 4 4 4 4 7 7 7 7 7 5 5 5 8 8 8 8 4 4 4 4 4 7 7 7 7 7 5 5 5 5 5 8 8 8 8 4 4 4 4 4 7 7 7 5 5 5 5 5 8 8 8 8 4 4 4 5 5 5 5 5 8 8 8 5 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 6 6 6 . . . 6 6 6 6 6 . . . . . 6 6 6 6 6 6 6 6 . . . . . 1 1 1 6 6 6 6 6 6 6 6 6 . . . . . 1 1 1 1 1 6 6 6 6 6 6 6 . . . 1 1 1 1 1 6 6 6 6 1 1 1 1 1 6 6 6 1 1 1 ...... 2 2 2

0.1 2 2 2 2 2 0 0 0 2 2 2 2 2 0 0 0 0 0 2 2 2 0 0 0 0 0 0 0 0 0 0 0 0 0 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 . . . 4 4 4 4 4 4 4 4 4 . . . . . 4 4 4 4 4 4 4 . . . . . 4 4 4 4 . . . . . 4 4 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

0 11 10 9 8 7 6 5 4 3 2 Chemical Shift (ppm)

1 Figure C7. H NMR Spectrum (500 MHz, CDCl 3 ) of citreohybriddione D.

107

HDAC-F2-1F_13C_dd800.esp

0.020

0.015

0.010 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 7 7 . . . 7 7 7 7 . . . . . 7 7 7 7 . . . . . 7 7 7 7 ...... 8 8 . . 8 8 8 8 . . . . 8 8 8 8 . . . . 8 8 8 8 . . . . 8 8 . . 6 6 6 6 6 6 6 6 6 6 6 6 6 1 1 1 6 6 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7 7 5 5 7 7 7 7 5 5 5 5 7 7 7 7 5 5 5 5 7 7 7 7 5 5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 2 2 2 2 2 2 2 . . 2 2 2 2 2 . . . . 2 2 2 2 2 . . . . 1 1 1 2 2 2 . . . . 1 1 1 1 1 . . 1 1 1 1 1 . . . 1 1 1 1 1 . . . . . 1 1 1 ...... 4 4 4 4 4 4 3 3 3 4 4 4 4 3 3 3 3 3 4 4 4 4 3 3 3 3 3 4 4 3 3 3 3 3 3 3 3 6 6 6 9 9 6 6 6 6 6 9 9 9 9 6 6 6 6 6 9 9 9 9 9 9 9 6 6 6 9 9 9 9 9 9 9 9 9 9 9 9 6 6 6 9 9 9 9 9 6 6 6 6 6 8 8 8 9 9 9 6 6 6 6 6 8 8 8 8 8 6 6 6 6 6 8 8 8 8 8 6 6 6 8 8 8 8 8 2 2 8 8 8 4 4 4 2 2 2 2 4 4 4 4 4 2 2 2 2 . . . 4 4 4 4 4 2 2 2 2 . . . . . 4 4 4 4 4 2 2 . . . . . 4 4 4 5 5 5 5 5 . . . . . 5 5 5 5 5 5 5 5 5 5 5 5 5 1 1 5 5 5 5 5 5 5 5 1 1 1 1 5 5 5 5 5 1 1 1 1 . . . 5 5 5 5 5 1 1 1 1 . . . . . 5 5 5 1 1 . . . . . 2 2 2 . . . . . 2 2 2 2 2 . . . 2 2 2 2 2 8 8 8 2 2 2 2 2 8 8 8 8 8 2 2 2 8 8 8 8 8 8 8 . . . . . 8 8 8 8 8 8 8 8 8 . . . . . 6 6 6 8 8 8 8 . . . . . 6 6 6 6 6 8 8 8 8 . . . 6 6 6 6 6 8 8 6 6 6 6 6 8 8 8 6 6 6 8 8 8 8 8 . . . 6 6 6 8 8 8 8 8 . . . . . 6 6 6 6 6 8 8 8 8 8 . . . . . 6 6 6 6 6 8 8 8 . . . . . 6 6 6 6 6 6 6 6 . . . . . 6 6 6 6 6 6 6 6 . . . . 6 6 6 6 6 . . . . 6 6 6 6 6 1 1 1 1 1 . . . . 1 1 1 1 1 . . . 1 1 1 1 1 . . . . . 1 1 1 . . . . . 6 6 6 . . . . . 6 6 6 6 6 . . . 6 6 6 6 6 6 6 6 6 6 1 1 1 6 6 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 8 8 8 1 1 1 8 8 8 8 8 5 5 5 5 5 8 8 8 8 8 5 5 5 5 5 0 0 8 8 8 1 1 1 6 6 6 5 5 5 5 5 0 0 0 0 1 1 1 1 1 6 6 6 6 6 5 5 5 0 0 0 0 1 1 1 1 1 6 6 6 6 6 0 0 0 0 1 1 1 1 1 6 6 6 6 6 0 0 1 1 1 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 . . . 7 7 7 7 7 2 2 2 . . . . . 7 7 7 2 2 2 2 2 . . . . . 2 2 2 2 2 . . . . . 2 2 2 2 2 . . . 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 4 4 3 3 1 1 1 4 4 4 4 4 3 3 3 3 4 4 4 4 4 3 3 3 3 4 4 4 4 4 3 3 3 3 4 4 4 3 3 2 2 2 3 3 3 2 2 2 2 2 3 3 3 3 3 2 2 2 2 2 3 3 3 3 3 2 2 2 . . . 3 3 3 3 3 . . . . . 3 3 3 ...... 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 4 5 5 5 5 5 4 4 4 4 4 5 5 5 4 4 4 4 4

0.005 4 4 4 4 4 4 4 4 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 4 4 4 6 6 6 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 8 8 8 0 0 0 0 0 8 8 8 8 0 0 0 0 0 8 8 8 8 0 0 0 0 0 8 8 8 0 0 0 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 5 5 5 5 5 6 6 6 6 6 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 9 9 9 2 2 2 9 9 9 9 2 2 2 2 2 . . . 9 9 9 9 2 2 2 2 2 . . . . . 9 9 9 9 5 5 5 2 2 2 2 2 . . . . . 9 9 9 5 5 5 5 2 2 2 . . . . . 5 5 5 5 ...... 5 5 5 5 . . . . 5 5 5 7 7 7 . . . . 7 7 7 7 7 ...... 7 7 7 7 7 7 7 7 . . . . 7 7 7 7 7 7 7 7 . . . . 7 7 7 7 7 . . . . 7 7 7 7 7 7 7 7 . . . 7 7 7 7 7 7 7 4 4 4 7 7 7 7 4 4 4 4 7 7 7 7 . . . 4 4 4 4 7 7 7 . . . . . 4 4 4 4 . . . . . 4 4 4 . . . . . 0 0 0 6 6 6 . . . 0 0 0 0 0 6 6 6 6 0 0 0 0 0 8 8 8 6 6 6 6 0 0 0 8 8 8 8 6 6 6 8 8 8 8 7 7 7 8 8 8 8 7 7 7 7 7 8 8 8 6 6 6 7 7 7 7 7 6 6 6 6 1 1 1 7 7 7 7 7 6 6 6 6 1 1 1 1 1 7 7 7 6 6 6 6 . . . 0 0 0 1 1 1 1 1 6 6 6 . . . . 0 0 0 0 1 1 1 1 1 . . . . 3 3 3 0 0 0 0 1 1 1 . . . . 3 3 3 3 0 0 0 0 2 2 2 . . . 6 6 6 3 3 3 3 0 0 0 2 2 2 2 2 6 6 6 6 6 3 3 3 2 2 2 2 2 6 6 6 6 6 7 7 7 2 2 2 6 6 6 6 6 7 7 7 7 7 . . . 6 6 6 7 7 7 7 7 . . . . . 7 7 7 7 7 ...... 7 7 7 ...... 3 3 3 ...... 5 5 5 3 3 3 3 ...... 5 5 5 5 5 3 3 3 3 ...... 5 5 5 5 5 2 2 2 3 3 3 3 . . . . 5 5 5 5 5 2 2 2 2 2 3 3 3 3 3 3 . . . 5 5 5 2 2 2 2 2 3 3 3 3 2 2 2 2 2 3 3 3 3 0 0 0 3 3 3 3 0 0 0 0 0 0 0 0 0 0 0 0 7 7 7 0 0 0 0 0 0 7 7 7 7 7 0 0 0 0 0 7 7 7 7 7 . . . 0 0 0 0 0 7 7 7 7 7 . . . . . 0 0 0 0 0 1 1 1 7 7 7 2 2 2 . . . . . 0 0 0 1 1 1 1 1 2 2 2 2 2 . . . . . 1 1 1 1 1 2 2 2 2 2 . . . 1 1 1 1 1 3 3 3 2 2 2 2 2 1 1 1 3 3 3 3 2 2 2 7 7 7 3 3 3 3 6 6 6 6 6 7 7 7 7 3 3 3 3 . . . . . 6 6 6 6 6 7 7 7 7 . . . . . 6 6 6 6 6 7 7 7 . . . . . 6 6 6 . . . 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 . . . 2 2 2 . . . . . 2 2 2 2 2 2 2 2 . . . . . 2 2 2 2 2 2 2 2 2 2 . . . . . 2 2 2 2 2 2 2 2 2 2 7 7 7 7 7 . . . 2 2 2 2 2 3 3 3 7 7 7 7 7 3 3 3 3 7 7 7 7 7 6 6 6 3 3 3 3 7 7 7 6 6 6 6 3 3 3 3 4 4 4 6 6 6 6 3 3 3 4 4 4 4 4 6 6 6 6 4 4 4 4 4 6 6 6 4 4 4 4 4 4 4 4 2 2 2 2 2 2 2 2 2 2 2 7 7 7 2 2 2 7 7 7 7 7 7 7 7 7 7 5 5 5 7 7 7 7 7 5 5 5 5 5 7 7 7 5 5 5 5 5 5 5 5 5 5 5 5 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7 7 7 1 1 1 1 1 1 1 7 7 7 7 7 1 1 1 1 7 7 7 7 7 1 1 1 7 7 7 7 7 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0 240 220 200 180 160 140 120 100 80 60 40 Chemical Shift (ppm)

13 Figure C8 . C NMR Spect rum (200 MHz, CDCl 3 ) of citreohybriddione D.

108

1 13 Figure C9. H - C gHSQCAD NMR Spectrum (800 MHz, CDCl 3 ) of citreohybriddione D.

109

100

120

140

160

180

200

F2 Chemical Shift (ppm) 10 8 6 4 2 0

1 13 Figure C10 . H - C gHMBCAD NMR Spectrum (500 MHz, CDCl 3 ) of citreohybriddione D.

F2 Chemical Shift (ppm) 9 8 7 6 5 4 3 2 1

1 1 Figure C11. H - H gCOSY NMR Spectrum (600 MHz, CDCl 3 ) of citreohybriddione D.

110

F2 Chemical Shift (ppm) 10 8 6 4 2 0

1 1 Figure C12 . H - H NOESY NMR Spectrum (800 MHz, CDCl 3 ) of citreohybriddione D.

111