Kyle Planck Report 2016

ISOLATION OF BIOACTIVE NATURAL PRODUCTS FROM MARINE INVERTEBRATES

Kyle Planck

University of Notre Dame Department of Chemistry & Biochemistry

Summer 2016 University of California, San Diego

STARS Program Mentor: Professor Tadeusz Molinski, Ph.D.

ABSTRACT

Natural products derived from marine organisms have garnered the attention of drug discovery scientists because of their unique chemistry and therapeutic potential. Compounds from marine bacteria and invertebrates are under investigation in clinical trials, and some have already received FDA approval as anticancer and analgesic drugs. The search for natural products is ongoing as researchers harvest specimens from different marine invertebrate species to determine if their contents have useful pharmaceutical applications. This project used nanomolar-scale protocols to examine sponge and tunicate samples from the ocean around Micronesia and Western Australia for biological activity and to investigate the unique compounds they contain. Compounds were extracted using progressive solvent partitioning, separation and purification were carried out by HPLC, and the structures of these molecules were elucidated using LC-MS, 1H NMR, and IR spectroscopy. As a result of these experiments, a number of natural products were successfully identified. These compounds, as well as those from other marine invertebrate samples, will continue to be examined for new, exciting bioactive natural products.

BACKGROUND Natural products chemistry is an extensive and significant discipline within modern drug discovery. Natural products, broadly defined, are compounds created by living organisms that have a useful scientific application. Most of these are cytotoxic secondary metabolites produced by symbiotic microorganisms and represent the evolution of defense mechanisms; for this reason, many of them possess attractive anti-cancer and antibiotic properties1. Although natural products derived from terrestrial organisms like penicillin and morphine have been known for a long time, it took much longer for marine natural products to become a viable field of research. This is due to many factors, but the predominant obstacles to marine natural products discovery until recent years were prohibitively high exploration costs, inaccessible specimen habitats, and a lack of appropriate harnessing and analytical technology. The development of new equipment and techniques like robot collection systems, aquaculture, and high-throughput screening has led to a renaissance of marine natural products research2. Both the discovery and development of compounds from the ocean that have pharmacological potential are daunting tasks, but identifying molecules of interest from marine sources has yielded important discoveries and generated multiple drug leads that are being developed in industry and academia2. My summer research project was an attempt to isolate appreciable amounts of bioactive compounds from various marine invertebrate species. This approach has had much success in finding molecules with unique chemical properties and high therapeutic potential; one molecule composed of non-standard amino acids—jamaicensamide A—was actually discovered by researchers in the Molinski lab just this year3. The first FDA-approved medicine derived from a marine natural product is ziconotide, a treatment for chronic pain inspired by a toxic peptide from Conus spp. Many other promising compounds have been derived from organisms of the sea, such as anti-cancer agents halichondrin B and aplidine, anti-inflammatory agent topsentin, anti-mitotic agent dolastatin 10, and immunosuppressant (+)-discodermolide, to name only a few2,4. These compounds come from a variety of organisms and have an even wider array of chemical structures and functions, illustrating the importance and wide applicability of marine natural products in drug discovery efforts. During the summer, I became familiar with several important analytic and synthetic techniques that are utilized in marine natural products research. Such skills include TLC, which is used to identify the Rf values of interesting molecules, LC-MS to approximate compound masses, HPLC to isolate compounds of interest, NMR to elucidate structural characteristics, and various synthesis reactions that used marine natural products as inspirations and starting points for creating novel molecules. I also learned extraction techniques to collect these compounds from animal samples. The specifics of these processes are outlined in the next section, Materials and Methods, followed by the results and implications of the work that I conducted in the lab. A list of abbreviations used in this report can be found after the Discussion section.

MATERIALS AND METHODS Compound Extraction

Compounds were extracted from frozen marine invertebrate samples using a protocol called progressive solvent partitioning (PSP). Frozen samples were cut up into small chunks, lyophilized overnight, and stirred in 500 mL of 1:1 MeOH:DCM solution for 24 hours. The solution was filtered to remove particulates, dried down, dissolved in 9:1 MeOH:H2O, and sonicated for 10 minutes. PSP was carried out on this sample as described in Figure 1 below by extracting with hexanes, DCM, and n-BuOH to obtain A, B, C, and D layers.

Figure 1. Progressive solvent partitioning of a type sample using hexanes, DCM, and n-BuOH.

This procedure was carried out at the beginning of each investigation in order to extract compounds of interest from each sample. Each layer was dried down and weighed, but investigations focused primarily on the B and C layers due to the favorable drug development properties of compounds that dissolve in those layers, such as a mix of hydrophobic and hydrophilic moieties that may lead to good bioavailability. The masses of material recovered from each layer of multiple invertebrate samples are presented in Table 3 of the Results section.

Isolation and Identification of Compounds For sample 01-09-031, TLC of each layer was performed in 90:10 DCM:MeOH and stained

with p-anisaldehyde in order to determine the Rf value of the major components. Then, LC-MS analysis (reversed phase, MeCN:H2O) was performed on the A, B, and C layers to determine the approximate molecular mass of compounds present in the mixtures. The A layer was dissolved in chloroform, the B and C layers were dissolved in methanol, and 1H NMR was performed to get an idea of the types of compounds present in each. Rough IR spectra were also obtained. The same procedures were carried out on sample 01-13-075. After PSP and initial analysis, reversed phase HPLC was performed on the B layer using a 10% to 100% MeCN gradient in H2O at a flow rate of 13 mL/minute. Six peaks were collected, and two were identified as bisabolene derivatives using LC-MS, 1H NMR, and IR. These are presented in Table 4 of Results. The other peaks were analyzed using 600 MHz 1H NMR, but nothing of interest was found in suitable quantities for further analysis.

Samples 93-07-101 had already been separated in to A, B, C, and D layers, so TLC was performed on each layer, and the B layer was again identified as the most promising for further investigation. Size-based column chromatography was carried out on the B layer using Sephadex LH-20 resin (GE Healthcare Life Sciences) and a drip of MeOH overnight. Reversed phase HPLC was used on the second LH-20 fraction (L2), which contained about 200 mg of material and was chosen because of its LC/MS profile. Three peaks were collected from L2 and submitted for LC-MS and 1H NMR. The next fraction to be separated using HPLC was L4, which yielded nine peaks that were submitted for analysis. LC-MS data and cross-referencing with MarinLit6 suggested that these compounds were previously known, so 1H NMR was taken in DMSO in order to accurately compare the spectra with those from the literature. High-resolution mass spectrometry was performed by the UCSD Molecular Mass Spec facility on all samples of known compounds. All of this data was taken together and resulted in the identification of four previously known compounds from the L4 sample. Finally, the L1 portion was analyzed using LC-MS and proton NMR. HPLC of this layer yield eight peaks, but later analysis of each peak revealed insufficient quantities of material to continue investigations. The next sponge sample to be investigated was the A (hexane-soluble) layer of 01-076, which had a very interesting, UV-active TLC profile. The layer was tested using TLC with 100% hexane, 50:50 hexane:isooctane, and 100% isooctane, and the best separation was obtained using 100% isooctane. A simple silica column was set up in a Pasteur pipet and primed with isooctane before loading with 22.70 mg of crude sample. Isooctane was pushed through the column as the mobile phase, and 20 fractions that contained 25 drops each were collected and analyzed by TLC using short-wave UV light. The fractions of interest were submitted to LC-MS and dissolved in CDCl3 for 1H NMR (600 MHz). The signal was too weak, so more sample was obtained using the procedure outlined at the beginning of the “Compound Extraction” methods section. The wet weight of this additional sample was 104.57 g, and the dry weight was 20.34 g. After PSP of this sample, the A layer was dried down and re-suspended in hexane. Some precipitation occurred, so the mixture was filtered using cotton and the soluble portion was dried down. 44.0 mg of this clear oil was loaded onto a primed Pasteur pipet column (100% isooctane mobile phase), and 36 fractions

consisting of 20 drops each were collected from the column. The fractions were analyzed using TLC, and the UV-active fractions of interest (F1-F4) were dried down and individually dissolved in CDCl3 for 1H NMR. Overnight crystallization of A-F4 was attempted using n-heptane but resulted in no precipitation. Synthesis of L-isoleucic Acid

L-isoleucic acid was made as part of a larger effort to synthesize the sponge-derived peptide jamaicensamide A, which was isolated from a sample of Plakina jamaicensis in the Bahamas3. A schematic of the reaction performed and the reagents used are shown below:

Figure 2. Synthesis of L-isoleucic acid

Table 1. Reagents Used for Synthesis of L-isoleucic acid

Reagent Formula Weight (g/mol)

Equivalence Amount of Reagent (mmol)

Amount of Reagent (mg)

L-isoleucine 131.18 1.0 2.93 383.9

Sodium nitrite

68.9 4.5 13.17 907.4

Sulfuric acid 98.08 0.5 M in H2O

7.5 mL of 0.5 M solution

L-isoleucic acid

(product)

132.16

1.0

2.93

386.8 (theoretical yield)

383.9 mg of starting material was dissolved in 7.5 mL of 0.5 M H2SO4 and cooled in an ice

bath for 30 minutes. Then, 907.4 mg of NaNO2 was dissolved in 1.5 mL of H2O, put into a syringe, and added to the reaction mixture over 4 hours in the ice bath. The reaction was removed from the ice bath, allowed to warm to room temperature, and left to stir for 20 hours. The following day, 3 mL of 1 M HCl were added to the reaction mixture, which was extracted with 10 mL ethyl acetate three times. The organic layer was washed with 10 mL of NaCl brine and dried over MgSO4. The liquid was filtered and concentrated, yielding 192.7 mg of a yellowish, clear oil in 49.8% yield. 1H NMR was conducted on a sample to ensure purity.

Coupling of Bisabolene Amine with Naproxen Isocyanate As part of an ongoing project in the Molinski Lab investigating naproxen derivatives,

6R,7S)-7-amino-7,8-dihydro-α-bisabolene (isolated from sample 01-075) was coupled to naproxen isocyanate. That reaction was performed on a small scale and is shown in Figure 3 and Table 2 below:

Figure 3. Coupling of Bisabolene Amine and Naproxen Isocyanate

Table 2. Reagents Used for Naproxen Isocyanate Coupling

Reagent Formula Weight (g/mol)

Equivalence Amount of Reagent (mmol)

Amount of Reagent (mg)

Bisabolene amine

220.21 1.0 0.00227 0.50

Naproxen isocyanate

227.27 1.1 0.00250 0.57

DCM 84.93 solvent ~1.75 mL

Coupling product*

448.65

1.0

2.93

386.8 (theoretical yield)

* 1-((S)-1-(6-methoxynaphthalen-2-yl)ethyl)-3-(6-methyl-2-(4-methylcyclohex-3-en-1-yl)hept-5-en-2-yl)urea

5.3 mg of the amine was dissolved in 2.65 mL DCM to create a 2 mg/mL solution, and 1.14 mg of naproxen isocyanate was dissolved into 2 mL DCM to create a 0.57 mg/mL solution. 250 µL (0.50 mg) of the amine solution and 1.0 mL (0.57 mg) of the isocyanate solution were combined with an additional 500 µL of DCM. The solution was allowed to stir in a closed vessel overnight at room temperature under nitrogen gas. The next day, TLC was performed, which indicated an incomplete reaction. LC-MS and 1H NMR were performed on the amine sample to verify purity of the starting material, but the reaction was not performed again during my time in the lab.

RESULTS

The amount of crude material obtained by PSP from each sample is presented in Table 3 below. The amount of material varied widely between sample and layer, but B and C layers yielded between 10 and 60 mg of crude, unseparated product.

Table 3. Crude Layers from Progressive Solvent Partitioning Sample Number

A Layer Mass (mg)

B Layer Mass (mg)

C Layer Mass (mg)

D Layer Mass (mg)

01-09-031 7.4 12.1 11.9 87.3

01-13-075 74.7 —* 57.1 198.0

01-13-076 43.9 —† —† —†

* this sample could not be quantified due to mishandling and contamination with soapy water. † only the A layer was of interest in this PSP, so the remaining layers were not separated.

The first compounds successfully identified from our samples were bisabolene derivatives.

These molecules were first isolated and characterized from Halichondria sp. and show appreciable antimicrobial activity5. Bisabolene derivatives isolated from the B layer of sponge 01-075 are presented below in Table 4.

Table 4. Bisabolene Compounds Identified from 01-075-B Fraction Name Structure Amount

Recovered (mg) 1

(6R,7S)-7-amino-7,8-dihydro-α-bisabolene

6.0

6

N,N'-Bis[(6R,7)-7,8-dihydro-α-bisabolen-7-

yl]urea

2.4

H2N

SCN

-Cl+H3N

HN

NH

O

Chemical Formula: C31H52N2OExact Mass: 468.41

Molecular Weight: 468.77m/z: 468.41 (100.0%), 469.41 (33.5%), 470.41 (2.7%), 470.41 (2.7%)

Elemental Analysis: C, 79.43; H, 11.18; N, 5.98; O, 3.41

6

5

4

3

Chemical Formula: C15H27NExact Mass: 221.21

Molecular Weight: 221.39m/z: 221.21 (100.0%), 222.22 (16.2%), 223.22 (1.2%)

Elemental Analysis: C, 81.38; H, 12.29; N, 6.33

Chemical Formula: C16H25NSExact Mass: 263.17

Molecular Weight: 263.44m/z: 263.17 (100.0%), 264.17 (17.3%), 265.17 (4.5%),

265.18 (1.4%)Elemental Analysis: C, 72.95; H, 9.57; N, 5.32; S, 12.17

Chemical Formula: C15H28ClNExact Mass: 257.19

Molecular Weight: 257.85m/z: 257.19 (100.0%), 259.19 (32.0%), 258.19 (16.2%), 260.19

(5.2%), 259.20 (1.2%)Elemental Analysis: C, 69.87; H, 10.95; Cl, 13.75; N, 5.43

Sullivan et. al. 1986Halichondria compounds

The B layer of sample 93-101 was the most fruitful source of compounds during our investigations. These compounds were identified, as were the bisabolene derivatives, preliminarily using LC-MS and MarinLit, followed by 1H NMR in appropriate solvents. The peaks of these spectra were compared to the literature and found to be consistent in the isolated compounds7,8,9. The five compounds recovered from 93-101-B are presented below in Table 5.

Table 5. Compounds Identified from 93-101-B Fraction Name Structure Amount

Recovered (mg)

L2-F3

Aplysamine-2

19.30

L4-F1

Aerophobin-1

3.17

L4-F3

Purpuroceratic acid A

1.60

L4-F4

Purpuroceratic acid B

2.15

L4-F6 Chloride salt of purpuroceratic acid A

PAAH+Cl- (location of H+ uncertain)

1.37

Select LC-MS data, HPLC chromatograms, and NMR spectra from various portions of these

experiments are shown in the “Supplemental Information” section and labeled appropriately. Many relevant chromatograms and NMR spectra are physically stored in manila folders in the Molinski lab.

N

O

Br

OH

NH

O Br

Br

O

N

Aplysamine-219.3 mg

O

N

HO

O

BrBr

NH

O

OH

O

N

HO

O

BrBr

NH

O

OH

O

O

Purpuroceratic acid A1.60 mg

Purpuroceratic acid B2.15 mg

O

N

OH

O

BrBr

NH

O

NHNAerophobin-1

3.17 mg

Compounds Identified (93-07-101)Kyle Planck

N

O

Br

OH

NH

O Br

Br

O

N

Aplysamine-219.3 mg

O

N

HO

O

BrBr

NH

O

OH

O

N

HO

O

BrBr

NH

O

OH

O

O



O

N

OH

O

BrBr

NH

O

NHNAerophobin-1

3.17 mg


N

O

Br

OH

NH

O Br

Br

O

N

Aplysamine-219.3 mg

O

N

HO

O

BrBr

NH

O

OH

O

N

HO

O

BrBr

NH

O

OH

O

O



O

N

OH

O

BrBr

NH

O

NHNAerophobin-1

3.17 mg


N

O

Br

OH

NH

O Br

Br

O

N

Aplysamine-219.3 mg

O

N

HO

O

BrBr

NH

O

OH

O

N

HO

O

BrBr

NH

O

OH

O

O



O

N

OH

O

BrBr

NH

O

NHNAerophobin-1

3.17 mg


N

O

Br

OH

NH

O Br

Br

O

N

Aplysamine-219.3 mg

O

N

HO

O

BrBr

NH

O

OH

O

N

HO

O

BrBr

NH

O

OH

O

O



O

N

OH

O

BrBr

NH

O

NHNAerophobin-1

3.17 mg


DISCUSSION The discovery and characterization of marine natural products is complex and challenging. Sizable amounts of invertebrate sample can yield very little amounts of compounds, and working with those compounds is an exercise in patience and scientific know-how. Despite these and other challenges, I was able to successfully isolate five compounds from various marine invertebrate samples, and I got a taste of the work involved in the marine natural products field. Although I did not end up discovering any new or exciting compounds, I gained a lot of experience with the basics of marine natural products discovery and followed the same steps that have led to important finds in the field. I was also able to isolate a considerable amount of natural products that may be used as starting materials in further syntheses conducted by the Molinski lab. The coupling of bisabolene derivatives and naproxen compounds is one such example. There are many experiments that I wish I had time to perform during my short summer at UCSD. Natural products isolated from animal samples are the starting point for myriad possibilities and further investigations, and I have a few ideas of how my research might be extended. First, I would like to continue developing assays that test these compounds for noteworthy biological activity. My colleague Maria Cabrera worked on carrying and optimizing protease inhibition assays using the enzymes chymotrypsin and thrombin in combination with marine invertebrate extracts, and I think that this is a useful test for desirable drug potential. The Molinski lab has also discovered that certain sponge extracts contain compounds that exhibit antifungal activity against C. gattii, a dangerous pathogen prevalent along the West Coast10. Developing and using assays that also test the antifungal activity of isolated marine natural products could also yield promising drug candidate molecules. Another future direction of this research is to continue the same processes on a larger amount of samples as well as a more diverse set of samples. Our sponge collection is already fairly diverse, containing specimens from all parts of the world and various collection years. However, ensuring a diversity of species for examination may result in the discovery of previously unknown or uncharacterized compounds with therapeutic potential. Since natural products discovery is essentially a random process, increasing the amount of invertebrate samples that are investigated by the lab will only increase the chance of finding something exciting. Lastly, the marine natural products that I isolated this summer could potentially serve as scaffolds for molecules with desirable drug properties. These compounds can be a source of ideas—or even simply starting material—for creating similar compounds with optimized moieties that increase their therapeutic potential. This is an area in which I have very little expertise, but it is of great interest to me, and I look forward to seeing marine natural products influence the medicinal and pharmaceutical chemistry fields in the future. Overall, I had a very instructive and enjoyable summer experience in the Molinski lab. I acquired a great amount of theoretical and technical knowledge, worked alongside incredible scientists in the medicinal chemistry field, and learned more about my research interests as I approach graduate school. I am truly thankful for this opportunity and look forward to seeing more of the Molinski lab’s work as I enter academia and/or the pharmaceutical industry.

ABBREVIATIONS USED PSP: progressive solvent partitioning FDA: United States Food and Drug Administration LC-MS: liquid chromatography-mass spectrometry HPLC: high performance liquid chromatography TLC: thin layer chromatography Rf: retention factor IR: infrared

NMR: nuclear magnetic resonance DCM: dichloromethane MeOH: methanol MeCN: acetonitrile DMSO: dimethyl sulfoxide n-BuOH: n-butanol CDCl3: deuterated chloroform

ACKNOWLEDGMENTS I would like to thank Dr. Tadeusz Molinski, my lab mentor, as well as Dr. Matthew Jamison and Dr. Christopher Gartshore, for their incredibly helpful guidance and patience as I worked alongside them this summer. I am also indebted to my lab partner Maria Cabrera, who motivated me, helped me, and was a great friend to me this summer. I am also grateful for Mariam Salib, Troy Bemis, Daniel Flores, and Dr. Xiao Wang for assisting me in various endeavors, helping me with lab procedures and equipment, and for creating a collaborative and welcoming learning environment in the lab. I would also like to thank the UCSD Molecular Mass Spectrometry facility for their role in definitively identifying the compounds that were isolated. Lastly, I would like to thank Dr. Elisa Maldonado and the UCSD STARS program for granting me this research opportunity and providing an abundance of helpful resources as I prepare for graduate school, as well as my graduate student advocate Aaron Ward, who was instrumental in helping me craft my abstract, personal statement, and final presentation.

REFERENCES

1. Hunter, P. “Harnessing Nature's wisdom.” EMBO Rep. 2008, 9, 838-840.

2. Molinski, T.F.; et al. “Drug development from marine natural products.” Nat. Rev. Drug.

Discov. 2009, 8, 69-85.

3. Jamison, M.T.; Molinski, T.F. “Jamaicensamide A, a Peptide Containing β-Amino-α-Keto

and Thiazole-Homologated η-Amino Acid Residues from the Sponge Plakina jamaicensis.” J.

Nat. Prod. 2016, 79, 2243-2249.

4. Faulkner, D. John. “Highlights of marine natural products chemistry (1972-1999).” Nat.

Prod. Rep. 2000, 17, 1-6.

5. Sullivan, B.W.; et al. “(6R,7S)-7-amino-7,8-dihydro-α-bisabolene, an antimicrobial metabolite

from the marine sponge Halichondria sp.” J. Org. Chem. 1986, 51, 5134-5136.

6. MarinLit database, http://pubs.rsc.org/marinlit, accessed June—August 2016.

7. Gunasekera, M.; Gunasekera, S.P. “Dihydroxyaerothionin and aerophobin 1. Two

brominated tyrosine metabolites from the deep water marine sponge Verongula rigida.” J. Nat.

Prod. 1989, 52, 753-756.

8. Kijjoa, A.; et al. “Dibromotyrosine derivatives, a maleimide, aplysamine-2 and other

constituents of the marine sponge Pseudoceratina purpurea.” Z. Naturforsch. B 2005, 60, 904-908.

9. Kottakota, S.K.; et al. “Synthesis and biological evaluation of purpurealidin E-derived marine

sponge metabolites: aplysamine-2, aplyzanzine A, and suberedamines A and B.” J. Nat.

Prod. 2012, 75, 1090-1101.

10. Jamison, M.T.; Dalisay, D.S.; Molinski, T.F. “Peroxide natural products from Plakortis

zyggompha and the sponge association Plakortis halichondrioides—Xestospongia deweerdtae:

antifungal activity against Cryptococcus gattii.” J. Nat. Prod. 2016, 79, 555-563.

SUPPLEMENTAL MATERIALS

Supplemental Figure 1. Collection sites of samples from which compounds were isolated.

Supplemental Figure 2. An enthusiastic summer student takes a proton NMR.

APLYSAMINE-2 DATA CASE STUDY (as presented on 8/11/16)

— This data is representative of the procedure that was followed for identifying each compound. —

Supplemental Figure 3. HPLC Chromatogram of 93-101-B-L2. Peak 3 (≈9.8 min) consisted of aplysamine-2.

Supplemental Figure 4. LC-MS trace of 93-101-B-L2-F3. Isotope pattern indicated Br3, m/z=648.

Supplemental Figure 5. MarinLit database search for criteria from LC-MS (m=647±5, Br3).

Compound ID: L3196

Molecular Formula: C H Br N O

Exact Mass: 647.97100

InChIKey: RCOAHKXJTMBQLI-VFCFBJKWSA-O

UV Value Log ε

266 nm 3.59

294 nm 3.52

Compound status: New

aplysamine-2

UV max ( in MeOH):

Compound 6 in original article

23 29 3 3 4

Compound ID: L12115



InChIKey: RCOAHKXJTMBQLI-VFCFBJKWSA-N


aplysamine-2 free base


23 28 3 3 4

Compound ID: L23435



InChIKey: QORPEZJCTMWCLO-FPOVZHCZSA-N

UV Value Log ε

280 nm

250 nm

218 nm


psammaplysin I

UV max ( in EtOH/water):


21 24 3 3 6

Compound ID: L15872



InChIKey: ZKAGSGYSVMKJAF-IUKFSEKMSA-N

UV Value Log ε

310 nm 4.10

244 nm 3.94


kuchinoenamine

UV max ( in MeOH):


23 25 3 2 5

Compound ID: L13070

suberedamine B

Compound search results | MarinLit http://pubs.rsc.org/marinlit/Search/CompoundSearchPrintResult?FullText...

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Supplemental Figure 6. 1H NMR of 93-101-B-L2-F3 (CD3OD). Numbered molecule of aplysamine-2 shown with corresponding peaks.

HIGH-RESOLUTION MASS SPECTROMETRY DATA — Provided by the UCSD Molecular Mass Spectrometry Facility —

This data describes three of the four compounds isolated from sample 93-07-101.

Supplemental Figure 7. Hi-res MS: aerophobin-1. HR-ESI-TOFMS, positive ion mode.

Supplemental Figure 8. Hi-res MS: purpuroceratic acid A. HR-ESI-TOFMS, negative ion mode.

Supplemental Figure 9. Hi-res MS: purpuroceratic acid B. HR-ESI-TOFMS, negative ion mode.

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Kyle Planck Report 2016