Isolation of Bioactive Natural Products from Marine Invertebrates

Isolation of Bioactive Natural Products from

Marine InvertebratesKyle Planck

UCSD STARS

Department of Chemistry & Biochemistry

August 11, 2016

Whataremarinenaturalproducts?Naturalproducts (NP)arechemicalcompoundsthatcomefromplantsandanimals.

◦ Oftensecondarymetabolites(notessentialfororganism’s survival)

◦ Mostderivedfrommicroorganismsandcytotoxic,havingevolvedprimarily for“chemicalwarfare”

◦ Highlyconservedmolecules thatfunctionwithinvariouspathwaysandorganisms

Marine NPsareuniqueforafewreasons:

◦ Accesshasbeenlimiteduntil recentyearsbecauseof limitson technology

◦ Evolutionaryenvironmentsaredifferent:landvs.sea

◦ Uniquechemistryoftheocean

2Hunter,HarnessingNature'swisdom.

TheMolinskiLab

3

• Naturalproductsdiscovery

• Samplecollection

• Extraction

• Characterizationandisolation

• Organicsynthesis

• Bioassaydevelopment

• Antifungalactivity

(againstCandidaalbicans, C.glabrata)

ProfessorTadeuszMolinski,Ph.D.

01-13-75

Unidentifiedspecies

Collectionsite:Pohnpei,Micronesia

93-07-101

4

SpecieswithinorderVerongida

Collectionsite:Exmouth Gulf,Australia

SelectedSamples

http://portphillipmarinelife.net.au/images/species/speciesHero_511827.jpeg

MaterialsandMethods

5

Progressivesolventpartition (PSP):

• A:hexanes

• B:DCM

• C:n-BuOH

• D:H2O

SAMPLE(9:1MeOH:H2O)

D

A

B

C

+H2O

- MeOH+H2O

hexanes aqueous

DCM aqueous

n-BuOH aqueous

Figure1.PSPofatypesample.

MaterialsandMethods

6

Liquidchromatography—massspectrometry (LC-MS):

Components:• UVdetector• Autosampler• Pump• Column• MassSpec

http://www.thyrocare.com/Liquid-Chromatography.html

Sample

UV Detector

Reversed phase

MaterialsandMethods

7

Nuclearmagneticresonance(1HNMR):

• Determinesarrangementofprotons inasample

MaterialsandMethods

8

Highperformanceliquidchromatography (HPLC):

• Reversedphase

• Mobilephase:H2O/MeCN

• Stationaryphase:C18 column

http://images.slideplayer.com/26/8465585/slides/slide_2.jpg

RESULTSN

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

9

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

(6R,7S)-7-amino-7,8-dihydro-α-bisabolene N,N’-bis[(6R,7S)-7,8-dihydro-α-bisabolen-7-yl]urea

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


Purpuroceratic acidA

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


Aplysamine-2

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


Aerophobin-1Purpuroceratic acidB

01-13-75:

93-07-101:

RESULTSN

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


10

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

(6R,7S)-7-amino-7,8-dihydro-α-bisabolene N,N’-bis[(6R,7S)-7,8-dihydro-α-bisabolen-7-yl]urea

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


Purpuroceratic acidA

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


Aplysamine-2

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


Aerophobin-1Purpuroceratic acidB

01-13-75:

93-07-101:

CaseStudy:Aplysamine-2

11

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


Results:HPLC

12

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


1

2

3

Results: LC-MS

(HPLCPeak3)

13

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


indicatesBr3

Results:MarinLit

14

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


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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Results: 1HNMR

15Kottakota, Suresh K., et al.

Results: 1HNMR

16

26

5

13,17

Kottakota, Suresh K., et al.

Results: 1HNMR

17

18

OCH3

7

2010

MeOH

N(CH3)2

1911

Kottakota, Suresh K., et al.

Conclusions

• Sixcompoundsisolatedandpositivelyidentifiedfromdifferentspongesamples

• StructureswereelucidatedusingNMRandLC-MS

• Experimentaldatacomparedtoliteratureinordertoverifystructures

18

FutureDirections

• Antifungalassaysonisolatedcompoundstodeterminetheirbiologicalpotential

• Continuingsimilarisolationandelucidationprocessesonmorespongesamples

• Synthesisofpromisingnaturalproductscaffolds

19

References1. Hunter,Philip."HarnessingNature'swisdom." EMBOreports 9.9(2008):838-840.

2. http://portphillipmarinelife.net.au/images/species/speciesHero_511827.jpeg

3. http://www.thyrocare.com/Liquid-Chromatography.html

4. http://images.slideplayer.com/26/8465585/slides/slide_2.jpg

5. Kottakota,SureshK.,etal."Synthesisandbiologicalevaluationofpurpurealidin E-derivedmarinespongemetabolites:aplysamine-2,aplyzanzineA,andsuberedamines AandB." JournalofNaturalProducts(2012):1090-1101.

6. Kijjoa,Anake,etal."Dibromotyrosine derivatives,amaleimide,aplysamine-2andotherconstituentsofthemarinespongePseudoceratina purpurea."Zeitschrift für Naturforschung B 60.8(2005):904-908.

7. Gunasekera,Malika,andSarath P.Gunasekera."Dihydroxyaerothionin andAerophobin 1.TwobrominatedtyrosinemetabolitesfromthedeepwatermarinespongeVerongula rigida." JournalofNaturalProducts 52.4(1989):753-756.

8. Sullivan,BrianW.,etal."(6R,7S)-7-amino-7,8-dihydro-.alpha.-bisabolene,anantimicrobialmetabolitefromthemarinespongeHalichondria sp." TheJournalofOrganicChemistry 51.26(1986):5134-5136.

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Acknowledgments

Dr.TadeuszMolinskiDr.ChristopherGartshoreDr.MatthewJamisonMariaCabreraMariamSalibTroyBemis

DanielFloresXiaoWangMolecularMassSpectrometryFacilityDr.ElisaMaldonadoAaronWardUCSDSTARSProgram

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Isolation of Bioactive Natural Products from Marine Invertebrates

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