isolation of bioactive natural products from marine invertebrates
TRANSCRIPT
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Molecular Formula: C H Br N O
Exact Mass: 646.96310
InChIKey: RCOAHKXJTMBQLI-VFCFBJKWSA-N
Compound status: New
aplysamine-2 free base
Compound 2 in original article
23 28 3 3 4
Compound ID: L23435
Molecular Formula: C H Br N O
Exact Mass: 650.92170
InChIKey: QORPEZJCTMWCLO-FPOVZHCZSA-N
UV Value Log ε
280 nm
250 nm
218 nm
Compound status: New
psammaplysin I
UV max ( in EtOH/water):
Compound 5 in original article
21 24 3 3 6
Compound ID: L15872
Molecular Formula: C H Br N O
Exact Mass: 645.93150
InChIKey: ZKAGSGYSVMKJAF-IUKFSEKMSA-N
UV Value Log ε
310 nm 4.10
244 nm 3.94
Compound status: New
kuchinoenamine
UV max ( in MeOH):
Compound 3 in original article
23 25 3 2 5
Compound ID: L13070
suberedamine B
Compound search results | MarinLit http://pubs.rsc.org/marinlit/Search/CompoundSearchPrintResult?FullText...
1 of 2 8/9/2016 1:04 PMCreate PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
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.
20