new approaches for the dereplication, marine isolation and...

New Approaches for the Dereplication, Isolation and Structural Elucidation

of New Bioactive Marine Natural Products.

Marcel Jaspars

ToolsChromatographySpectroscopyMolecular biologySynthesisMolecular modellingNew software

Marine invertebratesMarine microorganisms

& Symbionts

FunctionsChemical ecologySymbiosisBiosynthesisBioinorganic chemistry

Applications:PharmaceuticalCancerInfectionInflammationParasitesEpilepsyAlzheimer’s

PeopleProf Marcel JasparsDr Rainer EbelDr Hai DengDr Wael HoussenProf Jörg FeldmannDr Eva KruppDr Laurent TrembleauDr Andrea RaabMr Russell Gray

The Marine Biodiscovery Centre

Aim: The discovery of novel pharmaceuticalcandidates and research tools from extremeenvironments.

Natural Product ChemistryPharmacy & PharmacognosyChemical BiologySynthetic BiologyAnalytical MethodsAnalytical MethodsOrganic SynthesisMass spectrometryNuclear Magnetic Resonance

The Biodiscovery Pipelinem

Development

Sampling

Curation

Biomass

Extraction

Purification

Active NCE

BottleneckAccess to resources(Physical and legal)

Taxonomic identificationStorage with associated metadataWhole organism collection Microbial cultivation/cryptic pathwaysExtraction processEarly DereplicationAssay throughput vs information obtainedFalse positives/negativesPurification processRemoval of nuisance compounds

Structure determinationLate Dereplication

SUPPLYScale-up/process intensification

reCompound Isolation

Solvent-solvent partition Size-exclusion chromatography

Medium pressure liquid chromatography

HighPressureLiquid Chromatography

Bioassay Guided Isolation

S1Inactive

S2Inactive

S3Inactive

S4Inactive

S6F1Inactive

S6F2Inactive

S6F3Inactive

S6F4H1Inactive

S6F4H2Inactive

Pure CompoundActive

S6F4Active

S5Active

S6Inactive

Crude extractActive Partition fraction

Size exclusion

Column chromatography

Problems with bioassay guided isolation

• Poor assay reproducibility using crude/semi-purified extracts

• False positives/negatives• Long times between cycles of

purification/bioassay are common• Repeated rediscovery of known

compounds (bioactives)

One Solution – Compound Librariesm

Collection/TaxonomyCuration/Informatics

Crude extracts

Purified extracts

Pure compounds

Pure compoundswith structure

Library size

Library generation

Library value

DereplicationHas your compound been reported before and

how do you find out?m

Isolation of known compounds is time consuming and costly

John Blunt et al. in Handbook of Marine Natural Products, 2012

Available databases:

• 30,000 - MarinLit (RSC)• 36,000 - Antibase• 50,000 – AntiMarin• 160,000 – Dictionary of

natural products

Early stage dereplicationIdentifying known compounds in extractsPrioritising samples for further work

Late stage dereplicationAfter compounds have been purified.

Hyphenated DataUV (DAD)

ES+ (HR)

MS/MS (LR)[Crude] = 0.5 mg/ml[Pure] = 0.05 mg/ml

Sample submission

• Coupling of LC and MS increases resolution of both LC and MS methods

• Can monitor selected ions• Can monitor selected reactions

• Using high resolution, molecular formula can be determined and searched in database.

• Useful for early dereplication – at extract stage

Targeted DereplicationSearching for Every Database Compound in the LC-MS

Unidentified Peak

Aspergillus indologenus extractUse database of all known Aspergillus and Penicilliumcompounds and retention timesto identify known peaks and locate unknowns

Tetrahedron Lett. 2015, 56 1847

MBC Streptomyces Database5070 Streptomyces compounds

Jioji Tabudravu

MBC Spectroscopic Databasem

665 natural products from marine and terrestrial sources representing several classes of natural products including peptides, alkaloids, terpenes and others

Case StudyDereplication of Streptomyces albus, ΔlgnC

Tamarindus indica, Legon, Ghana

Legonmycin A

Streptomyces albus

Angew. Chem. Int. Ed. 2015, 54, 12697

reKnowns and Unknowns

6 new possible compound candidates

reNew Compounds

Known Known

reMBC Spectroscopic Database

Similarity Match

reRoss Sea Pseudomonas sp. BTN1

Ross Sea

re Untargeted DereplicationSearching Every LC-MS peak in the Database

Analysis of the Blank

Jioji Tabudravu

re Untargeted DereplicationBlank and Medium Removed

re Untargeted DereplicationBlank, Medium and PharmaSea Database

(28,000 compounds) removed

533.368

re Untargeted DereplicationAfter Removal of Blank/Medium/Knowns,

Remainder are New Compounds

Marine Drugs, 2016, 14, 83

reMonorhamnolipids

Activity Against Burkholderia

OC7H15O

OC9H19O

OC7H15OC9H19

Mar. Drugs 2016, 14, 83

Novelty, Complexity and Diversity Metrics

Jioji Tabudravu

Novelty, Complexity and Diversity Metrics

Novelty for FijianInvertebrate Extracts

0.90Ta

Fractions

New Compound Discoveredm

• Analysis MS/MS data is time consuming• No publicly available dereplication databasesGNPS• Compares fragmentation patterns• Clusters compounds with similar

fragmentation patterns based on similarity• Dereplication by comparing newly generated

MS/MS data with deposited dataSylvia Soldatou/Kevin Miranda

Molecular Network of Marine Endophytic Aspergillus sp. Extract

Node (parent ion)

Edge (cosine similarity score)

Sylvia Soldatou/Kevin Miranda

Molecular Network of Marine Endophytic Aspergillus sp. Extract

GNPS spectral library hit: Emericellamide A

Confirmed by MS-MS Fragmentation

Circles: potentially newanalogues

Sylvia Soldatou/Kevin Miranda

Late Stage DereplicationFrom Prioritising Crude Extract to

Identifying Pure Compound

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5

JTSD114_FD_1H_CDCl3JTSD114_FD_1H_CDCl3

102030405060708090100110120130140150160170180190200210

JTSD114_FD_13C_CDCl3JTSD114_FD_13C_CDCl3

Crude 1H NMR Crude 13C NMR

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5

JT_SD114_FD_S1_F9_H5JT_SD114_FD_S1_F9_H5

0102030405060708090100110120130140150160170180190200210

JT_SD114_FD_S1_F9_H5_13CJT_SD114_FD_S1_F9_H5_13C

Pure 1H NMR Pure 13C NMR

1 .62 .43 .24 .04 .85 .66 .47 .2f1 (ppm )

water CD3OD

Using Simple Features

-CH(Me2)

reChemical Structure

2D Framework1 Carbon skeleton

2 Functional groups

3 Location of functional groups

3D Framework 1 Relative stereo

2 Absolute stereo

3 Conformational features

But what about:

reStructure Determination Steps

Molecularformula

Functionalgroups

Substructures

Very secure3D molecular

structure

MS, NMR

NMR, IRUV

UnsaturationNumber

Working 2D

structures

List of working

2D structures

Reasonable3D molecular

structure

New 2Dmolecularstructure

NMRORD Total

synthesis Molecularmodeling

Knownmolecularstructure

Dereplicate by MF

Dereplicate

Draw all isomers

NMR, MS, IR, UV

by structure

Purecompound

Strategy Based on C-H Connectivity

H1JCH &

C C C&

2-3JCHH

C C C C C C

MF = C10H18O

sp3: Me Me Me

sp3: CH2 CH

sp3: CH; sp2: CH

sp3: C-OH; sp2: C

1.) Obtain C/H pairs from HSQC

2.) Determine functional groups(NMR, IR etc)

1JCHHC C

4.) Assemble working structures

5.) Make structural proposal

2-3JCHHC C C

reStructure Determination Steps

Me MeOH

3.) Generate substructures3JHHHC C

Determining the MF224

UV, 1.4min #97

575.2114

622.0290

277.1190299.1007

KSA1_P1-A-3_01_12578.d: +MS, 1.4min #201

168.0808

186.0915

204.1023214.0867

230.0815

247.1082 277.1188

KSA1_P1-A-3_01_12578.d: +MS2(277.1190), 18.6-46.6eV, 1.4min #202

187.0944

208.0738

226.0847269.0916

299.1000

KSA1_P1-A-3_01_12578.d: +MS2(299.1007), 18.9-47.3eV, 1.5min #203

Intens.[mAU]

5x10Intens.

Intens.

1.04x10

Intens.

200 250 300 350 400 450 500 550 600 m/z

200 220 240 260 280 300 320 340 360 380 Wavelength [nm]

Data:Kojo Acquah

2030405060708090100110120130140150160170f1 (ppm)

13C NMR Data

Atom 13C/ppmA 172.75B 136.13C 132.61D 126.45E 120.74F 118.33

Data: Kojo Acquah

HSQC Data - C-H (1 bond)

2.42.83.23.64.04.44.85.25.66.06.46.87.27.6

KSA032_WB_SF8_G, HSQC, 303K, DMSO-d6

N N’

n n’Red = CH/CH3Blue = CH2

Atom 13C/ppm Mult 1H/ppmA 172.75CB 136.13CC 132.61CD 126.45CE 120.74CH 7.06F 118.33CH 6.98G 117.29CH 7.42H 111.15CH 7.36I 107.77CJ 70.51CH 4.18K 62.94CH2 3.60, 3.54L 56.09CH 3.62M 54.65CH 4.43N 24.62CH2 3.04, 2.69

2.3.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)

1H NMR Data

NH g h e fm

k k’

l n n’

Data: Kojo Acquah

COSY Data - H-C-H, H-C-C-H

2.42.62.83.03.23.43.63.84.04.24.44.6f2 (ppm)

KSA032_WB_SF8_G, COSY, 303K, DMSO-d6 m

m jk k’

ln n’

n-n’

COSY Data - H-C-H, H-C-C-HAtom 13C/ppm Mult 1H COSYA 172.75 CB 136.13 CC 132.61 CD 126.45 CE 120.74 CH 7.06 f, hF 118.33 CH 6.98 e, gG 117.29 CH 7.42 fH 111.15 CH 7.36 eI 107.77 CJ 70.51 CH 4.18 m, k

K 62.94 CH2

3.60, 3.54 j

L 56.09 CH 3.62 nM 54.65 CH 4.43 j, n

N 24.62 CH2

3.04, 2.69 l, m

Data: Kojo Acquah

6.87.27.68.08.48.89.29.610.010.410.8

KSA032_WB_SF8_G, HMBC, 303K, DMSO-d6

HMBC Data - C-C-H, C-C-C-H

NH g h e f

B-g B-e

D-h D-f

HMBC Data - C-C-H, C-C-C-HAtom 13C/ppm Mult 1H COSY HMBCA 172.75 C l, nB 136.13 C HN, g, eC 132.61 C HN, m, j, nD 126.45 C HN, h, f, nE 120.74 CH 7.06 f, h g, fF 118.33 CH 6.98 e, g hG 117.29 CH 7.42 f e,fH 111.15 CH 7.36 e f, g, eI 107.77 C HN, g, m, nJ 70.51 CH 4.18 m, k m, k

K 62.94 CH2

3.60, 3.54 j j, m

L 56.09 CH 3.62 n nM 54.65 CH 4.43 j, n l, k

N 24.62 CH2

3.04, 2.69 l, m l

Data: Kojo Acquah

Substructuresh

COSY Data

C14H16N2O4

HMBC Data

Combining the PiecesHO

reComputer Aided Structure Elucidation

HSQC analysis - assign C-H

Tabulate data

# dC dH

1 173.3 9.272 121.5 6.873 110.8 6.09

COSY/HMBC analysis

Generate connectivities

H3C C O O NH NH

92.2 134.4

59.0 160.1

= MF (C10H10N2O2)

Structuregeneration

NHOMeO

& 386 others

173.3118.7

109.8110.8

Calculate matchwith 13C shif ts

Correct structure

The Determinator

• Fast• Easy to use• Generally

applicable• Reliable• Inexpensive

Solving StructuresUsing Atomic Force Microscopy

Nat. Chem. 2010

The Determinatorm

AFM of Unknown

Applying the Determinatorto Breitfussin A

Prerequisite

C15H8N3O1

Br I OMe

Applying the Determinatorto Breitfussin A

“If you have a strange substance and you want to know what it is, you go through a long and complicated process of chemical analysis. ……. It would be very easy to make an analysis of any complicated chemical substance; all one would have to do would be to look at it and see where the atoms are.”

Plenty of Room at the Bottom Richard P. Feynman

December 1959

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