New Approaches for the Dereplication, Isolation and Structural Elucidation

of New Bioactive Marine Natural Products.

Marcel Jaspars

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*

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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

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Development

Sampling

Curation

Biomass

Extraction

Assay

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

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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

HPLC

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)

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One Solution – Compound Librariesm

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Collection/TaxonomyCuration/Informatics

Crude extracts

Purified extracts

Pure compounds

Pure compoundswith structure

Library size

Library generation

Library value

SCRE

EN(in

-hou

se/p

artn

er)

DereplicationHas your compound been reported before and

how do you find out?m

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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)

LCMS

ES+ (HR)

ES-

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

Sample submission

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• 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

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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

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Jioji Tabudravu

MBC Spectroscopic Databasem

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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

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Tamarindus indica, Legon, Ghana

1' NH

3N

4

7 1

OH O

8

O

4'

5'

Legonmycin A

Streptomyces albus

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

X

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reKnowns and Unknowns

6 new possible compound candidates

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reNew Compounds

1 2

N

OH

NHO

ON

OH

NHO

O

N

O

ONH

OHO

O

N

O

ONH

OHO

O

3 4

1

3468

9 11

12

13

14

Known Known

New

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reMBC Spectroscopic Database

Similarity Match

N

OH

NH

O

O

N

O

ONH

OHO

O

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reRoss Sea Pseudomonas sp. BTN1

Ross Sea

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re Untargeted DereplicationSearching Every LC-MS peak in the Database

Analysis of the Blank

Jioji Tabudravu

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re Untargeted DereplicationBlank and Medium Removed

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re Untargeted DereplicationBlank, Medium and PharmaSea Database

(28,000 compounds) removed

533.368

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re Untargeted DereplicationAfter Removal of Blank/Medium/Knowns,

Remainder are New Compounds

O O

O OH

O

OHO

HOOH

Marine Drugs, 2016, 14, 83

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reMonorhamnolipids

Activity Against Burkholderia

O OH

OC7H15O

13

C6H13

1

4

6

O OH

OC9H19O

C4H9

O OH

OC7H15OC9H19

2 3

1

AB

C

OHO

OHHO

OHO

OHHO

1

23

56

OHO

OHHO

O

O O

Mar. Drugs 2016, 14, 83

Novelty, Complexity and Diversity Metrics

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Jioji Tabudravu

Novelty, Complexity and Diversity Metrics

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Novelty for FijianInvertebrate Extracts

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0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90Ta

varu

a-2-

50

Lau-

1-50

Tava

niko

-5-5

0

Tava

niko

-4-5

0

Buag

o-7A

-25

Tava

niko

-7-5

0

Eden

-4-5

0

Tava

rua-

2-10

0

Eden

-5-1

00

Pina

ccle

-5-5

0

Buag

o-B-

50

Buag

o-B-

100+

TFA

Buag

o-7A

-100

+TFA

Pina

ccle

-5-2

5

Tava

rua-

8.9-

100+

TFA

Eden

-5-1

00+T

FA

Balo

lo- 3

.14-

100

Lau-

1-10

0+TF

A

Buag

o-7A

-100

+TFA

Tava

niko

-5-1

00

Nam

-2-2

5

Tava

niko

-4-1

00

Tava

niko

-5-2

5

Nam

-2-1

00

Pina

ccle

-5-1

00+T

FA

Tava

niko

-4-1

00+T

FA

Eden

-6-1

00

Eden

-6-2

5

Nov

elty

inde

x

Fractions

New Compound Discoveredm

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• 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

ONH

O

NHO

NHO

NH

ONHO

O

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 )

6.9

8

0.9

2

1.1

4

1.1

2

2.0

31

.13

3.8

3

3.8

9

2.8

5

3.9

1

3.8

7

1.8

71

.08

0.9

9

0.9

3

0.8

9

0.9

5

1.9

8

2.2

9

1.9

6

2.0

0

10.0

0

water CD3OD

Using Simple Features

N-Me

S-Me

-CH(Me2)

N

HN

HN

NH

NH

O

OO

OO

HN

O

HN

O OH

SMe

HO

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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

Br N

O

N

OHBrO

Br

NN

O

HO

Br

O

OH

OH

NHN

S

HN

SN

NH

O

O

O N

N

O

O

OK

But what about:

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reStructure Determination Steps

Molecularformula

Functionalgroups

Substructures

Very secure3D molecular

structure

MS, NMR

NMR, IRUV

NMR

X-RAY

UnsaturationNumber

(UN)

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

1JCH

H

C C

H1JCH &

3JHH

H

C C

H

C C

1JCHH

C C C&

2-3JCHH

C C C C C C

MF = C10H18O

sp3: Me Me Me

sp3: CH2 CH

2 CH

2

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

H1JCH

OH

OH

OH

4.) Assemble working structures

5.) Make structural proposal

2-3JCHHC C C

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reStructure Determination Steps

H

Me MeOH

MeHH

H

H HHH

3.) Generate substructures3JHHHC C

H

Determining the MF224

272

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

0

100

Intens.[mAU]

0.0

0.5

5x10Intens.

0

2

45x10

Intens.

0.0

0.5

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

AB

CD

EFG H

IJ

KLM N

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

20

30

40

50

60

70

80

90

100

110

120

f1 (

ppm

)

KSA032_WB_SF8_G, HSQC, 303K, DMSO-d6

EFG

HI

N

E

e

F

f

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

j

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)

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

4.4

4.6

f1 (

ppm

)

KSA032_WB_SF8_G, COSY, 303K, DMSO-d6 m

jk

k’

ln

n’

m jk k’

ln n’

j-m

k-j

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

105

110

115

120

125

130

135

KSA032_WB_SF8_G, HMBC, 303K, DMSO-d6

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

B

C

D

EFG

H

I

NH g h e f

B-NH

C-NH

B-g B-e

D-h D-f

H-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

e

f

g

HOOH

jk k'

mn n'

l

COSY Data

HOOH

JK

MN L

OH

O

A

HN

C14H16N2O4

HMBC Data

HNB C

DE

FG

H

I

Combining the PiecesHO

OHJ

K

MN L

OH

O

A

HN

HNB C

DE

FG

H

I

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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

HC

C

HC C

HC

CCH

CH

H3C C O O NH NH

173.3

118.7

92.2 134.4

124.7

109.8

110.8

121.5

59.0 160.1

= MF (C10H10N2O2)

Structuregeneration

NH

HN O

HOMe

NH

HN O

HMeO

NH

NHOMeO

H

& 386 others

173.3118.7

92.2

134.4

124.7

109.8110.8

59.0

160.1

Calculate matchwith 13C shif ts

NH

HN O

HOMe

Correct structure

The Determinator

• Fast• Easy to use• Generally

applicable• Reliable• Inexpensive

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Solving StructuresUsing Atomic Force Microscopy

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Nat. Chem. 2010

The Determinatorm

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AFM of Unknown

Applying the Determinatorto Breitfussin A

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Prerequisite

C15H8N3O1

Br I OMe

Applying the Determinatorto Breitfussin A

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Applying the Determinatorto Breitfussin A

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Applying the Determinatorto Breitfussin A

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Applying the Determinatorto Breitfussin A

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Applying the Determinatorto Breitfussin A

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Br

I

“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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