Rapid, Small-scale Dereplication of

Bioactive Extracts

John BluntUniversity of Canterbury

New Zealand

1

~ 145,000 known natural products

Probability for rediscovery very high

Efficient dereplication processes required

is differentiating extracts that contain novel metabolites from those with known natural products

Dereplication

Bioactive Discovery

2abc

Scale of Dereplication Exercise

0.5 – 2 mg extract

4 mL agar slopePetri dish

Bioassay and HPLC/UV/MS/NMR evaluation

100 mg sponge

3a

~0.6 mg crude extract separated on analytical C18 column

DAD & ELSD detection

Eluant collected in microtitre plate1 mL/min, 250 L/well

Daughter plate submitted to bioassay to locate bioactive components

(Master plate submitted for automatedES-MS analysis of each well)

4

Separation of 600 g fungal extract (F8095) using acetonitrile/methanol gradient

Jackson Lin Sun 5

6

7

F8095.3G94M

m /z150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

%

0

100

SLJ0273 223 (2 .516) C m (198:223) 1: TO F M S ES+ 3.17e3407.5368

281.3798

253.4369402.5803282.3919

791.9871

408.5374

408.7168

770.0074409.5392

793.0001

793.9875

814.0095

M+Na

HPLC peak F8095-1

8

Search against ~44,000 unique compounds in AntiMarin for those with M+Na = 407

AntiMarin – combination of compound data from

MarinLit (~20,000 marine natural products)

and AntiBase (~33,000 microbial natural products)

All compounds coded for 1H NMR-recognisable features

9 ab

10

ppm1.52.02.53.03.54.04.55.05.56.0

solvent

3 x doublet methyls1H CapNMR spectrum of

15 g of F8095-1

CD3OD, 2 min, presat

11a

12

Only 1 compound found- details match with F8095-1 13 a

Now examine contents of microtitre plate well containing F8095-214

F8095-4G191M

m /z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950

%

0

100

SLJ0291 120 (2 .405) C m (118:120) 1: TO F M S ES- 106192.8424

386.6739

193.6537

306.7098

238.8127238.7910

238.7622

306.6853

239.7081

261.7245

308.7327

308.7737

309.7589

328.6413

386.6923

386.7290

387.6937

387.8132

580.5323422.6362 588.2189

M-H-

+ve ion MS of F8095-2 not useful, but –ve ion MS suggests M-H = 193

15

1H CapNMR spectrum of6 g of F8095-2

CD3OD, 2 min, presat

Recognise 1 doublet methyl signal, and 2 aromatic singletsprobably indicating 1,2,3,5-tetrasubstitution pattern

ppm2.02.53.03.54.04.55.05.56.0

solvent

1 x doublet methyl

2 x aromatic singlet H

16

Search in AntiMarin for 1 doublet CH3, 1,2,3,5-substituted benzene, and Mr = 194One hit only – data matches that for F8095-2

17

Now examine contents of microtitre plate well containing F8095-718

F8095.9G100M

m /z150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

%

0

100

SLJ0279 225 (2 .545) C m (210:225) 1: TO F M S ES+ 862669.8434

647.8329

253.4294

219.3950157.2350

217.3993 337.5758279.4392

391.6849

465.7622

669.8797

669.9039

670.8597

671.9132

685.8550

864.0764686.8834

M +Na

+ve ion MS of F8095-7 shows M+Na = 669

Search in AntiMarin gives 19 hits

19 a

1H CapNMR spectrum of13 g of F8095-7

CD3OD, 2 min, presat

Recognise 5 doublet methyl signals

ppm1.52.02.53.03.54.04.55.05.56.0

solvent

5 x doublet methyls

20

Search in AntiMarin for 5 doublet CH3s, and M+Na = 669

One hit only – data matches that for F8095-721

7 compounds from F8095 identified by HPLC-microtitre-MS-NMR/database method

15 g6 g 16 g

8 g12 g 12 g

12 g

OO

O

O

O

HO

OH

OH

OHO

OOH

HO O

OO

O

O

O

O

OO

OH

HO

OH

OH

OH

HO

OH

O

O O

O

O

O

O

O

O

OOH

HO

HO

OH

O

O O

O

O

O

O

O

O

OOH

HO

HO

OH

OH

O

O

O

O

O

O

O

O

O

O

HO

OH

OH

HOOH

O

OO

O

O

O

O

OO

OH

HO

OH

OH

OH

HO

OH

22

time

cost

The pathway to bioactive compound identification

complete structure/dereplication

sampleextract

bioassay

scale-up & extraction

bioassay-guided fractionation

pure compound ~1 mg

spectroscopic analysis

HPLC-Bioassay-UV-MS ~5-50 gHPLC-bioassay-(UV)-(MS)-NMR ~500 g crude extract

database searching dereplication

spectroscopic analysis 2-50 g

complete structure

J. Nat. Prod., 2008, 71, 1595-99.

23 abcdef

Acknowledgements

Development of the concept and techniques for the use of HPLC-microtitre plate-capillary NMR:

John Blunt & Murray Munro (UC)Kirk Gustafson (MTDP, NCI, Frederick MD)

Development of the concept of and construction of databases for use in dereplication:

John Blunt & Murray Munro (UC)Hartmut Laatsch (University of Göttingen)

Preparation of samples for demonstration of techniques:

Gill Ellis, Sultan Sadia, Jackson Lin Sun

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