rapid dereplication using capillary nmr and a database of structures

Rapid Dereplication using Capillary NMR and a Database of

StructuresJohn Blunt

University of Canterbury, NZ

(A presentation given at the Gordon Research Conference on Marine Natural Products in Ventura, CA, February 2006. Several of the concepts contained herein had been presented earlier at

other conferences and institutions during 2003-2005.)

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 Goettingen)

Preparation of samples for demonstration of techniques:

Gill Ellis, Gerhard Lang, Jackson Sun Lin, Maya MitovaRichard Phipps & Sonia van der Sar (UC)

Dereplication

Determining which of the bioactive components in an extract are known compounds, or are likely to be new and therefore worthy of further attention.

Active extracts analysed by HPLC HPLC eluant collected into microtitre plate Daughter plates assayed for activity

Microtitre HPLC Analysis

Obtain UV and Mass spectral data from the bioactive region(s) of the microtitre plate

Dionex HPLC

Foxy Jr

DADELSD

C18 HPLC of extract frommarine-derived Cephalosporium sp.

DAD detection

Bioactivity profile

Provides retention time, UV and molecular weight of the active compound

With possible taxonomy, UV and molecular weight of the active compound(s) known, databases can be consulted to establish if the bioactive is unique or known.

• MarinLit - for 16,303 compounds originating from marine organisms. (Blunt/Munro;

University of Canterbury)

• AntiBase - for 29,253 compounds of microbial origin. (Laatsch; University of Goettingen/Wiley)

• AntiMarin - a combination of the two databases (43,324 compounds). (Blunt/Munro/Laatsch)

Search for MW=518 and UV 410-419 and 290-299

AntiMarin

Only 1 hit

•Problem – not much UV data in AntiBase

• Solution – use 1H NMR data

All structures in AntiMarin are coded with the numbers of each structural feature that could be deduced from 1H NMR spectra

eg #s of CH3 of different types (s,d,t)

Problem:

Preparing an HPLC/microtitre plate from200 – 500 g extract will provide 2 – 40 gcompound/peak spread over 1, 2 or 3 wells.

How to obtain meaningful 1H spectra?

Solution:

Use a Protasis CapNMR probe. Sample can be taken up in 6 L solvent, introduced into probe, and spectral acquisition commenced within 2 minutes.

Protasis CapNMR Probe

1.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0

-200

0

200

400

600

800

1,000

1,200

1,400

1,600

1,800

2,000 CAP PROBE #28 F4470 Int_Chan_1mV

min

Flow: 1.000 ml/min

water: 0.0 %

acetonitrile: 10.0 %

75.0

MeOH: 0.0 %

HPLC with ELSD detection

of 600 g extract of

unidentified endophytic fungus

E9,E10,E11 G4

250 L/well

500 MHz8 g in 6 L CD3ODPRESAT 1.5 min

Recognisable features: 5 CH3 groups, of which 1 methoxyl,3 singlet CH3-C, 1 doublet CH3-C; 1 aldehyde

AntiMarin search for all compounds containing1 aldehyde

and 5 methyl groups, of which 1 is methoxyl,

3 are singlet CH3-C, and 1 is doublet CH3-C

only 1 hit

1.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0

-200

0

200

400

600

800

1,000

1,200

1,400

1,600

1,800

2,000 CAP PROBE #28 F4470 Int_Chan_1mV

min

Flow: 1.000 ml/min

water: 0.0 %

acetonitrile: 10.0 %

75.0

MeOH: 0.0 %

E9,E10,E11 G4

250 L/well

500 MHz5 g in 6 L CD3OD

1.0 min

Spectrum very similar to that of auranticin B except no aldehyde peak.MW 2 amu more than for auranticin B. New peak at 4.8 ppm.

11 hits with search in AntiMarin for 5 CH3, 3 singlet CH3-C, 1 doublet CH3-C, 1 methoxyl and 1 sp3-methylene.

Only 1 hit if MW = 440 included in search.

1.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0-200

0

200

400

600

800

1,000

1,200

1,400

1,600

1,800

2,000 Cap probe #5 F4045 Int_Chan_1mV

min

HPLC with ELSD detection

of 400 g extract of

unidentified endophytic fungus

E9,E10

F12

F2,F3

Bioactivityprofile

500 MHz17 g in 6 L CD3OD

16 sec

Recognisable features: 4 CH3 groups, of which 1 methoxyl and 3 singlet CH3-C; 1 aldehyde

1 hit only if MW = 346 included in search

1.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0-200

0

200

400

600

800

1,000

1,200

1,400

1,600

1,800

2,000 Cap probe #5 F4045 Int_Chan_1mV

min

HPLC with ELSD detection

of 400 g extract of

unidentified endophytic fungus

E9,E10

F12

F2,F3

500 MHz2 g in 6 L CD3OD

PRESAT 2 min

Recognisable features: 3 CH3 groups, of which 1 methoxyl and 2 singlet CH3-C; 1 aldehyde. MW = 332

only 1 hit

1.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0-200

0

200

400

600

800

1,000

1,200

1,400

1,600

1,800

2,000 Cap probe #5 F4045 Int_Chan_1mV

min

HPLC with ELSD detection

of 400 g extract of

unidentified endophytic fungus

E9,E10

F12

F2,F3

500 MHz14 g in 6 L CD3OD

PRESAT 1 min

Spectrum similar to that of phomosine A, but no aldehyde, and MW 2 amu more than for phomosine A.

(New compound – CH2 presumably at 4.9 ppm, lost in solvent presat)

O

O

HO

OHOH

OH

O

1.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0-200

0

200

400

600

800

1,000

1,200

1,400

1,600

1,800

2,000 Cap probe #7 F6312 Int_Chan_1mV

min

HPLC with ELSD detection

of 500 g extract of

unidentified endophytic fungus

F2,F3,F4

H11

500 MHz30 g in 6 L CD3OD

2 min

MW 684 – unknown peptide

TOCSY40 min

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0-500

0

500

1,000

1,500

2,000

2,500

3,000

3,500 Endophytes #124 F5584.NCI MT UV_VIS_1mAU

min

WVL:210 nm

HPLC with UV detectionof 250 g extract ofendophytic fungus

Bioactive region

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0

-200

0

200

400

600

800

1,000

1,200

1,400

1,600

1,800

2,000 Endophytes #124 F5584.NCI MT Int_Chan_1mV

min

ELSD detectionE1,E2,E3

1H 500 MHz NMR spectrum, 30 s

~20 g from wells E1-E3 in 6 L CD3OD, 24 sec

2 x 1,2,3-trisubstituted benzenes

COSY 8 min

AntiMarin search with MW = 320, 0 CH3,2 x 1,2,3-trisubstituted benzenes

Only1

hit

(Not this compound, but gives clue as to structural type)

Other search strategies

2 x 1,2,3-trisubstituted benzenes 198 hits

plus 0 x CH3 89

plus 0 x sp3-methylene 35

plus 1 x sp3-methine 3

(none of these fit data, but all contain 1,8-dioxonaphthalene fragment)

gHSQC45 min

gHMBC11 hr

O O

OH

O

O

Spiromycin A

Sonia van der Sar, John W Blunt & Murray H G Munro

Org Lett 2006 in press

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0

-200

0

200

400

600

800

1,000

1,200

1,400

1,600

1,800

2,000 Endophytes #124 F5584.NCI MT Int_Chan_1mV

min

ELSD detection

D4,D5

well D5, ~3 g, 8 min

COSY 25 min

O O

OH

OH

O

Spiromycin C

Summary Rapid dereplication of bioactives :

• 250-500 g bioactive extract separated by analytical HPLC into microtitre plate wells

• In-well bioassay to locate active components

• UV and MS data obtained from active wells

• 1H NMR spectra obtained from active components (> 2 g) (CapNMR)

• Recognisable structural features searched for in AntiMarin or MarinLit

• If compound unknown, collect 2D NMR data for structure determination

Note – all masses of samples given in this presentation are the masses of compound in the CapNMR microcell – these are ~65% of the amounts originally in the microtitre plate well(s) from which they were taken. The masses have been estimated from a calibration of the CHD2 solvent peak integral in a solution of a known compound of known concentration.