rapid, small-scale dereplication of bioactive extracts john blunt university of canterbury new...
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Rapid, Small-scale Dereplication of
Bioactive Extracts
John BluntUniversity of Canterbury
New Zealand
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~ 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)
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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
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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
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
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
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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
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Search in AntiMarin for 1 doublet CH3, 1,2,3,5-substituted benzene, and Mr = 194One hit only – data matches that for F8095-2
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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
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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
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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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