Rapid Dereplication using Capillary NMR and a Database of Structures
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Transcript of Rapid Dereplication using Capillary NMR and a Database of Structures
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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.)
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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)
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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.
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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
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C18 HPLC of extract frommarine-derived Cephalosporium sp.
DAD detection
Bioactivity profile
Provides retention time, UV and molecular weight of the active compound
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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)
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Search for MW=518 and UV 410-419 and 290-299
AntiMarin
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Only 1 hit
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•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)
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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.
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Protasis CapNMR Probe
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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
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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
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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
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only 1 hit
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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
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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.
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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.
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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
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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
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1 hit only if MW = 346 included in search
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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
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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
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only 1 hit
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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
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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
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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
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500 MHz30 g in 6 L CD3OD
2 min
MW 684 – unknown peptide
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TOCSY40 min
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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
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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
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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
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AntiMarin search with MW = 320, 0 CH3,2 x 1,2,3-trisubstituted benzenes
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Only1
hit
(Not this compound, but gives clue as to structural type)
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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)
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gHSQC45 min
gHMBC11 hr
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O O
OH
O
O
Spiromycin A
Sonia van der Sar, John W Blunt & Murray H G Munro
Org Lett 2006 in press
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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
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200
400
600
800
1,000
1,200
1,400
1,600
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2,000 Endophytes #124 F5584.NCI MT Int_Chan_1mV
min
ELSD detection
D4,D5
well D5, ~3 g, 8 min
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COSY 25 min
O O
OH
OH
O
Spiromycin C
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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
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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.