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E-SPE : Explorative Solid-Phase-Extraction for Accelerated Natural Product Discovery and Purification
Månsson, Maria; Phipps, Richard Kerry; Gram, Lone; Larsen, Thomas Ostenfeld; Nielsen, Kristian Fog
Publication date: 2009 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit
Citation (APA): Månsson, M., Phipps, R. K., Gram, L., Larsen, T. O., & Nielsen, K. F. (2009). E-SPE : Explorative Solid-PhaseExtraction for Accelerated Natural Product Discovery and Purification. Poster session presented at Nordic Natural Products Conference, Reijkavik, Island, .
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E-SPE : Explorative Solid-Phase-Extraction for Accelerated Natural Product Discovery and Purification Maria 1Center
1* Johansen ,
Richard K.
1 Phipps ,
Lone
2 Gram ,
Thomas O.
1 Larsen
& Kristian F.
1 Nielsen
for Microbial Biotechnology, Department of Systems Biology, 2National Institute of Aquatic Resources. Technical University of Denmark. *
[email protected]
Background
Method validation
Many NP labs have developed internal strategies for standardised extract screening and purification, only publishing parts of the overall setup1,2. Most modern NP purification methods are based on reverse-phase (RP) due to its versatility3,4,5. However facing complex extracts with many components, a simple RP strategy can lead to poor recoveries. This is especially a problem when dealing with low-output extracts, e.g. from the marine environment6. For these types of extracts, orthogonal purification strategies are requisite7. So, we have developed a so-called Explorative SolidPhase Extraction (E-SPE) method, which in a fast and easy way will indicate the optimum purification strategy on a small scale in the exploratory stage of the discovery process. This will allow a more rational approach to the purification process.
The E-SPE method was tested on 25 different marine bacteria with antibacterial activities such as growth and quorum sensing inhibition. A few well-researched filamentous fungi were included as further validation. Each organism was tested in triplicates to ensure reproducibility. The method was found to be widely applicable on both bacterial and fungal samples, however very dependent on the matrix. Especially for the marine samples, extensive desalting was necessary to ensure reproducibility. Further tests are necessary to investigate the robustness of the method as well.
Ps. luteoviolaceae
Broth extraction with XAD7/HP20
Unretained acidics and neutrals
Unretained neutrals
Medium polar acidics
Polar acidics
C-18 separation: Korormycin? Total of 11 compounds awaiting final NMR confirmation
To obtain maximum complementarity, four different columns were selected. Together they provide information on size, charge and polarity of the active components needed to develop a purification strategy. • Strong anion-exchanger (SAX): Presence of carboxylic acids • Mixed-mode (RP) anion-exchanger (MAX): Presence of acids, enols and phenols Relative polarity (25, 60, 100% AcCN)
Example - Pseudoalteromonas luteoviolaceae: A marine bacterium representing a highly complex extract with multiple known antibacterials. Nonetheless, the E-SPE bioactivity profile against Vibrio anguillarum revealed the presence of a potential novel bioactive present in a fraction with no known bioactives. E-SPE also provided the information that this compound is medium polar with an acidic functionality other than COOH. SAX
MAX
SCX
CH3
LH20 H3C
O
O O
OH
O
NH H3C
• Strong cation-exchanger (SCX): Presence of basic amines
Korormicin 1c vs. marine gram neg.? H N
HO
• LH-20 for size-exclusion: Relative size
O
N H O
HN
H N O
H
CH3
N
H N
HO
O
N H O
O
Indolmycin MW 257
Size-exclusion by LH-20: Indolmycin Violacein Red pigment Other yellow
N H
Violacein MW 343
Above: UV chromatogram of retained fraction from cation-exchange, further separated by LH20 to give pure indolmycin and violacein (ID confirmed by CapNMR) as well as two minor, unknown pigments.
Each of the purified compounds were present in submilligram quantities and were therefore subjected to CapNMR for increased sensitivity. Besides a series of 3butyric acid oligomers, the known antibacterial compound indolmycin was identified for the first time in a nonStreptomyces strain. We are currently awaiting final NMR confirmation of a series of unknown compounds as well as the rest of the active components.
N H
Violacein vs. gram pos.
A total of 15 fractions for bioassay is generated from each extract (1 agar plate or 50 mL of liquid culture).
Apolar acidics
C-18 separation: 3-hydroxybutyric acid polymers 10 other compounds awaiting final NMR confirmation (incl. potential novel active phenol/enol)
H3C
Method setup
Retained basic compounds
Potential new bioactive vs. marine gram neg.! Above: E-SPE bioactivity profile for Ps. luteoviolaceae showing the distribution of active known and unknown compounds against gram-negative Vibrio aguillarum in a well-diffusion assay. Bioactivity profile shown as elution matrix from Cardellina et al. 1.
Method implementation As a proof-of-concept, a purification strategy was developed based on the E-SPE activity profile for Ps. luteoviolaceae and carried out on extract from 8 L of culture.
Conclusions E-SPE is a successful strategy to reveal novel, bioactive natural products from both fungal and bacterial sources. The advantages of E-SPE are many: Finding a purification strategy on small amounts before engaging semi-preparative purification Identifying possible pit-falls (stability, synergy etc.) Reducing false positives (media components, salt etc.) Reducing number of candidates for dereplication (at CMB by LC-UV-HRMS)
Basic/neutral
Unmasking potential candidates
Basic/neutral + medium polar
Enables target-guided isolation rather than bioguided fractionation
Basic
Standardising the analysis of samples (SOP) Medium size
Results obtained on a small scale (a single agar plate or 50 mL of culture) can readily be translated into bigger scale (200 plates, 10 L culture) for preparative results. The E-SPE strategy has proven it-self to be fast, easy and reproducible in use and has therefore been implemented as a standard screening procedure at CMB when dealing with new extracts.
Each of the active fractions are subjected to comparative dereplication by LC-HRMS. The MS chromatograms for all active fractions are compared, and the MS spectra are extracted for all peaks in common. The accurate mass is then used as query in a database search (AntiBase, AntiMarin or similar). This provides a full list of potential candidates as well as their likelihood to be novel.
References 1: Cardellina et al. J. Nat. Prod. 56(7):1123-1129, 1993 2: Samuelsson et al. J. Etno. Pharm. 14(2-3):193-201, 1985 3: Lang et al. , J. Nat. Prod. 71:1595-1599, 2008 4: Bugni et al., Mol. 13(6):1372-1383, 2008
____________________________
Acknowledgements
5: Appleton et al., Chimia 61(6):327-331, 2007 6: Molinski et al., Nat. Rev. Drug. Discov. 8(1):69-85, 2009 7: Dufresne et al., in Methods in Biotechnology vol. 4: Natural Products Isolation, ed. R.J.P. Cannell, 1998
The Danish Strategic Council for Food and Safety (FøSu) is acknowledged for their funding to the project. The Marine Chemistry Group at University of Canterbury, New Zealand with professors Murray Munro and John Blunt is greatly appreciated for their help with running CapNMR.
