Natural Product Chemistry
NGE Choy Eng, CHING Kuan Chieh
Post Doc Reseach Fellows
YANG Lay Kien
Senior Research Officer
CHEW Yi Hong, SNG Xin Yi Amanda
The primary focus of my research group is to explore the chemical diversity of the A*STAR Natural Product Library (A*STAR NPL) from plants and microbes as a source of new natural products derived compounds, enzymes and drug candidates to advance our knowledge of the nature and development of new pharmaceuticals, agrochemicals, functional foods, flavourings, cosmetics and personal care products. These studies require an efficient integration of fractionation methodologies (extraction and chromatography), analytical techniques (spectroscopy e.g. High Resolution Mass Spectrometry, 1H and 2D Nuclear Magnetic Resonance) and chemical modification (degradation, derivatization and biotransformation), supported by a wide array of fermentation, biochemical or cellular assays, genomic analysis and data mining. The compounds isolated from these natural sources, form the NP Compound Library, which is also managed and maintained by my group. The increasing sophisticated analytical platforms generate large and complex data structures, and we are continuously exploiting advance bioinformatics tools for metabolites studies.
Platforms for Natural Product Metabolites Discovery
- Bio-assay Guided Purification
- Chemometrics (Differential Metabolomics Analysis)
Since 1993, the Natural Product Collection has been used for the discovery of small organic bioactive molecules. This is accomplished through the high throughput screening of the extracts through biochemical or cellular assays to identify biological activities of interest. Once active extracts have been identified, they will be subjected to a two-phase chemical characterization process. Phase I entailing dereplication and Phase II comprising of fractionation, compounds isolation and structure elucidation.
Phase 1, Dereplication
The purpose of bio-assay guided dereplication characterisation is to rapidly identify known compounds which account for the activity observed in a particular extract. This is achieved by simultaneously analyzing the extract mixtures by analytical HPLC coupled to a high-resolution mass spectrometry and collecting 38 fractions directly into a deep well microtiter format block for biological testing. A schematic of the process and the equipment used at Natural Product Chemistry Laboratory is shown in Figure 1.
Compounds observed within the active fractions are matched against NPL HRMS/MS database of natural products which had been acquired under the same LC/MS conditions for identities confirmation (see confirmation of hypoxanthine via HRMS/MS). The NPL chemistry has established an accurate mass (HR-MS) and tandem mass (HR-MS/MS) spectral library which comprises of 2,622 compounds, including most commonly isolated natural products. Most of these compounds are available at NPL in its pure form, which can be tested for confirmation of the compound activity in the crude sample. In conjunction, we have also computed an accurate mass libraries (HR-MS) where the high-resolution MS data are "coupled" with the Dictionary of Natural Products or Chemspider search, to enable dereplication of compounds not represented in the NPL database and detection of target compounds (new compounds) for isolation.
Phase 2, Fractionation & Structure Elucidation
Phase II analysis involves fractionation and structure elucidation of the purified active compounds (Figure 2). We have developed an efficient high-throughput process whereby the secondary metabolites of crude extracts are enriched using vacuum-assisted chromatography followed by automated fractionation using preparative reverse phase (C18) HPLC. Aliquots of each fraction are tested for biological activity and active fractions are selected for high resolution mass spectrometer and 1H-NMR analysis. Subsequent purification steps are performed on the active fractions using HPLC separations or orthogonal separation techniques such as normal phase (silica and diol), size-exclusion or ion-exchange chromatography.
After each separation step, fractions are tested for biological activity and analysed by HR-MS and 1H-NMR. Fractions of sufficient purity and amount will then be further analysed using HR-MS/MS and 2D-NMR techniques, as appropriate, to confirm compound identity or to solve novel structures. Tandem mass spectral data of novel compounds will also be acquired to populate the A*STAR NPRL MS/MS database for future annotation.
Our group performs two strategies for the examination of metabolites: 1) targeted metabolomics (hypothesis-driven), or 2) untargeted metabolomics (unbiased approach, hypothesis generating). Multivariate statistical methods such as PCA, tests of significance, Venn diagram and Hierarchical clustering etc. are used to perform data analysis to distinguish the metabolites which have been significantly altered in samples. Identities of known metabolites can be confirmed by matching their fragmentation patterns with tandem mass spectral libraries. For unknown compounds which lack ready standards, purification and structure elucidation is undertaken to efficiently confirm their identities.
Collaborations and Services
One of the challenges in the natural product discovery is the difficulty and time required to isolate pure chemicals from the complex mixtures.
Our dereplication platform has proven to be an efficient downstream process to the high-throughput screenings. Besides in-house biological assays in NPB, we also support various research groups in A*STAR, such as discovery of SALL4 inhibitors (Justin Tan, GIS) and anticancer compounds (Siew Bee, BII and Boon Tin, IMCB), supply of compounds for chemogenomics (Prakash, BII) and Sortase Inhibitor (Hao Fan, BII), as well as cross functional collaboration project to identify bioactives (Prof Ganesan, NUH-NUS-BII). We have also attracted industrial partners who have committed into collaborative research programmes or engaged our capabilities on a fee for service basis confidential contract research.
We believe that augmenting the currently available information of the A*STAR Natural Product Collection with genome sequence and biosynthetic pathway annotation will greatly increase the utility of the Collection for diverse applications. We had isolated anthracimycin and analogues from a non-Streptomyces actinobacterial microorganism, providing validation evidence to its biosynthetic gene cluster identified by genomic mining strategies (Brigit and Frank, BII). In addition, we had undertaken LC-MS analysis and compounds purification, which provided useful insights into the characterization of Notonesomycin A analogues biosynthetic gene cluster from A793 by gene disruption experiments (Shawn Hoon, Wong Fong Tian and Zhang Mingzi, MERL and MEL).
Using the QTOF HRMS, our highly versatile discovery platform has found wide applications in both in-house and other research groups. We conducted analysis ranging from evaluation of effects of fungal cocultivation and gene disruption on actinomycetes (NPB), strains selection and process optimization for production of target chemical classes (BioTrans), to confirmation of modified analogues and products of enzyme digestion (IMCB). We are also expanding our analytics capability into answering biochemical questions. In collaboration with IMCB groups, we are establishing metabolomics platform to study the effect of gene knockout on mice (Sebastian, BII and Philipp, IMCB) and tumour cells (Wee Wei, IMCB).
More collaborative researches are also in the pipeline. We are involved in the metabolites profiling and identification for the study of Modesof-Action (MoA) of Phthalates and Diketones using the Toxicity MoA Discovery (ToxMAD) Platform (Lit Hsin, BII). We will also profile, isolate and characterise alkaloid compounds for the project to build a Microbial Alkaloid Production Platform for Biotechnology (Ang Ee Lui, MERL).
Natural Product Chemistry Members
Dr. Yoganathan KANAGASUNDARAM
|Dr. KANAGASUNDARAM Yoganathan||Principal Investigator|
|Dr. CHING Kuan Chieh||Postdoctoral Fellow|
|Dr. NGE Choy Eng||Postdoctoral Fellow|
|Ms. SEOW Chwee San, Deborah||Research Officer|
|Ms. YANG Lay Kien||Research Officer|
|Ms. Amanda SNG Xin Yi||Research Officer|
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