Natural products dereplication

Natural products offer a source of unique chemical diversity for the pharmaceutical industry. Numerous successful drugs derived from natural products have been introduced for the treatment of cancer (Hung et al., 1996 Pettit et al, 1994), immunosuppression (Perico et al., 1996), cardiovascular therapy (Nadin and Nicolaou, 1996 Tomoda et al., 1994), and antiinfective therapy (Turner and Rodriguez, 1996). 

Traditional approaches for natural product screening in drug discovery involve the testing of crude extracts obtained from microbial fermentation broths, plants, or marine organisms. When activity above a certain level is detected, active components are isolated and purified for identification. This process is often time consuming where the physicochemical characteristics of the active components are determined, known compounds are identified (dereplication), and the novel compounds are scaled-up for more detailed investigation. 

Shorter discovery timelines and accelerated development expectations have hindered the traditional approaches for natural products research. Furthermore, emphasis on chemical diversity presents a great challenge in this area, particularly because traditional natural products screening programs focus on one source of chemical diversity such as microorganisms or plants. Still, the primary issue remains how to assay this ideal source of new, biologically active compounds within the current timeframe necessary for modern drug discovery research. At the heart of this issue is the fact that traditional isolation and scale-up procedures are inefficient and often become the bottleneck in natural products dereplication. 

A specific area of drug discovery research, which required an immediate need for highly sensitive and rapid analysis, dealt with 

Most antibiotics come from secondary metabolites of soil microorganisms that inhibit bacteria or fungi. Large-scale screening of microorganism fermentations followed by isolation and structure elucidation is required. Because many natural products have been previously identified, approaches that avoid time-consuming isolation and provide quick elucidation are essential. 

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Lead identification Natural products identification Natural products dereplication Ackerman et al., 1996a... 

Using this approach for natural products dereplication, data are routinely obtained from 40 gg of crude extract. Performance examples include the identification of 16 analogs of teicoplanin and 12 analogs of phenelfamycin from separate samples. The summary of results obtained for phenelfamycin is shown in Table 6.4. The correlation of fraction, retention time, and molecular weight provides the essential information for rapid dereplication and identification. The time required to dereplicate natural product samples is about 1 week with this LC/MS-based method compared to several weeks by previous methods that involve traditional isolation steps. The use of this LC/MS-based methodology results in greater clarity and confident decisions for proceeding with the full structural study of an active component derived from a culture. 

When a new impurity is encountered during chemical process research, retention time and molecular weight information are compared to the database for rapid identification. This approach is similar to the procedure described for natural product dereplication. If the compound is not contained in the structure database, then the corresponding LC/MS/MS analysis is performed to obtain substructural detail and the proposal of a new structure. 

Analysis strategies that use on-line ESI-LC/MS approaches provide an integrated format for natural product dereplication by combining traditional fraction collection, sample preparation, and multi-component analysis into a single step. In this way, crude extracts are screened without extensive purification and chemical analysis. Furthermore, less material is required due to the sensitivity of the technique and chromatographic resolution is retained. 

Gilbert, J.R. Lewer, P. Carr, A.W. Snipes, C.E. Balcer, J.L., Gerwick, W. Natural Product Dereplication and Structural Elucidation Using LC/MSn Combined With Accurate Mass LC/MS and LC/MS/MS, in Proceedings ofthe 47th ASMS Conference onMass Spectrometry and Allied Topics, Dallas, Texas, June 13-17, 1999. 

The last two decades have seen tremendous breakthroughs in each of these technologies as well as in information transfer. This chapter attempts to outline the scope of the dereplication problems and show how these technological advances, along with long-established methods, can be applied to these processes of natural product dereplication and partial identification. 

A technology that is likely to be of great use for natural product dereplication, once It becomes more established, is capillary electrochromatography (CEC)... 

One difficulty in employing mass spectrometry in natural product dereplication is the lack of a universal ionization condition under which any unknown compound could be expected to be ionized. Optimization of ionization conditions involves many factors. In addition to the mode of ionization, the pH modifiers used in the HPLC solvent can be critical to the ionization process, and the choice of ion source and strength of electric field are all critical parameters. 

To illustrate how useful the unit-molecular-weight information is in natural product dereplication, Fig. 3A shows the unit-molecular-weight distribution of the approx 78,000 known natural products contained in the Chapman and Hall database (32). It can be seen that, for the reported natural products, determination of the molecular weight will narrow the reported structural possibilities to <800, and the largest number of known natural products that share a common unit molecular weight is only 759 (mol wt = 318). For that molecular weight, there are known natural products that exhibit 45 different empirical (or molecular) formulas, each of which adds up to approx 318 Dalton. 

Since natural product dereplication is an attempt to minimize effort by using that which is already known, efficient use of literature databases is critical to most dereplication procedures. The use of commercially available databases for natural products dereplication has been reviewed, and it has been estimated that a 300 investment in literature searches could avoid a 50,000 investment in reisolation and identification of a complex compound already described in the literature (64). 

Some of the most useful databases for natural product dereplication with their particular attributes are summarized in Table 4. The search mechanics vary between databases and those details should be obtained directly from the database vendors. However, a successful typical overall process is described below,... 

Lessons learned from natural products dereplication and prioritization in high-throughput assays can also be applied to the deconvolution problems, faced by combinatorial chemists when assaying pools of compounds. With limited resynthesis capabilities, not every active pool can be followed up, and the use of prioritization methods will be required, similar to those outlined above. 

R76 J.-L Wolfender, G. Marti and E. F. Queiroz, Advances in Techniques for Profiling Crude Extracts and for the Rapid Identification of Natural Products Dereplication, Quality Control and Metabolomics , Curr. Org. Chem., 2010, 14, 1808. 

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