Dereplication

Wolfender, J. L., Waridel, P., Ndjoko, K., Hobby, K. R., Major, H. J. and Hostettmann, K. (2000). Evaluation of Q-TOF-MS/MS and multiple stage IT-MSn for the dereplication of flavonoids and related compounds in crude plant extracts. Analysis 28 895-906A. 

The isolation and structural determination of natural products is a time-consuming and expensive process, even using modern methods. It is most important therefore to recognize and exclude known compounds at the earliest possible stage, a process which is called dereplication. 

For this task, easily accessible properties of mixtures or pure metabolites are compared with literature data. This may be the biological activity spectrum against a variety of test organisms. Widely used also is the comparison of UV [90] or MS data and HPLC retention times with appropriate reference data collections, a method which needs only minimal amounts and affords reliable results. Finally, there are databases where substructures, NMR or UV data and a variety of other molecular descriptors can be searched using computers [91]. The most comprehensive data collection of natural compounds is the Dictionary of Natural Products (DNP) [92], which compiles metabolites from all natural sources, also from plants. More appropriate for dereplication of microbial products, however, is our own data collection (AntiBase [93]) that allows rapid identification using combined structural features and spectroscopic data, tools that are not available in the DNP. 

Modern drug discovery approaches involve HTS, where, applying full automation and robotics, hundreds of molecules can be screened using several assays within a short time, and with very little amounts of compounds. In order to incorporate natural products in the modern HTS programmes, a natural product library (a collection of dereplicated natural products) needs to be built. Dereplication is the process by which one can eliminate recurrence or re-isolation of same or similar compounds from various extracts. A number of hyphenated techniques are used for dereplication, e.g. LC-PDA (liquid chromatography-photo-diode-array detector). 

An effective way of dereplicating known or otherwise uninteresting compounds. 

The nature of the separation problem varies considerably, from the isolation of small quantities for dereplication study (analytical scale, milligram or less) to the isolation of larger quantities for structure elucidation and comprehensive biological testing (semipreparative scale, 5 mg or more). For these purposes, a good selection of different techniques and approaches is essential. 

The system relies upon preliminary fractionation of the microbial crude extract by dualmode countercurrent chromatography coupled with photodiode array detection (PDA). The ultraviolet-visible (UV-Vis) spectra and liquid chromatography-mass spectrometry (LC-MS) of biologically active peaks are used for identification. Confirmation of compound identity is accomplished by nuclear magnetic resonance (NMR). Use of an integrated system countercurrent chromatography (CCC) separation, PDA detection, and LC-MS rapidly provided profiles and structural information extremely useful for metabolite identification (dereplication, Figure 14.1). 

HSCCC fractions containing known metabolites as determined by dereplication were eliminated from further study and only fractions with novel structures and high potency were designated "hits."... 

In conclusion, the process allowed early recognition of lead novelty and has become an effective filtering function after a preliminary dereplication. In addition, the procedure provided adequate amounts of pure metabolites which allow proper chemical and biological characterization. The operation is semiautomated which makes leads generation faster, cheaper, and more efficient. 

Brust A, Garson MJ (2004) Dereplication of Complex Natural Product Mixtures by 2D NMR Isolation of a New Carbonimidic Dichloride of Biosynthetic Interest from the Tropical Marine Sponge Stylotella aurantium. ACGC Chem Res Commun 17 33... 

Moreover, such conventional off-line approaches often result in the reisolation ( replication ) of already known compounds. Here, LC-NMR offers an unique insight into the composition of mixtures at an early stage of the analytical process for identifying ( dereplicating ) unwanted or already known compounds and thus guide the targeted isolation of potentially new substances. 

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

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. 

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. 

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