Structure dereplication

Gas chromatography is a powerful technique to dereplicate low to moderate polarity natural products especially when it is coupled with ESI-MS. The reproducibility of ionization and fragmentation pattern from GC/MS make it one of the most efficient and reliable techniques in structure dereplication. There are different GC/MS libraries commercially available that contain mass spectra of hundred thousands of compounds. Unfortunately, approximately 80% of all known natural compounds are nonvolatile or thermally unstable and therefore incompatible with GC/MS methods. Due to the diversity of HPLC columns and the broad selections of solvent combinations and gradients, HPLC is capable of separating almost any kind of natural products. The development of HPLC detectors and... 

Fig. (7). Structure dereplication results of COX inhibitory HTP fractions from the aqueous extract of green tea.

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. 

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."... 

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. 

Bradshaw et al. have disclosed a rapid and facile method for the dereplication of a purified natural product library [58]. The method integrates the molecular weight from low resolution MS data with the exact count of the number of methyl, methylene and methine groups obtained from ]H-13C NMR correlation data. Those structure properties were converted into a searchable text file that could be downloaded into a customized software program with chemical structure information in a specific format - SMILES. In the program, more than 120,000 unique structures were derived from commercial databases, such as Dictionary of Natural Products and Beilstein. 

Carter, G.T. 1998. LC/MS and MS/MS procedures to facilitate dereplication and structure determination of natural products. In Natural Products Drug Discovery II New Technologies to Increase Efficiency and Speed. Sapienza, D.M. and Savage, L.M., Eds. Southborough, MA IBC Communications, pp. 3-19. 

In general terms, dereplication refers to the differentiation of a compound of interest from nuisance compounds, e.g. a novel structure from known natural products. 

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