Natural product Purification

Computer-aided inhibitor design is a relatively new and powerful approach for the development of novel, potentially potent, nonsubstrate-analogue enzyme inhibitors. Computer-aided methods and biological screening can each lead to new classes of novel inhibitors. However, computer-aided design methods can focus the search for inhibitors, thereby circumventing much of the time-consuming synthetic and natural product purification procedures for those compounds they find unlikely to function as inhibitors. 

Foncanlt, A.P. and Chevolot, L., Connter-cnrrent chromatography instrumentation, solvent selection and some recent applications to natural product purification, J. Chromatogr. A, 808, 3, 1998. 

The six chromatographic modes available in HPLC for the isolation and purification of natural products are described in more detail in Section 9.02.2. Since the groups of natural products differ in their molecular properties, certain chromatographic modes have been shown to work better with particular natural product groups. However, in order to take full advantage of a specific HPLC mode for a separation task and to effectively utilize time and resources, comprehensive method development should be performed. An example of such method development from the analytical to the preparative stage is described for reversed-phase chromatography (HP-RPC), the most frequently employed mode in natural product purification, in Section 9.02.3. 

Molecular distillation is used in the separation and purification of vitamins and other natural products, and for the distillation of high-boiling synthetic organic compounds. 

Human growth hormone was originally manufactured by isolation of the natural product from human pituitaries and subsequent purification of the protein. Since 1985, manufacture of hGH has been almost exclusively by recombinant DNA technology. 

Interest in synthetic naphthenic acid has grown as the supply of natural product has fluctuated. Oxidation of naphthene-based hydrocarbons has been studied extensively (35—37), but no commercially viable processes are known. Extensive purification schemes must be employed to maximize naphthene content in the feedstock and remove hydroxy acids and nonacidic by-products from the oxidation product. Free-radical addition of carboxylic acids to olefins (38,39) and addition of unsaturated fatty acids to cycloparaffins (40) have also been studied but have not been commercialized. 

Ethanolamines. These are produced by the reaction of ethylene oxide and ammonia (see Alkanolamines). Approximately one-third of the production is used in detergents. Other appHcations include natural gas purification, cosmetics, metalworking, textiles, and chemical intermediates (282). 

Isolation and identification of substances (natural products from nature, protein purification and characterisation, etc). 

Flash chromatography is widely employed for the purification of crude products obtained by synthesis at a research laboratory scale (several grams) or isolated as extracts from natural products or fermentations. The solid support is based on silica gel, and the mobile phase is usually a mixture of a hydrocarbon, such as hexane or heptane, with an organic modifier, e.g. ethyl acetate, driven by low pressure air. (Recently the comparison of flash chromatography with countercurrent chromatography (CCC), a technique particularly adapted to preparative purposes, has been studied for the separation of nonchiral compounds [90].)... 

From the pioneering studies of Ito et al. [117], CCC has been mainly used for the separation and purification of natural products, where it has found a large number of applications [114, 116, 118, 119]. Moreover, the potential of this technique for preparative purposes can be also applied to chiral separations. The resolution of enantiomers can be simply envisaged by addition of a chiral selector to the stationary liquid phase. The mixture of enantiomers would come into contact with this liquid CSP, and enantiodiscrimination might be achieved. However, as yet few examples have been described in the literature. 

Structure determination of luciferin. Once a luciferin is obtained in a sufficient purity, the determination of luciferin structure should be attempted most of the important properties of luciferin are usually already obtained during the course of purification as a necessity. The structural study is considerably more straightforward than the extraction and purification, due to the availability of advanced methods, such as high-resolution mass spectrometry and various NMR techniques. If help or collaboration is needed in structure determination, the attractiveness of a luciferin will make it easy to find a good collaborator. However, the purified luciferin is usually an extremely precious material considering the effort spent in preparing it. To avoid accidental loss of the purified material, the chosen collaborator must have solid knowledge and experience in structure determination a criterion to be considered is that the person has successfully done the structure determination of at least one new natural product. 

Many natural products are charged substances, and can be isolated by IEC methods. Dufresne has published a comprehensive review describing various resins and column operating conditions applicable to purification of natural products from fermentation broths or crude extracts.168 Among natural products, antibiotics are of special interest due to their widespread use in humans and animals. Sample cleanup by IEC prior to analysis by other LC methods for quantitative determination of antibiotics in biological fluids is frequent.I69171 Also, IEC followed by TLC appears useful for the quantitation of fumonisin Bl, a mycotoxin found in agricultural products.172... 

One of the cornerstones of combinatorial synthesis has been the development of solid-phase organic synthesis (SPOS) based on the original Merrifield method for peptide preparation [19]. Because transformations on insoluble polymer supports should enable chemical reactions to be driven to completion and enable simple product purification by filtration, combinatorial chemistry has been primarily performed by SPOS [19-23], Nonetheless, solid-phase synthesis has several shortcomings, because of the nature of heterogeneous reaction conditions. Nonlinear kinetic behavior, slow reaction, solvation problems, and degradation of the polymer support, because of the long reactions, are some of the problems typically experienced in SPOS. It is, therefore, not surprising that the first applications of microwave-assisted solid-phase synthesis were reported as early 1992 [24],... 

Extracellular or intracellular nature of product. If the biopharmaceutical is an extracellular product then the less complex the media composition the better, in order to render subsequent product purification as straightforward as possible. 

Since 1988, the methods that we use to isolate cDNAs of alkaloid biosynthesis have become ever more facile and sensitive, allowing for more efficient cDNA identification. We do not, however, yet understand enough about the cellular localization of alkaloid formation or about the nature of the catalysts to move completely away from enzymology and biochemistry and to use only molecular genetic techniques to dissect these biosynthetic pathways. Even our most recently successful cDNA isolations and identifications involved classical protein purification. We are beginning now to use proteomics and EST sequencing to identify natural product biosynthetic cDNAs, but these approaches are more feasible when a specialized cell/tissue type in which secondary metabolite biosynthetic pathways are active, can be isolated and used as a protein or RNA source. 

Absorption is a commonly applied operation in chemical processing. It is used as a raw material or a product recovery technique in separation and purification of gaseous streams containing high concentrations of organics (e.g., in natural gas purification and coke by-product recovery operations). In absorption, the organics in the gas stream are dissolved in a liquid solvent. The contact between the absorbing liquid and the vent gas is accomplished in countercurrent spray towers, scrubbers, or packed or plate columns. 

---