Some Recent Aspects in the Structure Elucidation of Natural Products

Although in the past 20 yr a large number of new synthetic approaches have been developed methods of isolation and structural elucidation have changed more dramatically in the last two decades than have those of synthesis. For the purification of natural products more and more different chromatographic techniques like paper, thin-layer, column, and gas—liquid chromatography are being used. Characterizations of natural products by elemental analysis, melting points, optical rotation values, and degradation and derivatization reactions are being increasingly supported by parameters of spectroscopic tools like proton nuclear magnetic resonance, carbon nuclear magnetic resonance, phosphorus nuclear magnetic resonance, fluorine nuclear magnetic resonance, infrared spectroscopy, Raman spectroscopy, absorption spectroscopy, optical rotatory dispersion, circular dichroism, magnetic circular dichroism and computer analysis. The principle of one of the most efficient methods for the structural elucidation of natural products, pulse Fourier transform '3C NMR spectroscopy, is discussed in detail. The usefulness of this method is demonstrated using examples from the peptide, carbohydrate and terpenoid field. The structural elucidations by the application of different physicochemical tools for the three following most recently isolated natural products are given: Hypothalamusreleasing hormones, terpenoids from Melia azadirachta Linn., and substances, isolated from urines of sick children. Natural product's chemistry can be divided into three different fields: methods of isolation, methods of structure elucidation and synthetic approaches. Though in the last two decades numerous new types of chemical reactions have been found the methods of isolation and structure elucidation have changed more fundamentally than those of synthesis. For the isolation and purification of organic compounds the different methods of chromatography (paper,'3 thinlayer,'6 column,79 and gas chromatography,'°2 have been developed. One of the most recent and fruitful developments in the field of chromatography, which surely will find great applicability for the separation of natural products, is that of high performance liquid chromatography. Some examples, mainly investigated in our laboratory, are given below. If a natural product's chemist is convinced that he has isolated a compound of high purity he can begin with its structure elucidation. Only two decades ago this work was mainly based on chemical reactions like those of degradation and derivatization. Only few physico-chemical parameters were available at that time characterizing organic molecules: melting points, solubilities, values of elemental analysis, molecular weights, or specific rotations. Since then different physicochemical instruments like those of absorption spectroscopy (ABS), optical rotatory dispersion (ORD), circular dichroism (CD), mass spectrometry (MS), and nuclear magnetic resonance (NMR) were developed which allow recording of spectra routinely and thus the basis of correlation between spectral characteristics and structure for organic molecules was laid. The enormous amount of data accumulated in the last years in the ABS, ORD, CD, MS and NMR laboratories will force us in future time to lookfor ways of storingthese parameters on computerfiles. In the following the principal fundamental newer developments in some of these fields are discussed an finally some practical examples investigated in our laboratory are given. 2. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY'3'14 The isolation of a natural product can be considered already as part of its structure elucidation and therefore it is a necessity for a natural product's chemist to know about the theory, the limits and applicability of the different chromatographic methods. As already mentioned before, high performance liquid chromatography seems to have become the most efficient and suitable method for the separation of complex mixtures of natural products. This idea caused us to develop a system for the separation of carbohydrates respectively amino acids and peptides, as our research group works on the synthesis of peptides and carbohydrates. (a) Principle of a high resolution Carbohydrate analyzer More than 20 yr ago the first column chromatographic separations of carbohydrates were reported. Since then three different principles were applied: 1. adsorption chromatography15'7 2. partition chromatography'2° and 3. ion exchange chromatography.224 The efficiency of the latter method prompted us to construct an apparatus (Fig. 1) applying this principle. Borate complexes of carbohydrates specifically interact with anion exchange resins and are therefore separable on columns filled with this material.26 A Milton Roy—Dosapro micro pump pumps borate buffer from a gradient-generating system (Grad., Fig. 1) respectively borate buffer reservoirs (A, B, Fig. 1) via a prewashing column on a Biotronik (Frankfurt, Germany) glass-jacketed high pressure liquid chromatographic column, filled with an anion-exchange DA-X 4 resin from Durrum Chemical Corporation, Palo Alto, U.S.A. The DA-X 4 resins are 4% cross-linked polystyrenes of 10—20 j.m particle size. To the effluent of the column orcinol sulfuric acid reagent is added and the mixture passes a 20 m x 0.7 mm Teflon coil which is placed in a heated (98—100°C) water bath. Under these conditions 105 PAC, Vol. 48, No. 1—H tCommunication 120 about structures of natural products and organic molecules. 106 WOLFGANG VOELTER sugars from furan derivatives which react with orcinol