The Systematic Identification

Transcription

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2 T. J. Mabry, K. R. Markham and M. B. Thomas The Systematic dentification of Flavonoids With 325 Figures Springer-Verlag Berlin Heidelberg. N ew Y ork 1970

3 TomJ. MABRY Professor of Botany K. R. MARKHAM M.B. THOMAS The Cell Research nstitute and Department of Botany The University of Texas at Austin SBN SBN (ebook) DO / This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concemed specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under 54 ofthe German Copyright Law where copies are made for other than private use, a fee is payable to the publisher, the amount of the fee to be determined by agreement with the publisher. by Springer-Verlag New York nc Library ofcongress Catalog Card Number Softcover reprint of the hardcover 1 st edition 1970 The use of general descriptive names, trade names, trade marks etc. in this publication, even if the former are not especially identified, is not to be taken as a sign that such names, as understood by the Trade Marks and Merchandise Marks Act, may accordingly be used freely by anyone. Title No. 1622

4 The authors wish to acknowledge contributions and comments by Drs. Heinz Rösler and J acques Kagan

5 Preface About 1958, the late Professor R. E. ALSTON and Professor B. L. TURNER, both of the Department ofbotany, The University oftexas at Austin, initiated a general systematic investigation ofthe legurne genus Baptisia. They found that flavonoid patterns, as revealed by two-dimensional paper chromatography, were valid criteria for the recognition of the Baptisia species and for the documentation of their numerous natural hybrids. Later, they showed that the flavonoid chemistry could be used for the analysis of gene flow among populations. At that time no attempt was made to even partially identify the flavonoids which were detected chromatographically. Neverthe1ess, it soon became apparent that the full value of the chemical data for systematic purposes required knowledge of the structures of the flavonoids. n 1962, one of us (T.J.M.) in collaboration with Drs. ALSTON and TURNER beg an the chemical analysis of the more than 60 flavonoids which had been chromatographically detected in the 16 Baptisia species. n the intervening years, a number of chemists and botanists, inc1uding Drs. K. BAETCKE, B. BREHM, M. CRANMER, D. HORNE, J. KAGAN, B. KROSCHEWSKY, J. MCCLURE, H. RÖSLER, and J. WALLACE, participated in the development of techniques and procedures for the rapid identification of known flavonoids and in the structure determination of new flavonoids. n addition, the flavonoid chemistry of many plants other than Baptisia was investigated. Two of us (K. R. M. and M. B. T.) joined the group in 1965 and were recipients of Post-doctoral Fellowships from the University of Texas at Austin during the period ( ) when most of the information presented in this volume was assembled. This volume presents, for the most part, procedures wh ich were most useful in our flavonoid studies, together with our collection of ultraviolet and nuc1ear magnetic resonance spectra of flavonoids. Thus, no attempt has been made to describe all the information available in the literature regarding the isolation and identification of flavonoids (J.B. HARBORNE'S "Comparative Biochemistry of the Flavonoids" provides an excellent summary of the literature up to i966). Moreover, a number of c1asses of flavonoids are either not treated at all (anthocyanins) or are only covered briefly (for example, cha1cones and aurones). The quantity of data presented for each of the various c1asses of flavonoids corresponds roughly to the frequency with which we have encountered them. The book is divided into three parts (, 11 and 111); the first deals mostly with the isolation and purification of flavonoids while the second and third comprise a spectra section in which flavonoid UV and NMR spectra are discussed. Before an analysis of the flavonoids in a given plant is initiated, we place in the University of Texas at Austin Herbarium a voucher specimen representing the plant population under investigation. The importance of properly vouchering the plant material before beginning the chemical studies cannot be over emphasized for only in this way can later investigators ascertain with certainty the plant for which the chemical results are reported. Our first step in a typical investigation of the flavonoids in a plant is to extract the flavonoids from a few dried leaves with methanol or aqueous methanol; the extract is then used to determine the two-dimensional paper chromatographic flavonoid pattern.

6 V Preface Chapter describes in detail the two-dimensional paper chromatographic analysis of flavonoids. Although sufficient pure material can usually be eluted from the paper chromatograms to obtain the ultraviolet spectra, in some instances the purification of the flavonoids can only be achieved by other techniques such as thin-ayer or column chromatography. n Chapter the column and thin-ayer chromatographic procedures which are commonly employed in our laboratory for the separation of flavonoids are described. Chapter, the last chapter in Part, presents gas and paper chromatographic procedures for the identification of the sugar moieties in flavonoid glycosides together with comments on the various methods available for determining the structures of flavonoid aglycones. Part presents data and procedures for the ultraviolet spectral analysis offlavonoids. Once a pure flavonoid is obtained, its ultraviolet spectra in methanol alone and methanol with each of five diagnostic reagents are always recorded. Chapter V outlines the steps for obtaining the UV data while Chapters V, V and V present, respectively, the UV spectral curves with interpretations for flavones and flavonols; isoflavones, flavanones, and dihydroflavonols; and chalcones and aurones. For each of the 175 flavonoids examined in the present investigation a set of six UV spectra are presented along with Re values in the solvents used for two-dimensional paper chromatography and spot colors when viewed on paper under ultraviolet light alone and utraviolet light in the presence of ammonia vapor. Finally in Part we have discussed procedures for obtaining and interpreting NMR spectra offlavonoids (Chapter V) as wel as presenting 128 NMR spectra (Chapter X). Most of the NMR spectra were determined for the trimethylsilyl ethers of the flavonoids, all of which are soluble in carbon tetrachlqride. Only with two flavonoids, hymenoxin and scaposin, both of which are highly oxygenated and methoxylated flavones, have we found it necessary to resort to the total synthesis of a flavonoid in order to establish its structure; in alm ost all other cases the information recorded here was sufficient for the complete structure analysis. We wish to acknowledge a number of people who, either in correspondence or by providing flavonoid sampies, helped us complete this volume: E. M. BCKOFF, J. CHOPN, J. W. CLARK-LEWS, P. CRABBE, E. DEEDS, S. E. DREWES, D. L. DREYER, L. FARKAS, T.A. GESSMAN, B. HARBORNE, M. HASEGAWA, J. HERRAN, W. HERZ, W.E. HLUS, L. HÖRHAMMER, R.M. HOROWTZ, P.R. JEFFERES, L. JURD, N.KAWANO, AR. KDWAl, B.H. KOEPPEN, M. KOMATSU, P. LEBRETON, AC. NESH, R. NEU, A NLSSON, F.S. OKU MURA, W.D.OLUS, W. RAHMAN, D.G. Roux, M. SANSBURY, M.K. SEKEL, T.R. SE SHADR, E. SONDHEMER, H. SUGNOME, T. SWAN, T. TOMNAGA, E. W. UNDERHLL, H. WAGNER, J.E. WATKN, S.H. WENDER, E. WONG. Finally, we are grateful to a number of individuals who helped in the preparation ofthe manuscript and the running ofthe UV spectra: SUSAN WOODLAND, LNDA McMA HAN, SDNEY MORRS, GENE BRACKENRDGE, FRANCS HA YNES, SHARON SUTHERLAND, JAMES MEARS and JUDy"AUTREY. January 2, 1970 T.J.MABRY K. R. MARKHAM M.B. THOMAS

7 Contents Part The solation,purification and Preliminary dentification of Flavonoids Chapter 1. The Two-Dimensional Paper Chromatographie Analysis of Flavonoids Reagents and Materials Experimental Proeedures for the Two-Dimensional Paper Chromatographie Analysis of Flavonoid Mixtures The Determination of Re Values for Flavonoids The EfTeets of Flavonoid Struetural Variations on Re Values Relationships between Spot Color and Flavonoid Strueture The solation and Purifieation of Flavonoids by Preparative Two Dimensional Paper Chromatography The One-Dimensional Paper Chromatographie Purifieation of a Partially Purified Flavonoid ;.. 14 Chapter 11. The Separation offlavonoids by Column and Thin Layer Chromatography Preliminary Purifieation of Flavonoids in a Crude Plant Extraet U sing Chareoal The Separation offlavonoids by Polyamide and Siliea Gel Column Chromatography The Separation of Flavonoids by Siliea Gel and Polyamide Thin Layer Chromatography Chapter The Aglyeone and Sugar Analysis of Flavonoid Glyeosides Proeedures for the Acidic and Enzymatie Hydrolysis offlavonoid Glyeosides The Gas and Paper Chromatographie Proeedures for dentifying the Sugars Obtained by Hydrolysis of Flavonoid Glyeosides.. 26 The dentifieation of the Aglyeone and Loeation of the Sugar in Flavonoid Glyeosides The dentifieation of the Sugars in C-Glyeosylflavonoids Part 11 The Structure Analysis of Flavonoids by Ultraviolet Spectroscopy Chapter V. Reagents and Proeedures for the Ultraviolet Speetral Analysis of Flavonoids V -1. Preparation of Reagent Stoek Solutions and Solids

8 x Contents V -2. Procedures for Determining the Ultraviolet Absorption Spectra of Flavonoids Chapter V. The Ultraviolet Spectra of Flavones and Flavonols V-. The UV Spectra of Flavones and Flavonols in Methanol 41 V-2. The UV Spectra of Flavones and Flavonols in the Presence ofnaome V-3. The UV Spectra of Flavones and Flavonols in the Presence of NaOAc V-4. The Detection of Ortho-dihydroxyl Groups in Flavones and Flavonols by the Effect of NaOAc/H 3 B0 3 on the UV Spectrum. 50 V-5. The UV Spectra of Flavones and Flavonols in the Presence of ACl 3 and AC 3 /HC V-6. ndex ofultraviolet Absorption Spectra offlavones and Flavonols 57 Chapter V. The Ultraviolet Spectra of soflavones, Flavanones, and Dihydroflavonols V-. The UV Spectra ofsoflavones, Flavanones and Dihydroflavonols in Methanol V-2. The UV Spectra ofsoflavones, Flavanones and Dihydroflavonols in the Presence of NaOMe V-3. The UV Spectra ofsoflavones, Flavanones and Dihydroflavonols ' in the Presence of NaOAc V-4. The Detection of A-Ring Ortho-dihydroxyl Groups in soflavones, Flavanones and Dihydroflavonols by the Effect ofnaoac/h 3 B0 3 on the UV Spectrum V-5. The UV Spectra ofsoflavones, Flavanones and Dihydroflavonols in the Presence of Alel 3 and AC 3 /HC V-6. ndex ofultraviolet Absorption Spectra ofsoflavones, Flavanones and Dihydroflavonols...' Chapter V. The Ultraviolet Spectra of Chalcones and Aurones V-. The UV Spectra of Chalcones and Aurones in MeOH V-2. The UV Spectra of Chalcones and Aurones in the Presence of NaOMe V-3. The UV Spectra of Chalcones and Aurones in the Presence ofnaoac V-4. The Detection of Ortho-dihydroxyl Groups in Chalcones and Aurones by the Effect ofnaoac/h 3 B0 3 on the UV Spectrum V-5. The UV Spectra of Chalcones and Aurones in the Presence of ACl 3 and AC 3 /HC V-6. ndex ofultraviolet Absorption Spectra ofchalcones and Aurones 230 Part The Structure Analysis of Flavonoids by Proton Nuclear Magnetic Resonance Spectroscopy Chapter V. The Determination and nterpretation of NMR Spectra of Flavonoids. 253 V-. ntroduction

9 Contents X V-2. The Use ofdmso-d 6 as Solvent for Flavonoid NMR Spectroscopy 254 V-3. Preparation oftrimethylsilyl Ether Derivatives of Flavonoids V-4. nterpretation of the NMR Spectra of Fully and Partially Trimethylsilylated Flavonoids Chapter X. The NMR Spectra of Flavonoids. 274 Subject-ndex 345

10 Skeletons and N umbering Schemes for the Classes of Flavonoids Discussed in this Volume 3' 2' o Flavones o F1avonols zr 3' 3' o sofia vones Flavanones o Dihydrofiavonols 3' :CQ='i 1 2' }-' " 0 Aurones o Cha\cones