Preface Plants possess the ability to biosynthesize a large variety of steroids, but it was not until 1979 that steroids with plant hormonal activity were discovered. American scientists were able to isolate a new steroidal lactone called brassinolide from bee-collected pollen of Brassica napus L. Two years later, castasterone was found in insect galls of Castanea crenata spp. To date, more than 40 structurally and functionally related steroids in plants are known, and this group of compounds is now characterized as brassinosteroids (BS). BS are present m nearly every part of the plant, with the highest concentration in the reproductive organs (pollen and immature seeds). They demonstrate various kinds of regulatory actions on growth and development of plants, such as stimulation of cell enlargement and cell division, accretion of biomass, yield and quality of seeds, and plant adaptability. At the molecular level, BS change the gene expression and the metabolism of nucleic acids and proteins. For these reasons, BS are now considered a new group of plant hormones. The extremely high activity of BS has attracted the attention of many specialists in the fields of analytical and synthetic chemistry, biochemistry, plant physiology, and agriculture. Several problems connected with BS had to be solved after their discovery. First, it was necessary to elaborate methods for isolation and identification as well as for biological testing to guide the isolation procedure. Also very important was progress in the chemical synthesis of BS and their analogs. This was necessary to elucidate the structures, to study the biosynthesis, and to clarify the structure-activity relationships in the BS series. Later, synthesis became important for obtaming sufficient quantities for IX X PREFACE biological testing and for searching for stable, economically attractive compounds with high activity for practical application in agriculture. It is very important to realize that BS are naturally occurring compounds, ubiquitous in plants that are used for human and animal nutrition. BS are metabolized m the usual ways, and they have played roles in the long combined evolution of plants and animals. This fact provides relative assurance of the safety of BS in the very low doses found naturally in plants. Chapter II reviews all the BS known today, and a new system of abbreviated names related to their structures is proposed. The isolation procedures and spectroscopic structure determination of BS are documented in Chapters III and IV. The biosynthesis of BS and their metabolic transformations are treated in Chapter V. Synthetic approaches to BS and the syntheses of then- most important natural representatives and analogs are discussed in Chapters VI, VII, and VIII. Much knowledge has been gathered about the physiological mode of action of BS, and the present state of the art is described in Chapter IX. Special attention has been devoted to investigations carried out in the former Soviet Union and described in Russian journals and patents. This literature is practically inaccessible to an international readership, and this monograph will be of help in filling this void. To obtain a more precise understanding of the structural requirements for high activity in BS, much attention has been given to structure activity relationships in Chapter X. This will allow researchers to predict the activities of new analogs and to design BS with the best synthetic cost/activity ratio. Chapter XI covers the substantial amount of data indicating that the application of BS allows a significant increase in the yields of most crops. Parallel with increasing crop yields, a substantial improvement in their quality takes place in many cases. Based on these properties, the natural phytohormone epibrassinolide (EBl) has been approved for agricultural use in countries of the former Soviet Union since 1992. PREFACE xi The world literature on BS now includes more than 1000 publications. These are spread among one monograph, conference proceedings, and general and specialized reviews on the distribution of BS in plants; their isolation, structure, and synthesis; plant physiology; and practical application of BS. To the best of our knowledge, the bulk of this literature has been considered in compiling this monograph. The authors thank the many people who have contributed to this work. Fu-st, we sincerely thank Dr. Natalia Khripach, not only for the way she took care of our mood and health but also for her substantial contribution to the biological part of the book and for many stimulating discussions. We also acknowledge the valuable contributions of Prof Dr. Linus van der Plas and the staff of the Laboratory of Plant Physiology of the Wageningen Agricultural University and Dr. Harro Bouwmeester of the AB-DLO Institute in Wageningen for reading the chapters on plant physiology. We extend our appreciation to Dr. Margarita L Zavadskaya for help in the preparation of the manuscript and for her kind and fruitfiil discussions throughout this work. Thanks are also due to Drs. Natalia N. Malevannaya, Ivan K. Volod'ko, Alexander I. Zabolotny, Alexander N. Vedeneev, Natalia N. Vlasova, Ludmila N. Kalituho, and Zhanna E. Mazets for generously supplying data and participating in discussions on the particular problems of BS action in plants. We also thank Dr. Vladimir B. Petukhov for fruitful discussion on the prospects of studying BS in vertebrates, Dr. Alexander S. Lyakhov for discussion and for providing of X-ray analysis data, and Dr. Han Zuilhof, Bep van Veldhuizen, and Dr. Maarten Posthumus for their contributions in the areas of NMR and mass spectroscopy. Abbreviations Substances, Reagents, and Solvents ABA Abscisic acid AD-mix-p A mixture of (DHQD)2-PHAL, K20s02(OH)4, K3Fe(CN)6, and K2CO3 AIBN Azobis(isobutyronitrile) 9-BBN 9-Borabicyclo[3.3.1 ]nonane BS Brassinosteroids CHP Cumene hydroperoxide CSA Camphor-10-sulfonic acid DBU 1,5-Diazabicyclo[4.3.0]non-5-ene DCC 1,3-Dicyclohexylcarbodiimide DEAD Diethyl azodicarboxylate DHP Dihydropyran DIB AH Diisobutylaluminum hydride DMAP 4-(Dimethylamino)pyridine L-DET (L)-Diethyl tartrate DME 1,2-Dimethoxyethane DMF A^^-Dimethylformamide DMSO Dimethyl sulfoxide DHQD CLB Dihydroquinidine p-chlorobenzoate DHQDNAP Dihydroquinidine 9-(r-naphthyl) ether DHQD PHN Dihydroquinidine 9-phenanthryl ether DHQ CLB Dihydroquinine /?-chlorobenzoate (DHQD)2-PHAL Dihydroquinidine 1,4-phthalazinediyl diether DPTBSCl Dipropyl-reA•^butylsilyl chloride GA Gibberellic acid HMPA Hexamethylphosphoramide lAA Indole^3-acetic acid XIU XIV ABBREVIATIONS LDA Lithium diisopropylamide MCPBA w-Chloroperbenzoic acid NCS A^-Chlorosuccinimide NMO A^-Methylmorpholine A^-oxide PCC Pyridinium chlorochromate PPTA 4-Phenyl-1,2,4-triazoline-3,5-dione PPTS Pyridinium toluene-p-sulfonate Py Pyridine TBAF Tetrabutylammonium fluoride TDTAP Tetradecyltrimethylammonium permanganate TFD Methyl(trifluoromethyl)dioxirane THF Tetrahydrofuran Protecting Groups andLigands Ac Acetyl acac Acetylacetonate Bn Benzyl Bu «-Butyl Bz Benzoyl dmpe 1,2-Bis(dimethylphosphino)ethane dppp 1,3-Bis(diphenylphosphino)propane EE 1-Ethoxyethyl MEM (2-Methoxyethoxy)methyl MOM Methoxymethyl Ms Methanesulfonyl (mesyl) Ph Phenyl Pr ^-Propyl TBDMS rer/-Butyldimethylsilyl Th Thienyl THP Tetrahydropyranyl TMS Trimethylsilyl Ts /7-Toluenesulfonyl (tosyl) Tol Tolyl (4-MeC6H4) CHAPTER INTRODUCTION Until recently it was assumed that the main plant growth processes were controlled by 5 types of phytohormones, namely the auxins, the cytokinins, the gibberellins, abscisic acid, and ethylene (Fig. 1). CH2C00U H H indole-3-acetic acid gibberellic acid kinetine (an auxin) (a gibberellin) (a cytokinin) H 2 C = C H 2 COOH ethylene abscisic acid Fig. 1. The structures of some phytohormones. 2 BRASSINOSTEROIDS : A NEW CLASS OF PLANT HORMONES The role of steroids as hormones of mammals has been known since 1930 and steroidal hormones have been found also in insects and fungi. Plants possess the ability to biosynthesize a large variety of steroids and a hormonal function was repeatedly postulated for plants also. However, it was not until 1979 that steroids with hormonal functions were discovered in plants. In that year American scientists reported a new steroidal lactone called brassmolide from bee-collected pollen of Brassica napus L. Two years later castasterone was isolated from insect galls of Castanea crenata spp. (Fig. 2). To date, more than 40 structurally and functionally related steroids have been isolated from natural sources, and this group of compounds is indicated now as brassinosteroids (BS) and considered as a new group of plant hormones. BS demonstrate various kinds of regulatory activities on the growth and development of plants, such as stimulation of cell enlargement and cell division, lamina inclination, bending of leafs at the joints, and changes in membrane potentials. At the molecular level BS change the gene expression and the metabolism of nucleic acids and proteins. BS are widespread in nature and are found in gymnosperms, monocotyledons, dicotyledons, and algae. BS are present in nearly every part of the plant, with the highest concentrations in the reproductive organs (pollen and immature seeds). OH OH HO, HO. . HO' • \ o HO'"^ % brassinolide castasterone Fig. 2. The first brassinosteroids. INTRODUCTION H Other countries D Italy B Belarus • China • Germany r^ DUSA O) • Japan Year Fig. 3. The number of articles on BS. Treatment of plants with BS at the appropriate stage of their development results in an increase of crop yield and, in some cases, in an increase of its quality. These effects can be achieved by applying BS at doses of 20-50 mg/ha and that is much less than those for the usual plant growth stimulators. For practical application the ability of BS to increase the resistance of plants to unfavorable factors of the environment, such as extreme temperatures, salinity, drought, or pesticides, is important also. The extremely high activity of BS has attracted the attention of many specialists in the fields of chemistry, biology, and agriculture (Fig. 3). As result of large-scale scientific programs, started initially in the U.S. and Japan and later in the USSR, Germany, and China (Fig. 4), many problems connected with BS Other countries Japan USA Germany Fig. 4. The contribution of scientists from different countries to the total number of articles. 4 BRASSINOSTEROIDS: A NEW CLASS OF PLANT HORMONES chemistry and biology have been solved since 1980. Plants contain BS at such low concentrations (10"^-10"^^%) that elaboration of specific methods for their analysis became necessary. Although the first BS were identified after isolation as pure compounds, later on the presence of many BS was proven by their synthesis as reference compounds, in combination with GC-MS analysis. Progress in BS chemistry and biology would have been impossible without sensitive biological tests. In the beginning tests originally developed for other phytohormones were used. When research on BS was well established, a number of specific tests were developed for this group of phytohormones. Because of the low BS contents in plants, the only source of these compounds for biological studies and practical purposes is chemical synthesis. The progress in the chemical synthesis of BS and their analogs has been important and has led to economically feasible approaches that have brought their practical application in agriculture within reach. To date, only one attempt has been made to realize the total synthesis of BS but also the partial synthesis of these complex compounds, with 10-13 chiral centers, is a rather difficult task. Investigations in this direction have led to the elaboration of many new synthetic methods, especially for the construction of the side chain. Much attention has been devoted to syntheses of BS starting from abundantly available natural steroids. A number of sterols can be used for this purpose, and the best choices are of course those that contain the same carbon skeleton as the final compound. In this way carbon-carbon bond-forming reactions can be avoided and the synthesis can be performed in a more simple and less expensive way. This is especially important for BS that have chances for practical application in agriculture. Since their discovery in 1979, a great deal of information on BS has been accumulated, and the necessity for a monograph in this field has become evident. The data presented here form a reliable basis for fiirther development of BS INTRODUCTION 5 research in a theoretical and practical way. Further investigations will contribute to theories about the role of steroids in the exchange of biological information in all living organisms. BS research is based on a vast experience in the field of steroids and phytohormones. Many problems proved to be common for steroids and phytohormones and the results obtained with BS are of interest for many scientists working in related disciplines. The present book is the first monograph in English that covers all aspects of BS research, including isolation and identification of BS, distribution in the plant kingdom, biosynthesis and metabolism, synthesis, biological activity, and practical application. In this book also much information on this subject from countries of the former Soviet Union has been made available for international readership. The authors hope that this book will stimulate scientists to do research on brassinosteroids, being one of the new and promising subjects in the area of natural product chemistry and plant physiology.