. “um. w. . HT w 4.5?- I ; . fl (u . . op... . (N ( v. f... uldfii‘ ..u....v. l‘... ..... ow ‘ u v ,. 1.. 1!. 3.... Q ... Gun-n. . . a... _ . - .“I.O‘.n l 75.305 a. Pink». r? . Kill/5.9 r/Jmi. .. ‘ {5.1.9. .. .hu. . A. (it JEN: I“ 1.411?! A «A R J. A“ ichigm State 1 University L..- This is to certify that the thesis entitled presented by Y") are? L. “warm has been accepted towards fulfillment of the requirements for I‘l‘. . D . mwi-wv hz'wr'. Wan-i- degree in " " H 3 «Ba; )L; Major professor Date 7J3" /?% 0-7 639 g magma BY IIOAS & sous" am mum mc. LIBRARY BINOE RS SPRIIBPIIT. IMIEII ABSTRACT INDOLE COMPOUNDS IN SEEDS 0F ZEA MAYS By Axel Ehmann I GAS-LIQUID CHROMATOGRAPHIC ANALYSIS OF INDOLE-B-ACETIC ACID MYOINOSITOL ESTERS IN MAIZE KERNELS By Minori Ueda, Axel Ehmann and Robert S. Bandurski An improved method of fractionating the myoinositol esters of indoleacetic acid (IAA) from maize kernels by gas-liquid chromato- graphy has been developed. Mass spectrometry was employed as an aid in identification of the esters. Maize kernels contain three groups of esters of IAA: (a) IAA myoinositols, (b) IAA myoinositol arabino- sides, and (c) IAA myoinositol galactosides. Each group has three chromatographically distinguishable isomers. The glycosylinositols described are unique in that carbon 1 of the sugar is attached to the hydroxyl at C-5 of the myoinositol. II PURIFICATION OF INDOLE-3-ACETIC ACID MYOINOSITOL ESTERS ON POLYSTYRENE-DIVINYLBENZENE RESINS By Axel Ehmann and Robert S. Bandurski A method for the purification of indole-3-acetic acid and esters of indole-3-acetic acid and myoinositol on Dowex SON-X2 and on partially sulfonated polystyrene resins is described. The method has been applied to the analysis of the indolylic compounds present in crude acetone-water extracts of the kernels of Zea mays. Sufficient purification and enrichment of these compounds is obtained by a single column chromatographic step so that the column eluates can be examined by thin-layer chromatography, gas-liquid chromatography or combined gas-liquid chromatography-mass spectrometry. III THE ISOLATION OF DI-O-(INDOLE-3-ACETYL)wMYO‘INOSITOL AND TRI-O—(INDOLE-3-ACETYL)-MYO-INOSITOL FROM MATURE KERNELS OF ZEA MAYS By Axel Ehmann and Robert S. Bandurski Di-O-(indole-B-acetyl)—myo-inositol and tri-O-(indole-3- acetyl)-myo-inositol have been isolated from kernels of Zea mugs and identified by gas-liquid chromatographic-mass spectrometric analysis of their trimethylsilyl ethers. IV THE ISOLATION OF 2-0-(INDOLE-3-ACETYL)-D-GLUCOPYRANOSE, 4-0-(INDOLE-3-ACETYL)-D-GLUCOPYRANOSE AND 6-0-(INDOLE-3-ACETYL)- D-GLUCOPYRANOSE FROM MATURE SWEET CORN KERNELS OF ZEA MAYS By Axel Ehmann A new ester of indole-3-acetic acid and glucose has been isolated from mature sweet corn kernels of Zea mays. Two isomeric forms of this ester were resolved by thin-layer chromatography with Rf-values distinct from that of authentic l-O-(indole-B-acetyl)-B-D- glucopyranose. Analysis of the trimethylsilyl-ethers of the two iso- mers by combined gas-liquid chromatography-mass spectrometry showed that the esters have a free carbonyl function. The labeling of the carbonyl carbon with an O-methyloxime group, and the analysis of the trimethylsilyl O-methyloxime derivatives by gas-liquid chromatography- mass spectrometry allowed the unambigous identification of the new ester of indole-3-acetic acid and glucose as a mixture of 2-0-(indole- 3-acetyl)-D-glucopyranose, 4-0—(indole-3-acetyl)-D-glucopyranose, and 6-0-(indole-3-acetyl)-D-glucopyranose. V N-(p-COUMAROYL)-TRYPTAMINE AND N-FERULOYL-TRYPTAMINE IN MATURE SWEET CORN KERNELS OF ZEA MAYS By Axel Ehmann N-(p-coumaroyl)-tryptamine and N-feruloyl-tryptamine were isolcated from aqueous acetone extracts 0f ground kernels of Zea mays by successive column chromatography on partially sulfonated styrene- divinylbenzene copolymer resin, lipophilic Sephadex and preparative thin-layer chromatography. Identification of these compounds was made by combined gas-liquid chromatography-mass spectrometry of their trimethylsilyl-derivatives and the trimethylsilyl-derivatives of their acid hydrolysis products. INDOLE COMPOUNDS IN SEEDS OF ZEA MAYS By Axel Ehmann,é'~af>«— A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY' Department of Botany and Plant Pathology 1973 To Jan and Isa 11' ACKNOWLEDGMENTS I am deeply indebted to Professor Robert S. Bandurski, my major professor, and to the members of my guidance committee, Professors William B. Drew, Norman E. Good and William W. Wells for their continued encouragement and advice during the course of these studies. I am also very grateful to Professor Charles C. Sweeley for the use of the mass spectrometer facility and his helpful discussions on the interpretation of some of the mass spectral data, and Mr. Jack Harten and Mr. Norman Young for their technical assistance in operating the mass spectrometer and on-line computer system. The financial support from the National Science Foundation (GB-18353X) and the Michigan Agricultural Experiment Station in the form of a Research Assistantship during the course of this work is greatly appreciated. iii TABLE OF CONTENTS LIST OF TABLES ....................... LIST OF FIGURES ...................... INTRODUCTION ........................ LITERATURE REVIEW ..................... A. Heliotropism and the growth hormone concept B. The isolation and identification of "free" auxins C. Isolation and identification of bound auxins . . . . D . Isolation and identification of bound auxins in seeds of maize .................. EXPERIMENTAL ........................ I. Gas-liquid Chromatographic Analysis of Indole-3- acetic acid Myoinositol Esters in Maize Kernels II. Purification of Indole-3-acetic acid Myoinositol Esters on Polystyrene-Divinylbenzene Resins III. The Isolation of Di-O-(indole-3-acetyl)-myo- inositol and Tri-o-(indole-3-acetyl)-myo- inositol from Mature Kernels of Zea mays ..... IV. The Isolation of 2-0-(indole-3-acetyl)-D-gluco- pyranose, 4-0-(indole-3-acetyl)-D-glucopyranose and 6-0-(indole-3-acetyl)-D-glucopyranose from Mature Sweet Corn Kernels of Zea mays ...... V. N-(p-coumaroyl)-tryptamine and N-feruloyl-trypta- mine in Mature Corn Kernels of Zea mays ..... CONCLUSIONS ' ........................ BIBLIOGRAPHY ........................ iv Page vii 24 28 3T 3T 36 46 LIST OF TABLES LITERATURE REVIEW 1. Summary of the important experiments which lead to the growth hormone concept ............. 2. Naturally occurring indole compounds in plants . . . . EXPERIMENTAL I l. Gas chromatography of TMS derivatives of the IAA myoinositols .................... 2. Gas chromatography of TMS derivatives of the IAA myoinositol glycosides ............... 3. Mass spectrometry of TMS derivatives of IAA myoinositols and their glycosides ......... 4. Summary of the origin of the nine GLC components from the Salkowski-positive spots on TLC ...... 5. Gas chromatography of TMS derivatives of myo- inositol glycosides ................ 6. Mass spectrometry of TMS derivatives of myo- inositol glycosides ................ 7. Component analysis and stoichiometry ......... Comparison of the ratios of galactose to arabinose and inositol hexoside to inositol pentoside 9. Gas chromatography of permethylated sugars and pentamethyl myoinositols .............. 10. Mass spectrometry of pentamethyl myoinositols EXPERIMENTAL II 1. Quantitative detennination of IAA liberated by alkaline hydrolysis in eluent fractions from a sample of crude B fraction chromatographed on Dowex SOW-XZ resin ................. EXPERIMENTAL III 1. Stoichiometry of HRF-3 and HRF-4 ........... V Page 18 32 33 33 33 33 33 33 34 34 34 41 65 Page EXPERIMENTAL IV 1. Relative retention times of TMS-derivatives of HRF-l and HRF-Z, and their MO-TMS-derivatives ....... 97 2. The contribution of HRF-l and HRF-2 to the Ehrlich- positive spots on TLC, the five peaks of the TMS- derivatives of a mixture of HRF-l and HRF—Z, and to their MO-TMS-derivatives ............. 98 CONCLUSIONS 1. The endogenous concentrations of known indole com- pounds isolated from plant material ......... l26 vi LIST OF FIGURES LITERATURE REVIEW 1. The reactions of B-substituted indoles with p-dimethyl aminobenzaldehyde and the colors of the complexes . . EXPERIMENTAL I 1. Flow sheet for the preparative procedure described in the text ..................... 2. Thin layer chromatographic patterns of the eight preparations on silica gel F-254 .......... EXPERIMENTAL II 1. .Flow sheet of the extraction procedure of dry corn kernels ....................... 2. Elution profile of IAA and partially purified IAA esters chromatographed on acidic Dowex 50W-X2 resin . 3. Elution profile of IAA and partially purified IAA esters chromatographed on neutral Dowex SON-X2 re51n ........................ 4. TLC patterns of the distribution of IAA esters in eluent fractions from the acidic (A) and neutral (B) Dowex SOW-XZ column chromatography ....... 5. Elution profile of IAA and partially purified IAA esters chromatographed on "low capacity" resin 6. Elution profile of IAA and IAA esters from crude plant extract chromatographed on "low capacity" resin ........................ 7. Gas-liquid chromatogram of a mixture of TSIM-IAA ester derivatives .................. EXPERIMENTAL III 1. Plot of the Rf-values of B HRF-3 and HRF-4 . B . versus the ogarithm of mole? of IAA per mole of myoinositol ........ . ............. vii Page 16 32 37 39 4o 43 44 67 Page 2. Mass spectra of the TMS-ethers of B : I1QL;l-0- (indole-3-acetyl)-myo-inositol (spectrum I) and 32: 2-0-(indole-3-acetyl)-myo-inositol (Spectrum II) . 68 3. Mass spectra of peak 3 of the TMS-ethers of HRF-3 f ' (Spectrum III), of the intact TMS-ether of HRF-4 (Spectrum IV) and of the thermal degradation product of the TMS-dg-ethers of HRF-4 ........ 69 4. Mass spectra of peak 3 and peak 5 of the thermal degradation product of the TMS-dg-ethers of HRF-4 (Spectrum V) ..................... 70 EXPERIMENTAL IV l. Thin-layer chromatograms of HRF-l and HRF-2 follow- ing "low capacity" resin (A), Sephadex LH-20 (B) and preparative thin-layer (C) chromatography . . . . 102 2. Mass spectra of the first three peaks (Spectra I— III) of the TMS-derivatives of a mixture of HRF-l and HRF-2 ...................... l03 3. Mass spectra of the last two peaks (Spectra IV and V) of the TMS-derivatives of a mixture of HRF—l and HRF-2 ...................... l04 4. Mass spectra of the first SS ectrum VIII), second (Spectrum VII), and thir ISpectrum IX peak of the MO-TMS-derivatives of a mixture of HRF-l and HRF-2, silylated with BSTFA ............. 105 5. The mass spectra of the MO-TMS-derivatives of D- glucosamine silylated with TSIM (Spectrum X) and BSTFA (Spectrum XI) ................. l06 6. Mass chromatograms for the intensities of the ion pairs m/e 130 and m/e 202, m/e 157 and m/e 229, m/e 290 and m/e 362, m/e 494 and m/e 566, and m/e 347 and m/e 419, compared with the total ion in- tensity of the same scans (bottom panels) ...... 107 7. Mass spectra of the first (Spectrum XIII), second (Spectrum XIV) and third (Spectrum XV) peak of the MO-TMS-derivatives of a mixture of HRF-l and HRF-2 silylated with TSIM .............. 108 8. Mass spectra of the MO-TMS-derivatives of D-galact- uronic acid (Spectra XVI and XVII) and the TMS— derivative of the same compound (Spectrum XVIII) . . . 109 9. The structures of 2-0-(indole-3-acetyl)-D-gluco- pyranose (A), 4-0-(indole-3-acetyl)-D-glucopyranose (B), and 6-0-(indole-3-acetyl)-D-glucopyranose (C) . . llO viii Page EXPERIMENTAL V l. The mass spectra of the TMS- derivatives of compound (1) (Spectrum A) and compound (II) (Spectrum B) .................... l24 ix INTRODUCTION In 1935 Boysen-Jensen wrote in the foreward to his book "Die Wuchsstofftheorie": Twenty-five years have passed since the discovery of the growth substance in Avena coleoptiles. The development of research on the growth substance in this period of time is characterized by a con- tinually growing mass of publications. The growth-substance literature, in a narrow sense, includes at present about 200 individual papers." In 1961 Pilet published his comprehensive treatise on "les phytohormones de croissance” containing some 2500 original research papers on the subject. Today (1972/1973) it is safe to state that the number of original research reports on auxins is in excess of 5000. Despite this overwhelming accumulation of scientific data little is known about the mode of action of the auxins, and even less about their chemical nature. There is convincing experimental evidence that probably all endogenous auxins exist in bound forms, but only a few such bound auxins have to date been isolated and identified. The work presented under EXPERIMENTAL has been undertaken to complete the structural characterization of essentially all the extract- able, bound auxins in mature kernels of Zea mays. The experimental work is in the form of five individual research papers, two of which have been published, while the remaining three are in manuscript form. ADDENDUM: The first reprint is included only for the conven- ience of the reader. Parts II, III, IV, and V constitute the 'corpus' of the dissertation. LITERATURE REVIEW A. Heliotropism and the growth hormone concept De Candolle (1832) first used the term heliotropism (today called phototropism) to describe the phenomenon of plant movement in response to the changing position of the sun, observed on flowers of Helianthus, apical stems and leaves of many vegetable and garden plants. It was however the apparently unrelated phenomenon, called geotropism (Frank, 1868), that may be considered the beginning of auxin physiology and auxin chemistry. Ciesielski (1872) was the first to study the nature of the geotropic response of roots. He observed that the developing radicle of the germinating seed responds to gravity only at the tip (Table l-a). He decapitated the horizontally oriented roots and noticed that the roots had lost the ability to respond -u: gravity. The replacement of the root tip however restored the geotropic response. Since the actual bending of the root occurred some distance from the tip Ciesielski concluded that a "stimulus" must exist in the root tip that had to get to the zone of growth and there trigger the observed geotropic response. Darwin (1880), stimulated by Ciesielski's experiments per- formed the first* experiments on heliotropism under controlled *De Candolle (1832) writes on page 831: "Les anciens naturalistes et la plupart des agriculteurs ont attribué cet effet a 2 Table l. A summary of the important experiments on geotropism and heliotropism which lead to the growth hormone concept. Plant Experiment noigtion Reference :3 CR [ I: IIJCZ> [::::::J Avena Dark a Ciesielski (1872) c::1> ca 0' Avena +— Darwin (1880) Phalaris hv Rothert (1892, 1894) E::._... D Avena I] a 0 ‘—— W e Rothert (1892, 1894) phalam's hv ,____ Avena hv d Fitting (1907, 1908) .____ Avena hv e Boysen-Jensen (1910, [l ._ Avena [I hv U f Paal (1919) Table 2. Continued. - . Text Plant Exper1ment notation Reference Coax g Paal ( 1919) Avena Dark Nielsen (1924) .J Avena Hordeum 227 Avena k h Stark (1921) tissue extract Dar Seubert (1925) o———— ° Stark and Drechsel hv (1922) Zea mays j Stark (1924) Dark Cholodny (1924) A211 r-‘r- "' 2““ Avena Dark 1: k Sod1ng (1925) Li L Avena $5ding (1925) .1 a Table 2. Continued. Text Plant Exper1ment notation Reference Z Avena m Cholodny (1928) Zea mays W Zwv R A Avena n Nielsen (1928) Dark 0 Went (1928) 'O Cholodny (1935) laboratory conditions. He exposed young seedlings of Avena and Phalaris to unilateral illumination and, within a few hours, observed the bending of the coleoptiles towards the light source. (Table l-b). When he removed the tips from the coleoptiles before the illumination no curva— ture was observed. Shielding of the stem from the light did not prevent the heliotropic response, whereas shielding of the tips abolished the heliotropic response. Darwin (1880) concluded "that when seedlings are exposed freely to a lateral light some influence is transmitted from the upper to the lower part causing the latter to bend." The scientific community was very hesitant to accept this idea (Wiesner, 1881), and it was not until 1894 that Darwin's conclusions were confirmed. Rothert (1892, 1894) repeated the experiments with a large number of coleotpiles (Table l-c) under carefully controlled conditions. He found that the cutting of the vascular bundles just below the tip did not prevent a heliotropic curvature, and he concluded that the stimulus must be traveling in the living parenchyma. He estimated this basipetal transmission of the stimulus to be around 2 cm per hour. (Almost forty years later van der Weij (1932) published a value of 1-1.5 cm per hour for the rate of transport of the stimulus.) ce que les plantes cherchent l'air libre. M. Tessier a démontré la fausseté de cette opinion par une expérience simple et ingenieuse: il a placé des plantes vivantes dans une cave qui avait deux sortes d'ouvertures: d'un c6té, des soupiraux formés par des vitrages donnaient passage 3 la lumiére, et non a l'air; de l'autre, des soupiraux ouverts dans un hangar vaste et obscur, qui donnaient passage 3 l'air, et non a lumiere. Si les plantes eussent cherché l'air, elles se seraient dirigées vers ces derniéres ouvertures; mais elles se sont, au contraire, toutes tournées du c0té des soupiraux clos et éclairés. Il est donc bien certain que, quand les plantes se dirigent vers les croisées des maison ou les clairieres des foréts, c'est la lumiere et non l'air qu'elles vont chercher." Rothert (1894) also observed that the loss of the heliotropic response could be overcome by continued unilateral illumination of the coleop- tile stumps for 3-24 hrs. He concluded that the stump, at the cut end, is able to regenerate (physiologically speaking) the tip, Fitting (1907, 1908) extended these experiments by removing portions of the coleoptile tip from the illuminated side (Table l-d). He found no measurable effect of the cutting itself, and concluded that curvature of the coleoptiles towards the light is due to differ- ential growth: rapid cell elongation on the dark side and lack of cell elongation on the illuminated side. Boysen-Jensen (1910, 1913) showed that decapitated coleoptiles which had their tips replaced could respond to a light stimulus (Table l-e). He further demonstrated that physical continuity between the tip and the basal part of the coleop- tile is necessary only on the dark side of the coleoptile for a heliotropic response to occur. The insertion of a thin piece of mica into a cut on the illuminated side did not prevent curvature, whereas the insertion of the mica into a cut on the dark side completely pre- vented the heliotropic response. The insertion of a small disc of calamus (a reed with very large vessels) into the cut on the dark side did not abolish the heliotropic response. He concluded that the phototropic "Reizleitung"* is located in the tip and that it has to travel basally on the dark side before a heliotropic response can be observed. *"Reizleitung" is used in the early german literature to mean "the transmission of the phototropic stimulus." Reizleitung is often translated as "stimulus", but "growth-stimulus" is more correct, since the stimulus in phototropism is the light, which induces the movement of the growth-stimulus from the Avena tip to the basal part of the coleoptile. These experiments of Boysen-Jensen (1910, 1913) show clearly that the phototropic "Reizleitung" is a substance which is transmitted by diffusion. A few years later Paal (1919) confirmed Boysen-Jensen's experiments and showed that the "Reizleitung" is able to travel through a non-living entity, a block of gelatine (hydrogel) (Table l-f). He concludes that "die phototropische Reizleitung durch die Gelatineschicht nicht durch elektrische Strbme, sondern durch gelbste Stoffe vermittelt wird - Trager der Wachstumskorrelation ist zugleich Vermittler der phototropischen Reizleitung." That this is indeed the case is shown by Paal's experiment (Table l-g) where he used tips from Coix and placed them assymmetrically on Cbix hypocotyl sections. Within a few hours in the dark he noted a strong curvature of the hypocotyl section away from the assymmetrically placed tip. Paal (1919) postulated, for the first time, a hypothesis for the phenomenon of heliotropism: "The coleoptile tip is the center of growth regulation. This center pro- duces a substance (or mixture of substances) which is internally secreted and moves basally in the living tissue with an equal distri- bution on all sides. Once the substance reaches the zone of growth it induces uniform growth. (This was confirmed a few years later when deing (1925) discovered that decapitated Avena coleoptiles and de- capitated coleoptiles with the tips replaced showed a significant (difference in straight growth (Table l-k) in the absence of light. lJnilateral illumination of the coleoptiles resulted in negative curva- tnlre (Table l-l)). If however the movement of the substance is Ilartially or totally prevented growth slows down or is stopped alto- QEther. If the movement of the substance is prevented unilaterally growth decreases on the side of interruption, whereas the noninter- rupted side continues to grow, resulting in a curvature of the coleop- tile towards the side of the out (see Table l-g). (Nielsen (1924) confirmed this by placing Avena coleoptile tips unilaterally on decap- itated coleoptiles, causing a positive curvature in the dark (Table l-g)). If the intact coleoptile is however exposed to unilat- eral illumination, the production of the growth-stimulating substance stops, or the substance is photochemically destroyed on the illuminated side. This would then result in a decrease of growth on the illumi- nated side, that is, a curvature of the coleoptile towards the light." This no doubt represented a major step towards the isolation and identification of the substance (or substances) responsible for phototropism. Stark (1917) studying traumatotropism (the movement of plants in response to physical and chemical stimuli) pursued this idea. He speaks of a "chemical polarity" in the tip created by the light stimu- lus, and proposes two possibilities which could bring about this chemical polarity: 1) different substances for different kinds of stimuli (such as geo-, photo-, and traumato-tropism), and 2) internal chemotropism (different stimuli leading to the same result, curvature). It should therefore be possible to isolate a substance which when applied unilaterally should give the same result regardless of whether light, gravity or other stimuli evoked the growth response. A few Years later he performed such an experiment (Stark, 1921) and found that coleoptile extracts, solidified in agar blocks and applied uni- laterally to decapitated Avena coleoptile cylinders resulted in a 10 negative curvature in the dark (Table l-h). In 1924 Stark showed that the tips of geotropically stimulated coleoptiles could induce curvature in decapitated, vertically oriented coleoptiles (Table l-j). Cholodny (1924) repeated Ciesielski's (1872) experiments on decapitated roots which no longer respond to gravity . The replace- ment of the root tip resulted in a geotropic response. Cholodny then replaced the root tips with coleoptile tips, and observed a normal geogrophic response (Table 1-j). He concluded that (at least in maize) the growth hormone* is identical in coleoptile tips and root tips. Stark and Drechsel (1922) also showed, that the substance transmitted from the tip to the coleoptile base is not species- specific. They placed tips of Avena on decapitated coleoptiles of Hardeum and Triticum and Herdeum tips on Avena coleoptiles, and observed a phototropic response (Table l-i). Cholodny (1928) confirmed this non-specificity of the growth-promoting substance, when he showed that the "growth hormone from maize tips could accelerate the growth of the decapitated Avena coleoptiles (Table l-m). Seubert (1925) found that the ethanol extract of human saliva could induce curvatures in decapitated Avena coleoptiles (Table l-h). She observed that high concentrations of the extract in the applied agar blocks induced posi- tive curvature. But upon making serial dilutions of the extract she found that the degree of positive curvature decreased, and at very low L *Fitting (1909 and 1910) introduced the term hormone (Bayliss and Starling, 1904) to plant physiology when he referred to the growth-promoting substance in orchid pollen as "Wuchsstoffhormon." 11 extract concentrations the curvature was negative. Nielsen (1928) demonstrated that culture medium extracts from Rhizopus and Absidia contain growth-promoting substances. Using decapitated Avena coleop- tiles he noted a curvature by applying the extract concentrate in agar blocks (Table l-n). Similar studies by Nielsen (1931, 1932) and Boysen—Jensen (1931a, 1931b, 1932) on growth-promoting substances from yeast, Boletus edatis, Aspergillus niger and several bacteria supported the non-specificity of the growth-promoting substance (or substances) as measured by the Avena curvatures.test. Until a method was found for the quantitative assessment of the biological activity of the growth-promoting substances from differ- ent sources it was impossible to relate their activity to the activity of the endogenous growth hormone in the coleoptile tip. Went (1928) found that coleoptile tips from Avena placed on agar apparently transferred the growth hormone to the agar. Following the diffusion of the hormone from the tips into the agar he cut the agar into small cubes and applied them to decapitated Auena coleoptiles (Table l-o). He found that the amount of diffusate in the agar block was proportional to the degrees of curvature, or straight growth, of the coleoptile per unit time. This agar block diffusion method also allowed Went (1928) to estimate the apparent molecular weight (=380) of this growth hormone, which he found to be heat-stable). The quantitative bioassay of potential growth promoters developed by Went (1928) stimulated an extensive search for these substances in plants and their identification. 12 B. The isolation and identification of "free" auxins K691 (1932) first introduced the term "auxin" (from the Greek word auxo--to grow) for growth substances that bring about cell enlargement, and it has become a standard term in the plant growth hormone literature. The experiments by Stark (1921), Seubert (1925), Nielsen (1928, 1930, 1931, 1932) and Boysen-Jensen (1931a, 1931b, 1932) on the existence of growth-promoting substances in crude extracts of Avena coleoptiles, human saliva, and culture media of Rhizopus, Boletus edutis, saccharomyces, AspergiZZus.niger and bacteria have already been mentioned. Purification of the growth-promoting substances from the extracts was however not accomplished until later and then only from Rhizopus. K691 et a1. (1933) were the first to isolate two highly active compounds: auxin a and the lactone of auxin a CH CH 1 3 3 CH3CH2-CH g CH-CHZ-CH3 CHOH-CHz-(CHOH)2 COOH $H3 1H3 CH3CH2-CH——— CH-CHZ-CH3 CHOH-CHz-fi-CHZ-COOH O 13 in crystalline form from human urine. The extraction and purification of 600 liters of urine from pregnant women gave 29.4 mg of crystalline auxin a and 6.9 mg of its lactone. Subsequent attempts to re-isolate these two auxins from urine of various sources have failed. K691 et a1. (1934) isolated another active auxin from urine which to their surprise contained nitrogen. Chemical analysis of this auxin showed it to be identical with indole—3-acetic acid which had been isolated by Salkowski and Salkowski (1880) from urinary albumin and synthesized by Ellinger (1904). 8 CH -C-OH ll2 Having found a compound with known chemical structure (indole-3-acetic acid) and high biological activity K691 and Kostermans (1935) began to study the structural requirements for biological acti- vity. They found that the free carboxyl group and the double bond between carbons 2 and 3 of the indole ring were necessary for highest activity. Similarly substitution of the ring nitrogen and other ring carbons resulted in complete loss of biological activity. The previous year K691 and Kostermans (1934) had already shown that the growth- promoting substance isolated from Rhizopus and AspergiZZus had a mole- cular weight similar to that of IAA. Thimann (1935) obtained convinc- ing experimental evidence that the growth substance from Rhizopus was 14 indeed IAA. These experimental results seemed to indicate that the growth hormone present in coleoptile tips might also be IAA. Many reports have since been published on the isolation and identification of free IAA from plant material. Hamilton (1960) has tabulated 50 such reports, but in no case has the auxin been positively identified. Identification was usually based on bioassay of regions from paper chromatograms which co-chromatographed with synthetic IAA. Unfortunately most of the chromatograms were developed in ammonia- water solvent systems which promote hydrolysis of IAA esters. Wildmann and Bonner (1948), Terpstra (1953) and other research groups (see Hamilton, 1960) have isolated a highly active substance from coleoptile tips by solvent extraction or diffusion into agar. The probable identity of this substance with IAA was based on its chromogenic properties (Hinsvark et aZ., 1954). The colorimetric detection of "IAA" was done with Salkowski (1885), van Urk (1929) or Ehrlich (1901) reagent. Though relatively specific for compounds containing an indole ring these color reagents also react with aromatic acids such as phenolics (Platt and Thimann, 1956; Steelink, 1959). Hartley-Mason and Archer (1958) have reported that p-dimethylaminocin- namaldehyde is more sensitive than p-dimethylaminobenzaldehyde (Ehrlich reagent). A careful study of these two reagents with a number of indole compounds showed, however, that the Ehrlich reagent is more specific for indole compounds, and also more sensitive. The Ehrlich reagent is about ten times more sensitive than Salkowski reagent, and for this reason has been used widely to identify IAA and its deriva- tives on paper chromatograms (PC), which had become a very useful 15 technique in the investigation of natural plant growth substances (Stowe and Thimann, 1954; Stowe, et al., 1956). However the "specific" color reaction for any given indole compound depends largely on the simultaneous interaction of at least three distinct color reactions of the indole moiety with p-dimethylaminobenzaldehyde. Dibbern and Rochelmeyer (1963) have studied these reactions and identified color reaction products, which is illustrated in Figure 1. They also found that a number of simple indoles do not react with the reagent: indole-3-aldehyde, indole-3-carboxylic acid and indole-3-carboxa1dehyde. Indoles with a substituted carbon 2 cannot react with p-dimethylaminobenzaldehyde, and any functional group on carbon 3 has to be removed by at least one methylene group. If the functional group is alpha to the 3-position the blue color does not develop. This shows that one should not solely rely on the chromogenic behavior of indoles for their identification (Denffer et aZ., 1952a, 1952b; Denffer and Fischer, 1952; Clark et aZ., 1959; Teubner, 1953; Isogai et aZ., 1963a and 1963b; Hofinger et aZ., 1970; Augier, 1970; KegleviC and Pokorny, 1969; Davies, 1972; Fellows and Bell, 1971). With the development of new techniques such as thin-layer chromatography (TLC), Fluorometry, gas-liquid chromatography (GLC) and mass spectrometry (MS) in the late fifties and early sixties and more recently affinity chromatography, it finally became possible to purify and identify endogenous auxins in sub-microgram amounts (Burnett and Audus, 1964; Dullaart, 1970; Venis, 1971; Bosin and Wehler, 1973). The combined GLC-MS technique offers the added 16 :=Ctl R ‘jl + O + '11 CH3—'N—CH3 ’/ O O weakly acidic strongly OCIdIC OWE—D g / \ | I cna—w—cn3 CH —N—CH colorless 3 11+ 3 I yellow oxidation dilution with solution H c1-13—w—c113 w .1 blue CHa—rD—Cfla violet— red Figure 1. The reactions of B-substituted indoles with p- dimethylaminobenzaldehyde and the colors of the complexes (from Dibbern and Rochelmeyer, 1963). 17 advantage that relatively crude, but volatile extracts can be analyzed directly and usable spectra may be obtained from as little as 50-100 ng of substance (Grunwald et aZ., 1967; Grunwald and Lockard, 1970; Hutzinger and Jamieson, 1970; Jamieson and Hutzinger, 1970; Raj and Hutzinger, 1970; Hutzinger, et aZ., 1972). using these techniques a number of free auxins have now been identified (Table 2). Greenwood et al. (1972) purified the diffusate from 15000 coleoptiles from maize and identified it as IAA. IAA has also been isolated from root tips of maize (Greenwood et aZ., 1973), from young fruits of citrus (Igoshi et aZ., 1971), from the young thalli of the marine alga undaria primatifida (Abe et aZ., 1972) and the immature seeds of Helianthus annus (Abe and Marumo, 1972). Indole-3-ethanol was isolated from the green stems of Cucumis sativus by Rayle and Purves (1967). Tamura et aZ. (1970) and Abe et aZ. (1972) identified methyl- indole-3-acetate in clubroots of Brassica pekinensis infected with Plasmodiophora brassicae and the alga Undaria primatifida. Marumo et aZ. (1968a, 1968b, 1971) have identified two chlorinated compounds: 4-chloro-indole-3-acetic acid and methyl-4-chloro-indole-3-acetate from immature seeds of Pisum sativum. This is the first time that halogenated indole compounds have been isolated from plant material. Indole-3-acetamide, an artifact of many earlier isolations, has now been demonstrated in clubroots of Brassica pekinensis (Tamura et aZ., 1970) and young fruits of citrus unshiu (Igoshi et aZ., 1971). Indole- 3-acetonitrile, N-methoxy-indole-3-acetonitrile and 4-methoxy-indole- 3-acetonitrile have also been isolated from the clubroots:of chinese 18 Amem_v .Ns as aa< m: .>: .ooe P__ege mesa» z ,//, ”seabeazfia 839$ _ _ m N Aoum_v .Ne no sesame m2 .045 muoogaapu 200% :9. \\\ mommamxwxoa oowmmoxm I 2 Legacy m¢>a=a ace a_»ea m: .ooa .04» weave cmaam N N _ _ umzawuem mwsxoxo :0 :0 I A_~va cancez can mn< age .>: .oap mummm mesons new Hazard oxxuxawNom A_Nmpv .Ns as egmoma mz .uau .>= .ooe meezac maze» "xwxmez oxmuwb Ammo—v .Ne em wn< m2 .>= .uah % PPPezw acaoz I "gm. wfigtw UHFNUNVED \z/ / AnanV .Ne em voozcmogw one .>: .oae mpooc _ . :0 N1. Aommpv weammh uau uap muwmm w a \\\ Amempv .Ns as noozcmaee m: .oa we?“ mpwpaompoo names com mucmgmaom ecowumomawpcmuH ougaom “cap; uczoqeou Lo aweeeeeu .mpzmpg cw muczogEoo mpoucw mcwcgsooo appogzpmz .N m_nek 19 /// Aon_v .Ns as sesame m: .azz .mH .>= .aae mmeoenape N _ _ umwmrmiwxm cowmmokm 20 :0 \\\ I z AONm_v .Ns as weasee m: .aoe meooan=_e xx, xx/ ”mentorwxom eommmegm N N _ _ 12% :o - \\\\ A_Nm_v .Ne as wcmomH m: .uoe mpwaaw mesa» "xwekméx «gens I z A_Nm_ .nmem_ m N _ _ .emompv .Ns as caste: m2 .NH .>= .04» memmm agape; zoom zu new "Exoweem Exmwm I _u _\,_/_ / apnop .nwmmp m .emmmpv .Ns as cases: mz .aH .>= .ase mamas mazes; row :0 \\\\ new "sxeweeo sxmwm no mucmcmwmm «cowpmowmwpcmuu mocsom u=m_a uczoqeou co seemeeeu .uazceeeoo .N m_eee 20 AFNm—V ozoum ccm HOVPFm A_empv cocmpgw> use cwpoew A_Nmev .Ns as xzouaaom as: .mH .uaw .>: .uap .ma .um mH .>: .uap m: .mzz .mH .>: .oah mw>mw_ "ogzuonwn mwnomN mm>mm_ mczoz HomooaoNo uowmmaxm mEmpm puma—mm "owoneENon Ezuomwxom \ / i: a I 12 / Aonmpv .Ne no mg:EmH m: .mZZ .mH .>: .QAH muoognspo zuwzu _ _ nowmroxwxom movemeam m \\\ oo mzuo —\\z /// Aonmpv .Ne no mgzsmh mz .mZZ .mH .>: .oap muoocnspo zumzu _ _ umwmrmtvxom cowomaxm \\\\ scowuwowmwpcovH mucwcwmmm we mmcmpwsu moszom ucmpa ucsoasou .uoacwucou .N opnmh 21 . Io ANNN_ mem_v .xmaaeeem use new: m: .uaa o: A_Nmpv .Ns as appeauez «:2 .>= .uae ANem_v .__o;o.z :o I 38: .6 6 839%. >2 6.: meme... 2 ogzume Homes com o: A_Nm_v mzoem ace eoe__m mzz .uae m .aH .>= .uae .ma .ua me=e_a a_o:z -om 83$ . . . 38:3 2 / azoem new “OFF,“ azz «H >3 one me>eap w _ _ nakzuoxvn owuomH /uN:o , mome-z _\\\ A_Nmpv ezz .NH .osa mzoo mzopm UCM HOP—Fm .>D non—H «mm «on mw>mw_. Omouz—p Z / Nonsense» mwoemw m _ ANem_V /UN=s _ 5:3,»; 28 535 E .>: .9: mo>mmp 959a momouz c \ "oooonoNo dowmmonm u wocmamewm scowumuwewecmuH mucsom pcmpa vasanou to seaweeeo .ea==_e=oa .N mpnee 22 Acmsmwpnaaczv meczucem ace mum: m2 .uaw Aon_v .Ns as new: m2 .usa .aoe Ammmpv .Ns as magmas; >> .oae mama. mszwE Homes 6mm Aumcmwpnsaczv wxmczucmm use mum: m: .uag Amempv Fxmaaucmm can mum: Aommpv .Ne em mocmnma >3 .04> mummm ouw \\\\ assume names new «cowumuwewucmuH mococmmwm Lo eeeeeeeu wocsom vamp; uczoaeou .vmacwucou .N mFth 23 .xgumsoguomgm mmms "m2 magamgmopmeoaso uwacwpnmmm "gnu “monocomoc ovumcmms cmoFusc "mzz mchmsocuomam nocumewcw ”mH mxcpmsocpooam uw>o>>umcp>3 ">2 "Azamcmoumsocgo swampucwnu "04> mmwmmcocnocuumpm comma ”ma mxgnmcmoumsoccu gonna "um « : z mUPFococa was _ emu: m . — ¢+>m noummzo ANNm_v .xmes Io nucmm use chcoxmwa m: .oao .uoh mvmmm menace Home: cow : :o z o: :0 N: Aumgmwpnzaczv :o : exmeaecem was new: m: .04a 0: z Ao>mPV .Ne em mum: m2 .uaw .uo» mummm N _ ‘ «capes names emm cum :u mucmgmamm ecowpmowwwpcmuH mocsom pcmpm ucaoasou to ameaeeea .casceecou .N sense 24 cabbage by Tamura et al. (1970). Recently Bourdoux et al. (1971) identified indole-3-carboxa1dehyde in aereal stems of Equisetum teZmateia. C. Isolation and identification of bound auxins Cholodny (1935) published a paper which for some unknown reason has been completely omitted from the auxin literature, and yet presents for the first time experimental evidence that the biologically active auxin (IAA) exists in the plant tissue in the form of an inactive precursor (bound IAA). Cholodny (1935) believed that the germination of seeds was hormonally controlled, and he asked the question whether this hormone was already present in the dry seeds. It was known at the time that the large endosperm in many members of the Gramminacae served as a food reserve for the developing embryo, and he concluded that the most likely tissue to look for such a hormone would be the endosperm. He cut small sections out of the endospenn of branless, dry karyopses of Avena sativa and attached them to the sides of decapitated, etiolated Avena coleoptiles (Table 1-p). He found that within 1.5 hours at 203 C in the dark the coleoptiles had developed a 30-40° negative curvature. In a series of experiments he was able to show that endosperm sections from progressive stages of germination produced decreasing degrees of coleoptile curvature. Endosperm sections from seeds in the third day of germination did not produce any curvature at all. Steam-treated endosperm from dormant seeds failed to induce curvature. He then extracted half seeds by soaking them in 96% ethanol (a good solvent for free IAA) and was unable to detect any growth hormone activity in the extract. In a similar experiment he imbibed the half seeds in distilled 25 water for 12 hours. Then he concentrated the water and mixed it with gelatine, applied it in small cubes to the decapitated coleoptiles, and observed a negative curvature. The control experiment done with intact seeds gave no curvature. Steam-treated endosperm from pre- viously imbibed seeds however could induce negative curvature. Cholodny (1935) concluded from these experiments that the endosperm of Avena, Zea mays and other members of the Graminaceae contains a growth hormone which is liberated during the initial stages of germination. This hormone must be present in dormant seeds, since alcohol and steam- killed endosperm was still able to produce a growth response, provided the endosperm tissue was imbibed for several hours after the treatment. Laibach and Meyer (1935), Pohl (1935), Hatcher and Gregory (1941) and Hatcher (1943, 1945) also found that the growth hormone (possibly the same as the growth hormone found in coleoptile tips) is most concent— rated in dormant seeds, and that the level decreases rapidly during germination, reaching an extremely low level during the vegetative phase. Brunner (1932) cultured embryos of Pinus maritima in defined nutrient media and found that normal embryo development required the addition of an aqueous endosperm extract to the medium. Avery et a2. (1940) studied the growth hormone content of maize endosperm at different stages of germination and confirmed the findings reported by Cholodny (1935), Laibach and Meyer (1935) and Pohl (1935). Van Overbeek (1941) observed a large discrepancy between the amount of auxin obtained by exhaustive diffusion from Avena and maize coleoptile tips and exhaustive solvent extraction of the tips. He demonstrated that at the moment of cutting the tips only about 26 5% of the total diffusible auxin is available, and concluded that the auxin (IAA) is not free, but bound in the form of a precursor. Simil- arly Ohwaki (1970) has shown that twice as much IAA could be obtained from rice coleoptiles by diffusion into agar than by solvent extraction. That the major portion of the auxin was not free, but bound was conclusively shown by Avery et aZ. (1942a, 1942b). They found that the content of free auxin in wheat and maize kernels of various stages of maturation never accounted for more than 12% of the total auxin which could be liberated from the tissue by mild alkaline hydrolysis. This was independently confirmed by Haagen-Smit et aZ. (1942) and Thimann and Byer (1942) and Hemberg (1955). Haagen-Smit et aZ. (1942) obtained 122 mg of crystalline auxin which showed no melting point depression when mixed with synthetic IAA. Haagen-Smit et aZ. (1946) reextracted the auxin from maize kernels in the late milk stage and proved by elemental composition that it was IAA. This was the first time that IAA had been positively identified as an endogenous plant auxin. It also showed that the bound auxin was IAA in ester linkage with an unknown component. Tandler (1960) has reported the isolation of a high molecular weight auxin from Acetabularia which gives substantial amounts of IAA upon alkaline hydrolysis. Several other reports have been published on the isolation of high molecular weight IAA-protein complexes from wheat grain (Gordon, 1946), peas ($1991 and Gordon, 1953; Meudt and Galston, 1962a and 1962b) and soluble leaf protein (Wildmann and Gordon, 1942). One of these IAA-complexes has been identified as an 27 IAA-tRNA complex by Davies and Galston (1971), but it appears now that this complex is an artifact of isolation (Davies, 1972). Two interesting indole derivatives have been isolated from Brassica oZeracea: glucobrassicin (Gmelin and Virtanen, 1961) and neo- glucobrassicin (Gmelin and Virtanen, 1962) (see Table 2). Elliot and Stowe (1970, 1971) have identified glucobrassicin-l-sulfonate in Isatis tinctura. Recently Josefsson (1972) has demonstrated that glucobrassicin is formed in stem segments of Sinapis aZba incubated with indole-3- acetaldehyde oxime. In connection with glucobrassicin it is interesting to note that Henbest et aZ. (1953) first reported the isolation of IAN from Brassica oZeracea. Prochazka and Sanda (1960), Kutacek et al. (1962), Kutacek and Kefeli (1968) and Gmelin (1963) have demonstrated that Brassica oZeracea contains an enzyme, myrosinase, that rapidly hydrol- izes glucobrassicin to IAN and ascorbic acid. Gmelin (1963) has pointed out that inactivation of the enzyme by boiling prior to the solvent extraction of the tissue is necessary for the isolation of gluco- and neo-gluco-brassicin. Failure to inactivate the enzyme results in high yields of IAN. A number of conjugation products of exogenously applied IAA isolated from plant tissue have been reported. Andreae and Good (1955), Good et al. (1956) and Andreae and Ysselstein (1956) have incubated pea stem sections with ‘4 C-IAA and identified the conjugation product as N-(indole-3-acetyl)-aspartic acid. Klambt (1960) has isolated a conjug- ate from wheat coleoptiles which co-chromatographed with authentic IAA-aspartic acid on paper. 28 Hutzinger and Kosuge (1968) have grown Pseudamonas savastanoi in defined medium containing 0.04% IAA, and isolated an1 indole com- pound from the medium. TLC, UV and IR analysis showed it to be identical with indole-3-acetyl-e-L-lysine. Several esters of IAA and a sugar have been isolated from plants. Shantz and Steward (1957), Steward and Shantz (1959) and Srivastava (1963, 1964) reported the isolation of an ester of IAA and arabinose from immature sweet corn kernels. Zenk (1961) first isolated IAA-glucose from pea epicotyls and leaves of Cblchicum neapolitanum after incubating the tissues in the presence of 14C.-IAA. The IAA- glucose showed characteristics typical of an ester linkage on carbon 1 of the glucose, and Zenk (1961 and 1964) assigned to this ester the structure 1-0-(indole-3-acety1)-B-D-glucopyranose. Following the 14MM K16mbt (1961) isolated an incubation of wheat coleoptiles with ester of IAA and glucose, tentatively identified as l-0-(indole-3- acetyl)-B-D-glucopyranose. KegleviC and Pokorny (1969) isolated a compound from Avena coleoptiles which co-chromatographed with authentic l-0-(indole-3-acetyl)-B-D-glucopyranose. Unfortunately none of these esters of IAA and glucose have been further characterized. D. Isolation and identification of bound auxins in seeds of maize Berger and Avery (1944a and 1944b) extracted dormant seeds of sweet corn with acetone-water (1:1) and showed that the bound auxin consisted of two fractions: a water-soluble fraction and a water- insoluble fraction. Berger and Avery (1944b) studied the 29 water-insoluble fraction and found that mild alkaline hydrolysis lib- erated IAA, and concluded that it might be an IAA-protein complex. But they could not explain the low nitrogen content (about 5%) of this complex. It was only recently that a partial characterization of this IAA-complex has been accomplished. Piskornik and Bandurski (1972) showed that this complex consists of a heterogenous mixture of glucans with varying chain lengths. The glucan comprises between 35 and 50% of the dry weight of this IAA-complex. Complete and partial acid hydrolysis of the glucan revealed that it was a pure 8 1+4 glucan (Table 2). Chromogenic properties of this IAA-complex indicated that it also contains phenolic components. These phenolics may be impurities or a component of this complex. Piskornik and Bandurski (1972) con- cluded that the IAA is esterified directly to the glucose units of the glucan, but this has not yet been confirmed. The water-soluble fraction of Berger and Avery (1944b) has been studied by Labarca (1965) and Labarca et aZ. (1966). They were able to isolate four esters of IAA and myo-inositol from this fraction: two isomers of indole-3-acetyl-myo-inositol (B1 and 82) and two isomers of indole-3-acetyl-myo-inositol-arabinose (B3 and 84). Based on the chromatographic behavior of B and B2 on Sephadex, silicic acid, paper 1 and silica gel thin-layers, and the observed conversion of B1 to B2 and B2 to 81 by acyl migration Nicholls (1967) tentatively identified 82 as 2-0-(indole-3-acetyl)-myo-inositol. B1 is therefore most likely 1-§;;1-0-(indole-3-acetyl)-myo-inositol (Nicholls, 1967). Nicholls et al. (1971) confirmed this configuration of 82 by comparing the NMR 3O spectra of authentic and naturally occurring 82. They showed that both esters exhibited the same chemical down shifts for the equatorial proton. The enzymatic removal of the arabinose moiety from 83 and 84 gave mainly B and 82, and Labarca (1965) and Labarca et al. (1966) 1 concluded that 83 and B4 are the arabinosides of B1 and 82 respectively. Ueda and Bandurski (1969b) have identified an additional ester, indole-3-acetyl-myo-inositol-galactoside (85) in the water- soluble fraction. They also showed that the bound auxin of Berger and Avery (1944b) is composed of about 50% water- and 50% water-insoluble auxin, and determined the total amount of alkali-labile IAA in both fractions to be 60-80 mg IAA/kg dry weight of corn kernels. These esters of IAA and myo-inositol and myo-inositol- glycosides have been characterized by TLC, GLC and combined GLC-MS by Ueda and Bandurski (1969a and 1973) and Ueda et al. (1970) permitting the following assignments of configuration: 81 lzgg-l-0-(indole-3-acetyl)-myo-inositol B2 2-0-(indole-3-acetyl)-myo-inositol B l1QL75-0-B-L;arabinopyranosyl-l-0-(indole-3-acetyl)- myo-inositol B 5-0-81L7arabinopyranosyl-2-0-(indole-3-acetyl)-myo- inositol 85 5-0-81;;galactopyranosyl-2-0-(indole-3-acetyl)-myo- and represents the first characterization of any group of naturally occurring bound auxins in plants. EXPERIMENTAL I Gas-Liquid Chromatographic Analysis of Indole-3-acetic Acid Myoinositol Esters in Maize Kernelsl Received for publication June 1, 1970 MINORU UranA2 AXEL EHMANN AND ROBERT S. BANDURSKI , 7 Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48823 ABSTRACT An improved method of fractionating the myoinositol esters of indoleacetic acid (IAA) from maize kernels by gas-liquid chromatography has been developed. Mass spec- trometry was employed as an aid in identification of the esters. Maize kernels contain three groups of esters of IAA: (a) IAA myoinositols, (b) IAA myoinositol arabinosides, and (c) IAA myoinositol galactosides. Each group has three chromatographically distinguishable isomers. The gly- cosylinositols described are unique in that carbon 1 of the sugar is attached to the hydroxyl at C-5 of the myoinositol. Previous publications from this laboratory have reported the occurrence of four esters of indole-3-acetic acid and myoinositol as well as several unidentified alkali-labile IAA compounds (3, 5, 6, 8, 9). Two of the four esters identified were IAA myoinositol isomers (B, and B2) and two were isomers of IAA myoinositol arabinoside (B3 and B4). One of the unidentified and newly de- tected spots on thin layer chromatograms was given the designa- tion of Bs. Collectively, these esters were designated as the B group. The purpose of this report is to present an improved method of fractionating the esters of the B group by gas-liquid chromatography and to present some mass spectrometric data helpful in identifying the esters. A partial characterization of the B, area demonstrated that it contained a previously undescribed compound, IAA myoinositol galactoside. METHODS Preparation of Samples for GLC3 Analysis. A schematic sum- mary of the procedures utilized is presented in Figure 1. A 2.5—kg sample of dry sweet corn kernels (Zea ma ys L., cultivar, Stowell’s Evergreen Hybrid) was ground to 20 mesh, and extracted with 5 liters of 1:1 acetone-water for 48 hr at room temperature with occasional stirring. The liquid phase was separated, and the residue was re-extracted with another 5 liters of l :1 acetone-water for 48 hr. The combined liquid phases were filtered and concen- trated under reduced pressure to 16 5 of the original volume, and 1 This work was supported, in part, by National Science Foundation Grant GB 6709 X. Journal Article 5109 from the Michigan Agricul- tural Experiment Station. 3 On leave from the Sumitomo Chemical Company, Ltd, Japan. 3 Abbreviation: GLC: gas-liquid chromatography; TLC: thin layer chromatography; TMS: trimethylsilyl; MS: mass spectrometry. a gummy precipitate was removed by centrifugation.‘ The super- natant fluid was partitioned against an equal volume of l-butanol twice. The water phase was chromatographed on a 43- X 4.8-cm column of Sephadex G-10 with water as eluant. Two fractions containing B, + Ba and B, + B, + B, were pooled. The B, to B, fraction was rechromatographed on the same column. Each frac- tion was lyophilized, redissolved in a small amount of water, streaked on Whatman No. 1 filter paper, and irrigated descend- ingly, with n-butanol-acetic acid-water (5:1 :2.2) for 20 hr. From the chromatogram of the B, + B2 fraction, a broad band con- taining B1 and B2 was cut out and extracted with water. From the chromatogram of the B, to B, fraction, three bands, correspond- ing to B3, 8., and B.-,, were extracted separately. The fractions B, + B2, B3, B4, and B, thus obtained were chromatographed individually on a 49- X 3.4-cm column of silica gel. The column was eluted with 500 ml of pure acetone, followed successively by 500 ml each of 20:1, 10:1, and 5:1 acetone-water mixtures. B, and 32 were eluted with 10:1 solvent and B; to B, with 5:1 sol~ vent. The B, and B2 fractions were lyophilized, redissolved in 3 ml of warm acetone, and centrifuged, and the IAA compounds were precipitated from solution by adding 5 ml of ether. The B, to B, fractions were first washed with acetone, then dissolved in 0.3 ml of water, diluted with 3 ml of acetone, and precipitated by adding 5 ml of ether. 'lhin Layer Chromatography. Preparations were routinely examined on silica gel thin layer plates using an acidic and a neutral solvent as previously described (9). Gas-Liquid Chromatography. Analysis was carried out on an F & M model 402 equipped with a hydrogen flame ion detector. The column types used are indicated in the legends and include: 1: 3%, OV-l, 19 inch X 6 feet on Chromosorb W, 2: 2‘2, OV-l, 3g inch X 6 feet on Gas-Chrom Z, 3: 3.8 9'}, UC-W98 lg inch X 4 feet on Diatoport, 4: 3.7%, HiEfl-SBP, lg inch X 6 feet on Gas-Chrom Z. These stationary phases are available from Anspec Co., Ann Arbor, Michigan. The flow rate of the carrier gas was 60 ml /min. Combined gas-liquid chromatography-mass spectrometric (GLC-MS) analysis was conducted with an LKB-9000 analyzer unit. Trimethylsilylation. A mixture of hexamethyldisilazane and trimethylchlorosilane in pyridine, 2:1 :10 (7) was satisfactory for derivatization of B1, B2, myoinositol, and monosaccharides. 4 Recent studies, currently in progress in this laboratory, indicate that considerable purification and enrichment of the B esters can be attained at this stage by chromatographing the concentrated aqueous supernatant fluid adjusted to pH 2.5, on a Dowex 0 column which has been previously equilibrated with sodium citrate buffer, pH 2.5. The B esters are then eluted with water and are contained in the frac- tions appearing between 4 and 13 bed volumes. The remaining pro- cedure may then be followed as indicated or the silica gel column may be used as the next step. 715 31 32 CORN KERNELS Extracted with 50% acetone Filtered Concentrated Centrituaed i T StPERNATANT LIOUiD PRECIPITATE Partitioned with n-hieanai l 1 serum. PHASE wares Chrornatogrmhed on Sephadex 640 water as eluent l | at ‘ 92 93* 34* 95 Filter chromatographed Chrarnatoaraahed on Sephadex anon: HOAc: H20 G-io. water as etuont one: new. Grandma-apnea on Paper chromatographed Silica get 300": HOAc: H20 Acetamwater ' i 1 9i .2 g : Slice aei Silica gel Silica get i l 8.3 8‘ a? 85 . é 3' 4 ‘5 is i a FIG. 1. Flow sheet for the preparative procedure described in text. Eight preparations were obtained as numbered at the bottom. The yields were: preparation 1 (1.03 mg), preparation 2 (0.12 mg), prepa- ration 3 (0.50 mg), preparation 4 (0.27 mg). preparation 5 (0.27 mg), preparation 6 (1.50 mg), preparation 7 (0.75 mg), preparation 8 (0.18 mg) as 1AA. BuOH and HOAc are abbreviations for l-butanol and acetic acid respectively. Silylation of B3 to B5 and myoinositol glycosides was accom- plished with trimethylsilylimidazole in pyridine, 1.5 meq /ml. Usually 20 to 50 ul of the reagent were added to an aliquot con- taining 20 to 50 pg of the ester as IAA. Silylation was for 0.5 to 1.0 hr at room temperature. Alkaline and Acidic Hydrolysis. For identification of inositol glycosides, B; to B, rich samples, equivalent to 50 pg of IAA, were hydrolyzed with 1 N NaOH for 15 min at room temperature or with 1 N NH40H for 10 min at 60 C. When a sugar analysis was desired, the alkali-hydrolyzed samples were further hy- drolyzed with 3% HNO; for 3 hr at 100 C. The acid-hydrolyzed solution was neutralized and desalted with a mixed cation-anion exchange resin (Amberlite-MB-3) prior to chromatography. Sugar Determination by GLC. The hydrolyzed solution, after neutralization, was concentrated to a small volume, 20 pg of a-methylmannoside were added as an internal standard, and the mixture was lyophilized. Silylation was carried out for exactly 30 min before analysis. The area of the peaks on the recorder chart of the chromatogram was measured by weighing. For monosaccharides, the two anomeric peaks were combined on the assumption that the detector responses were the same for the two anomers. Determination of the Position of Sugars on Myoinositol. The method of Ballou and Lee (2) was followed with slight modifica- tions. Inositol glycosides were permethylated with methyl iodide in dimethylformamide with silver oxide as catalyst and methano- lyzed with 5% BC] in methanol, and the resultant pentamethyl- inositol was compared with standard substances by GLC. The standards 1,3,4,6-tetramethylinositol and l,4,5.6tetramethyl- inositol were supplied by Dr. C. E. Ballou, and glycosylinositol was provided by Dr. H. E. Carter. Galactinol was purchased from Calbiochem. Detection of IAA. IAA-containing fractions from the column were detected after alkaline hydrolysis, by Salkowski reagent as previously described (9), except that the ether extraction step was omitted. IAA-containing compounds on thin layer and paper chromatograms were detected with a Salkowski reagent spray (9). RESULTS AND DISCUSSION Examination by TLC. The TLC patterns of each preparation show two discrete spots for each IAA-inositol compound (Fig. 2). Since isomerization of the compounds in aqueous solutions is a rapid and continuous process, no special attempt was made to isolate a single isomer. With acidic solvents, preparations 7 and particularly 8 consist mostly of one isomer, but with neutral solvents the major component of 7 and 8 migrates like 8.. Thus 13, behaves like B; and B. in terms of acyl migration. GLC Analysis of TMS-B Esters. Retention times and relative magnitude of the peaks for the various preparations are sum- marized in Tables I and 11. Each peak was examined in the mass spectrometer and was found to contain TMS-inositol and the IAA moiety (Table III). The m/e 697 signal is the molecular weight of penta-TMS-IAA inositol, m/e 973 is the molecular weight of hepta-TMS-IAA inositol pentoside, and m/e 1075 cor- responds to the molecular weight of octa-TMS-IAA inositol hexoside. The peaks P-5, P-6, and P-8 yielded the same kinds of fragment ions, and among these were ions due to pentose. P-4, P-7, and P—9 showed fragment ions due to hexoside. The other ions were common to all three peaks except that P-7 lacked m/e 1075 and had m /e 1060 instead. An m /e of 1060 would be pro- duced by loss of CH;’ (1075 — 15). It is important to note that by TLC only two isomeric com- pounds are detected, but by GLC an extra isomer is found for each of the pairs, 81-82, and 83-84. The Bs-rich preparations con *A- -9- 3 § 0 o g 8.9922)" : a'Qi'azié e e .2 .2 :6 § 8:83 88 e e a a 83'38 '0- 80 8850 t: 83B .383837. i?3233§3 Preparation Number Preparation Number FIG. 2. Thin layer chromatographic patterns of the eight prepara- tions on Silica Gel F-254. A: Acidic solvent: ethyl acetate-methylethyl ketone-formic acid-water (5:3:lzl). B: Neutral solvent: ethyl acetate- methylethyl ketoneethyl alcohol-water (5:3:l:l). Table 1. Gas Chromatography of TMS Derivatives oft/re IAA Myoinositols Column was 2% OV- l, ,V inch X 6 feet on Gas- Chrom Z, and the temperature was 245 C. Retention Time Peak ! .. _ ____ _ ._ Preparation 1 Preparation 2 ’- u N .. 3 P-l 3.8 3.8 (main) P2 5.8 (m ain') 5_8 P-3 6 8 (minor) 68 (trace) 33 tained three isomers of IAA inositol hexoside, together with the three isomers of IAA inositol pentoside. For our present pur- poses, we wish to retain the nomenclature B. and B, for the two previously described IAA inositols and B; and B. for the two previously described (cf. Ref. 3) IAA inositol arabinosides and retain the designation B. for the IAA inositol hexosides described here. However, as will be discussed below, the existence of three, or possibly more, isomeric forms for each compound necessitates a new system of abbreviations. Table IV summarizes the data on the distribution of the nine esters obtained by GLC from the five spots detectable by TLC. Thus, for example, elution of the B. area of a TLC plate will yield peaks 5 and 8 on the gas chromatograph, while the B5 region yields peaks 4, 7, and 9. The assignment of the structures 1- (or 3-) 0-ester to B. (P-2) and 2-0—ester to B. (P—l) is endorsed Table II. Gas Chromatography of TMS Derivatives of the IAA Myoinositol Glycosides Column was 2% OV-l, 1g inch X 6 feet on Gas-Chrom Z, and the temperature was 270 C. Retention Time Peak Preparation 3 Preparation 6 Preparation 7 min P—4 12.2 (main) P-5 13.2 13.2 (main) 13.2 P-6 15.0 (main) 15.0 (minor) 15.0 (minor) P-7 . . . . . 16.9 P-8 21.3 21.3 21.3 (minor) 1x9 . 23.7 i Table 111. Mass Spectrometry of TMS Derivatives of IAA Myoinositols and Their Glycosides Peak Highest m/r Second Highest m/c m/e 433' 153430., P-l 697 523 (weak) Weak 1.0 P-2 697 607 (strong) Weak 1.6 P-3 697 607 (weak) Strong 2.4 P4 1075 (weak) 985 Strong 0.64 P—5 973 (weak) 958 Strong 0.75 P-6 973 (strong) 958 . . . . . . P-7 1060 (weak) 985 Weak 0.84 P8 973 (strong) 868 Weak .0 P-9 1075 (strong) 1060 Weak 1 mz‘e 433 is derived from the inositol moiety. 2 mge 130 and 157 are derived from the IAA moiety. Table IV. Summary of the Origin of the Nine GLC Components from the Salkowski-positive Spots on TLC Peaks Obtained on GLC Column Region of TLC Plate Muted Major Secondary B, P-2 P-3 B, P-1 (P-3‘)1 B; P-6 P-8 B. P-5 (P-8)1 B. P-4 P-7, P-9 ' P-3 and P—8 are thought to be located at an RF between B._-—Bl and B.—B,., respectively. by the MS data (8). From P-l the IAA moiety is easily liberated, producing the ion m/e 523, whereas from P-2 (CH3), SiOH is easily eliminated, yielding the ion m /e 607. These results can be explained by the more labile character of the linkage between the substituent and the axial oxygen of inositol. It seems likely that for P-l, the IAA moiety is folded parallel to the inositol ring, rendering the molecule more compact and thus decreasing the boiling point of the ester or its affinity for the GLC column. To explain the third isomer P-3, it is necessary to assume that IAA can be esterified to any of three hydroxyls of inositol. The assign- ment of the 2-0-ester structure to P-5 (B.) is reasonable because the B. fraction is mainly composed of P-5 and can be hydrolyzed to produce 8:. It is diflicult to determine which of the two isomers, P-6 and P-8, corresponds to the 1- (or 3-)0 ester. It is interesting to note, however, that the longer the retention time the higher the ratio of 157 / 130 becomes. Since P-4 occurs in greater amount than the other isomers and has a low 157/130 ratio, the analogy would predict that it is a 2-0—ester. GLC Analysis of TMS-inositol Glycosides. The inositol glyc0« sides obtained by the alkaline hydrolysis of preparations 3 to 8 were studied by means of GLC-MS (T ables V and VI). While the B. or B. fractions yielded only one peak of TMS-inositol pentoside, the B. fraction yielded two peaks. One of them (G-l) was a pentoside peak, but the other (G-Z) had a slightly longer retention time and a different MS pattern than the former. The Table V. Gas Chromatography of TMS Derivatives of M yoinositol Glycosides Column was 3.8% UC-W98, 3g inch X 4 feet on Diatoport, and the temperature was 230 C. Retention Time Peak _ 1 ‘ l . Prepag'ationiPrepagationi Prepayratton Galactinol Glycosylinositol l min G-l 9.9 9.9 9.9 G-2 10.9 . . G-3 10.9 . . . (3-4 12.9 Table VI. Mass Spectrometry of TMS Derivatives of M yoinositol Glycosides Peak Highest m/c Secong‘iltighest m/c 433‘ m/e 3493 m/e 451' G-l 888 873 + + — G-2 990 885 + — + G-3 990 975 + — + G-4 990 885 + — + ‘ m/e 433 is derived from the inositol moiety. ’ m/e 349 is derived from the pentose moiety. 3 m/e 451 is derived from the hexose moiety. Table V”. Component Analysis and Stoichiometry Compound I Preparation 6 I Preparation 7 1 Preparation 8 , mole ratios of compound to inositol i". 1AA ’ 1.09 | 0.72 i 0. 71 Ara binose 0.95) i 0. 28“ . 0. 35? » 2 - ' . 1 .- . Galactose 0.0711'0 0.761”1 04 i 0.63.0 98 Inositol 1.00 i 1.00 | 1.00 34 highest mass obtained from this peak was 990 and is due to nona- TMS-inositol hexoside, a mass also found in the mass spectra of galactinol and glucosylinositol. The ion m/e 451 and related ions due to the hexose moiety are also observed. Although G-2 has the same retention time as G—3 (galactinol) on nonpolar columns, G-2 and galactinol are separated on a polar column. The different Table VIII. Comparison of the Ratios of Galatose to Arabinose and Inositol Hexoside to Inositol Pentoside Preparation 7 Preparation 8 2.7 1.8 2.5 2.1 Table IX. Gas Chromatography of Permethylated Sugars and Pentamethyl M yoinositols Galactose/‘ara binose , Inositol hexoside/inositol pentoside Retention Time 1211251113., Compound Formed .. 0,, - 161.92 mu Arabinose Tetra-Me-arabinose 3.0 3.0 1.9 Galactose PentavMe-galactose 8. 8 8. 1 4. 3 Glucose Penta-Me-glucose 7. 5 7. 5 3. 4 1,3,4,6-Me Hexa-Me-inositol 8. 7 8. 2 3.2 inositol l,2,3,4,6-Me-inositol 11.1 10. 7 9.6 1,3,4,5,6-Me-inositol 12.1 11.7 10.5 1,4, 5,6-Me- Hexa-Me-inositol 8. 7 8. 2 3. 2 inositol l,2,4,5,6-Me-inositol 8.7 8.2 6.6 1,3,4,5,6-Me-inositol 12.1 11.7 10.5 Galactinol Penta-Me-galactose 8.8 8. 1 4. 3 1,2,4,5,6-Me-inositol 8.7 8.2 6.6 Glucosyl- Penta-Me-glucose .5 7.5 3.4 inositol 1,2,3,4,5-Me-inositol 11.9 12.4 10.5 Preparation Tetra-Me-arabinose 3 .0 3 . 0 1.9 6 1,2,3,4,6-Me-inositol 11.1 10.7 9.6 Preparation Tetra-Me-arabinose 3. 0 3 . 0 1.9 7 Penta-Me-galactose 8. 8 8. 1 4. 3 1,2,3,4,6-Me-inositol 11.1 10.7 9.6 gas chromatographic behavior combined with the different MS pattern of G-2 compared with G-3 or G-4 suggests that this new inositol hexoside is neither galactinol nor glucosylinositol. Examination of the MS data shows that it is C. of the pentose or hexose that is linked to inositol. Determination of Sugars. The preparations 6, 7, and 8 were hydrolyzed first by alkali and then acid and examined by TLC with the solvent l—butanol-acetic acid-ether-water, 9:6:3:1 (4). Preparation 6 contained L-arabinose and myoinositol with a trace of D-glucose. Preparations 7 and 8 were found to contain D-galactose as well as arabinose and inositol, and also small amounts of glucose. GLC analysis showed that the stoichiometry between (arabinose plus galactose) to inositol was good for all preparations (Table VII). The stoichiometry between inositol and IAA was 1.09 for the arabinoside-rich preparation but was low (0.72) for the galactoside-rich fraction. A possible explana- tion of this low stoichiometry is that our TLC preparations are contaminated by an unknown inositol glycoside. Owing to its chromatographic properties the contaminant would be, most likely, an aromatic inositol glycoside. In Table VIII the peak ratios of galactose to arabinose and inositol hexoside to inositol pentoside are shown. These data together with the MS data strongly suggest that the B5 fraction contains three isomers of indole-3-acetyl myoinositol galactoside. Position of the Sugars on Inositol. Table IX shows the retention time on GLC of the peaks obtained from methylated sugars and methylated inositol. It is evident that methanolysis of permeth- ylated inositol glycosides (both the arabinoside and the galacto- side) yields 1,2,3,4,6—pentamethylinositol. Thus, the arabinose or galactose was linked to the 5-0 of inositol. The MS data, as presented in Table X, confirm this conclusion. It can be seen that the four isomers of pentamethylinositol give characteristic MS patterns and that the pentamethylinositol from the B esters is comparable to the spectra obtained from 1,2,3,4,6-penta- methylinositol. Concluding Dimion. By the use of combined GLC-MS analysis, the IAA esters of the B group have been fractionated into nine rather than the five components previously detected by TLC (8). These nine components are divided into three groups: IAA inositols, IAA inositol arabinosides, and IAA inositol galactosides. In our previous paper (9) it was our implicit assump- tion that IAA could only migrate facilely between neighboring cis-hydroxyl groups of inositol, and this was supported by the fact that only two chromatographically distinguishable isomers of IAA inositol could be detected. Now that other isomers have been found, we must conclude that IAA can be esterified to still other hydroxyls of myoinositol, either by enzymatic reactions in vivo or by acyl migration between trans-hydroxyl groups during the fractionation procedure. Migration of phosphoric acid be- tween trans-hydroxyl groups has been reported by S. J. Angyal Table X. Mass Spectrometry of Pentamethyl Myoinositols The numbers represent relative amounts of fragment ions of the indicated mass when m/e 101 is normalized to 300. 1- 231316113” 1, 2, 4, 5, 61118111651101 1, 3, 1. 5, trite-16651161 1, 2,3,1,61~1e-1ms.1161 m/el (13.82138? "#123313?” 6.11.6.1... "13.3333?“ " lti‘ifil" " $1.338 Preparation 6 Preparation 7 250 2.1 1.0 0.8 1.7 2.1 0.1 + 0.2 249 .> 0.3 0.3 0.4 0.2 + . > + 218 0.2 8.9 11.7 1.5 1.2 6.8 1 4.6 5.2 186 3.4 0.6 0.6 3 0 3.3 0.3 l + 0.3 144 22.9 31.9 27.8 21 2 23.4 6.8 - 6.7 6.5 130 24.7 10.2 12.8 22 9 22.7 11.9 12.2 13.9 ‘ m/e 250 is the molecular ion. m/e 218, 186, 144, and 130 are odd electron ions characteristic of pentamethyl myoinositols. and A. F. Russell (1). If our assumptions are correct, three isomers may exist for the arabinoside and the galactoside, be- cause position 5 is occupied by the sugar. This is in accord with the present data. If, however, optical isomers are taken into ac- count, then theoretically six isomers of IAA inositol, five isomers of IAA inositol arabinoside, and five isomers of IAA inositol galactoside can exist. If isomers other than the 2-0 ester are the products of asymmetric enzymatic synthesis, the whole mixture of such isomers should be optically active. Optical inactivity does not, however, necessarily mean that such isomers are formed by acyl migration. This remains a subject for future work. In order to establish the structures of the IAA esters predicted above, each isomer must be obtained in substantial amount. The above considerations of the numbers of possible isomers leads us to make the following recommendations concerning trivial nomenclature. The general designation, B group, should be retained for the water-soluble IAA esters of inositol. The lipid- soluble esters, previously described (5, 9), will be called the A group. Within the B group, in those cases where the position of the IAA on the cyclitol ring is known, it will be indicated by a number; thus B2 designates indole-3-acetyl-2-0-myoinositol. In cases where the point of substitution is unknown, as for example, the galactosides here described, we suggest they be referred to as IAA-inos-gal., IAA-inos-galb, and IAA-inos-galc. Since we found that the sugars are linked to the 5-0 of myo- inositol, our next interest is in whether the sugars are in the alpha or beta form. If IAA inositol arabinoside and galactoside can function as glycosylation reagents in cell wall synthesis, 3 35 beta linkage would be reasonable. So far, however, there is no knowledge of the function of the IAA inositol esters. More extensive MS data concerning these esters will be pub- lished elsewhere. Acknowledgments—We thank Professors W. W. Wells and C. C. Sweeley for their kind help in the gasliquid chromatographic and mass spectrometric analysis, and Professors C. E. Ballou and H. E. Carter for supplying precious samples of tetra~ methylinositols and glucosylinositol. Mr. Jack E. Harten rendered technical assistance in the mass spectrometric analysis. LITERATURE CITED 1. ANGYAL, S. J. AND A. F. RUSSELL. I969. Cyclitols. XXVIII. Methyl esters ofinositol phosphates. The structure of phytic acid. AuSt. J. Chem. 22: 383-390. 2. BALLOU. C. E. AND Y. C. Lee. 1964. The structure of a myoinositol mannoside from M ycobacterium tuberculosis glycolipid. Biochemistry. 3: 682—685. 3. BANDURSKI, R. S., M. UEDA, AND P. B. Nlcnous. 1969. Esters of indole 3-acetic acid and myo-inositol. Ann. N. Y. Acad. Sci. 165: 655-667. 4. HAY, G. W., B. A. LEWIS, AND F. SMITH. 1963. Thin-film chromatography in the study of carbohydrates. J. Chromatogr. 11: 479-486. 5. LAaAaCA, C., P. B.N1cao1.ts, AND R. S. BANDURSKI. 1966. A partial characterization of indoleacetylinositols from Zea mays. Biochem. Biophys. Res. Commun. 20: 641-646. 6. NICHOLLs, P. B. 1967. The isolation of indole-3-acetyl-2-0-myo-inositol from Zea mays. Planta 72: 258-264. 7. Swasuav, C. C., R. BENTLEY, M. MAKITA, AND W. W. WELLS. 1963. Gas-liquid chromatography of trimethylsilyl derivatives of sugars and related substances. J. Amer. Chem. Soc. 85: 2497-2507. 8. UEDA, M. AND R. S. BANDURSKI. 1969. Gas chromatographic and mass spectrometric analysis of indole-3-aoetic acid-myo-inositol esters. Plant Physiol. (Suppl) 44: 27. 9. UEDA, M. AND R. S. BANDURSKI. 1969. A quantitative estimation of alkaliJabile indole-3-acetic acid compounds in dormant and germinating maize kernels. Plant Physiol. 44: 1175-1181. II Reprinted from Journal of Chromatography Elsevier Publishing Company, Amsterdam '- Printed in The Netherlands CHROM. 6170 PURIFICATION OF INDOLE-3-ACETIC ACID MYOINOSITOL ESTERS ON POLYSTYRENE—DIVINYLBENZENE RESINS' AXEL EHMANN AND ROBERT S. BANDURSKI Department of Botany and Plant Pathology, Michigan State University, East Lansing, Mich. 48823 (U.S.A.) (Received May 23rd, 1972) SUM MARY A method for the purification of indole-3-acetic acid and esters of indole-3-acetic acid and myoinositol on Dowex 50W-X2 and on partially sulfonated polystyrene resins is described. The method has been applied to the analysis of the indolylic compounds present in crude acetone—water extracts of the kernels of Zea mays. Sufficient purification and enrichment of these compounds is obtained by a single column chromatographic step so that the column eluates can be examined by thin- layer chromatography, gas—liquid chromatography or combined gas—liquid chroma- tography—mass spectrometry. INTRODUCTION Work in this laboratory has been concerned with the isolation and chemical characterization of esters of indole-3-acetic acid (IAA) and myoinositol and myo- inositol glycosides1 4. The purification of these esters was accomplished by subjecting small samples of plant extracts to successive Sephadex G-10 column chromatography, silica gel column chromatography and preparative thin-layer chromatOgraphy (TLC). Only small amounts of these esters could be prepared by such methods, as they are present in low concentrations in plant tissues (35—45 mg/kg), are chemically diverse and undergo acyl migration during preparation. For preparative purposes, it was. desirable to develop a technique that could be used to purify and concentrate these compounds as a group. In the present communication, we present a column chromatographic technique involving the use of styrenedivinylbenzene copolymer resins capable of a large and relatively non-selective enrichment of the IAA esters from crude plant tissue extracts. To the best of our knowledge, there are no previously published single-step methods that permit qualitative and quantitative analysis of indolylic compounds in plant tissue extracts. A prior attempt had been made to use sulfonated polystyrene resin in the identification of growth-promoting substances in plant tissue extracts’. A brief report of our studies has been made", and subsequently there has been a study of the separation of some simple indole derivatives on neutral polystyrene resin’. ‘This work was supported, in part, by the National Science Foundation (GB-I8353X). Journal Article No. 5931 from the Michigan Agricultural Experiment Station. j.Chromatogr., 72 (1972) 61—70 36 37 62 A. EHMANN, R. s. BANDURSKI MATERIALS AND METHODS Extraction The extraction of ground sweet corn kernels of Zea mays L'. (cultivar, Stowell's evergreen hybrid) was carried out as described previously“, except that batches Wool-n Ital-ml: mun-uh Mount “Tm-u nation max ’1 "hint. Roldan our ' Pinion Residue | m “bl/fl flail-duh sauna-u Ina-ad ~M I "his!” Midi» upwind-III at I m .. Butanolphou Waiorphou mam Fig. 1. Flow sheet of the extraction procedure of dry corn kernels. of I 5 kg were used and the filtered acetone—water extract was concentrated to 50 ml per kilogram of extracted corn meal“. Fig. I summarizes the extraction procedure used. Column chromatography Dowex 50W-X2 (H+form), 200—400 mesh (‘Bakers Analysed' reagent) was allowed to swell in distilled water, and washed exhaustively with water. The resin was then washed successively with I, 2 and 5 N NaOH followed by washing with water until the pH of the eluate was stable at 5.5. The resin was then washed with 5 N HCl followed by water until the pH of the eluate was stable at 6.0. The resin was packed into a glass column of 1.8 cm ID. The bed volume (VB) was 66.5 ml and the void volume (V0), determined with blue dextran, was 20.5 ml. When the resin was to be used at an acidic pH, the packed column was washed with 0.1 M sodium citrate bufler (pH 2.5), followed by washing with water until the pH of the eluate was stable at 4.2. The sample was then eluted with 1.0 mM sodium citrate buffer at pH 3.3. For use at a more neutral pH, the packed column was washed with 0.1 mM sodium citrate buffer (pH 5.6). Then the column was washed with water until the pH of the eluate was stable at 4.7. The sample was eluted with 1.0 mM sodium citrate bufier at pH 6.2. . The experimental “low capacity" resin, a styrene—divinylbenzene copolymer, I % cross-linked, 100—200 mesh, and only 20 % sulfonated (Dow Chemical Co., ]. Chromatogr., 72 (1972) 61-70 38 PURIFICATION OF IAA MYOINOSITOL ESTERS 63 Midland Division, Midland, Mich. 48640, U.S.A.) was prepared in the same manner as the Dowex 50W-X2 resin. The resin was packed into a glass column of 0.55 cm I.D., VB = 3.5 ml and V0 = 1.8 ml. The sample was eluted with 1.0 mM sodium citrate buffer of pH 3.3, followed by water, and finally a linear acetone—water gradient from pure water to 50 % acetone. Thin-layer chromatography Crude extracts, at the stage of purity of fractions A and B (Fig. 1), contain too much dry matter to allow their examination by TLC. Following Dowex 50W-X2 or “low capacity” resin chromatography, TLC is possible. The developing solvents and TLC plates used were as described previously“. Indolylic compounds were made visible on the TLC plates with a modified Ehrlich reagent”. Within 5 min of Spraying with the reagent, the IAA esters and free IAA spots developed a pink-red color that slowly changed to blue-purple, and reached a maximum intensity after 5-8 h. This color reaction is considerably slower than that with the Salkowski reagent5, but is superior in that the color appears to be indefinitely stable. After full color develop— ment had been reached, other non-indolylic compounds on the same plates were made visible by spraying with concentrated (37 %) H2504 and charring for 20 min at 105°. After charring, the Ehrlich-positive spots lost their colors and intensities, and sometimes were no longer distinguishable from other charred matter. However, the initial blue-purple color of the indolylic compounds could be restored and inten- sified by simply submerging the TLC plate in water for 2-4 min. The plates were then dried at 35° and stored with no detectable changes in color with time. Gas—liquid chromatography Silylation with N—trimethylsilylimidazole (TSIAI). A samme containing between 0.05 and 0.2 pmole of IAA esters from the appr0priate column chromatographic eluant fraction was dried over anhydrous calcium sulfate in a 1.0—ml serum vial, and sealed with a silicone-rubber serum cap. Pure, dry N,N-dimethylformamide (Io—20 pl) was added with a syringe through the serum cap to dissolve the dry residue. Then 20—40 lul of TSIM (Regis Chemical Co., Chicago, Ill. 60610, U.S.A.) were added. The vial was shaken for several seconds and allowed to stand at room temperature for 30 min. This silylating reagent completely derivatizes the free hydroxyl groups of the inositol and glycoside moieties of the IAA esters. Silylation with bis(trimethylsilyl)trifluoroacetamide (BS TFA). The preparation of the trimethylsilyl (TMS) derivative with BSTFA (Regis Chemical Co.) was the same as with TSIM except that the Silylation was carried out at 50° for I h. Under these conditions, combined gas—liquid chromatography (GLC)—mass spectrometric analysis showed that all the hydroxyl groups were silylated as well as the nitrogen atom of the indole nucleus. The derivatized samples were analyzed on an F & M Model 402 gas chromatograph equipped with flame ionization detectors, with nitrogen as carrier gas at a flow-rate of 60 ml/min. Two columns were used, a 6 ft. X 3.0 mm ID. U-shaped glass column packed with SE30, 3 % 0n Supelc0p0rt (Supelco Inc., Bellefonte, Pa. 16823, U.S.A.), and a 6 ft. x 6.0 mm ID. U-shaped glass column packed with OV-I, I % 0n Gas-Chrom Z (Applied Science Lab. Inc., State College, Pa. 16801, U.S.A.). Combined GLC—mass spectrometry was carried out on a LKB— 9000 instrument. ]. Chromatogr., 72 (1972) 61—70 39 64 A. EHMANN, R. s. BANDURSKI RESULTS AND DISCUSSION Chromatography on Dowex 5oW-X 2 resin Dowex 50W-X2 is a sulfonated polystyrene—divinylbenzene copolymer and, as a strong cation-exchange resin, would not retain IAA or its esters. However, the structure of the resin suggested that the salt of IAA would be excluded, whereas IAA, as the undissociated acid, would partition between the stationary resin phase and the moving solvent phase. Further myoinositol has a single axial and five equatorial groups and it is known that the sterically less hindered equatorial hydroxyl groups are more strongly absorbed to polar absorbants than axial hydroxyl groups“. There- fore, a non-polar stationary phase, such as a polystyrene—divinylbenzene copolymer, Should have a greater affinity for equatorially acylated IAA—myoinositol esters. The axially acylated -2-O- esters should be eluted from the column first, followed by the equatorial esters, in order of decreasing polarity. That this does in fact occur is shown by the results in Figs. 2 and 3. The elution profiles were obtained when aliquot mix- tures of partially purified, axially and equatorially acylated IAA—myoinositols, IAA—myoinositol glycosides, unesterified IAA and [I-“C]IAA (only detectable radiologically) were chromatographed on Dowex 50W-X2. The samples were eluted with 1.0 mM sodium citrate buffer at pH 3.3 (Fig. 2) or at pH 6.2 (Fig. 3). IAA, as detected colorimetrically, or by radioactivity, was eluted between 7.5 and 12 bed volumes (0.5 and 0.8 1) from the column at pH 3.3, whereas at pH 6.2 IAA was eluted from the column between 0.3 and 1.2 bed volumes (0.02 and 0.08 l). Dissociated IAA was excluded from the column while the partially undissociated IAA was partitioning into the resin. The IAA esters were found to elute in three broad, overlapping peaks at 2.1-4 (0.14—0.27 l), 4.2-6.0 (0.28—0.40 l), and 6.1—8.3 (0.41-0.55 1) bed volumes . x": .. W - -°1i_‘-"c]m 40> r... I. IO > 4!” A--A4 ........ 0.1 0.2 0.3 0.4 0.: 0:0 or 0.0 0.0 . Isl-no at clout (I) Fig. 2. Elution profile of IAA and partially purified IAA esters chromatographed on acidic Dowex 50W-X2 resin. For conditions of column chromatography, see text. The IAA of the IAA esters was measured as described previously“. The -2-0- esters of IAA and myoinositol and myoinositol glycosides (3,. B‘ and B5) are eluted ahead (see Fig. 4A) of the equatorially acylated IAA esters. The numbers under the peaks indicate the fractions used for examination by TLC (Fig. 4A). j. Chromatogr., 72 (1972) 61—70 40 PURIFICATION OF IAA MYOINOSITOL ESTERS -o— lu-utm . xiii-g .. W = - we 65 Wm M mu A; 04 0-2 0.0 0-7 DJ M 0.: 0.: loll-o at clout (I) Fig. 3. I‘llution profile of IAA and partially purified IAA esters chromatographed on neutral Dowex 5o\\'-.\12 resin. For conditions of column chromatography and explanation of figure, see text and Fig. 2. The TLC pattern of the eluted fractions is shown in Fig. 4B. Fig. 4. TLC patterns of the distribution of IAA esters in eluent fractions from the acidic (A) and neutral (B) Dowax sow-X2 column chromatography. Aliquots, containing between 2 and 20 pg of IAA from the respective peak areas (see Figs. 2 and 3). were spotted on Silica Gel F,“ TLC plates. The plates were developed in ethyl acetate—methyl ethyl ketone—ethanol—water (5 :3 : I :I), made visible with Ehrlich reagent and charred with concentrated H,SO‘. J. Chromatogr., 72 (1972) 61—70 41 66 A. EHMANN, R. s. BANDURSKI from both the acidic and the neutral column. Therefore, pH had no effect on the elution profile or the elution volume of the IAA esters. Fig. 4 shows the TLC profiles of the material eluted from the columns. It can be seen that the axially acylated -2-O- IAA—myoinositols and IAA—myoinositol glycosides occurred in the first peak (labeled axial). The equatorially acylated esters were in the second and third peaks. The position of the hydroxyl group of myoinositol to which IAA is esterified is not known for all the equatorial esters. The compounds to which we have assigned the -I- or -3-0- structures were present only in the second and third peaks“. Further characterization of these compounds will be presented elsewhere. Most important, however, in terms of the applicability of the Dowex 5oVV—X2 resin for the purification of the IAA—myoinositol esters is the fact that the esters are eluted essentially as a group. In contrast, Sephadex G-10 chromatography separates IAA—myoinositols from IAA—myoinositol glycosides? Purification of crude extracts on Dowex 50W-X 2 resin The above behavior of IAA esters on Dowex 5oW-X2 resin has been studied with extracts that had been partially purified by two successive Sephadex 010 column chromatographic steps. It was therefore desirable to determine the behavior and degree of purification obtainable when a crude extract (crude B fraction, Fig. I) is chromatographed on this resin. Provided that the IAA esters are retained sufficiently long, it should be possible to wash mono—, oligo- and polysaccharides and organic acids (present in large amounts in the crude extracts) through the column. Amino acids would be only partially retained and, if absorbed, would remain on the column while the neutral esters are eluted. We tested this theory by using the acidic column and found that the column behaved as eXpected. A sample of crude B fraction (Fig. 1) of 47.5 g dry weight containing 6.10 mg of esterified IAA (0.0123 % 0f the dry weight) dissolved in 60.0 ml of water was applied to the column (conditions as in Fig. 2). As before, when using pre-purified material, the esters were eluted in three poorly TABLE I QUANTITATIVE DETERMINATION OF IAA LIBERATED 13v ALKALINE HYDROLYSIS IN ELUENT FRACTIONS FROM A SAMPLE or CRUDE B FRACTION CHROMATOGRAPHED 0N DOWEx 5oW-X2 RESIN Volume of Dry weight Alkali —labile Dry weight Purification eluent fraction of fraction IAA of IAA factor‘ (ml) (mg) (mg) (% 244—336 386 1.26 0.33 26 377—480 113 1.80 1.59 124 481-640 94 1.58 1.68 131 641—800 103 0.80 0.78 61 801—860 153 0.43 0.28 22 Total 849 5 .87 0.69 54 ' The purification factor is the IAA content as a percentage of the dry weight of the fraction divided by the IAA content as a percentage of the dry weight in the crude fraction. separated peaks within I 3 bed volumes. Table I shows the IAA content of the pooled fractions. Recovery of the IAA esters was 96 %. The total dry weight of the material J. Chromatogr., 72 (1972) 61-—70 42 PURIFICATION OF IAA MYOINOSITOL ESTERS 67 recovered was 849 mg containing 5.87 mg of IAA (0.69 % of the dry weight). This represents an overall 54-fold purification of the IAA-esters in a single column-step. Chromatography on “low capacity” resin Owing to the limited porosity of the non—sulfonated divinylbenzene copolymer of which Dowex 50 is made, the native resin is probably not suitable for chromato- graphy of IAA esters. Resins of low cation-exchange capacity, which show increased molecular sorption as a result of partial sulfonation“, however, should be suitable for the purification of IAA esters. A small sample of a divinylbenzene copolymer with only 20 % sulfonation was made available to us. The elution profile of a mixture of pre-purified IAA-myoinositol glycosides (Ba—B, mixture and IAA, plus a small amount of [I-“C]IAA) is shown in Fig. 5. Washing the column with 23 bed volumes ‘1; IL ‘ u u -°- Ill - «tan -o-(i J‘fl m -—--fnat-—--— -o- III . I.» 2 [i .. 5 s \ ‘ =4» l‘ L .~ ,' “i w 1' l 405 HO. 1% '1 °’\ >3: 1 R "as a II Hominid“) Fig. 5. Elution profile of IAA and partially purified IAA esters chromatographed on "low capacity" resin. For conditions of column chromatography, see text. Samples from the indicated eluent fractions (1-4) were chromatographed by TLC as shown in the insert. Conditions for TLC were the same as for Fig. 4. ' (80.0 ml) did not elute IAA or the IAA esters. Free IAA, however, was eluted with water between 1.4 and 10 bed volumes (5.0 and 35.0 ml). Most interestingly, the IAA esters could be eluted only with an aqueous acetone gradient from 6 % (v/v) to 46 % acetone. The TLC profile of the eluted material is shown in the insert of Fig. 5. Although not as marked as for the Dowex 50W-X2 resin with aqueous eluant, there is nonetheless a tendency for the axial IAA esters to be eluted ahead of the equatorial esters. The advantage of this partially sulfonated divinylbenzene copolymer lies in the high sorption capacity for the IAA—myoinositol glycosides and presumably also the IAA—myoinositols. Purification of crude extracts on "low capacity” resin In view of the results obtained with the pre-purified IAA esters, the suitability of this resin for the purification of crude extracts was tested. A sample of crude B J. Chromatogr., 72 (1972) 61—70 43 68 A. EHMANN, R. s BANDURSKI fraction (Fig. I) of 4.45 g dry weight containing 570 pg of esterified IAA together with free IAA and [I-“CHAA dissolved in 4.0 ml of water was applied to the column (conditions as for Fig. 5). The elution profile of this mixture of IAA and IAA esters is shown in Fig. 6. Again, the esters are not eluted with aqueous buffer Or water, but Jl Al r II -o- I“ -estm 14 WE - c] m L..__m.n———— '85” 20’ 3 3 .. 3 I" r-llmr Ia‘tu Acetone-[later ,_ g; g. 5,. QB; .1... I ‘l‘ 4 -" ( _12» ——0riua——— . 1200 . l >- i so . i g x. =1 '4 4. I” a .3. g 1 a» I >20; - m . I 4° I u" l -\ A A A1 4 u zit is so u Vol-no of eluent ml! Fig“. I». Elution profile of IAA and IAA esters from crude plant extract chromatographed on “low capacity" resin. For conditions of column chromatography, see text and Fig. 5. only with the aqueous acetone gradient. Surprisingly, IAA could not be eluted with water, but co-eluted with the IAA esters. This indicates that IAA in the crude extracts not only partitions between the resin and the mobile solvent phase, but also between a mobile phase and solute of the sample which apparently shows a high degree of solvation for IAA. Similar behavior of IAA upon column chromatography of crude B fraction has been observed before”. The insert in Fig. 6 shows the TLC profile of fractions taken from the respective regions of the single peak. The -2-0- esters are again eluted ahead of the equatorially acylated esters. Recovery of the indolylic compounds was 97 ‘31,. The dry weight of the pooled fractions (0.5-50.0 ml) was 34.4 mg containing 553 pg of IAA. Therefore the IAA content increased from 0.0128 to 1.60 %, which represents a 125-fold purification in this single column step. Gas—liquid chromatography It has been shown above that the material recovered from both column systems is suitable for direct TLC analysis. More important, however, is the fact that the same material may be used for GLC, and combined GLC—mass spectrometric analysis without additional purification steps. An example of the GLC behavior of a small sample taken from the pooled fraction of the ‘low capacity’ column is Shown in Fig. 7. The GLC profile shows the ccmplete series of IAA—myoinositols (Bl—B2 peaks), IAA—myoinositol arabinosides (B3—B,i peaks) and IAA—myoinositol galactosides (B5 peaks). The identity of these peaks was established by comparison of their mass ]. Chromatogr., 72 (1972) 61—70 44 PURIFICATION OF IAA MYOINOSITOL ESTERS 69 I. B, B, B, B. i .. B, 3 k if " 50 T in Mil-Isl Fig. 7. Gas—liquid chromatogram of a mixture of TSIM—IAA ester derivatives. A pooled sample containing between 0.05 and 0.2 pM of IAA from the appropriate eluent fractions of crude plant extract chromatographed on “low capacity" resin was prepared for GLC (using BSTF A) as described in the text. The derivatized sample was run isothermally at 250° with a carrier gas flow-rate of 60 ml/min. spectra with the previously published spectra for these compounds“. The results from the combined GLC—mass spectrometric analysis will be published elsewhere. CONCLUSIONS A sulfonated polystyrenedivinylbenzene COpOIymer (Dowex 50W-X2) column effectively purifies (54-fold) and concentrates indolylic compounds from extracts of kernels of Zea mays in a simple single column step. After column chromatography, IAA and esters of IAA and myoinositol or myoinositol glycosides are of sufficient purity to be analyzed by TLC, GLC or com- bined GLC—mass spectrographic analysis. A 20 % sulfonated polystyrene-divinylbenzene copolymer resin was found to be even more efficacious than Dowex-5o in the purification (125-fold) of indolylic compounds. Both Dowex-5o and the partially sulfonated resin Show lower affinity for axially acylated than for equatorially acylated esters of IAA and myoinositol or myoinositol glycosides. ACKNOWLEDGEMENT We are deeply indepted to Dr. H. SMALL, Dow Chemical Co., Midland, Mich., for supplying the 20 % sulfonated polystyrene-divinylbenzene c0polymer. REFERENCES 1 C. LABARCA, P. B. NICHOLLS AND R. S. BANDURsxl, Biochem. Biophys. Res. Commun., 20 (1966) 641. 2 P. B. NICHOLLS, Planta, 72 (1967) 258. j. Chromatogr., 72 (1972) 61—70 45 A. EIIMANN, R. s. BANDURSKI R. S. BANDURSKI, M. UEDA AND P. B. NICHOLLS, Ann. N.Y. Acad. Sci., 165 (1969) 655. M. UEDA AND R. S. BANDL'RSKI, Plant Physiol. Suppl., 44 (1969) 27. M. UEDA AND R. S. BANDURSKI, Plant Physiol., 44 (1969) 1175. M. UEDA, A. EHMANN AND R. S. BANDURSKI, Plant Physiol., 46 (1970) 715. G. BEAUCHESNE, Plant Growth Regulation, Iowa State University Press, Ames, Iowa, 1961, p. 607. A. EIIMANN AND R. S. BANDURst, Plant Physiol. Suppl., 47 (1971) 3. A. NIEDERWIESER AND P. GILIBERTI, ]. Chromatogr., 61 (1971) 95. E. STAIIL AND H. KALDEWEY, Hoppe-Seyler's Z. Physiol. Chem, 323 (1961) 182. T. POSTERNAK, The Cyclitols, Holden-Day Inc., San Francisco, 1965, p. 20. R. N. SARGENT AND D. L. GRAHAM, Ind. Eng. Chem. Process Des. Develop, I (1962) 56. ]. Chromatogr., 72 (1972) 61—70 III The isolation of Di-O-(indole-B-acetyl)-myo-inositol and Tri-O-(indole- 3-acetyl)-myo-inositol from mature kernels of Zea mays*. Axel Ehmann and Robert S. Bandurski Department of Botany and Plant Pathology Michigan State University East Lansing, Michigan 48823 *This work was supported by the National Science Foundation (GB-18353X). Journal Article No. from the Michigan Agri- cultural Experiment Station. 46 47 ABSTRACT Di-O-(indole-3-acetyl)-myo-inositol and tri-o-(indole-B- acetyl)-myo-inositol have been isolated from kernels of Zea mays and identified by gas-liquid chromatographic-mass spectrometric analysis of their trimethylsilyl ethers. INTRODUCTION Mature kernels of Zea mays contain between 20-100 mg/kg of "bound" indole-3-acetic acid (IAA).“5 that is hydrolyzable by dilute alkali. Studies from this laboratory established that the "bound" IAA (about 65 mg/kg) is composed of approximately one-half water soluble esters of IAA and myo-inositol, myo-inositol arabinoside and myo- 6'10 and one-half water-insoluble cellulosic- inositol galactoside glucan containing esterified IAA“. In the present work we have characterized by thin-layer chromatography (TLC), gas-liquid chromato- graphy (GLC) and combined gas-liquid chromatography-mass spectrometry (GLC-MS) two new, less polar, IAA esters present in trace amounts in butanol extracts of the water soluble fraction: di-O-(indole-3-acetyl)- myo-inositol and tri-O-(indole-B-acetyl)-myo-inositol. RESULTS AND DISCUSSION Ueda and Bandurski9 have partitioned the aqueous condensate of the acetone-water (l:l) extract of mature sweet corn kernels with l-butanol. They found trace amounts of two unidentified esters of IAA, designated HRF-l and HRF-2 (Ueda and Bandurskig, Figure 6) in the water-soluble, dried l-butanol fraction. HRF-l and HRF-2 have 48 recently been identified as esters of IAA and glucose12. Preliminary TLC analysis of the remaining water-insoluble l-butanol fraction, which was soluble in ethanol-water (l:l) showed that it contained IAA, the previously identified esters of IAA and myo-inositol, and myo- inositol-glycosides, and minute amounts of a new unknown indole com- pound, designated HRF-3. Ammonolysis of HRF-3 yielded IAA and indole- 3-acetamide (IAAM). HRF-3 is therefore an ester of IAA. The extraction of 10.5 kg of mature sweet corn kernels yielded 510 ug of HRF-3 (as IAA) and 210 ug (as IAA) of a second unknown indole compound, desig- nated HRF-4. Ammonolysis of HRF-4 also showed that it was an unknown ester of IAA. HRF-3 and HRF-4 gave a single spot each on TLC (Figure I, TLC insert, Samples l and 2), but following storage in 50% ethanol both HRF-3 and HRF-4 had an additional minor spot on TLC (Figure l, TLC insert, Sample 3). These minor components result from acyl migration, as has previously been observed with IAA esters of myo-inositol6’7, 9 and glucoselz. No attempt was made to separ- myo-inositol glycosides ate these components and all experiments were performed on HRF-3 and HRF-4 samples which were isomeric mixtures. Cbmponent analysis and stoichiometry of’HRF-3 and HEP-4: Ammonolysis of HRF-3 and HRF-4 yielded two Ehrlich-positive products which co-chromatographed on TLC with IAA and IAAM. Identification was confirmed by GLC and combined GLC-MS analysis of the trimethylsilyl (TMS) ethers of the hydrolysis products. HRF-3 and HRF-4 were there- fore esters of IAA. Charring of the TLC plates with concentrated sulfuric acid revealed an additional component which co-chromatographed with myo-inositol on TLC and was identified as myo-inositol by GLC and 49 combined GLC-MS analysis of the TMS ether. Thus the hydrolysis pro- ducts of HRF-3 and HRF-4 were identical with those of previously characterized IAA-myo-inositol esterss“7 but the Rf values on TLC were greater suggesting that HRF-3 and HRF-4 contained an additional lipophylic moiety. Since the only hydrolysis products were IAA and myo-inositol the additional lipophylic component must be one, or more, additional moles of IAA per mole of inositol. Plotting the Rf values of the two isomers of IAA-inositol l-Q;;l-0-(indole-3-acetyl)-myo- inositol (Bl) and 2-0-(indole-3-acetyl)-myo-inositol (Bz), HRF-3 and HRF-4 against the logarithm of the number of moles of IAA per mole of inositol (see Figure 1) indicated that HRF-3 had two moles of IAA per mole of inositol and HRF-4 three moles of IAA per mole of inositol. Incomplete hydrolysis of HRF-3 should yield HRF-3, IAA-myo-inositol and IAA, whereas HRF-4 should yield HRF-3, IAA-myo-inositol and IAA. Preliminary experiments with a mixture of 2-0-(indole-3-acetyl)-myo- inositol and QL;l-0-(indole-3-acetyl)-myo-inositol in ethanol-water (l:l) showed that hydrolysis with 4.6% NH 0H hydrolyzed about 20% of 4 the esters after three minutes at room temperature and 70% after thirty minutes. Under these hydrolysis conditions only free IAA and the transesterification product, the ethyl ester of IAA, were formed. IAAM is not formed with dilute NH40H although it accounts for about 50% of the IAA when hydrolyzed with 28% NH40H. Hydrolysis of a sample of HRF-3 with 2.l5% NH40H for five minutes resulted in IAA, its ethyl ester a mixture of B1 and B , and intact HRF-3 (Figure l, TLC insert, 2 Sample 4), whereas similar hydrolysis of HRF-4 yielded IAA, the ethyl ester of IAA, B1 and 32 and HRF-3 (Figure l, TLC insert, Sample 5). 50 This shows that HRF-4 can be hydrolyzed to HRF-3 by loss of IAA and that both HRF-3 and HRF-4 can be hydrolyzed to yield IAA-myo-inositol. The stoichiometry of the hydrolysis products of HRF-3 and HRF-4 is given in Table l and show that HRF-3 and HRF-4 are di-o-(indole-B- acetyl)-myo-inositol and tri-O-(indole-3-acetyl)-myo-inositol, respectively. Gas-liquid chromatography: The TMS ethers of HRF-3 and HRF-4 were prepared as described previously13. GLC analysis of both com- pounds on several liquid phases, 0V-l, 2%; SE-30, 3%; 0V-l7, 3% in a 1.8 meter or 1.2 m glass column showed only two peaks, corresponding to the mono-substituted myo-inositols B1 and 82 indicating thermal insta- bility of the di- and tri-o-(indole-B-acetyl)-myo-inositol esters. Using a 0.4 m column decreased the temperature for the elution of 2-0-(indole-3-acetyl)-myo-inositol from 230° to 190°, and the TMS ether of HRF-3 could be chromatographed on this column at 270° yielding three peaks (see Figure 3, GLC-profile in spectrum III). GLC analysis of the TMS derivative of HRF-4 was not possible. It decomposed to HRF-3 and two isomers of HRF-3, P4 and P5, (Figure 4, GLC insert in spectrum VI). Mass spectrometry: Combined GLC-MS was performed on the TMS-ethers of HRF-3 and HRF-4 to confirm the stoichiometry and to detenmine, insofar as present methods permit, the position of substitu- 14 tions of IAA on myo-inositol. Ueda, et at. studied the GLC behavior of IAA-myo-inositol, the parent compound of HRF-3 and HRF-4. IAA-myo- inositol yields two spots on TLC designated B1 and B2 (see Figure 1, Sample 6) and Nicholls et al.15 showed by NMR that B is 2-0-(indole-3- 2 acetyl)-myo-inositol. Since 82 is known to isomerize to B1 and acyl SI migration presumably occurs more readily between vicinal groups, it has been concluded that B1 is, most likely, l-Q;:l-0-(indole-3-acetyl)- 14 observed three peaks of the TMS-ethers of 13 myo-inositol. Ueda et at. B1 and B2 by GLC, and, more recently, Ehmann and Bandurski resolved four of the six possible isomeric esters (two pairs of mirror images and two optically inactive esters). The GLC inserts in spectrum I of Figure 2 show these four isomers. B2 prepared by preparative TLC gives rise to P1 and P2. P], the predominant compound, is therefore the 2-0-ester whereas the minor peak, P2, is probably the 5-0-ester. B1 on the other hand gives rise to P3 and P4 and by analogy P3 is the l1ggr l-O-ester and P4 possibly the l:QL;4-0-ester. However the configuration of peaks 2 to peaks 4 have not yet been confirmed. Ueda et al.14 and -16 Ueda and Bandurski studied the TMS ethers of B and B2 by combined 1 GLC-MS where the imino-group of the indole ring was not substituted with a TMS-group. They noted that 32’ the 2-0-ester fragments preferentially by loss of the acyl group whereas B1 the lzggyl-O-ester fragments by loss of TMSOH. The mass spectra of the fully trimethylsilylated derivatives of the major peaks of B1 and 82 have not previously been published and are shown in Figure l (Spectra I and II). Both spectra were recorded at about equal sample pressure and identical conditions to facilitate comparison of the fragmentation patterns. It is apparent that the spectra of both compounds in the mass range from 30 to 350 amu are almost identical. The ions m/e l30, m/e 157, m/e 202, m/e 229 and m/e 247 characteristic for the indole moiety have been described12 as have the ions due to the fragmentation of the inositol moiety16. 52 Mass spectrum of B] ( I zi-I -0-( “indol e-3-acetyl )-myo- inositol): The introduction of a TMS group on the imino group of the indole moiety results in a very stable molecular ion M5: at m/e 769. M? loses 72 amu to give rise to the ion m/e 697 which involves the transfer of a proton to the indole nucleus from one of the methyl groups of the TMS group on the imino group with subsequent loss of the neutral -CH2(CH3)2 Si radical. -+ o Iz-o-'c'--CH2 I I ) N R—O-O-CF-CH-Ej 'I' /| ___. H—CI— S|i—Mo and AEZBCHz f. AA. R=rny04nommol AA. + M’ ml. 769 ml. 697 I Loss of TMSOH from Mr leads to the ion m/e 679, a characteristic ion for B], followed by the loss of a neutral oCH2(CH3)2 Si radical to give rise to m/e 607. The ion m/e 597 results from the loss of the indole moiety from MT. If charge retention is on the leaving group an ion is found at m/e 202. O Iz-o-c'f-CH2 / I -' R-O-CEO or + T Sfihhoa + M' mle 769 mle 597 rule 202 53 The ion m/e 507 is the result of the elimination of a 'CH3 radical from MT followed by the loss of the acyl group. Charge retention on the acyl group leads to the ion m/e 247 by proton transfer from the myo- inositol skeleton to the leaving groupIz. This is confirmed by the shift of 9 amu of this ion to m/e 256 in the spectrum of the perdeuterotrimethylsilyl (TMS-d9) analogue. SiMoa ml. 754 ml. 247 _ h-k The MIL ion may also fragment by ring cleavage of the inositol moiety and loss of a three-carbon unit to give rise to m/e 462. Fragmentation of the inositol moiety and indole moiety have been described16. 17 The ion at m/e 36l, a characteristic ion of TMS-glycosides , is not norm- ally observed in TMS derivatives of cyclitols. Its origin may be explained by ring opening of the inositol moiety followed by the loss of a four carbon unit (408 amu) with charge retention on the indole moiety. 54 Mass spectrum of'BZ (2-0-(indoIe—3-acety1)-myo-inositol): The loss of TMSOH from MT is suppressed, but an ion is found at m/e 574 which comes from M1L by the loss of two molecules of TMSOH and a °CH3 radical. The ions m/e 679, m/e 607 and m/e 36l are absent and‘ therefore distinguish B2 from 81 which together with the different retention times on GLC permit identification of B1 and 82. Mass spectrum of'HRF-s: The mass spectrum of the TMS deriva- tive of HRF-3 is shown in Figure 3, Spectrum III. TMS-HRF-3 yields three peaks on GLC with peak 3 accounting for more than 80% of the total peak area. The mass spectra of P1 and P2 have also been studied and are virtually identical with the mass spectrum of P3. The ion found at m/e 926 corresponds to the molecular ion of di-o-(N-TMS-myo indole-3-acetyl)-0-tetra-0-TMS-myo-inositol, confirming the stoichio- metry of 2 IAA per inositol found for HRF-3. The molecular ion loses ~CH3 to yield a low intensity ion at m/e 911. MT also loses 72 amu to yield m/e 854 which fragments to m/e 697 by the loss of the indole- 3-ketene radical. 55 This transition is supported by the presence of a small metastable ion found at m/e 568.5 (calculated 568.9). Loss of the corresponding ketene (158 amu) from the TMS-d9 analogue confirms this (see Figure 4, Spectrum V). M? can also lose an N-TMS-indole-3-acetoxy group to yield m/e 697 directly. The metastable peak found at m/e 524.5 (calculated 524.6) supports this transition. The ion m/e 911 may lose 72 amu to give the ion m/e 839. The ion m/e 782 arises from the loss of two ~CH2(CH Si radicals from the molecular ion. A low intensity 3)2 ion at m/e 681 probably comes from the loss of the N-TMS-indole-3- ketene radical from Mt. The presence of m/e 726 in the TMS-d9 analogue (see Figure 4, Spectrum V) supports this fragmentation step. The ion m/e 681 in turn loses 72 amu (-CH2(CH3)2 Si) to give rise to m/e 609. The strong intensity ion at m/e 697 fragments to m/e 625, a small intensity ion by the loss of a 'CH2(CH3)2 Si radical and to m/e 607 and m/e 592 by the successive loss of TMSOH and 'CH3. The ion m/e 664 corresponds to m/e 507 of the mass spectra of B1 and B2 by having one TMS-group replaced by a N-TMS-indole-3-acetoxy group. The ion m/e 592 56 then loses one TMSOH to give m/e 502. The low intensity ion m/e 475, which is not present in the mass spectra of B1 and B2 probably origin- ates from M? by inositol ring cleavage and the loss of 451 amu. .+. E UT {fl/m3 Sfifln'b (JSHNMQB nub4rzs. The shift of this ion in the mass spectrum of the TMS-d9 analogue to m/e 502 supports this assumption. The ion found at m/e 463 does not belong to any fragmentation pathway and examination of the UV recording of the mass spectrum shows that m/e 463 was accompanied by ions at m/e 463.5, m/e 464 and m/e 464.5. This is evidence that m/e 463 is the doubly charged molecular ion found at m/e 926. The presence of the corresponding ion at m/e 490 in the TMS-d9 analogue of HRF-3 confirms this (Figure 4, Spectrum V). The mono-acylated parent compounds B1 and 82 do not show a doubly charged molecular ion suggesting that in HRF-3 charge localization may occur simultaneously on both acyl groups at the carbonyl oxygen. 57 .+ , 9 9+ C H-C— . - -c H (I o’ll ‘\HAA.3 WI l AA“ Si 3 SNAA.5 «-smnne3 M. ',mI. 463 In comparing Spectrum II (32) with Spectrum III (HRF-3) it is apparent that the fragmentations from M? are identical, and it may be concluded that peak 3 of HRF-3 is the di-0-(indole-3-acety1)-myo-inositol which has one of the indole-3-acetyl groups on the 2-0- position of myo- inositol. This is supported by the fact that HRF-3 thermally decom- poses to mainly B1 upon GLC on 1.2 and 1.8 m columns by the preferred elimination of the axial 2-0- substituent. The position of the second indole-3-acetyl group is however not certain. It is very likely on the 1-Q;;l-0- position since no steric hindrance exists between the acyl groups vicinal to each other, and since no major mono-acylated peaks were found which would correspond to the 5-0-ester (P2) or the 11QE74-0-ester (P4) on GLC. Mass Spectra of’HRF—4: The composition and stoichiometry of HRF-4 showed three moles of IAA esterified to one mole of inositol. GLC of the TMS ethers of HRF-4 on the 0.4 m column showed five peaks, three of which (P1, P2, P3) corresponded in retention time to the three peaks of HRF-3. Peaks 4 and 5 were not observed with HRF-3. As 58 mentioned previously this indicates that HRF-4 thermally decomposes to HRF-3 and two isomers not previously observed. Combined GLC-MS of the TMS-ethers of HRF-4 confirmed this assumption. Peaks 1 through 5 all showed the same molecular ion at m/e 926. The mass spectra of the first three peaks were indistinguishable from the mass spectra of the TMS-ethers of HRF-3. The mass spectra of the TMS-d9 analogue of HRF-4 are shown in Figure 4; Spectra V and VI. Comparison of these spectra with Spectrum III (HRF-3) confinns the fragmentation pathways of HRF-3 by the corresponding mass shifts in the spectra. Spectrum V is almost identical with Spectrum III and it may therefore be concluded that at least two acyl groups in HRF-4 occupy the same position as in HRF-3, one acyl group on the 2-0-position and the second one on either the l-QL;l-0- or 1-g;;4-0- position of myo-inositol. Peaks 4 and 5 by virtue of their longer retention times are thus probably diacyl isomers of the following types: 110;;1,4—0-, 110;;1,6-0-, 11QL72,3-0-, 4.6—0 or 112L71,5-0-, since there are fifteen theoretical di-O-(indole-3- acety1)-myo-inositols, six pairs of mirror images and three symmetrical esters. At the present time it is not possible to assign P5 of HRF-4 to any one of these possible configurations. Merely by analogy to the B]-B2 series it may be inferred that P4 and P5 belong to the 81 series. By first converting HRF-4 to the TMS ether and subsequent MS analysis by direct probe it was possible to obtain the mass spectrum of the intact compound. The spectrum is shown in Figure 3; Spectrum IV. The ion at m/e 1083 corresponds to the TMS ether of the tri-O-(indole-B- acetyl)-myo-inositol confirming the assignment of three moles of IAA per mole of inositol. The molecular ion fragments to m/e 881 by losing 59 one N-TMS-indole-3-methylene radical (202 amu). Mf also loses one TMS-indole-3-ketene radical (229 amu) yielding m/e 854 or m/e 836 by the loss of N-TMS-indole-B-acetic acid. A small ion at m/e 926 is the result of a rearrangement of the odd electron molecular ion leading to the elimination of an indole-3-acetoxy radical. + M',ml.1083 R-indoIeo-acotyl ml. 926 The ion m/e 361 is intense compared to Spectrum I indicating that inositol ring cleavage is more frequent in HRF-4 than in HRF-3 or B]. The remaining ions have been discussed in connection with B], 32 and HRF-3. It may be assumed that the mass spectrum of HRF-4 is a com- posite of at least two or more isomers. Two isomers of HRF-4 have been found by TLC and the thermal degradation on GLC showed at least five isomers of the twenty theoretically possible esters: 8 pairs of mirror images and four symmetrical esters. By analogy with the major breakdown products of B1 and HRF-3 it may however be concluded that in HRF-4 the acyl groups predominantly occupy the 1,2,3-0- and 2,4,6- o-positions of myo-inositol. 60 EXPERIMENTAL Extraction and Purification: The extraction of 10.5 kg of ground corn kernels of Zea mays L. (cultivar Stowell's Evergreen hybrid) has been described previously9’13. The condensed aqueous extract was partitioned with l-butanol, the butanol-phase dried and redissolved in water. The water-insoluble residue was removed by filtration and dried. The dry weight of this HRF fraction was 2.89 g with a total IAA 9"2"3) of 4.35%. This content (determined as described previously fraction was dissolved in 1.5 ml of ethanol-water (1:1) and chromato- graphed on partially sulfonated styrene divinylbenzene copolymer (column 1.0. = 9.0 mm, bed volume = 38.2 ml, void volume = 13.5 ml) with ethanol-water (1:1) as eluent, collecting 2.0 m1 fractions. Small aliquots of the fractions (lo-50 ul) were monitored on TLC as described before13, and the HRF-3 and HRF-4 containing fractions (tubes 25-80) were pooled. The pooled sample was dried, redissolved in 1.5 m1 of ethanol-water (1:1) and re-chromatographed on Sephadex LH-20 (column 1.0. = 9.0 mm, bed volume = 38.5 ml, void volume = 8.5 ml) with ethanol- water (1:1) as eluent. The 2.0 ml fractions were again monitored on TLC, and the fractions containing HRF-3 and HRF-4 (tubes 40-70) were pooled and dried. The dry weight of the residue was 2.3 mg and the total IAA was 31.6%. The residue was dissolved in 1.0 ml ethanol-water (1:1) and chromatographed again on the same Sephadex LH-20 column. The fractions containing mainly HRF-3 (tubes 43-55) and HRF-4 (tubes 56-72) were pooled and further purified by preparative TLC as described pre- viousiy‘z, Following this step 510 ug of HRF-3 and 210 ug of HRF-4 were obtained. 61 Ammonolysis: Samples of HRF-3 and HRF-4 from the last col- umn step and following preparative TLC were hydrolyzed in 14% NH40H and the hydrolysis products analyzed by TLC, GLC and combined GLC-MS 12’13. Stoichiometry of HRF-3 and HRF-4 was as described previously performed on samples from the last Sephadex LH-20 column step and fol- lowing preparative TLC. IAA was determined in two ways: spectrophoto- 14 metrically at 221 nm and 280.5 nm, and by quantitative GLC Inositol was determined by quantitative GLC14. Incomplete hydrolysis with 2.15% NH40H in ethanol-water (1:1) was carried out exactly as with 14% NH40H. After 1, 5 and 30 minutes the reaction was stopped by rapidly drying in vacuo. The hydrolysis products were assayed by TLC and visualized with Ehrlich reagent and concentrated sulphuric acid as described elsewhere13. Gas liquid chromatography: Silylation of B], 82, HRF-3 and HRF-4 with N-O-bis-trimethylsilyl-trifluoroacetamide or N-(trimethyl- d9 silyl) imidazole was performed as described before12’13. The TMS ethers were analyzed on a F and M Model 402 Gas Chromatograph equipped with flame ionization detectors with nitrogen as carrier gas at a flow rate of 60 ml per minute. Glass columns of 1.8 m, 1.2 m and 0.4 m x 3.0 mm ID were used, packed with either 0V-17, 3% on Gas chrome Q (mesh 100/120), 0V-l, 2% on Gas chrome Z (mesh 100/120) (Applied Science Lab Inc., State College, Penn., 16801, USA) or SE-30, 1% on Supelcoport (mesh 100/120) (Supelco Inc., Bellefonte, Penn. 16823, USA). Mass spectrometry: Combined GLC-MS was performed on an LKB-9000 Mass Spectrometer with a 0.3 m x 3.0 mm ID glass column 62 packed with 0V-1, 2% on Gas chrome Z (mesh 100/120) and helium as carrier gas at a flow rate of 25 ml/min. The ionizing energy was 70eV, the flash heater 250°, the molecular separator 250° and the ion source temperature 290°. The mass spectra were recorded with an on-line data 18 acquisition and processing program The mass spectrum of HRF-4 was recorded at a probe temperature of 135°. ACKNOWLEDGMENTS Dr. M. Ueda kindly provided the butanol concentrates of IAA- cyclitols used in the early stages of this work. The mass- spectrometry facility made available to us by Professor 0. C. Sweeley was supported by a grant from the National Institutes of Health ( ). We thank Professor H. Wells for his helpful discus- sions concerning the thermal instability of HRF-3 and HRF-4 and Mr. Jack Harten for technical assistance in operating the mass spectro- meter. 10. 11. 12. 13. 14. 15. 16. 17. REFERENCES G. S. Avery, H. B. Creighton and B. Shalucha, Amer. J. Bot. 27 (1940) 289. 6.75. Avery, J. Berger and B. Shalucha, Amer. J. Bot. 29 (1942) 65. J. Berger and G. S. Avery, Amer. J. Bot. 31 (1944) 199. J. Berger and G. S. Avery, Amer. J. Bot. 31 (1944) 202. A. J. Haagen-Smit, N. D. Leech and w. R. Bergen, Amer. J. Bot. 29 (1942) 500. ('3 . Labarca, P. B. Nicholls and R. S. Bandurski, Biochem. Biophys. Res. Commun. 20 (1966) 641. v . B. Nicholls, Planta 72 (1967) 258. JO . S. Bandurski, M. Ueda and P. B. Nicholls, Ann. N. Y. Acad. Sci. 165 (1969) 655. M. Ueda and R. S. Bandurski, Plant Physiol. 44 (1969) 1175. M. Ueda and R. S. Bandurski, Plant Physiol. Suppl. 44 (1969) 27. z. Piskornik and R. s. Bandurski, Plant Physiol. so (1972) 176. A. Ehmann (unpublished). A Ehmann and R. S. Bandurski, J. Chromatogr. 72 (1972) 61. M.7Ueda, A. Ehmann and R. S. Bandurski, Plant Physiol. 46 (1970) 5. t .2367Nicholls, B. L. Ong and M. E. Tate, Phytochem. 10 (1971) M. Ueda and R. S. Bandurski (unpublished). U . C. DeJongh, T. Radford, J. D. Hribar, S. Hanessian, M. Bieber, G. Dawson and C. C. Sweeley, J. Amer. Chem. sec. 91 (1967) 1728. 63 64 18. C. C. Sweeley, in Introduction to lipid Chemistry, edited by R. M. Burton, Bi-Science International, Webster Groves, Inc. (1973) in press. 65 Table 1. Stoichiometry of HRF-3 and HRF-4. INOSITOLa IAA-UV IAA-GLC IAA/INOSITOL HRF-4 HRF-3 b CC -samp1e TLCc-sample CC-sample TLC-sample 1.00 3.15 3.04 3.10 mole ratios of IAA 1.00 3.06 2.88 2.97 to myo-inositol 1.00 2.14 1.94 2.04 1.00 2.05 1.97 2.01 a. Myo-inositol was determined by quantitative GLC14 b. This sample was purified by the two column chromatographic steps described in the text. c. These samples were prepared by the two column steps described and by further purification on TLC. 66 LEGENDS FOR FIGURES Figure 1. Plot of the R -va1ues of B], 32’ HRF-3 and HRF-4 f versus the logarithm of moles of IAA per mole of myo-inositol. The insert shows the TLC behavior of HRF-3 (l), HRF-4 (2), the isomers of HRF-3 and HRF-4 (3), and the incomplete hydrolysis products of HRF-3 (4) and HRF-4 (5). Sample (6) is a mixture of standards: l-QL;0- (indole-3-acety1)-myo-inositol (B1), 2-0-(indole-3-acetyl)-myo-inositol (B2), indole-3-acetic acid (IAA) and ethyl-indole-B-acetate (IAE). The samples were chromatographed on silica gel plates (Merck, Darmstadt) in methylethylketone-ethylacetate-ethanol-water (3:5:l:1) and visualized with a modified Ehrlich reagent‘3. Figure 2. Mass spectra of the TMS-ethers of B1: 1-QL;1-0- (indole-3-acetyl)-myo-inositol (Spectrum I) and B2: 2-0-(indole-3- acetyl)-myo-inositol (Spectrum II). The spectra were recorded as described in the EXPERIMENTAL section at a column temperature of 230°. The inserts show the GLC-profiles and the shaded area under peak 1 and peak 3 represent the contribution of the peak to the individual spectra obtained. Figure 3. Mass spectra of peak 3 of the TMS-ethers of HRF-3 (Spectrum III) and of the intact TMS-ether of HRF-4 (Spectrum IV). Spectrum III was recorded at a column temperature of 260°. Spectrum IV was obtained at a probe temperature of 135°. Figure 4. Mass spectra of peaks 3 and 5 of the thermal decomposition products of the TMS-dg-ethers of HRF-4 (Spectra V and VI). For conditions see EXPERIMENTAL. 00 moles of IAA/mole of INOS N 1 67 ____Fl‘Oflfl‘ WE'RE -_ . a,“ 43,4; M .. .4 ‘6; 411':- «i- 4.144 5‘: 44 434444 * c‘:. 4.? 24.444? an NIH 123456 / “GAO/00* L ##J 0.4 0.6 relative R, values -_..-o..,...-__-- / 0.8 68 pH 00 ooh 0mm Oom 0mm oom Om¢ 00¢ 0mm DP F F;F F FVF b b bF rTPF >>F F F>F>F _ F F F hint-bF h F 511F1PIPPF>> F>F F b > 000 F F F17 0mm ms CD... 1% h! «3411 3 ocul>><< W. JWHR duster mx Fr {*4 a 5n FbPhFVFPF>F> ooh F1} >F 0mm F>b>>>F>F 00m 0mm oom om¢ p11tFleblFrPF>> F>FDD>F>>>FthFtFDF>F>F>F>>> 00¢ 0mm h FLP>F>F >>>FPP F b 5 000 b 0mm Db h? 1 Com omfl F F b p E FDF b b b h1F F b F n .— OOH .. ti FF 4 4 b .6. \\ k: .35 141 h! \ \ 41 «Iv 23 2» sauna V H he. ox Db {44> 4. Us H u: 41 10m OOH (I) 69 omofi OOOH 00m 0mm 00m OOF 0mm 00w 0mm. Gum. F>F>F >F>->F>.1EL,F.L b11F1F L1F FLFL 14 s u \., a +2 .24 mm L 7 4 100. 1 1 4m. J .‘ T .TU my. 1 - 11 4:1 - Odd 00¢ oma 00m 0mm OON Om” “OH mm 1.): ttfterFtthih1F1F>F PhtFpF1l—4F F FtFLFbeF FinF k» >F>F4 F1fF F F F>L$F>F F11F1>F FLF h F F4» _111 x 1 11:1 J1 141 1 1.11 4 141d 4 4314.141 14 11 _ A 141414 1 4411 K. «31$ 3. a. A 141 PH 5 are .— 5: 8. 4m 8. .8 a.» sun \ 8. J §§A a g 1 ,114, _ In a! , 1— \ B 8 1 41. J _\ 8. Am I. I. L\. +r_xll..r1lI n87): ex 2 on a (.11 cmm 00m 0mm 00m 09. 09. 0mm 0mm (mm 00m F>1F1r F F 1F1PF L P F P1b1b F F F1157 F F F F11F F1F b F 4 F F L11R; FL1P > F » 7» FL F Np 1— a \ .. A I. ”8 If .—48.o\.voo3 \3. «S I. 8.. 1 0m. 8. L +1 s .144 J ”1:11;; 1 d .1 a mm by Omfl (1.0.. mm 1D): 111? F F F F F m. F1F lmur F1.1»141F F u a i e. e 8. _ 1 .. an on R - \ r L I f L. a «I (NU .\ 1 L B lam .. 08.32 mx .1 l 1 11 1 SHE}? on. R -_ _- 50 300 200 150 100 50 U) 850 BOO SSO 800 MWHMO 800 850 X2 750 700 650 X2 550 600 I 500 IV The isolation of 2-0-(indole-3-acetyl)-D-g1ucopyranose, 4-0-(indole-3-acetyl)-D-glucopyranose and 6-0-(indole-3-acety1)- D-glucopyranose from mature sweet corn kernels of Zea mays*. Axel Ehmann Department of Botany and Plant Pathology Michigan State University East Lansing, Michigan 48823 *This work was supported, in part, by the National Science Foundation (GB-18353X). Journal Article No. from the Michigan Agricult- ural Experiment Station. 71 ABSTRACT A new ester of indole-3-acetic acid and glucose has been isolated from mature sweet corn kernels of Zea mays. Two isomeric forms of this ester were resolved by thin-layer chromatography with Rf-values distinct from that of authentic 1-0-(indole-3-acety1)-B-D- glucopyranose. Analysis of the trimethylsilyl-ethers of the two iso- mers by combined gas-liquid chromatography-mass spectrometry showed that the esters have a free carbonyl function. The labeling of the carbonyl carbon with an O-methyloxime group, and the analysis of the trimethylsilyl o-methyloxime derivatives by gas-liquid chromatography- mass spectrometry allowed the unambigous identification of the new ester of indole-3-acetic acid and glucose as a mixture of 2-0-(indole— 3-acety1)-D-glucopyranose, 4-0-(indole-3-acety1)-D-glucopyranose, and 6-0-(indole-3-acetyl)-D-glucopyranose. INTRODUCTION Several esters of indole-3-acetic acid (IAA) and a sugar have been isolated from plant material. Zenk1 first isolated IAA- glucose from pea epicotyls and leaves of Colchicum neapoZitanum after incubating the tissues and labeled IAA. The IAA-glucose showed char- acteristics typical of an ester linkage on the carbon 1 position of k1,2 the D-glucose and Zen assigned the 1-0-(indole-3-acetyl)-B-D- glucopyranose structure to it. Following the incubation of wheat 72 73 3 isolated an ester of IAA and coleoptiles with labeled IAA Klambt glucose which he tentatively identified as 1-0-(indole-3-acetyl)-B-D- glucopyranose. A compound was isolated from Avena coleoptiles by Keglevié and Pokorny4, which behaved on thin-layer chromatography (TLC) like authentic l-0-(indole-3-acetyl)-B-D-glucopyranose, but needs further characterization before a definitive assignment can be made. The isolation of naturally occurring esters of IAA and arabinose from immature corn grains has been reported by Shantz and Stewards, Steward 6 and Srivastava7’8. However no chemical characterization and Shantz of these IAA esters has been published. Esters of IAA and myo-inositol, myo-inositol-arabinosides, and myo-inositol-galactosides have been isolated from mature corn kernels of Zea mays and characterized by paper chromatography and 14 TLC9’13, nuclear magnetic resonance studies , gas-liquid chromato- graphy (GLC) and combined gas-liquid chromatography-mass spectrometry (GLC-MS)12’13’15’16’17. In addition to these IAA esters of myo- inositol and myo-inositol-glycosides Ueda and Bandurski12 have isolated two unidentified esters of IAA designated HRF-l and HRF-2 in trace amounts from mature kernels of sweet corn. The present report des- cribes the isolation, purification and identification of these esters as three isomers of IAA-glucose by TLC, GLC, and combined GLC-MS analysis. RESULTS AND DISCUSSION The extraction of 10 kg of dry sweet corn kernels of Zea mays L. (cultivar Stowell's Evergreen Hybrid) yielded 420 ug of HRF-1 and 74 480 ug of HRF-2. This concentration (about 100 ug/kg dry weight) is low compared to the IAA-cyclitol esters (about 65 mg/kg dry weight) previously identified‘z. Following purification on "low capacity" styrene divinylbenzene copolymer resin13 (Figure l-A), Sephadex LH-20 (Figure l-B) and separation of the two IAA esters into HRF-l and HRF-2 by preparative TLC it became apparent that HRF-l and HRF—2 intercon- vert, presumably by acyl migration18'24. As is seen in Figure l-C, rechromatography of HRF-1 (band 3: central region of HRF-l) or HRF-2 (band 2: central region of HRF-2) always yields a small amount of the other isomer HRF-1 from HRF-2, or HRF-2 from HRF-l. This phenomenon of acyl migration has previously been observed with IAA esters of myo- 19’10 12. Keglevié18 synthesized inosito and myc-inositol glycosides 1-0-(indole-3-acety1)-o+B-D-glucopyranose, and reported acyl migration of the a anomer to 2-0-(indole-3-acety1)-D-glucopyranose under neutral and acidic conditions. This conversion was not quantitative, indicat- ing that an equilibrium exists. The acyl migration observed in IAA- myo-inositol esters and HRF-1 and HRF-2 also does not result in a quantitative conversion of one isomer into another, indicating an equilibrium condition. The ammonolysis products of HRF-l and HRF-2 were identified by TLC as IAA, indole-3-acetamide (IAAM) and D-glucose, and by GLC and combined GLC-MS as IAA, IAAM and a+B-D-g1ucopyranose. Using authentic l-O-(indole-3-acetyl)-B-D-glucopyranose as a working standard the stoichiometry of IAA+IAAM/glucose was found to be 0.98/1.00 for HRF-1 and 0.97/1.00 for HRF-2. HRF-1 and HRF-2 were thus identified as two isomers of indole-B-acetyl-D-glucose. It can be seen in Figure 1-C 75 that the Rf values of HRF-l and HRF-2 are different from that of authentic l-0-(indole-3-acetyl)-B-D-glucopyranose indicating that IAA in HRF-1 and HRF-2 is esterified to glucose on carbons other than carbon 1. Gas-liquid chromatography. GLC of the trimethy1si1y1 (TMS)- ethers of a mixture of HRF-1+2 (tube #22 from the Sephadex LH-20 column; Figure l-B) gave five peaks, the relative retention times of which are shown in Table 1. Besides the liquid phases listed in Table 1 (0V-l, 2%, and 0V-l7, 3%) other liquid phases (SE-30, 3%; 0V-210, 3%; and SP-2401, 5%) were tried, but none of them improved the resolution of the overlapping peaks. Subsequent GLC of the TMS-ethers of HRF-1 and HRF-2 (band #3 and band #2; Figure l-C) showed that HRF-1 gives rise to peaks 2 and 4 (main peaks), and peaks 1, 3 and 5 (minor peaks). HRF-2 on the other hand gives peaks 1, 3 and 5 (main peaks) and peaks 2 and 4 (minor peaks). A correlation of the Ehrlich-positive HRF spots on TLC with the peak areas observed by GLC of the TMS-ethers of HRF-1 and HRF-2 is shown in Table 2. HRF-1 and HRF-2 may therefore be assigned to peaks 2 and 4, and peaks 1, 3 and 5 respectively. By contrast GLC of the TMS-ethers of l-0-(indole-3-acetyl)-B-D-glucopy- ranose gave one peak with a retention time distinct from that of peaks 1 to 5. This, together with the TLC data, demonstrates that HRF-l and HRF-2 are esters of IAA and glucose with the ester linkage on carbons other than carbon 1. Mass spectrometry. The mass spectra of the five peaks of the TMS-derivatives of HRF-1 and HRF-2 are presented in Figure 2 (Spectra I to III) and Figure 3 (Spectra IV and V). The mass spectrum of the 76 TMS-derivative of 1-0-(indole-3-acetyl)-B-D—glucopyranose is shown in Figure 3; Spectrum VI. Since the ester linkage is known for Spect- rum VI, its interpretation might permit the location of the ester linkage for the compounds of Spectra I to V. Mass spectrum of the TMS-derivative of.1-0-(indoZe-3-acetyZ)- B-D-glucopyranose (Spectrum VI). A peak for the molecular ion (M1) is found at m/e 697. The frequently observed "M-15" ion in TMS-deriva- 25 is absent. MT fragments by the elimination tives of carbohydrates of one molecule of trimethylsilanol (TMSOH) to m/e 607 a very low intensity peak, followed by the loss of a methyl radical (4CH3) to give the ion at m/e 592. The mass spectrum of the perdeuterotrimethylsilyl (TMS-d9)-ether of l-0-(indole-3-acetyl)-D-glucopyranose (unpublished results) showed that the hydrogen atom of the hydroxyl group of the 25. The ion m/e 592 TMSOH comes exclusively from the carbon skeleton further fragments to m/e 502 by loss of another TMSOH. An ion is found at m/e 653 which must arise from the mole- cular ion by the loss of 44 amu. This ion is not present in the other spectra of the TMS-derivatives of HRF-1 and HRF-2, and is thus char- acteristic of the ester linkage in l-0-(indole-3-acetyl)-B-D-glucopy- ranose. It probably involves the expulsion of C02 from the odd- electron molecular ion. This transition is supported by a metastable peak found at m/e 611.5. 77 1412cosnvio3 “"1“: | anIN53 The presence of m/e 450 rather than m/e 451 as in tetra-0- TMS--hexopyranoside525’32 suggests that the N-TMS-indole-3-acetyl group is lost as N-TMS-indole-3-acetic acid from the molecular ion. This requires the transfer of a hydrogen atom to the leaving group prior to the cleavage of the ester linkage. This hydrogen could come from either a methyl group of one of the TMS- groups on the glucose ring or from the carbon skeleton of the ring. Charge retention on the leaving group gives rise to m/e 247, and charge retention on the glucose ring results in the ion m/e 450. The spectrum of the TMS-dg-derivative of l-0-(indole-3-acety1)-B-D-glucopyranose (unpublished results) showed however that the hydrogen atom comes from the carbon skeleton. o-+ CH2 .3. 444 .. UC 78 The ion m/e 450 fragments further to m/e 435 and m/e 345 by the successive loss of -CH3 and TMSOH. A second fragmentation pathway leads from m/e 450 to m/e 361, an ion of high relative intensity in Spectrum VI, through the loss of a trimethylsilyloxy (TMSO-) radical. A metastable peak at m/e 289.5 supports this transition from m/e 450 to m/e 361. The ion m/e 361 loses another TMSOH to give rise to m/e 271. Inhofiuil .nmgjz71 The ion m/e 361, common for TMS-derivatives of hexopyrano- 25,32 sides has been shown to consist of all six carbons of the hex- osezs. The high relative intensity of this ion could, in part, be due to a fragment which consists of the ester-linked substituent on carbon 1 and carbon 2. However, the shift of m/e 361 in the spectrum of the TM-d9S-analogue to m/e 388 shows that the ion m/e 361 retains three intact TMS-groups, and does not retain the indole-3-acety1- group. As a consequence the low intensity ion m/e 204 is not due to the shift to m/e 361 by the A amu of the substituent on carbon 1. 0n the other hand, earlier findings have shown that substitution on 79 carbons 1 or 6 of the hexopyranose ring has little effect on the relative intensities of m/e 204 and m/e 21725’33'35'35. The mass spectra of TMS-ethers of pyranoses25 are character- ized by a high relative intensity ion m/e 204, whereas the furanoses show a high intensity ion at m/e 217. The ratio of m/e 204/217 is therefore characteristic of the ring size25. The presence of a low intensity ion at m/e 204 would therefore imply that the hexose is in the furanose form. However, Keglevic and Pokorny8 who have synthesized this compound, have shown that the glucose is in the pyranose form. The presence of the indole-3-acety1- group on carbon 1 (and also on other carbons of the ring) must suppress the formation of m/e 204 in favor of m/e 217. The ion m/e 316 is of uncertain origin. A possible fragment- ation pathway leading to its formation could involve the loss of the indole-3-methylene radical from the molecular ion, followed by the elimination of one molecule of TMSOH and one TMSO- radical. The corres- ponding ions in the spectrum of the TMS-dg-analogue support this fragmentation. Similarly, the ion m/e 331 may be due to the loss of a (CH3)ZSi0' radical in the last step of this fragmentation pathway. The remaining ions 243, 191, 147, 117, 103 and 73 are common fragments from TMS-derivatives of hexoside525’32. The strong peaks at m/e 229 and m/e 202 are characteristic of N-TMS-indole-3-acetyl esters. 80 (“*2 \\\_ 21“ or @074 i. 1' '1 1‘ Sfihfleb SHAA.3 Sfihnoa ImM.2K12 nudeZSD In comparing the mass spectra of the TMS-derivatives of HRF-1 and HRF-2 with that of the TMS-derivatives of 1-0-(indole-3-acety1)-B~ D-glucopyranose it is apparent that the glucose of HRF-l and HRF-2 is not substituted at carbon 1. Again all the spectra show a strong molecular ion at m/e 697, the molecular weight of (N-TMS-indole-3- acetyl)-0-tetra-0-TMS-D-glucose. This confirms the stoichiometry of IAA/glucose of HRF-l and HRF-2. The spectra of peak 2 and 4 (Spectra II and IV) are nearly identical with only slight differences in the relative intensities of the ion fragments. The same is true for the spectra of peaks 1, 3 and 5 (Spectra I, III and V). mass Spectra II and IV (HRF-1: peaks 2 and 4). Both spectra show a strong molecular ion. The loss of one molecule of TMSOH from M1 is more pronounced in these spectra than in Spectrum VI. This transition is supported by a metastable peak found at m/e 528.5 (cal- culated: 528.6). A very small peak is found at m/e 625 in Spectrum IV. It is formed from the molecular ion by the loss of 72 amu. Elimina- tion of 72 amu from the molecular ion is also observed in the mass 81 spectra of (N-TMS-indole-3-acetyl)-0-penta-0-TMS-myo-inositol deriva- tives (unpublished results). It involves the transfer of a hydrogen atom from one of the methyl groups of the N-TMS group to the indole ring with subsequent loss of the neutral 4CH2(CH3)ZSi radical. "t i? R--O-C--CZl-l2 I l 7|- 1? N R-O-C-Cl-l _. M. H-C—Si—M. N/ ls'-CH2 J. 1 AA. AA“ Rsll1iuoqpymanoo. 4. M', ml. 697 ml. 625 Two low intensity ions at m/e 489 and m/e 474 are not pre- sent in Spectrum VI. The ion m/e 489 is formed by the loss of one molecule of TMSOH from M1, followed by the elimination of carbon 1 and the ring oxygen (118 amu). The ion m/e 489 then loses a -CH radical 3 which gives rise to m/e 474. No ion is found at m/e 450, but m/e 435 is present, which loses one molecule of TMSOH to give m/e 345. The ion m/e 333 is not found in Spectrum VI. It is probably formed from the molecular ion by the loss of carbon 1 and the ring oxygen (118 amu), followed by the loss of the N-indole-3-acetoxy radical. The ion m/e 333 can then lose a TMSOH to give rise to the fragment m/e 243. 82 SflN~e3 (DR M', ml. 697 MIG 579A—0 fill. 333 It is of interest to note that the ion m/e 217 is more intense than m/e 204, which should be characteristic of a hexo-furanose 25. 25 and consists mainly ring The ion m/e 204 is a two-carbon fragment of carbons 2 and 3, and carbons 3 and 4. Since the IAA in Spectra II and IV is not esterified on carbon 1, the low intensity of m/e 204 must be the result of the substitution as mentioned above. The presence of the very intense ion m/e 129 in both spectra suggests that it is a doublet. Selective Deuterium labeling of D- 25 that this fragment is a doublet, 75% of which re- glucose has shown tains carbon 1 and four hydrogens of the glucose molecule. In addition this ion could also retain a carbon other than carbon 6. Substitution of carbons 2, 3 and 4 may, therefore, result in the preferred formation of m/e 129. AA’rnfioffl97' R1¢urRgcurflhphmdbioéranotyl 83 Mass Spectra I, III and V (HRF-2; peaks Z, 3 and 5). These spectra are characterized by a high intensity ion m/e 204, and corres— pondingly less intense peaks at m/e 217 and m/e 361. This is strong evidence that HRF-2 is not substituted at carbons l, 2, 3 and 4. Since the m/e 204/217 peak ratio agrees with the pyranose form, the only other carbon available for substitution is carbon 6. An ion supporting this conclusion is found at m/e 375, a fragment which is not found in the spectra of HRF-1 or Spectrum VI. This ion may result from the molecular ion by a cleavage of the pyranose ring between carbon 5 and the ring oxygen, and carbons 3 and 4 with charge retention on the leaving group. Unfortunately, no other characteristic fragments are present in these spectra that could support the localization of the substitu- tion on carbon 6, and further evidence is needed to confirm the substi- tution on carbon 6. A number of published mass spectra of TMS-deriva- 33’34’35 were studied. The tives of carbon 6 substituted glucopyranoses ion corresponding to m/e 375 was present, but this by itself did not allow a definite localization of the substitution to carbon 6. The 84 mass spectrum of the TMS-derivative of D-galacturonic acid (Figure 8, Spectrum XVII) shows an ion at m/e 233 which corresponds to the frag- ment m/e 375 in the Spectra I, III and V. The presence of the strong ion at m/e 292 which is a characteristic rearrangement ion in TMS- derivatives of hydroxyldicarboxylic acids36’37 may be diagnostic for galactaronic acid. It may, therefore, be said that the assignment of the ester linkage to carbons 2, 3 or 4 for HRF-1, and carbon 6 for HRF-2 based on the mass spectral data is tentative at best. 0n the other hand, the interpretation of the spectra has conclusively shown that HRF-1 and HRF-2 have a free carbonyl function and can, therefore, be converted to their M0-TMS derivatives. Gas-liquid chromatography-mass spectrometry of’the MD-TMS- derivatives Of'HFF-I and HRF—2. Laine and Sweeley38’39 have demonstra- ted the usefulness of MO-TMS derivatives of sugars in locating substit- uents on the carbon skeleton. Preliminary experiments with 1-0-(indole- 3-acety1)-B-D-glucopyranose which cannot react with methoxylamine-HCl showed that the ester linkage is unaffected during the reaction. Sub- sequent silylation with BSTFA yielded l~0-(N-TMS~indole-3-acetyl)-2, 3,4,6-tetra-0-TMS-B-D-glucopyranose quantitatively. The MO-TMS-derivatives of HRF-1 and HRF-2 gave only three peaks on GLC with complete resolution of the individual peaks. The relative retention times for these peaks are listed in Table l. A very small shoulder (less than 20% contribution to the total peak area) accompanied each peak at the downslope. Laine and Sweeley39 have shown that these -syn- and -anti- forms of the o-methyloxime function had similar mass spectra. This was confirmed by the comparison of the two 85 mass spectra of the M0-TMS-derivatives of D-galacturonic acid, which showed two completely resolved peaks on GLC: a major peak (Spectrum XVIII) followed by a minor peak (Spectrum XVII). No attempt was there- fore made to separate these two forms. Since the O-methyloxime function labels the carbonyl carbon the interpretation of the acyclic MO-TMS- derivatives of HRF-l and HRF-2 should allow the positive determination of the location of the ester linkage. It may be assumed that HRF-l and HRF-2 both have the pyranose ring, thus the four following acyclic structures are possible with IAA esterified either at carbon 2, 3, 4 and 6: HC=N0CH3 HC=N0CH3 HC=N0CH3 HC-NOCH3 H 0-IAA HCO-Si(CH ) HCO-Si(CH ) H 0-Si on u 317 l __ u=150 _ ! __----§-§ _____ H 150 g 3 3 w 150 l ( §Z§__ l o 409 I o 555 l o 555 1 o 555 (CH3)3SiO H AAI-OCH (CH3)3Si-OCH (CH3)3Si-0 H 1 x 419 I x 419_ 1 _ x 252 1 _x_g§g_ c 307 H10 Si(CH ) c 307 HIo 51(CH ) c 454 l c 454 . - - H 0-IAA HCO-Si(CH ) v 521 3 3__ v 521 1 3 3 _____ v_§g1 I _____ v 354. ____§_§___ B 205 ”‘0 s1(cn ) 5 205 HIo $1( ) 5 205 l ( ) 5 352 : - - CH H 0-Si cu HCO-Si(CH ) z=gg§ _ _ 3 3 z 523 1 §_§ _____ z 523 I 3 3 z 455 __-§_§___ A 103 ’ A 103 I A 103 A A 250 2HC0-Si(CH3)3 2HC0-Si(CH3)3 2H 0-Si(CH3)3 ZHCO-IAA M Themass spectra ofkthe three—peaks of the MO-TMslderivativegr of HRF-1 and HRF-2 are shown in Figure 4 (Spectra VII and IX). All the spectra show an ion at m/e 726, which corresponds to the molecular ion. This allows the immediate conclusion that HRF-l and HRF-2 comprise three isomers of esters of IAA and D-glucose. Initial fragmentation results in the loss of -CH to give the ion at m/e 711. The ion at 3 m/e 694 differs from M? by 32 amu, and may be explained by the 86 following McLafferty rearrangement of the odd-electron molecular ion, which subsequently leads to the loss of the neutral HOCH3 molecule. c H3 \0 /N+ c/ lief/jgiff' -———9 u can! chycpl c ,c\ 3 (1: O R Measio/ \n 4441.594 + Akgndblifli The ions m/e 202 (L) and m/e 229 (M) have been described in connection with the mass spectra of the TMS-derivatives of HRF-1 and HRF-2, and are due to cleavage of the N-TMS-indole-B-methylene and N- TMS-indole-3-acetyl group from the carbon skeleton with charge reten- tion on the leaving group. In addition, a number of ions, m/e 73, 117, 129, 147, 204, 127, 247, 291, 305 and 319, are common ions of TMS- ethers of carbohydrates25’39. Mass spectrum of'peak 1 (Spectrum VIII). The presence of ion w at m/e 160 arises by a homolytic cleavage between carbons 2 and 3 with charge retention on w, and implies that carbon 2 is not substi- tuted35. This is confirmed by the presence of the corresponding ion 0 at m/e 566 and its homologues D’ (D-TMSOH) and D” (D-2TMSOH). Peaks found at m/e 103 (A) and m/e 205 (B) identify the substitution on carbon 4. This is confirmed by the presence of ion C, m/e 464 and its homologue C’ at m/e 374, which arises by a homolytic cleavage be- tween carbons 3 and 4. The ion Y at m/e 521 supports this. A small 87 ion is found at m/e 274, derived from ion Y by loss of N-TMS-indole- 3-acetic acid. Similarly, the ion m/e 358 could be derived from the ion Z; m/e 623 (not present) by the loss of the same group. Two ions, m/e 434 and its homologue m/e 344, could arise from the successive loss of two -CH3 radicals from the ion C. Indeed, a small intensity ion at m/e 449 supports this fragmentation pathway. Mass spectrum of’peak 2 (Spectrum VII). The spectrum is characterized by the absence of m/e 160, but has the ions m/e 205 (A), m/e 205 (B) and m/e 307 (C), providing evidence that the substitution is on carbon 2. The ions m/e 419 (X), m/e 521 (Y) and m/e 623 (Z) confirm this conclusion. The ion pair w and D is missing, suggesting that the homolytic cleavage between carbon 2 and 3 is suppressed in favor of the cleavage between carbon 2 and the N-TMS-indole-B-acetyl group. This results in the rather intense ion N at m/e 246. The corresponding ion at m/e 480 with charge retention on the carbon skele- ton confirms this fragmentation. The ion m/e 173 is probably derived from ion X by losing the N-TMS-indole-3-acetyl group. 88 Mass spectrum of'peak 3 (Spectrum IX). The presence of the ion series w (m/e 160), X (m/e 262) and the corresponding ions 0 (m/e 476), C (m/e 464) and B (362) are conclusive evidence that the sub- stitution is on carbon 6. By analogy to D (m/e 409) in Spectrum VII, the ion A (m/e 260) is suppressed in favor of the preferred cleavage between carbons 4 and 5. To support this reasoning the M0-TMS-deriva— tive of the model compound D-galacturonic acid was studied. Its spect- rum is shown in Figure 8 (Spectrum XVI). As expected, the substituted carbon 6 completely suppresses the homolytic cleavage between carbons 5 and 6 in favor of the cleavage between carbons 4 and 5, resulting in the ions B at m/e 248 and Y at m/e 364. The mass spectra of the MO-TMS-derivatives of HRF-l and HRF-2 were obtained by silylating the MO-derivatives with N,0-bis-(trimethy- silyl)-trifluoroacetamide (BSTFA), resulting in the quantitative sub- stitution of the imino-group of the indole nucleus by a TMS-group. It was known from other experiments that N-trimethylsilyl-imidazole (TSIM) at 25° will not react with primary or secondary amines, and it should, therefore, be possible to obtain the MO-TMS-derivatives of HRF-l and HRF-2 retaining a free imino-group. All indole-3-acetyl containing ion fragments would thus be identified in the mass spectra by a down-shift of 72 amu, thereby providing additional evidence as to the location of the ester linkage. First the model compound 2-amino-2-deoxy-D-glucose was converted to the MO-derivative and then trimethylsilylated with TSIM, and a duplicate sample derivatized with BSTFA. It was found that 40 the BSTFA-derivative had a longer retention time on GLC To illus- trate the usefulness of the selective labeling of the amino-group, the 89 mass spectra of both MO-TMS-derivatives of D-glucosamine are shown in Figure 5 (Spectrum X and XI). The ions m/e 87 (N), m/e 189 (X) and the corresponding ions m/e 159 (N) and m/e 261 (X) differ by 72 amu, which locates the amino-group positively on carbon 2. Silylation of the MO-derivatives of HRF-l and HRF-2 with TSIM on the other hand resulted in three doublet peaks on GLC, the second peak of each doublet corresponding to the BSTFA-analogues. This indi- cated that about one half of the derivatives had a substituted imino- group. Utilizing the mass spectrometer-computer data acquisition system from repetitive scans41 the mass chromatograms shown in Figure 6 were produced. The bottom panels show the total ion intensity versus the mass spectrum scan number of a mixture of imino-substituted and imine-non-substituted MO-TMS-derivatives of HRF-l and HRF-2 (solid line). The total ion intensity versus mass spectrum scan number of the three corresponding peaks of the imino-substituted derivatives is shown by the broken line. The panels above show the mass chromatograms for the intensities of the ion pairs m/e 130 and m/e 202, m/e 157 and m/e 229, m/e 290 and m/e 362, m/e 494 and m/e 566, and m/e 347 and m/e 419 versus mass spectrum scan number. These mass chromatograms clearly demonstrate that the first peak of each doublet has the free imino- group. As can be seen from the mass chromatograms of the total ion intensities, there is poor resolution of the doublet peaks. However, by computer subtraction of the second peak in each doublet from the first peak, the three spectra of the MO-TMS-derivatives of HRF-l and HRF-2 with a non-substituted imino-group were obtained. The spectra 90 are shown in Figure 7 (Spectra XIII, XIV and XV). To facilitate the comparison of the two sets of ion series they are listed in Table 3 along with their relative intensities. The table clearly shows that the selective labeling of the indole-3-acetyl group confirms the con- clusions reached earlier. The location of the ester linkage on carbons 2, 4 and 6 for peaks 2, l and 3 has therefore been conclusively estab- lished. The structures of these three isomers of indole-3-acetyl-0-D- glucopyranose are shown in Figure 9. One remaining uncertainty was the fact that only two isomers are found by TLC, whereas GLC and combined GLC-MS analysis of the TMS- and MO-TMS-derivatives of HRF-l and HRF-2 definitively shows the exist- ence of three isomers. By preparing these derivatives from HRF-l and HRF-2 separately, it could be shown that HRF-l (one spot on TLC) con- sists of 2-0-(indole-3-acetyl)-D-glucopyranose (37%) and 4-0-(indole- 3-acetyl)-D-glucopyranose (63%). HRF-2 on the other hand consists of only 6-0-(indole-3-acetyl)-D-glucopyranose (Table 2). The two GLC peaks (peaks 2 and 4) of the TMS-ethers of HRF-1 presumably represent the B anomers of 2-0-(indole-3-acetyl)-D-glucopyranose and 4-0-(indole- 3-acetyl)-D-glucopyranose which could not be separated on any liquid phase studied. The TMS-ether of HRF-2 has three GLC peaks (peaks 1, 3 and 5), two of which may be attributed to the a and B anomers of 6- 0-(indole-3-acetyl)-D-glucopyranose. The TMS-ether of D-galacturonic acid also has three GLC peaks (see Figure 8; Spectrum XVIII: insert). Thus, the third GLC peak of the TMS-ether of HRF-2 probably represents a distinct anomeric form due to the substitution on carbon 6. 91 EXPERIMENTAL Extraction and purification. The extraction of 10 kg of ground sweet corn kernels of Zea mays L. (cultivar, Stowells Evergreen hybrid) was carried out as described before13. The 1-butanol phase was taken to dryness under reduced pressure at 45°. Water (40 ml) was added to the residue, and the water-insoluble fraction removed by fil- tration. The filtrate was lyophilized and redissolved in 8.0 m1 of ethanol-water (1:1). The sample was chromatographed on "low capacity"13 styrene-divinylbenzene copolymer resin (column I.D.=9.0 mm, bed volume= 38.2 ml, void volume- 13.5 ml) with ethanol-water (1:1) as eluent, collecting 2.0 ml fractions. Aliquote (10-50 ul) of the fractions were monitored on TLC13 (Figure l-A), and the Ehrlich-positive regions of HRF-1 and HRF-2 pooled (tubes 15-28). The pooled sample was dried and redissolved in 4.0 ml of ethanol-water (1:1) and rechromatographed on Sephadex LM-20 (column I.D.=9.0 mm, bed volume=38.5 ml, void volume= 8.5 ml) with ethanol-water (1:1) as eluent. The 2.0 m1 fractions were again monitored by TLC (Figure l-B). The fractions containing HRF-l and HRF-2 (tubes 20424) were pooled, dried and redissolved in 0.2 ml of ethanol-water (1:1). HRF-l and HRF-2 were separated by preparative 12’13 into sample #1 (HRF-1+2), TLC on precoated silica gel plates sample #2 (HRF-2) and sample #3 (HRF-l). Small aliquots were re- chromatographed on TLC (Figure l-C), and it was apparent that acyl migration had occurred during the elution of the compounds from the silica gel with ethanol-water (l:l). No attempt was made to further purify HRF-l and HRF-2. 92 Ammonolysis. Small samples of #2 and #3 (containing 150 pg of IAA) were hydrolized in 14% NH40H for fifteen minutes at 65° in sealed ignition tubes. The reaction mixture was then dried repeatedly from ethanol-water (1:1), and the residue dissolved in 100 ul of ethanol-water (1:1). The reaction products were then analyzed by TLC, GLC and combined GLC-MS. Authentic l-O-(indole-3-acety1)-B-D-glucopy— ranose served as internal standard for the stoichiometry determinations of HRF-l and HRF-2, which was done by quantitative GLC as described before‘s. Free IAA and the a and B anomers of D-glucose were also used as internal standards. The amount of HRF-l and HRF-2 was determined by measuring quantitatively the IAA and IAAM liberated during ammonolysis by GLC. The amount of HRF-l and HRF-2 isolated from 10 kg of corn kernels was 420 pg and 480 pg respectively. Preparation of'O-methyloximes. The conversion of HRF-1 and HRF-2 to their corresponding O-methyloximes was done according to Laine and Sweeley36’37 with slight modifications. Samples containing 50 pg of IAA were dried in 1.0 ml screw top, Teflon-lined vials, and 50 pl of dry pyridine containing 100 pg of methoxylamine-HCl (Supelco, Inc. Bellefonte, Penn. 16823, USA) added, the mixture was allowed to react at 80° for two hours. N,0-bis-trimethylsilyl-trifluoroacetamide (BSTFA) (100 pl) or 100 pl of trimethylsilylimidazole (TSIM) (Regis Chemical Co., Chicago, 111. 60610, USA) was added to the reaction mix- ture and heated for an additional fifteen minutes at 80°. To facili- tate sample handling for repeated GLC and GLC-MS the Teflon liner was TM replaced by a Flurorcone septum (Pierce Chemical Co., P.0. Box 117, Rockford, Ill. 61105, USA) which allowed the direct and repeated 93 withdrawal of reaction mixture aliquots. Samples stored in this way were stable for at least one month at 4°. The O-methyloximes of D- glucosamine and D-galacturonic acid were prepared as described above. Gas-liquid chromatography. The Si1y1ation of the samples 13. The deriv- with BSTFA or TSIM was done exactly as described before atized samples were analyzed on a F+M model 402 gas chromatograph equipped with flame ionization detectors with nitrogen as carrier gas at a flow rate of 60 ml/min. Two columns were used, a 1.8 m X 3.0 mm 1.0. U-shaped glass column packed with 0V-17, 3% on Gas-Chrom Q (100/ 120 mesh), and a 1.2 m X 3.0 mm 1.0. U-shaped column packed with OV-l, 2% on Gas-Chrom Z (100/120 mesh), (Applied Science Lab Inc., State College, Penn. 16801, USA). Mass spectrometry. Combined GLC-MS was performed on a LKB- 9000 mass spectrometer with a 1.8 m X 3.0 mm 1.0. glass column packed with SE-30, 1% on Supelcoport (100/120 mesh), (Supelco Inc., Belle- fonte, Penn. 16823, USA) and Helium as carrier gas with a flow rate of 25 m1/min. The ionizing energy was 70 eV, the flash heater 250°, the molecular separator 250°, and the ion source temperature 290°. The mass spectra and mass chromatograms were recorded with an on-line data acquisition and processing program4]. ACKNOWLEDGMENTS I am grateful to Dr. C. C. Sweeley for his help in the interpretation of the mass spectral data, Dr. R. S. Bandurski for his continued interest and advice, and Dr. D. KegleviC for the generous gift of l-0-(indole-3-acetyl)-B-D-glucopyranose. I would like to 94 thank Mr. Jack Harten for technical assistance in operating the mass spectrometer, and Mr. Norman Young who developed the mass spectrometry computer system for the acquisition of data from repetitive scans and production of mass chromatograms. 95 LEGENDS FOR FIGURES Figure 1. Thin-layer chromatograms of HRF-l and HRF-2 fol- lowing "low capacity" resin, (A), Sephadex LH-20, (B), and preparative thin-layer, (C), chromatography. l-0-(indole-3-acetyl)-B-D-glucopy- ranose (b) was used as a standard (S) along with free IAA (a). Condi- tions for TLC and the visualization of the indole compounds were 13 described before The relative Rf values are based on the Rf value of free IAA. The relative Rfs for HRF—1 and HRF-2 reported by Ueda and Bandurski12 were 0.81 and 0.71, compared to 0.80 and 0.71 reported here. Figure 2. Mass spectra of the first three peaks (Spectra I- III) of the TMS-derivatives of a mixture of HRF—l and HRF-2. The spectra were recorded as described in the EXPERIMENTAL section isotherm- ally at 230°. The inserts show the gas-liquid chromatograms, and the shaded area under each peak represents the contribution of the peak to the individual spectra obtained. Figure 3. Mass spectra of the last two peaks (Spectra IV and V) of the TMS-derivatives of a mixture of HRF-l and HRF-2. Spectrum VI is that of authentic 1-0-(indole-3-acetyl)-B-D-glucopyranose. Condi- tions are as in Figure 2. Figure 4. Mass spectra of the first (Spectrum VIII), second (Spectrum VII), and third (Spectrum IX) peak of the MO-TMS-derivatives of a mixture of HRF-l and HRF-2, silylated with BSTFA. The inserts show the ion series resulting from simple homolytic cleavage of the carbon skeleton. Conditions are the same as in Figure 2. 96 Figure 5. The mass spectra of the MO-TMS-derivatives of D- glucosamine silylated with TSIM (Spectrum X) and BSTFA (Spectrum XI). Spectrum XII is that of the TMS-derivative of D-glucosamine following silylation with BSTFA. Conditions as in Figure 2. Figure 6. Mass chromatograms for the intensities of the ion pairs m/e 130 and m/e 202, m/e 157 and m/e 229, m/e 290 and m/e 362, m/e 494 and m/e 566, and m/e 347 and m/e 419, compared with the total ion intensity of the same scans (bottom panels). The mass chromato- grams were obtained from the repetitive scans of a mixture of MO-TMS- derivatives of HRF-l and HRF-2 having both a free and TMS-substituted imino-group (solid lines), and the same mixture with no free imino- group (broken lines). Conditions are as described in Figure 2 and EXPERIMENTAL. Figure 7. Mass spectra of the first (Spectrum XIII), second (Spectrum XIV) and third (Spectrum XV) peak of the MO-TMS-derivatives of a mixture of HRF-1 and HRF-2 silylated with TSIM. Conditions as in Figure 2. Figure 8. Mass spectra of the MO-TMS-derivative of D-gal- acturonic acid (Spectra XVI and XVII), and the TMS-derivative of the same compound (Spectrum XVII). Conditions as described in Figure 2. Figure 9. The structures of 2-0-(indole-3-acetyl)-D-glucopy- ranose (A), 4-0-(indole-3-acetyl)-D-glucopyranose (B), and 6-0-(indole- 3-acetyl)-D-glucopyranose (C). 97 Table 1. Relative retention times of TMS-derivatives of HRF-1 and HRF-2, and their MO-TMS-derivatives . . . Relative retention time Der1vat1ve Liquid phase OV-l, 2% 0V-17, 3% (1.2 m, 220°) (1.8 m, 227°) HRF-l and HRF-2 Peak 1 0.70 0.68 Peak 2 0.75 0.73 Peak 3 0.98 0.96 Peak 4 1.03 1.01 Peak 5 1.14 1.09 1-0-(indole-3- acetyl)-B-D- b c glucopyranose 1.00 1.00 Methoximes Peak 1 0.62 Peak 2 0.77 Peak 3 0.90 aThe imino-group of the indole nucleus is substituted with a TMS- group. bRetention time 9.65 minutes. cRetention time 12.6 minutes. 98 Table 2. The contribution of HRF-la and HRF-2a to the Ehrlich-positive spots on TLC, the five peaks of the TMS-derivatives of a mixture of HRF-l and HRF-2, and to their MO-TMS-derivatives. Preparative TLC sample number naéfgrgngf R: or R: 3:HRF-l 2:HRF-2 HRF-2 % peak area % peak area TLCb: HRF-l 0.81 20 9o HRF-2 0.71 80 10 GLCC: TMS-deri- vatives Peak 1 0.68 17.3 3.4 Peak 2 0.73 8.6 38.7 Peak 3 0.96 41.2 10.1 Peak 4 1.01 13.7 47.8 Peak 5 1.09 19.2 MO-TMS-de- rivatives Peak 1 0.62 21.3 49.2 Peak 2 0.77 6.4 26.8 Peak 3 0.90 72.3 24.0 aBand eluted from the HRF-1 and HRF-2 regions (see Figure l). b% spot intensity was estimated visually from reference spots of known amounts of IAA. c% peak area was determined as described before13. de values from Figure l. eRt values from Table l. 10. 11. 12. 13. 14. 15. 16. 17. REFERENCES M. H. Zenk, Nature 191 (1961) 493. M. H. Zenk, Colloq. Int. Cent. Nat. Rech. Sci. Paris 123 (1964) 241. H. D. K1ambt, Planta 56 (1961) 618. E. M. Shantz and F. C. Steward, Plant Physiol. Suppl. 32 (1957) viii. F. C. Steward and E. M. Shantz, Ann. Rev. Plant Physiol. 10 (1959) 379. B. I. S. Srivastava, Plant Physiol. 38 (1963) 473. B. I. S. Srivastava, Colloq. Int. Cent. Nat. Rech. Sci. Paris 123 (1964) 179. D. KegleviC and M. Pokorny, Biochem J. 114 (1969) 827. C. Labarca, P. B. Nicholls and R. S. Bandurski, Biochem. Biophys. Res. Commun. 20 (1966) 641. P. B. Nicholls, Planta 72 (1967) 258. R. S. Bandurski, M. Ueda and P. B. Nicholls, Ann. N. Y. Acad. Sci. 165 (1969) 655. M. Ueda and R. S. Bandurski, Plant Physiol. 44 (1969) 1175. A. Ehmann and R. S. Bandurski, J. Chromatog. 72 (1972) 61. P. B. Nicholls, B. L. 0ng and M. E. Tate, Phytochem. 10 (1971) 2297. M. Ueda, A. Ehmann and R. S. Bandurski, Plant Physiol. 46 (1970) 715. A. Ehmann and R. S. Bandurski, Plant Physiol. Suppl. 47 (1971) 3. M. Ueda and R. S. Bandurski, unpublished. 99 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 100 D. Keglevié, Carbohyd. Res. 20 (1971) 293. W'UUDII'H U—i 2 Cat; £63 . Fischer, M. Bergmann and A. Rabe, Ber. 53 (1920) 2362. . Oh1e, Ber. 57 (1924) 403. Helferich and w. Klein, Ann. 450 (1926) 219. Brigl and H. Grfimmer, Ann. 495 (1932) 67. . M. Mann, N. D. Maclay and C. S. Hudson, J. Amer. Chem. Soc. 61 (1939) 2432. . N. Cawley and R. Letters, Carbohyd. Res. 19 (1971) 373. . C. DeJongh, T. Radford, J. D. Hribar, S. Hanessian, M. Bieber, G. Dawson and C. C. Sweeley, J. Amer. Chem. Soc. 91 (1967) 1728. . A. McCloskey, R. N. Stillwell, and A. M. Lawson, Anal. Chem. 40 (1968) 233. . K. Kochetkov, 0. S. Chizhov and N. V. Molodtsov, Tetrahedron 24 (1968) 5587. . Karkkfiinen, Carbohyd. Res. 11 (1969) 247. . Vink, J. J. de Ridder, J. P. Kamerling and J. F. G. Vliegen- thart, Biochem. Biophys. Res. Commun. 42 (1971) 1050. . Dawson and C. C. Sweeley, J. Lipid Res. 12 (1971) 56. . N. Brinkley, R. C. Dou herty, D. Horton and J. D. Nander, Carbohyd. Res. 17 (1971) 127. . A. Karlsson, I. Pascher, B. E. Samuels son and G. 0. Steen, Chem. Phys. Lipids 9 (1972) 230. . M. Kim and C. C. Sweeley, Carbohyd. Res. 5 (1967). . J. Harvey and M. G. Horning, J. Chromatogr. 76 (1973) 51. . Zinbo and w. R. Sherman, J. Amer. Chem. Soc. 92 (1970) 2105. . Petersson, Org. Mass Spectrom. 6 (1972) 565. . Petersson, Org. Mass Spectrom. 6 (1972) 577. . A. Laine and C. C. Sweeley, Anal. Biochem. 43 (1971) 533. . A. Laine and C. C. Sweeley, Carbohyd. Res. (1973) in press. 101 40. J. Kdrkkainen and R. Vihko, Carbohyd. Res. 10 (1969) 113. 41. C. C. Sweeley, in Introduction to Lipid Chemistry, edited by R. M. Burton, Bi-Science International, Webster Groves, Inc. in press. 102 U ullllc_m_..011 m P N a a .5 IIIIII 2.9.". .1 11 1|Il£mtOIll m cu ON or m cm. 111111 29.". ill < Illl£mt01|1 m an «N 9 . mam” er 5 as... % a in. lllll finch“ IIIJ 1133 OH Fa ooh 0mm 00M 0mm con OWN A 41 1-4 .43 g .3. m “w b1 +2 1- an «x oop omm oom omm com omq A No. 1 I4. P. g I +1 3.". .ofl ooh omm oom own oom one a j- .- .. s B: . +2 an”. mx .8. oow 0mm com on.‘ 004 one cow 0mm com on" 00" 0m 13): , 00F 0mm cow 0mm 00m htFirF FDF FPFPF I FPFPF‘F F F F 104 # - h: F: +2 0m: . . 0mm 000 own 00m 0? 00¢ on» 000 0mm com on" 00" on .11): 4 L» L: 1 J - q 1 .r a. u. .5 A a yawn“. s 4 8. In wow I H o .3 so . N +2 Tom r .8 338; o! 00H 105 ................................................................ ‘ 4 J . i d S. as. \afq A «if; .3 o In I. ha ‘ 3.3. n.- o . 3 a: n g . nu ow 83>: Q B. x- a 02. 0mm 00w 0mm com on? 0 0mm 00m 0mm oom om~ oofi owns :1: 7 a. C. 4. awaflajhr1 4 1 3.5.9 + 1! 3. > 5U .3 an '8 K— 3 3. 5 E o b I .u an 3 a. < , .u 7 4 us (4.0 vicill ( 10¢ _ OD é ....... a n J. ... . .8 4. sings w aux . --|- n A 2 _=> , oak-3: a.“ 2 WH L[ cop. omw oom 0mm com. 00¢ .00.» .omw oom 0mm. com 09 9: on HIS: p L. .2. i. ... W. x... an.» a SAEfi J: ’/ +2 ‘N u ”I... 8\ 3 8. .8 In I“. < .130 an .0.» .. in)!“ z 106 Z 9 _ .03 _ L .03 . . _oom _ hm.» o? 08 com omm com 09 08 om .l.\e _ w“. a... ... a. HM:HHH1afi a .8 Ba 70¢ N a. 18 Iom ,i «x as; .. =X l 09 mx can; con om¢ 00¢ 0mm 00m 0mm OON 0m“ OOH om [0): > r 4 PPbbbhb-pbbtbbhhnrbh‘mhbkLth-nbb“hbh>h>h>b>nblh1lb>hpbhh.bpwbhflqn n‘flJHhh‘bi hprblPLthbb flhihfi- 01th“ ¥ R: u E. x TS : g 1 ( . ION .3 3. 3 1 ‘I. a I? w . n N . flow , A H x e o x 8152 c mH OOH 107 II. III .. 7 \158 a. a V /.I../ ..\ ill..- « \..\. ‘ ..//(.\ l-/\-\\ H H ._. /\ omN ,.. \ N NON ¢m¢ wmm Cum mfw LN] 00» 0mm 00w 0mm 00m 0m.» 00¢ 0mm 00m 0mm 00m 09 00" on In): 108 FFFFFFFF 4‘. 0 b b b h \P A!» b L F ¢ b P by b h brdh {b 1 L!’ #L— dbl ‘5, F ‘b :IAQLL 5 FL I D 41"" ha .1» 4b! 5 Lit Kb 5 D E , a N. do. A can x3 4.822 _=H_ ______ + x O 0’ tom 1 z a J. 0.x}: 3 b. .0619 x ”c 3. am: To.» .809... u b Illa"... “ w a Vow subs-.1 an .18... 3 mx .8 1.6L... 30:32 an. R >x d on: 00» 0mm 00w 0mm 00m 09v 00¢ 0mm 00m 0mm OON 0m“ 00" on In): J 4 .b P hrhbb PL h b‘h PDF‘ h DVB b ,h hDPb‘b - bebhbh rr>}\-DD~.-?hdi1qu‘-d>dbb brhbbih 1P bfih bIHVlebkaihbb hbbid‘b F DFF bP‘DDqul‘db ¥ D10 .11“pr H ... A .H. .H. A d a mm. x a. a... I. > b a z 3 13 0 «on .8 .ON +2 2 l.— 10¢ : a .0m .. 8--.. as 13‘ 8. mx a .8 .9451 9. 00a OOP 0mm 00m 0mm 00m 0m¢ 00:. 0mm 00m 0mm 00m 0m" 00— on IE 1 11 4"}.Pbb . b be I b?br}Pfib>4>’P bbbib>th z. .r .H. a A A .5. 4 1 A s h .. +2 03 on» > la 3 Z ‘ 3 B 8. ,om .3 x on n 9. > b ( 10¢. B. U 10m 5% a a EX ,8 s A .. 00H 109 08 03 com 0? 00¢ 0mm oom omw oom om“ 09 cm IS: .H. A arc...» 1th. Hi an: 11 A I... s can [ON A: - 10¢ W 80" a - -8 . 8. low a: 2 a =_>X 2 8H omm oom omm com 09 02 on IS. A :_f__.4..<.+.v.:.JeIi: +.1... I ~ > .na 9 ION . ,3 w - 18 . Tom «is ; =>X 2 3 c 02 08 03 com 0?. 00¢ 03 oo omm oom cm" 08 om IS. _ L p . . — . . — I. — —4—- 4. nd b in h h <1» DJI- _ p p 1 _ p ‘. - — — b - h 1.14-‘1» h n =4. 0 J \g .3. C. a H if I In! 0H5 “\\a ‘> ”N .3 ION ‘ - a. a. ,9» w o 0! fi . 3 om . _>X g as}: mx 2 m: on: 110 / , INJ OH N-(p-coumaroy1)-tryptamine and N-feruioyl-tryptamine in mature sweet corn kerne1s of Zea mays*. Axe] Ehmann Department of Botany and Plant Pathology Michigan State University East Lansing, Michigan 48823 *This work was supported in part by the National Science Foundation (GB-18353X). Journa] Artic1e No. from the Michigan Agri- cu1tura1 Experiment Station. 111 112 KEY WORD INDEX Zea mays; Graminaceae; Sweet corn; N-(p-coumaroyl)-trypta- mine; N-feruloyl-tryptamine; mass spectra. ABSTRACT N-(o-coumaroyl)-tryptamine and N-feruloyl-tryptamine were isolated from aqueous acetone extracts of ground kernels of Zea mays by successive column chromatography on partially sulfonated styrene- divinylbenzene copolymer resin, lipophilic Sephadex and preparative thin-layer chromatography. Identification of these compounds was made by combined gas-liquid chromatography-mass spectrometry of their tri- methylsilyl-derivatives and the trimethylsilyl-derivatives of their acid hydrolysis products. INTRODUCTION Cinnamic acid1 and its hydroxy-, and hydroxymethoxy- derivatives, p-coumaric acid2 and ferulic acid3, have been isolated from pi neapple4, barley seeds and barley embryoss, members of the family Cycadaceaes, and in trace amounts from etiolated oat l. DUMAS, J. and PELIGOT, E. (l834) Justus Liebigs Ann. Chem. 12, 24. 2. HLASIWETZ, H. (l865) Justus Liebigs Ann. Chem. l36, 3l. 3. HLASINETZ, H. and BARTH, L. (1966) Justus Liebigs Ann. Chem. l38, T— —— 4. GORTNER, w. A., KENT, G. and SUTHERLAND, G. K. (1958) Nature 181, 630. >. SUMERE van, C. F., COTTENIE, J., DeGREEF, J. and KINT, J. (1972) Recent Advances in Phytochemistny 4, l65. i. WALLACE, J. w. (l972) Amer. J. Bot. 59, 1. coleoptiles, pea and sunflower seedlings . acids exist in plants as esters of glucose as esters of quinic aci Heupe and spinach leaf chloroplasts and shown it to be p-coumaroyl-meso- tartaric acid20’2]. 113 7 Most of these phenolic 7'13, as B- glucosidess’, ’8 or 'd4’5’14'17, Tadera et al.18, and Oettmeier and 119 have isolated a p-coumaryl derivative from spinach leaves Recently Tanguy and Martin22 have identified feruloyhmwlic acid and cafeoyl-malic acid in the cotyledons of bean 7. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19H 21). 221. £32. OETTMEIER, w. and HUEPEL, A. (1972) Z. Naturforsch. 27b, 586. _Ilfllgflfifi, .J. and MARTIN, C. (1972) C. R. Acad. Sci. TOMASZENSKI, M. (1964) CollocL. Intern. Centre Natl. Rech. Sci. Paris 123, 335. HARBONE, J. B. and CORNER, J. J. (1961) Biochem. J. 81, 242. ======== ======= 1 KOSUGE, T. and CONN, E. E. (1961) J. 3101. Chem. 236, 1617. EL- BASYOUNI, S. Z. , NEISH, A. C. and TOWERS, G. H. N. (1964) Phyto- chem. 3, 627. EL-BASYOUNI, S. Z. and NEISH, A. C. (1965) Phytochem. 5, 683. KOJIMA, M. and URITANI, I. (1972) Plant Cell Physiol. 13, 1075. HARTLEY, R. D. (1973) Phytochem. 12, 661. ,HULME, A. C. and WILKINS, A. H. BRADFIELD, A. F, FLOOD, A. E. 119521 Nature 170,168. LEVY, C. C. and ZUCKER, M. (1960) J. 3101. ROHRBAUGH, L. M. and WENDER, S. H. Chem. 235, 2418. (1965) Phytochem. BRAGT van, J., ‘4, 977. HANSON, K. R. (1966) Phytochem. 5, 491. T_A____DERA, K, _S_____UZUKI, Y., Riki/g, F. and M, H. (1970) Agr. Biol. C__h__em. 34,517. OETTMEIER, w. and H__L1_E_P_E__L_, A. (1972) Z. Naturforsch. 27b Chem. 35, 1431. , 177. m, K. and MITSUDA, H. (1971) Agr. Bio1. Ser. D 274, 3402. have an amide linkage. cosyl)-histamine cinnamoyl-histami ne2 moyl-amino)-4-guani di nobutane) 27 114 Rmnd phenolic acids have been found in plant material which These are casimirvedine (N-cinnamoyl-N-(glu- 23,24 . . . 25 , adenocarpin (N-c1nnamoyl)-tetrahydroanabas1n) , , p-coumaroyl-agmatine (l-(trans-p-hydroxycinna- , and N-feruoylglycyl-L-phenylalanine28. The latter occurred as a peptide in barley globulins. In this paper we wish to report the isolation and identifica- tion from mature sweet corn kernels of two new phenolic acid derivatives in amide linkage with tryptamine; N-(p-coumaroyl)-tryptamine (I) and N-feruloyl-tryptami ne (II) . RESULTS AND DISCUSS ION Compounds (I) and (II) were isolated during the course of a study of esters of indole-3-acetic acid (IAA) and myo-inositol and myo- inositol glycosides29 from kernels of Zea mays and purified by column chromatography on partially sulfonated styrene-divinylbenzene copolymer 23. 24. 25. 26. 27. 28. 29. DJERASSI, C., BANKIENICZ, C., KAPOOR, A. L. and RINIKER, B. (1958) Tetrahedron 2 , 168a . RAMAN, S., REDDY, J., LIPSCOLB, N. N., KAPOOR, A. L. and DJERASSI, C. (1962) Tetrahedron Lett. 357. SCHUTTE, H. R., KELLING, K. L., KNOFEL, D. and MOTHES, K. (1964) Phytochem. 3, 249. W, J. S. (1964) Aust. J. Chem. 17, 375. STOESSL, A. (1965) Phytochem. 4, 973. SUMERE van, C. F., DePOOTER, H., ALI, H. and van BUSSEL, D. (1973) Pfiytochem. 12, 407. UEDA, M., EHMANN, A. and BANDURSKI, R. B. (1970) Plant Physiol. 46, 715. ll—. n 115 min”, Sephadex LH-20 and preparative ti n-layer chromatography (TLC). Both compounds yielded a single spot on TLC and gave bluish-green colors with Ehrlich reagent. The Rf values were greater than the Rf value of IAA in two solvent systems (see Experimental), and the com- pounds did not yield IAA following mild alkaline hydrolysis. This indicated that (I) and (II) were not esters of IAA or related compounds. The trimethylsilyl (TMS) ethers of (I) and (II) were prepared as des- cribed before30 and subjected to gas-liquid chromatography (GLC). Both compounds showed two peaks on GLC, a major first peak and a minor second peak (see GLC-chromatogram inserts in Figure l), which permitted the direct analysis of (I) and (II) as their ethers by combined GLC- mass spectrometry. Mass spectral analysis of compound (I): The mass spectrum of the TMS-derivative of (I) is shown in Figure l-A. A molecular ion (Mt) is found at m/e 522. The correspond- ing molecular ion of the direct probe analysis of the underivatized compound at m/e 306 shows that (I) has three TMS-groups. M? fragments to m/e 507 by the elimination of a methyl radical followed by the loss of a neutral -CH2(CH3)ZSi radical to give rise to m/e 435. The direct elimination of 72 amu from the molecular ion predominates yielding the abundant ion at m/e 450. This elimination of 72 amu from the molecular ion has also been observed in the mass spectra of (N-TMS-indole-3- acetyl)-0-penta-0-TMS-myo-inositol derivatives (unpublished results), 30. EHMANN, A. and BANDURSKI, R. S. (1972) J. Chromatogr. 72, 61. F 116 and involves the transfer of a hydrogen atom from one of the methyl groups of the N-TMS group to the indole ring with subsequent loss of the neutral ~CH2(CH3)ZSi radical. This means that compound (I) must have two nitrogens (nitrogen rule) each of which is substituted with one TMS-group. The third TMS-group is on a hydroxyl oxygen. The loss of 202 amu from M1 which gives rise to m/e 320, and the intense ions at m/e 202 and m/e 215 confirm the presence of N-substituted trypta- mine“. The low intensity ions at m/e T30 and We l43 are the corres- ponding tryptamine fragments without the TMS-group on the imino-group, and support the structure proposed. A model compound 5-methoxy-N- acetyl-tryptamine (melatonin) was examined and found to yield analagous ion fragments at m/e 160, 232 and 245, thus confirming (I) as an N-acyl derivative of tryptamine. The ion at m/e 2l9 arises from M4f by loss of the neutral tryptamine fragment with the structure 5"”: +. \ l l —. i 551le3 OSiMo3 Mun/0522 M219 with charge retention on the acyl group. This ion further fragments” This to give m/e 203, a part of which is the isotope of m/e 202. NARASHIMHACHARI, N., SPAIDE, J. and HELLER, B. (197l) J. Chroma- togr. Sci. 9, 502. 31. 117 This transition is supported by the presence of a small metastable peak at m/e 188.1 (calculated: 188.1). The fragment m/e 219 also gives rise to the ion m/e 191 by the loss of CO, a transition supported by the small metastable peak found at m/e 166.5 (calculated: 166.6). Horman and Viani32 have described the same series of eliminations from m/e 219 for the fragmentation of the TMS-derivatives of m-coumaric and p-coumaric acid, suggesting that the acyl group of compound (I) could be a hydroxy-cinnamic acid. The presence of the ion m/e 249, a char- acteristic rearrangement ion in the mass spectra of the TMS-ethers of hydroxy-, and hydroxy-methoxy-cinnamic acid533, supports this conclu- sion. The ratio of the relative intensities of m/e 219 and m/e 24932 allows a distinction between m-coumaric acid and p-coumaric acid. The ratio found in the mass spectrum of compound (I) for these two ions suggests that the acyl group is a p-coumaryl group. To obtain additional evidence concerning the identity of the acyl group, compound (I) was hydrolyzed with 2.0 M trifluoroacetic acid (TFA) and the hydrolysis products analyzed by GLC and combined Two compounds were found which on GLC co- GLC-MS as the TMS-ethers. Their chromatographed with TMS-tryptamine and TMS-p-coumaric acid. mass spectra were identical with the mass spectra of tryptamine and p-coumaric acid. Compound (I) is thus identified as N-(p-coumaroyl)- tryptami ne. W, I. and LIAM, R. (1971) Org. Mass Spectrom. 5, 203. 32. DALLAS, F. C. and KOEPPL, K. G. (1969) J. Chromatogr. Sci. 7, 565 33. 118 Mass spectral analysis of'compound (II): The mass spectrum of the TMS-derivative of (II) is shown in Figure 1-B. The molecular ion is at m/e 552, and the corresponding molecular ion of direct probe analysis of the underivatized compound is at m/e 336. The presence of the ions m/e 215, 202, 130 and 143 identify the indole as an N-substituted tryptamine. The difference of 30 amu between compounds (I) and (11) indicates the presence of a meth- oxy group on the acyl moiety. This is confirmed by the shift of 30 amu of m/e 320 and m/e 450 of compound (I) to m/e 350 and m/e 480. The presence of a strong intensity ion at 249 with a concommittant low intensity ion at m/e 219, is diagnostic of TMS -derivatives of ferulic- and isoferulic acid32. The acyl group of compound (II) is therefore either a feruloyl- or iso-feruloyl group. GLC analysis of the acid hydrolysis products of compound (II) as their TMS-ethers disclosed two compounds which co-chromatographed with authentic TMS-ferulic acid and tryptamine. Combined GLC-MS yielded mass spectra identical with those obtained from the authentic TMS-acid and tryptamine. Compound (II) is therefore identified as N-feruloyl-tryptamine. To our knowledge this is the first time that N-(p-coumaroyl)- tryptamine and N-feruloyl-tryptamine have been isolated from plant material. 119 EXPERIMENTAL Isolation of compounds (I) and (II): Extraction of 10 kg of ground kernels of Zea mays L. (cult- ivar, Stowell '5 Evergreen hybrid) was carried out as previously des- cribed29’30. The resulting n-butanol phase was dried at reduced pressure at 45°, the residue taken up in 40 ml of water, and the water- insoluble fraction removed by filtration. The water-insoluble fraction was dissolved in 10 ml of 50% ethanol and chromatographed on a 20% sulfonated30 styrene divinylbenzene co-polymer resin (column 1.0. = 9 0 - 13.5 ml) with 50% ethanol as m, bed volume = 38.2 ml, void volume - eluent, and collecting 2.0 ml fractions. Compounds (I) and (II) emerged between 0.6 and 2.9 bed volumes. Small aliquots of each fraction (10-50 pl) were chromatographed on thin-layer plates (TLC)30 (Merck, Darmstadt) and the fractions containing the Ehrlich-positive compounds (I) and (II) were pooled. The pooled fractions were dried, redissolved in 4.0 m1 of 50% ethanol and chromatographed on Sephadex - 38.5 ml, void volume = 8.5 ml) LH-20 (column 1.0. = 9.0 m, bed volume - using 50% ethanol as eluent. Fractions of 1.5 ml were collected and Compound (I) eluted between 2.7 - 3.5 bed volumes, monitored on TLC. Both compounds were and compound (II) between 3.5 - 4.3 bed volumes. further purified by preparative TLC using two solvent systems: a) methylethylketone-ethylacetate-ethanol-water (3:5:lzl) and b) chlor- oform-methanol-water (85:14:1). In both solvent systems the relative Rf values (Rf of IAA =1.00) of compounds (I) and (II) were in a) 1.02 and 1.02, and in b) 1.95 and 1.80 respectively. The total amount of 120 compound (I) obtained was 1400 ug/10 kg (140 ug/kg) and 400 ug/10 kg (40 pg/kg) of compound (II) as determined colorimetrically with Ehrlich reagent and quantitative GLC of the TMS-derivatives. Acid hydrolysis of compounds (I) and (II): Small samples (20-50 pg) of both compounds were hydrolyzed in 2.0 M trifluoroacetic acid (TFA) in sealed ampules at 50° for two hours. This was found to give about 50% hydrolysis with a minimum loss of the phenolic acid fractionss. TFA was used since it is volatile and could be removed to pennit derivatization of the hydrolysis pro- ducts for GLC and GLC-MS. Gas-liquid—chromatography-mass spectrometry: The TMS-derivatives of compounds (I) and (II), their hydrol- ysis products and the standards melatonin, tryptamine, p-coumaric acid (133’34 were prepared as described before30. The TMS- and ferulic aci ethers were chromatographed on a F and M model 402 gas chromatograph equipped with flame ionization detectors, and using nitrogen as carrier gas at a flow rate of 60 m1/min on a 1.2 m X 3.0 mm 1.0. glass column packed with 0V-1, 2% on Gas chrom Z (100/120 mesh) (Applied Science Lab., Inc., State College, Penn. 16801, USA). Combined GLC-MS was performed on a LKB 9000 mass spectrometer with a 1.2 m X 3.0 mm 1.0. glass column packed with 0V-1, 2% on Gas chrom Z (100/120 mesh) and helium as a carrier gas at a flow rate of 25 ml/min. The ionizing 34. STEELE, J. w. and BOLAN, M. (1972) J. Chromatogr. 71, 427. 121 energy was 70 eV, the flash heater 250°, the molecular separator 250° and the ion source temperature 290°. The mass spectra were recorded 35. Mass using an on-1ine data acquisition and processing program spectra of the underivatized compounds (I) and (II) were also recorded using a solid probe in-let system. 35. SNEELEY, C. C. (1973) Introduction to Lipid Chemistry (BURTON, R. M. ed.), Webster Groves, Mo., in press. 122 LEGENDS FOR FIGURES Figure 1. The mass spectra of the TMS-derivatives of com- pound (I) (Spectrum A) and compound (11) (Spectrum B). The inserts show the GLC-profiles, and the shaded area under peak 1 represents the con- tribution of the peak to the individual spectra obtained. The second peak in the GLC-profiles corresponds to the TMS-derivatives of compounds (I) and (II) which have a free amide nitrogen. Structures: N-(p-coumaroyl)-tryptamine (I) N-feruloyl-tryptamine (II) 123 “WIND CH30 o “M \ H“ L I / N H 124 0mm oom omq 00¢ 0mm oom 0mm oom omfi OOH 0m 4— JR» 4 W3 («.5 (on FM our mm. \u/(mfll am we «an E I . o3 . a. . a... w a: .om I can 1 ocu «ca 10¢ w .... . B .5 ,._ -om . a! L I .3 fl. anon}: m x as om a om n 09 0mm oom om¢ 00¢ 0mm oom 0mm oom omfi 00H om I u. 4 a. ._.. .w. .4. - a}. sfiaw/ei i 4.3.. 4 «no 0: Op - o - m 0’32.“ 08 [ON 4 +2 Or“ I «8 10¢ w .3. a. can , BUY? 8 ,8 1 tom coca .._ among: ax 2 ON 09 CONCLUSIONS With the identification of the three isomers of IAA-glucose, di-0-(indole-3-acetyl)-myo-inositol, tri-O-(indole-3-acetyl)-myo- inositol and the two tryptamine derivatives N-(p-coumaroyl)-tryptamine and N-feruloyl-tryptamine the characterization of all the water-soluble, Ehrlich-reactive compounds of the kernels of Zea mays has been accomp- lished. This is the first time that the chemical nature of a group of indolylic compounds from a plant material has been elucidated. The amounts of these extractable indole compounds from mature kernels of sweet corn are listed in Table l. The concentrations of the esters of IAA and myo-inositol and myo-inositol-glycosides are high and 5 to 1.3 x 10'4 M. It is interesting to note that range from 1.8 x 10' the content of these esters varies with the harvest. Ueda and Bandurski (1969) have determined the content of these esters (the B-group) in seeds harvested in 1963 (five-year-old seeds) and 1967 (one-year-old seeds), and found that the 1963 seeds had a significantly higher ester content. The same was observed for free IAA. The free IAA content in the 1963 seeds was 6 x 10'4 6 M, whereas it was only 2.8 x 10' M in seeds from the 1967 harvest. It could be that during the aging of the seeds the free IAA content increases, as has been suggested by Ueda and Band- urski (1969). Ehmann and Bandurski (1972) have re-extracted the total free IAA from seeds of the 1967 harvest (the seeds were then four years 6 old) and found a value of 7.4 x 10' M, which is in close agreement with 125 126 muoos azpu mpwcpwcopwum Acumpv .Ne om measmp muopx~.m me o.m "memeorwxom sommmcsm umumpoccwuxxogpmsue mpoos capo mpwsuwcoumum Ao~m_v .Ns as sesame m-o_xs.m ms o.o_ "assesessss sssssssm -m-m_oecw-sxo;sas-_ mpoog a:_u Acumpv .Ns um menace ¢-o_xm._ me o.o~ "mememewmom movemesm mpwspwcopmumnmumpoucH msmum cameo AummFV mm>sza ucm mpzmm o-o_xu._ «me m.o "mxawuum mwexosm _ocmcpmumumFoncH mpoos nape mpmpmum Aosm_v .Ns cs sesame o-opxm.m as o.P "assessesss assasssm -m-mpoucw-usepmz mu_:s$ Apmmpv .No no wcmomH muopxn.m «as m.m mcsoz "seams: mssuwb Amom_v Peseep m-opxm.w me m.s mummm message? Ammmpv wxmczucmm ucm mum: wuopxo.m me m.o_ vpo meow» m Awumpv wxmsancmm cam :cmscu o-o_x¢.n as m.~ upo memo» N Ammva mesaucmm ucm mum: mIOFxm.~ as m.o upo Low» F AmmmFV reams» ouo_xc.e me n.o mumwm menace names com neon uwpmumumumpoucH cowumcpcwucou a: xgu ax mucmgwemm sm—oz \ucmucou mugzom “cups uczoasou .FMVmemE pcwpn seem wmumpomw muczoaeou wpoucw czocx mo mcowumgpcmucoo mzocwmovcm on» ._ anmp 127 Ammmpv messucmm can mum: muopxm.m as N.@ upo meow» m Ammmpv wxmszvcmm use new: m-o_xm._ ms F.m vpo Lam» F Fopwmocwuome-apzpmwm mummm sesame names 3mm -m-m—oucwv-oufievperrp Amompv wxmeaucmm use mum: e-o_xm.— ms m.mN upo meow» m Ammmpv megancmm new mum: muopxo.¢ as o.~ upo sum» _ _ouwmocwuoms mummm menace names com -A_xumumumnmpoucpv-elm msmum Pomsmm muxnmupm Apnmfiv .Ns so xzougzom m-oFx¢.m ms o.m newmussgmu essemwswm -xongmuumnmponcH mpcmpa mpocz :wuwm Aomm—V ozoum can wavy—m N-o_xm.p m o.n "ssxnoswu seesaw -mmenou=_muo;aF:m-F Amway mm>mmp mcaoa ._mmpv cmcmpsw> use cwpmaw Nuopxm._ m o.N_ ”emoesoNo movemesm :wuwmmmsnoozpo Apem_ .n wmm_ muamm assume asapmue-m-mpou=w .mmmmpv .Ne so oases: N.opxoé a: m.om [Ev "ssawuem ssmwm -oeopzuuanpzspmz A_Nmp .nmmm_ mumsm assume clue uwpmoe-m .mwompv .Ns so oases: muopxn.m m: o.e~ new ”esawuem esmwm -m—oucpuosoFLu-v mpoos a: o AonFV .Ne em menace muopxm.m me o._ ”memzozmxm cowmmeam muwsmumomumumpoucu cowpmspcmucou #3 age ox mocmsmemm capo: \pcmpcou mugzom peep; ucaoqsou .ussctseoo ._ s_nse 128 mmocmgaaouszuc MW.2sz a; o.FF -FFsssue-m-aFou=Fv-s-~ -oFxs.s a: o.m~ FooFmocF-sss-FFFusue m -m-mFos=FF-s-FeF 4Fm uanmz smmsm mg» Eosm ummezuqu coma mm; pszmz Fen mst FquxF.F m: o.o¢ wcFEmFaxguuFaoFagmwnz mcFEanxgp FquxN.¢ m: o.o¢F -FFxosmE:oolqvuz mmocmszaouszlo F-0Fxs.F ms o.ms -FFsque-m-mFoseFv-o-m mmocmcaqooszuo mqux¢.F a: o.m~ -FFAFwomlmumFochvus-e coFumgucmocou p3 zen ax mucmsmme smFoz \pcmpcou moszom ucmFa uczoasou .vwacFucou .F anmF 130 the value reported by Ueda and Bandurski (1969). It does therefore not seem likely that the age of the seed significantly influences the con- tent of free IAA. The observed variation in free IAA and the IAA ester content is likely to be due to seasonal variations in the different harvests. With regard to free IAA it cannot be decided at the present time whether the amounts found reflect the actual concentration of the endogenous free IAA. It is quite possible that the actual endogenous concentration of IAA is much lower. One reason is the fact that some of the IAA esters are quite labile, and IAA is liberated from these esters during the isolation procedure. It was found that the esters of IAA and glucose and the di- and tri-O-(indole-B-acetyl)-myo-inositol esters are more labile than the mono-acyl inositols. The liberation of IAA from these esters stored at -20°C has also been observed by Ueda and Bandurski (1969). Compared to the high molar concentration of the IAA-esters of myo-inositol and myo-inositol-glycosides, the concentration of the newly identified esters is lower by 2 to 3 orders of magnitude. The only other indole compounds found at this low concentration in plant material are 4-chloro-indole-3-acetic acid and its methyl ester, isolated from immature pea seeds (Table 1). The concentrations of the other tabulated indoles are of the same magnitude as those found for the IAA esters of myo-inositol and myo-inositol-glycoside found in corn. An exception to this are the glucobrassicons isolated from the young leaves of Brassica oleracea (Table 1). The high concentration of these compounds suggests that they are not functioning as auxins. 131 With regard to free IAA it should be mentioned that the values given by Tafuri (1966) reflect the early findings by Berger and Avery (1944b) and Hemberg (1955), who observed that the amounts of free IAA decreases rapidly as the seeds mature. This is presumably due to the esterification of IAA with inositol and inositol-glycosides. The capacity of the maize endosperm to incorporate free IAA does however not cease with the seeds reaching maturity. Hemberg (1955) has shown that imbibition of dormant seeds with free IAA in water results in a substantial conversion of free IAA to bound IAA. However, the IAA- conjugates have not been identified and it remains to be seen whether IAA is incorporated into the known esters or unknown esters. In this connection, it should be mentioned that the presence of l-0-(indole-3-acety1)-D-glucopyranose could not be demonstrated in dormant seeds of maize. l-O-(indole-B-acetyl)-D-glucopyranose is a major conjugation product in Colchicum and wheat (Zenk, 1961; Klfimbt, 1961). Recently Gibson et al. (1972) were also unable to isolate any labeled IAA-glucose or any other conjugate from tomato and barley shoots after feeding large amounts of 14 C-IAA. The large amounts of free IAA taken up by the tissue did not greatly increase the pool size of IAA, which suggests that the level of free IAA is tightly controlled. The so-called detoxification of IAA via conjugate formation (Andreae and Good, 1955) may therefore not be operating in many plants. Instead the IAA may be rapidly degraded to indole-3-a1dehyde or other inactive indoles (Gibson et al., 1972; Gibson and Schneider, 1972; Glombitza and Hartmann, 1966; Rajagopal, 1971; Rajagopal and Larsen, 1972). 132 At present the function of these indole compounds in seeds of maize is not known. They are only found (in detectable levels) in the endosperm (Laibach, 1935; Avery et al., 1940; Hemberg, 1955; Piskornik, unpublished). Since the endosperm in the Graminaceae re— presents a food reservoir for the developing embryo it could be inferred that these indole esters play a role (as of yet unknown) during the early stages of germination. Laibach (1935), Avery et al. (1940) and Ueda and Bandurski (1969) have shown that the total IAA content and the content of the IAA—esters decreases rapidly during seed germination. After 96 hours of germination the total IAA had decreased some 10-fold (Ueda and Bandurski, 1969) and Avery et al. (1940) observed a 300-fold decrease of IAA during the first five days of germination. This indi- cates that IAA is not only liberated from the esters, but must be rapidly metabolized. The mode of action of IAA as a hormone, despite the great efforts being made, is not understood. Some experimental evidence suggests that it might act directly on the genome via a protein-hormone complex intermediate (Mathysse and Phillips, 1969; Mathysse and Abrams, 1970; Mathysse, 1970). Some experimental results indicate a link of IAA with RNA metabolism (O'Brien et al., 1968; Hardin et aZ., 1970; Fellenberg, 1970; Essuault, 1970). Whether the esters of IAA found in the endosperm of dormant seeds are therefore precursors of IAA (assum- ing that only free IAA is biologically active) or whether the bound forms of IAA are themselves the active forms cannot be answered at the present time. Owing to permeability characteristics, the biological activity of the esters may be difficult to determine. 133 The presence of mono-, di-, and tri-O-(indole-B-acetyl)-myo- inositols in the dormant seeds of Zea mays tempts one to compare them with the phosphate esters of inositol, and in particular the hexa- phosphate ester phytic acid (Anderson, 1912, 1914), which functions primarily as a phosphate donor during the early stages of germination. From a steric point of view, it is possible to have tetra-, penta-, and hexa-IAA-inositol, which however could not be demonstrated in the water- soluble extracts. The fact that the tri-O-ester is more labile than the di-o-ester indicates that the higher homologues (if they are present in corn) would probably not survive the isolation procedures. The low con- centration of these two esters makes it unlikely that they are degrada- tion products of hexa-IAA-inositol. The increased lipid solubility of the di-o- and tri-O-esters could be important in view of the fact that the esters are exclusively located in the endosperm, and that they have to be transported to the embryo (even though it is not known whether this is actually the case). In this connection, it should also be pointed out that the relatively high concentration of the IAA esters of myo-inositol and myo-inositol- glucosides could conceivably function in transglycosylation and/or transport of carbohydrates across the endosperm-scutellar space. The IAA moiety would thus not only provide increased lipid solubility of the compound, but also specificity. Luttge et al. (1972) have obtained experimental evidence that IAA can significantly modify the membrane fluidity of membranes isolated from leaves of Muium. Unlike the esters of IAA which have been shown to be present only in the endosperm in the dormant seed, the location of the two 134 tryptamine derivatives N-(o-coumaroyl)-tryptamine and N-feruloyl- tryptamine is not known. It is quite possible that they are found predominantly in the embryo. Sumere et al. (1972) have shown that bound cinnamic acid and its hydroxy-, and methoxy-derivatives are more con- centrated in the embryos of barley and beans. Sumere et al. (1972) studied the distribution and possible function of these bound and free phenolics and concluded that the phenolics probably function as germi- nation inhibitors by inhibiting the transport of amino acids and subsequent protein synthesis in the seed. 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