STUDIES IN THE METABOLISM OF PLANTS
A thesis submitted for the degree of Doctor of Philosophy
by
David John Bourne
School of Biochemistry
University of New South Wales
December 1990 UNIVERSITY OF N.S.W. -4 JUL 1031 LIBRARY DECLARATION
The work described in this thesis was carried out by the author on a part and full-time basis between July 1983 and August 1990 while I was employed as a part-time Technical Officer in the School of Biochemistry and a full-time
Professional Officer in the Biomedical Mass Spectrometry Unit, University of
New South Wales.
This thesis represents original research which has not been previously submitted for examination for any other degree. All work was carried out by the author unless otherwise acknowledged.
David Bourne ACKNOWLEDGEMENTS
I wish to thank my supervisors, Professor B. V. Milborrow and
Associate Professor K. D. Barrow for giving me the opportunity to undertake this work. Their enthusiasm for science was infectious and greatly appreciated. I am also very grateful for their assistance and constructive input of ideas during this research. Most critical to any progress was the bioassays performed by Dr D. J.
Osborne and this is gratefully acknowledged.
I would also like to recognise the contributions made by Dr. A. G.
Netting for advice on HPLC, Dr. A. M. Duffield and Mr. R. O. Lidgard for providing some of the mass spectral data and Dr. G. T. Vaughan for the ingenious construction of the Sep-Pak apparatus. Also greatly appreciated support shown by Ms Anne Tibbett over the period taken to complete this thesis. Her patience in contending with the late nights and a certain lack of the authors good humour is a debt that will be hard to repay. ABSTRACT
Extracts from yellowing bean leaves (Phaseolus vulgaris) were
fractionated to yield fractions which stimulated the senescence of petiolar explants
of beans and whose chromatographic behaviour was distinct from those of other
known stimulators of abscission and ethylene production such as abscisic, jasmonic, indoleacetic or aminocyclopropane carboxylic acids. Evidence was
found which shows that the activity is caused by a small aliphatic acid or hydroxy
acid of less than eight carbon atoms. This acid was probably volatile in the free
state and very volatile after methylation. This property was a significant factor in
frequent poor reproducibility of bioassayed extracts. There was some indication
that the active compound, as well as existing in the free state, may also be
conjugated to a carrier group such as caffeic acid or glucose.
Two unusual plant metabolites were isolated, purified and identified as the
monoethyl monomethyl esters of o-methoxybenzoylaspartate. The ethylation of
the aspartate moiety and the methylation of the ring hydroxyl (to form the methoxy
group) was shown to be a result of extraction and storage and derivatisation
procedures. The native compound was isolated, derivatised with pentafluorobenzyl
bromide and shown to carry three pentafluorobenzyl groups; one on the aromatic
ring hydroxyl and one on each of the carboxyl groups. The endogenous compound
was thus shown to be salicyloylaspartate.
Salicyloylaspartate (S A) was present in bean and tomato leaves as the free
acid and in a bound state from which SA could be released by acid hydrolysis.
Assays were performed on five different vegetables and all contained some SA in
either or both of these two forms.
The relative ethylation of the a and p carboxyls of S A in the stored bean
leaf extracts was found to be different from the proportion of esters formed by
acid-catalysed esterification. The identity of each of the synthetically derived
monoethyl esters was established by MS, MSMS and NMR. These studies showed that in acid-catalysed esterification the major product was the (3-ethyl ester whereas the compound isolated from the bean leaf extracts was esterified in the a position. It was postulated that this ethylation was the result of trans-esterification by acidic ethanol of another conjugating group attached to the a-carboxyl.
Tomato explants, when fed with salicylic acid lost turgor and showed outward signs of phytotoxicity whereas SA fed explants showed no such effects.
Some of the applied salicylic acid was metabolised to SA by these explants. When the aqueous residue after extraction of neutrals and acids was examined there were other compounds with the SA chromaphore present. These compound were not
sufficiently volatile to ionise on solid probe MS and may have been sugar conjugates of salicyloylaspartate. ABBREVIATIONS
2D Two dimensional
3D Three dimensional
ABA Abscisic acid
ACC Aminocyclopropane 1-carboxylic acid
AMU Atomic mass unit
BBOT 2, 5 bis (5-tert-butyl-2-benzoxazolyl) thiophene
BSTFA N, O-bis (trimethylsilyl) trifluoroacetamide
Cl Chemical ionisation
CIMS Chemical ionisation mass spectrometry
D Deuterium
DMP Dimethylphaselic acid
DNA Deoxyribonucleic acid
El Electron impact
EIMS Electron impact mass spectrometry eV Electron volts fw Fresh weight
GA Gibberellic acid
GCMS Gas chromatography mass spectrometry
GLC Gas-liquid chromatography
HPLC High pressure liquid chromatography
IAA Indole-3-acetic acid
IPA Isopropyl alcohol
JA Jasmonic acid
M Molecular ion
MBA Methoxybenzoylaspartate
MeJ Methyl jasmonate
MHz Megahertz MS Mass spectrometry
MS MS Mass spectrometry - mass spectrometry (secondary fragmentation spectrometry)
NIST National Institute of Standards Technology
NMR Nuclear magnetic resonance
OBA Ortho-hydroxybenzoic Acid
PFB Pentafluorobenzyl
SA Salicyloylaspartate
SAM S - adenosy lmethionine
SF Senescence factor
SIM Selective ion monitoring
TLC Thin-layer chromatography
TMCS Trimethylchlorosilane
UV Ultraviolet PUBLICATIONS
Bourne, D. J., Barrow, K. D. and Milborrow, B. V. Salicyloylaspartate as an endogenous component in the leaves of Phaseolus vulgaris . Submitted to
Phytochemistry.
Bourne, D. J., Barrow, K. D. and Milborrow, B. V. The existence of bound and free forms of salicyloylaspartate in the leaves of various plants and the evidence for the site of conjugation. In preparation. CONTENTS
DECLARATION
ACKNOWLEDGEMENTS
ABSTRACT
ABBREVIATIONS
CHAPTER 1
1.1 INTRODUCTION
1.2 MATERIALS
1.2.1 Chromatographic Materials
1.2.2 Chemicals and Solvents
1.2.3 BBOT Scintillant
1.2.4 Diazomethane
1.2.5 Analytical Gas-Liquid Chromatography
1.2.6 Preparative Gas-Liquid Chromatography
1.2.7 High Performance Liquid Chromatography
1.2.8 Mass Spectrometry X
1.2.9 Nuclear Magnetic Resonance Spectrometry 11
1.2.10 Infrared Spectroscopy 12
1.3 METHODS 13
1.3.1 Extraction 13
1.3.2 Column Chromatography 13
1.3.2.1 AUS 1 13
1.3.2.2 AUS 2 14
1.3.2.3 AUS IS 14
1.3.2.4 AUS 11C 14
1.3.2.5 AUS 12A 15
1.3.2.6 AUS 27A 15
1.3.2.7 AUS LM1 15
1.3.3 Sep-Pak Purification of Fractions from AUS Columns 16
1.3.3.1 Fraction 6 AUS 12A 16
1.3.3.2 Fraction 3 AUS 27A 16
1.3.3.3 Fraction 2 AUS 27A 16
1.3.4 Determination of ABA 17
1.3.5 Determination of Methyl Jasmonate 17
1.3.6 Fractionation of Crude Extract on Activated Charcoal 17
1.3.7 Purification of Fractions 1, 2, 3, 4 (AUS 27A) on 18
Sephadex LH20
1.3.8 Hydrolysis of Active Fractions from AUS 12A 18 x i
1.3.9 Hydrogenation of Fractions 1, 2 and 3 AUS 2 18
1.3.10 Hydrolysis and Extraction of Fractions 1 to 4 AUS 2 19
1.3.11 Sep-Pak Purifications 19
1.3.11.1 Reverse-Phase of Crude Acid Fraction 19
1.3.11.2 Reverse-Phase of a Methylated Fraction 20
from Sep-Pak Purification of Crude
Acid Fraction
1.3.11.3 Reverse-Phase of Hydrolysed Crude Acid 21
1.3.11.4 Reverse-Phase of Hydrolysed Fractions 21
1, 2, 3 and 4 (AUS 2)
1.3.12 Thin-Layer Chromatography 21
1.3.12.1 30% Ethanol Reverse-Phase Sep-Pak Eluent 21
1.3.12.2 Fraction 40H 22
1.3.12.3 Acetylated Methylated 40H 22
1.3.12.4 Zones Rf 0-0.2 and 0.2-0.4 from TLC 22
Purification of 30% Ethanol Sep-Pak
1.3.13 GC Purification and Analysis 22
1.3.13.1 Fraction 6 AUS 12A 22
1.3.13.2 Fractionation of 3 AUS 12A 23
1.3.13.3 Fractionation of Hydrogenated 23
Fractions 1, 2, 3 AUS 4
1.3.13.4 Fractions 30H, 30HAF, 40M20, 80HAF, 23
40M20H and 40M30K
1.3.13.5 Fractions 1, 2, 3 and 4 AUS 27A 24
1.3.14 Preparation of Samples for Normal-Phase HPLC 24
1.3.15 HPLC Purification 25 x i i
1.3.16 Identification of the Unknown Peak 25
1.3.16.1 Instrumental 25
1.3.16.2 Hydrolysis and GCMS 25
1.3.17 Some Chromatography with ACC and a Derivative 26
1.3.17.1 Preparation of N-Acetyl-ACC 26
1.3.17.2 Reverse-Phase Sep-Pak of 26
N-Acetyl-ACC and ACC
1.3.17.3 Other Derivatisation of ACC 26
1.3.18 Preparation of Samples for Bioassay 27
1.3.18.1 Column Chromatography Samples 27
1.3.18.2 GCTrappates 27
1.3.18.3 HPLC Fractions 27
1.3.18.4 TLC Fractions 27
1.3.18.5 General 28
1.3.19 Bioassay 28
1.3.20 Determination of Ethylene 28
1.3.21 Synthetic Work 29
1.3.21.1 3,4 Dimethoxycinnamic Acid Methyl Ester 29
1.3.21.2 3, 4 Dimethoxycinnamic Acid 29
1.3.21.3 3,4 Dimethoxycinnamoylmalate 29
1.3.21.4 Purification of Dimethoxycinnamoylmalate 31
1.3.21.5 1 -Methoxysuccinic Acid Dimethyl Ester 31
1.3.21.6 HPLC Purification of 1-Methoxysuccinate 33
Methyl Ester XIII
1.4 RESULTS 34
1.4.1 Early Results 34
1.4.2 Extraction of Hydrolysed Fractions 1 to 4 (AUS 2) 34
1.4.3 Silica Column Purifications 37
1.4.4 Silica Sep-Pak Purifications of AUS Column Fractions 39
1.4.5 Other Column Purifications 39
1.4.6 Senescence Activity, Ethylene and ABA 42
1.4.7 Senescence Activity and Methyl Jasmonate 47
1.4.8 ACC, N-acetyl-ACC Bioassay, Chromatography 49
1.4.9 GC Trapping of AUS Fractions 51
1.4.10 GC Trapping of Hydrogenated AUS 2 Fractions 54
1.4.11 Reverse-Phase Sep-Pak Purifications 54
1.4.12 Thin-Layer Chromatography 62
1.4.13 GC Trapping of Sep-Pak Fractions 64
1.4.14 Normal-Phase Sep-Pak Purification 66
1.4.15 HPLC of Methylated 40M20 66
1.4.16 Identification of the Unknown Compound 69
1.4.17 Bioassay of Synthetic Compounds 82
1.4.18 Relationship of Activity and the Cinnamoylmalate 82
Derivative XIV
1.4.19 Identification of a Major Peak as a Succinate Diester 83
1.4.20 GLC Traps of Hydrolysed FI, 2, 3, 4 AUS 27A 86
1.4.21 Identity of Other Compounds in the Ethyl Acetate 88
Extract of Hydrolysed FI, 2, 3 and 4 AUS 27A
1.5 DISCUSSION 91
1.5.1 Early Work 91
1.5.2 Activity and Ethylene S timulation 91
1.5.3 Activity and Known Stimulatory Compounds 92
1.5.4 Chromatography 94
1.5.5 A Phaselic Acid Derivative in an Active Fraction 99
1.5.6 Problems in the Isolation of SF 100
1.5.7 Suspected Structural Characteristics of SF 102
1.5.8 Further Work 103
CHAPTER 2
2.1 INTRODUCTION 105
2.2 MATERIALS 111
2.2.1 General 111
2.2.2 Chemicals and Solvents 111 XV
2.3 METHODS 112
2.3.1 Solvent Extraction 112
2.3.2 Sep-Pak Purification 112
2.3.3 Methylation 115
2.3.4 HPLC Purification of the Unknown 115
2.3.5 GC Trapping 115
2.3.6 Proton NMR of the Unknown 116
2.3.7 Synthesis of Meta, Ortho and Para MBA 116
2.3.7.1 General Aspects 116
23.1.2 Para MBA 116
2.3.7.3 Ortho and Meta MBA 116
2.3.8 Preparation of the Monomethyl-Monoethyl, Dimethyl 118
and Diethyl Esters
2.3.8.1 Para 118
2.3.8.2 Ortho and Meta 120
2.3.9 Thin-Layer Chromatography 120
2.3.10 HPLC of the Ethylated Ortho, Para and Meta MBA 120
Preparations
2.3.11 Spiking the Plant Extract with Synthetic Isomers 120
2.3.12 GCMS 121 XVI
2.4 RESULTS 122
2.4.3 Preliminary Rationale 122
2.4.2 Isolation and Purification 125
2.4.2.1 Reverse-Phase Sep-Pak Steps 125
2.4.2.2 HPLC Steps 126
2.4.2.3 Preparative Gas-Liquid Chromatography 127
2.4.3 Monitoring Progress of the Purification by GCMS 130
2.4.4 Proton NMR of Trappate 5+6 BX;C 130
2.4.5 Synthesis of the Methoxybenzoylaspartic Acids 132
2.4.5.1 General Aspects 132
2.4.5.2 Para 132
2.4.5.3 Ortho and Meta 133
2.4.6 Purification 133
2.4.7 Formation of the Monoethyl Esters 134
2.4.8 Mass Spectra of Ethylated, Methylated MBA 134
Preparations
2.4.8.1 Para 134
2.4.8.2 Ortho and Meta 139
2.4.8.3 Comparison of MS Data of Standards and 139
Unknown
2.4.9 GCMS of Standard-Spiked 30M20; 10IPA 143
2.4.10 HPLC Purification of the Ethylated Methylated MBA 147
Preparations XVII
2.4.11 Proton NMR of Synthetic MBA 147
2.5 DISCUSSION 154
CHAPTER 3
3.1 INTRODUCTION 156
3.2 MATERIALS 159
3.2.1 Purification of Thionyl Chloride 159
3.2.2 High Performance Liquid Chromatography 159
3.2.3 GCMS 159
3.2.4 Chemicals and Reagents 159
3.2.5 0.4M Tetraethylammonium Hydroxide 159
3.3 METHODS 160
3.3.1 Synthesis of OBA 160
3.3.1.1 Formation of Acetylsalicylic Acid 160
3.3.1.2 Formation of Acetylsalicylic Acid Chloride 160
3.3.1.3 Formation of the Salicyloylaspartate Derivative 160
3.3.2 Preparation of o-Methoxybenzoylaspartic Acid 162
3.3.3 Extraction of Plant Material 162
3.3.3.1 Free SA 162 XVIII
3.33.2 Bound SA 163
3.3.4 HPLC Purification 163
3.3.5 Extraction, Purification of Vegetable Leaves for SIM- 164
GCMS
3.3.6 Derivatisation 164
3.3.7 Purification of Derivative 165
3.3.8 Full Scan Negative Ion GCMS of the PFB Derivative of 165
Synthetic Salicyloylaspartate and Plant Extracts
3.3.9 SIM-GCMS of Derivatised Vegetable Leaf Samples 166
3.3.10 Deuteration of Salicyloylaspartate 166
3.4. RESULTS 167
3.4.1 Purity and Identity of the Salicyloylaspartate Derivatives 167
3.4.2 Formation of PFB Derivatives of Synthetic SA 167
3.4.2.1 Salicyloylaspartate 167
3.4.2.2 o -Methoxybenzoylaspartate 175
3.4.3 GCMS of Bean and Tomato Leaf Extracts 175
3.4.4 Approximate Quantitation of SA in Bean and Tomato Leaf 181
Extracts
3.4.5 SIM GCMS of Vegetable Leaf Extracts 181
3.4.6 GCMS of Deuterated Salicyloylaspartate 181 XIX
3.5 DISCUSSION 189
CHAPTER 4
4.1 INTRODUCTION 192
4.2. MATERIALS 194
4.2.1 HPLC 194
4.2.2 Mass Spectrometry 194
4.2.3 NMR 194
4.2.4 pH Measurement 195
4.2.5 Solvents and Chemicals 195
4.3. METHODS 196
4.3.1 Determination of pKa of Synthetic S A 196
4.3.2 Synthesis of Monoethyl SA 196
4.3.3 Reverse-Phase HPLC 196
4.3.3.1 Determination of the Ratio of Monoethyl 196
Esters
4.3.3.2 Separation of Monoethyl Esters 197
4.3.4 Methylation 197
4.3.5 Normal-Phase HPLC 197
4.3.6 Time Course of Ethylation of SA 197 XX
4.3.7 Separation and Purification of Endogenous SA 198
4.3.8 Proton NMR 198
4.3.9 Preparation, Purification of Deuteromethyl SA Esters 198
4.3.10 Trans-esterification of Diethyl S A 199
4.3.11 Chemical Ionisation GCMS 199
4.3.11.1 Bean Leaf Fraction 30M20;10IPA 199
4.3.11.2 Labelled Dimethyl S A Esters 199
4.3.11.3 S A Diethyl Ester Trans-esterification 200
Mixture
4.3.11.4 Various HPLC Column Eluents 200
4.3.12 MSMS Studies of the Isomeric SA Diesters 200
4.4. RESULTS 201
4.4.1 pKa Determination 201
4.4.2 Ratio of Monoethyl Esters 201
4.4.3 Identity of the Monoethyl Esters 205
4.4.4 Cl Mass Spectra of Methylated Monoethyl Esters 208
4.4.5 Studies with Deuteromethyl, Methyl Esters of S A 211
4.4.6 Reverse-Phase HPLC on a Preparative Column 213
4.4.7 Proton NMR of the Methylated Monoethyl Esters 214
4.4.8 High Resolution EIMS and MSMS of SA Diesters 214
4.4.9 GCMS of 30M20;10IPA 221 XXI
4.4.10 Time Course of Ethylation 224
4.4.11 Trans-esterification 224
4.5 DISCUSSION 227
CHAPTER 5
5.1 INTRODUCTION 231
5.2 MATERIALS 232
5.2.1 Reverse-Phase HPLC 232 5.2.1.1 General 232 5.2.1.2 Acid Extract Mobile Phase 232 5.2.1.3 Sep-Pak Eluent Mobile Phase 232
5.2.2 Mass Spectrometry 232
5.2.3 Chemicals and Solvents 233
5.3 METHODS 234
5.3.1 Feed Solutions 234
5.3.2 Tomato Feeding 234
5.3.3 Pre-treatment of the Aqueous Residue Before 235
Reverse-Phase Sep-Pak
5.3.4 Sep-Pak Fractionation of Aqueous Residue 235 XXII
5.3.5 HPLC 235 5.3.5.1 Acid Extracts 235 5.3.5.2 20% Ethanol Sep-Pak Eluents 236
5.3.6 GCMS 236
RESULTS 237
5.4.1 HPLC 237 5.4.1.1 Acid Extracts 237 5.4.1.2 20% Ethanol Sep-Pak Eluents 237
5.4.2 GCMS 241
DISCUSSION 243
5.5.1 Role of Salicylic Acid in Plants 243
5.5.2 Aspartate Conjugation 244
5.5.3 Salicylic Acid Metabolism in Tomato Explants 247
REFERENCES 251 CHAPTER 1
Purification of senescence-active fractions from bean leaves 1
LI INTRODUCTION
The history of the discovery plant hormones is a very intriguing one, particularly on consideration of the remarkably similar procedures that were followed leading to the discovery of each hormone or class of plant hormone. After the notion developed that endogenous plant compounds could be responsible for the control of some physiological processes the general approach in each case was very similar. Firstly extracts from various sources were assayed in an attempt to mimic the natural plant response to a particular stimulus and these extracts were then fractionated and purified to maximise this bioassayed activity. In this way the active compounds were eventually characterised. Interestingly, in some cases the hormones were first identified and characterised from sources other than plants and then were finally isolated from plant extracts.
The auxins were the first group of hormones to be pursued with great vigour by numerous research groups. Charles and Francis Darwin carried out a series of experiments in phototrophism late last century that were the first to show that the observed effect of light on growing shoots (the bending towards light) was mediated by the tip and somehow transmitted to the tissues below. The Darwins probably did not realise the significance of their experiments - that it was a plant hormone eliciting the response of the plant to light. Further experimentation by other researchers confirmed that the coleoptile tip was the origin of this response to light but it was not until Went in 1928 (as outlined in Marumo, 1986) showed that the effects of the light-induced response could be were transferred from an excised coleoptile tip to a decapitated stump via an agar block that it was realised that a compound originating in the tip of the shoot was responsible for bending of the coleoptiles towards light. Went first excised coleoptiles, attached these to agar discs, and then applied these discs to one side of another excised coleoptile. He found that the shoots grew faster on the side where the agar disc was placed. Other agar discs prepared from dark-grown shoots or shoots excised further towards the roots had no effect. The procedure involving the treatment of agar discs and the application to decapitated shoots evolved to become the Avena coleoptile assay - a test that was subsequently used to bioassay compounds or extracts suspected to have this biological activity. This group of compounds became known as the auxins.
Intensive efforts by many research groups followed, with auxin-active compounds being extracted from such diverse sources as human urine, plant and fungal tissue. During this period numerous structures were proposed for auxins extracted from these sources and clearly there was more than one active compound isolated. However the first successful isolation and unequivocal identification of the compound now known to be universal in distribution in the plant kingdom from plant extracts was achieved by Haagin-Smit et al. (1946). This compound, 3- indoleacetic acid (IAA) had previously been isolated from other tissues; from yeast by Kogl's group in 1934 (see Marumo, 1986) and fungus (Thimann, 1935) but this was the first time the free acid had been extracted from plant tissue.
Undoubtedly, the work on IAA served to stimulate the search for phytohormones that controlled other processes in plant growth and development.
Another plant hormone that was characterised long after its effects were recognised was ethylene. The use of smoke and other gaseous agents to promote fruit ripening had been known since late last century (Yang, 1983) but the gas responsible for this effect was not identified as a natural plant constituent until the mid-thirties when
Gane (1934) showed that ripe apples produce ethylene.
Aside from the auxins and ethylene, three other groups of plant hormones are now known. These are the gibberillins, cytokinins and abscisic acid.
The isolation and identification of these hormones followed a similar sequence to that which occurred with IAA. Firstly it was recognised that extracts of either plant or fungal tissues contained compounds which elicited characteristic physiological responses when applied to plants. Infection of rice seedlings with the fungus Gibberella fujukuroi was long known to result in tall unhealthy plants and isolation of two active compounds from this source was first reported by two 3
Japanese researchers in 1938 (see Takahashi, 1986). Later, various research groups including Curtis and Cross (1954) and Stodola et al. (1955) succeeded in increasing the number of known GAs from fungal extracts. The earliest evidence that gibberellins were endogenous plant compound was provided by Mitchell et al.
(1951) and Radley (1956) so that up to 1986 the family of fungal and plant
gibberellins has grown to sixty-eight (Takahashi et al., 1986). The accumulation of knowledge on cytokinins also followed the well used paths for the other plant hormones: firstly a physiological effect was hypothesised to be controlled by a hormone, extracts from various sources were bioassayed as active and finally active endogenous compounds were characterised. Miller et al. (1956) isolated and identified a compound they termed kinetin from autoclaved
herring sperm DNA. This compound was not a natural plant product but other related substances were later discovered to occur endogenously (Letham, 1963). Like the gibberellins, many compounds with the same basic chemical structure that have cytokinin activity (principally the promotion of cell division) are known to exist in plants. The discovery of abscisic acid (ABA) grew from a belief that probably two distinct plant hormones were responsible for two separate processes - the inhibition of bud development and the stimulation of abscission. Two research groups
working independently in these areas isolated and identified the same compound.
Addicott and his co-workers found an inhibitory compound in of immature cotton fruit which stimulated the abscission of older cotton fruit (see Hirai, 1986). This compound, abscisin II, was isolated by Ohkuma et al. (1963), the structure established by Ohkuma et al. (1965) and the identity confirmed by synthesis
(Comforth et al., 1965a).
Concurrently a group led by Wareing were working towards the isolation of a compound called domain that was isolated from Acer leaves (see Hirai, 1986).
Comforth et al. (1965b) extracted and purified dormin from Acer leaves and found that this was the same compound as abscisin II. 4
Assigning in-vivo physiological effects to particular plant hormones is not as simple as suggested by the bioassay systems used to identify them. There are, of course, the obvious problems that responses induced by the hormone applied to explants, sections or tissue culture may or may not be as important to the growth and development of the whole plant as implied by the results of exogenous
application. Further complication can be caused by differences in response of the
same tissues dependant on factors such as age, or the fact that different concentration of the same hormone applied to the same tissue may illicit contrary responses, or that the responses to a hormone may be very tissue-specific. Additional complication can be introduced when hormone mixtures are considered and the fact that responses to particular plant hormones is sometimes species- dependant. Although some plant responses have been directly related to changes in hormone levels it is generally accepted that in the absence of direct conclusive evidence that the physiological effects induced after treatment with a plant hormone might, not will be duplicated in-vivo. Some of the probable roles of LAA include stimulation of rooting, cell division and cell elongation, induction of parthenocarpy and inhibition of leaf senescence, fruit ripening and abscission. Gibberellins have been implicated in the stimulation of cell division, cell elongation and fruit
ripening, the breaking of seed dormancy and the maintenance of juvenility in
leaves. Cytokinins promote cell division, seed germination and leaf growth and
seems to play a role in differentiation while ethylene has strong links with
abscission and fruit ripening. Abscisic acid may be central to the inhibition of germination, promotion of leaf abscission, maintenance of turgor and control of stomatal closing. One of the less convincing aspects of hormonal control of abscission is the
role of ABA in this process. Abscission of leaves is always accompanied by an
increase in ethylene production and several groups such as Morgan et al. (1972) have demonstrated the correlation between abscission and increased ethylene 5 production while others such as Jackson and Osborne (1972) have shown that ethylene production is regulated by levels of ABA and IAA. Although there is some evidence that ABA has a stimulatory effect on ethylene production this is by no means conclusive. Clearly another plant hormone may be implicated in this process. Milborrow (1974) has suggested that the level of ABA required to promote abscission is very high and that physiological amount of ABA have little or no effect on abscission in most plants. Zeevaart and Creelman (1988) make no mention of of abscission or ethylene production during senescence as a physiological role of ABA in plants.
Nonetheless there is little doubt that ethylene at least plays a central role in abscission. The findings of Jackson et al. (1973) typify what many others have found before and since - that ethylene increases just prior to senescence and increases during senescence.
The notion that a hormone could be responsible for the induction of senescence in plants initially arose with the discovery by Osborne (1955) that exudates from senescent petioles of various plants was found to accelerate abscission of bean leaf explants and to antagonise the effects of exogenously applied auxins. Osborne (1959) later stated that the active compound did not appear to be any of the common sugars, amino acids, purines or pyrimidines.
Other workers (van Steveninck 1959, Scott and Leopold 1966) verified that a non volatile abscission accelerator was present in plants.
Later Osborne et al. (1972) showed that aqueous diffusates from green bean leaves both accelerated abscission and stimulated ethylene production: a combination of effects that were distinct from those caused by applications of two known plant hormones, IAA and ABA (the former stimulates ethylene production but not abscission while the latter stimulates abscission but has little effect on ethylene formation). These workers also found that ethanolic extracts of yellowing and green bean leaf mimicked the action of the aqueous diffusates of yellowing leaves. 6
Studies by the same group showed that diffusates and ethanol extracts could be fractionated on silica TLC plates to give two broad zones of activity, one close to the origin and another from Rf 0.65 to 0.95. The faster migrating zone was later shown by Milborrow and Osborne (unpublished results) to contain a compound with potent senescence-inducing characteristics. When this zone was eluted, acetylated and rerun in a different solvent system the activity was confined to a region closer to the solvent front compared to the un-acetylated material. This
suggested that the active compound contained an acetylatable group, probably a hydroxyl group. Chromatography of an ethanolic extract of bean leaves on a DEAE cellulose column gave three active zones, one of which was purified by TLC to give an active zone as described above. This zone was fractionated by preparative GLC after acetylation and methylation. The abcission-accelerating activity was concentrated under one peak which was trapped and assayed. No structure has been published. Other compounds have been found to induce senescence and/or promote ethylene production. One of these, the methyl ester of jasmonate acid was isolated from wormwood leaves, identified by Ueda and Kato (1980) and found to be a senescence inducing compound. However these workers used the yellowing (loss of chlorophyll) of oat leaf segments as a measure of senescence activity. Jasmonic acid and its methyl ester were inactive under the conditions employed by Osborne et al. (1972) in both stimulation of abscission and promotion of ethylene production.
A compound that is directly involved in ethylene biosynthesis is 1- aminocyclopropane 1-carboxylic acid (ACC). This compound, isolated and identified by Burroughs (1957) was first postulated to be an ethylene precursor by Adams and Yang (1979) who showed that labelled ACC accumulated when 14C labelled L-methionine was fed to apple slices under anaerobic conditions designed to prevent ethylene production. Methionine was known to be a precursor of ethylene (Lieberman M., 1979).
The aim of this project was to attempt to find the same SF as Osborne et al. (1972) and isolate sufficient quantity for identification. This meant that other methods had to be developed since earlier procedures would have been tedious for the large quantities of bean leaves it was planned to extract. This SF activity and the coincident ability of this compound to induce ethylene production had to be distinct from other known abscission accelerators or ethylene stimulators such as IAA, ABA, ACC and jasmonic acid. 8
1.2 MATERIALS
1.2.1 CHROMATOGRAPHIC MATERIALS Silica for column chromatography, Keiselgel G (type 60), was supplied
by Merck (Darmstadt, FGR), dried overnight at 110°C and then cooled before use. Charcoal powder and Celite were obtained from BDH (Poole, England) while Sephadex LH20 was supplied by Pharmacia (Uppsala, Sweden). Glass backed plates (200 x 200 mm coated with a 0.25 mm of Kieselgel 60 F254.) from Merck were dried at 110°C for 10 min and cooled to room
temperature before use. Sep-Paks used in purification steps were obtained from Millipore
(Milwaukee, USA). These were supplied pre-packed with either Ci8 or silica (10 pm particle size) and were used singly or in series. Phase choice, number of Sep- Paks and eluting solvents are stipulated in the appropriate Methods sections.
1.2.2 CHEMICALS AND SOLVENTS Methyl jasmonate was kindly donated by Ueda and Kato, University of Osaka Prefecture, Osaka, Japan. Palladium on carbon (10%) was supplied by
Merck, BBOT by Prolabo (Paris, France) and while Fluka (Buchs, Switzerland)
supplied the N-methyl-N-nitroso-/? -toluenesulphonamide. o-acetoxybenzoic acid was prepared from salicylic acid (BDH) by treatment with excess 1:1 pyridine/acetic anhydride. After heating at 80°C for two hours the reagents were evaporated under at reduced pressure, yielding oacetoxybenzoic acid as a
solid, mp. 133-135°C. 1-aminocyclopropane 1-carboxylic acid was kindly donated by Dr. D. J. Osborne (see Methods 1.3.19)
Radioactively labelled compounds [U-14C] palmitic acid (50 pCuries/pmole) and [2-14C]ABA (10 pCuries/pmole) were obtained from Amersham (Buckinghamshire, England). Tritiated methyl jasmonate 9
(approximately 5fiCuries/pmole) was prepared by catalytic exchange at the
Chemistry department of this University.
Deuterochloroform (99.96%D) for NMR spectra was supplied by
Aldrich (Milwaukee, USA)
All other compounds and solvents were obtained from local sources.
1.2.3 BBOT SCINTILLANT
Scintillant was prepared by dissolution of BBOT (12 gm) and napthalene (160 gm) in 2-methoxyethanol (80 ml) and toluene (1200 ml).
1.2.4 DIAZOMETHANE
Ethanol (10 ml) was added to 60% potassium hydroxide (4 ml) in a round bottom flask fitted with a dropping funnel and condenser. A solution of N-methyl-
N-nitroso-p -toluenesulphonamide (9.2 gm) in diethyl ether (40 ml) was slowly added through a dropping funnel while the contents of the flask were constantly stirred. The solution was maintained at 50°C until distillation ceased and the ethereal distillate collected in a receiving flask cooled in ice. The distillate contained approximately 1.2 gm of diazomethane.
1.2.5 ANALYTICAL GAS LIQUID CHROMATOGRAPHY
Gas liquid chromatography was carried out on a Pye Unicam GCV chromatograph equipped with a flame ionisation detector and an electron capture detector with a 63Ni source. Injector and detector temperatures were set at
200°C and 260°C respectively. Signals were recorded on a National VQ-068 chart recorder. Open tubular glass columns (1.5 m x 4 mm) containing 3% QF1 on chromasorb W HP80-100 were used with nitrogen as carrier gas. Gas flow rates were set to 30 ml/min for nitrogen, 30 ml/min for hydrogen and 380 ml/min for air.
Oven temperatures are stipulated in the appropriate sections. 10
1.2.6 PREPARATIVE GAS-LIQUID CHROMATOGRAPHY
Conditions of preparative GC are the same as used for analytical runs with the exception of gas flow rates. For nitrogen, hydrogen and air the flow rates were
30, 10 and 150 ml/min respectively. An outlet splitter diverted approximately
l/25th of the gas stream to a flame ionisation detector and the rest to a manual trapping port.
The outlet stream was passed through glass U-tubes (20 cm x 5 mm ED) loosely packed with glass wool. These U-tubes were partially immersed in a mixture of dry ice and ethanol.
Oven temperature profiles are mentioned in the appropriate sections.
1.2.7 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
For isocratic separations a Waters Model 6000A pump fitted with a U6K injector was used. Normal phase separations utilised a Waters pPorasil column
(300 x 7.8 mm, particle size 10p.m).
Detection system included a Hewlett Packard 1040A Diode Array interfaced with a HP 9000 series 300 computer and a HP 9133 disc drive.
Chromatograms were recorded at 220 nm, 293 nm and 330 nm with a bandwidth of 8 nm. Reference wavelength was set at 550 nm with a bandwidth of 100 nm.
UV spectra and chromatograms were plotted with a HP Model 7470A plotter.
1.2.8 MASS SPECTROMETRY
Chemical ionisation mass spectra were conducted on a Finnigan 3200 quadrapole GCMS interfaced with a T0.2300 Incos data system. Ion source was maintained at 110°C by filament emission (100 mA) and source pressure was 10-3
Torr.
GC runs were carried out using a 1.6 m glass tubular column (ED 2 mm) packed with 3% QF-1 on Chromasorb HP 100/120 and methane carrier/reagent gas at a flow rate of 20 ml/min. Oven temperature conditions are outlined in the appropriate sections. High resolution MS of the unknown dimethoxycinnamoyl conjugate was carried out by solid insertion probe on an AEI MS 12 single focussing mass spectrometer. Source temperature was 200°C, filament emission energy was 70 eV and accelerating voltage was 8 kV.
Other high resolution electron impact mass spectra utilised a VG
AUTOSPEC Q with VG Opus software and a VAXstation 2000 computer from Digital (Chatswood, NSW). Positive ion electron impact was used with filament emission energy of 70 eV and a source temperature of 180°C. The instrument was tuned to a resolution of 10,000 and each peak automatically mass measured. The mass spectrometer was interfaced with a Hewlett Packard 5890 gas chromatograph. A 25 m DB-5 bonded phase polyimide coated vitreous silica column (0.25 mm ID, 0.25 |im film thickness) from J&W (Folsom, USA) was used with helium carrier gas at a flow of 2 ml/min. Oven temperature was raised from an initial temperature of 50°C to a final temperature of 280°C at a rate of 6°C/min.
1.2.9 NUCLEAR MAGNETIC RESONANCE SPECTROMETRY
Proton NMR spectra were obtained at 300 Hz on a Bruker CXP-300 operating in the pulsed Fourier transform mode with quadrature detection. 90° radio frequency pulses of 6.5 to 7.5 jisec duration were used with a pulse repetition of 2 sec, 8K data points and a spectral width of 4000 Hz. Chemical shifts were measured digitally using the chloroform proton as a reference. Samples were contained in 99.96% deuterochloroform in 5 mm OD glass tubes maintained at 27°C.
Other NMR spectra were recorded on a Bruker 500 MHz spectrometer using the same tubes and solvent as outlined above. Typical spectral parameters 12 were: pulse width 5.8 psec, pulse repetition 2 sec, sweep width 4000 Hz, and 8K data points.
1.2.10 INFRARED SPECTROSCOPY
Samples dissolved in dichloromethane were pipetted into 1 ml liquid cells with NaCl windows and spectrum recorded under the following conditions: normal slit width, dual beam mode, time constant 1 sec, scan range 4000 to 800 cm-1 and total scan time 12 min. The instrument used was a Pye Unicam SP2000. 13
1.3 METHODS
1.3.1 EXTRACTION
About 200 kg of senescent yellowing leaves of French bean (Phaseolis vulgaris, var Dwarf Pioneer) were collected over a period of months from market gardens at Dural, New South Wales after beans had been harvested.
The leaves were stored at -20°C and extracted in batches in large 50 1 metal bins. Leaf material in the bins was submerged in 0.5% acetic acid in 95% aqueous ethanol (301), left standing at room temperature for two days and then the filtered through cheese-cloth. Each batch of leaf material was extracted twice in this fashion and the combined extracts evaporated under reduced pressure to a largely aqueous residue (about 50 1 in total).
This aqueous residue was then treated in 2 1 lots. Each batch was neutralised with solid sodium bicarbonate, extracted three times with an equivalent volume of diethyl ether and the neutrals discarded. The aqueous extract was then acidified to pH 2.5 and extracted three times with an equal volume of ethyl acetate.
The acid fractions were combined and evaporated to near dryness and stored in the dark at 4°C. Final volume of this extract was approximately 2.5 1. Small and equal aliquots of the neutrals, acid fraction and aqueous were submitted for bioassay (Methods 1.3.14).
1.3.2 COLUMN CHROMATOGRAPHY
1.3.2.1 AUS 1
10 ml of the acid fraction concentrate was added to the top of a silica column (25 cm x 7 cm diameter) pre-conditioned with hexane (200 ml). This column was eluted with toluene/ethyl acetate/acetic acid (50/30/2), and fifty fractions (100 ml) collected. Fractions were evaporated to dryness and l/3rd of every third fraction starting with fraction 3 was submitted for bioassay. 14
1.3.2.2 AUS 2
The base of a large sintered glass column (15 cm diameter) was coated with a 1 cm layer of celite. Silica was added and compressed by tamping until the silica layer was about 8 cm thick. The outlet of the funnel was connected via a buchner flask to a water pump and toluene (400 ml) drawn through the column.
About 100 ml of acid fraction concentrate was added to the column and the vacuum applied until the entire fraction was absorbed onto the column material. The column was then eluted with toluene/ethyl acetate/acetic acid (50: 30: 2) and forty fractions (100 ml) collected. l/10th of each third fraction starting with fraction 1 from one of these runs (AUS 2) was submitted for bioassay and ethylene assay.
1.3.2.3 AUS IS
40 ml of acid fraction concentrate was hydrolysed overnight at room temperature by addition of a 2/1 mixture of 95% ethanol/60% aqueous potassium hydroxide (25 ml). After extraction of neutrals with diethyl ether the solution was acidified to pH 2.5 - 3 with 2M sulphuric acid and extracted three times with ethyl acetate (100 ml). This acid extract was evaporated under reduced pressure to low volume, spiked with about 20,000 dpm of ABA and chromatographed by the same procedure used with column AUS 2. Forty fractions (100 ml) were collected and 1 ml of each was added to 10 ml of BBOT scintillant and counted. 1 ml of each fraction was also submitted for bioassay.
1.3.2.4 AUS 11C
The first four 100 ml fractions from six separate runs of column AUS 2 were evaporated, neutralised with saturated sodium bicarbonate and extracted with diethyl ether (3 x 50 ml). After adjustment to a pH 2.5 - 3 with 2M sulphuric acid the aqueous residue was extracted three times with ethyl acetate (50 ml). l/200th of the ether extract and l/2000th of the ethyl acetate extract were submitted for bioassay. The remainder of the ethyl acetate extract was evaporated to near 15 dryness and chromatographed under the same conditions used for column AUS 2. l/500th of fractions 1, 3, 5, 7 and 9 were bioassayed.
1.3.2.5 AUS 12A
The first four fractions from AUS 11C were combined, evaporated and acetylated at room temperature overnight with pyridine and acetic anhydride (1:1,
10 ml). This mixture was diluted to about 100 ml with toluene and absorbed onto a silica column of the same size and composition as the ones used for AUS 2 and
AUS 11C. The column was then flushed with toluene/ethyl acetate/acetic acid
(15/3/1) and forty 100 ml fractions collected. l/500th of each fraction was bioassayed.
1.3.2.6 AUS 27A
The first 400 ml eluting from thirteen separate runs of column AUS 2 were combined, evaporated to near dryness and acetylated overnight with pyridine and acetic anhydride (1:1, 50 ml). After evaporation the residue was chromatographed by the same procedure used for AUS 12A. l/1000th of each fraction was bioassayed.
1.3.2.7 AUSLM1
One-half of fraction 2 AUS 27A was methylated with excess etherial diazomethane and loaded onto a small silica column (10 cm x 2.5 cm diameter) pre conditioned with hexane (100 ml). The column was then eluted with hexane (100 ml), 20% and 60% dichloromethane in hexane (150 ml) followed by dichloromethane (150 ml) and acetone (150 ml). l/100th of each fraction was prepared for bioassay in the usual manner. Jasmonic acid methyl ester (1 mg) was chromatographed under the same conditions on a separate run and the same size fractions collected. 16
1.3.3 SEP-PAK PURIFICATION OF FRACTIONS FROM AUS COLUMNS
1.3.3.1 FRACTION 6 AUS 12A
Active fraction 6 was evaporated, acetylated overnight with pyridine and acetic anhydride (1:1, 10 ml) and methylated with an excess of diazomethane.
After evaporation to near dryness the sample was dissolved in dichloromethane (10 ml) and the solvent forced through a single silica Sep-Pak cartridge attached to a 10 ml syringe. The Sep-Pak was washed with a further aliquot of dichloromethane
(70 ml) followed by methanol (40 ml) and acetone (40 ml). The three fractions were evaporated, hydrolysed overnight with a 2/1 mixture of ethanol and 60% aqueous potassium hydroxide (0.5 ml). Each sample was then acidified to pH 2.5
- 3 and extracted with ethyl acetate (2 x 20 ml). About 1/10th of each was submitted for bioassay and tested for stimulation of ethylene production.
1.3.3.2 FRACTION 3 AUS 27A
One-tenth of fraction 3 ALTS 27A was acetylated, methylated, absorbed onto a single silica Sep-Pak and eluted with dichloromethane/methanol (2/1). Ten fractions (20 ml) were collected as well as a methanol and an acetone purge (20 ml of each). Each fraction was then prepared for bioassay in the manner described in
Methods 1.3.14.
1.3.3.3 FRACTION 2 AUS 27A
One-tenth of fraction 2 AUS 27A was acetylated, methylated and loaded onto a single silica Sep-Pak ( pre-washed with 10 ml of hexane ) on the outlet of a
10 ml syringe. The Sep-Pak was then eluted with hexane (20 ml) followed by the same volume of 10%, 20%, 60% dichloromethane in hexane, dichloromethane, and methanol. Methyl jasmonate (1 mg) was added to fraction 2 before chromatography and its distribution between fractions determined by GC. Each eluent was prepared for bioassay in the usual manner. 17
1.3.4 DETERMINATION OF ABA
Fractions 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, and 50 from AUS 1 were evaporated to near dryness, methylated and made up to one ml.
One (il of each fraction was analysed by GC on a 2 m glass column filled with 3%
XE60 on chromasorb W (100 to 120 mesh). The column was maintained at
198°C,and an electron capture detector with a 63Ni source used with a purge gas flow of 40 ml/min. Other relevant GC conditions are outlined in Materials 1.2.5.
1.3.5 DETERMINATION OF METHYL JASMONATE
Appropriate fractions from the duplicate methyl jasmonate spiked runs of
AUS LM1 and the Sep-Pak purification of fraction 2 AUS 27A were analysed by
GC on the same QF1 column used for the trapping of AUS column fractions. Gas flow conditions were identical to those used for the ABA determination. Detection was by flame ionisation and the column was maintained at a temperature of 130°C.
1.3.6 FRACTIONATION OF CRUDE EXTRACT ON ACTIVATED
CHARCOAL
Bean leaf (50 gm) was extracted by immersion in 1 % acetic acid in ethanol
(41) on an 80°C water bath for 2 hr. The mixture was then filtered, evaporated to near dryness, redissolved in 850 ml of 10% aqueous acetone, combined with activated charcoal (220 gm) and stirred overnight at room temperature. The mixture was then poured into a sintered glass filter and the 10% acetone run off. The column was then eluted with 400 ml each of 20%, 40%, 60%, 70%, 80%, and
100% acetone. Each fraction was evaporated down to 50 ml, neutralised with solid sodium bicarbonate and extracted with diethyl ether (2 x 100 ml). After acidification to pH 2.5 - 3 with 2M sulphuric acid the aqueous residues were extracted with ethyl acetate (2 x 100 ml). l/20th of both the neutrals and acid extracts were submitted for bioassay. 18
1.3.7 PURIFICATION OF FRACTIONS 1. 2. 3. 4 ( AUS 27A ) ON SEPHADEX LH20 Sufficient Sephadex LH20 was mixed into a slurry with the elution solvent of 95% ethanol/chloroform (9/1) to prepare a column with the dimensions 320 mm x 22 mm. About one-tenth of fractions 1, 2, 3 and 4 from AUS 27A were evaporated to near dryness, redissolved in the elution solvent (10 ml) and loaded onto the column. This mixture was then absorbed onto the column, elution solvent added and the column run at a flow rate of about 1 ml per minute. Twenty 10 ml samples were collected and l/40th of each prepared for bioassay in the usual fashion. Prior to this purification two standards, [U^C] palmitic acid and acetyl-/?
-hydroxybenzoic acid were chromatographed under the same conditions.
1.3.8 HYDROLYSIS OF ACTIVE FRACTIONS FROM AUS 12A The active fractions from AUS 12A were hydrolysed overnight at room temperature by the addition of 10% aqueous NaOH (2 ml). After acidification to pH 2 with 2M H2SO4 the solution was extracted with ethyl acetate (3 x 100 ml).
The ethyl acetate extracts were evaporated, redissolved in a small volume of methanol and methylated with excess diazomethane.
1.3.9 HYDROGENATION OF FRACTIONS 1. 2 AND 3 AUS 2
l/20th of fractions 1, 2 and 3 AUS 2 were dissolved in methanol (50 ml) and added to a round bottomed flask containing of palladium on carbon (4 mg).
The contents were then stirred under an atmosphere of hydrogen for two hours. The solution was then filtered through Whatman no. 4 paper and the filtrate evaporated to dryness.
A solution of jasmonic acid methyl ester (50 mg) in methanol (50 ml) was hydrogenated under the same conditions as a check on the efficiency of reaction. 19
1.3.10 HYDROLYSIS AND EXTRACTION OF FRACTIONS 1 TO 4 AUS 2
The first four fractions from six separate runs of AUS 2 were combined, evaporated and hydrolysed with 60% aqueous KOH (5 ml). After standing overnight at room temperature the mixture was extracted with pentane (2 x equal volumes) and then ethyl acetate (2 x equal volumes). The pH was then lowered to
2 and the solution left standing for one hour to encourage lactonisation of any compound prone to do so. This acidified mixture was then extracted with ethyl acetate (2 x equal volumes) and the organic layer combined and washed twice with
50 ml of saturated NaHC03. The bicarbonate wash was back-extracted with ethyl acetate (2 x 50 ml) and then acidified to pH 2 and extracted again with the same volume of ethyl acetate. Each organic extract and the aqueous residue were evaporated and l/100th of each submitted for bioassay.
1.3.11 SEP-PAK PURIFICATIONS
1.3.11.1 REVERSE-PHASE OF CRUDE ACID FRACTION
Crude acid fraction (10 ml) was made up to 50 ml with 0.2% aqueous acetic acid and the resultant suspension filtered and re-filtered through a #4 glass sinter until completely clarified. Then l/5th of this filtrate was transferred to a
100 ml separating funnel with a set of four Ci8 Sep-Paks attached to the outlet.
The solution was pushed through the Sep-Paks with the aid of a small peristaltic pump connected to the inlet of the separating funnel and the flow adjusted to a rate of about 5 ml/min. After the clarified acid extract has been completely pumped through, the Sep-Paks were eluted in turn with 20 ml each of 5%,
10%, 20%, 30%, 40%, 50% and 80% ethanol in 0.2% aqueous acetic acid and finally with 20 ml of 100% ethanol. The remaining filtrate was treated in the same fashion and each corresponding set of eluents combined. l/100th of each fraction was submitted for bioassay. 20
1.3.11.2 REVERSE-PHASE OF A METHYLATED FRACTION FROM SEP-PAK PURIFICATION OF CRUDE ACID FRACTION The combined 30% ethanol fractions from Methods 1.3.10.1 were evaporated to remove ethanol, acidified to pH 2 and extracted with ethyl acetate (3 x 100 ml). The organic layer was evaporated, redissolved in methanol (2 ml) and methylated with excess diazomethane (1 hr, room temperature). After evaporation the residue was dissolved in 95% ethanol (5 ml) and diluted to 50 ml with 0.2% acetic acid. 10 ml of this solution was then pumped through the four pre-conditioned Sep-Paks on the apparatus mentioned in the previous paragraph (Methods 1.3.11.1) and the eluent collected. The Sep-Paks were then washed with 30%, 40%, 50%, 60% and 70% ethanol in 0.2% aqueous acetic acid followed by 95% aqueous ethanol and each of these fractions collected. Four additional runs were conducted with the remaining 40 ml of extract and each appropriate ethanol eluent combined. l/100th of each fraction was then hydrolysed overnight (room temperature) with 2 drops of 60% KOH, evaporated to an aqueous residue, acidified to pH 2 with 2M H2SO4 and extracted three times with ethyl acetate (20 ml). Each ethyl acetate extract was evaporated and 1/100th submitted for bioassay.
20%, 30% and 40% eluents from separate reverse phase Sep-Pak purifications of crude acid fraction (Methods 1.3.11.1) were processed in the manner of the 30% eluent above. Differences were: the period of methylation was thirty minutes rather than overnight, the initial solvent for application to the Sep- Paks was made up to 20%, 30% and 40% ethanol for the 20%, 30% and 40% series respectively and the eluent solvents covered the range initial solvent to 100% ethanol. 1/100th of each Sep-Pak fraction was submitted for bioassay. 21
1.3.11.3 REVERSE-PHASE OF HYDROLYSED CRUDE ACID FRACTION To crude acid fraction (10 ml), 60% aqueous KOH was added (1 ml) and the mixture left at room temperature overnight. After adjusting the pH to 7 with 2M H2SO4 the solution was diluted to 150 ml with 0.2% acetic acid and treated in the same manner as the crude acid fraction (Methods 1.3.10.1).
Fractions were eluted with 5%, 10%, 20%, 30%, 40%, 60% and 80% ethanol in 0.2% acetic acid,then 100% ethanol, and each of these fractions were extracted and bioassayed (l/100th of each fraction) in the usual way. Fractions were labelled 5HAF, 10HAF, 20HAF, 30HAF, 40HAF, 60HAF, 80HAF and 100HAF.
1.3.11.4 REVERSE PHASE OF HYDROLYSED FRACTIONS 1. 2.
3. AND 4 (AUS 2) Fractions 1 to 4 inclusive an AUS 2 purification sequence (Methods 1.3.2.2) were treated in identical manner to the hydrolysed crude acid fraction (Methods 1.3.10.3). The resultant Sep-Pak eluents were 5H, 10H, 20H, 30H, 40H, 60H, 80H and 100H. In this case l/50th of each fraction was bioassayed.
1.3.12 THIN-LAYER CHROMATOGRAPHY
1.3.12.1 30% ETHANOL REVERSE-PHASE SEP-PAK ELUENT
About l/30th of the 30% ethanol extract from Methods 1.3.11.1 was applied to a silica TLC plate and dried under a stream of warm air. The plate was then run with toluene/ethyl acetate/acetic acid (15/3/1). When the solvent front had moved to about 2 cm from the top edge, the plate was removed and air dried. The plate was then divided into five equal regions; Rfs 0-0.2, 0.2-0.4, 0.4-0.6, 0.6- 0.8, 0.8-1.0. Each of these regions was scraped, eluted and l/3rd of each submitted for bioassay. 22
1.3.12.2 FRACTION 40H
l/100th of fraction 40H (Methods 1.3.11.4) was treated in the same fashion as the 30% ethanol fraction above (Methods 1.3.11.1) except the developing solvent was hexane/ethyl acetate/acetic acid (50/50/1) and only four equivalent zones were scraped from the plate, eluted with methanol (5 ml) and bioassayed.
1.3.12.3 ACETYLATED METHYLATED 40H One quarter of fraction 40H was acetylated and methylated in the usual manner and l/20th of this mixture applied to the origin of a silica TLC plate. After running in hexane/ethyl acetate (60/40) the plate was divided into six equivalent zones and each scraped into separate glass sintered filters and eluted twice with methanol (5 ml). The methanol solutions were then shaken for a few minutes with a two drops of 60% KOH and the eluents then left overnight to hydrolyse. The methanol was then evaporated and the aqueous layers acidified to pH 3. The mixtures were then extracted with ethyl acetate (2 x 20 ml) and these extracts evaporated under a stream of nitrogen and the samples submitted for bioassay.
1.3.12.4 ZONES Rf 0-0.2 AND 0.2-0.4 FROM TLC PURIFICATION OF 30% ETHANOL SEP-PAK Half of zones 1 and 2 (Rf 0-0.2, 0.2-0.4) after TLC purification of the 30% ethanol Sep-Pak fraction were combined, acetylated, rerun and eluted under the same conditions as outlined in Methods 1.3.12.1. The one exception was that four equivalent zones were collected and bioassayed.
1.3.13 GC PURIFICATION AND ANALYSIS
1.3.13.1 FRACTION 6 AUS12A A small amount (l/10th) of fraction 6 AUS 12A was reacetylated, methylated and fractions collected through a manual trapping port. A 2 m column 23 packed with 3% QF 1 on chromasorb W (100/120 mesh) was used with a nitrogen flow rate of 30 ml/min. All runs were isothermal (130°C). Three zones were trapped; from solvent front to 10.3 minutes, 10.3 to 13.0 minutes, and 13.0 to 17.0 minutes. Samples were collected at the appropriate times in glass U-tubes packed with glass wool and cooled by immersion in a mixture of dry-ice and ethanol. U-tubes were washed with aliquots of dichloromethane (3x3 ml) and these washings combined. The three fractions were submitted for bioassay.
1.3.13.2 FRACTIONATION OF 3 AUS 12A A small portion (l/25th) of active fraction 3 from AUS 12A was acetylated, methylated and fractionated by multiple injections using the same column and U-tubes as above (Methods 1.3.13.1) but different oven temperature. An initial temperature of 135°C was maintained for 12 min and then raised to 180°C at a rate of 6°C per min. Nine zones were collected and eluted in the same manner as outlined for fraction 6 (AUS 12A). These zones are shown in Figure 1.4.9.
1.3.13.3 FRACTIONATION OF HYDROGENATED FRACTIONS 1. 2. 3 AUS 4 The hydrogenated extract was acetylated, methylated and aliquots fractionated by the method used in Methods 1.3.13.1. After trapping the five samples were prepared for bioassay in the usual manner.
1.3.13.4 FRACTIONS 30H. 30HAF. 40M20.80HAF. 4QM20H AND
40M30K
About l/40th of fraction 30H was acetylated, methylated by the usual methods and purified by GC under the conditions of Methods 1.3.13.1. Oven temperature profile in this case was 130°C for 10 min and a rise to 190°C at 8°C per minute. Six fractions were collected in multiple injections of each crude solution (the zones are shown in Figure 1.4.16) and then hydrolysed, extracted and bioassayed in the usual way. Methyl jasmonate and penta-acetylglucose
(racemic) were injected beforehand in an attempt to localise activity with respect to these compounds. Fraction 40M20H was first hydrolysed overnight before acetylation, methylation while 40M30 was acetylated and then methylated after addition of 2 |il of 60% KOH (this becomes 40M30K). All the other extracts:
30HAF (l/40th), 40M20 (l/20th) and 80HAF (l/40th), 40M20H (l/20th) and
40M30K (l/20th) were trapped in the same manner as 30H.
1.3.13.5 FRACTIONS 1. 2. 3 AND 4 AUS 27A
60% aqueous KOH (2 ml) was added to 40 ml of combined fractions 1, 2,
3, and 4 from AUS 27A and this mixture was left to stand for 24 hr at room temperature. After adjustment to pH 2 with 2M H2SO4 the solution was extracted twice with ethyl acetate (100 ml). This extract was evaporated, methylated with diazomethane and evaporated again to a syrupy residue.
Some of this residue was extracted of hexane (1 ml) and part of this hexane extract purified by trapping on preparative GC (QF1, 50/6°C) in an attempt to identify at least one of the early eluting components. Solid insertion probe MS and IR analysis of a dichloromethane solution of the trapped peak were conducted to identify the compound.
100 pi of the ethyl acetate extract was also purified by GC (QF1, 40/6°C) and half treated appropriately before submitting for bioassay.
1.3.14 PREPARATION OF SAMPLES FOR NORMAL-PHASE HPLC
Samples arising from reverse phase Sep-Pak fractionation were pre-treated with a silica Sep-Pak before normal phase HPLC. Dried fractions were dissolved in warm isopropanol (1 ml) and hexane (9 ml) was added to this solution. In most cases the solutions became very turbid. The resultant 10% isopropanol in hexane mixture was then forced through a single silica Sep-Pak attached to a syringe. This
Sep-Pak was then eluted with 10% isopropanol in hexane (10 ml) followed by 25
20% isopropanol in hexane (10 ml), isopropanol (10 ml) and finally methanol (10 ml). Equal aliquots of each was sent for bioassay.
1.3.15 HPLC PURIFICATION
Half of fraction 40M20 was purified on a silica semi-prep column (300 mm x 7.8 mm ED, 10 Jim particle size) using 5% isopropanol in hexane as mobile phase. Detector monitor wavelength was 330 nm. Other details of the instrumentation are outlined in Materials 1.2.7. Five fractions were collected and l/10th of each bioassayed. The monitored chromatogram, trapped regions and activity of each trappate are shown in Figure 1.4.17 along with the retention time of methyl-ABA run under the same conditions.
1.3.16 IDENTIFICATION OF THE UNKNOWN PEAK
1.3.16.1 INSTRUMENTAL After four injections of 40M20 (about 1/4 of the total fraction) onto the silica HPLC column and collection and combination of the relevant peaks the elutant was evaporated and analysed first by IR, then proton NMR and 2D proton
NMR and finally by Cl and high resolution El GCMS. Conditions for these analyses are outlined in Materials 1.2.8, 1.2.9 and 1.2.10.
1.3.16.2 HYDROLYSIS AND GCMS
A small amount of the peak trapped by HPLC was dissolved in methanol (0.1 ml) and then 0.4 ml of water and one drop of 60% KOH added. The solution was left overnight at room temperature, acidified to pH 2 and extracted with ethyl acetate (5 ml). This extract was evaporated to dryness, redissolved in a few drops of methanol and methylated. The methylated hydrolysate was then analysed by
GCMS. 26
1.3.17 SOME CHROMATOGRAPHY WITH ACC AND A DERIVATIVE
1.3.17.1 PREPARATION OF N-ACETYL-ACC A few milligrams of ACC was dissolved in of pyridine (10 jil), acetic anhydride (10 jil) added and the mixture heated at 80°C for two hours. The pyridine and acetic anhydride was then removed by evaporation under reduced
pressure at 50°C. The residue was dissolved in methanol (30 |il), 1/10th applied to the origin of a silica TLC plate and eluted with hexane/ethyl acetate/acetic acid (50/50/1). The chromatographed material was visualised under a UV lamp at 312 nm. About 0.5 mg of ACC was also co-chromatographed on the same plate. Another similar preparation from 2 mg of ACC was submitted for bioassay.
1.3.17.2 REVERSE-PHASE SEP-PAK OF N-ACETYL-ACC AND
ACC The remainder of the synthetic N-acetyl-ACC solution was diluted to 5 ml with 0.2% aqueous acetic acid was applied to the Sep-Pak apparatus (Methods 1.3.11.1) and eluted with 10 ml of 5%, 10%, 20% 30% 40% 50% and 100% ethanol in 0.5% aqueous acetic acid. Each eluent was evaporated to dryness, redissolved in methanol, spotted onto a silica plate and visualised under a UV
lamp.
One mg of ACC was treated in the same manner.
1.3.17.3 OTHER DERIVATISATION OF ACC
Attempts were made to analyse ACC by formation of the methyl and silyl
derivatives and GCMS. About 100 ng of ACC was mixed with 0.5 ml of various solvents and excess diazomethane added. Another 100 ng sample was mixed with
30 |il of trimethylchlorosilizane and heated to 80°C for 30 min. 27
1.3.18 PREPARATION OF SAMPLES FOR BIOASSAY
1.3.18.1 COLUMN CHROMATOGRAPHY SAMPLES
Eluents from the various AUS columns were simply evaporated under a stream of nitrogen. Samples from both charcoal and Sephadex LH20 columns were also treated in this manner.
1.3.18.2 GC TRAPPATES
The dichloromethane washings from the glass wool packed U-tubes were shaken for a few minutes with a few drops of 10% aqueous sodium hydroxide and after standing at room temperature overnight the dichloromethane was removed under a stream of nitrogen. The aqueous layer was acidified to pH 3 with 2M
H2SO4 and extracted twice with ethyl acetate (5 ml). These extracts were combined and then evaporated slowly under nitrogen.
1.3.18.3 HPLC FRACTIONS
Ethanol/water eluents were first concentrated under reduced pressure to remove ethanol, then acidified to pH 2 and extracted twice with equivalent volumes of ethyl acetate. Derivatised fractions were hydrolysed with 10M KOH before initial evaporation of the ethanol. Hexane/isopropanol eluents were hydrolysed if necessary with 10M KOH, the organic layer evaporated followed by acidification to pH 2 and extraction with ethyl acetate. These extracts from both normal and reverse phase HPLC were then evaporated carefully under nitrogen.
1.3.18.4 TLC FRACTIONS
The relevant bands were scraped from the plate into small sintered glass tubes and washed twice with 5 ml of methanol. Underivatised and acetylated samples were evaporated and submitted for bioassay while methylated samples were first hydrolysed by addition of a few drops of 60% aqueous KOH before evaporation, and extraction with ethyl acetate (twice, triple volumes). These ethyl acetate extracts were then evaporated as above. 28
1.3.18.5 GENERAL All samples for bioassay were finally transferred to small smoked-glass tubes (25 mm x 5 mm) and the transfer solvent (usually methanol) evaporated to near dryness. The tubes were then stoppered with the proper polymer lids and these lids secured shut with adhesive tape. The samples were then sent by air mail to Dr D. J. Osborne who carried out all bioassays.
1.3.19 BIOASSAY
All bioassays were carried out by Dr. D. J. Osborne at the Unit of Developmental Botany, University of Cambridge and later at the Weed Research Organisation and Department of Plant Sciences, Oxford University. Bean seedlings (about 14 days old) were used in the bioassays. The method of Osborne et. al., (1972) was used with one change; dried samples were used rather than agar diffusates. These samples were dissolved in 25% aqueous methanol (100 q.1) and 10 (ll of this solution applied to the agar discs on the pulvinar side of the abscission zone. Usually 10 explants were treated with the same solution. After a period of 24 hrs the explants were lightly tapped with a pencil at the excised section so that positive abscission scores were recorded if the abscission zone fractured. A total score was recorded out of ten (if ten plants were used) or out of the total number of plants used in the assay.
As well as samples scores were determined for 25% methanol and a 5 x 1(H M solution of ABA. Toxic responses were also noted.
1.3.20 DETERMINATION OF ETHYLENE
Ethylene assays were also performed by Dr D. Osborne. Ethylene produced by petiole segments was trapped by the method in Osborne et. al. (1972) and assayed by GLC following the procedures outline by Jackson and Osborne (1970) and Porter and Van Steveninck (1966). 29
1.3.21 SYNTHETIC WORK
1.3.21.1 3.4 DIMETHOXYCINNAMIC ACID METHYL ESTER
An outline of the method is given in Figure 1.3.1.
Caffeic acid (trans isomer, 10 gm) was first purified by dissolving in boiling distilled water and cooling to room temperature. The pale yellow crystals which formed were filtered, washed with cold water (50 ml) and dried at low pressure over anhydrous CaCl2-
Recrystallised caffeic acid (200 mg) was dissolved in methanol (10 ml), excess etherial diazomethane added and the solution left at room temperature for 2 hrs. The preparation was then evaporated under reduced pressure. The residue was then dissolved in ethyl acetate (50 ml) and washed with saturated NaHCC>3
(10 ml). The ethyl acetate layer was then separated, evaporated and the residue dried at reduced pressure over anhydrous CaCl2 and stored. Some of this compound was dissolved in chloroform for IR analysis.
1.3.21.2 3. 4 DIMETHOXYCINNAMIC ACID
One preparation from above (Methods 1.3.21.1) was redissolved in a small volume of ethanol and 2M NaOH (50 ml) added. This solution was heated to 80°C for four hours. After cooling, the aqueous residue was extracted with ethyl acetate (2 x 100 ml), acidified to pH 2 with 2M H2SO4, and re-extracted with ethyl acetate (2 x 100 ml). The second ethyl acetate extracts were combined dried with solid anhydrous Na2S04, filtered, evaporated, dried over CaCl2 and stored.
1.3.21.3 3.4 DIMETHQXY CINNAMQ YLM AL ATE
The dimethoxycinnamic acid (Methods 1.3.21.2) was dissolved in oxalyl chloride (5 ml) and heated slightly (40°C) and mixed occasionally over a period of one hour. After evaporation of the excess oxalyl chloride, malic acid (70 mg in 10 ml of pyridine) was added and the mixture heated to 80°C for 4 hrs. This solution was then evaporated to dryness under reduced pressure. 30
OH ■OCH, HC= HC=CH> Methanol ------> CH2N2 in diethyl ether ( 2 hrs)
CAFFFIC ACID 2M NaOH, 80 C, 4 hrs
;—OH
Malic Acid
Pyridine
O— HC=CH
3.4 DIMETHOXYCINNAMOYLMALATE
FIGURE 1.3.1 Synthetic scheme for the synthesis of 3,4 dimethoxycinnamoylmalate. 31
1.3.21.4 PURIFICATION OF DIMETHOXYCINNAMOYLMALATE
About l/20th of the preparation (Methods 1.3.21.3) was purified on a reverse phase semi-prep column of the dimensions used in Methods 1.3.15.
Mobile phase was 40% ethanol in 0.2% aqueous acetic acid and the peaks eluting with apexes at 5 and 6.5 min was collected. The larger peak following these had a retention identical to dimethoxycinnamic acid.
The eluent containing the first peak was evaporated to dryness and redissolved in methanol (500 |il). Sufficient diazomethane was then added for the solution to maintain a pale yellow colour. After standing for a few minutes the solution was evaporated, redissolved in 10% IPA in hexane and purified by normal phase HPLC. A silica semi-prep column of the same dimensions as above was used together with a mobile phase of 5% IPA in hexane. The peak eluting between
8.5 and 10 minutes was collected and the solvent removed. The residue was redissolved in 1 ml of deuterochloroform and a proton NMR performed on this sample. The same sample was later analysed by solid probe MS.
1.3.21.5 1 -METHOXYSUCCINIC ACID DIMETHYL ESTER
The preparation of this compound was based on the method of Purdie
(1885) except that the amount and proportion of reactants was changed, see Figure
1.3.2 for an overview. Maleic anhydride (2 gm) was refluxed in methanol (60 ml) for 6 hrs and after cooling was added to a prepared mixture of sodium (1 gm) in methanol (10 ml). After standing for 30 minutes water (5 ml) was added and the solution refluxed for two hours to destroy residual sodium methoxide. This solution was then cooled, filtered, evaporated, acidified to pH 2 with 2M H2SO4 and extracted with diethyl ether (2 x 50 ml). The ether extract was evaporated and some of the residue redissolved in methanol for purification by HPLC. 32
OH
Methanol
Reflux, 6 hrs
Sodium Methoxide
MALEIC ANHYDRIDE
och3 H,C^/
wo
1 -METHOXYSUCCINATE DIMETHYL ESTER
FIGURE 1.3.2 Synthetic scheme for the formation of methoxysuccinate dimethyl ester from maleic anhydride. 33
1.3.21.6 HPLC PURIFICATION OF 1-METHOXYSUCCINATE METHYL ESTER A sample of the residue was purified using a reverse phase semi-prep column and a mobile phase of 30% methanol in 0.2% acetic acid. After injection
the methanol concentration was increased to 100% over a period of 10 minutes. Detector wavelength was set to 210 nm and each of the three major peaks were
collected separately. The eluents were evaporated to dryness redissolved in methanol, methylated with diazomethane and analysed by GCMS. A small portion (about 100 mg) was hydrolysed, extracted and sent for bioassay. 34 1.4 RESULTS
1.4.1 EARLY RESULTS
Table 1.4.1 shows the activity resulting from the initial fractionation steps of the crude acid fraction. It can be readily seen that activity can be extracted from the aqueous residue after evaporation of the ethanolic bean leaf extract. Most activity is extracted into the ethyl acetate layer after the removal of neutrals and ethyl acetate was more efficient at this partitioning than diethyl ether. Some activity remains in the aqueous layer despite the fact that this layer was exhaustively extracted until the ethyl acetate was colourless. The initial acid extract with ethyl acetate was invariably dark brown or a brownish yellow colour.
It was later found that more activity in the aqueous layer could be extracted into ethyl acetate if the aqueous layer, previously extracted with ethyl acetate, was first hydrolysed with KOH overnight, acidified and than re-extracted. It was decided then that some fractions would be hydrolysed before further purification.
Methylation of the crude acid fraction was found to reduce activity markedly while acetylation has no measurable effect.
1.4.2. EXTRACTION OF HYDROLYSED FRACTIONS 1 TO 4 (AUS 2)
Bioassay results for these hydrolysed fractions are shown in Table 1.4.2.
The pentane and ethyl acetate neutral extracts showed very low activity as did the aqueous residue while the ethyl acetate extract of the bicarbonate wash before acidification was inactive. After acidification the ethyl acetate extract of the bicarbonate wash gave very high activity so this confirmed that the active compound(s) were indeed acidic, extractable into ethyl acetate and not prone to lactonisation.
Ethylene assays of the very active ethyl acetate fraction showed it to be slightly stimulatory in the short term (up to 1.5 hr) and strongly stimulatory over a longer period (5 hr). 35
FRACTION ACTIVITY
Crude Ethanol Extract Very Active
Neutral Ether Extract Inactive
Ether Acid Extract Slightly Active
Ethyl Acetate Acid Extract Very Active
Aqueous Residue Active
(After Neutral and Acid Extraction)
TABLE 1.4.1 Activity of fractions from preliminary
purifications of the ethanolic bean leaf extracts.
Activity scale is: Very active ->70% abscission,
Active: 30 to 70%, Slightly active: <30%. Inactive:
not significantly different from 25% methanol
control. 36
FRACTION ACTIVITY
Pentane Neutrals S*
Ethyl Acetate Neutrals S^
Ethyl Acetate - Bicarbonate Washed m
Ethyl Acetate Extract of Bicarbonate Wash VA
Aqueous Residue S\
TABLE 1.4.2 Bioassay results for the extracts of hydrolysed AUS 2
fractions 1 to 4 (Methods 1.3.10). SA: Slightly
active, VA: Very active, NA: Not active. Activity scale
is: Very active ->70% abscission, Active: 30 to
70%, Slightly active: <30%. Inactive: not
significantly different from 25% methanol control. 37
1.4.3 SILICA COLUMN PURIFICATIONS
An outline of the interrelationship between some silica columns together with activity of the various fractions is shown in Figure 1.4.1. In general these large scale purifications showed that under the conditions used some activity elutes successfully from silica columns.
AUS 2 used the same elution solvent as AUS 1 but the column was larger and consequently capable of purifying greater amounts of crude acid fraction.
AUS 11C was a re-purification of fractions 1, 2, 3 and 4 from AUS 2 under the same conditions used for AUS 2. Here the capacity of the column has been increased because the very polar compounds (eg carbohydrates, polyaromatic phenolics) have been previously removed by AUS 2.
Acetylated active fractions from columns AUS 2 and AUS 11C were chromatographed on AUS 12A and 27A respectively. Both used the same elution solvent but the loading on AUS 27A was greater. Not surprisingly, the bioassay results were similar for these two columns: active compound(s) eluting in the early fractions.
GC studies of methylated fractions from AUS 2, 11C, 12A and 27A
(results not shown) illustrated that there was not a high level of effective purification. For example the active or very active fractions from AUS 2 contained probably more than 80% of the compounds from the crude acid fraction that could pass through the QF1 column under the conditions used (isothermal, 135°C).
Additionally there was little difference between the chromatograms of these first four fractions.
However the main purpose of these early purifications was to remove very polar compounds and to provide a store of raw materials for further purification. 38
AUS 1 (10 mis) of crude acid fraction ------Fractions 5 to 30 - Active
AUS 2 (100 mis of crude acid fraction)
f1 f2 f3 f4 f7 VA VA VA VA SA
(COMBINED) Fractions Fractions From 13 Runs From 6 Runs of AUS 2 of AUS 2
Acetylation
AUS 27A AUS 11C
f1 f2 f3 f4 f11 A VA VA A SA SAVA VA A A
(COMBINED) I Acetylation
AUS 12A (f1, f2, f3 and f4)
f1 f2 f3 f4 f5 f6 A A VA A A A
FIGURE 1.4.1 Silica column purifications of crude acid fraction from the ethanolic extract of bean leaf. Activity scale is: VA - >70% abscission, A - 30 to 70%, SA - <30% abscission. All other fractions (not shown) were inactive. 39
1.4.4 SILICA SEP-PAK PURIFICATIONS OF AUS COLUMN FRACTIONS
The normal phase Sep-Pak purification of fraction 6 (AUS12A) showed that activity bound quite tightly to the silica, is not eluted by dichloromethane but is displaced by methanol.
Using an equivalent fraction from AUS 27A (fraction 3) and an eluting solvent of dichloromethane/methanol (2/1) the activity was spread over three zones. The first two 10 ml fractions were active along with the sixth and the tenth fraction.
1.4.5 OTHER COLUMN PURIFICATIONS
Two other small scale columns, activated charcoal (Methods 1.3.6) and
Sephadex LH20 (Methods 1.3.7) were conducted in an attempt to uncover some structural insight into SF. Activated charcoal has a great affinity for certain compounds especially carbohydrates, while Sephadex LH20 binds aromatics more strongly than aliphatics of similar molecular weight.
Figure 1.4.2 shows the bioassay results for the acetone eluents from the activated charcoal column. In this case there are two quite distinct zones of activity, the first in the 60% fraction and the second in the 80 and 100% elutants.
This result suggests that there is probably a polar active compound and another less polar substance responsible for senescence activity. The occurrence of two zones raised the possibility that the more polar compound may be a glucose or carbohydrate ester of the less polar compound. This was however a very tentative conclusion based mainly on the premise that sacharrides such as glucose commonly form esters with a wide variety of plant metabolites. These conjugates are extremely common in the plant world and span a range of compounds including the phenolic acids like caffeic acid (Andary et al., 1982) and the plant hormones like ABA (Koshimizu et. al, 1968).
Figure 1.4.3 shows the relative amounts of standards (palmitic acid and p
-acetoxy ben zoic acid) in comparison with SF activity in the fractions eluted from 40
1.0 - EJ Activity
0.8 -
Relative 0.6 - abscission rates
0.4 -
0.2- 26
0.0-
40 60 70 80 100
Percentage of aqueous acetone
FIGURE 1.4.2 Relative abscission scores for aqueous acetone eluents from the activated charcoal column (Methods 1.3.6) 41
[U ortho-ecetoxybenzoic acid H Senescence activity H Palmitic acid
1.00 -
0.80 -
Relative Amounts
Standards and Senescence activity
0.20 -
Fraction Numbers
FIGURE 1.4.3 Relative abscission scores and relative amounts of standards (14C-Palmitic acid and o -acetoxybenzoic acid) of fractions eluted from a Sephadex LH20 column (Methods 1.3.7) 42 the Sephadex LH20 column (Methods 1.3.7). Here the distinction between the palmitic acid and the acetoxybenzoic acid band is very clear as the aliphatic compound elutes earlier than the aromatic compound although they have similar molecular weights (palmitic acid 256 daltons, acetoxybenzoic acid 180 daltons). A large proportion of SF activity co-elutes with the palmitic acid standard so again a tentative observation could be made that the band of activity was due to an aliphatic compound. The non-aromatic nature of SF was earlier suggested by Milborrow
(unpublished results) because the compound suspected to be responsible for activity did not have significant UV absorption or mass spectral properties consistent with an aromatic compound. In El mode the low mass fragments did not include m/z 77, 91 or 105 (typical of a simple aromatic) and additionally the complex array of ions and the lack of intense parent or other high mass ions was more indicative of an aliphatic compound.
1.4.6. SENESCENCE ACTIVITY. ETHYLENE AND ABA
One of the first concerns after senescence activity was found to elute reproducibly from silica columns was to confirm that abscission activity and stimulation of ethylene production were present in the same fractions and that abscission activity was not due to ABA (a known accelerator, Porter and
Steveninck, 1966).
Fractions 1, 2, 3 and 4 from silica column AUS 2 were found to be both very active in the stimulation of abscission and the production of ethylene as were some of the active fractions from AUS 1 (5 to 20). The ABA content and elution behaviour from these two columns were determined in two ways; analysis of endogenous ABA by GC with electron capture detection for fractions from AUS 1 and the addition of 14C-labelled ABA to the crude acid fraction before elution from a silica column (AUS IS - equivalent to AUS 2) followed by comparison of cpm with senescence activity. The results of the ABA analysis in fractions from silica column AUS 1 (Figure 1.4.4) clearly show that the abscission activity is 43
ABA (ng/fraction) i Senescence activity (arbitary units)
Senescence Activity and ABA Content
Fraction Numbers
FIGURE 1.4.4 Endogenous ABA content and senescence activity for fractions from silica column AUS 1. 44 distinguishable from ABA. Significant SF activity spreads from fractions 7 to 19 while ABA occupies a larger span (fractions 7 to 35) but elutes later. The most significant difference is the discrimination between the peaks of senescence activity and ABA concentration. A major peak of activity occurs at fraction 12 together with a minor peak at fraction 27. Neither peak is coincident with the highest ABA concentration (fraction 21).
A similar result occurred after the experiment with labelled ABA and AUS
IS. This is represented graphically in Figure 1.4.5. Again the distinction between
ABA elution and senescence activity is obvious as maximum senescence activity occurs in fraction 2 and the ABA concentration peaks in fraction 4.
Ethylene assays were conducted on various fractions from the large scale silica column purifications and it was found that the active fractions did promote ethylene production. More evidence of a more substantive link between SF and ethylene promotion was found after further purification of some active (and ethylene stimulating) fractions from AUS 2. Fractions 1, 2, 3 and 4 from AUS 2 were re-purified on AUS 11C, the first four fractions taken, acetylated and chromatographed on AUS 12A. Fraction 6 from this column was purified by Sep-
Pak (Methods 1.3.3).
The ethylene production of the resultant three fractions together with a control are shown in Figure 1.4.6. It can be seen that when water is applied to the agar disc 1.5 hrs after incision the ethylene production falls over the next two measurement periods. The acetone eluent follows a similar path even though one experiment shows a small rise 1.5 hr after treatment with the eluent. Both the dichloromethane and methanol eluents show similar responses after application of the respective eluents; a rise in ethylene production measured at 3 hr followed by a fall at 4 hr to an amount that was still higher than the pre-treatment ethylene level.
The one exception was experiment 2 with the methanol eluent where there was a slight rise in ethylene at 4 hr. Comparison of these two eluents reveals that in the one experiment there is less stimulation with the methanol eluent and in the other 45
ABA (cpm x 1/1000) per fraction
Senescence activity (arbitary units) Senescence Activity and ABA Counts
Fraction Numbers (AUS 1S)
FIGURE 1.4.5 Senescence activity and 14C ABA cpm for fractions from silica column AUS 1S. 46
Production Oil)
Tim* (hr*) Tim* (hr*)
□ Experiment 1 0 Experiment 2
VERY ACTIVE Acetone - INACTIVE Beginning of treatment
Ethylene Beginning o( treatment Production Qil)
Tim* (hr*)
FIGURE 1.4.6 Ethylene production by excised bead explants after treatment with water, and three Sep-Pak fractions from a purification of Fraction 6 AUS 12A. Two experiments were conducted with each extract and evolved ethylene sampled and measured 1.5, 3 and 4 hrs after excision. Each eluent was applied 1.5 hrs after excision. 47 there is a greater effect. However the amount of total fraction used in the treatment was different, twice the dichloromethane eluent was used compared to the methanol so it suggests that the greater SF activity was coincident with greater promotion of ethylene production.
1.4.7 SENESCENCE ACTIVITY AND METHYL JASMONATE
Earlier work by Ueda and Kato (1980) demonstrated that jasmonic acid and methyl jasmonate possessed senescence-inducing properties. Consequently a few experiments were carried out to determine if JA and MeJ were present in the active fractions prepared in these experiments and if the induction of senescence could be attributed to the JA content of these fractions.
One small silica column (AUS LM1) and a normal-phase Sep-Pak purification of methylated fraction 2 AUS 12A were eluted as described in Methods
1.3.2.7 and 1.3.3.3 and the methyl jasmonate assayed by GC (Methods 1.3.5).
The results are shown in Figure 1.4.7. The AUS LM1 column (top graph) shows two fractions, 20% DCM and acetone eluents have good SF activity while methyl jasmonate appears in only one; the acetone eluent. This suggests that there are at least two compounds responsible for activity and one at least co-elutes with MeJ using these particular solvents. However co-elution with acetone is not particularly significant in this case since acetone is really a column purge which has undoubtedly washed off a range of compounds with quite different polarities.
The lower graph makes matters clearer since activity elutes in fractions quite distinct from MeJ. Activity eluted mainly in the hexane fraction with a smaller amount in 20% DCM and a little with 60% DCM. Methyl jasmonate elution is however spread over the 60% DCM and methanol eluents. At this stage it appeared that there maybe a little coincident activity but there is definitely SF activity quite separate from MeJ. The active compound(s) in these bean leaf fractions also show chromatographic properties quite different from those found 48
1.^ "
LM1 actons 1.0- PI ^ Senescence Activity ■
Relative 0.8- PI Methyljasmonate Content Senescence Activity ?o% and Methyljasmonate 0.6- Content
0.4-
0.2- 100% hexane 60%
0.0- m m
Sep-Pak hexane 60%
Hi Senescence Activity
Relative m Methyljasmonate Content Senescence Activity and Methyljasmonate Content methanol
20%
10%
FIGURE 1.4.7 Elution of senescence activity and methyl jasmonate from two purification sequences. Top graph represents the AUS LM1 column purification (Methods 1.3.2.7) while the bottom graph shows the results of the Sep-Pak purification of methylated fraction 2 AUS 27A (Methods 1.3.3.3). Percentage figures refer to percent dichloromethane in hexane. 49 earlier (Milborrow, personal communication) consequently the active moiety is a different compound but may be a precursor of the Milborrow and Osborne SF.
1.4.8 ACC. N-ACETYL-ACC BIOASSAY. CHROMATOGRAPHY
The bioassay results for N-acetyl-ACC (prepared as in Methods 1.3.17) and ACC are shown in Table 1.4.3. ACC is as active as ABA at an equivalent concentration of 10'3 M whereas N-acetyl-ACC has to be 100 times more concentrated (10_1 M) to produce similar abscission scores.
Silica TLC under the conditions outlined in Methods 1.3.17 gave an Rf for N-acetyl-ACC of 0.35-0.42 while ACC did not move from the origin.
Reverse phase Sep-Pak partitioning of ACC and the acetyl derivative
(Methods 1.3.17.2) showed that both elute before the major active zones following the same procedure with the crude acid extract of the bean leaves. ACC elutes with
5% ethanol in 0.5% aqueous acetic acid while N-acetyl-ACC elutes with the 20% fraction. Most active Sep-Pak fraction from the crude acid fraction is the 30% ethanol eluent.
Attempts to quantify ACC so that it could be unequivocally rejected as the active compound in particular fractions was not successful. Methylation of ACC was difficult since the free acid is barely soluble in such solvents as methanol, diethyl ether, ethyl acetate or toluene, all of which are suitable as a medium for methylation with diazomethane. Silylation using BSTFA, or TMCS was also unsuccessful for similar reason (insolubility) and although a mass spectrum was obtained of a product which was assumed to be the monosilyl derivative, the yield of product was very low. The use of pentafluorobenzyation was not explored but since this procedure has been used very successfully by Netting and Duffield
(1985) on other amino acids it would probably be very useful for sensitive ACC quantitation. 50
CONCENTRATION N-ACETYL-ACC AX ABA
10"1 M 19.5 NT NT
10‘2 M 1.5 NT NT CO o 0 19.5 1 7
10‘4 M 0 NT NT I 01
0 0 0.5 NT
TABLE 1.4.3. Abscission scores (out of 20) on bioassay of ACC,
synthetic N-acetyl ACC (Methods 1.3.17) and ABA.
Scores were the mean of two experiments. NT - not
tested. 51
1.4.9 GC TRAPPING OF AUS FRACTIONS
Milborrow (unpublished work) has shown that senescence-inducing activity can be trapped using a QF1 column after acetylation and methylation of a purified bean leaf extract and that activity can be detected after hydrolysis and bioassay. So attempts were made to reproduce this chromatography. Fraction 6
(AUS12A) was treated in the fashion outlined in Methods 1.3.13.1, the three zones collected and the GC trace are shown in Figure 1.4.8. Under these conditions the second trappate was expected to show some activity. This didn't happen, weak activity was found only in the first trappate. On the positive side, however, these results indicated that some activity could be trapped using GC methods.
A more comprehensive trapping sequence was developed for fraction 3
(AUS 12A) so that a narrower elution time could be defined (see Figure 1.4.9).
The bioassay results again indicated that activity was not coincident with elution of
MeJ but was mainly in a more volatile fraction, fraction 1 was slightly active but other very weak activity was spread over another three trapping zones. Another run was done under slightly different conditions (results not shown) but the early eluting activity was not present this time and there was some later eluting activity.
However, overall abscission scores for all the GC trapping experiments were much lower than expected and this was puzzling since in each case the amount of active fraction used was more than sufficient to provide very active bioassay results. The possibilities causing loss of activity at this stage were considered to lie in four areas. Either the active component was too involatile to pass through the column, the compound was thermally labile, the compound in question was so volatile that major losses were occurring during post column hydrolysis and extraction, or the SF activity in these fractions was due to two or more compounds which were not very active alone but in were very active in combination. Oven Temperature: 135 0 O !> o o a o c 03 co 03 o> 03 FIGURE 1.4.8 GC profile and trapping zones for fraction 6 (AUS 12A) and the bioassayed act > Methods 1.3.13.1. 52 Oven Temperature: 135°C
FIGURE 1.4.9 GC profile and trapping zones for fraction 3 (AUS 12A). The bioassayed activity is also shown. GC given in Methods 1.3.13.2. 53 54
This lack of reproducibility was a major factor in the decision to try other purification methods.
1.4.10 GC TRAPPING OF HYDROGENATED AUS 2 FRACTIONS
Hydrogenated fractions 1, 2 and 3 (AUS 2) were fractionated by preparative GC under the conditions outlined in Methods 1.3.12.3. Abscission scores followed the same pattern as the trappates from fraction 3 (AUS 12A) which was treated in identical fashion (Methods 1.3.12.1). That is, activity was present in only the first fraction, the others were inactive.
This indicated that the active compound was either unaffected by hydrogenation so did not have any double bond (or was extremely resistant to hydrogenation) or that the active compound in hydrogenated form still retains activity at about the same level. The second and third proposals are unlikely since there are no precedents for this behaviour in other hormones. Consequently the first explanation is preferred.
1.4.11 REVERSE-PHASE SEP-PAK PURIFICATIONS
Bioassay results for the Sep-Pak fractionation (Methods 1.3.11.1) are shown in Table 1.4.4 and the mean abscission scores of the five experiments represented graphically in Figure 1.4.10. There are two major zones of activity; the water eluent and the 10%, 20%, 30% and 40% ethanol fractions. Clearly the fact that the 5% ethanol fraction was inactive means that at least two senescence active compounds are present in the bean leaf extract.
After methylation of the 30% ethanol fraction, re-purification on the same
Sep-Pak apparatus (Methods 1.3.11.2), hydrolysis and extraction the bioassays showed activity in some of the eluents. These results are shown in Figure 1.4.11.
Significant activity was restricted to the 30%, 40% and 100% fractions. The high activity in the 100% ethanol fraction was a surprise finding since methylation resulting in the simple conversion of an acid to a methyl ester should not make 55
SEP-PAK ABSCISSION SCORE ETHYLENE
FRACTION EXPERIMENT NO: STIMULATION
1 2 3 4 5
WATER 1 0 1 0 1 0 8 1 3 None
5% 0 - 0 - - Not Tested
10% 9 9 2 8 1 8 Not Tested
20% 1 0 - 6 6 1 8 Not Tested
30% 1 0 1 0 1 0 9 1 9 Positive
40% 7 - 8 - 5 Not Tested
50% 0 0 - - - Not Tested
80% 0 0 - - - Not Tested
100% 0 0 - - - Not Tested
25% Methanol 0 1 0 - 6 None
5X1 O'4 M ABA 8 6 8 - 9 None
TABLE 1.4.4 Bioassay results for the Sep-Pak fractions
(Methods 1.3.10.1), the 25% methanol control and
50%, 80% and 100% ethanol in 0.2% aqueous
acetic acid. The ethanol used was 95% constant
boiling point mixture. Values are shown for five
experiments, 1 and 2 were conducted on one day, 3
and 4 on another and 5 on another. Experiments
1, 2, 3, and 4 used 10 explants. Experiment 5
used 20 explants. 56
Q Mean Abscission Score 10 - S / 7] / B / / / / / / / / / / / / / / / / / 6 - / / / / / / / / / [A / y / / 30% / / ki / Water / / 4 - V\ / 20% / / 40% 10% / / / / / y\ / ]A / / / / / 2 - / / / / / / / / 80% / / / / / / / / / / / 50% 1 00% a
Sep-Pak Fractions, Control (A) and 0.0005 M ABA (B).
FIGURE 1.4.10 Abscission scores (out of 10) for Sep-Pak fractions (Methods 1.3.11.1) from the purification of crude acid fraction, control (25% methanol) and 5 x10-4 M ABA. Values are the mean of five experiments shown in Table 1.4.4. Error bars show standard deviation. 57
Mean Abscission Score (out of 10)
Sep-Pak Fractions, Control (A) and 0.0005M ABA (B)
FIGURE 1.4.11 Abscission scores (out of 10) for Sep-Pak fractions from the purification of 30% ethanol eluent from the first Sep- Pak fractionation of the crude acid extract. The 30% ethanol eluent was methylated overnight before Sep-Pak elution (Methods 1.3.11.2). Scores for control (25% methanol) and 0.5 mM ABA are also shown. 58 such a dramatic difference to the polarity. Experience in this laboratory has shown that such a conversion necessitates elution by a solvent with a 10% greater proportion of ethanol. For instance jasmonic acid will elute from this four Sep-Pak system with 30% ethanol whereas methyl jasmonate needs 40% for complete displacement. Consequently there had to be some other process or processes involved - possibly trans-esterification or methylation of more than one functional group.
A repeat of this procedure using a shorter methylation time and a different range of eluting solvents (Methods 1.3.11.2) gave quite different results. There was no activity in the 100% ethanol fraction, rather an expected shift of the major activity to an eluent at a slightly higher (by 10%) ethanol concentration. The bioassay results for the Sep-Pak purifications of this methylated 30% fraction as well as the 20% and 40% eluents from the reverse phase purification are shown in
Figure 1.4.12.
Of these fractions with significantly greater activity than any other were
40M20 and 40M30 so further purifications used these fractions. Hydrolysis of a
10 ml aliquot of the crude acid extract and reverse phase Sep-Pak fractionation (the
HAF fractions from Methods 1.3.11.3), and bioassay gave a spread of activity illustrated by Figure 1.4.13. Clearly there is a somewhat different pattern than similar bioassays done on the crude acid fraction (Figure 1.4.10). There is activity both before and after hydrolysis in fractions 10%, 20%, 30% and 40% but after hydrolysis the activity in later fractions (60%, 80% and 100%) increased relative to this early activity. At this stage the situation was probably further complicated by the likelihood that there may be more than one compound responsible for promotion of abscission and that these compounds are released from conjugates by hydrolysis.
Very similar results were obtained with the H series (Figure 1.4.14), ie the reverse phase Sep-Pak runs with hydrolysed Fractions 1, 2, 3 and 4 AUS 2
(Methods 1.3.11.4). The only significant difference was the slightly lower activity 59
20% Sep-Pak
30% Sep-Pak
40% Sep-Pak Relative Abscission Scores
30%
50% 60% 70%
1 00%
Sep-Pak eluents
FIGURE 1.4.12 Relative abscission scores for three separate Sep-Pak experiments. The first involves elution of the 20% Sep-Pak fraction from the crude acid extract purification (Methods 1.3.11.1) with various ethanol- aqueous acetic acid eluents, after methylation (Methods 1.4.11.2). Results for the 30% and 40% Sep-Pak fractions from the same purification sequence are shown. Note that the lowest percentage of ethanol in the eluents varies for the three runs (eg. 20% for the 20% Sep-Pak fraction, 30% for the 30% Sep-Pak fraction and 40% for the 40% Sep-Pak fraction). All other elutants are the same. In this case ABA and methanol controls are not shown. GO
4.0
Scores 10)
3.0-
2.0-
1.0-
0.0
Sep-Pak Fractions, Control (A) and 0.0005M ABA (B)
FIGURE 1.4.13 Abscission scores (out of 10) for Sep-Pak fractions in the purification of hydrolysed crude acid extract (Methods 1.3.11.3), control (25% methanol) and 5 X10*4 M ABA. Values are the result of one experiment. 61
E3 Abscission Scores (out of 10)
30%
40%
20%
100% A
Sep-Pak fractions, 25% Methanol Control (A) and 0.0005M ABA (B)
FIGURE 1.4,14 Abscission scores (out of 10) for Sep-Pak fractions from the purification of hydrolysed fractions 1, 2, 3 and 4 (AUS 2). Control (25% methanol) and 0.0005 M ABA are also shown. 62 in the 20% eluent and more importantly the much less active 80% and inactive
100% ethanol eluents. Fractions 30H and 40H were the most active just like the corresponding fractions in the HAF series.
1.4.12 THIN-LAYER CHROMATOGRAPHY
Bioassayed activity, scraped and eluted from the various TLC plates in the purifications in Methods 1.3.12 are illustrated in Figure 1.4.15. The material applied to the plates is illustrated below and the chemical form of the active compound is shown in brackets.
Plate A - l/30th of the 30% ethanol Sep-Pak eluent from purification of
crude acid fraction (free acid)
Plate B - l/100th of fraction 40H from Sep-Pak purification of combined
fractions 1, 2, 3, and 4 (AUS 2) after hydrolysis of these
fractions (free acid)
Plate C - l/100th of fraction 40H after acetylation and methylation (methyl
ester, acetyl derivative if OH group present)
Plate D - 1/2 of combined zones 1 and 2 from TLC A, after methylation
and acetylation (methyl ester, acetyl derivative of OH group)
Most obvious correlation between plates is that each shows activity near the origin, a strong indication that an active compound is very polar. Derivatisation of an acid (by methylation) or hydroxyl group (by acetylation) would reduce the polarity of the active compound so that it should migrate further towards the solvent front than the underivatised material if the same solvent is used. When active zones 1 and 2 were scraped from plate A, eluted, acetylated, methylated, re applied to another plate and run with the same solvents there was still no difference in the Rf of the activity. Either the derivatisation was unsuccessful, a very unlikely scenario since pyridine/acetic anhydride acetylations and diazomethylation are very 63
(Methods (Methods
ethanol ethanol and
30%
Sep-Pak of
1.3.11.3) ethanol
(Methods purification
30% of first 40H
the
shown. from
purification methylated
- are
0.4)
A - and
Rfs 0
runs
(Rf
2
TLC acetylated
- and
Approximate
C from 1
zones zones
1.3.11.2), 1.3.11.4).
eluted the methylated
(Methods Methods
for
A, and
40H
-
B results (Plate
acetylated
-
D 1.3.11.1), Bioassay Sep-Pak
1.4.15
FIGURE 64 straightforward procedures, or activity has been lost due to some other factor
(volatility or extremely strong binding to the silica).
If the plant extract is first hydrolysed before application to the plate, the activity still does not migrate away from the origin to a significant extent (Plate B).
Only Plate C has activity in any other zone. The fraction applied to this plate had been acetylated and methylated so it would be expected that this derivative would move away from the origin. The wide spread of activity across half the plate indicated either that a single compound has chromatographed badly or that there was more than one compound in those zones responsible for abscission activity. This result was different from that obtained with the derivatised fraction chromatographed on Plate D where there was no significant migration of activity away from the origin. This indicated that after hydrolysis of the plant extracts the derivatisation does markedly reduce the polarity of the active compound whereas derivatisation does not have this effect on unhydrolysed fractions.
1.4.13 GC TRAPPING OF SEP-PAK FRACTIONS
Six active fractions from different sources (Methods 1.3.13.4) were trapped by GLC under the identical conditions in an attempt to find whether the same compound is likely to be responsible for the abscission acceleration in these different extracts. Figure 1.4.16 shows the GC trace of one (acetyl, methyl 30H) together with the bioassay results for all six.
Most obvious problem here was basically that the bioassays, with one exception gave weak activity. The one exception was trap 1 (40M30K) which was considerably more active than any of the other fractions.
The low activity was puzzling since l/40th of the entire fractions 30HAF,
80HAF and l/20th of the other fractions had been trapped by GC and bioassayed.
In the untrapped state l/50th of 30H and 1/100th of the other fractions were sufficient for clear activity in the bioassay. Obviously significant losses have 65
FRACTIONS
3 OH 30HAF 80HAF 40M20 40M20H 40M30K
I I l NA - Not Active SA - Slightly Active (0 to 30% abscission) A - Active (30 to 70% abscission)
Initial Temperature: 130°C Initial Time: 10 minutes Programme Rate: 8°C/min
Penta-acetyl glucose Methyl jasmonate
TRAP 6 TRAP 5 TRAP 4 TRAP 3 TRAP 2 TRAP 1
24.0 22.0 20.0 18.0 16.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0 0.0 Time (mins)
FIGURE 1.4.16 GC profile of acetylated, methylated fraction 30H. Retention times of marker compounds methyl jasmonate and penta-acetylglucose are shown together with the abscission scores for each trappate from fractions 30H, 30HAF, 80HAF, 40M20, 40M20H and 40M30K. 66 occurred. Again, either GC instability, involatility or high volatility were suspected at this stage.
Despite the fact that most of the fractions gave unconvincingly low results there still appeared to be some pattern and correlation between the various fractions. The slight activity for trap 4 of 30HAF and 40M20H and trap 5 of 30H,
80HAF and 40M20 could represent the same compound if the GC conditions were slightly different and the trapping zones overlapped. Trap 1 of 80HAF may have reflected a stronger activity in the same trap of 40M30K. However the very weak activity found compared with the relatively large amounts of activity applied means that most was lost and the results here cannot be considered satisfactory.
1.4.14 NORMAL-PHASE SEP-PAK PURIFICATION
One purification sequence, the one leading to fraction 40M20 was found to be give reproducible activity after a number of separate runs on the Sep-Pak apparatus using reverse phase Sep-Paks (Methods 1.3.10.2) so it was decided to concentrate further on this fraction. After silica Sep-Pak treatment of this fraction
(Methods 1.3.14) only the 10% isopropanol fraction after bioassay showed strong activity so this was used for HPLC purification. The isopropanol and methanol eluents were invariably inactive.
1.4.15 HPLC OF METHYLATED 4QM20
Figure 1.4.17 shows the HPLC trace, trapping regions and abscission scores for the HPLC purification of the 10% isopropanol in hexane eluent of
40M20 after silica Sep-Pak purification (Methods 1.3.14). Activity was spread between four of the five trappates with the most active being trap 4. Figure 1.4.17 also shows the elution time of methyl-ABA run under the same conditions.
Further examination of this extract using diode array detection showed that the chromaphore of the most active peak was very like that of trans 3,4 dimethoxycinnamic acid methyl ester (Figure 1.4.18). The overall shape of the 67
Eluent: 5% isopropanol in hexane Flow rate: 4mls/min Monitor Wavelength: 330 nm
METHYL ABA
Absorbance
Trap 1 - Slightly Active Trap 2 - Slightly Active Trap 3 - Very Active Trap 4 - Active Trap 5 - Not Active
2 l3l 4
I I I | I 0.0 4.0 8.0 12.0 16.0 Time (mins)
FIGURE 1.4.17 HPLC chromatogram and trapping zones of the 10% I PA in hexane Sep-Pak eluent of 40M20. The activity associated with each zone and the retention time of ABA methyl ester are shown.
68
methyl conditions HPLC same the under separately run ester 1.3.15). (Methods
S I A I L RE U HG UV 1.4.16) Figure 3, (trap peak unknown the of apex the at spectra aci< dimethoxycinnamic 3,4 and eoueqjosqv
UMO 09 chromaphore is maintained in the two compounds: two maxima at low wavelength followed by a pronounced shoulder before the highest intensity peak at around 320 nm. Both also showed the identical maxima at the higher wavelength end of 297 and 323 nm.
1.4.16 IDENTIFICATION OF THE UNKNOWN COMPOUND
The previous section showed that the UV of the unknown compound was very similar to that of 3,4 dimethoxycinnamic acid methyl ester. This congruence extended to the infrared spectrum. Figure 1.4.19 shows the IR of the unknown and the cinnamic acid derivative. Basic features are much the same for the two compounds although there are some wavelength shifts, particularly the carbonyl band. For the unknown the carbonyl maxima occurs at 1680 cm'1 while the cinnamic derivative has a corresponding band at 1720 cm'1.
The fact that the two IR spectra were so similar strongly precluded a combination of dimethoxycinnamic with a substituent having some other strongly absorbing functional group. In addition the comparative broadness of the carbonyl band of the unknown suggested that there may be another or other ester groups in the unknown.
GCMS results under chemical ionisation conditions are shown in Figure
1.4.20. Bottom trace is the total ion chromatogram of the peak trapped on normal phase HPLC (Methods 1.3.15) while the upper trace is the mass spectrum of the major peak. The mass spectrum is rather simple with a base peak m/z 191 and low intensity peaks at m/z 366 and 367. Very little else is of any importance aside from minor fragments at m/z 321 and 335. There were no obvious methane adducts
(M+29, M+41) to point to a definite molecular weight but the high mass peaks at
366 and 367 were significant.
The appearance of two peaks on the total ion chromatogram provided further indication that the unknown was an ester of a cinnamic acid derivative. The mass spectrum of the earlier eluting peak was almost identical to that of the later 70
0% 100%
I i i * i ' i 1 ~i >------1------»------r 3500 3000 2500 2000 1800 1600 1400 1200 1000
Wavenumber (cm'1)
FIGURE 1.4.19 Infrared spectra of the dichloromethane solutions of 3,4 dimethoxycinnamic acid methyl ester and the unknown peak on the HPLC purification of 40M20 (Methods 1.3.12). Total Ion Current ro VO O' CM O' o >
— >*
FIGURE 1.4.20 Total ion chromatogram (top) and mass spectrum of the peak centred at scan #842 (bottom). Mass spectra have been summed from scans #830 to #854 with scans #851 and #828 subtracted as background. 71 72 eluting peak aside from very minor differences in the relative intensities of the major ions. The proportion of this earlier eluting peak rose with time and exposure to light. Cinnamic acid derivatives are known to isomerise very readily from the native trans form to the cis form so the early eluting peak was undoubtedly the cis isomer formed by light-induced isomerisation.
High resolution EI-GCMS duplicated to some extent the results from the CLMS (Figure 1.4.21). Main fragments were m/z 366, 208, 191, and 164. Like the CIMS the base peak was m/z 191. Mass measurement of the m/z 366 by a peak timing method developed by Brophy et al (1979) gave an exact mass to four decimal places of 366.1286. Most likely molecular formulas for this mass were
C14H18N6O6, C13H22N2O10, C15H14N10O2 and C18H22O8. In the absence of any IR evidence to suggest that the unknown does contain nitrogen it was assumed at this stage that the unknown was most likely comprised of only carbon, oxygen and hydrogen. Thus a molecular formula of C18H22O8 fitted very well the result of the mass measurement. The UV spectrum suggested that the chromaphore is 3,4 dimethoxycinnamic acid so the unknown was probably an ester of this acid as represented below.
O ll c—oc7h„o4 100.0 191
1 S4
208 29 147 77 119 225
lllllllll|lllll
40 60 80 100 120 140 160 180 200 220
Relative Intensity
100.0
50.0 -
JTTTTT^TTTJTTITrrnTprriTTlTFl jTT Ik ■ 1111 j 11111111 • 111 H i • H > 11II111V i • ^ 111111411111 m 111 i 111111111111
260 280 300 320 340 360 380 400 420 440
m/z
FIGURE 1.4.21 Direct insertion probe electron impact MS of unknown compound. The accurate mass measurement of the molecular ion is also shown. 74
To test this hypothesis a small amount of the unknown was hydrolysed, methylated and analysed by GCMS. The results are shown in Figure 1.4.22.
After this treatment there were three major peaks and a number of minor ones in the total ion chromatogram. Three major peaks gave mass spectra shown at the right side of the figure. Peaks B and C had the same retention and mass spectra as the cis and trans isomers of 3,4 dimethoxycinnamic acid methyl ester. The two isomers are present since the native trans compound readily isomerises in light to give the cis isomer. This result clearly showed that the notion that the unknown was the structure shown above was correct. However, this compound was not
SF.
The mass spectrum of the other peak (A) had major ion species m/z 69,
75, 89, 103, 117, 145, 177, 205 and 217. The last two ions correlate with the methane adducts of a molecular ion 176 (176 + 29, 176 + 41) so the molecular weight was 176 daltons. This fitted very well with the expected molecular weight of the ester portion of the unknown (C7H11O5). Fragmentation gave some clue of the structure. The ion m/z 145 representing a loss of 32 (methanol) from M+l indicated that the compound was a methyl ester and a further loss of 28 from that fragment or 59 from M suggested that the entire methyl ester group was lost during fragmentation. A further loss of 14 from this fragment (117) also suggested that there was a methylene attached to the methyl ester group. Not much else of significance could be gleaned from the fragmentation pattern.
However one possible structure that fitted the IR data and had a MW of
176 daltons was 1-methoxysuccinic acid dimethyl ester. Attempts were made to synthesise this compound and the results of the synthesis and purification of the product are shown in Figure 1.4.23. The top part of the figure is the GCMS trace of the reaction mixture after extraction, purification by reverse phase HPLC and methylation (Methods 1.3.21.5 and 1.3.21.6). This GCMS trace indicated that probably three compounds were present in the methylated extract and the major one had a mass spectrum shown in the bottom half of the figure. The mass spectrum is 75
the
1.3.13.2). across
scans
(Methods
summing
of methylation
result after scans.
the
are unknown
trace background the
of few
a current
ion hydrolysate total
subtracting the the
and for
C)
above
and
B
shown
(A, chromatogram
peaks spectra ion
major Mass Total
1,4,22
FIGURE 76
100.0 -1 53210
Relative Intensity
205 217
Initial Temperature: 60°C Programme Rate: 6°C/min
600 800 10:00 13:20 Scan Number Time (min:sec)
FIGURE 1.4.23 Total ion chromatogram (bottom) from the methylation of the methoxysuccinic acid preparation (Methods 1.3.21. 5) after HPLC purification (Methods 1.3.21.6). Mass spectrum of the major peak is also shown (top) Spectra been summed from scan #399 to #485 with about 30 scans of background subtracted. virtually identical to that shown for peak A in Figure 1.4.21. Retention times of
the unknown and the major product of the synthesis also match well, at least within the expected range of error.
The discovery that methoxysuccinate was formed after hydrolysis and methylation of the unknown did not suit the hypothesised structure for this cinnamoyl ester. Clearly it was necessary that the ester moiety contain a free hydroxyl group.
Obviously the ester portion of the unknown is a diacid and after further consideration of the Cl mass spectrum of the unknown before hydrolysis (Figure
1.4.19) a possible route was uncovered for the generation of methoxysuccinate.
Ions at m/z 321 and 335 were most likely formed by neutral losses of ethanol and methanol from the parent compound, a fact which indicates that the unknown is a methyl ethyl ester and that the hydrolysis will release malic acid. Methylation of malic acid generally will only produce dimethylmalate but in this case conditions must have favoured the methylation of the hydroxy group and the formation of methoxysuccinate dimethyl ester (MW 176 dal tons). The methylation of the hydroxy group by diazomethane is an unlikely event since this group is not very acidic and attempts to mimic this reaction with synthetic malic acid were unsuccessful. However there is no question that the reaction did occur and that the unknown is a malic ester of dimethoxycinnamic acid.
NMR studies of the unknown compound and the synthetic dimethoxycinnamoylmalate dimethyl ester offered further proof that the hypothesised structure was correct. Proton NMR of the intact cinnamoyl ester showed, as expected, aromatic resonances very similar to 3,4 dimethoxycinnamic acid methyl ester (see Figure 1.4.24). All the aromatic protons have very similar chemical shifts to the corresponding methylcinnamate derivative. This confirms the notion that dimethoxycinnimate is an important part of the structure.
Additional common features of the two spectra are the methoxy proton resonances (both 3.92 ppm) and other singlets in the same region. The methyl 78
Unknown
i « ■ i ■ i « i « » i • • 1 i i i i » > i » ■ 1 i ] ■ ' ' * r 7.0 6.0 5.0 4.0 3.0 2.0 1.0
3,4 dimethoxycinnamic acid
Chemical Shift (ppm)
FIGURE 1.4.24 Proton NMR of the unknown compound (top) and 3,4 dimethoxycinnamic acid methyl ester (bottom). The former analysis was performed on a 500 MHz instrument, the latter at 300 MHz. 79 ester protons for the methoxycinnamate derivative produced a singlet resonance with a chemical shift of 3.81 ppm and there is a minor peak at 3.74 ppm attributed to the methyl ester protons of the cis isomer. The unknown has four other singlets, apart from the methoxy protons, in the same region of the spectrum. The minor peaks (3.72 and 3.77 ppm) were at this stage assumed to be due to the cis isomer of the unknown and the major resonances 3.79 and 3.74 ppm attributed to its a and p methyl ester protons. Other resonances unique to the unknown were multiplets at 1.26, 2.98, 4.23 and 5.65 ppm. The 2D NMR provided more information to allow assignment of these multiplets centred at 5.65, 4.23, 2.98 and 1.26 ppm (Figure 1.4.25). The full plot (top) shows that coupling occurs between the multiplets at 1.26 and 4.23, and the ones at 5.65 and 2.98 ppm. These results together with other observations:
a) the former pair of multiplets resembles the typical quartet/triplet pattern of an ethyl group b) the latter is basically a doublet/triplet pattern of a CH2-CH combination although there is obvious secondary coupling c) the two singlets at 3.79 and 3.74 ppm occur in the region expected of methyl esters
and d) the likely molecular formula of the ester moiety is C7H11O5 all suggested that the unknown was 3,4 dimethoxycinnamoylmalate ethyl methyl ester. The fact that there were the two methyl ester resonances also indicated that the two isomers of the monomethyl monoethyl esters were present in the unknown sample.
Additional proof of the identity of the unknown came with the proton
NMR of synthetic 3,4 dimethoxycinnamoylmalate dimethyl ester (Methods
1.3.21.1, 1.3.21.2, 1.3.21.3 and 1.3.21.4). Figure 1.4.26 shows the spectrum together with the structure and assigned resonances. The monoethyl monomethyl 80
FIGURE 1.4.25 2D proton NMR of the unknown. Top trace is the 2D plot while the bottom section shows the corresponding 1D spectrum. No chemical shift scale is shown. The dotted lines connect the satellite bands with the coupled resonances. The important couplings occur between multiplets A(1.26ppm) and B(4.23 ppm), C(2.98 ppm) and D(5.65 ppm). 81 shown
Also ester. dimethyl
resonances. dimethoxycinnamoylmalate
3,4
assigned
and synthetic
of
MHz)
compound
(300 this
of
NMR
Proton structure
1,4-2$
FIGURE 82 esters were not prepared (and hence not analysed by proton NMR) since treatment with ethanol/HCl caused hydrolysis of the ester linkage between the malic and cinnamoyl parts and this led to very low yields. In this case it was not considered necessary to try alternative methods since other evidence clearly supported the hypothesised structure. For instance the Cl mass spectra of the unknown showed losses of 46 and 32 mass units from M+l, very characteristic of methyl and ethyl esters where neutral losses of methanol or ethanol occurs. Another significant feature common to the Cl mass spectra of the synthetic dimethyl and the endogenous monoethyl monomethyl ester was the unusual ratio of M and M+l for a compound of this type. The relative abundances of the parent ion (M) and M+l were consistently very similar and this served as a diagnostic pointer to compounds of this type. All the possible combinations of ethyl and methyl esters all demonstrated this unusual feature. Typically the major fragment ions and relative abundance for the synthetic dimethyl ester were: 173(2), 191(100), 209(18), 321(10), 352(12), 353(13).
1.4.17 BIOASSAYS OF SYNTHETIC COMPOUNDS
About 100 |ig of synthetic 3,4 dimethoxycinnamoylmalate, methoxysuccinic acid, and hydrolysed malate conjugate submitted for bioassay were all totally inactive when dissolved in methanol (200 p.1) and applied to the agar discs in 2 p.1 lots.
1.4.18 RELATIONSHIP OF ACTIVITY AND THE CINNAMOYLMALATE
DERIVATIVE
Osborne (unpublished results) found that after repeated HPLC purifications of an active bean leaf extract the activity was gradually lost and moreover that the major constituent of the purified fraction was caffeic acid. So initially it was suspected that the UV absorbing peak on HPLC of fraction 40M20 (Figure 1.4.17) was responsible for the trapped senescence activity since it did 83 have the characteristic caffeic acid chromophore. However the hydrolysates of the synthetic compounds were not active so obviously another co-eluting compound was the cause of the bioactivity. Hydrolysis of the trapped peak failed to indicate that other compounds (apart from the components of the cleaved cinnamoylmalate conjugate) were present but it is likely that the senescence active compound was present at a concentration to low to detect by the methods used for analysis.
Interestingly the major peak following the active peak on HPLC was later identified as the dimethyl ester of 3,4 dimethoxycinnamoyl malate and that this trapped region was the next most active of all the fractions collected during this purification. The relative amount of activity collected in these two fractions corresponded well to the relative absorbance maxima of the two different esters.
This may mean that the active compound is very similar to malate (a small hydroxy diacid) and that this unknown compound is esterified to a caffeic acid derivative.
1.4.19 IDENTIFICATION OF A MAJOR PEAK AS A SUCCINATE DIESTER
One of the major peaks from the GC trapping experiment from the hexane extract (Methods 1.3.13.5) was analysed by direct insertion probe MS and by IR
(in dichloromethane). These results are shown in Figure 1.4.27 (GC trace of whole fraction and MS of trapped peak) and Figure 1.4.28 (IR spectrum of trapped peak).
Chemical ionisation mass spectrometry revealed a number of features of the unknown. The appearance of a strong ion (base peak) at m/z 161 together with ions at m/z 189 and 201 indicated that the unknown had a molecular weight of 160.
The fragmentation pattern also gave some indication of the chemical structure.
Neutral losses of 32 (methanol) and 46 (ethanol) from M+l suggested that the compound was a methyl ethyl ester. An M-28 fragment is commonly the result of the loss of ethylene or a carbonyl group but previous experience suggested that this could not arise from fragmentation of a methyl ethyl ester. Generally these esters fragment by neutral losses of methanol from the methyl ester and ethanol from the 84
100.0 -|
Relative Intensity
m / z
Initial Temperature: 60 C Programme Rate: 6°C/min
Trapping Region
20.0 16.0 12.0 8.0 4.0 Time (mins)
FIGURE 1.4.27 GC chromatogram of hexane extract (Methods 1.3.13.5) and trapping region (bottom). Solid insertion probe Cl mass spectra of the trapped peak is shown in the top section. 100% o £= to FIGURE 1.4.28 Infrared spectrum of the trapped peak in dichloromethane. GC chromatogram and trapping r< cn
shown in Figure 1.4.27. 85 86 ethyl ester. The infrared spectrum showed a strong band at 1745 cm-1 which suggested that the compound contained either a carbonyl or carboxyl group. The sharpness of this band also suggested that both types of carbonyl (ester, ketone or aldehyde) could not be present. There was little evidence from the IR spectra that could be used to identify other functional groups.
Assumption of the presence of both a methyl and ethyl ester group together with the knowledge that the compound had a molecular weight of 160 left only a small number of alternative structures and the most likely one from plant sources would be succinic acid ethyl methyl ester. This notion was proved by the coincidence of GC retention and the similarity in the infrared and mass spectra of the unknown and a synthetic diester produced by the methylation of monoethyl succinate.
Two questions still needed to be answered. Is the ethyl ester an endogenous compound and what loss could have produced the M-28 peak?
Neither question was pursued with any vigour but it was thought that the occurrence of an ethyl ester group was due to storage in acidic ethanol and the 28
AMU loss from the parent ion was due to a McLafferty-type rearrangement resulting in the loss of ethylene from the ethyl group. The dimethyl ester of succinic acid does not lose 28 mass units from the parent ion so obviously the ethyl ester group is the source the ethylene lost during fragmentation.
1.4.20 GLC TRAPS OF HYDROLYSED FI. 2. 3. 4 AUS 27A
The GC traces and trapping regions for the ethyl acetate extract of this fraction as treated in Methods 1.3.13.5 are shown in Figure 1.4.29. Also shown is the retention time of a marker compound (dimethyl succinate) and the bioassay scores for each trappate. It can be seen quite clearly that activity elutes earlier than dimethyl succinate. 87
Trap 1 - Inactive Trap 2 - Very Active Trap 3 - Active Trap 4 - Slightly Active Trap 5 - Inactive Trap 6 - Inactive
Succinic Acid Dimethyl Ester
TRAPS
I I I I I I I I 28.0 24.0 20.0 16.0 12.0 8.0 4.0 0.0
Time (mins)
FIGURE 1.4.29 GC Chromatogram and trapping zones for the ethyl acetate extract (Methods 1.3.13.5). Also shown is the retention time of a marker compound (succinic acid dimethyl ester) and the bioassay scores for each zone. 88
1.4.21 IDENTITY OF OTHER COMPOUNDS IN THE ETHYL ACETATE
EXTRACT OF HYDROLYSED FI. 2, 3 AND 4 AUS 27A
Having discovered that the active fraction contained succinate esters further MS studies were undertaken to identify other compounds in that fraction.
The molecular weights of other compounds, determined by MS using the Finnigan
Cl instrument, together with the fragmentation patterns, gave results suggesting the presence of compound such as malonate, fumarate and glutarate in the form of dimethyl, diethyl and monoethyl monomethyl esters. Analysis of the same fraction on the VG high resolution instrument with a NIST library confirmed the presence of some of these compounds. Compounds identified after library search routines and the mass measurement of the parent ion were the dimethyl esters of malonate, malate, fumarate and succinate, the monoethyl monomethyl esters of these diacids and the methyl esters of ortho methoxybenzoic acid and a dimethoxybenzoic acid
(isomer unknown). Figure 1.4.30 shows the mass spectrum of one example: fumaric acid dimethyl ester with the best two library fits and the accurate mass measurement of the parent ion together with the assignment of a molecular formula.
However all of these compounds eluted after senescence activity. In the active zone the major components were compounds which were shown by CIMS to have molecular weights of 118,132 and 146. Tentative identification of the first compound as dimethyl oxalate was confirmed by coincidence of retention time and the mass spectrum while the others have not been positively identified as yet. The fragmentation pattern of these two other unknowns shows losses of 32 (methanol) and 42 (acetate group minus one proton) so it was probable that these compounds were acetyl methyl glycollate and similar derivatives of lactate or p- hydroxypropanoic acid. CIMS produced ions with a relative intensity of 5% or greater for dimethyl oxalate of m/z 159(5) and 119(100). Data for the other compounds follows. Unknown (MW 132, probably acetylglycollic acid methyl ester): m/z 133(13), 101(100), 91(18) and 61(7). Unknown (MW 147, probably File:ETACEY Ident:402_407-395_395 Win 20PPM Acquired: 9-FEB-90 14:19:26 AutoSpecQ Ionization:EI+ Function:Magnet
100_, 113 m 00' m . h - -CM . — -co
o O o i o X ■u -u £ € • U O •rl G X m rH 13 PS r- O CM •H ^ k r-i -H A XJ r* 2 -P CO oo CM (0 c 4J l£> 2 fa U X •• co 0 e a < CM -H 2 ” •H -P -ri -H T — *0 w (0 a a) n t-4 C 0 - - 0 >o 1 in* a> onoi o •o *> ^ O to (SI 04 , O O) m |-CM h <*> tn *H O ro m (SJ co o -00 o H4- CM ■rl M XI r- o PS CTl PI iH rH TT co -H k ^ x: V 2 4-1 oo ■^r m fcl 4-1 co Si 2 fa •• X o •• m o <1) •• * • CM -H •H z 4-> x) -H X -H x) — — V n -H tsa 4-1 a) 4-1 O c a) P o o <43 V I g a) >1 a) a) m U i - cTS 04 m ' 1 -CM - — -00 O O *H O CO tH o o i — 4 u Li_ =) QC LU ■sf CO CH X sz "5 03 CD CO o C Bioassays of these compounds have not yet been undertaken however it is unlikely that either oxalate, lactate or glycollate would act as a senescence hormone considering the other roles these compound have in the primary metabolism of plants. However, the possibility that P-hydroxypropanoic acid is present in the extract is rather interesting. Early debate on the biogenesis of ethylene centred mainly around two possible pathways; synthesis from methionine or p-alanine. The second alternative is now largely discredited as most researchers favour the pathway from SAM to ethylene via ACC. It is relevant to note though, that the scheme involving p-alanine, as proposed by Spencer (1969) was presumed to proceed via p-hydroxypropionate and acrylic acid. These compounds may have some effect on abscission and ethylene production when applied to bean explants but this has yet to be tested. Intuitively a sequence involving dehydration of p- hydroxypropionoic acid followed by dehydration of the resultant acrylic acid could not be considered unusual. Precedents include the p-oxidation of fatty acids. 91 1.5 DISCUSSION 1.5.1 EARLY WORK Previous work by Osborne et. al., 1972 and Milborrow (unpublished results) had shown that a compound was present in both extracts and diffusates of bean and cherry laurel leaves which accelerated abscission of bean explants and various properties of this compound (SF) had been determined. It was demonstrated that the compound was an acid and had properties distinct from ABA or IAA. Initial efforts concentrated on confirming these early findings and developing a method whereby large scale separations could be applied to isolate SF. The first set of bioassays confirmed that the neutral extract of bean leaves used in these experiments was inactive and that the ethyl acetate acid fraction was the most active. A similar ether acid extract was only about 1/1 Oth as active as the ethyl acetate extract and the aqueous solution after extraction of neutrals and acids was active but less so than the ethyl acetate acid fraction. This followed the same pattern as the earlier (Milborrow, unpublished results) extracts. A more comprehensive extraction sequence of alkaline hydrolysed fractions from AUS 27A showed that activity did not partition efficiently into a non-polar solvent (pentane) and that the active compound did not form a stable lactone on acidification. 1.5.2 ACTIVITY AND ETHYLENE STIMULATION One relevant concern was the coincidence of senescence activity and ethylene production. The coincidence of ethylene production with relative senescence activity was clearly shown by the fractions from AUS 2 and the elutants from the silica Sep-Pak purification of fraction 6 (AUS 12A). In both cases the maximal ethylene production was coincident with maximum abscission activity. 92 1.5.3 ACTIVITY AND KNOWN STIMULATORY COMPOUNDS Another relevant concern was the distinction between senescence activity and other known stimulatory compounds present in the same fractions. Obviously it was necessary to exclude, as far as possible, the likelihood that abscission activity and ethylene stimulation produced by the bean extracts were not due to compounds such as ABA, IAA, ACC or JA. The distinction between senescence activity and ABA was made on the basis of two separate chromatographic experiments. In the first, direct ABA assays by electron capture GC were performed on fractions eluting from a run of AUS 2 and in the second tritiated ABA was added to some hydrolysed crude acid fraction and purified as per AUS 2. In both cases the assayed ABA and the counts in each fraction due to added 14C ABA rose to maximum amounts in later fractions than the bioassayed senescence activity. For jasmonic acid and its methyl ester the results offered similar chromatographic distinction. Column AUS LM1 showed that there was two zones of activity for methylated fraction 2 from AUS 27A, one distinct from jasmonic acid (and less polar) and one which co-eluted with jasmonic acid in acetone, virtually a column wash. Sep-Pak purification of the same fraction after methylation compared to methyl jasmonate showed that most of the activity was less polar than methyl jasmonate. Additionally there was other evidence to suggest that methyl jasmonate or jasmonic acid were not responsible for the abscission activity in the fraction from column AUS 27A. Both jasmonic acid and its methyl ester (at a concentration of 10'3 M) were found to be inactive as an abscission accelerators or ethylene stimulators under the conditions used in the bioassay. This does not contradict the findings of Ueda and Kato (1980) that methyl jasmonate was active in oat leaf chlorophyll bleaching assays but simply indicates that methyl jasmonate or JA are involved in other processes in senescence than abscission or ethylene stimulation. 93 The distinction between ACC and senescence activity was made in a less direct manner. ACC is now accepted to be a direct precursor of ethylene and as such is quite active using Osborne's bioassay procedure. However, the ethylene stimulatory action caused by the AUS 2 column active fractions (fractions 1 to 4) was found to be quite different from ethylene production induced by the application of ACC to the excised bean explants. This was demonstrated by the fact that after treatment described in Methods 1.3.10 the active extract was not stimulatory for ethylene production in the short term. There was no measurable stimulation of ethylene production after 5.5 hrs. By contrast ACC promoted ethylene production in 30 minutes. Intuitively it would also not be expected that ACC would elute in the earlier fraction from AUS 2. Under those conditions the acetic acid would ensure that the amino nitrogen is protonated and would bind very strongly to the silica. So ACC was considered to be out of contention as an active component in the AUS 2 column early fractions by virtue of its polarity and the fact that the ethylene production was not stimulated soon after application of the extracts. However this did not rule out ACC altogether. A major metabolite of this acid is also likely to be present in the crude plant extract. N-malonyl-ACC, discovered by Amrhein et al. (1982) and Hoffman et al. (1982) is also likely to be active as an ethylene promoter. This feature is complicated by the fact that the malonyl ester of ACC is not stable under some extraction conditions. Koester et al. (1983) found that special techniques had to be used to prevent cleavage of the ester group or trans-esterification in the presence of alcohol. Presumably some of the malonyl groups may also readily decarboxylate under acidic conditions (pH 2, during acid extraction step) to produce the corresponding acetyl derivative. Bioassays of the synthetic N-acetyl-ACC and ACC demonstrated good activity at levels of 10-3 M (Table 1.4.4), the acetyl being less active than the free acid. Conceivably N-acetyl-ACC or any remaining N-malonyl-ACC would also elute in the early fractions of the AUS 2 column but the possibility of this compound being responsible for significant abscission activity was removed by the results of the reverse-phase Sep-Pak purification of crude acid fraction compared to similar runs with ACC and N-acetyl-ACC. ACC and N-acetyl-ACC elute mainly in the 5% and 20% ethanol eluent respectively while the most active abscission accelerating and ethylene promoting fraction in the purification of the crude acid extract was the 30% ethanol fraction. Any N-malonyl-ACC not decarboxylated would undoubtedly elute from the Sep- Paks in an equivalent or more polar fraction than the N-acetyl-ACC because of the greater polarity of the malonyl derivative. IAA was not considered to be a likely cause of senescence activity for a number of reasons - primarily that it does not stimulate abscission under our system of bioassay. Additionally IAA will tend to decompose after any prolonged storage at low pH. Any slight chance that IAA was responsible for ethylene stimulation was ruled out by GC retention. It was found (results not shown) that IAA methyl ester eluted far later than the major band of activity trapped from the QF1 column. Clearly then there is another compound or compounds other than ABA, ACC, IAA or jasmonic acid responsible for the activity in the AUS columns and the Sep-Pak fraction. 1.5.4 CHROMATOGRAPHY GC analysis of the acetylated and methylated fractions from the large silica column purifications (AUS 2) showed that there was very little difference between active fractions and substantially lower amounts of material in other non-active fractions. Consequently this was not considered a very efficient method of purification. Conversely similar studies of the reverse phase Sep-Pak fractious (Methods 1.3.11.1) showed considerably greater differentiation between the various fractions even though some compounds eluted over a range of 30% ethanol 95 the amounts varied substantially between each different fraction. It was then decided that the Sep-Pak system would be a better method for purification of the crude bean acid extract even though there were some disadvantages; a lower capacity than the large scale silica columns and consequent need for many more runs to purify the same amount of crude acid fraction. The first Sep-Pak fractionation of the crude acid fraction showed a peak of activity in the water eluent and then a relatively broad spread of activity from 10% to 40% ethanol (see Figure 1.4.10). The activity of the water fraction could have been due to ACC but it is more likely that this activity was caused by overloading and the consequent elution of unbound active compounds. This notion was supported by consequent assays of the water eluent which showed widely varying activity from run to run until strict control was maintained over the amount of extract applied to the Sep-Paks. The activity in the later fractions peaked at the 30% level and this fraction was a very effective stimulator of ethylene production. Hydrolysis of the crude acid fraction before fractionation using the same conditions gave similar results showing a spread of activity between the 10% and 40% fractions and a peak at 30% ethanol. However there was additional later eluting activity over fractions 60%, 80%, and 100% (see Figure 1.4.12). This late eluting activity also occurred with further fractionation of the 30% ethanol eluent from the crude acid extract purification after overnight methylation. In fact, the 100% ethanol eluent was equally active with the most active 30% ethanol eluent (see Figure 1.4.11). A similar sequence with a 30% Sep-Pak fraction after a 30 minute methylation time did not mimic this result. Rather, the bulk of the activity just shifted to the 40% ethanol eluent, a result expected when there is a simple methylation of an acid group. The 20% fraction from the initial Sep-Pak purification after 30 minute methylation behaved just like the 30% fraction did, an indication that the same compound may be present in the original 20% and 30% fractions and that this compound was responsible for abscission activity. 96 The difference caused by longer methylation times was at first puzzling but the same sort of distinction existed between the crude acid extract and the hydrolysed crude acid extract. That is, after hydrolysis there was a tendency under reverse phase conditions for activity to elute later in less polar eluents. It was realised later that one possible reason for the shift in polarity of the active zone could lie in the conjugation of an active non-polar compound with a polar carrying group. Hydrolysis of this conjugate would release the active moiety and this would result in the less polar compound binding more tightly to the reverse-phase Sep-Paks. Methylation over a long period may perform a similar feat to hydrolysis. If the diazomethane reagent does contain some KOH (carried over during the distillation of the diazomethane) then this may encourage trans- esterification leading to the splitting of the conjugate and the formation of the small active compound in methylated form. The long methylation procedure did not lead to significant late eluting activity if the etherial diazomethane was redistilled over water before use which suggested that KOH carry-over may have been a factor. Further complication was added by the fact that if the crude acid fraction was first purified by silica column chromatography, hydrolysed and run through the Sep-Pak apparatus (Methods 1.3.11.4) then the activity does not appear to any significant degree in the later fractions (see Figure 1.4.14). TLC purification of various fractions did confirm this finding although the results were frequently not reproducible. Analysis of the active 30% ethanol Sep- Pak fraction gave one very active zone which barely moved from the origin and after elution of this band acetylation and re-analysis under the same conditions the activity was still restricted to the same region. This would tend to suggest that the compound responsible for the activity in the 30% Sep-Pak eluent is very polar and this polarity is not greatly reduced by acetylation. Most polar conjugates are carbohydrate or monosacharride esters or ethers and these may not change Rf appreciably under these conditions especially if acetylation is incomplete. 97 Purification of 40H using only a slightly less polar solvent system produced the same result, activity did not migrate to any great extent. Acetylation and methylation of 40H followed by TLC (Methods 1.3.12.3) gave three zones of activity, one at the origin, and two others at Rf 0.33 - 0.5 and Rf 0.67 - 0.83. The activity at the origin may have been due to the same compound as in the 30% ethanol Sep-Pak fraction while the others undoubtedly arise because of the hydrolysis step and derivatisation step. Obviously the derivatisation has been successful in reducing the polarity of the active compounds to the degree that they have migrated well up the TLC plate. This did not happen with the acetylated 30% Sep-Pak fraction. A similar experiment with acetylated and methylated 30H gave the same result as with 40H. So it appeared that hydrolysis did release two active compounds much less polar than the precursor. This suggests that SF in this case may be bound to two carrying groups and that one of these may be very polar, and the other less polar. Thus the activity eluted from the plate with derivatised 40H could contain X-Y-SF on the origin, Y-SF further up the plate and SF closest to the solvent front. This would be the case where X is a very polar group which is either difficult to acetylate and methylate or when derivatised is still very polar (eg an aspartyl or glutamyl group is difficult to acetylate and will bind very strongly to silica if the amino group is underivatised), Y is a non-polar group that does not acetylate and methylate or a more polar group which is derivatisable. An example of this type of pairing was found (see Section 2) where the equivalent SF part is still unknown. Much effort in the early stages was expended in finding the active peak on GC using a column with the same packing (different batch) and occasionally the same column as used by Milborrow in his attempt to isolate sufficient SF for identification. Numerous active fractions from silica column and Sep-Pak purifications were acetylated, methylated and analysed by GC to find a peak with the appropriate relative retention - without success. More effort was spent screening purified active fractions by GCMS for a compound in the right retention 98 zone which had a fragmentation pattern that was even vaguely similar to Milborrow's active peak - again without success. MeJ itself was only found when a HPLC purification of 40% ethanol Sep- Pak fraction was purified by HPLC (using synthetic MeJ as a marker) and derivatised with pentafluorobenzy bromide and compared with a similarly derivatised standard on GCMS. The very low jasmonate levels were presumed to be a result of picking the bean leaves at an earlier stage than the Milborrow crop. Jasmonate seems to be associated with yellowed senescent leaves. This raised the possibility that the Milborrow SF was also present at very low levels and that the active compound was in the form of a precursor or a conjugate of this precursor. GC trapping experiments also failed to produce any activity at the appropriate retention. However early GC trapping experiments did confirm that active compound(s) were eluting from the QF1 column but at different retentions to those expected if the same compound(s) were responsible for SF activity. The earlier and later activity trapped from the QF1 column purification were initially ignored as a great amount of material was processed in attempts to find activity at the appropriate retention. Part of the reason for this persistence was that some other compounds such as salicylic acid, and caffeic acid have weak activity under our bioassay system and the retention of these compounds falls outside the appropriate senescence zone. The methyl ester of salicylic acid elutes before a methyl jasmonate marker while methyl caffeate elutes later. Other similar compounds can display weak growth inhibitory behaviour (Kefeli and Dashek, 1984) and the retentions of these in the methylated state does not match the retention properties of the original active compound. Another reason for this persistence lay in the primary aim of this project; to find and purify the compound found by Milborrow to promote abscission and ethylene production. 99 1.5.5 A PHASELIC ACID DERIVATIVE IN AN ACTIVE FRACTION After consistent failures to duplicate the activity results of Milborrow's earlier work it was decided to pursue other avenues of purification. HPLC was one option followed. A normal phase separation (Methods 1.3.12) of the fraction 40M20 gave rise to a UV absorbing peak which on bioassay was found to be very active. The chromophore was in shape and wavelength maxima to almost identical to trans- 3,4 dimethoxy cinnamic acid methyl ester. IR spectrum of the unknown was very similar to that of the cinnamic acid derivative apart from a broader carbonyl peak. Hydrolysis of the trapped compound and methylation gave trans- 3,4 dimethoxy cinnamic acid methyl ester and another compound of molecular weight 176. The identity of this unknown product was suspected to be methoxy succinic acid dimethyl ester and this was shown by congruence of retention and mass spectrum on GCMS to the synthetic methoxy succinic acid dimethyl ester. Methoxysuccinate was assumed to be the product of methylation of malic acid (an unlikely event since the malate hydroxyl is not very acidic) and this reaction was not repeatible on synthetic malic acid. However the identity of the cinnamoyl conjugate was confirmed by synthesis and comparison of mass spectral and proton NMR data. Bioassay of the synthetic compound after hydrolysis was negative as was the activity of synthetic methoxy succinic acid. The synthetic cinnamoyl malate was also tested as free acid and found to be inactive. Clearly the active constituent in the HPLC purification of 40M20 was not phaselic acid but did co-elute with it. This was an intriguing result since the Osborne groups attempts at purification of SF eventually yielded nothing more than caffeic acid which is at most only slightly active. Most activity was gradually lost during the purification procedure, a procedure which included some of the methods used here, particularly HPLC, hydrolysis and methylation. Perhaps SF was also a conjugate of caffeic acid but is present in considerably smaller amounts than phaselic acid. 100 The likelihood that SF was present as a conjugate with a cinnamoyl derivative was also indicated by the split of activity on GC purification. Almost without fail the activity occurred in two zones, an early and a late zone. This activity corresponded reasonably closely to dimethylmalate and 3,4 dimethoxycinnamoyl malic acid dimethyl ester. The later eluting activity (on GC) was spread across a large retention zone, a feature which fits very well with the notion that SF is conjugated with a cinnamic, ferulic or caffeic acid carrying group. These aromatics isomerise from the natural trans form to the cis form in light and there is significant retention difference between the two isomers. For instance the trans form of 3,4 dimethoxycinnamoylmalate monoethyl monomethyl ester has a retention time of 28 min while the cis form elutes earlier at 24 min 52 secs (see Figure 1.4.19). 1.5.6 PROBLEMS IN THE ISOLATION OF SF Obviously the path to the isolation of a bioactive compound is not a straightforward exercise. The problems faced are numerous and clearly the fact that a compound remains uncharacterised despite attempts in this field reflects the extent of the difficulty. One problem faced in the attempt to isolate SF was the time lag between preparation of samples for bioassay and the receipt of the results. Unfortunately the assays had to be performed in England since Dr Osborne was very skilled in this procedure and it was not practical to do them at this University. Consequently there was a delay of two to four months before further purifications of active fractions could be done with any clear direction. However this problem was not the major impediment. The greatest frustration was the irreproducibilty of activity for the same fractions after the same derivatisation and fractionation steps were carried out. While the unmethylated, unacetylated or unhydrolysed crude acid extract was fractionated the bioassay 101 results were consistent whether large-column silica, Sep-Pak or TLC methods were used. However, after derivatisation activity was not consistent from experiment to experiment, and in some cases this could have led to some false negatives. That is, many fractions could have returned negative results through losses before bioassay. This possibility did not become obvious before the realisation of the significance of the early activity on GC. Later GC trapping experiments showed that activity is in a very volatile area after methylation. After one major constituent of a fraction from the purification of AUS 12A was identified as succinic acid dimethyl ester this compound was used as a marker and activity was found to elute before this diester. Thus the active compound was exceedingly volatile in methylated form. This explained some of the problems with the irreproducibility of some purification sequences and the fact that in some cases activity was entirely lost For instance, activity eluting from TLC purification sequences of acetylated methylated fractions were invariably lower than expected. One extreme case involved an experiment utilising a chromatotron and a silica plate. In this instance the combined 30% ethanol fractions from three separate Sep-Pak runs were methylated and eluted with a variety of solvents including hexane, ethyl acetate and methanol. None of the fractions collected showed any activity at all, surprising since the total activity from the three 30% ethanol Sep-Pak eluents should have been extremely high. Presumably the active compound was lost during evaporation of the solvents. One other possible cause of loss could lie with the suspected structure of SF. Some of the compounds present in or near the active GC zone were dimethyl esters of oxalate, malate, succinate and fumarate as well as the methyl esters of glycollic and lactic acids. In the free state most of these acids particularly malate and glycollate, do not partition very effectively into organic solvents from aqueous solution. Methylated fractions needed to be hydrolysed in base, acidified and then 102 extracted into ethyl acetate before bioassay. Perhaps at this stage there was significant losses. Instability of SF was not a cause of activity losses. Storage at low and high pH and prolonged storage at neutral pH did not diminish activity to any significant extent 1.5.7 SUSPECTED STRUCTURAL CHARACTERISTICS OF SF Early results showed that SF was an acid and that this acid was inactive after methylation, but activity could be regenerated after alkaline hydrolysis. It was also shown that alkaline hydrolysis of the ethanolic bean leaf extract released additional extractable activity. This promoted the idea that there may be a polar conjugate of SF which will not partition into ethyl acetate. Further evidence to support the presence of a very polar conjugate was supported by the acetone eluents from the charcoal column (Methods 1.3.6). Here, a substantial amount of activity eluted with the 60% aqueous acetone fraction, a result which suggested that the active compound(s) has quite high polarity. Results from this column also indicated that there was two clear zones of activity (see Figure 1.4.2). Other chromatographic properties suggested that there is, at the very least two active compounds; a very polar active compound and a nonpolar form. This splitting of activity into more than one zone was a common feature during any of the various purification sequences. For instance the Sep-Pak purification of fraction 3 (AUS27A) gave three zones (Results 1.4.3), TLC of acetylated and methylated fraction 40H gave three zones (Results 1.4.11) and GC trapping of fraction 80HAF gave two zones of activity (Results 1.4.12). Late eluting GC activity and the co-elution of activity with dimethoxy cinnamoyl malate monoethyl monomethyl ester on normal phase HPLC and GC suggests that a compound of intermediate polarity exists as well. This compound may also be a cinnamoyl ester. These three forms may be related and may thus 103 contain a very polar carrying group, a cinnamoyl, caffeoyl or feruloyl group and the small active acid. GC trapping results suggest that this small active acid has about six carbons and may also have a hydroxyl group. Further, hydrogenation of a combination of three fractions from AUS 2, followed by GC trapping gave activities very similar to the un-hydrogenated fractions so it is likely that the active compound is aromatic or does not contain any CHCH double bonds. The notion of an aromatic was readily dispelled by two pieces of evidence, elution behaviour of activity in comparison to acetoxybenzoic acid and GC behaviour. In the first case activity from the Sephadex LH20 column co-eluted with palmitic acid while the aromatic acid eluted in later fractions. In addition the simplest aromatic acid (benzoic acid methyl ester) had a longer retention on GC than bioassayed activity. 1.5.8 FURTHER WORK After the realisation that significant losses were occurring during purification and that the volatility of the methylated form was a major problem greater care was taken with the manipulation of active methylated fractions. At this stage one fraction which has been shown to have very strong early eluting GC activity is being stored until more efficient methods of fractionation are devised. It is possibly to just purify this fraction by GC trapping but this would be tedious and would undoubtedly result in the loss of significant activity. One option yet to be investigated is the hydrolysis of the methyl esters, formation of much less volatile phenacyl derivatives and separation of these derivatives on normal phase HPLC. These compounds have the advantages that a strong chromophore (e ~ 14000) allows sensitive determination by UV detectors and that the phenacyl group can be removed under quite mild hydrolytic reactions. Phenacyl esters of fatty acids have been prepared by Borch (1975) while bromophenacyl and napthylacyl esters of carboxylic acids have been synthesised by 104 Durst et al. (1975) and Cooper and Anders (1974) respectively. The separated phenacyl compounds could then be hydrolysed and bioassayed and if the activity appears to coincide purely with one HPLC peak then the identity of this unknown should be relatively simple using mass spectroscopic and NMR techniques. Among the derivatisation reactions outlined in Knapp (1979) is one specific for a-hydroxy acids that could be used to test the probability that the active compound is a small a-hydroxy acid. N-butylboronic acid will form cyclic boronate derivatives from a-hydroxy acids and if this derivative was prepared then the products could be analysed by GCMS. Boron compounds give a characteristic isotope pattern (18.98% 10B, 81.02% nB) which would allow an easy distinction between these derivatives from other unreacted acids. CHAPTER 2 Isolation of an m-methoxybenzoylaspartate derivative from bean leaf 105 2.1 INTRODUCTION During their divergent evolutionary paths plants have developed entirely different morphology, shape and function from the animal kingdom and indeed some plant biochemical processes are as divergent as these evolutionary paths. Most are aware that plant cells have polysaccharide cell walls and that plants do not possess a discreet organ mediated hormonal system. One lesser known but extremely important difference is the universal distribution of secondary products. Some members of the animal kingdom synthesise secondary products for use as defence agents or pheromones but a huge proportion of known secondary products are of plant origin (Swain 1977). Plants contain an incredibly wide range of phenolic compounds, most numerous are the flavanoids, quinone, lignans, xanthones, and coumarins (Harbome, 1980). A smaller but nonetheless substantial group, the phenolic acids serve as biogenic precursors of most of these phenolics and also themselves contribute to the pool of plant secondary products. The elucidation of the central role of the phenolic acids in the derivation of the phenolics reached a critical stage almost four decades ago when various workers found that mutant strains of E. coli would not grow unless particular aromatics such as tyrosine, phenylalanine, p-aminobenzoic acid, p-hydroxybenzoic and tryptophan were added to the growth medium. The dependence on these aromatic acids could be abolished by the addition of only shikimic acid. By the mid seventies, knowledge of the role of shikimic acid had grown to the extent that Stafford (1974) recognised that shikimic acid served a pivotal role in the biosynthesis of a major class of aromatic secondary products. So essentially the pathway leading to these aromatic plant secondary products evolves around the formation of shikimic acid from carbohydrate precursors and consequent oxidation and rearrangement to produce cinnamic acid. This acid can then undergo various hydroxylation and methylation reactions to form coumaric, caffeic, ferulic, and sinapic acids - the most common of the 106 phenylpropenoic acids. This group of phenolic acids can undergo a variety of reactions to form esters, amides, alcohols, and flavanoids. Benzoic acids arise either by side chain degradation of the corresponding cinnamic acid derivatives or more directly by the aromatisation of particular shikimate derivatives. The structure of some of the common benzoic and phenyl propenoic acids are shown in Figure 2.1.1. Cinnamic acid derivatives occur as free acids or more commonly are esterified to glucose (Harbome et al., 1961; El-Basyouni and Neish, 1966) or quinic acid (Levy and Zucker, 1960; van Bragt et al., 1965) and these esters are of widespread occurrence. Quinic and glucose esters of cinnamic acid derivatives occur in leaves (van Bragt et al., 1965), fruit (Moller and Herrmann, 1983), vegetables (Brandi and Herrmann, 1983a, 1983b) while the less common glucosides have also been reported as constituents of some vegetables (Winter and Herrmann, 1986). More complex oligosaccharide esters and glycosides of caffeic acid have been characterised by Andary (1982), of sinapic acid by Hamburger and Hostettmann, (1985) and of ferulic acid by Kato et al., (1983) and Fry (1982). Malic acid esters of the hydroxylated cinnamic acids also seem to be quite widespread. Caffeoylmalic acid has been found in red clover (Yoshihara et al., 1974), more recently in radish cotyledons (Strack and Dahlbender, 1984), and mature radish leaves (Brandi and Herrmann, 1984). The dimethylated derivative in ethyl, methyl ester form has been separated in quantity from beans in this work (Chapter 1). Additionally other small hydroxylated aliphatic diacids have been found as esters of substituted cinnamic acids. Some of these include tartronic acid (Strack et al., 1985), glutaric acid (Ellinger et al., 1981) and meso-tartaric acid (Strack et al., 1987). In short there is a bewildering array of these conjugates discovered over the past two decades and the enzymes that are involved in the biosynthesis of these compounds seem to be specific for the phenylpropanoid and the particular conjugating group. For instance, studies by Strack and Gross (1990) on a purified chlorogenic acidiglucaric acid caffeoyl transferase demonstrated that 107 Cinnamic Acid C02H OH OH p-Coumaric Acid Salicylic Acid C=C C02H Ferulic Acid o-Methoxy Benzoic Acid OH Caffeic Acid p-Hydroxy Benzoic Acid FIGURE 2.1.1 Some Commonly Occurring Free Phenolic Acids 108 this enzyme is most active with 5-O-caffeoylquinic acid and glucaric acid, much less active with 5-O-caffeoylquinic and galactaric acid and inactive with other hexose acids (eg glucuronic acid) and other chlorogenic acid isomers (eg 4-0- caffeoylquinic acid). A short overview showing some phenolic acid conjugates is shown in Figure 2.1.2. While the conjugation of phenylpropenoic acids seems to be very widespread in both occurrence and quantity the incidence of similar benzoic acid derivatives is comparatively limited. Nonetheless benzoyl conjugates are still quite common in some plant families and the characterized structures exhibit great diversity. Derivatives of salicylic acid and glucose have been discovered in the Salicacea (willow) by Pearl and Darling (1970) and substituted benzoic acids have been found esterified to alkaloids (eg. aconitine, Challis et al., 1968), terpenes Wani et al., 1971), and cyanogenic glycosides (Nahrstedt and Williams, 1976). The largest group of substituted benzoic acid derivatives comprises the gallic acid esters (Haslam, 1980) where the parent phenolic acid is linked with glucose, phenols and glycosides (Haslam, 1982). Substituted cinnamic amides at present offer less structural diversity than the malic, quinic, glucose or small aliphatic hydroxy-diacid esters of hydroxylated cinnamic acid derivatives but nonetheless make up a significant and widespread population. One of the earliest known phenyl propenoic acid conjugates (p- coumaroylagmantine) was characterised by Stoessl (1965). Later Mizusaki et al., (1970) isolated paucine (caffeoylputrescine) in extracts from the tissue culture of tobacco cells and since then the number of hydroxylated cinnamoyl-putrescine derivatives has increased to include feruloylputrescine, coumaroylputrescine, dicoumaroyl and diferuloylputrescine. Similar derivatives of spermidine and tryptamine (Ehmann, 1973) have also been characterised while some complex dicinnamoyl amide derivatives such as cononocarpine and chaenorhine are outlined by Smith (1977). Other amide conjugates have been found to be associated with the cell wall. Hahlbrock and Scheel (1989) describe two compounds, p- 109 COzH R,-OKR2-H R, - R2 - OH R, - OK Rj - OCtij I caffeoyltartronate leruloyltartronate CH = CH —C02 -CH p- coumaroyltartronate (Strack at. al.. 1085) (Strack «L at.. 1085) (Strack at. at.. 1085) R, - R2 - OH R, - OK R2 - OCH, cafleoylmalate feruloytmalate (Tanguy and Martin. 1072) (Tanguy and Martin. 1072) COjjH I (CH2OH)3 R, - R2 -OH R, - OK R2 - OCHj I caffeoylglucarate feruloylglucarate (Ellinger et. al., 1981) (Nagels et. al.. 1982) C02-CH I COjH R, - Rj _ OH R, - OK Rj - H cattelc add glucose aster p-coumaric acid glucose e6ter CH = CH (Hartoome at. at.. 1961) (El-Basyounl et. al.. 1966) CHjOH R, - OH. R* - H R, - OK R2 - OCKj N-(p-coumaryl)tryptamine N-feruloyltryptamlne (Ehmann, 1073) (Ehmann, 1073) NH R, - OH. Rj - H CH = CH — CO-NH-(CHJ*—NH — CH p-coumaryl agmantlne I (Stoessl, 1965) NHj FIGURE 2.1.2 Some examples of C6-C3 Phenolic acid derivatives 110 coumaroyltryamine and feruloyltryamine, compounds that are found in the cell walls of potatoes after wounding. Amino acids linked to either benzoic or cinnamic acid and their hydroxylated derivatives are rare. Bacteria offer a more fruitful source of substituted benzoylamino acid conjugates, and a number of amino acids have been found conjugated to hydroxybenzoic acid groups including including lysine (Corbin and Bulen, 1969) and serine (O'Brien and Gibson, 1970). In plants to date hippuric acid (benzoylglycine), omithuric acid (dibenzoylorthithine) have been discovered and more recently benzoylomithine and dibenzoylomithine have been separated and characterized from Vicea pseudo-orobus by Hatanaka et al., (1981). Imperato (1980) has separated N-p-coumaroylglutamic acid from black tea. A substituted dipeptide (N-feruloylglycyl-phenylalanine) has been isolated from barley husks (van Sumere et al, 1972) and an enzyme from barley embryos (N- feruloylglycine amidohydrolase) partially purified by Martens et al (1988). This chapter details the extraction, purification and identification of a phenolic amino acid conjugate from Phaseolus vulgaris, ortho- methoxybenzoylaspartate. Ill 2.2 MATERIALS 2.2.1 GENERAL For preparation of diazomethane, conditions used for MS, HPLC, TLC, preparative GC and NMR and the Sep-Pak purifications see Materials, Chapter 1. 2.2.2 CHEMICALS AND SOLVENTS Aldrich Chemicals (Milwaukee, USA) supplied the p-anisyl chloride and the oxalyl chloride. The o- and m-hydroxybenzoic acids were obtained from Merck (Dahlstadt, FGR). N-methyl-N-nitroso-p-toluenesulphonamide and aspartic acid was provided by Fluka (Buchs, Switzerland). HPLC grade hexane was supplied by Mallinkrodt Australia (Sydney, NSW) while other solvents used in HPLC were redistilled from either AR or laboratory grade stock supplied by Univar (Sydney, NSW). All other solvents and chemicals were AR grade obtained from local suppliers. 112 2.3 METHODS Isolation of the unknown compound involved extraction of plant material and Sep-Pak fractionation followed by HPLC and GC purification. The overall scheme is shown in Figure 2.3.1 and Figure 2.3.2. 2.3.1 SOLVENT EXTRACTION Senescent bean leaves Phaseolis vulgaris cv Dwarf Pioneer (200 kg) were extracted as outlined in Methods 1.3.1. 2.3.2 SEP-PAK PURIFICATION Some of the resultant acid fraction concentrate from the bean leaf extract (about 10 ml from a total volume of about 2 1) was then added to 0.2% aqueous acetic acid (200 ml) and this mixture filtered through a No.4. glass sinter until completely clarified. One-fifth of this extract was then transferred to a 100 ml separating funnel with a set of four Ci8 Sep-Paks attached to the outlet and the flow adjusted to a rate of about 5 ml/min. The solution was pushed through the Sep- Paks with the aid of a small peristaltic pump connected to the inlet of the separating funnel. After the clarified acid extract has been completely pumped through, the Sep-Paks were eluted in turn with 20 ml each of 5%, 10%, 20%, 30%, 40%, 50%, 80% and 100% ethanol in 0.2% aqueous acetic acid. The remaining acid extract was treated in the same fashion and the 20% ethanol fractions combined and evaporated. After methylation with diazomethane the 20% ethanol eluent was dissolved in 95% ethanol (20 ml) and diluted to a volume of 100 ml with 0.2% aqueous acetic acid. This solution was added to the Sep-Pak apparatus in two equal portions and these eluted separately with 30%, 40%, 50%, 60% and 100% ethanol in 0.2% aqueous acetic acid to give fractions 30M20, 40M20, 50M20, 60M20 and 100M20 respectively. The 30M20 fractions were combined, evaporated, dissolved in isopropanol (2 ml) and hexane (18 ml) added. The gelatinous precipitate formed 113 BEAM LEAF Extraction NEUTRALS AQUEOUS Reverse-ohase Seo-Paks WATER 10% 20% 30% 40% 50% 80% 100% Methvtatbn. revarse-ohase Sop-Paks 20M20 30M20 40M20 50M20 60M20 100M20 Norma khase Seo-Pak 30M20;1 OIPA 30M20;M ETHANOL FIGURE 2.3.1 Initial clean-up procedure for the extraction and isolation of MBA esters from bean leaves. 114 30M20; 10IPA Normal-Phase HPLC Before BX BX After BX Normal-phase HPLC 1BX 2BX 3BX 4BX 5BX 6BX 7BX 8BX Normal-phase HPLC 5+6BX;A 5+6BX;B 5+6BX;C GC trapping Purified OBA Monomethvl Monoethvl Ester FIGURE 2.3.2 Final HPLC and GC purification steps in the isolation of MBA esters from bean leaves. 115 after the addition of the hexane was allowed to settle overnight. The clear pale yellow solution was then decanted and loaded onto a single silica Sep-Pak. The eluent from the Sep-Pak was collected and the Sep-Pak washed with 10% isopropanol in hexane (10 ml) followed by methanol (10 ml). The 10% isopropanol eluents were combined, evaporated and redissolved in a small amount of isopropanol for HPLC purification. 2.3.3 METHYLATION Unless otherwise specified all methylations were carried out by dissolving the sample in a small volume of methanol and treating with ethereal diazomethane (five to ten fold excess, 2 hr, room temperature). 2.3.4 HPLC PURIFICATION OF THE UNKNOWN Purification of the 30% ethanol fraction after Sep-Pak fractionation (30M20, 10IPA) was carried out on a silica semiprep column with a mobile phase of 3% isopropanol in hexane at a flow rate of 4 ml per minute. Monitor wavelength was set at 293 nm with a bandwidth of 4 nm. The fraction eluting between 8.0 and 10.5 minutes (BX) was collected, evaporated and reinjected onto the same column under the identical conditions. The volume eluting between 9.3 and 10.5 minutes (5+6BX) was collected, evaporated, again reinjected and chromatographed under the same conditions. Three fractions were collected 5+6BX;A (7.5 to 8.3 min), 5+6BX;B (8.3 to 9.5 min) and 5+6BX;C (9.5 to 11.0 min). Each fraction was evaporated under a stream of nitrogen, for GCMS analysis. Fraction 5+6BX;C was further purified by preparative GC. 2.3.5 GC TRAPPING Fraction 5+6BX;C was injected onto a 3% QF1 column into a nitrogen stream flowing at 30 ml/min with the oven temperature maintained at 200°C. The major peak was collected on the surface of a glass capillary inserted into the manual 116 trapping port. The material condensed on the capillary tube was washed off with 99.96% deuterochloroform into an NMR tube. 2.3.6 PROTON NMR OF THE UNKNOWN The isolated trappate from fraction 5+6BX;C (Methods 2.3.5) was submitted for proton NMR under the conditions outlined in Materials 1.2.7. 2.3.7 SYNTHESIS OF META. ORTHO AND PARA MBA 2.3.7.1 GENERAL ASPECTS During the following synthetic procedures little attempt was made to quantify yields or purify intermediates since only small amounts of the final products were required and these could easily be identified by their behaviour on TLC, GC or HPLC. GCMS was routinely used to monitor the progress of the syntheses and to identify the various products. An overview of the method is shown in Figure 2.3.3. 2.3.7.2 PARA MBA Aspartic acid (1 gm, 7.5 mmoles) was dissolved in 2N sodium hydroxide (5 ml), chilled in an ice-bath and treated with p-anisyl chloride (1.35 gm) and 2N sodium hydroxide (5 ml) in five equal and alternate portions. After addition of the reagents the solution was allowed to slowly warm to room temperature with intermittent shaking. In about ten minutes a white crystalline material precipitated and this was removed by filtration, washed with ice-cold water and dried in air overnight. Most of the contaminating p-methoxybenzoic acid was removed by washing the crystals with dichloromethane. 2.3.7.3 ORTHO AND META MBA o- and m-hydroxybenzoic acid (1 gm, 6.5 mmoles) was treated separately. Each acid was dissolved in methanol (30 ml), treated with ethereal diazomethane 117 C-OH <&OH c—Cl OCH, FIGURE 2.3.3 Synthesis of ortho-methoxybenzoylaspartic acid. 118 containing approximately 1.2 gm (28 mmoles) of diazomethane and left at room temperature for two hours. After evaporation the residue was dissolved in 50% aqueous methanol (80 ml), 6% aqueous potassium hydroxide (10 ml) added and the mixture left to hydrolyse overnight. The methanolic solution was extracted with hexane (2 x 100 ml) to remove the unhydrolysed ester, acidified to pH 2.5 and extracted with ethyl acetate (3 x 200 ml). The ethyl acetate extracts containing a mixture of hydroxy and methoxybenzoic acids were evaporated to dryness. This mixture, in a 50 ml round bottom flask equipped with a calcium chloride guard tube, was dissolved in oxalyl chloride (5 ml) and heated in a 70°C waterbath - with occasional removal for sonication. The residue was then evaporated to dryness, cooled in an ice-bath and treated with an ice-cold solution of aspartic acid (1 gm) in 2N sodium hydroxide (10 ml). After slowly warming to room temperature the resultant crystalline mass was filtered and the precipitate washed with hot 95% ethanol (20 ml). This ethanol wash was evaporated and stored at 4°C. 2.3.8 PREPARATION OF THE MONOMETHYL-MONOETHYL. DIMETHYL AND DIETHYL ESTERS An outline of the method is shown in Figure 2.3.4. 2.3.8.1 PARA A few ml of the ethanol wash from the p-MBA preparation was evaporated to dryness, redissolved in absolute ethanol (5 ml), a few drops of concentrated hydrochloric acid added and the solution left to stand overnight The reaction mixture was then evaporated to dryness and redissolved in a small volume of ethanol prior to further purification. 119 O \ ch3ch2o"C C'ch-c/OCH3 cl /CHCV C-NH OCH. FIGURE 2.3.4 Synthesis of the monomethyl, monoethyl, and diethyl esters of MBA. 120 2.3.8.2 ORTHO AND META About 10 mg of the crystals from the respective o- and m-MB A preparations was dissolved in absolute ethanol and treated in the same way as the p-isomer. 2.3.9 THIN-LAYER CHROMATOGRAPHY The products from the ethylation of synthetic p-methoxybenzoylaspartate were applied to a silica TLC plate and run with hexane/ethyl acetate/acetic acid (10/5/1). Three strongly UV absorbing zones were scraped into small glass sinters and washed with methanol (30 ml). The methanol washes (TLC A, TLC B and TLC C) were evaporated, methylated and analysed by GCMS and the eluent from one of these zones (TLC A) used as a standard for the spiking experiment (Methods 2.3.11). Ethylated o- and p-isomers were treated in the same manner. 2.3.10 HPLC OF THE ETHYLATED ORTHO. PARA AND META MBA PREPARATIONS The ethylated o- and m-MBA preparations were methylated with an excess of diazomethane and then analysed by normal-phase HPLC under the same conditions used in Methods 2.3.4. Fraction TLC A (from the para preparation) was also analysed by HPLC under the same conditions. 2.3.11 SPIKING THE PLANT EXTRACT WITH SYNTHETIC ISOMERS A small amount of the products from the ethylation and methylation reactions (Methods 2.3.9) were added to Sep-Pak fraction 30M20;10IPA in an attempt to find which of the ortho, meta or para isomers was present in the plant extract The quantities of synthetic diesters added were adjusted by first performing GCMS analyses of 30M20;10IPA, TLC A (para isomer after methylation) and the products from the methylation of the unpurified ortho and meta 121 monoester preparations and then calculating the amount of each solution that would give rise to approximately equal total ion current response for each species. The calculated volumes of the each synthetic isomer solution were then added to three aliquots of 30M20 and GCMS performed on these mixtures. 2.3.12 GCMS GCMS was routinely used to check on the identity of compounds prepared in the synthetic schemes outlined in Methods 2.3.8. and to verify the identity of the compounds purified by HPLC for NMR analysis. Fractions collected during Sep-Pak and HPLC purifications of the bean leaf extract were also screened by GCMS for the presence of the unknown compound and analyses were run of the spiked bean extract (Methods 2.3.11) In all instances the column used had the dimensions outlined in Materials 2.2.1. The column packing used was 3% QF-1 on chromasorb W HP 80-100 and oven temperature was raised 6°C/min from 100 to 250°C. Temperature programme was activated 1 minute after injection. Methane at a flow rate of 20 ml/min was used as carrier/reagent gas. 122 2.4 RESULTS 2.4.3 PRELIMINARY RATIONALE The Erst clue of the possible existence of MBA came from the GCMS of a senescence active fraction, 40M20;10IPA. This particular fraction had previously been shown to contain very large amounts of monomethyl, monoethyl DMP and it was considered likely that other dicarboxylic acid conjugates of aromatic acids could be present in this fraction. One very minor constituent showed some very interesting characteristics. It appeared to have a very simple fragmentation pattern and an odd molecular weight (see Figure 2.4.1) Screening of the preceding and the subsequent fractions (30M20;10IPA and 50M20;10IPA) showed that 30M20;10 IPA was richest in this unknown. Even so the relative ion current was low and the molecular weight 309 peak was a barely discernible shoulder on the tail of another peak (Figure 2.4.2). Also, specific ion monitoring showed that in the mass ion range above m /z 60 the most abundant ions were m/z 135 and 310 (see Figure 2.4.2) The simplicity of the mass spectra gave some indication of the likely identity of the unknown metabolite, especially considering previous experience with monomethyl, monoethyl DMP. On positive ion mass spectra this compound displayed a weak but distinguishable parent ion and a very intense m/z 191, corresponding to the loss of the malic ester portion and the formation of a very stable aromatic moiety, [(CH30)2:C5H3:CH::CH:C0+]. It seemed quite likely that the m/z 135 ion could also comprise the aromatic part of a conjugate with a nitrogen containing diacid. Moreover the significantly greater relative abundance of the parent ion reflected a greater stability of the parent ion and lent credence to the notion that the the linkage between the benzoic moiety and the conjugating compound was probably an amide bond rather than an ester bond. Previous experience had shown that the parent ion of synthetic omethoxybenzoylmalate was typically less than 2% relative abundance. FIGURE RELATIVE INTENSITY RELATIVE INTENSITY 100.0 100.0 50.0 50.0 2.4.1 -i - -i - with total Summed 2.4.2 Spectrum about ion for chromatogram mass the consists 40 total scans spectrum m/z of ion of summed chromatogram. background of across fraction scan a small 30M20;10IPA. subtracted. #1280 shoulder to #1300 See r- r- in 9680 9680 the Figure 123 CO io E N 1341 1366 o CO E N DC ~ > o> o c I. U S o y- o CM T- CO o o 1- ^ o o o 91 GO <* o CM O CM T- t o CM CO ° 3 : l Si o ro E J DC *-« *-> c o £ E1S.URE 2,4.2 Specific ion traces for ions m/z 135 , m/z3io and totai ion chromatogram of fraction 30M20;10IPA 124 125 Various aromatic conjugating groups have been discovered in plants and the most common are ferulic, caffeic, p-coumaric, p-hydroxy (and methoxy)benzoic and o-hydroxy (and methoxy)benzoic acids. Of this group only an o-,p- (or m-) methoxybenzoyl derivative could, on loss of the conjugating group (including the amide nitrogen) during fragmentation, give an ion m/z 135: [CH3O: C6H4: CO+] The DMP isolated from fraction 40M20;10IPA was principally in the form of the monomethyl, monoethyl ester. Ethyl esters have been reported rarely in plants so the ethylation was presumed to be an artifact of isolation, probably acid catalysed esterification during the initial extraction stage and storage. If this is so it would follow that if the unknown was a diacid then it would probably also have been ethylated during the purification procedures. As the purification proceeded it became obvious that this was the case. 2.4.2 ISOLATION AND PURIFICATION 2.4.2.1 REVERSE-PHASE SEP-PAK STEPS After extraction of the acid fraction, the initial stages of the purification involved the separation from very polar acids and the strongly hydrophobic acids on reverse-phase Sep-Paks. Sep-Paks are small polyethylene tubes packed with silica coated with a hydrocarbon layer via a silanol linkages to the silica surface and these normally used for quick preliminary sample clean-up prior to HPLC. However if a number of these Sep-Paks are connected in series the overall capacity and resolution is sufficient to make them very useful for separation of mixtures into basic polarity groupings. Four Sep-Paks in series gives a reasonable loading capacity as well, something in the order of 4 mg. Following the first Sep-Pak fractionation, the 20% ethanol eluent was methylated before re-running on the same Sep-Pak system. 126 Methylation decreases the polarity of the acids in the mixture so the methyl esters require higher ethanol concentrations in the mobile phase than their corresponding acids for successful elution. Typically in practice, a monomethyl ester will elute with a mobile phase containing about 10% more ethanol (or methanol) than the free acid. In this case the free acid was washed from the Sep- Paks with 20% ethanol in 0.2% aqueous acetic acid whereas the methylated compound needed 30% ethanol. Crude plant acid extracts contain a great variety of compounds. Among the simpler ones such as small aliphatic acids, aromatic acids, fatty acids and amino acids there are the complex mixtures of denatured and degraded proteins and phenolics. Most of these polar unwanted compounds will readily pass through reverse-phase Sep-Paks but most will be strongly retained on silica. The next step, the normal-phase Sep-Pak fractionation of 30M20 results in the removal of most of these so consequently HPLC purification can be done isocratically without interference. Polyhydroxy phenolics typically form very broad tailing peaks during normal-phase HPLC in isopropanol/hexane mixtures that often completely mask peaks of interest. In this case a great proportion of polar compounds are removed simply by their insolubility in the elution solvent. Before the 30M20 fraction was loaded onto the Sep-Pak it was first dissolved completely in isopropanol (2 ml). The addition of 18 ml of hexane produces a gelatinous precipitate which compacts on standing overnight. The soluble material containing the unknown could then be decanted and loaded onto the Sep-Pak. 2.4.2.2 HPLC STEPS The 10% isopropanol in hexane eluent from the Sep-Pak was then purified by normal-phase HPLC and relevant fractions tested for the presence of the molecular weight 309 compound by GCMS. Firstly a series of runs was performed taking broad cuts (before BX, BX and after BX). Fraction BX was then evaporated, injected under the same conditions and eight fractions collected over a 127 14 min period (1BX to 8BX). The final HPLC step involved the re chromatography of fractions 5BX and 6BX and the separation into three parts; 5+6BX;A, 5+6BX;B and 5+6BX;C. The latter was found by GCMS to contain more than 95% of the unknown compound. The chromatogram from the three HPLC runs together with the trapping regions is shown in Figure 2.4.3. It should be noted that it is not usually good practise to utilise a purification sequence that involves retrapping over the same time period using exactly the same run conditions. However in this case the small amount of the unknown in the sample suggested that conservatism would be the best policy. One previous run was used in concert with GCMS to localize the presence of the unknown in fraction BX so there was no real advantage gained by using another system. Additionally the major contaminating material appeared to be phenolics which had by previous experience occasionally affected the retention of other compounds differently when mobile phase properties were altered. Figure 2.4.3 clearly shows that when the loading of these contaminants decreases the peaks shapes change quite dramatically. In any case a preparative GC step was planned so the phenolics would bind very strongly to the inlet end of the column and would not contaminate the unknown at the final purification step. 2.4.2.3 PREPARATIVE GAS-LIQUID CHROMATOGRAPHY The last stage was the trapping of the major peak detected by flame ionisation via a manual trapping port. At the appropriate time a glass capillary tube was pushed over the outlet and the compound condensed on the surface. The contents of the capillary were washed into an NMR tube with 99.96% deuterated chloroform and a proton NMR performed. See Figure 2.4.4 for the flame ionisation trace and trapping regions. FIGURE mRU mflu mflU 200 250- 300- 200 150- 1 1 - 1 M0- 120 1 00 00 G0- 00 20 2.4.3 - - - - * : - chromatogram The chromatogram purification, result and 5+6BX;C. BX of trapping reinjection Before T T the T I t I 1 mo mo mo BX represents of production sequence. BX to fraction (min. 4BX of fraction ) the 30M20;10IPA. of The last samples BX upper stage and trace After 5+6BX;A, in the Centre the shows 7BX, BX lower HPLC 6BX 5+6BX;B trace the HPLC is the 128 129 GC Conditions: Carrier Gas: Nitrogen Gas Flow Rate: 30mls/min Temperature: 210°C Isothermal Detector: Flame Ionisation Trapping Region 8 6 4 2 0 Time after Injection (mins) FIGURE 2.4.4 GC chromatogram and trapping region for fraction 5+6BX;C 130 2.4.3 MONITORING PROGRESS OF THE PURIFICATION BY GCMS At most points in the purification sequence a portion of each fraction from HPLC was analysed by GCMS. At the latter stages of the purification sequence, as the purity of the unknown increased the mass spectrum showed more detail. The MS of fraction 5+6BX:C showed the adduct ions m/z 338 (M+29, M+C2H5) and 350 (M+41, M+C3H5) and so confirmed that the molecular weight was 309. The appearance of ions at M-46 and M-32 indicated strongly that the compound was a methyl, ethyl ester since loss of methanol (32AMU) is typical of methyl esters while loss of ethanol (46AMU) is very common with ethyl esters. These findings combined with the strong suspicion that the unknown was a methoxybenzoyl amide suggested that the most likely structure would be a methoxybenzoylaspartic acid monomethyl monoethyl ester (MBA methyl, ethyl ester). 2.4.4 PROTON NMR OF TRAPPATE 5+6 BX:C The proton NMR of the GC trappate of 5+6 BX-C (see Figure 2.4.5) only gave some credence to the proposed structure. Due to the small quantity of material isolated from the extract the NMR signal was very weak despite an overnight accumulation of 25,000 scans. There were only a few obvious resonances distinguishable above background noise. The spectrum also showed an extremely strong resonance at 7.27 ppm (the proton from the chloroform in deuterochloroform) which tended to obscure any weak resonances due to aromatic protons that may have been present. However the occurrence of three discernible singlets in the region 3.6 to 4.2 ppm certainly did suggest that the proposed structure was most likely. According to (Silverstein et al., 1974) singlets in this range of chemical shift for a compound which is both acidic and aromatic are most commonly due to methyl protons from methoxy groups or methyl esters. >inglets in the chemical shift region expecte methoxy or methyl ester protons. EIGURE 2,4,5 Proton NMR of the GC trappate (see Figure 2.4.4) of HPLC fraction 5+6BX;C. Only part of the spectra is shown: 3.5 to 5.0 ppm. 131 132 2.4.5 SYNTHESIS OF THE METHOXYBENZOYLASPARTIC ACIDS 2.4.5.1 GENERAL ASPECTS A lack of conclusive evidence to the identity of the unknown together with the need for large quantities of raw materials to isolate very small amounts of the pure compound necessitated a different approach to the problem. If the methoxybenzoylaspartic acids could be synthesized relatively easily then a comparison of GC retention and mass spectra should clearly prove or disprove the proposed structure. N-benzoylation of amino acids is a well documented procedure which basically involves the condensation of the benzoyl chloride with the amino acid in ice cold 2M sodium hydroxide. Benzoyl chloride is a liquid at ice bath temperature (MP -1°C) so the reaction can proceed at a reasonable rate in the liquid phase at 0°C. However the monomethoxybenzoyl chlorides are solids at 0°C so the method of Greenstein and Winitz (1961) had to be modified slightly to compensate for the difference in the melting points of the substituted and unsubstituted benzoyl chlorides. In this case the acid chloride solidifies on addition of the aspartic acid / sodium hydroxide solution so the reaction mixture was slowly allowed to warm to room temperature. During this warming the acid chloride melted and reacted with the aspartic to form the desired product. Condensation was complete when the milky suspension formed by warming was transformed into a flocculant white precipitate. 2.4.5.2 PARA In the case of the para isomer the acid chloride was readily available so the reaction was straightforward and simply involved the addition of the aspartic acid solution to the acid chloride in sodium hydroxide, the reverse order of addition to that used in method 23.1.2 due to the solidification of the acid chloride at low temperature. 133 2.4.53 ORTHO AND META Before the condensation could take place the acid chloride of ortho and meta methoxybenzoic acid had to be prepared from salicylic and meta hydroxybenzoic acids respectively. This involved firsdy the methylation of the acid group and the formation of the appropriate methyl ether by reaction with diazomethane. The methyl esters were then cleaved by basic hydrolysis and after extraction with ether to remove any unhydrolysed esters the free acid was extracted into ether after acidification to pH 2. The mixture of hydroxy and methoxybenzoic acids were then treated with oxalyl chloride to form the acid chloride and after thorough removal of excess reagent by evaporation under reduced pressure the ice- cold sodium hydroxide solution was added followed by the aspartic acid solution. As with the preparation of the p- isomer the reaction mixture was left to slowly warm to room temperature so that the acid chloride could melt and condense with the aspartic acid. 2.4.6 PURIFICATION Since the objective was purely to isolate a small amount of the appropriate conjugates, attempts to monoethylate, methylate and then check for coincidence in retention and similarity of mass spectra on GCMS of one of these synthetic isomers with the naturally occurring compound then there was little point, at this stage at least, in undergoing a rigid purification sequence. In the case of the para isomer the p-anisic acid was filtered and the crystals washed with about 20 ml of ice cold ethanol. This ethanol wash was enriched with the conjugate compared to the crystalline material. Lower yields in the o- and m-MBA preparations meant that any similar purification procedure would result in greater proportional losses of the product so the crystals gathered in the filtration step were used directly. 134 2.4.7 FORMATION OF THE MONOETHYL ESTERS There were two major reasons for the preparation of the monoethyl esters of MBA, the first was to see if it was feasible that the esters had formed during isolation and storage and secondly to see if the mass spectra of the monoethyl ester after methylation matched that of the natural compound. Since the crude extract had been stored at a low pH for a prolonged period with traces of ethanol, it seemed feasible that the MBA could have been slowly esterified under these conditions. Most acids require dried ethanol/dry HC1 mixtures as an esterification medium but it was decided that since the extract was not totally anhydrous it would be more relevant to test for a tendency to esterify under conditions where at least some water was present (see Methods 2.3.6). After esterification the products were applied to a silica TLC plate and run under the conditions outlined in Materials 1.2.1 using ethyl acetate/hexane/acetic acid (5/10/1) as a developing solvent. Five major bands were obtained and after methylation, were shown by GCMS to be unreacted free acid, monoethyl esters (two discreet bands for the two possible products), diethyl ester and methoxybenzoic acid methyl ester. The Rfs of the isomeric monoethyl esters are shown in Table 2.4.1 2.4.8 MASS SPECTRA OF ETHYLATED. METHYLATED MBA PREPARATIONS 2.4.8.1 PARA The bands arising from the TLC purification after scraping, elution and methylation were subjected to GCMS analysis. The zone closest to the solvent front had the same Rf as authentic p-methoxybenzoic acid methyl ester so identification by MS was not necessary. Of the four other bands, TLC A and B gave two major peaks with molecular weights of 295 and 309, while TLC C contained a compound with the molecular weight of 323 (see Figures 2.4.6, 2.4.7, and 2.4.8 for the chromatograms mass spectra of the methylated TLC zones). 135 COMPOUND Rf para monoethyl 1 0.19 - 0.23 para monoethyl 2 0.26 - 0.32 meta monoethyl 1 0.15-0.20 meta monoethyl 2 0.21 - 0.27 ortho monoethyl 1 0.23 - 0.26 ortho monoethyl 2 0.28 - 0.34 TABLE 2.4.1 Rfs of para, meta and ortho monoethyl MBA on silica TLC with 10:5:1 (hexane:ethyl acetate: acetic acid). 136 1 00 -| 1412 Relative Ion Current 1000 1200 1400 16:40 20:00 23:20 Scan Number Retention Time (mln:sec) r17920 50.0- 510 250 2^4 278 I I T '"I , |”. I rtr I I 120 140 160 180 200 220 240 260 520 540 m/z FIGURE 2.4.6 Total ion current chromatogram of TLC A (Methods 2.3.9) and mass spectrum of the major peak. Spectra were summed from scans #1394 to #1426 with background subtraction of scan #1428 and #1394. 137 1 00 -! Relative Ion Current 1000 1200 1400 16:40 20:00 23:20 Scan Number Retention Time (mln:sec) r15824 = 50 X) - FIGURE 2.4.7 Total ion current chromatogram of TLC B (Methods 2.3.9) and mass spectrum of the peak centred at scan #1414. Spectra were summed from scans #1400 to #1429 with background subtraction of scan #1431 and scan #1391 to #1397. 138 1 oo i Relative Ion Current 1358 1 000 1 500 16:40 25:00 Scan Number Retention Time (mln:sec) r70144 - 50 JO - mtz FIGURE 2.4.8 Total ion current chromatogram of TLC C (Methods 2.3.9) and mass spectrum of the major peak. Spectra were summed from scans #1448 to #1543 with background subtraction of scan #1519 to #1523 and #1428 to #1442. 139 Clearly TLC A and B contained monoethyl esters which after methylation were converted into monoethyl monomethyl esters and dimethyl esters by trans esterification while TLC C contained the diethyl ester of p -MBA. 2.4.8.2 ORTHO AND META The o- and m- esterification mixtures were also methylated and then subjected to MS analysis. The chromatograms and mass spectra of these preparations are shown in Figures 2.4.9, and 2.4.10. 2.4.8.3 COMPARISON OF MS DATA OF STANDARDS AND UNKNOWN The fragmentation pattern of the p-monomethyl monoethyl ester was quite simple as the only ions above 5% relative abundance were the parent ion and the m/e+ 135 fragment. In contrast, the m- and o-MBA derivatives showed a greater abundance of other ions principally m/z 163, 264 and 278 and some variation in the population of the major species. Table 2.4.2 illustrates this point and suggests that there is a clear distinction between the p- and the o/m- pair, and that there should be sufficient difference between the m- and o- derivatives to allow at least a tentative identification of the unknown. The relative abundance of ions in the mass spectrum of the major peak in fraction BX is also included in Table 2.4.2 and it can be seen that the correlation between the unknown and the standards is not strong enough for definite identification. However these results indicated that the unknown would more likely be the o- or m- isomer. From experience with Cl mass spectra, it is very likely that the spectra of the same compound will show quite different relative abundances of ions from day to day. One of the variables involved is ion source pressure. If the operator is not careful to reset ion source pressures consistently then the ratio of high mass to low mass ions can show considerable variation. According to the Finnigan Users Manual for the Model 3200 (1975), the ratio relative abundance of the molecular ion for palmitic acid methyl ester varies from 100% at 1800 microns to 3.7% at 300 microns. Lower ion source pressures favour 140 est mixtui subtrac methylation reaction monomethyl after background crude of the 2.3.8.2) monoethyl scans for 30 and 324 to (Methods 20 and diethyl has 310 esters and 296, dimethyl, peak the 135, monoethyl of m/z spectra appropriate traces the ion mass the across specific are and summed shown ortho-methoxybenzoylaspartate been Also of has chromatogram ion spectra preparation Each the Total diazomethane. 2.4.9 FIGURE 141 2 IpCD o a C W CO X 1O= CD XJ£= E co 3 a itTw3 o 3 TT O CO CO CM CO TD Z> 2 O C — 03 c\J CO CO ■D c/3 - co o CD^ sz CO 03 a n 03 c CO CO 03 CM XI T3 co CD CD CD LO Q_ CO g£ 3 N 1 o CO *v_ E CD 2 CL •+-* O CO CO o CD r oj D. o co Q- Q. CO CL W co CO 03 J2m COUJ c= CO 3 o CO 8 E CO o o H- S ® b ‘o XD 3 0) >* CO X *o CO9- £o 03 ■D E c= E CO E z> CO CO E 03 c 5 03 E 03 03 -O SoCO CO E c co o o jc V— •— 03 CO J= CO C o CO co x: £= 03 o Q. II (0 Q- o XI 75 N o CD CO CO H a ^ LU c\i LU DC 3 o LL 142 RELATIVE ABUNDANCE ION BX TLC A m-MBA o-MBA SPECIES TRAP (para) PREP PREP 135 37 1 00 88 31 152 <1 1 8 8 3 1 63 <1 2 30 4 264 2 2 1 9 6 278 - <1 1 2 3 310 1 00 23 1 00 100 338 1 0 3 1 4 1 0 350 3 <1 5 4 TABLE 2.4.2 Relative abundance of the monoethyl monomethyl ester isomers on GCMS of methylated TLC A (para isomer, Methods 2.3.9) and crude mixtures of esters from the methylation of ortho and meta ethylation mixtures (Methods 2.3.8.2) and trappate BX (Method 1.3.4). 143 El type spectra with greater abundances of lower molecular weight ions. Other factors including the age of the filament, and the operators method of optimising response can lead to some discrepancies. The state of the ion source also has some effect. With the instrument used a clean source tended to accentuate the higher masses. It has been shown by Watson (1985) that sample pressure (related to amount of sample) in the source can also effect the distribution of high and low mass ions. These factors alone may not produce great differences but in combination the possibility of great variation is a real one. 2.4.9 GCMS OF STANDARD-SPIKED 30M20:10IPA One way that a distinction could be made would be to spike the extract richest in the unknown with standards to determine which of the peaks are coincident in retention with the unknown. This should also confirm whether the postulated structure is likely. Three small subsamples of 30M20;10IPA were spiked separately with the crude preparations of p-, m- and o-MBA monoethyl monomethyl esters and the results compared with a similar sample of unenriched 30M20:10IPA. The GCMS traces of the spiked fractions are shown in Figures 2.4.11., 2.4.12 and 2.4.13. Clearly these results indicate that the p- isomer is retained on the column longer than the endogenous compound and that the o- and m- isomers are both coincident with the unknown compound. The latter two isomeric methyl, ethyl esters are also not resolved using this system. Unfortunately the original extract was used over a period of years in an attempt to isolate a senescent active compound and there was little remaining of this crude fraction. Since the extract was stored at a relatively low pH in a mixture containing ethanol, the suspected ethylation reaction occurred over this period. Consequendy it was not practical to process more bean leaf in an attempt to mimic the original extraction and storage procedure. If this was practical then the spikes could have been repeated using a range of polar and nonpolar GC phases, and other run conditions. Undoubtedly the m- and o- 100~i _ O CM CD ^ o O CD O CO CD ^ ™ if) z O C TO E d o DC •- 1= ~ o fl) c C o E ® C CO O FIGURE 2.4.11 Total ion chromatogram and specific ion traces of m/z 135 and 310 for fraction 30M20;10IPA spiked wit methylated TLC A (Methods 2.3.9). 144 100- _ O CM ^ o in CD CO O ™ in CO 2: c O CO § DC •- |= o 0 c o C E c (/> fli (_> ) sz HOURS 2.4,12 Total ion chromatogram and specific ion traces of m/z 135 and 310 for fraction 30M20;10IPA spiked £ methylated reaction mixture from the preparation of ortho-MBA monoethyl esters (Methods 2.3.8.2). 145 100n o o o t CD o ° T- LO O O - CD o 04 o CM OO CD T. 5 to 04 9 ® sz FIGURE 2,4,13 Total ion chromatogram and specific ion traces of m/z 135 and 310 for fraction 30M20;10IPA spiked I methylated reaction mixture from the preparation of meta-MBA monoethyl esters (Methods 2.3.8.2). 146 monoethyl monomethyl MBA esters could eventually be separated by GC had a greater amount of material been available. 2.4.10 HPLC PURIFICATION OF THE ETHYLATED METHYLATED MBA PREPARATIONS A portion of the /?-, o- and m-MBA ethyl, methyl ester preparations were fractionated by normal-phase HPLC into the monoethyl monomethyl and dimethyl esters. Under the HPLC conditions used the two isomeric monoethyl, monomethyl esters of the o- and m- MBA resolved into distinguishable but fused peaks whereas the p- isomers were not resolved. The retention data (Table 2.4.3), shows both absolute retention and retention relative to 3,4 dimethoxy cinnamoyl malate dimethyl ester. The diodearray detection system also allowed the determination of UV absorbance maxima for each of the MBA esters. The appropriate maxima are shown in Table 2.4.4 along with UV data of the corresponding methyl benzoate derivatives. 2.4.11 PROTON NMR OF SYNTHETIC MBA Since the para isomer had been ruled out of contention through the different GCMS retention time one of the remaining options was to run NMR spectra of the purified o- and m- monoethyl, monomethyl esters for comparison to the native compound. Since the most likely observable difference between the o- and m- MBA isomers would involve the chemical shift of the methoxy group, NMR spectra were performed on the major products of the ethylation, methylation reactions. At this stage the major problem was the identification of the unknown as the o- or m- isomer and it was not necessary to obtain comprehensive NMR data. In the case of the o- isomer a mixture of the a-methyl P-ethyl (labelled A ester) and p-methyl a-ethyl (labelled B ester) esters were used and as the yield of ethylated product from the m- preparation was poor the dimethyl ester was 148 COMPOUND ABSOLUTE RELATIVE RETENTION (min) RETENTION p-methoxy-BA diME 14.5 1.5 o-methoxy-BA diME 11.4 1.26 m-methoxy-BA diME 10.8 1.1 p-methoxy-BA ME, EE 9.6* 0.98* o-methoxy-BA ME, EE 7.3, 7.9 0.74, 0.81 m-methoxy-BA ME, EE 8.9, 9.2 0.91, 0.93 3,4 dimethoxycinnamoylmalate 9.8 1.0 dimethyl ester (trans) Abscisic acid ME 5.8 0.6 TABLE 2.4.3 Retention data for dimethyl and monomethyl monoethyl esters of methoxybenzoylaspartate and two reference compounds on HPLC under the conditions outlined in Methods 2.3.4. * both isomers coelute, ME: methyl ester, EE: ethyl ester, BA: benzoylaspartate. 149 COMPOUND UV MAXIMA (nm) o-methoxybenzoate ME 229, 291 o-methoxy-BA diME 232, 287 o-methoxy-BA ME, EE 232, 287 m-methoxybenzoate ME 294, 233 m-methoxy-BA diME 290, 229 (sh) m-methoxy-BA ME, EE 290, 229 (sh) p-methoxybenzoate 253 p-methoxy-BA diME 250 p-methoxy-BA ME, EE 250 TABLE 2.4.4 UV maxima of dimethyl and monomethyl monoethyl esters of methoxybenzoyl aspartate and two reference compounds on HPLC under the conditions outlined in Methods 2.3.4. sh: shoulder, ME: methyl ester, EE: ethyl ester, BA: benzoylaspartate 150 submitted for NMR analysis. Both of the standards were purified by HPLC (Methods 2.3.10). The NMR spectra in the range from 2.0 to 6.0 ppm are shown in Figures 2.4.14, 2.4.15 and Table 2.4.5 contains a list of some assigned resonances. It would be expected that the chemical shifts of the methyl ester singlets of both meta and ortho isomers would be similar while the corresponding signals due to the methyl group of the methoxy moiety show a significant difference. On this basis the resonances at 4.01 and 3.86 ppm were assigned to the ortho and meta methoxy groups and the methyl ester protons for both isomers at 3.79 and 3.70 ppm. Distinction between the methyl esters is not possible with great certainty but it is most likely that the higher field resonance is due to the methyl ester on the a- carboxyl. 151 HPLC by purified MBA ortho of esters ppm. 6.6 to monomethyl 2.2 range monoethyl the in the of 2.3.10) NMR Proton (Methods 2.4.14 FIGURE 152 range o -s' the CO in 2.3.10) (Methods HPLC by purified meta-MBA of ester o LO dimethyl the of ppm. NMR 6.8 to Proton 2.2 O CD 2,4,15 FIGURE 153 CHEMICAL SHIFT (ppm) GROUP ORTHO META UNKNOWN >£H- 5.12 (A,B) 5.07 (A,B) - -QH?- 3.11 (A.B) 3.13 (A,B) - -(0)£H3 3.83 (A) 3.83 (A) 3.83 -(0)£H3 3.75 (B) 3.75 (B) 3.75 -CH2£H3 1.25 (A.B)* - - -CH9CHq 4.21 (AfB)* - - -0OCH3 4.04 (A,B) 3.86 (A,B) 4.03 TABLE 2.4.5 Assigned chemical shift for protons on ortho and meta monomethyl monoethyl esters of methoxybenzoylaspartate and the unknown compound (GC trappate 5+6BX;C, Methods 2.3.5). * no discernible difference in chemical shift. 154 2.5 DISCUSSION It is noticeable that the resonances for the methine, methylene and the methyl ester protons do not offer much distinction between the o- and m-isomers of the synthetic esters. More significant is the chemical shift of the methoxy protons which often allows a clear distinction to be made between ortho, para and meta methoxy protons. The p-isomer had been excluded from consideration since it did not coelute with the unknown on GCMS analysis so the choice of possibilities was limited to the ortho and meta isomers. As shown in Table 2.4.5 the resonance due to the methoxy protons occurs at 4.04 ppm for the ortho isomer and 3.86 ppm for the meta isomer. The unknown shows a resonance at 4.03 ppm so can be unequivocally identified as the ortho isomer. The unknown also has the two isomeric methyl ester protons at 3.79 and 3.70 ppm. So clearly the unknown compound has been identified as a mixture of the two isomers of o-MBA monoethyl, monoethyl ester. There is also another very interesting quantitative difference in the spectra of the endogenous and synthetic MBA monoethyl mono methyl ester mixtures. The ratio of the a-methyl and p-methyl singlets is different in the two spectra. The ratios are approximately 1:2 (3.8:3.7 ppm, endogenous) and about 6:1 (3.8:3.7 ppm, synthetic). This means that ethylation has occurred on different carboxyl groups in the synthetic and endogenous preparations. However, during the purification sequence there was not any concerted effort taken to include all of each isomer and this result was not at all conclusive. A derivative of ortho hydroxybenzoylaspartate has been isolated from the ethanolic extract of bean leaves. This compound, as isolated was found to be o - methoxybenzoylaspartic acid monomethyl monoethyl ester. The major position of ethylation was found to differ in the endogenous and synthetic compounds so it is most likely that the formation of the monoethyl ester in the extract was due to transesterification rather than straight forward acid catalysed ethylation. 155 Despite some uncertainty in the absolute determination of the structure (ie is the methoxy group endogenous or is it generated by methylation, and has there been conjugation on one of the carboxyl groups and if so which one?) the essential structure could be definitely assigned. CHAPTER 3 Studies to show that salicyloylaspartate is the endogenous species which exists as the free and bound form in a number of plant tissues 156 3.1 INTRODUCTION One of the possible problems in any isolation procedure is the occurrence of artifacts. It has already been shown that the likely cause of the ethylation of MBA is the storage of the plant extract at a relatively low pH in a solvent containing some ethanol. In fact there are many instances where the nature of the extraction or purification solvents has chemically altered the structure of an endogenous component and this has led to misinterpretation of results and incorrect assignments of structure. And it is not only the solvent used during purification and extraction that can cause the anomalies. If the compound of interest is present in only minute amounts and there are functional groups on the molecule then the most effective means of separation usually involves derivatisation. In this laboratory a standard procedure has been developed which works very well with most acids. The crude extract is first separated into acids and neutrals and the acid fraction is then is fractionated on reverse-phase Sep Paks followed by reverse-phase HPLC. The mixture is then methylated, separated on reverse-phase HPLC and then normal- phase HPLC. The most common reagent for methylating acids is diazomethane and this esterification agent is used very regularly for numerous reasons, principally the very high yield of product (>99%), the very short reaction times (instantaneous for most acids) and the ease with which the excess reactant can be removed. However diazomethane is not very specific and under some circumstances will react with other functionalities, for example double bonds and phenolic hydroxyl groups (Black, 1983) Methylation with diazomethane was used in this purification sequence because of the advantages and despite the disadvantages. In this case the disadvantage is the propensity of diazomethane to methylate aromatic hydroxyl groups. The tendency for this reaction depends on a number of factors particularly 157 the acidity of the hydroxyl group, and the solvent used for the dissolution of the compound to be methylated (Black, 1983). The hydroxy proton of salicylic acid is strongly hydrogen bonded to the carboxyl oxygen so the pKa of this proton is high (pKa = 13.8, Albert and Sergeant, 1971). Consequently this group methylates very slowly. Conversely the hydroxy proton of SA methylates very rapidly, as would be expected since this proton cannot interact as strongly with the carboxyl group. This relative ease of methylation indicated that the o-methoxy group of the isolated MBA probably arose through methylation by diazomethane and was not the endogenous species. Another question involved the mechanism giving rise to ethylation of the carboxyl group of MBA. It has been shown in Chapter 2 that the MBA readily reacts with ethanol under acidic conditions to form mono and diethyl esters. However comparison of the proton NMR of the endogenously derived monoethyl ester with that of the synthetic product indicated that the proportion of each isomeric ethyl ester is different in the two cases. This introduced the possibility that at least some of the ethyl ester could have arisen from the trans-esterification by ethanol of a conjugate of MBA which has an unknown group (possibility glucose) attached to one of the carboxyl groups. These questions are addressed in this chapter. Further studies on bean leaf and other plant tissue introduced some difficulty due to the large amount of plant material needed to detect any MBA as the dimethyl ester. Attempts to find any MBA in 1 kg of bean leaf was unsuccessful. Clearly since the structure of MBA has been determined (by combination of coincidence of GC retention, molecular weight and fragmentation pattern by MS, and some confirmatory NMR information) and the concentration in plant tissue is so low there was a definite need for a more sensitive assay system. On the advent of a better assay procedure the presence of MBA in beans could be confirmed, the presence in other species could be explored and the viability of utilisation of a deuterated internal standard for accurate analysis could be explored. 158 A revised method of synthesis of salicyloylaspartate was devised so that more than sufficient amount of compound was available for use in assays and markers in purification steps. 159 3.2 MATERIALS See also Materials Chapter 1 and Chapter 2. 3.2.1 PURIFICATION OF THIONYL CHLORIDE About 200 ml of analytical grade thionyl chloride was purified by the method in Vogel (1967). 3.2.2 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY All HPLC runs were performed under the conditions outlined in Materials 1.2.6. Elution solvent was 30% methanol in 0.5% aqueous acetic acid. 3.2.3 GCMS Negative ion chemical ionisation mass spectra were conducted as specified in Materials 1.2.7. GC run conditions are specified in the appropriate sections. 3.2.4. CHEMICALS AND REAGEANTS Pentafluorobenzyl bromide, silver (I) oxide and tetraethylammonium bromide were obtained from Fluka (Buchs, Switzerland), thionyl chloride and salicylic acid from BDH (Poole, England) 3.2.5. 0.4M TETRAETHYLAMMONIUM HYDROXIDE A solution of tetraethylammonium bromide (1.68 gm) in dry methanol (50 ml) was shaken overnight in a stoppered tube with silver (I) oxide (2 gm). The solution was then filtered to remove silver bromide and excess silver oxide and stored at 4°C. 160 3.3 METHODS 3.3.1 SYNTHESIS OF OBA See Scheme outlined in Figure 3.3.1 3.3.1.1 FORMATION OF ACETYLSALICYLIC ACID Salicylic acid (20 gm, 0.15 moles) was mixed with acetic anhydride (28 ml, 0.34 moles) and concentrated sulphuric acid (10 drops) added. The reactants were heated to 60°C with constant stirring and this temperature maintained for 15 minutes. At the end of this period the mixture was cooled to room temperature and stirred into distilled water (300 ml). Crystals formed in this step were gathered by filtration and recrystallised by dissolution in hot ethanol (60 ml) and warm water (150 ml). This solution was left to cool to room temperature for about 4 hr, after which the crystals were harvested, washed with cold water and dried for two days at reduced pressure over anhydrous calcium chloride. Yield 10 gm. (40%) 3.3.1.2 FORMATION OF ACETYLS ALICYLIC ACID CHLORIDE Dried acetylsalicylic acid (5 gm, 0.027 moles) was dissolved in purified, redistilled thionyl chloride (15 ml, 0.206 moles) in a 200 ml round bottomed flask equipped with a calcium chloride guard tube. After heating to 60°C for two hours the excess thionyl chloride was removed by evaporation under reduced pressure. Toluene (10 ml, dried with molecular sieve) was added to the acid chloride and evaporated. This step was repeated until all traces of sulphur dioxide were removed. 3.3.1.3 FORMATION OF THE SALICOYLASPARTATE DERIVATIVE This acid chloride mixture was added to a solution of aspartic acid (3.26 gm, 0.025 moles) in ice cold 2M sodium hydroxide (30 ml). After the acetylsalicylic acid chloride dissolved on warming to room temperature the mixture 161 Aspartic Acid in 2M NaOH Acetate FIGURE 3.3.1 Synthetic scheme for the synthesis of salicyloylaspartate from salicylic acid. 162 was heated to 50°C and maintained at this temperature for 30 minutes with occasional shaking. The solution was then acidified to pH 2.0 with 2M sulphuric acid and extracted with ethyl acetate (2 x 100 ml). These extracts were combined and evaporated. The residue was then dissolved in hot ethyl acetate (30 ml), filtered and cooled to 4°C. Crystals separated after storage overnight at this temperature and these were collected by filtration, washed with ice cold ethyl acetate (2 ml) and air dried. Yield of dry crystals was 1.5 gm (21%). 3.3.2 PREPARATION OF a-METHOXYBENZOYLASPARTIC ACID About 100 mg of salicyloylaspartic acid (from Methods 3.3.1) was dissolved in methanol (2 ml) and three times excess of ethereal diazomethane added. After standing for 30 minutes the solvents were removed under a stream of nitrogen. The residue was dissolved in methanol (2 ml), water (8 ml) added followed by 10M sodium hydroxide solution (4 drops). After standing at room temperature overnight the unhydrolysed ester was removed by extraction with diethyl ether (2 x 20 ml). The aqueous phase was then adjusted to pH 2.0 with 2M sulphuric acid and extracted with ether (2 x 20 ml). This ether extract was then evaporated to dryness under a stream of nitrogen. 3.3.3. EXTRACTION OF PLANT MATERIAL 3.3.3.1 FREE SA Leaf tissue from two plant species, bean (Phaseolus vulgaris cv Dwarf Pioneer, 8.6 gm fresh weight) and tomato (Lycopersicon esculentum cv Grosse Lisse, 14.6 gm fresh weight) was treated in identical fashion. Plant material was mixed with 1% acetic acid in acetone (100 ml) and crushed with acid washed sand in a mortar and pestle. This mixture was filtered through Whatman no. 1 paper and the filtrate evaporated to near dryness. The 163 residue was acidified to pH 2.0 and extracted with ether (2 x 60 ml). This ether extract was washed with saturated sodium bicarbonate (2 x 30 ml). The bicarbonate washes were combined, acidified to pH 2.0 and extracted with ether (2 x 100 ml). The ether extracts were combined and evaporated to dryness. 3.3.3.2 BOUND SA The aqueous residues from the first extraction step were diluted with sufficient 10M hydrochloric acid to give a final concentration of 2M HC1, then immediately extracted with ether (100 ml) to test the efficiency of the first extraction (Methods 3.3.3.1). Both tomato and bean extracts were then left in the resultant 2M HC1 to hydrolyse at room temperature. After standing overnight each hydrolysate was then extracted with ether (2 x 100 ml), the extracts then combined and evaporated to dryness. 3.3.4. HPLC PURIFICATION A few microlitres of a solution of salicyloylaspartate and o- methoxybenzoylaspartic acid in methanol (10 mg/ml of each) was injected onto a reverse-phase Ci8 column with the dimensions outlined in Materials 3.2.3. Each of the plant leaf extracts was then run under the same conditions and the eluents separately collected over the regions coincident to MBA and the corresponding methoxy derivative. The identity of the plant extracts is: BEANS Bla, Bib Initial acid extract B2a, B2b Acid extract after addition of HC1 B3a, B3b Extract of HC1 hydrolysate TOMATOES Tla, Tib Initial acid extract T2a, T2b Acid extract after addition of HC1 164 T3a, T3b Extract of HC1 hydrolysate (a relates to the HPLC trappate coincident with o-hydroxybenzoylaspartate, b relates to the trappate coincident with MBA) Each extract was evaporated to dryness for the derivatisation step (Methods 3.3.6). 3.3.5 EXTRACTION. PURIFICATION OF VEGETABLE LEAVES FOR SIM-GCMS Vegetable leaf from five sources was stripped of excess stalk material and extracted in the same manner as outlined in Methods 3.3.3. - with one variation; the solvent extraction step immediately after addition of HC1 was not performed. The ten samples were then dissolved in 95% ethanol (100 |il) and diluted to 10 ml with water. This solution was then passed through a single reverse-phase Ci8 Sep-Pak. The Sep-Pak was then washed with 5% and then 35% aqueous ethanol. The 35% fraction was collected for HPLC purification. After evaporation each Sep-Pak fraction was dissolved in a small amount of methanol and analysed by HPLC (Methods 3.3.4). Eluent was collected over a period corresponding to the elution of synthetic salicyloylaspartate and these fractions evaporated for derivatisation. 3.3.6. DERIVATISATION Pentafluorobenzyl esters of the synthetic MBA, the methoxy derivative and the eluents from the reverse-phase HPLC purification step were prepared using the method of Netting and Duffield (1985) developed for amino acid derivatisation. A solution of MBA in methanol containing 10-100 |Xg of compound was added to a 2 ml Reactivial and evaporated under a stream of nitrogen. Dimethylacetamide (50 p.1) was added to dissolve the sample and this solution was drawn up into a 100 p.1 syringe. 165 To a second Reactivial was added 0.4M tetraethylammonium hydroxide (4 |il) and pentafluorobenzyl bromide (2 pi) followed immediately by the sample. The reactants were stirred and after one minute hexane (100 pi) and water (100 pi) added in that order. The tube was shaken and the hexane layer removed. A further aliquot of hexane (100 pi) was added, the tube shaken again and the hexane layer combined with the first hexane extract. The hexane extracts were evaporated under a stream of dry nitrogen. The fractions prepared for SIM GCMS were derivatised in a slightly different manner. After samples were transferred to Reactivials, dimethylacetamide (50 pi) was added followed by simultaneous addition of 0.4M tetraethylammonium hydroxide (4 pi) and pentafluorobenzyl bromide (2 pi). The remaining derivatisation and purification sequence remained unaltered. 3.3.7. PURIFICATION OF DERIVATIVE The residue was dissolved in dichloromethane (5 pi) and added to a silica Sep-Pak previously activated dry hexane (10 ml). The Sep-Pak was eluted with dry hexane (10 ml) and this eluent discarded. The derivative was then eluted with dry dichloromethane (10 ml) and the solvent evaporated under a stream of dry nitrogen. 3.3.8 FULL SCAN NEGATIVE ION GCMS OF THE PFB DERIVATIVE OF SYNTHETIC SALICYLOYLASPARTATE AND PLANT EXTRACTS Each sample was dissolved in a few microlitres of ethyl acetate and analysed by negative ion GCMS under the conditions shown in Materials 3.2.3. With one exception all GCMS determinations in full scan negative ion mode were carried out using an open tubular 2 m glass column (2 mm ID) with 1.5% OV-1 on chromasorb W 80/120. Column oven was programmed to raise temperature 10°C per minute from an initial 100°C starting one minute after injection. Injector temperature was 200°C. 166 3.3.9 SIM-GCMS OF DERTVATISED VEGETABLE LEAF SAMPLES Selected ion monitoring of these fractions was performed in negative ion mode under the conditions outlined in Materials 1.2.7. Selected ion monitor negative ion GCMS utilised a column of the same dimensions as Methods 3.3.8 containing 2% OV-17 on chromasorb W 100/120. Initial temperature was set at 200°C and raised 10°C per minute to 300°C one minute after injection. Full scan GCMS of the PFB derivative of deuterated salicyloylaspartate (Methods 3.3.10) was performed under these conditions. 3.3.10 DEUTERATION OF SALICYLOYLASPARTATE A small sample (about 20 mg) of salicyloylaspartate was dissolved in 99.9% D2O (2 ml) and 35% DC1 in D2O (10 drops) added. This solution was maintained at 80°C for four hours and after cooling extracted with diethyl ether (10 ml). The organic extract was dried with anhydrous sodium sulphate, filtered and evaporated. 2 pi of this reaction mixture was transferred to a glass cup and dried for direct insertion MS. A few microgram of this deuterated salicyloylaspartate was derivatised by the procedure shown in Methods 3.3.6 for SIM vegetable leaf extracts and some of this analysed by full scan negative ion Cl GCMS under the conditions outlined in Methods 3.3.9. 167 3.4. RESULTS 3.4.1 PURITY AND IDENTITY OF THE SALICYLQYLAS PART ATE DERIVATIVES On silica TLC, the crystalline material isolated by the method outlined in Methods 3.3.1. ran as a single band (visualised under UV light, 256 nm, Rf=0.6) using 10% acetic acid in ethyl acetate as developing solvent. HPLC (Materials 3.2.3) of the salicyloylaspartate gave a single peak with a retention time 14 min. Methylation of the product with diazomethane gave a compound with a molecular weight of 295 and retention identical to o-methoxybenzoylaspartic acid dimethyl ester shown in Figure 2.4.11. This means that the crystals isolated did not retain the ortho acetyl group as this should be cleaved under the basic conditions of the condensation reaction. Since there wasn't a significant amount of self condensation of o-hydroxybenzoic acid chloride to produce polymeric salicyloylsalicylate esters in the acid chloride preparation the acetate was probably still intact at this point of the synthesis. Reverse-phase HPLC (Materials 3.2.3) of the crystalline material from the preparation of o-methoxybenzoylaspartate gave a single peak with a retention time of 18.7 min. 3.4.2. FORMATION OF PFB DERIVATIVES OF SYNTHETIC SA 3.4.2.1 SALICYLOYLASPARTATE The derivatisation reaction would be expected to produce either the di-PFB ester or the tri-PFB derivative (Figure 3.4.1) or a mixture of the two compounds. The total ion chromatogram (Figure 3.4.2) of the dichloromethane extract of PFB derivative of synthetic MBA shows six major peaks present in the preparation. Peaks at scans 135, 284, 417, and 694 result from impurities in reagents and side reactions between reagents while peaks at scans 947 and 1110 have high mass 168 Tri-PFB SA Di-PFB SA FIGURE 3.4.1 Structures of the di- and tri-pentafluorobenzyl- salicyloylaspartate. 100 n CO lO o> ■ m DC o o m > © LO o c o 3 - i - CM CO ■M" r -M- CD CM CO CD -M; o CM O CM O O O C/3 z O TO c 1 C 0 DC ~ i= © 0> c O C E iz O E V) o o O FIGURE 3,4,2 Total ion current chromatogram of the dichloromethane Sep-Pak eluent from the pentafluorobenzylation < micrograms of synthetic salicyloylaspartate (Methods 3.3.6). Figures above peak apices are scan number: CO 169 170 fragments in the range expected for the possible PFB derivatives of MBA, and neutral losses consistent with a successful derivatisation. The peak at scan number 947 has a peak-summed mass spectra shown in Figure 3.4.3. If the compound is a pentafluorobenzyl ester or ether then it would be expected to show losses of 181 daltons (-CH2C6F5), less commonly 180 daltons, (presumably from hydrogen transfer before cleavage of the ester or ether bond), or 197 daltons (-OCH2C6F5). The mass spectra shows a low intensity peak at m/z 612, and major ion species of m/z 432, 252 and 234. This fragmentation pattern is consistent with the scheme shown in Figure 3.4.4 where the parent substance, the di-PFB derivative, shows neutral losses of 181, 180 and 18. The ion at m/z 612 would result from loss of one hydrogen, a process that commonly occurs in negative ion mass spectrometry. Another diagnostic peak for a PFB derivative is the appearance of m/z 181, the pentafluorobenzyl fragment carrying a negative charge. The peak at scan 1110 shows some similarity in the population of the ion species (Figure 3.4.5). Major ions occur at m/z 612, 432, 414, 252, 234 and 181 and Figure 3.4.6 shows a likely route leading to the production of these fragments from the parent compound, the tri-PFB derivative of MBA. The loss of m/e 181 from the parent ion gives rise to m/z 612 which in turn loses 180, 180, and 18 mass units to produce ions m/z 432, 252 and 234. The ion m/z 414 would arise through the loss of water from m/z 432. In the case of the tri-PFB derivative the parent ion (m/z 792) or M-l (m/z 791) was not detected since these masses fall outside the mass range set on the mass spectrometer. Consequently the preparation of the PFB derivatives has led to a mixture of products at the concentrations used in the method. GCMS will, of course only show the derivatives with both acid groups esterified, so it is likely that other isomers such as the monoether monoester combination were produced. Further work however, showed that if less SA was used for the same amount of reagents then a single product, the tri-PFB derivative was formed. 100.0-1 I p 281600 FIGURE 3.4.3 Mass spectrum of peak with apex at scan #947 (Figure 3.4.2). Scans have been summed from #928 #968 with about 20 scans of background subtracted. 171 172 CD est di-pentaflurobenzyl salicyloylaspartate of route fractionation probable the showing Scheme 3.4.4 FIGURE 173 subtracted. background of scans 20 about with #1122 to #1100 3.4.5 FIGURE 3.4.2). (Figure #1110 scan at apex with peak of spectrum Mass fro summed been have Scans E 2 00 i 174 ether, pentafluorobenzyl salicoylaspartate of fractionation of route possible One 3.4.6 FIGURE 175 Amounts of 10 jig rather than 100 fig led to the exclusive production of tri-PFB MBA. 3.4.2.2 o - METHOXYBENZOYLASPARTATE The preparation resulting from derivatisation and purification of approximately 10 |ig of o-methoxybenzoylaspartate gave a single PFB derivative and this compound had the mass spectrum shown in Figure 3.4.7. 3.4.3 GCMS OF BEAN AND TOMATO LEAF EXTRACTS Figure 3.4.8 shows the total ion chromatogram of the bean leaf extract (Bla) in the negative ion, chemical ionisation mode and Figure 3.4.9 the mass spectrum of the peak at scan number 1116. This spectrum is clearly tri-PFB salicyloylaspartate by virtue of almost identical retention behaviour to the standard compound and great concurrence in the fragmentation pattern. The mass spectra shows ions at masses m/z 612, 414 and 234 which correlate to the important fragments of the synthetic tri-PFB MBA. The base peak (m/z 414) is not of significant intensity in the di-PFB derivative. The remaining bean extracts in the 'a' series (B2a and B3a) do not demonstrate the presence of any MBA derivative (Figure 3.4.10). Similar ion traces for the tomato extracts Tla, T2a and T3a are illustrated in Figure 3.4.11. In Tla there is a detectable amount of the tri-PFB derivative as shown by the occurrence of some m/z 414 at about scan number 1120. T2a does not contain MBA (absence of m/z 414) but T3a does contain significant amounts of tri-PFB MBA. Corresponding GCMS analysis of the extracts from the 'b' series for both bean and tomato failed to show the presence of any o-methoxybenzoylaspartate. 176 -O 03 t across summed pentafluorobenzyla of g VO been 2 vX> have chromatogram o Scans \D O GC/MS shown). a on 2 not r> is o N s #962 E subtracted. 10s scan at § (chromatogram background g apex of 2 with scans peak 20 of o 8 about spectrum with Mass peak ortho-methoxybenzoylaspartate >> © > « C © © cc c 3.4.7 FIGURE 177 (m ln:sec) Time Retention co CO LU OC => O Ll_ 122 FIGURE 3.4.9 Mass spectrum of the peak with apex at scan #1110 in the total ion chromatogram of fraction B1a (Figure 3.4.8). Scans have been summed across the peak with about 20 scans subtracted as background. 178 179 100 m/z 234 100-1 m/z 414 100-, Relative Ion Current 800 1200 13:20 20:00 Scan Number Retention Time (mln:sec) m/z 234 100-1 m/z 414 100-i Relative Ion Current 1200 1600 13:20 20:00 26:40 Scan Number Retention Time (min:sec) FIGURE 3.4.10 Total ion current chromatogram and specific ion traces of m/z 234 and 414 for pentafluorobenzylated fractions B2a and B3a (Methods 3.3.4). 180 Ralatlva Ion Currant 1200 1600 20:00 26:40 Ralatlva Ion Currant *-Ul 400 800 1200 1600 6:40 13:20 20:00 26:40 m/z 234 m/z 414 Ralatlva Ion Currant 6:40 13:20 20:00 Scan Number Retention Time (min:sec) FIGURE 3.4.11 Total ion current chromatogram and specific ion traces of m/z 234 and 414 for pentafluorobenzylated fractions T1a, T2a and T3a (Methods 3.3.4). 181 3.4.4. APPROXIMATE QUANTITATION OF MR A IN BEAN AND TOMATO LEAF EXTRACTS Although the amounts of detectable MBA in the plant extracts was small in most cases, approximate quantitation could still be made by comparison of the summed ion intensity of the m/z 414 fragment of the assays with that of a standard preparation. Table 3.4.1 shows the results from the extracts of the 'a' series and a standard MBA determination. 3.4.5 SIM GCMS OF VEGETABLE LEAF EXTRACTS Figure 3.4.12 shows the SIM traces for ions m/z 234, 414 and 612 for 1 fig of standard salicyloylaspartate and endogenous free acid in the tomato extract after pentafluorobenzylation. The results for the remaining plant leaf extracts are summarised in Table 3.4.2. These results clearly demonstrated that in all the plant species tested there was at least detectable amounts of free and bound SA. Strawberry leaves were shown to contain the greatest amount of bound SA and tomato leaves more free S A than any plants tested. 3.4.6 GCMS OF DEUTERATED SALICYLOYLASPARTATE Mass spectra of both the undeuterated acid and the deuterated form are shown in Figure 3.4.13. The unlabelled compound shows intense ions of m/z 254 (M+l), 236 (loss of water) and 121 (the aromatic portion after cleavage of the amide bond). Other significant peaks occur at m/z 208, 162, 134, 88 and 70. These ions probably arise from loss of one of the carboxyl groups from the parent (208), loss of both carboxyls and a hydrogen (162), cleavage at the amide bond and addition of two protons to the nitrogen (134), loss of the a-carboxyl and cleavage of the amide bond (88) and loss of water from m/z 88 (70). 182 SAMPLE TOTAL ION CURRENT WEIGHT AMOUNT OF PFB-OBA m/z414 LEAF (gm) (|j.g/kg fw) Standard (10jig) 2498 - - B1 a 606 45 56 T1a 1 3 60 0.9 T3a 20 60 1.4 TABLE 3.4.1 Total ion current for m/e- 414 for synthetic pentafluorobenzylated salicyloylaspartate (10p.g) and derivatised fractions B1a, T1a and T3a. Calculated amounts of derivative are also shown. Total ion current is expressed as peak heights. 183 1920 102912 57 -i Standard 2060453 m/z 234 33792 934272 m/z 414 1920 136448 3820540 m/z 612 1 700 1800 1900 2000 2100 2200 11:51 12:37 13:15 13:57 14:33 15:20 Scan Number Retention Time (mln:sec) First Tomato 100~i Extract m/z 234 1911 1350 22440 m/z 414 1909 2073 40496 m/z 612 1800 1900 2000 2100 12:37 13:15 13:57 14:33 Scan Number Retention Time (mln:sec) FIGURE 3.4.12 SIM traces of ions m/z 234, 414 and 234, 1jil of synthetic salicyloylaspartate and the first acid extract of tomato leaf (Methods 3.4.5). 184 SAMPLE WEIGHT OF AREA* AMOUNTOF LEAF (gms) SA (X102 ng/kg) Standard(lpg) - 3820540 - Tomato (f) 18.7 40496 5.7 Tomato (b) 18.7 D - Shallot (f) 20.7 21236 2.7 Shallot (b) 20.7 D - Corn (f) 16.0 28005 4.6 Corn (b) 16.0 23056 3.8 Radish (f) 19.1 1 2060 1 .6 Radish (b) 19.1 D - Strawberry (f) 16.4 13512 2.2 Strawberry (b) 16.4 62208 9.9 TABLE 3.4.2 SIM mass spectrum peak areas of ion m/e' 612 for vegetable extracts (Methods 3.3.5) and synthetic salicyloyl aspartate after pentafluorobenzylation. Weights of tissue and calculated amounts of SA are included. SA-Salicyloy! aspartate, f-free, b-bound, * areas are in arbitary computer generated units, D-a peak has been detected but was not intense enough to satisfy the parameters for calculation of peak areas. 185 SA r25872 Relative Intensity DSA 100.0 -i Relative Intensity m / z FIGURE 3.4.13 Solid insertion probe mass spectra of salicyloylaspartate (SA) and an aliquot of the deuteration mixture prepared by the procedure outlined in Methods 3.3.10 (DSA). 186 Some dehydration has occurred during heating on the solid probe before ionisation as evidenced by the presence of ions m/z 264 and 276, both of which are methane adducts of m/z 236. The spectrum of the deuterated acid shows the typical clustering of groups around the major ion species so clearly the salicyloylaspartate has been deuterated by acid mediated exchange with up to five deuteriums introduced. Comparison of relative intensities of the deuterated parent ions gives the following percent incorporations; DO (6%), D1 (18%), D2 (32%), D3 (27%), D4 (13%) and D5 (4%). Likely exchange sites are the protons on the two carboxyl groups, the ring hydroxy and the amide nitrogen. A small proportion of molecules carry a fifth deuterium probably on the aromatic ring. Examination of the labelling pattern of the fragment ions correlates with both the suggested fragmentation and the probable sites of deuteration. Maximum significant labelling of the parent substance is 4D, of the dehydrated fragment 3D, of the decarboxylated fragment 3D and of the salicyloyl fragment ID. Fragment ions m/z 70 and 88 in the mass spectrum of the undeuterated acid have one and two deuteriums respectively in corresponding deuterated acid mass spectrum. The fragment incorporating the aspartate group and the amide nitrogen has been labelled with three deuteriums. For the tetra-deuterated compounds only one would be retained after derivatisation by pentafluorobenzylation so if this procedure was to be useful for quantitation of salicyloylaspartate in plants by standard addition of the deuterated acid, derivatisation and SIM-MS then the label has to be stable under the basic reaction conditions. A clue that this was the case was underlined by an attempt to exchange deuterium under basic conditions, a procedure which failed to introduce any label. Additionally, it is always advantageous for the exchanged label to be stable on storage. Figure 3.4.14 shows the mass spectrum of the deuterated compound after pentafluorobenzylation. Clearly some label has been retained as m/z 613 is 27% of 187 CD across summed been have Spectra subtracted. 3.3.10). been have (Methods DSA background of scans 30 pentafluorobenzylated and of peak spectrum Mass appropriate © > > - ™ C © © 3.4.14 DC — C FIGURE 188 m/z 612. Undeuterated acid derivatised and analysed a little earlier gave an intensity of the 613 ion as 4% of m/z 612. So some incoiporation of a single deuterium has succeeded and further work could improve the amount of deuteration. Undoubtedly the best methodology involving the use of a deuterated internal standard demands that at least two deuteriums remain after derivatisation. This reduces the complication imposed by naturally occurring 13C. In this case acid catalysed deuteration does not fulfil this requirement so the best results would be obtained by the synthesis of SA from either 13C-salicylic acid and 12C-aspartic acid or 13C-aspartic acid and 12C-salicylic acid. Both of these 13C-labelled compounds are commercially available. 189 3.5 DISCUSSION The investigations in this chapter has answered some of the questions posed in the introduction. Pentafluorobenzylation proved to be a very useful method for the production of derivatives which could be determined at high sensitivity by negative ion GCMS. In full scan mode amounts as low as 70 ng (Table 3.4.1, sample T3a) were determined with reasonable accuracy while SIM analyses showed far smaller amounts could be measured with good precision. Under ideal conditions (stable electron multiplier, clean ion source etc.) a peak area of 1000 units is close to the practical limits for accurate quantitation. Consideration of the response of the standard salicyloylaspartate derivative (Figure 3.4.11) where 1 jig was equivalent to 3820520 area units means that a detectability of at least 250 pg is well within reach. One other advantage is afforded by the assay system used here. The tri- PFB derivative has a longer retention time than the other compounds present in the extract after HPLC purification and Figure 3.4.8 illustrates this quite clearly. Most (more than 99%) of the other compounds in the derivatised sample B la have lower retention than the tri-PFB SA This clear separation from interfering components would make GC/electron capture determinations easier to perform with satisfactory precision. Clearly the formation of these PFB esters in the plant extracts, their coincidence in GC retention and similarity in MS fragmentation has confirmed the findings in Chapter 2. Additionally the fact that the plant derivatives gained three PFB groups after derivatisation indicated that the endogenous compound was present in the o-hydroxy not the o-methoxy form as isolated in Chapter 2. The amounts determined in the full scan negative ion experiment were small and quite close to the limits of detection of the instrument so the negative result concerning the presence of the methoxy isomer does not completely rule out its existence. 190 It can be said with absolute certainty that salicyloylaspartate is present in plants as the free acid and that each plant tested by SIM-MS series did contain some free acid. Close to equivalent retention times on GC and similarity in ratios of the scanned ions for both standard and samples again offered good confirmatory evidence of the presence of SA. For instance, in Figure 3.4.11 the ratio of m/z 612 to 414 is 4.1 for the standard and 2.5 for the tomato extract. There is some variation but this is certainly well within expectation for chemical ionisation MS considering the much higher amounts of the standard derivative injected and previous comments about the effect of sample loading on relative ion intensity (Results 2.4.8.3). The mere presence of the ions m/z 612,414 and 234 in the plant extract at equivalent retention should be sufficient for positive identification. Another of the questions posed by the results of Chapter 2 was the possibility that the ethyl ester groups may have arisen by trans-esterification of some conjugate of OBA due to storage in ethanol at low pH. This notion was tested by hydrolysis of the plant extracts after extraction of the free acid. In the first series of plant extracts (bean and tomato leaf) there was no detectable derivative in B3a but T3a contained levels higher than Tla (free acid). Fraction T2a did not contain any detectable SA so this ruled out any possibility that the presence of SA in T3a was due to incomplete extraction of the free acid. The SIM experiment also indicated that hydrolysable conjugates were present in all the plants tested. This bound SA has been released by relatively mild acid hydrolysis which suggests that some salicyloylaspartate may be present as a glucose ester or diester or a carbohydrate linked compound. The ease of hydrolysis of the conjugate strongly precludes the existence of an amide linkage in similar form to the feruloyl glycine to protein combination discovered by Van Sumere et al (1973). Under the conditions of hydrolysis used (2M HC1, room temperature, overnight) it is unlikely that any significant cleavage of the SA conjugate would occur in this situation. So in this case a glucose ester (or carbohydrate ester) is the most likely scenario. 191 Preliminary work suggests that it would be quite possible to use deuterated SA-PFB as an internal standard for quantitation of SA in plant tissue. After initial incorporation of at up to four deuteriums on acid catalysed exchange only one survived the derivatisation reaction to give about 27% enrichment and this probably occurred on the amide nitrogen. Further studies would have to be done on the stability of the label but other methods can be used that may label the ring and these positions often seem less susceptible to exchange. Ideally three or four deuteriums or the use of 13C-labelled compounds are ideal for an internal standard. CHAPTER 4 Ethylation of purified salicyloylaspartate as an indication of the conjugation site of the endogenous compound 192 4.1 INTRODUCTION The results in Chapter 3 indicated that at least some of the endogenous SA is linked to a conjugating group at either the a- or p-carboxyl. Further, comparison of the NMR spectra of the isolated MBA and the synthetic monoethyl monomethyl ester preparation (Chapter 2, Figures 2.4.5 and 2.4.14) showed the relative ethylation of each carboxyl group is different in synthetic and endogenous MBA. The ratios are approximately 1:2 (3.8:3.7 ppm, endogenous) and 6:1 (3.8:3.7 ppm, synthetic). If the assignment of resonances was correct this indicates that in the case of the endogenous compound ethylation has occurred predominantly on the a- carboxyl. Results for the synthetic preparation demonstrate the opposite pattern of esterification, about six times more ethylation on the p-carboxyl. As mentioned earlier there was not a great amount of care taken to include all of both isomers during HPLC purification so the established pattern of ethylation may not follow this trend. With acid catalysed esterification of a dicarboxylic acid it would be expected that the group with the highest pKa would react more readily since acid catalysed esterification involves a protonated transition state. Other features influencing the rate of esterification are the tendency of the carbonyl oxygen to protonate and steric hindrance. Primary acids react more readily than secondary or tertiary acids so in this case esterification would favour the p-carboxyl. The pKa of the a- and p-carboxyl groups of aspartic acid are 2.0 and 3.9 (Stryer 1981) and if SA behaved in similar fashion then a divergence in pKa should promote a difference in ethylation rates favouring the p-carboxyl. Thus, if this does occur (the p-carboxyl is ethylated to a greater extent than the a-carboxyl) then ethylation of the endogenous compound could be due solely to esterification of the free acid and the discovery that conjugation also occurs is purely coincidental. The work in this chapter will be directed towards the exploration of the pKa values of the ionisable groups of SA, studies of the ethylation behaviour of this compound and the use of this information to determine whether acid-catalysed 193 esterification of the free acid or trans-esterification in acidic ethanol has converted a conjugate of SA to an ethyl ester - and ultimately whether the site of conjugation can be identified. Obviously one way to find the conjugation site would be to process leaf material, develop a purification sequence and finally identify the isolated conjugate (or conjugates) by NMR, MS and other methods. However, this would involve the extraction of at least 100 kg of plant material in order to obtain around 100 fig of conjugate from strawberry leaf, the best source of conjugate (Table 3.4.2). Purification from such a mass of plant tissue or the screening of a large number of plant species to find a better source would be a lengthy procedure not within the time constraints of this project. Consequently, the use of the following ethylation studies in concert with further analysis of the original bean extract was the best way to approach the problem of identification of the conjugation site. 194 4.2. MATERIALS See also Materials Chapters 1, 2 and 3. 4.2.1 HPLC Purifications were carried out with the same pumps, gradient controller, Diode Array detector, computer and peripherals oudined earlier (Materials 1.2.7). The columns used and run conditions are outlined in the relevant sections. 4.2.2 MASS SPECTROMETRY Chemical ionisation mass spectra were recorded using the Finnigan instrument described in Materials 1.2.8. Samples analysed by solid probe were transferred to a small glass cup, evaporated, placed in the probe and heated to 250°C at a rate of about 200°C per minute after insertion into the ion source. GC run conditions are outlined in the appropriate conditions. Electron impact analysis was undertaken on the AUTOSPECQ as described in Materials 1.2.8. with the following additional procedures. Resolution was set to 5000, and the dual quadrupole was used as a mass filter/analyser system for MSMS of the chosen parent ion. Magnet voltage was held constant to select the appropriate fragment ion resulting from the primary fragmentation in the ion source. This ion was fragmented in a collision cell with air as the collision gas and collision energy set at 30 eV. Quadrupoles were scanned at 20 secs per decade. Solid probe samples were treated in the same way as the Finnigan Cl samples except that no direct probe heating occurred. Compounds were volatilised with the aid of the temperature in the ion source (200°C) 4.2.3 NMR NMR spectra were recorded on a Bruker 500 MHz spectrometer under the conditions outlined in Materials 1.2.9. 195 4.2.4 pH MEASUREMENT All pH measurements were carried out with a platinum electrode and a digital display Model 671 Jenco pH meter. 4.2.5 SOLVENTS AND CHEMICALS Deuterated methanol (99.5% D) was obtained from Sigma Chemical Company (Poole, England) as was the 35% DC1 (99% D) in D2O. Solvents used during HPLC purifications were, with the exception of 95% ethanol, obtained from Mallinkrodt Australia (Sydney, NSW). Ethanol was laboratory grade redistilled over solid sodium hydroxide. All other solvents were analytical grade and obtained locally. 196 4.3. METHODS 4.3.1 DETERMINATION OF pKa OF SYNTHETIC SA Salicyloylaspartate (200 mg), prepared by the procedure shown in Methods 3.3.1 was dissolved in water (60 ml) and the pH adjusted to 1.6 with concentrated HC1. This solution was titrated against 0.2 M sodium hydroxide in 0.1 ml portions and the pH measured after the addition of each aliquot of alkali solution. 4.3.2 SYNTHESIS OF MONOETHYL SA Salicyloylaspartate (50 mg) was dissolved in absolute ethanol (20 ml) and enough concentrated HC1 added to lower the pH to 3.5. This mixture was heated to 60°C for 4 hrs. After cooling to room temperature, the solution was evaporated to dryness and redissolved in about 1 ml of ethyl acetate. An aliquot was removed for HPLC analysis. Hexane (20 ml) was added to the remaining ethyl acetate solution and this mixture applied to the top of a silica column (10 cm x 3 cm diameter) previously equilibrated with hexane (100 ml). The column was eluted with hexane (100 ml), followed by hexane/ethyl acetate/acetic acid (50/50/10, 100 ml) and 10% acetic acid in methanol (100 ml). The methanol/acetic acid eluent was evaporated and redissolved in methanol for HPLC purification. 4.3.3 REVERSE-PHASE HPLC 4.3.3.1 DETERMINATION OF THE RATIO OF MONOETHYL ESTERS An aliquot of the ethylation reaction mixture (Methods 3.2) was analysed isocratically on a Altech Ci8 Econosil analytical column (250 x 4.6 mm, 5 pm particle size) with 60% methanol in 0.2% acetic acid as the mobile phase at a flow 197 rate of 1 ml/min. The two monoethyl ester peaks were collected for MS analysis and peak areas determined by integration. A portion of each monoester was methylated for GCMS. 4.3.3.2 SEPARATION OF MONOETHYL ESTERS The hexane/ethyl acetate/acetic acid eluent from the silica column step was purified in six equal portions on a Whatman Magnum Ci8 reverse-phase column (600 x 8 mm, 10 pm). Mobile phase was 20% isopropanol in water adjusted to pH 4.0 with acetic acid and flow rate was 2 ml/min. The two monoethyl ester peaks were collected for methylation and normal- phase purification. 4.3.4 METHYLATION The two fractions from the HPLC purification step (Methods 4.3.3.2) were dissolved in methanol (2 ml) and enough etherial diazomethane added so that the solution retained a pale yellow colour. The solvents and reagent were immediately removed by evaporation under a stream of nitrogen to minimise trans- esterification. 4.3.5 NORMAL-PHASE HPLC Each of the methylated monoester preparations were purified on a Waters silica semi-preparative column (300 x 4.6 mm, 10 pm) using 10% isopropanol in hexane at a flow rate of 3 ml/min as the eluting solvent. The first peak from each run (the monoethyl monomethyl ester) was collected for NMR and MS. 4.3.6 TIME COURSE OF ETHYLATION OF SA An ethylation reaction was set up under the conditions of Methods 4.3.2 except that the reaction mixture was maintained at room temperature. Aliquots were 198 removed at various times over a period of twenty four hours and analysed by HPLC under the conditions of Methods 4.33.1. 4.3.7 SEPARATION AND PURIFICATION OF ENDOGENOUS SA A portion of bean leaf extract was purified by the procedure outlined in Methods 2.3.2. The Sep-Pak eluent of 30M20 was fractionated by HPLC in three equal portions on a Waters semiprep Cis reverse-phase column (300 x 4.6 mm, 10 Jim) with 40% ethanol in 0.2% aqueous acetic acid as the mobile phase. Flow rate was 2 ml/min. Solvent eluting at the retention times corresponding to a previously injected standard mixture of monoethyl monomethyl esters was collected, evaporated and redissolved in isopropanol. The isopropanol solution from the previous step was purified on a Waters silica semiprep column (300 x 4.6 mm, 10 Jim) with 6% isopropanol in hexane at 3 ml/min as the eluting solvent. Mobile phase eluting over the period of retention of the two synthetic monoethyl monomethyl ester isomers was collected and evaporated for GCMS. 4.3.8 PROTON NMR The two isomeric monoethyl monomethyl esters prepared and purified by the procedure outlined in Methods 4.3.2, 4.3.3, 4.3.4 and 4.3.5 were dissolved in 99.96% CD3CI and proton NMR performed. 4.3.9 PREPARATION. PURIFICATION OF DEUTEROMETHYL SA ESTERS Salicyloylaspartate (10 mg) was dissolved in 99.5% CD3OD (5 ml) and 35% DC1 in D2O (99% D, 2 drops) added. The tube was sealed and left overnight at room temperature. After evaporation the residue was dissolved in methanol and purified by reverse-phase HPLC using the method outlined in Methods 4.33.2. 199 After evaporation of the solvent the monomethyl ester preparations were methylated with diazomethane and submitted for GCMS analysis. 4.3.10 TRANS-ESTERIFICATION OF DIETHYL SA o-methoxybenzoylaspartate (10 mg) was dissolved in absolute ethanol (5 ml) and a few drops of concentrated HC1 added. After standing overnight the solution was evaporated, redissolved in ethyl acetate (1 ml) and hexane (9 ml) added. This solution was forced through a silica Sep-Pak previously equilibrated with hexane (10 ml). The Sep-Pak eluent was evaporated, redissolved in hexane (10 ml) and the Sep-Pak purification step repeated. A small portion of mixture was then submitted for GCMS. The rest was dissolved in CD3OD (2 ml) and a few drops of DC1 added. After standing this mixture was evaporated and an aliquot analysed by GCMS. 4.3.11 CHEMICAL IONISATION GCMS 4.3.11.1 BEAN LEAF FRACTION 30M20:10IPA A portion of the purified 30M20 was analysed by positive ion chemical ionisation GCMS under the following conditions: Column was a 12 m polyimide- clad vitreous-silica bonded-phase BP-1 from SGE Australia, flow rate was 2 ml/min of helium, makeup reagent gas was methane (20 ml/min) and column oven temperature was maintained at 180°C for one minute after injection and then raised 10°C per minute to 300°C. 4.3.11.2 LABELLED DIMETHYL SA ESTERS Each ester preparation after methylation with diazomethane was injected onto a 2 m 1.5% OV-1 packed column with helium carrier gas (30 ml/min) and helium reagent gas. Column temperature was held at 180°C for 1 minute and then increased to 300°C at 10°C per minute. 200 4.3.11.3 SA DIETHYLESTER TRANS-ESTERIFICATION MIXTURE An aliquot of the mixture from the trans-esterification of diethyl SA with deuteromethanol/DCl was analysed by GCMS under the conditions outlined above (Methods 4.3.10.1) with the exception of the temperature programme which was changed to: 150°C 1 min, 15°C per minute to 300°C. 4.3.11.4 VARIOUS HPLC COLUMN ELUENTS On occasion, various peaks from HPLC analyses or purifications were collected, evaporated and submitted for direct insertion or GC mass spectroscopy. This will be stipulated in the appropriate sections in the results. 4.3.12 MSMS STUDIES OF THE ISOMERIC SA DIESTERS The monoethyl esters were prepared by acid catalysed ethylation (Methods 4.3.2) , separated by reverse-phase HPLC (Methods 4.3.3.2), methylated with diazomethane and purified by normal-phase HPLC (Methods 4.3.5). Each isomeric diester was analysed by solid insertion probe EIMS under the conditions outlined in Materials 4.2.2. An ion resulting from the primary fragmentation of the diesters (m/z 176) was selected by setting the appropriate magnet voltage, further fragmented in the collision cell and analysed by quadrupole MS (See also Materials 4.2.2) . 201 4.4. RESULTS 4.4.1 pKa DETERMINATION The titration curve of salicyloylaspartate versus sodium hydroxide is shown in Figure 4.4.1. Only one point of inflection can be clearly seen from this curve. One of the other two expected points of inflection can be found by a plot of the first derivative of pH versus the volume of NaOH (Figure 4.4.2). The first derivative plot will show minima at the points of inflection and this plot shows one very clear minimum corresponding to a volume of 18.6 ml of NaOH solution. Another probable minimum at 14.8 ml can be confirmed by calculation of the amount of 0.2M NaOH needed to titrate 200 mg (0.79 millimoles) of SA. Titration of one of the three ionisable groups should consume 3.85 ml (0.79 millimoles) of 0.2M sodium hydroxide so the minimum at 14.8 is real. The point of inflection for the most acidic carboxyl group cannot be determined from either the titration curve or the first derivative plot. However subtraction 3.8 ml from the volume of NaOH corresponding to the minimum at 14.8 ml will give the experimentally derived pKa of the most acidic carboxyl. The measured pKas of the three ionisable groups of salicyloylaspartate are 2.7, 4.4 and 8.3. These values are uncorrected for activity effects and only serve to show that there is a clear distinction in pKa between the two carboxyl groups. 4.4.2 RATIO OF MONOETHYL ESTERS The monoethyl ester preparation from Methods 4.3.2 has a HPLC profile shown in Figure 4.4.3. The order of elution of the monoethyl esters is marked as monoethyl A and monoethyl B. Since the extinction coefficients of each isomer would certainly be very similar, if not identical, then clearly the amount of monoethyl B produced is much greater than monoethyl A. The integrated peak areas show a ratio A:B of about 1.0:3.0. pKai = 2.7 sodium hydroxide. Titrant was added in aliquots of 0.1 ml and pH measured after equilibration. Dotted lines show the volume of NaOH corresponding to the points of inflection on the titration curve determined by the first derivative plot (Figure 4.4.2) for pKa2 and pKa3. pKai is calculated by subtration of the appropriate volume of NaOH. 202 FIGURE Derivative 4.4,2 0.6 - cannot shown Graph are difference hydroxide. subtraction ml of shown 0.2M in of be Figure Volume the seen between by NaOH. of The first the 3.8 from 4.4.1 points of minima derivative ml the The this 0.2M versus of second of point graph titrant corresponding inflection NaOH volume of of but and (equivalent pH inflection (mis) can for third on of 0.2M be the the to minima). for 18.6 calculated to titration titration sodium the the and lower volume curve curve 14.8 by pKa 203 RETENTION TIME PEAK AREA (min) (arbltary uni CD 0 J l 088'8 88 * 0 I ( ~ ID 0 FIGURE 4.4.3 HPLC chromatogram of the ethylation mixture (Methods 4.3.2) performed under the conditions outlined in (Methods 4.3.3). Also included is a list of integrated peak areas. Identity of peaks was based on direct insertion chemical ionisation MS. 204 205 4.4.3 IDENTITY OF THE MONOETHYL ESTERS To glean any useful information on the ethylation behaviour of SA, and ultimately the identity of the predominant ethyl ester of SA isolated from bean leaf, these isomers have to be identified. Mass spectrometry offers one likely way to distinguish between the two isomers. With structures of this type the most likely fragmentation sequence would involve cleavage at the secondary carbon. Thus one of the monoethyl esters may lose the carboxyl group (45 AMU) and/or the ester group (87 AMU, -CH2COOCH2CH3) and the other 59 AMU (-CH2CO2H) and/or 73 AMU (-COOCH2CH3). See Figure 4.4.4 for a diagrammatic representation of these possibilities. The direct insertion probe of each isomer is shown in Figure 4.4.5. Both isomers have very similar fragmentation patterns and there is more congruence than expected. Readily identifiable cleavages for both monoethyl A and B include losses of 18 AMU (water), 45 AMU (carboxyl) and amide bond breakage to produce a fragment m/z 121 (aromatic side) and m/z 162 (amino acid side after nitrogen protonation). Of the side chain cleavages expected in Figure 4.4.4 only the loss the carboxyl group from both isomers (to give m/z 236) makes a marked contribution to the fragmentation pattern. However there is a minor ion with a low relative intensity that does indicate that monoethyl A is esterified at the a-carboxyl. Fragment m/z 208 (2%) only appears on the spectra of monoethyl A and could result from cleavage of COOCH2CH3 at the secondary carbon as there is no other primary cleavage that would give rise to this ion. Assuming this is the case other expected fragmentations; losses of 59 (from monoethyl A) and 87 (from monoethyl B) to ions m/z 222 and 194 respectively do not appear. Further indirect evidence to support this notion comes from studies of the El spectrum of diethyl aspartate. Biemann, as outlined in Hill (1966) showed that simple fission occurs only on one side of the secondary carbon (leading to a loss of -COOCH2CH3) and that cleavage of the -CH2COOCH2CH3 sidechain does not occur by simple fission. Rather, the fragmentation of this sidechain involves a multi-step rearrangement procedure beginning with the evolution of ethylene. Often 206 m/e4 208 'c OCH2CH3 o s'p? /OH rCH2 —C m/e* 222 1 UU/C\ /OCH2CH3 C —NH CH2—C OH m/e4 236 C “OH 0 / 1 CH C-NH OH m/e4 194 FIGURE 4.4.4 Possible fragmentation pathways of the two monoethyl esters of SA OC o © > ffi Intensity Relative Intensit 100.0 FIGURE 50.0- -I K yi ., y „. 80 ( . 88 4.4.5 "T 100 ” . 120 'V ...... ethylation monoethyl Direct . ... 149 ^ II — 160 Monoethyl 162 Monoethyl insertion |l-...... reaction esters m/z m/z o It — A 200 B probe J — of ...... mixture SA 220 J r ...... mass 236 eluted 240 (Methods spectra 260 T from 280 I L., HPLC of — 4.3.3.1). 300 300 ,....1 the 310 1 of — 320 320 two ...... the . - - 334336 63360 207 208 it is not very useful to translate similarities in El fragmentation into Cl fragmentation pathways but these findings do, nonetheless add a little more weight to the argument that monoethyl A is the a-ethyl ester. 4.4.4 Cl MASS SPECTRA OF METHYLATED MONOETHYL ESTERS The mass spectra of monoethyl A and B after HPLC purification (Methods 4.3.3.1) and methylation is shown in Figure 4.4.6. Analysis was carried out on a 12 m BP-1 (0.32 mm ID), initial oven temperature was held at 150°C for one minute then raised 10°C per minute to a maximum of 300°C. Again there is very significant congruence in the fragmentation patterns of the two isomers. Common ions occur at m/z 61, 89, 115, 129, 135, 152, 163, 174, 180, 202, 264, 278, 310, 338 and 350 AMU. However it is not the ion species shared by the two compound that is of greater interest but rather the ions that are present in one isomer but not the other. The fragment m/z 236 appears only in the spectrum of methylated monoethyl A whereas m/z 250 is exclusively present in the other isomer. Cleavages leading to the formation of these ions are reasonably easy to elucidate and these possibilities are shown in Figure 4.4.7. This figure shows that fragments m/z 236 and 250 can theoretically arise from both isomers and that the a- methyl p-ethyl ester could also lose the sidechain at the tertiary carbon to produce m/z 202. However both diesters have the m/z 202 fragment in their mass spectrum and for the a-ethyl p-methyl compound there is no way that this ion can be produced by simple fission. Losses of 59 AMU (-COOCH3) followed by 28 AMU (ethylene from the ethyl ester) could possibly contribute to the formation of m/z 202 in both cases. The exclusive presence of m/z 250 in the spectrum for one diester and m/z 236 means that only one of the possible two cleavages occurs adjacent to the carboxyl. For instance the a-ethyl p-methyl isomer could lose the ethyl ester moiety by cleavage at the tertiary carbon (to produce m/z 236) or the methyl ester by 209 Monoethyl A >« c © £ O > m © DC 236 i 350 — M 1 I I 80 120 140 160 220 240 260 m/z r19552 >. Monoethyl B TZ c© _c >© « © cc 264 250 | 278 350 TTrfrr. T LX 4——i*- r I I 80 100 120 140 160 220 240 260 300 320 m/z FIGURE 4.4.6 Direct insertion probe mass spectra of the two isomeric monoethyl monomethyl esters of SA after HPLC purification (Methods 4.3.3.2) and methylation. 210 C—NH oc-Fthyl B-Methvl MBA m/e 236 m/e* 250 C—NH m/e 236 0 1 MU-'0” .och2ch3 C—NH CH2 — g-Methyl B-Ethvl MBA och3 °S c — och3 m/e* 250 c — och3 CH C-NH/ Xch2 och3 m/e* 236 m/e 222 FIGURE 4.4.7 A few possible single step simple fission fragmentation pathways of the two monoethyl esters of SA after trapping on HPLC (Methods 4.3.3.1) and methylation. 211 cleavage of the bond adjacent to that carboxyl to give m/z 250. Only one of these fragmentation processes has occurred. So for both isomers either the fragmentation occurs between the tertiary carbon and the neighbouring carboxyl or at the secondary carbon one step along the sidechain. One clue was provided by the mass spectra of the monoethyl esters (Figure 4.4.5). Here both lose 45 AMU so there must be cleavage at both of these sites. However one isomer (monoethyl B) does not lose the ethyl ester group to give the ion m/z 208. Consequently the isomer giving rise to the m/z 236 in the spectum of the diesters is likely to be a-ethyl p-methyl MBA. Fragmentation adjacent to the secondary carbon on the carboxyl side may be a special case for the free acid which isn’t reflected in the spectrum of the diesters. 4.4.5 STUDIES WITH DEUTERQMETHYL. METHYL ESTERS OF SA The mass spectrum of the two deuteromethyl methyl esters prepared as in Methods 4.3.9 is shown in Figure 4.4.8. These samples were analysed using helium as the reagent gas in order to promote greater fragmentation and hopefully give more information to allow differentiation between isomers. Like the mass spectra of the mixed diesters under Cl conditions with methane reagent gas (Figure 4.4.6) there is a great congruence in fragmentation patterns. However again there is a telling difference which confirms the notion that cleavage of the ester group occurs at the same site on each compound. The mass spectrum of the monoester with the lower HPLC retention time (methods 4.3.3.2) after treatment with diazomethane, ie. the ester with the deuteromethyl group attached to the same carboxyl as monoethyl A, has an ion m/z 236, whereas the other isomer shows a fragment ion of m/z 239. Neither has both, a finding that confirms the earlier results with the methylated monoethyl esters that there is fragmentation exclusively at one site near the secondary carbon. FIGURE tr to > © 0 ) Intenslt 50 100.0 JO- 4.4.8 -I *T of appropriate methylation. Mass 100 SA spectra after 140 T~ HPLC peak Mass 160 of I the with purification 180 T spectra m/z m/z Monodeuteromethyl two Monodeuteromethyl some 200 I isomeric were 220 r background (Methods 4 260 240 summed deuteromonomethyl B A 4.3.3.2) 280 subtraction. across I ~T~ 300 and the esters 212 213 4.4.6 REVERSE-PHASE HPLC ON A PREPARATIVE COLUMN After trying various organic modifiers with 0.2% aqueous acetic acid the separation of monoethyl SA esters on semi-preparative or preparative columns was not to successful. With an analytical column baseline separation of the isomers could be achieved but purification of large amounts of compounds would have been very tedious. Better results were achieved using isopropanol/0.2% aqueous acetic acid as the mobile phase but baseline resolution was still not possible. Changing the pH of the elution solvent proved to be the solution. Retention times of the peak apices (minutes) on HPLC analysis of the hexane/ethyl acetate/acetic acid eluent from the silica column of the ethylation mixture of SA (Methods 4.3.2) run under three different pH conditions are shown below. Adjustment to the appropriate pH from the pH of 0.2% aqueous acetic acid (pH 3.0) was made with either glacial acetic acid or ammonia solution. Free Acid Monoester (B) Monoester (A) pH =2 5.8 11.4 11.4 pH = 3 7.1 17.3 18.2 pH = 4 6.9 15.5 27.0 As the results above clearly show at a pH of 2.0 the monoesters elute together, at pH 3.0 the peaks are partially resolved (but still fused) but at pH 4.0 a clear separation occurs. It should be noted that the first to elute under these conditions is monoethyl B. With the analytical column and a methanol/water/acetic acid mobile phase (Methods 4.3.3.1) the order of elution was reversed. The mass spectrum of the first monoethyl ester eluting from the Magnum column after methylation was 214 identical to the methylated monoethyl ester with the longer retention time from the analytical column. 4.4.7 PROTON NMR OF THE METHYLATED MONOETHYL ESTERS The monoesters collected by HPLC trapping of the methanol/acetic acid eluent from the silica column (Methods 4.3.2) after methylation were submitted for proton NMR. The spectra of the one isomer with assigned resonances is shown in Figure 4.4.9 and the other isomer in Figure 4.4.10. Resonances due to the ring protons, and protons of the methoxy group and the proton attached to the nitrogen have the same chemical shift in both structures but important differences are apparent in the rest of the molecule. This is where the basis for the assignment lies. It would be expected that the a-ester protons of ethyl and methyl origin would have higher chemical shifts than the (3-ester protons due to the closer proximity to the amide nitrogen. The spectrum in Figure 4.4.9 shows a quartet with further long range splitting centred at 4.26 ppm which is the resonance pattern expected for the ethyl methylene group while the triplet at 1.29 ppm would be due to the ethyl (methyl) group. The spectrum of the other isomer has corresponding resonances centred at 4.15 and 1.24 ppm. Methyl ester protons (singlets) have resonances at 3.70 (Figure 4.4.9) and 3.79 ppm (Figure 4.4.10). On this basis, the higher field resonances for both methyl and ethyl protons must belong to the a-carboxyl esters. So the spectrum monomethyl A in Figure 4.4.9 can be assigned to the structure shown, o-methoxybenzoylaspartate a- ethyl (3-methyl ester. 4.4.8 HIGH RESOLUTION EIMS AND MSMS OF SA DIESTERS The El mass spectrum of the monoethyl monomethyl esters (results not shown) displayed great congruence with the CIMS data. Most importantly, the appearance of the ions m/z 236 and m/z 250 followed the pattern shown by the Cl spectra. Thus the spectrum of methylated monoethyl A included an ion at m/z 236 215 CD B *♦— E . o Diagra o methylation. after q 4.3.3.2) (Methods E Q. HPLC Q. jr CO trapped o included. E ester o are x: O _ o monoethy! resonances second o the assigned of and NMR q Proton compound q 4,4,9 FIGURE CHjCHj o o o O xz C/5 -C a> E CL E CL b £ c o TO 03 co E FIGURE 4,4.1 Q Proton NMR of the first monoethyl ester trapped HPLC (Methods 4.3.3.2) after methylat 03 compound and assigned resonances are included. 216 217 but no significant m/z 250 ion while this situation was reversed with the other isomer. High resolution MS was specifically carried out to allow elemental formulas to be assigned to fragment ions so that appropriate ion could be chosen for further MSMS analysis. The MSMS experiments provided additional evidence to support the CIMS and NMR data regarding the identity of the two isomeric monoethyl monomethyl esters of salicyloylaspartate. The high resolution mass listing and the calculated molecular formulas for the mass spectrum of methyl monoethyl B are shown in Figure 4.4.11. Within the mass-variation limit set (20 ppm) the high mass ions do not show the expected formulas but in the mass range where the instrument was very carefully calibrated (m/z 160 to 180) the expected fragment formulas show less relative error and can be assigned with confidence. Thus the ions m/z 176 and m/z 174 have formulas C7Hi4N04+ and C7Hi2NC>4+ respectively. The other formulas in the listing are either trivial or could not possibly arise from fragmentation of the parent compound. The aim of the MSMS experiment was to induce fragmentation on both sides of the secondary carbon atom - a fragmentation that did not occur to any measurable extent in any of the CIMS or EIMS studies of the mixed esters. This was first attempted on the parent ion but again the result was negative, cleavage only occurred on one (probably the a-carboxyl side) of the secondary CH group. The fragment ion m/z 176 was then chosen as a possible candidate for MSMS analysis as the formula assigned by high resolution MS (C7Hi4NC>4+) fitted the notion that this ion arose from the cleavage of the amide bond, the loss of the salicyloyl group and a transfer of two protons to the nitrogen containing fragment Collision induced MSMS fragmentation is very much a chemical ionisation process (eg loss of methanol from methyl esters rather than - OCH3 in El conditions) so the appropriate possible cleavages for the m/z 176 parent from the two different monoethyl monomethyl isomers are shown in Figure 4.4.12 (a-ethyl) and 4.4.13 (p-ethyl). 218 Elemental Coiqposition File:D14 301 ldent:32 Acquired:13-MAR-90 21:17:03 +0:47 AutoSpecQ Ionization:EI+ Function:Magnet BpM:0 Bpl:3303725 TIC:8984343 File Text: Heteroatom Max: 20 Ion: Both Even and Odd Limits: 60.000 2.0 -0.5 0 0 0 0 350.000 100.0 20.0 20.0 200 400 10 10 Mas a %RA nOa PPM Calc. Masa DBK C H HO 309.114174 9.4 -0.2 -0.5 309.114022 16.5 20 13 4 0.4 1.2 309.114530 4.0 6 15 9 6 1.2 3.9 309.115364 16.0 22 15 1 1 -1.5 -4.8 309.112684 11.5 19 17 4 1.7 5.5 309.115872 3.5 8 17 6 7 -2.3 -7.5 309.111850 -0.5 3 17 8 9 -2.8 -9.2 309.111342 12.0 17 15 3 3 3.0 9.8 309.117210 8.5 9 13 10 3 3.0 9.8 309.117215 3.0 10 19 3 8 -4.2 -13.5 309.109999 12.5 15 13 6 2 4 . 4 14 .2 309.118552 8.0 11 15 7 4 4.4 14.2 309.118558 2.5 12 21 9 -5.5 -17.8 309.108662 7.5 14 17 2 6 -5.5 -17.9 309.108656 13.0 13 11 9 1 5.7 18.5 309.119895 7.5 13 17 4 5 264.092979 5.1 0.1 0.3 264.093066 4.0 5 12 8 5 0.9 3.5 264.093900 16.0 21 12 1.4 5.4 264.094408 3.5 7 14 5 6 -2.6 -9.8 264.090386 -0.5 2 14 7 8 2.8 10.5 264.095746 8.5 8 10 9 2 2.8 10.5 264.095751 3.0 9 16 2 7 -3.1 -11.7 264.089878 12.0 16 12 2 2 -3.9 -14.9 264.089043 0.0 12 10 7 4 .1 15.6 264.097088 8.0 10 12 6 3 -4 .4 -16.8 264.088535 12.5 14 10 5 1 251.106911 3.4 0.3 1.2 251.107205 10.5 17 15 2 -1.0 -4.2 251.105862 11.0 15 13 3 1 2.1 8.5 251.109050 3.0 4 13 9 4 -2.4 -9.5 251.104519 11.5 13 11 6 3.5 13.9 251.110393 2.5 6 15 6 5 -3.7 -14 .8 251.103182 6.5 12 15 2 4 4.8 19.2 251.111730 7.5 7 11 10 1 4.8 19.2 251.111736 2.0 8 17 3 6 250.105232 20.9 0.0 0.1 250.105248 7.5 9 12 7 2 0.0 0.1 250.105253 2.0 10 18 7 -1.3 -5.3 250.103911 2.5 8 16 3 6 -1.3 -5.3 250.103905 8.0 7 10 10 1 1.4 5.4 250.106591 7.0 11 14 4 3 -2.7 -10.7 250.102568 3 .0 6 14 6 5 2.7 10.8 250.107933 6.5 13 16 1 4 -4 .0 -16.0 250.101225 3.5 4 12 9 4 4.0 16.1 250.109271 11.5 14 12 5 176.089821 5.5 -0.2 -1.3 176.089598 2.5 3 10 7 2 1.1 6.4 176.090940 2.0 5 12 4 3 -1.6 -8.9 176.088255 3.0 1 8 10 1 2.5 14.0 176.092283 1.5 7 14 1 4 174.074997 2.8 0.3 1.7 174.075290 3.0 5 10 4 3 -1.0 -6.0 174.073948 3.5 3 8 7 2 1.6 9.4 174.076633 2.5 7 12 1 4 -2.4 -13.7 174.072605 4.0 1 6 10 1 3.0 17.1 174.077970 7.5 8 8 5 136.023727 14.7 -0.5 -3.5 136.023255 2.0 1 4 4 4 0.9 6.4 136.024592 7.0 2 8 0.9 6.4 136.024597 1.5 3 6 1 5 2.2 16.2 136.025935 6.5 4 2 5 1 135.028591 100.0 -0.6 -4 .3 135.028006 2.0 2 5 3 4 0.8 5.6 135.029343 7.0 3 1 7 0.8 5.6 135.029348 1.5 4 7 5 -1.9 -14.3 135.026663 2.5 3 6 3 2.1 15.5 135.030686 6.5 5 3 4 1 FIGURE 4,4,11 Mass list from m/z 309 to m/z 135 (inclusive) for ions with a relative abundance greater than 5% in the high resolution electron impact mass spectrum of methylated SA monoethyl ester (monoethyl B). Also shown are the calculated formulas for each fragment. Shown enclosed in brackets is the fragment ion used for further secondary fragmentation. 219 O Jl CH2^-C—OCH, -(59 + H) nh3+- CH A m/z = 116 ^C— OCH2CH3 O II ,ch2—c—och3 / -(73 + H) nh3+- ■CH B m/z = 102 X c —OCH2CH3 O II ^ch2-c—och3 -(73 + H) NH3+- ■CH C m/z = 102 ^c— och2ch3 II o O Ik ^ch2—c-|-och3 -(31 + H) nh3+- ■CH D m/z = 144 ^C—OCH2CH3 O II CHP—C—OCH3 / -(45 + H) NH3+------CH E ------m/z = 130 \^-OCH2CH3 or FIGURE 4.4.12 Possible fragmentation processes in the secondary MS of aspartate diester (a-ethyl) ion formed by El cleavage of the corresponding salicyloyl derivative. 220 ^ch2|-c—och2ch, -(73 + H) nh3+- •CH A m/z = 102 ^C—OCH. ch2—c—OCH2CH3 / -(59 + H) NH3+------CH . B m/z = 116 C —OCH, ch2—c—och2ch3 X -(87 + H) nh3+- ■CH c m/z = 88 ^C—OCH. CH2—C-|-°CH2CHC / -(45 + H) nh3+- ■CH m/z = 130 \ C —OCH, ch2—c—och2ch3 / -(31 + H) nh3+- ■CH m/z = 144 \ c-£-och3 oir FIGURE 4.4.13 Possible fragmentation processes in the secondary MS of aspartate diester (p-ethyl) ion formed by El cleavage of the corresponding salicyloyl derivative. 221 Figure 4.4.14 shows the daughter ion spectra of the two diesters monoethyl A and monoethyl B after methylation and purification on normal-phase HPLC (Methods 4.3.5). There are some common ions (m/z 102 and 116) but the significant feature is the appearance of m/z 88 in the spectrum of monoethyl A. This coincides with the process C in Figure 4.4.13 so this isomer must be the P- ethyl a-methyl mixed ester. There is no simple fragmentation path leading to this ion with the other isomer. Interestingly another difference between isomers indicates that the ester group is lost from the p-carboxyl exclusively so the a-ethyl ester loses methanol to produce m/z 144 while the p-ethyl ester loses ethanol to produce m/z 130. 4.4.9 GCMS OF 30M20:10IPA A GCMS analysis of purified 30M20;10IPA was performed under the conditions outlined in Methods 4.3.11.1. Figure 4.4.15 shows the specific ion traces for m/z 236, 250 and 310 as well as the total ion current chromatogram over the range 550 to 650 scans. Peak areas are also included. The congruence of retention time of peak apex for the three ions and the total ion current and the absence of any other peaks with these ions mean that the calculation of peak areas is not skewed in any way due to other interfering compounds. The ratios of ions m/z 236 to 250 is 3.6:1 compared to a ratio in the synthetic esters (Figure 4.4.6) of 2:1, both normalised to the base peak m/z 310. This clearly indicates that the ethyl methyl ester which fragments to produce this ion is present in almost twice the amount of the other isomer. The ester which does have m/z 236 in its mass spectrum is the one which gives the NMR spectrum shown in Figure 4.4.9. Thus this compound is a-ethyl P-methyl isomer. FIGURE 4.4.14 Relative Intensity Relative Intensity 100 95. 75. 90. 85 80. 70 50. 55 65 45 60. 40 35 30 25. 20j 10J 15j was fragmentation p-ethyl bond methylation. (-CH Daughter 5j j 2 m/z of COOCH Monoethyl 80 ester. the 176, ion 68 SA spectra 2 pattern 102 Primary the CH derivative 3 H6 ion B + 120 of of H) m resulting ion / monoethyl the from z and lio chosen monoethylated m/z transfer from lio 176 ’ for B the includes so of lio further cleavage this two isomers 200 isomer 200 protons. L6.9E6 L7.3E6 L4.6E6 .5.4E6 fragmentation .4.2E6 .0. .3. a .7. .1.2E6 .1.5E6 .2. .3. 7. 5. 6.1E6 6.5E6 5.0E6 3.5E6 3.1E6 2.7E6 1.9E6 0 7E6 8E6 8E6 3E6 . 7E5 OEO 8E5 loss OEO M/Z of M/Z is of the of the SA The 88 amide after AMU 222 591 266 Oi s .N CO O a: CD O EiGURE 4.4,1$ Total ion current chromatogram and specific ion traces m/z 236, 250 and 310 for fraction 30M20;10IPA after HPLC purification (Methods 4.3.8). Values above peaks represent scan number of peak apex (upper) and peak are CO 223 224 4.4.10 TIME COURSE OF ETHYLATION Peaks areas of the two isomeric monoethyl esters were measured in the six samples taken over the twenty four hour period. These results are shown in Table 4.4.1 along with calculated ratios of monoethyl A to monoethyl B. 4.4.11 TRANS -ESTERIFICATION The mass spectum of the monomethyl monoethyl esters is shown in Figure 4.4.16. The ion m/z 239 results from loss of the a-carboxyl (and ester) and thus represents the amount of diethyl ester that has been trans-esterified in the Im position while m/z 250 is the fragment also resulting from the loss of the a-ester group which in this case is labelled. The ratio of m/z 239 to 250 1.7:1 which is not greatly different from the usual ratio of these ions (2.0:1). Basically this means that the rate of trans-esterification may be slightly greater at the [3-carboxyl the difference is not significant. 225 TIME PEAK AREA PEAK AREA RATIO (hr) (monoethyl A) (monoethyl B) (A/B) 0.17 _ 98 _ 0.34 98 206 0.48 0.58 1 35 315 0.43 2.0 285 708 0.40 4.0 426 1 045 0.41 12.0 613 1747 0.35 24.0 951 2568 0.37 TABLE 4.4.1 Peak areas of the two monoethyl esters of SA on ethylation over a twelve hour period and the ratios of the isomers. 226 135 100.0 -l r- 14928 50.0 - Relative I ntensity r200X pi.ox 100.0 -I r 14928 50.0 - m / z FIGURE 4.4.16 Chemical ionisation mass spectrum of the peak eluting at 7.23 minutes (a mixture of the isomeric monoethyl deuteromonomethyl esters of SA) in the chromatogram of the trans-esterification mixture prepared as in Methods 4.3.11. GC conditions are outlined in Methods 4.3.12.2. The important feature is the relative abundances of m/z 239 and 250. 227 4.5 DISCUSSION Behind this series of experiments lay one particular aim, to find whether the ethylation has simply been the result of acid catalysed attachment of ethanol or whether the endogenous MBA has been trans-esterified from another conjugate and if so what was the most likely site of ethanolysis. Central to this aim was the need to distinguish between the two carboxyls of SA so a measure of the ratio of ethylation at the two carboxyls could be determined. This distinction between esters was made on the basis of pH measurements, HPLC retention data, mass spectral and NMR determinations. Measurement of pKa shows that the two carboxyl groups have divergent pKas (2.7 and 4.4) and like aspartic acid the a-carboxyl will have the lower pKa due to its closer proximity to the electron donating nitrogen of the amide group. Both values for SA are lower than the corresponding pKas of aspartic acid due to the presence of the electron withdrawing aromatic ring and as expected the (3- carboxyl is not affected to the same degree as the a-carboxyl since there is an extra methylene group separating that carboxyl from the benzene ring. Presence of the benzoyl group raises the pKa 0.7 units for the a-isomer and 0.5 units for the 13- isomer. Since the pKa values are different then it would also be expected that over a certain pH range this should be reflected in the the relative mobilities on reverse- phase HPLC of the monoethyl esters assuming there are no gross differences in the 3D structures. Using the experimentally derived values of pKa the relative protonation of each carboxyl can be determined. This is shown below: Percent Protonation a-carboxyl (3-carboxyl pH=2.0 86 >99 pH=3.0 39 95 pH=4.0 6 67 228 Essentially this would mean that at pH 4 the carboxyl with the greater degree of protonation (p-carboxyl) should interact more strongly with the hydrophobic reverse-phase packing and thus elute later than the other isomer. Further lowering the pH should induce smaller differences in the elution times. The table in Results 4.4.6 (pg 213) does show that at the lower pH the two monoethyl esters elute more closely and at pH 4.0 there is a large difference in retention times. On this basis it would be expected that the isomer with the lower retention time (monoethyl B) is the isomer with the free a-carboxyl, ie. the p-ethyl ester of SA. The fact that the order of elution at pH 4 on the preparative column is most likely to be p-ethyl, a-ethyl ester was further supported by NMR and MS evidence. The proton NMR of the ethyl ester isomers from preparative reverse-phase HPLC after methylation from showed significant differences which again suggest that the first eluted from preparative HPLC (monoethyl B) is the p-ethyl isomer. Most crucial was the resonances due to the methyl and ethyl ester protons. As outlined in Results 4.4.7 the isomer with the higher field signals for both methylene and methyl protons on the ethyl ester group would be the a-carboxyl. Conversely, protons on the a-methyl ester would be expected at higher field than the p-isomer. This indeed is the case. Monoethyl B was initially identified as the p-ethyl ester by its lower retention time on reverse-phase HPLC. After methylation of this isomer the proton NMR showed the resonance assigned to the methyl ester protons (3.79 ppm) was at higher field than the corresponding singlet for the other isomer (3.70 ppm). Conversely the resonances for ethyl ester protons (both methylene and methyl) of methylated monoethyl B were shifted to lower field (1.24 and 4.15 ppm) than the other isomer (1.29 and 4.26 ppm). This concurs with the notion that the first eluting ester is the P-ethyl ester because the ester protons closer to the nitrogen have resonances at higher field. Some of the early mass spectral data on the monoethyl esters also supported this notion. The appearance of m/z 208 in the spectrum of monoethyl A (Results 4.4.3) suggested that this compound is the a-ethyl isomer. 229 Final confirmation that monoethyl B is the p-ethyl ester came with MSMS studies of the both monoethyl monomethyl SA esters. A fragment ion (m/z 176) from the primary El spectrum of monoethyl B on further collision-induced cleavage gave a daughter ion of m/z 88 which suggested the loss of the entire sidechain, ie. (-CH2COOCH2CH3). The other isomer did not have this fragment but did show the corresponding loss of the same (but methylated) sidechain (-CH2COOCH3) to give m/z 102. The higher relative intensity of m/z 102 (for monoethyl A) indicated that this ion arose through two separate fragmentation processes (B and C, Figure 4.4.12). Monoethyl B has only one primary mechanism to produce this ion (A, Figure 4.4.13). Having identified the esters there was one critical difference in Cl mass spectral fragmentation which allowed quantitation of the ratios of each in the original bean leaf extract. After methylation, the a-ethyl ester (monoethyl A) had a mass spectrum that was almost identical to the methylated p-ethyl ester isomer - the difference was the exclusive occurrence of ion m/z 236. The corresponding fragment for the p-isomer, m/z 250 was also exclusive to that compound. Confirmation that these fragments were exclusive and had arisen from cleavage at the secondary carbon and loss of the ester group came with examination of the mass spectrum of the mixed deuteromethyl methyl esters. When the a-carboxyl was deuteromethylated the spectrum included an ion at m/z 236 but not 239. The converse occurred when the other carboxyl carried the deuteromethyl group. This appearance of either m/z 236 or 250 (but not both) in the mass spectrum of each isomer promoted a mechanism for determination of the ratios of each in the bean extract - comparison of the ratio of these ions in the purified endogenous ester. Taking into account the differences in intensity of these ions compared to the base peak (m/z 310) the a-ethyl P-methyl ester is present in the bean extract at about twice the concentration of the other isomer (Results 4.4.8). It has also been shown that under the conditions of storage of the bean extract that the synthetic p-ethyl ester formed at about three times the rate of the a- ethyl ester and that this ratio did not change greatly over a twelve hour period. If anything the formation of the (3-isomer is favoured at the later stages, perhaps because the ethylation of the side chain carboxyl somehow stimulated a greater rate of esterification of the a-carboxyl consequently removing more of the P-isomer from the reaction mixture. Additionally it was found that the rates of trans-esterification of each isomer was not significantly different. Treatment of the diethyl ester with deuteromethanol/DCl did not favour the formation of one deuteromethyl ethyl ester over the other (Results 4.4.10). Bearing in mind that the P-isomer esterifies at about three times the rate of the a-isomer, the rates of trans-esterification were not significantly different and that the plant extract contained about twice the quantity of the a-ethyl ester then it was very clear that the conjugate (or conjugates) of SA which have been destroyed in acidic ethanol have been esterified mainly or completely on the a-carboxyl group. Of course a way to confirm without doubt that this is the case is to isolate and purify the conjugate and then examine it by various spectroscopic methods. However the results presented here offer very strong evidence that the conjugating group is attached to the a-carboxyl of salicyloylaspartate. CHAPTER 5 Feeding studies with salicyloylaspartate 231 5.1 INTRODUCTION It has been shown earlier (Chapter 2) that S A is an endogenous component in bean leaves and that extraction, derivatisation and storage conditions led to the formation of the two isomeric monoethyl monomethyl esters of o -methoxybenzoic acid. This process occurred either through acid catalysed esterification or ethanolysis. Chapter 3 offered evidence that the natural form of the compound was o-hydroxy rather than o-methoxy and that this compound was also present in tomato leaves. SIM-MS was used to measure levels of free SA and SA released on acid hydrolysis in a variety of other vegetables. This indicated that salicyloylaspartate and its conjugate (or conjugates) were of widespread occurrence. In Chapter 4 some MS, HPLC and NMR techniques provided a means of unequivocally distinguishing between the two isomeric monoethyl esters of SA. Further measurements performed on one of the Sep-Pak eluents of the stored bean leaf extracts (30M20) showed that the ethylation occurred predominantly on the oc- carboxyl whereas the (3-carboxyl is favoured in acid-catalysed ethylation at moderately low pH. This offered additional evidence that SA conjugation does occur and suggested that the site of conjugation was the a-carboxyl of the aspartate moiety. To further investigate the metabolism of salicyloylaspartate a series of feeding experiments were performed with tomato seedlings. These experiments were aimed to promote formation of SA and its conjugate(s) to a level where unknown compounds could possibly be identified or at least their presence confirmed. 232 5.2 MATERIALS See also Materials Chapter 1,2, 3 and 4 5.2.1 REVERSE-PHASE HPLC 5.2.1.1 GENERAL HPLC purifications were performed using an Applied Biosystems (Foster City, USA) configuration consisting of two Model 400 pumps, a 1000S Diode Array detector and a 491 Dynamic Mixer with a Rheodyne 7125 injector. Hard copy output utilised a Hewlett Packard ColorPro plotter (Rockville, USA). An Applied Biosystems Spheri-5 RP-18 column (220 x 4.6 mm, 5|im particle size) was used with a solvent flow rate of 1 ml/min. 5.2.1.2 ACID EXTRACT MOBILE PHASE The mobile phase conditions were as follows: solvent A (5% methanol in 0.5% aqueous acetic acid adjusted to pH 4 with 30% aqueous NH3) for 10 minutes, then a linear gradient to solvent B (methanol) over a period of 30 minutes. Signal output was monitored at a wavelength of 293 nm with a bandwidth of 5 nm. 5.2.1.3 SEP-PAK ELUENT MOBILE PHASE The mobile phase conditions were as follows: solvent A (0.5% aqueous acetic acid adjusted to pH 4 with 30% aqueous NH3) then a linear gradient to solvent B (methanol) over a period of 50 minutes. Signal output was monitored at a wavelength of 293 nm with a bandwidth of 5 nm. 5.2.2 MASS SPECTROMETRY Chemical ionisation GCMS was carried out on the Finnigan Model 3200 system under the conditions outlined in Materials 1.2.8. A 12 metre BP-5 vitreous- silica column (SGE Ringwood, Victoria, Australia) was used with helium carrier gas at a flow rate of 2 ml/min and methane reagent gas (20 ml/min). Oven 233 temperature was raised from 60°C to 280°C at 10°C per minute with the temperature program commenced 1.5 minutes after injection. 5.2.3 CHEMICALS AND SOLVENTS Suppliers and grades of the chemicals and solvents used have been outlined in previous sections (Materials Chapter 1 and 2). Diazomethane was prepared by the method outlined in Materials 1.2.4. 234 5.3 METHODS 5.3.1 FEED SOLUTIONS Three feeding solutions were prepared as outlined below: a) salicylic acid (20 mg) dissolved in 6 drops of ethanol (100%) and made up to 10 ml with water. b) synthetic salicyloylaspartate (20 mg, prepared as in Methods 3.3.1) treated in the same way as the salicylic acid solution. c) a control solution of 6 drops of ethanol (100%) in 10 ml of water. The use of ethanol was essential since salicylic acid is barely soluble in water. For consistency ethanol was added to each solution. 5.3.2 TOMATO FEEDING Mature tomato plants were used in this experiment. Stems were excised about 15 cm from the apical buds and three or four of these segments put into tubes containing the feed solutions. These explants were left in the solutions for two days with water added from time to time to maintain the volume at about 10 ml. After removal from the feed solution the stems were cut about two cm above the level of the solution and the plant material thoroughly washed with distilled water. The tomato leaves were then stripped from the stems and equal weights of each transferred to a mortar containing about 20 ml of acetone and 2 gm of acid washed sand. The leaf material was ground with the pestle and washed into a 250 ml flask with acetone (100 ml) and left standing overnight at room temperature. The acetone extract was evaporated to an aqueous residue. Solid NaHC03 (1 gm) was added to ensure neutrality, the residue extracted with two aliquots of ethyl acetate (50 ml) and these extracts discarded. The aqueous residue was then acidified to pH 2 with 2M H2SO4 and extracted with ethyl acetate (3 x 50 ml). 235 These ethyl acetate extracts were combined, evaporated and redissolved in 100 |il of methanol for HPLC analysis, methylation and GCMS. 5.3.3 PRE-TREATMENT OF THE AQUEOUS RESIDUE BEFORE REVERSE-PHASE SEP-PAK The aqueous residue from the salicylic acid treated plants was extracted three more times with ethyl acetate (50 ml) and the third extract evaporated and methylated for GCMS. This step was performed to ensure that the aqueous residue did not contain any free salicylic acid or SA. 5.3.4 SEP-PAK FRACTIONATION OF AQUEOUS RESIDUE After pre-treatment, the yellow aqueous residue from the SA treated plant extract was loaded into a 10 ml syringe and pushed through one reverse-phase Sep- Pak that had been previously pre-conditioned with 95% ethanol (5 ml) and then 0.2% aqueous acetic acid (5 ml). The eluent from the first Sep-Pak was pushed through another pre-conditioned Sep-Pak and this procedure repeated until the final eluent was colourless. Five Sep-Paks were needed. Each Sep-Pak was then washed with 20% ethanol in 0.2% aqueous acetic acid and these eluents combined, evaporated and redissolved in a small volume of methanol. A few mg of NaHCC>3 was added to this ethanol solution to prevent any acid catalysed esterification occurring. 5.3.5 HPLC 5.3.5.1 ACID EXTRACTS 10 ml of each extract (control, salicylic and salicyloylaspartate treated tomato leaf) was analysed by HPLC under the conditions outlined in Materials 5.2.1.1. 236 5.3.5.2 20% ETHANOL SEP-PAK ELUENTS One-tenth of the 20% ethanol Sep-Pak eluent from the aqueous residue after acid extraction of the SA treated plants was analysed by HPLC under the conditions outlined in Materials 5.2.1.2. 5.3.6 GCMS Another 10 ml of the ethyl acetate acid extracts from the normal and salicylic acid treated plants (Methods 5.3.2) was methylated with an excess of etherial diazomethane and analysed by GCMS under the conditions outlined in Materials 5.2.2. A small amount of synthetic SA was also methylated and analysed. 237 5.4 RESULTS 5.4.1 HPLC 5.4.1.1 ACID EXTRACTS The chromatograms of the control, salicylic acid and salicyloylaspartate treated tomato leaves are shown in Figure 5.4.1. In the area of interest (15 to 27 minutes) the chromatograms of the control and salicylic acid treated plants show very similar profiles apart from the intense peak eluting between 15 and 17 minutes, differences in the resolution of the peaks eluting 20.5 to 23 minutes and a small peak at a retention of 17.3 minutes which is a barely discernible shoulder in the control. The intense peak appearing in the extract from the salicylic acid treated plants has the same retention and UV spectrum as salicylic acid while the small peak (retention 17.3 minutes) has the same UV spectrum and retention time as synthetic salicyloylaspartate (Figure 5.4.2). Interestingly the early broad peaks which appear in the control but not the salicylic acid treated plants all have the characteristic caffeic acid chromaphore (see Figure 1.4.18). The chromatogram of the salicyloylaspartate treated plants (Figure 5.4.1) shows a greater proportion of these caffeic chromaphores, a large peak of salicyloylaspartate, salicylic acid and another peak (retention 20.8 minutes) which does not appear in either of the other two extracts. This latter peak corresponded in retention and UV spectrum to synthetic p-ethyl salicyloylaspartate. 5.4.1.2 20% ETHANOL SEP-PAK ELUENTS The chromatogram between retention times 19 and 29 minutes is shown in Figure 5.4.3. Peaks labelled A, B, C and D all have the chromophore of salicylic acid and SA, also shown in this figure. One of these peaks (A) was identified as SA by virtue of its coincidence in retention with synthetic SA and the fact that solid 238 -0.0001 0.0000 0.3200. Tine (Minutes) FIGURE 5.4.1 HPLC chromatograms of control (A), salicylic acid (B) and SA treated tomato explants (C) over the retention period 15 to 20 minutes. Monitor wavelength was 293 nm. Peaks with caffeic chromaphores are labelled CA, salicylic acid is labelled S and salicyloylaspartate SA. 239 of peak the of spectrum the to spectrum the identical to is A identical is B spectrum The spectrum (B). the while 5.4.1) 5.4.1). salicyloylaspartate Figure Figure and (A, C, (A) and mins acid (B 16.1 at salicylic minutes of 17.3 eluting at spectra peak UV eluting the s}{un aoueqjosqv 5.4.2 FIGURE 0.03481 To > FIGURE 5.4.3 HPLC chromatogram of the 20% ethanol Sep-Pak eluent from the purification of the aqueous residue after remo of neutrals and acid fractions (Methods 5.3.5.2). Peaks labelled A, B, C and D have a UV spectrum like SA. Peak T3 A was identified as SA by virtue of its UV spectrum and retention time coincidence with the synthetic compoun 240 241 probe Cl mass spectrum contained all the major ions of the synthetic compound (results not shown). The other peaks (B, C and D) were also collected and analysed by solid probe CLMS. However the spectra obtained were either very weak (suggesting that the compound was involatile) or not similar to any "salicylic" type compound encountered during this work. 5.4.2 GCMS Figure 5.4.4 shows the total ion current and the specific ion trace of m/z 135 and 296 for the methylated acid extracts for the control and salicylic acid fed plants respectively. The retention time and the ratio of the ions m/z 135 and 296 for methylated synthetic SA (not shown) are very similar to the peaks at scan #1564 and #1565 for the control and salicylic acid treated plant extracts so clearly these peaks can be identified as methylated SA. The ratio of peak areas in the salicylic acid treated and control plant extracts is approximately 1:7 so obviously feeding the tomato explants with salicylic acid has dramatically increased the SA content. 242 1564 6864 m/z 135 1 564 5157 m/z 296 1565 61261 m/z 135 RIC l ' I i i 1 1 1564 47860 m/z 296 RIC I T ^ 1 1 1 “I 1 540 1560 1580 1600 14:12 14:23 14:34 14:45 Scan Number Retention Time (min:sec) FIGURE 5.4.4 Specific ion scans of m/z 135 and 296 for the methylated acid extracts from control (top) and salicylic acid treated (bottom) tomato explants. Mass spectrometer was in positive ion chemical ionisation mode. Values above peaks represent the scan number corresponding to the peak apex and the integrated peak area. Note that the area of the protonated parent ion (m/z 296 of dimethyl SA) from the salicylic acid treated plants is about nine times greater than the control. 243 5.5 DISCUSSION 5.5.1 ROLE OF SALICYLIC ACID IN PLANTS The occurrence of the phenylpropanoic acids and the processes of metabolism, conjugation and enzymology have been studied comprehensively (see Introduction, Chapter 2) and these compounds and their conjugates are ubiquitous in the plant kingdom (Harbome, 1980). The central role of the phenylpropanoic acids in lignin biosynthesis has undoubtedly contributed to the great concentration of effort on this group of phenolic acids. Comparatively, the benzoic acid derivatives have received much less attention - perhaps a consequence of their more sporadic occurrence and the lower perceived importance to the overall metabolism of the plant. Salicylic acid is widespread in angiosperms and is a common constituent in Ericaceae (Harbome, 1980) but the conjugates of this benzoic acid derivative are scarce and confined mainly to Salix sp. Free salicylic acid has been implicated as a phytoalexin by various studies including one by Robertson et al. (1968) which showed that the concentration of this acid, along with p -hydroxy ben zoic and 4- hydroxy-3-methoxybenzoic acid, increased after fungal infection. The latter two acids, with four other unknown phenolic compounds were found to increase in quantity in apples after attack by Sclerotinia fructigena (Fawcett and Spencer, 1967). Related to this is the discovery by Lyons et al. (1990) that there is an accumulation of caffeoyl esters in the leaves of Zea mays after inoculation with a fungus. Chandramohan et al. (1973) found that the quantities of acids such as p - coumaric, p -hydroxybenzoic, vanillic acid and three other unidentified phenolics decreased in soil after application of nitrogenous fertilisers while plant growth increased dramatically. This indicated that these compounds may be involved in growth inhibition. Intuitively it could be expected that salicylic acid may have been one of the unknown compounds if the work of Debell (1969), as described in Muller and Chou (1972), is to be considered. Debell found that aqueous leachates 244 of the leaves of Quercus falcata contained two compounds, the most abundant of which was salicylic acid. This compound decreased dramatically in soil after cutting of the the trees. Concurrent with the loss of foliage and the decline in salicylic acid levels was greater re-growth of seedlings in the area. Salicylic acid is listed as a growth inhibitor by Takashaki (1986) but the overall scheme of growth regulatory abilities of this compound and other phenolic acids is not a straightforward one but rather a more complex picture involving synergistic and antagonistic effects. Harbome (1980) provides an example of stimulation and inhibition of ethylene biosynthesis by two related phenolic acids. One of these compounds, p -coumaric acid, was shown to be a co-factor for an enzyme involved in the biosynthesis of ethylene from methionine. The other, caffeic acid, was shown to inhibit the same enzyme. Another example showed that gibberellin activity was moderated by interaction with phenolic acids. 5.5.2 ASPARTATE CONJUGATION It is now accepted that free phenolics are rarely present in plant tissues due to the toxicity of these compounds and that conjugation with various groups affords a means of de-toxification (Harbome 1979). Conjugation with glucose is universal among plant phenolics but conjugation with amino acids is much rarer and seems to be mainly confined to endogenous and synthetic auxins. As early as the mid-fifties, Andreae and Good (1955) discovered that LAA is converted to IAA-aspartate when this hormone was fed to pea seedling epicotyl sections. Later Andreae and Good (1957) also showed that benzoic, indolebutyric, indoleproprionic, and 2, 4 dichlorophenoxyacetic acid form aspartic conjugates when the precursor acids are incubated with pea epicotyl sections. Formation of aspartic conjugates was later found to be stimulated by pre treatment with the natural auxin IAA and synthetic non-physiological auxin NAA. Sudi (1964) demonstrated that pre-treating pea tissue with IAA, NAA, 2, 4 D and 2, 3, 6 T increased the amount of IAA-aspartate and NAA-aspartate produced by 245 this tissue and later Sudi (1966) showed that pre-treatment with unlabelled IAA and NAA increased the amount of aspartate conjugates formed by a factor of six when 14C-labelled IAA and NAA were add 2 hrs after the pre-treatment. Pre-treatment with other acids which do not show any auxin effects and which form aspartate conjugates did not increase the rate of formation of these conjugates. Venus (1972) showed that pre-treatment of pea stem sections with IAA or NAA also promoted the formation of the aspartate esters of IAA, NAA and benzoic acid. Additionally it was also shown that pre-treatment with 2, 4, 5 trichlorophenoxyacetic acid increased the synthesis of benzoylaspartate when benzoic acid was added to pea stem sections. Interestingly, Venus also found that pre-treatment with non-auxin acids stimulated the formation of malate esters but did not stimulate the formation of aspartate esters. Aranda et al. (1984) expanded on the earlier findings with pea stem sections (that the major metabolite after NAA is the aspartate conjugate) to include tobacco mesophyll protoplasts. Other amino acids were also found to form conjugates with exogenously supplied 2, 4 D. Feung et al. (1973) found that alanine, valine, isoleucine, phenylalanine and tryptophan all form conjugates in soybean callus tissue culture. Later Feung et al. (1976) described the identification of a series of IAA conjugates with the amino acids glycine, alanine, valine and glutamate in tissue cultures of crown gall (Parthenocissus tricuspidata). Conjugation of IAA with aspartate and other amino acids is not a de activation pathway as these compound still retain activity in Avena coleoptile elongation assays. Feung et al. (1977) synthesised twenty L-amino acid conjugates of IAA and found that most retained auxin activity and some, notably the aspartate conjugate, had the same stimulatory activity as free IAA but a longer half-life. Inactivation of IAA occurs mainly by oxidation of the indole ring to ultimately form such compounds as 3-methyloxindole and indolealdehyde (see Morumo, 1986). The role of the amino acid conjugates in plants is still not understood although if a parallel could be drawn with myo -inositol-IAA then these compound 246 may be implicated in storage of IAA in seeds. Cohen and Bandurski (1982) contended that the evidence available at that stage supported the notion that this monosaccharide conjugate is transported to the seed and on germination hydrolytic enzymes cleave the conjugate to provide IAA for growth stimulation. IAA-aspartate has not been implicated in this process and indeed this compound has not yet been found as an indogenous component in seeds. However this lack of positive evidence does not preclude a similar role for the aspartate conjugate. Cohen and Bandurski (1978) and others did show that conjugates of IAA including IAA-aspartate were resistant to peroxidative oxidation and surmised that conjugation is a protection mechanism. To extend the findings and hypothesised role of aspartate conjugation in the metabolism of IAA to salicyloylaspartate obviously demands some caution. To this point there has not been any studies on the metabolism of this compound and there is one significant difference in the conjugation of IAA and salicylic acid to aspartate. The results in Chapters 2, 3, 4 indicate that the aspartate group is further conjugated to some unknown compound. This has not been reported for IAA- aspartate. However conjugation of phenolics to compounds which impart water solubility have been suspected to be part of a process leading to the ultimate transport and sequestration of this compound in vacuoles (see Rhodes, 1985 and Harbome, 1985). It is not unreasonable to surmise that this sequestration is a universal process considering the known functions of vacuoles. Matile (1978) contended that vacuoles commonly store hydrophilic compounds and provide pools of metabolites that serve as a buffer against environmental changes which would otherwise deplete cytoplasmic pools of these substances. The difficulty in the experimental localisation of phenolic compounds in vacuoles was outlined by McClure (1979) who also intoned that the weight of evidence supported the notion of sequestration of phenolics in vacuoles. Yamaki (1984) found that the isolated vacuoles from apples contained almost all of the phenolic compounds present 247 whereas cytoplasmic phenolics were negligible by comparison. Interestingly Strack and Sharma (1985) produced evidence to show that the synthesis of the malate conjugates of hydroxycinnamic acids occurs in the vacuoles of protoplasts from radish leaves. 5.5.3 SALICYLIC ACID METABOLISM IN TOMATO EXPLANTS The HPLC and GCMS results show clearly that the application of salicylic acid and S A to tomato explants has led to significant uptake of both compounds and some interconversion between these compounds. The explants have been shown to readily take up substantial amounts of salicylic acid and moreover these extracts contained more SA than the control extracts. Also the chromatograms of the acid extracts of the SA treated plants illustrated that SA is readily taken up and about 10% is converted to salicylic acid. A surprising result was the discovery of the (3-ethyl monoester of salicyloylaspartate in the acid extract from the SA treated explants. There was ethanol (about 3%) in the feed solution so it was likely that ethanol would exert some effect on the metabolism. There was no detectable levels of the a-monoester or the diethyl ester in the HPLC chromatogram of the acid extract. The lower proportion of the a-monoester was later confirmed by methylation of the acid extract followed by GCMS analysis (results not shown). It was found that the ion m/z 236 which arises exclusively from the a-monoethyl ester was less than 2% relative abundance compared to m/z 250 (exclusive to the (3-isomer, see Chapter 4) which had a relative abundance of 5%. Normalised to m/z 310, the ratio of these ions in a 1:1 mixture of monoethyl isomers is m/z 236:m/z 250 / 2:1 so the ratio of a- to P-monoethyl esters is about 1:5. This is a similar ratio to that of the synthetic monoester formation (1:7 / a:p, see Chapter 4) so acid catalysed ethylation is a likely cause - even though the pH of the feedant solution is not low (pH 5). Of course the occurrence of enzyme-mediated esterification is not excluded. Endogenous ethyl ester formation is extremely rare and ethyl chlorogenate is one of 248 the few reported exceptions. This compound was isolated by Abe and Marumo (1972) but its occurrence as an ethyl ester may be artifactual as ethanol was used as in the extraction medium. The pKa of this acid is 2.7, a much lower figure than the p-carboxyl of SA (pKa 4.4), a group that has been demonstrated to ethylate rather easily. However there is still likely to be some esterification during extraction and storage of these extracts if precautions are not taken to prevent this happening. Methyl esters are more common generally and one compound, an aspartyl ester of 4-chloro-IAA was reported by Hattori and Marumo (1972) to be an endogenous constituent of immature pea seeds. The methodology used in this work leaves some doubt about the actual composition of the aspartate conjugate. The unknown compound was first isolated, purified, methylated and identified as the dimethyl ester by comparison with a synthetic 4-chloro-IAA aspartate dimethyl ester. The existence as a monomethyl ester was deduced through the chromatographic behaviour of the compound before methylation. The authors contended that at pH 4.1 the compound behaved like a mono-carboxylic acid but this is likely to be the case with the free acid at that pH if the pKa of the a and p carboxyls are similar to those of salicyloylaspartate. At pH 4.1 the p-carboxyl of SA is about 70% protonated so would behave more like a mono-carboxylic acid in most chromatographic systems. One of the obvious differences in the physiological effects of feeding salicylic acid and SA to tomato explants was the state of the treated explants at the end of the feed period. The salicylic acid treated explants showed some characteristic signs of toxicity, particularly a loss of turgor and the brown discolouration of the leaves. Conversely the SA treated explants showed none of these traits and appeared to be no different from the control explants at the end of the feeding period. Hence the hydrolysis of SA (non-toxic) to salicylic acid (toxic) seemed unusual. Probably since the amount of SA taken up by the explants was so high a significant amount was hydrolysed by non-specific hydrolases present in the plant tissue. 249 The toxicity of salicyclic acid to the explants and the contrary lack of similar physiological effects after feeding with SA indicates that the formation of the aspartate derivative may be a de-toxification mechanism. The concentration of salicylic acid in the feed solution was very high but the explants were exposed to the same concentration of SA without obvious adverse effects - despite the conversion of some SA to salicylic acid. One other notable difference between the results obtained in this feeding experiment and previous attempts to quantify SA was the greater amounts of the conjugate in the control explants. In previous attempts to isolate SA from plant tissue, the amount of the endogenous compound was insufficient to be detectable by GCMS after methylation. These extracts, made from young plants (Chapter 3), had to be assayed by formation of pentafluorobenzyl derivatives followed by analysis by negative ion CLMS, a very sensitive method. The difference in age of the tomato plants was significant and this is not without precedence for aspartate conjugates. Davidonis et al. (1978) first demonstrated that the metabolism of 2, 4 D after application to soybean callus tissue changed dependent on the age of the culture. Older tissue produced greater amounts of the aspartate conjugate. Davidonis et al. (1980) later found that in young tissue the amount of free 2,4 D increased proportionately to the concentration of 2, 4 D whereas in older tissue the aspartate conjugate increased along with the exogenously supplied herbicide. Other workers have shown that there is a correlation between age and the distribution of conjugates. Linschield et al. (1980) found that during the development of radish cotyledons the drop in sinapoylglucose is matched by a rise in the concentration of sinapoylmalate. Strack et al., (1985) have also shown that the quantity of feruloyl, coumaroyl and caffeoyltartronate rises in developing mung bean leaves. One interesting result was the suppression of caffeic acid conjugates after feeding with salicylic acid. Probably the plants cells have redirected resources 250 toward de-toxifying the salicylic acid by hydrolysis of the conjugating moieties (eg malate, glucose). These compounds could then be used to esterify with the salicylic acid. 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