Plant Growth Substances - ACS Publications - American Chemical

2 Aspects of G i b b e r e l l i n Chemistry

PETER HEDDEN

Downloaded by STANFORD UNIV GREEN LIBR on September 25, 2012 | http://pubs.acs.org Publication Date: September 27, 1979 | doi: 10.1021/bk-1979-0111.ch002

Department of Biology, University of California, Los Angeles, CA 90024

Since their discovery as secondary metabolites of the phytopathogenic fungus, Gibberella fujikuroi, gibberellins (GAs) have been identified from numerous species of angiosperms and some gymnosperms. In addition there are many more reports of GA-like substances (detected by biological assays) occurring in species of both groups. It seems probable that GAs are ubiquitous in seed plants. There are also reports of GA-like substances occurring in lower organisms, including fungi other than G. fujikuroi, algae and bacteria, but none of these have been conclusively identified as GAs. Gibberellins elicit a variety of physiological responses in seed plants and are well established as hormones controlling plant growth and development. Gibberellic acid (GA3) is used extensively in agriculture, and is produced commercially from large scale cultures of G. fujikuroi. Other GAs have been found to have specific agricultural applications where they are more effective than GA3. There is therefore interest in methods for producing GAs, other than GA3, in commercially useful quantities. GAs are also required for research purposes, both for testing their biological activity and as standards for GA identification and quantitation. In most cases it is impractical to extract sufficient quantities of GAs from their plant sources and they must be prepared chemically or microbiologically from more-accessible compounds. T h i s review discusses aspects of GA chemistry which may be u s e f u l to p l a n t p h y s i o l o g i s t s or b i o c h e m i s t s . I t covers GA i d e n t i f i c a t i o n and q u a n t i t a t i o n , p a r t i c u l a r l y d i f f i c u l t tasks c o n s i d e r i n g the low l e v e l s of GA i n p l a n t t i s s u e s . However the problems o f GA a n a l y s i s have been c o n s i d e r a b l y a l l e v i a t e d by recent technology, p a r t i c u l a r l y combined gas chromatography-mass spectrometry. A l s o d e s c r i b e d are methods f o r p r e p a r i n g l e s s a c c e s s i b l e GAs and f o r i s o t o p i c a l l y l a b e l i n g GAs f o r metabolism s t u d i e s . F i n a l l y some c o n s i d e r a t i o n i s given to s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s . Such c o r r e l a t i o n s may shed l i g h t on the mechanism o f a c t i o n of GAs at the molecular l e v e l and

0-8412-0518-3/79/47-lll-019$09.50/0 © 1979 American Chemical Society

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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P L A N T G R O W T H SUBSTANCES

suggest how the GA s t r u c t u r e might be m o d i f i e d t o produce com pounds w i t h enhanced b i o - a c t i v i t y or s p e c i a l i z e d a p p l i c a t i o n .


Structure The g i b b e r e l l i n s (GAs) are a c l a s s o f t e t r a c y c l i c d i t e r penoid a c i d s o f which 53 members (Figure 1) have been i d e n t i f i e d from higher p l a n t s or the fungus, G i b b e r e l l a f u j i k u r o i . For con venience, each f u l l y - c h a r a c t e r i z e d , n a t u r a l l y - o c c u r r i n g GA i s a l l o c a t e d a number ( 1 ) , thus the GAs are r e f e r r e d to as GAi GA53. S t r u c t u r a l l y , the GAs can be subdivided i n t o two groups, the C20 Ο19 GAs. The C20 GAs c o n t a i n the e n t - g i b b e r e l l a n e s k e l e t o n as i s t y p i f i e d by the simplest member o f t h i s group, GA12 (Figure 2 ) . The C19 GAs have an ent-20-norgibberellane s k e l e t o n i n which carbon-20 has been r e p l a c e d by a hydroxyl group. With one exception, the C19 GAs c o n t a i n a 19*10 γ-lactone, as, f o r example, i n GAo (Figure 2 ) . W i t h i n these two groups the GAs d i f f e r mainly i n the degree and p o s i t i o n o f o x i d a t i o n o f the b a s i c s k e l e t o n . The C19 GAs are d e r i v e d b i o s y n t h e t i c a l l y from the C20 GAs by an as y e t unknown mechanism. C20 GAs are found n a t u r a l l y having carbon-20 at each p o s s i b l e o x i d a t i o n l e v e l and Cô, GA b i o s y n t h e s i s may i n v o l v e successive o x i d a t i o n o f t h i s carbon atom. GAs w i t h an a l c o h o l f u n c t i o n at carbon-20 are i s o l a t e d as the 19,20 δ-lactones i n which carbon-20 i s prevented from f u r t h e r o x i d a t i o n (2). I t i s l i k e l y that t h i s l a c t o n e i s formed during i s o l a t i o n and t h a t these GAs occur as f r e e a l c o h o l s i n v i v o . Those GAs w i t h C-20 at the aldehyde o x i d a t i o n l e v e l appear t o e x i s t as the 19>20 l a c t o l s i n the s o l i d s t a t e and as an e q u i l i brium mixture o f l a c t o l and aldehyde i n s o l u t i o n ( 3 ) . The C20 and C19 GA r i n g systems can be hydroxylated at a number of p o s i t i o n s , 2β, 33 and 13 hydroxyled GAs being encountered most f r e q u e n t l y . Hydration o f the 16,17 double bond i s observed i n G. f u j i k u r o i t o g i v e the s a t u r a t e d C-l6 a l c o h o l . Other l e s s frequent f u n c t i o n a l i t i e s i n c l u d e a double bond at e i t h e r the 1,2 or 2,3 p o s i t i o n s i n C]_a, GAs; ketone and epoxide f u n c t i o n s . a n ( i

Stability The g e n e r a l chemistry of the GAs has been reviewed (h). Many o f the GAs c o n t a i n a h i g h c o n c e n t r a t i o n o f f u n c t i o n a l groups rendering them s u s c e p t i b l e t o rearrangement and degradation. Therefore, as a g e n e r a l r u l e high temperatures and extremes o f pH should be avoided when working w i t h them. In m i n e r a l a c i d 13-hydroxy GAs undergo a Wagner-Merwein rearrangement o f the c/D r i n g system (Figure 3) . When the 13-hydroxyl group i s absent the 16,17 double bond may be isomerized by a c i d t o the e n d o c y c l i c 15,16 p o s i t i o n or may be hydrated to g i v e the s a t u r a t e d l 6 a l c o h o l . Four GAs w i t h the l 6 - h y d r o x y l group (GA2, GAô, GAuJL, GA^) have been i d e n t i f i e d from G• f u j i k u r o i (6,7,8). Since the fungus i s u s u a l l y grown at a c i d i c pH, these GAs c o u l d be



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Gibberellin Chemistry

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the r e s u l t o f non-enzymatic h y d r a t i o n . GAs such as GA^ o r GA^ w i t h o n l y a 33-hydroxyl group i n t h e A r i n g a r e s e n s i t i v e t o d i l u t e aqueous a l k a l i , underoing epimeri z a t i o n a t thfe 3 p o s i t i o n t o g i v e a mixture o f epimers. A r e t r o a l d o l mechanism has been proposed f o r t h i s e p i m e r i z a t i o n ( F i g ure k) ( 9 ) , a mechanism supported b y the f i n d i n g t h a t t h e 3orhydrogen i s r e t a i n e d i n t h e rearrangement (10). The epimerizat i o n does not occur i f there i s a l s o a 2-hydroxyl group o r a 1,2 double bond i n the A r i n g . In t h e l a t t e r case there i s a s h i f t o f t h e 1,2 double bond t o the 1,10 p o s i t i o n and t h e formation o f a 19,2 l a c t o n e . T h i s i s o m e r i z a t i o n i s r a t h e r f a c i l e and can occur during gas chromatography o f GAs, such as GA3 or GAy, r e s u l t i n g i n broad double peaks. Many GAs i n aqueous s o l u t i o n a r e s l o w l y degraded, the process b e i n g a c c e l e r a t e d a t h i g h e r temperatures a s , f o r i n s t a n c e , during a u t o c l a v i n g . A f t e r h e a t i n g aqueous s o l u t i o n s o f GA3 i n an autoclave a t 120° f o r 20 minutes o n l y 1-2$ GA3 remains (11). The chemical processes i n v o l v e d i n t h i s degradation o f GA3 have been s t u d i e d i n some d e t a i l (12). The proposed pathway o f decomposition i s shown i n F i g u r e 5. The major products a r e i s o g i b b e r e l l i c a c i d , g i b b e r e l l e n i c a c i d , a l l o g i b b e r i c a c i d , 9-epia l l o g i b b e r i c a c i d and 9,11-didehydroallogibberic a c i d . The l a s t compound i s formed from g i b b e r e l l e n i c a c i d v i a a proposed t r i e n e intermediate by an o x i d a t i o n which appears t o i n v o l v e hydroperoxide i n t e r m e d i a t e s . Q u a l i t a t i v e and Q u a n t i t a t i v e A n a l y s i s E x t r a c t i o n and P u r i f i c a t i o n . Many methods have been used t o e x t r a c t and p u r i f y GAs from p l a n t m a t e r i a l , the procedure o f t e n depending on the t i s s u e s b e i n g e x t r a c t e d . Graebe and Ropers (13) i n t h e i r review on GAs have c r i t i c a l l y d i s c u s s e d e x t r a c t i o n and p u r i f i c a t i o n techniques. The c o n c e n t r a t i o n o f GAs i n h i g h e r p l a n t t i s s u e s v a r i e s from about 10 p,g per g f r e s h weight i n seeds o f c e r t a i n species t o l e s s than 1 ng p e r g f r e s h weight i n v e g e t a t i v e t i s s u e s . The extent o f p u r i f i c a t i o n r e q u i r e d w i l l depend on t h e p a r t i c u l a r p l a n t t i s s u e under i n v e s t i g a t i o n . T y p i c a l l y the m a t e r i a l i s homogenized i n a watermethanol mixture (about 75$ methanol) a t low temperature. Acetone has been used as the organic solvent but can cause problems due t o t h e formation o f acetonides w i t h v i c i n a l d i o l s i n s l i g h t l y a c i d c o n d i t i o n s (1^*15) · A f t e r t h e aqueous methanol e x t r a c t i o n , t h e homogenate i s f i l t e r e d and the methanol removed from the f i l t r a t e under reduced pressure a t h0° o r below. A t t h i s stage i t i s common t o b u f f e r t h e aqueous r e s i d u e , u s u a l l y w i t h potassium phosphate. With some t i s s u e s , f o r example l i q u i d endosperm, i t i s convenient t o e x t r a c t d i r e c t l y with a b u f f e r s o l u t i o n a t about pH 8 r e s u l t i n g i n a c l e a n e r e x t r a c t ( l 6 ) . The b u f f e r e d aqueous e x t r a c t i s a d j u s t e d t o pH 8 and n e u t r a l and b a s i c compounds a r e e x t r a c t e d w i t h an organic s o l v e n t ,



22 PLANT GROWTH


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2. HEDDEN

Gibbereïlin Chemistry


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24 PLANT GROWTH SUBSTANCES



2. HEDDEN Gibberellin Chemistry


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2. HEDDEN Gibberellin Chemistry 27

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Figure 2. Structures of GA (a C o GA) and GA (a C GA) possessing the ent-gibberellane and ent-20-norgibberellane skeletons, respectively. The numbering system and ring designations are shown also. 12

2

9

19



HEDDEN


Figure 3. Acid-catalyzed Wagner-Merwein rearrangement of the C/D ring 13-hydroxy GAs (5)



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Figure 4.

SUBSTANCES

Proposed retro-aldol mechanism for the hase-catalyzed epimerization of ^-hydroxy GA's (9)



HEDDEN


9,11-didehydroollogibberic acid

Figure 5.

9 / ? - H ; allogibberic acid 9 a - H ; 9-£/?/-allogibberic acid

Proposed pathway for the decomposition of GA in aqueous solution (12) 3



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u s u a l l y e t h y l a c e t a t e . Some o f the l e a s t p o l a r GAs, p a r t i c u l a r l y GA9 and GAi2> and GA-glucosyl e s t e r s are a l s o e x t r a c t e d i n t h i s f r a c t i o n . Most GAs a r e e x t r a c t e d i n t o e t h y l a c e t a t e a f t e r adjustment o f the pH t o 3.0 w i t h h y d r o c h l o r i c a c i d * Very p o l a r GAs and GA-glucosides can be e x t r a c t e d w i t h n-butanol. It i s important t h a t the a c i d i c e t h y l a c e t a t e and b u t a n o l e x t r a c t s be washed w i t h water before being concentrated t o dryness. Otherwise t r a c e s o f a c i d (phosphoric a c i d i f phosphate b u f f e r was used) w i l l be concentrated l e a d i n g t o rearrangement o r h y d r a t i o n o f any GAs present. Many methods have been used f o r a d d i t i o n a l p u r i f i c a t i o n o f the a c i d i c e x t r a c t s . I f the weight o f the e x t r a c t i s not too l a r g e then t h i n - l a y e r chromatography, e i t h e r a d s o r p t i o n chromatography on s i l i c a g e l o r p a r t i t i o n chromatography on k i e s e l g u h r , i s most convenient, although the s e p a r a t i o n obtained by t h i s method i s poor. B e t t e r r e s o l u t i o n has been obtained u s i n g part i t i o n chromatography on columns o f s i l i c i c a c i d Q^>l8>19) o r sephadex (17,20). R e c e n t l y high performance l i q u i d chromatography has been used w i t h GAs and gives e x c e l l e n t s e p a r a t i o n (21). The disadvantage o f chromatographic methods i s t h a t they separate the GAs from each other as w e l l as from other components i n the e x t r a c t . Thus numerous f r a c t i o n s are generated, each o f which has t o be analyzed s e p a r a t e l y f o r GAs. The most s a t i s f a c t o r y method a v a i l a b l e f o r i d e n t i f y i n g microgram o r l e s s q u a n t i t i e s o f GAs i n a complex mixture i s combined gas chromâtographymass spectrometry (GC-MS), a technique which i t s e l f contains a s e p a r a t i o n s t e p . T h e r e f o r e , i d e a l l y , p u r i f i c a t i o n methods p r i o r to GC-MS a n a l y s i s should separate the GAs as a group from other components* Short columns o f c h a r e o a l - c e l i t e (22), g e l f i l t r a t i o n chromatography on sephadex (23), and anion-exchange chromatography (2*+,25) have been used f o r t h i s purpose. I n s o l u b l e PVP i s o f t e n used t o remove phenolic compounds (26) and can be added t o the aqueous e x t r a c t b e f o r e p a r t i t i o n a g a i n s t organic s o l v e n t . The use o f a f f i n i t y chromatography i n which a n t i bodies s p e c i f i c t o GAo were bound t o sepharose has been i n v e s t i g a t e d (27) and c o u l d provide a r a p i d method f o r p u r i f y i n g GAs i f a n t i b o d i e s t o a l l GAs c o u l d be developed. G i b b e r e l l i n g l u c o s y l ethers and e s t e r s are d i f f i c u l t t o analyze by GC-MS although they can be gas chromatographed as t r l m e t h y l s i l y l (TMS ) ethers (28,29). The conjugates a r e g e n e r a l l y hydrolyzed e n z y m a t i c a l l y i n the crude e x t r a c t and the f r e e GAs subsequently p u r i f i e d and analyzed* Commercial c e l l u l a s e (30) o r p e c t i n a s e (31) have been used f o r the enzyme h y d r o l y s i s w i t h v a r y i n g success. A c i d o r base h y d r o l y s i s i s a l s o p o s s i b l e but may l e a d t o rearrangement o f the GAs. This complicates t h e i d e n t i f i c a t i o n u n l e s s the rearranged products f o r each GA are a v a i l a b l e f o r comparison (Ik). I d e n t i f i c a t i o n . Combined GC-MS has advanced p l a n t hormone r e s e a r c h g r e a t l y i n recent years and w i t h the i n t r o d u c t i o n o f



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computerized systems (32) the s e n s i t i v i t y and v e r s a t i l i t y o f t h i s technique have been i n c r e a s e d s t i l l f u r t h e r . The g e n e r a l methodology f o r GA a n a l y s i s by GC-MS has been reviewed r e c e n t l y (33, 3*0 « The v o l a t i l i t y o f GAs i s i n c r e a s e d p r i o r t o GC by forming the methyl e s t e r s w i t h diazomethane. Hydroxylated GAs are o f t e n converted t o t r i m e t h y l s i l y l (TMS) ethers a f t e r m e t h y l a t i o n . The mass s p e c t r a o f GA methyl e s t e r s TMS ethers f r e q u e n t l y c o n t a i n i n t e n s e molecular ions and c h a r a c t e r i s t i c fragmentation p a t t e r n s , which are e a s i e r t o i n t e r p r e t than those o f the f r e e hydroxy compounds. When recovery o f GAs i s r e q u i r e d a f t e r GC, GA TMS ether e s t e r s are a convenient d e r i v a t i v e s i n c e the f r e e GA can e a s i l y be recovered a f t e r h y d r o l y s i s i n water. Combined GC-MS-computer systems w i t h r e p e t i t i v e scanning can l e a d t o the i d e n t i f i c a t i o n o f GAs as minor components o f complex e x t r a c t s at l e v e l s down t o ÎCT- - g. In such cases mass fragmentograms can be c o n s t r u c t e d i n which the d i s t r i b u t i o n o f ions o f p a r t i c u l a r m/e values are p l o t t e d throughout a GC-MS r u n . Thus i f the presence o f a p a r t i c u l a r GA i s suspected, charact e r i s t i c ions i n the mass spectrum o f the d e r i v a t i z e d GA are p l o t t e d . An i d e n t i f i c a t i o n can be made i f the ions peak at the same r e t e n t i o n time as the GA and have the same r e l a t i v e i n t e n s i t y as i n the mass spectrum o f the a u t h e n t i c compound (see F i g ure 6 ) . In t h i s way GAs can be detected which are masked i n the GC t r a c e by other compounds o f s i m i l a r r e t e n t i o n time ( c f . 33). In order f o r an i d e n t i f i c a t i o n t o be made from mass fragmentograms s u f f i c i e n t ions (at l e a s t s i x ) must be scanned. I t i s sometimes p o s s i b l e t o o b t a i n a f u l l , interprétable mass spectrum i n such cases by background s u b t r a c t i o n . The scan (spectrum) i n which the ions due t o the compound o f i n t e r e s t are at a maximum i s determined from the mass fragmentograms. T h i s spectrum i s then "cleaned up" by s u b t r a c t i o n o f those ions c o n t r i b u t e d by the contaminant. I t i s a l s o p o s s i b l e t o compare the backgrounds u b t r a c t e d spectrum d i r e c t l y w i t h a u t h e n t i c s p e c t r a s t o r e d i n the instrument. The s e n s i t i v i t y o f GC-MS can be i n c r e a s e d s t i l l f u r t h e r by s e l e c t i v e i o n c u r r e n t m o n i t o r i n g (SICM) whereby o n l y a l i m i t e d number o f c h a r a c t e r i s t i c ions i n the mass spectrum o f the compound are monitored. Therefore the time f o r which each i o n i s monitored, and hence the s e n s i t i v i t y , i s i n c r e a s e d so that the amount o f an i d e n t i f i a b l e GA i s reduced t o Î C T ^ g. As i n mass fragmentometry, s u f f i c i e n t ions must be monitored f o r an i d e n t i f i c a t i o n t o be made. 1

1

1

Q u a n t i t a t i o n . Combined GC-SICM has been used mainly f o r q u a n t i t a t i o n . For a p a r t i c u l a r GA the absolute i n t e n s i t y o f a c h a r a c t e r i s t i c i o n i n i t s mass spectrum i s r e l a t e d t o the amount o f GA p r e s e n t , u s i n g standards t o c a l i b r a t e the instrument. Frydman et a l . (35) used t h i s " e x t e r n a l standard method" t o measure the l e v e l s o f a number o f GAs throughout the development o f pea seeds. An a l t e r n a t i v e and p r e f e r a b l e approach employs



34 PLANT GROWTH SUBSTANCES


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Figure 6. Mass fragmentograms of the acidic fraction from an extract of Marah macrocarpus endosperm. Ions in the mass spectrum of GA Me TMS (shown above) were examined. The extract was run as the Me TMS derivative on a MS 902 spectrometer coupled to a Varian 2700 GC via a membrane separator. GCMS conditions—3% OV-17 on 100-120-mesh Gas Chrom Q in a 2 m X 0.2 cm i.d. column. Temperature—200°C for 5 min; then programmed at 4°C/min. Heliumflow—16.5cm /min. Electron energy—70 eV; accelerator potential— 2.9 KV; separator temperature—226°C; source temperature—250°C. 4

3


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PLANT GROWTH

SUBSTANCES

the use of an internal standard such as an isotopicallylabeled analog of the GA being quantitated. A known amount of the standard i s added to the plant extract at any early stage of the purification procedure so that account i s taken for losses, which can be quite considerable. The most conveniently prepared standards are deuterated GAs (see l a t e r ) . The natural and deuterated GA have almost the same GLC retention time and the group of ions i n the region of the molecular ion (M") of the natural GA are monitored throughout the mass peak (see Figure 7 ) . The relative intensities of the ions at m/e M and M + X, where X i s the number of deuterium atoms i n the standard, are calculated and, after correction for natural heavy isotopes i n the M + X ions, the relative amounts of the natural and deuterated GAs are determined. Sponsel and MacMillan (36,25) have i l l u s t r a t e d the use of GAs labeled with deuterium and tritium to quantitate GAs and also to follow their metabolism. In one experiment (25) they injected a measured amount of [ H][3H]GA29 (both species labeled at the same position) into immature pea seeds. Some seeds were extracted immediately and others at regular time intervals thereafter so that the metabolism of GA29 could be studied. The [3H] label was present to determine now much of the added GA29 remained unmetabolized after a particular time. Then by comparison of the relative amounts of the natural and deuterated GA29 mass spectrometry, the amount of endogenous GA29 was calculated. Using this method they also compared the rates of metabolism of exogenous and endogenous GA29.


4

2

Structure determination for new GAs. Mass spectrometry i s a useful tool for identifying GAs whose structures have been previously determined, i n which case comparison of mass spectra is s u f f i c i e n t . In contrast, the characterization of GAs of unknown structure is a much more d i f f i c u l t and time-consuming task. In these cases mass spectrometry can give information such as molecular weight and some indications of structure. For instance i n the mass spectra of the methyl esters TMS ethers a strong ion at m/e 129 indicates that the A ring contains a single hydroxyl group at the 1 or 3 positions. 13-Hydroxy GAs produce ions at m/e 207/208 and v i c i n a l alcohol functions, such as i n GA8, result i n a strong ion at m/e lk7 (37). If the structure of a GA can be inferred from i t s mass spectrum the suspected compound may be synthesized and i t s mass spectrum compared with that of the unknown. Thus the structures of GAJ4.6 and G A I ^ Y were confirmed by the p a r t i a l synthesis of their methyl esters from GA1+ (38). This synthesis w i l l be discussed i n detail l a t e r . GAl^ was identified i n immature seeds of Pyrus communis (pear) i n an analogous manner by comparison of i t s mass spectrum with that of a product obtained from incubating ent-15or-hydroxykaurenoic acid with a mutant of the fungus G. fujikuroi (29).



2.

HEDDEN


6A

2 0

PEA SEED E X T WITH H - G A METMS

METMS H

M/E 418 419 420 421 422

37

INTY 100,0 31.5 8.4 1.7 0.2

G A

2" 20

M/E 418 419 420 421 422

2

M E T M S

2

INTY 1.2 17.5 100.0 30.8 8.2

M/E 418 419 420 421 422

2 0

INTY 30.3 25.2 100.0 31.3 10.1

Plant Growth Regulator Working Group Figure 7. An example of the use of deuterated GA's as internal standards for the quantitation of GAs in plant extracts. [2a- H ]GA was added to an extract of young pea seeds in order to quantitate GA (cf. 25). Ions in the region of the molecular ion were scanned. The acidic fraction from the pea seed extract was run as the Me TMS derivative on a MS 30 coupled to a Pye 104 GC via a singlestage silicone membrane separator (34). 2

1

29

29


PLANT GROWTH

38

SUBSTANCES

Where i t has been possible to obtain sufficient material, GAs of previously unknown structure have been f u l l y characterized and their structure determined by a combination of chemical and spectroscopic methods. Proton Nuclear Magnetic Resonance (NMR) spectroscopy provides a great deal of structural information (k). 1 3 NMR promises to be a very powerful technique for both structure determination and metabolism studies of GAs . Yamaguchi et a l . (h2) used a combination of proton and 1 3 c NMR to determine the structure of G A ^ Q (2 by formation of the toluene-psulfonate and treatment of this with b o i l i n g collidine ÇJ+2). Thus the structures of C19 GAs are ultimately related to that of G A q , whose structure was o r i g i n a l l y determined by degradation (tf) and absolute stereochemistry has been confirmed by X-ray diffraction ( ^ > ^ , ^ U 8 ) . Recently the t o t a l synthesis of GA3 has been completed by Corey and co-workers (kg). The structures of C20 GAs were related ultimately to entkaurene v i a 7P-hydroxykaurenolide ( 5 0 ) . However, the two classes of GAs have now been related directly by the oxidative decarboxylation of GA^ to give GA^ ( 5 1 * 5 2 ) · Bearder and MacMillan ( 5 1 ) treated GA^^ with lead tetra-acetate to obtain a mixture of GAlj. and the isomeric 2 0 , 4 lactone (Figure 8 ) . Murofushi et a l . ( 5 2 ) employing a more lengthy procedure, decarboxylated the dimethyl ester of GAv* with lead tetra-acetate and lactonized the resulting o l e f i n with iodine (Figure 8 ) . The t o t a l syntheses of GA15 ( 5 3 ) and GAj_2 ( 5 ^ > 5 5 ) have also been reported.


C

The Preparation of Less-readily Available

GAs

The isolation of significant quantities of many of the less-accessible GAs from plant tissues i s usually impractical. GAs are required as standards, both for qualitative and quantitative analysis, as substrates for metabolism studies (often isotopically labeled) and for b i o l o g i c a l assays. The most practical methods for preparing these compounds are the chemic a l or b i o l o g i c a l conversion to the more available GAs or entkaurenoids. Gibberella fujikuroi produces a number of GAs ( 5 6 ) , of which GA3, GAip GA7, GA13 and GA^ can be obtained i n r e l a t i v e l y large amounts. These fungal GAs are the starting point for the p a r t i a l synthesis of less-accessible GAs by r e l a t i v e l y simple chemical procedures. Also microbiological methods have been developed to convert GAs and GA-precursors (or analogs of these) of both fungal and higher plant origin to useful products.



HEDDEN

GA

13


dimethyl ester

Figure 8.

Methods for the oxidative decarboxylation of GA

13

to GA (51,52)


h


PLANT GROWTH SUBSTANCES



Figure 9. 3h

h0

7

51

Chemical methods for the partial syntheses of the methyl esters of GA GA , GAj , and GA from GA 4 (38,42)


42

PLANT GROWTH

SUBSTANCES

Chemical Methods. As an example, Figure 9 shows a scheme for the preparation of four 2-hydroxy GAs from GA^. Two of the products are less abundant fungal GAs (GA^Q and GAI4.7) and the others (GAo^ and G A 5 1 ) occur i n higher plants. The preparation of the methyl esters of GAI±Q GA^y and GAolj. was described by Beeley and MacMillan ( 3 8 ) and the conversion of G A ^ Q to GAi^ by Yamaguchi et a l . (k2). The starting point was a mixture of GAJ4. and G A y , GAs which are not easily separated. This mixture was treated with OsOlj. and NalO^ to convert GA^ to the 1 7 - n o r - l 6 ketone which is easily separated from the G A y product i n which the 1,2-double bond i s also oxidized. The exocyclic double bond is thus protected as the norketone and can be restored later by the Wittig reaction. This reaction also gives an opportunity to introduce a label at the 17 position (see l a t e r ) . To prevent complications due to the presence of a free carboxylic acid group, the GA^ norketone was methylated and subsequent reactions were carried out on the methyl esters. Demethylation i s achieved for the 3-deoxy products by methanolic NaOH treatment (57) and for the 3-hydroxy GAs using the method described by Nagata et a l . (53)· The reduction of GA^Q-ketone with NaBHlj. gives a mixture of the 2or (GA^Q) and 20 ( G A c ^ ) alcohols which can be separated by preparative TLC (k2)• A similar series of reactions has been used ( 3 8 ) to convert the C 2 0 GA, GAô, to GAJ4.3 and GAj^g, both of which occur i n the Cucurbitaceae ( 5 8 ) .


9

The synthesis of GA-60-aldehydes i s of particular interest since GA^-aldehyde i s the immédiate product of ring Β contrac tion i n GA biosynthesis ( 5 9 ) . GA]_2-aldehyde also appears to be a substrate for hydroxylation i n G . fujikuroi and at least one higher plant ( 6 θ ) . GA^-aldehyde was f i r s t synthesized from 70-hydroxykaurenolide, a metabolite of G . f u j i k u r o i , by treating the 7ff-toluene-p-sulfonate with KOH i n methanol ( 6 1 ) . The y i e l d was poor and the methyl ester was obtained as product. The y i e l d has been improved using t-butanol as solvent (59) with a small amount of water ( 6 2 ) • S t i l l higher yields were obtained with p-bromobenzenesulfonate as the leaving group ( 6 3 ) . The aldehydes of GA]_lj. and GA53 have also been prepared from 3$ 7B- and 7 3 , 1 3 dihydroxykaurenolides, respectively, using the same basic method ( 6 4 , 6 5 ) . The proposed mechanism for ring contraction of the kaurenolide i s shown i n Figure 1 0 ; the reaction requires an antiperiplanar relationship for the migrating 5>6-bond and the leaving group. Thus the 73-alcohol of the kaurenolide must be epimerized to the or position before formation of the sulfonate ester. The i n i t i a l product i s the 6cy-aldehyde but epimerization occurs under the basic conditions of the reaction to give the thermodynamically-favored 6g-aldehyde. The γ-lactone i s not essential for the reaction and the opened hydroxy acid w i l l also undergo ring contraction on treatment with base (collidine or sodium hydride). The reaction involves abstraction of the 6-hydroxy proton but requires the free 19-oic acid. It was proposed that the proton was abstracted from the s t e r i c a l l y 9



HEDDEN


Figure 10. The conversion of 7β-hydroxykaurenolide to GA -aldehyde showing the proposed mechanism of the reaction (59,). Τs = toluene-p-sulfonyl. 12


PLANT

44

GROWTH

SUBSTANCES


hindered 6OR-hydroxy group by internal attack of the carboxyl anion ( 6 2 ). Node et a l . ( 6 6 ) i n a detailed investigation of the mechanism of ring contraction concluded that opening of the lactone precedes bond migration so the reaction i s not concerted. Microbiological Methods. The low substrate s p e c i f i c i t y of many of the enzymes involved in GA biosynthesis i n Gibberella fu j ikuroi has been u t i l i z e d for the preparation of higher plant GAs. Suitable analogs of the natural GA-precursors are converted by the fungus to the corresponding GA analogs. It i s usual to prevent the synthesis of the natural GAs i n order to f a c i l i t a t e purification of the unnatural products. A mutant strain, B l - 4 L A , in which GA biosynthesis i s blocked early i n the pathway ( 6 7 ) (between ent-kaurenal and ent-kaurenoic acid) has been used. It i s also possible to block GA synthesis chemically using i n hibitors such as AMO-L6L8 ( 6 8 ) , or the quaternary ammonium i o dide compound i n Figure 1 1 X & 9 , 7 0 ) , which block ent-kaurene formation. Steviol, the 13-hydroxy analog of ent-kaurenoic acid, occurs naturally as the glucoside, stevioside, i n leaves of the shrub, Stevia rebaudiana. Steviol i s converted by B l - 4 L A to a number of 13-hydroxylated GAs (71). Although 13-hydroxylation i s a normal process i n fungal GA biosynthesis, i t i s the f i n a l step i n the pathway so that the end product, G A 3 , i s normally the only 13-hydroxy GA formed i n large amounts. When steviol i s incubated with B l - U l a the major products are GA]_ (equivalent to the natural fungal metabolite, GA^) and GAj£ (equivalent to GAi^). Other GAs such as G A 5 3 , GAô,, and G A 2 0 are also produced. There i s no GA3 because the presence of a 13-hydroxy group i n hibits formation of the 1 , 2 double bond. When steviol acetate was fed the major GA-products were the acetates of G A 2 0 i G A 1 7 (72) since the presence of a 13-acetoxyl group prevents 33-hydroxylation. Thus i t was possible to predetermine which products were obtained. Relative yields of products could also be manipulated by changing the concentration of the substrate. The B l - Û l a mutant was found also to metabolize ent-ljpor-hydroxykaurenoic acid to a number of 150-hydroxylated GAs (J.R. Bearder & K. Kybird, unpublished information), one of which, 5B-hydroxy GA9, was subsequently identified as a new GA, GAj^.5, i n seeds of Pyrus communis (39)• Other fungi have also proved useful for preparing GAs. GAo, methyl ester was hydroxylated by Rhizopus nigricans to a number of products among which the methyl esters of G A ^ Q (2orhydroxylation), GA o (13-hydroxylation), GA45 (158-hydroxylation) and a 12-hydroxy G A n of undetermined stereochemistry were identified (73). Interestingly the free acid was metabolized only to GA "by hydration of the 1 6 , 1 7 double bond. While the individual yields i n the above conversions were not high, another species, R. Arrhizus, 13-hydroxylates GAs and GAprecursors i n very high y i e l d ( 6 5 ) . a n (

2

1Q



HEDDEN


Figure 11. Inhibitors of GA biosynthesis: AMO-1618 (2'-isopropyl-4'-(trimethylammonium chloride)-5'-methylphenylpiperidine-l-carboxylate) and Ν, N, N-irimethyl-l-methyl-(2ifi\6'-trimethylcyclohex-2'-en^'-^^^ iodide.



46 The Preparation of Labeled GAs


Is otopically-labeled GAs and GA-precursors are required for metabolic studies and as internal standards for quantitation by mass spectrometry. Numerous chemical methods have been used for the synthesis of labeled GAs from readily-available GAs. Labeled GAs have also been prepared biochemically using either fungal cultures or cell-free preparations from higher plants. Chemical Methods. has been introduced by chemical means exclusively at carbon-17 by the Wittig reaction. The compound to be labeled i s oxidized to the 17-nor-L6-ketone with osmium tetroxide and sodium metaperiodate. The ketone i s then reacted with the labeled y l i d , [l^C-methylene]triphenylphosphorane. There are many examples of the use of this method i n which the y l i d was generated using [^"C-methyl]triphenylphosphonium iodide and η-butyl lithium as base (e.g. 5 9 ) . Alterna tive methods which give higher yields have been published re cently by Bearder et a l . ( 7 2 , 7 3 ) . They used potassium t-butoxide as base or salt-free y l i d prepared from methyltriphenylphosphonium bromide and excess sodium hydride i n tetrahydrofuran. The Witting reaction has been used also to introduce into GAs by reaction of the norketones with [3IÎ2-methylene] triphenylphosphorane. has an advantage over ^J+C i n being less expensive and obtainable with a higher specific radioactivity. Bearder et a l . ( 7 2 ) labeled the methylphosphonium bromide by exchange with 3 H 0 i n tetrahydrofuran containing triethylamine. In the f i n a l product there i s scrambling of label between the 1 7 and 1 5 positions. The strongly basic conditions required i n the Wittig reaction necessitate the protection of base-labile groups. Thus the 3 - and 13-hydroxy groups can be converted to t r i m e t h y l s i l y l ethers ( 3 8 ) or tetrahydropyranyl ethers which are then easily removed by mild acid treatment. T r i t i a t e d GAs of very high specific radioactivity have been prepared by catalytic reduction. The 1 , 2 double bond of GAo can be selectively reduced, using a p a r t i a l l y poisoned palladium catalyst, to give [l,2- H2]GA ( 7 4 , 7 5 * 7 6 ) > although some reduction of the 1 6 , 1 7 double bond and the lactone also occurs ( 7 6 ) . Introduction of % at sites other than carbon atoms 1 and 2 has also been found ( 7 6 ) . [^HlGA^ has been prepared from GAy by a similar method T 7 7 ) . [ 3 H ] G A I was converted to [ H]GA by eldjriination of the 3-toluene-p-sulfonate ( 7 8 , 7 9 ) . Murofushi et a l . ( 8 ) protected the 1 6 , 1 7 double bond of GA5 methyl ester by forming the epoxide with metachloroperbenzoic acid. After catalytic reduction of the 2 , 3 double bond they restored the exomethylene group by treatment with a mixture of sodium iodide, sodium acetate and zinc and hydrolyzed the methyl ester to obtain [ 2 , 3 - 3 H 2 ] G A O * Yakota et a l . ( 8 1 ) prepared [ 2 , 3 - 3 H 2 ] G A Q , from GA^ by an analogous method v i a 2 , 3 dehydro G A 9 . Selective catalytic reduction of the 3-methane2

3

1

3

5

2


2.

HEDDEN

47


sulfonate of GA3 methyl ester was used by Murofushl et a l . to prepare [ I - S H J G A C , from which [1-3H]GA8 was obtained by treatment with osmium tetroxide ( 8 0 ) . A convenient method for the specific introduction of H or 3H (or both) into a molecule i s by ketone reduction with labeled metal hydride. Beale and MacMillan (10) have u t i l i z e d this method for the preparation of GAs labeled at the 1, 2 or 3 positions from GA3 or GAy (Figure 1 2 ) . One point of interest i s the lithium borohydride reduction of the enone formed by manganese dioxide oxidation of GA3 or GAy. When the reaction i s carried out i n anhydrous tetrahydrofuran i t proceeds i n two steps. I n i t i a l l y the lithium enolate i s formed which incorporates a proton at carbon-2 from the acid used i n the work-up, forming the 3-ketone. This ketone i s reduced to the 3o?-alcohol by the borohydride which i s decomposed more slowly than i s the lithium enolate. Thus i t i s possible to introduce two different labels i n a single reaction. Acid or base exchangeable protons can be easily labeled with 3H or H. Bearder et a l . (67) labeled the 15 and 17 positions of ent-kaurene by treatment with CF3C003H( H). A mixture of the 16,17 and 15,16 double bond isomers i s obtained and they are separated by AgN03 TLC. This method could be used with some 13-deoxy GAs although separation of the resulting isomers would be more d i f f i c u l t than for ent-kaurene. The 6-hydrogen i n GAi2~ lûehyde and GA^k-aldehyde has been labeled by treatment with Me0 H( H) or 3 H ( H ) 0 and sodium methoxide (62,64).


2

2

2

a

3

2

2

2

2

Biological Methods. Microbiological methods have been used i n conjunction with chemical synthesis to convert chemicallylabeled precursors to labeled GAs. Hanson and Hawker (82) prepared [17-^0] GAQ by incubating chemically-synthesized TTf-l^C] GA^p-7-alcohol with G. fujikuroi cultures. In this case the product was diluted by endogenous GA3. The method could be improved by using cultures i n which the endogenous GA levels are reduced, either by mutation (Bl-4la) or with inhibitors of GA biosynthesis (70). Bearder et a l . (72) used the G. fujikuroi mutant, Bl-4la, to prepare [17-^H2TgA o with high specific radioactivity by feeding [17-^H ] steviol acetate. This method has the potential for the preparation of a number of labeled 13-hydroxy GAs (see 71,72) · Cell-free systems provide a rapid and convenient method for preparing GA and GA-precursors with high specific radioa c t i v i t y , although on a r e l a t i v e l y small scale. [l^C]-labeled ent-kaurenoid precursors of GAs have been obtained from [2-l^C] mevalonic acid by incubating with c e l l - f r e e systems from endosperm of Marah macrocarpus (83) or Cucurbit a maxima (84,85). The £. maxima system can be used also to prepare labeled C o GAs from L^C]mevalonic acid (86). 2

2

2

American Chemical Society Library 1155 16th St. N. W. In Plant Growth Substances; Washington, D. C.Mandava, 20036N.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


PLANT GROWTH SUBSTANCES



2. HEDDEN Gibberellin Chemistry


49

50

PLANT

GROWTH

SUBSTANCES

Structure-Activity Relationships Gibberellins vary greatly i n the degree of response they e l i c i t i n biological assays, and indeed, of the 53 naturally occurring GAs, less than half have appreciable bio-activity i n the standard assays. The relationship between the structure of a GA and i t s bio-activity has attracted considerable attention and the available information has been extensively reviewed


(13,87,88).

Two explanations for the different responses of plants to a particular GA have been suggested (87). The f i r s t assumes that the GA must bind to a specific receptor s i t e to give a response, the degree of response being related to the binding efficiency. Variations i n the structure of the receptor from one plant species to another would then explain the differences in the bio-activity of a GA i n different assays. The second p o s s i b i l i t y i s that the b i o - a c t i v i t y of some GAs i s due to their metabolism to active products by the assay plants. The presence or absence of bio-activity i n an assay would then reflect the a b i l i t y of the assay to metabolize the applied GA. The struc ture of the GA-receptor and the a b i l i t y of the assay plant to metabolize the applied GA probably both influence the result of a bioassay, and i t i s i n fact d i f f i c u l t to distinguish between these p o s s i b i l i t i e s . Thus those C Q GAs which show bio-activity may do so because they are converted to C^y GAs. However, C Q GAs with the 19,20 δ-lactone are probably active per se since they are protected from farther oxidation at carbon-20 and therefore probably from conversion to C 1 9 GAs. It has been pointed out by several workers that i t i s often misleading to compare bioassay data from different laboratories since the s e n s i t i v i t y of a response can be very dependent on the bioassay technique used (13,88)· Therefore the publications of Brian et a l . (89), who compared the bio-activities of 134 GArelated compounds i n four bioassays under the same conditions, and of Crozier et a l . (90), who compared the bio-activities of 26 GAs i n nine bioassays, are very useful. Thus i t i s possible to make some general observations about the structural features of the GA molecule which are necessary for high biological a c t i v i t y . A free carboxyl group on the B-ring appears to be essential. The γ-lactone characteristic of C]_a, GAs i s required for high bio-activity but substantial, although reduced, a c t i v i t y is exhibited by C Q GAS with an aldehyde at carbon-20 or with the δ-lactone. GAs with a methyl or carboxyl group at carbon20 have l i t t l e a c t i v i t y . Reeve and Crozier (87) suggested that the δ-lactone and δ-lactol formed from the C - 2 0 aldehyde might mimic the γ-lactone of the C19 GAs. The isomeric 20,4-lactone of GAlj. was found to have a c t i v i t y equal to GkU i n some assays and only s l i g h t l y reduced a c t i v i t y i n others (57). It was con cluded that the lactone was necessary only because of the shape i t conferred on the molecule. The slight change i n the position 2

2

2



2.

HEDDEN


51

of the lactone i n GAo from 19,10 to 19,2 has no effect on bioa c t i v i t y (89,91). In most bioassays a 30-hydroxyl group increases bio-activity as does a 13-hydroxyl group. An exception i s the cucumber hypocotyl assay i n which 13-hydroxy GAs have lower a c t i v i t y than the equivalent 13-deoxy compounds. In general the most active GAs have both 30- and 13-hydroxyl groups and a 1,2 double bond or some combination of these. Interestingly, when the 30-hydroxyl group i s epimerized to the 3or position bio-activity i s v i r t u a l l y eliminated (89). The effect of hydroxylation at positions other than 30 or 13, with the exception of the 20-position, i s d i f f i cult to assess because of insufficient examples. 20-Hydroxylation causes loss of bio-activity and i s quite possibly a deactivating process i n higher plants (60). The deactivation i s f a i r l y stereospecific since 2a-hydroxylation, while reducing bio-activity, does not eliminate i t (57). The idea that higher plants may use 20-hydroxylation as a deactivation mechanism has led to methods for producing GA-derivative s with very high biological a c t i v i t y . 20-Methyl GAlj. has been synthesized i n an attempt to prevent 20-hydroxylation from taking place (M. Beale and J . MacMillan, personal communication). This compound was found to have higher bio-activity than GA^, especially when the duration of the bioassay was increased (J. MacMillan et a l . , unpublished information). A more dramatic effect was seen with 2,2-dimethyl GA^ which, i n the dwarf-5 maize assay, i s a hundred times more active than G A 3 . The enhanced a c t i v i t y i s much less marked i n short-duration bioassays. The unexpected result that dimethyl GA\± i s more active than the monomethyl compound complicates the interpretation and more derivatives need to be tested. However the preliminary results indicate that blocking the 2 position leads to higher (or prolonged) bio-activity. 20-Methoxylation, as i n 20-methoxy GA9, reduces a c t i v i t y as effectively as hydroxylation ( j . MacMillan et a l . , unpublished information). Conclusions Since the f i r s t attempts to determine the structure of GA3, the chemistry of GAs has been the subject of a large number of publications. Chemically, the GAs have proved to be d i f f i c u l t compounds to work with, a consequence of the high number and arrangement of functional groups i n the molecule. G A 3 i s particularly l a b i l e and i t i s only recently that i t s t o t a l synthesis has been completed (j+9), more than twenty years after i t s structure was established. Total chemical synthesis i s not a feasible method for preparing useful quantities of GAs; G A 3 and some of the other GAs produced by Gibberella fuj ikuroi are more practically obtained from cultures of this fungus. However, preparatively useful chemical methods have been developed for the p a r t i a l synthesis



52


of some less-accessible GAs from more abundant precursors, such as the fungal GAs. Microbiological conversion, using G. f u j i kuroi and other fungi, i s also a promising method for obtaining higher-plant GAs from readily-available substrates. The identification and quantitation of GAs i n plant extracts are particularly d i f f i c u l t problems due both to the very low amounts of GAs present i n plant tissues and to the large number of different GA structures that can be encountered. When only a limited number of GAs were known, identification was often based on co-chromatography of the unknown with standards on thin-layer plates. It i s now realized that comparison of chro matographic behaviour with that of standards i n any system i s not sufficient basis for identification. Furthermore bioassays have proved to be very unreliable methods for GA-quant i t at ion. Combined gas chromatography-mass spectrometry has the advantage of giving conclusive identification on very low amounts of com ponents in complex mixtures. It i s therefore being used i n creasingly for the detection of GAs as well as other plant hormones. It also provides an accurate means for quantitation. The mechanism of action of GAs at the molecular l e v e l s t i l l eludes plant physiologists. There have been reports of stereospecific binding of GAs to protein ( 9 2 , 9 3 ) and other c e l l frac tions (9k) but i t has not been demonstrated that the binding i s associated with a physiological response. However, correlations of the biological a c t i v i t i e s of GAs with their structures are one possible method for obtaining information on the site of action. Furthermore, a possibly valuable "spin-off" from structure-activity studies i s the design of GA-like molecules which, because of increased bio-activity or specific physio l o g i c a l properties, may have important agricultural applications. Acknowledgment s The preparation of this a r t i c l e was supported by a grant from the N.S.F. to B. 0. Phinney. The author wishes to thank Professor B. 0. Phinney for helpful comments on the manuscript. Literature Cited 1. 2.

3.

MacMillan, J . , Takahashi, N. Nature, 1968, 217, 170-171. Graebe, J.E., Hedden, P. In "Biochemistry and Chemistry of Plant Growth Regulators"; Schreiber, Κ., Schütte, H.R., Sembdner, G., Ed. Institute of Plant Biochemistry: Halle, G.D.R. 1974, pp. 1-16. Harrison, D.M., MacMillan, J . J . Chem. Soc. (C), 1971, 631-636.

4.

MacMillan, J . , Pryce, R.J. In "Phytοchemistry"; M i l l e r , L.P., Ed. Van Nostrand-Reinhold: New York, 1973, pp. 283326.


2.

HEDDEN

5.


53

6. 7. 8.

Grove, J.F., MacMillan, J . , Mulholland, T.P.C., Turner, W.B. J . Chem. Soc. 1960, 3049-3057. Grove, J.F. J . Chem. Soc. 1961, 3545-3547. Hanson, J.R. Tetrahedron. 1966, 22, 701-703. Bearder, J.R., MacMillan, J . J.C.S. Perkin Trans. 1. 1973,

9.

MacMillan, J . , Pryce, R.J.

2824-2830.

J . Chem. Soc. (C), 1967,


740-742.

10. 11. 12. 13.

MacMillan, J. Pure & Appl. Chem. 1978, 50, 995-1004. Pryce, R.J. Phytochemistry. 1973, 12, 507-514. Pryce, R.J. J.C.S. Perkin Trans. 1. 1974, 1179-1184. Graebe, J.E., Ropers, H.-J. In "Phytohormones and Related Compounds: A Comprehensive Treatise. Volume 1." Lethem, D.S., Goodwin, P.B., Higgins, T.J.V., Ed. Elsevier/ North Holland Biomedical Press: Amsterdam, Oxford, New York. 1978,

14.

pp. 107-203.

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15. 16. 17. 18. 19. 20. 21.

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RECEIVED

June 19, 1979.


Plant Growth Substances - ACS Publications - American Chemical

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