The Chemistry of Allelopathy - American Chemical Society

5 The Involvement of Allelochemicals in the Host Selection of Parasitic Angiosperms Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 31, 2015 | http://pubs.acs.org Publication Date: December 17, 1985 | doi: 10.1021/bk-1985-0268.ch005

DAVID G. LYNN Department of Chemistry, The University of Chicago, Chicago, IL 60637 Several compounds have now been found which are capable of inducing the d i f f e r e n t i a t i o n of the haustorium—a spec i a l i z e d organ i n p a r a s i t i c angiosperms which functions to attach the parasite to its host. A detailed description of the chemical nature of these compounds i s presented. Measurements of the levels of these compounds exuded by a host have been made and correlated with levels required for haustorial induction. The data support a s t r i c t s t r u c t u r a l requirement for the factors involved i n the induction of the haustorium. Although more than one s t r u c t u r a l class may be involved, the compounds appear to be linked with the host's allelochemicals and constitutive a n t i b i o t i c s . The evidence for synergistic a c t i v i t y among several d i f f e r e n t components i n host exudate suggests the p o s s i b i l i t y of a more highly sophisticated mechanism of host selection through haustorial d i f f e r e n t i a t i o n than previously anticipated. As so many natural products chemists before us, we have become i n trigued with the seemingly endless array of organic structures which are produced i n nature. Over the past t h i r t y years, questions d i rected primarily at the structures of these compounds and at their effects on man's physiology have led to profound technological advances. Man can now synthesize v i r t u a l l y any organic compound which exists i n nature and has used these talents to develop new methods to study and combat human disease. We are now i n a position to explore a fundamentally d i f f e r e n t f r o n t i e r — t h e physiological and ecol o g i c a l significance of these compounds to the organisms which produce them. The greater our understanding of the chemical communication and chemical defenses inherent i n a l l l i v i n g organisms, the better we w i l l be able to understand and maintain the i n t r i c a t e chemical nature of the world which surrounds us (1). A need for this understanding i n the plant kingdom, the area on which so much of our food and oxygen supply i s dependent, i s becoming p a r t i c u l a r l y apparent. We have focused our interest on the mechanisms of host selection i n p a r a s i t i c angiosperms—plants which have evolved the capab i l i t y of p a r a s i t i z i n g other plants. An e a r l i e r proposal (2) that 0097-6156/85/0268-0055$07.75/0 © 1985 American Chemical Society

In The Chemistry of Allelopathy; Thompson, Alonzo C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 31, 2015 | http://pubs.acs.org Publication Date: December 17, 1985 | doi: 10.1021/bk-1985-0268.ch005

56

T H E C H E M I S T R Y OF A L L E L O P A T H Y

p a r a s i t i c plants, l i k e herbivorous insects, may key on host defense chemicals as recognition cues suggested that a study of this system may not only provide some access into the control of attachment i n some of these a g r i c u l t u r a l l y devastating parasites, but also may un cover aspects of the development and s p e c i f i c i t y of the host's a l l e lochemistry. The heterogenous group of plants that are c l a s s i f i e d as parasi t i c angiosperms are found across eight different families (for r e cent reviews see r e f . 3). Most, but not a l l , are photosynthetic and capable of maturing to seed set without a host; however, i t i s rare i f ever that f i e l d c o l l e c t i o n s reveal plants that are devoid of a t tachments. The implication i s that these attachments are uniquely important to the v i a b i l i t y of a l l of these parasites, and i t should be anticipated that s p e c i f i c early developmental events have evolved to f a c i l i t a t e rapid and e f f i c i e n t host attachment. Other papers i n this Symposium w i l l deal with s t r i g o l and derivatives of s t r i g o l which function as s p e c i f i c and highly sensitive stimulants of germi nation i n Striga a s i a t i c a (Witchweed). Germination appears to func tion as one viable l e v e l of chemical recognition i n host selection. However, the developmental feature that i s uniquely common to a l l the p a r a s i t i c angiosperms i s the haustorium (Figure 1) - the organ which forms the physiological and morphological attachment between host and parasite. These organs have been shown not to form when Agalinis purpurea (4_) or Striga (5) are grown axenically. The development of these organs can, however, be very rapidly induced i n the presence of host roots or host root exudate. Laboratory cultures of both A g a l i nis purpurea and Striga a s i a t i c a have been developed and maintain ed. (4) When induced, the haustoria are f u l l y formed within 6 to 12 hours* This system has been developed for both quantitative and q u a l i t a t i v e bioassays. Using these bioassays to d i r e c t the i s o l a tion, several haustorial inducing p r i n c i p l e s have now been character ized. This report d e t a i l s the chemical characterization of these haustorial inducing factors and the present knowledge about the role that these factors play i n host selection and host allelopathy. More s p e c i f i c a l l y , I have focused on the chemical, spectroscopic and bio l o g i c a l methods which we have developed during the course of this work with the hope that these methods w i l l be of some general use to other s c i e n t i s t s . Plant Materials and Bioassays Agalinis purpurea (L.) Raf. (Scrophulariaceae) seeds were collected by Professor Lytton Musselman, Old Dominion University, and grown i n s t e r i l e culture i n 60 χ 15 mm P e t r i plates on Murashige-Skoog medium as previously reported (4_) . Lespedeza sericea (Leguminosae) was grown i n vermiculite under greenhouse conditions. Bioassays for haustorial induction were carried out by either spotting fractions on 0.5 cm f i l t e r paper discs and inserting the discs into agar i n which 2 to 3 week old A g a l i n i s plants were growing or removing the plants i n d i v i d u a l l y and placing 4 to 5 each into depression slides contain ing d i s t i l l e d H 2 O and known concentrations of inducer. The haustoria i n each case were read after 48 hours. The transfer of the plants from agar to water introduces some stress, and the s e n s i t i v i t y of the assay i s diminished although the s p e c i f i c i t y seems unaltered.


5. LYNN

Parasitic Angiosperms: Allelochemicals and Host Selection

57


Haustorial Inducing Factors from Gum Tragacanth After finding that primary metabolites and hormones f a i l to i n i t i a t e haustorial development i n Agalinis (4) and r e a l i z i n g that these pa r a s i t e s must be cuing on substances exuded from the potential host, several commercially available plant exudates were screened for ac t i v i t y . Gum tragacanth, an exudate of Astragalus gummifer (Leguminosae), showed potent a c t i v i t y i n e l i c i t i n g haustorial development and was far more readily available than host exudate. Hoping that the tragacanth e l i c i t o r s would provide some insight into the kinds of molecular species involved i n haustorial induction, John Steffens and Dr. Mike Thompson focused their e f f o r t s on the factors present i n gum tragacanth (6). Soxhlet extraction of the dry gum (250 g) s e r i a l l y with hexanes, ether, and methanol resulted i n an a c t i v i t y r i c h ether f r a c t i o n (600 mg, dried i n vacuo) that was partitioned between and 50% aqueous MeOH. The dried aqueous layer (300 mg) was applied d i r e c t l y to drop l e t countercurrent chromatography (DCC), CfcHfc/MeOH/CHC^/^O (2:3:2:1, upper phase mobile). The application of large quantities of this crude f r a c t i o n d i r e c t l y to DCC proved c r i t i c a l to the p u r i f i cation and resolved two separate bands of a c t i v i t y . These bands, further p u r i f i e d by normal and reverse phase chromatography, existed as 8xl0~5% and 3.2x10"^% of the dry gum and were named xenognosin A,_l and B,2^ respectively. The UV (Xmax 260, MeOH) of xenognosin A showed an hydroxystyrene chromophore and ^H-NMR (100 MHz, acetone-d^) double resonance experiments i d e n t i f i e d four isolated proton spin systems; a charac t e r i s t i c A B (δ 7.20, 2H, d, J=8.5 Hz; δ 6.74, 2H, d, J=8.5 Hz) system, a 1,2,4-trisubstituted aromatic system (H-6, δ 6.90, d, J=8.4 Hz; H-5, δ 6.35, dd, J=8.4, 2.4 Hz; H-2, δ 6.44, d, J=2.4 Hz) with oxygen f u n c t i o n a l i t y at positions 2 and 4, a 1,3-substituted trans propene (δ 6.315, d, J=15 Hz; δ 6.16, dt, J=15, 5.6 Hz; δ 3.34, 2H, d, J=5.6 Hz) and a methoxy singlet (δ 3.77). The small sample size complicated these proton assignments and they were confirmed by a synthetic model system prepared by David Graden [2-(4'-methoxy)-3(2 ,4'-dimethoxy)-diphenylpropene] (7) and spectral simulations. For example, the double t r i p l e t (δ 6.16) was obscured due to the poor signal-to-noise and the severe second order coupling at 100 MHz. Simulations (7,8) of the o l e f i n i c protons were c r i t i c a l to these as signments (Figure 3). Two s t r u c t u r a l questions remained i n the characterization of xenognosin A. The propene unsaturation could be conjugated with e i ther of two possible aromatic phenolic chromaphores. Answers to re lated problems have been addressed through Dverhauser experiments (9) but i n this case the sample was too small to obtain s u f f i c i e n t s i g nal-to-noise to detect the enhancements. P a r t i a l l y relaxed ^H-NMR proved to be a f a r more sensitive method of detecting regiochemical proximities i n this system. Figure 4 details the downfield region of the lH -NMR spectrum and compares i t to a selected p a r t i a l l y relaxed spectrum, [180°-t-90°]n. The must faster relaxation rate of the doublet at δ 7.2,relative to the other half of the A 2 B 2 system or the protons of the other aromatic ring, confirms i t s proximity to the o l e f i n i c protons. This same d i s t i n c t i v e difference was observed i n several model systems including 4-methoxy styrene,which under similar

CCI4

2

2

1

9



58

THE CHEMISTRY OF ALLELOPATHY

F i g u r e 1. H a u s t o r i u m on A g a l i n i s p u r p u r e a 24 hours a f t e r R e p r i n t e d w i t h p e r m i s s i o n from Dr. Vance B a i r d . )

induction.

6.44(d,J=2.4)H

HO-

OChL 3 77(s) H

H

334 (2H,d,J=5.6)

6.35 (dd,J=8.4,24) 6.90(d,J = 84)H , χ ^ ^ Η 6.315 6.16 Η η (d,J=15) (m, J=15,5.6)

Η \ ^ ^ ^ Η 720

(2H,d,J:

Π

OH

683(d,J = 2)H H O ^ ^ O ^ H 697,dd JL (J=9,2)H^Y^ 8.05(d,J=9)H

85)

ff

( s )

H 649 (m, J = 9,2) OCH 378(s) H 649(m,J = 2) 3

6.74 (2H,d,J=8.5)

F i g u r e 2. S t r u c t u r e s o f x e n o g n o s i n A, 1, and B 2. (100 MHz, acetone-dfc s i g n a l s a r e a s s i g n e d . )

H-NMR


LYNN



I

V(1)« 6.315 V(2)»6.16 J(1,2) «15 H,

0. I

6.8

1

I

6.6

—I

1

6.4

1

6.2

1

1

6.0

'

I

5.8

PPM

F i g u r e 3. ( a ) The c o m p u t e r - s i m u l a t e d spectrum o f the o l e f i n i c p r o t o n s u s i n g c h e m i c a l s h i f t s o f 6.3 and 6.1 ppm and a c o u p l i n g c o n s t a n t ( J ) o f 15 Hz. ( b ) The o l e f i n i c r e g i o n o f the 100 MHz ^H-NMR spectrum o f the o r i g i n a l l y i s o l a t e d x e n o g n o s i n A. The marked r e s o n a n c e s c o r r e s p o n d e x a c t l y w i t h the resonances o f the s i m u l a t e d spectrum.

F i g u r e 4. P a r t i a l l y r e l a x e d F o u r i e r t r a n s f o r m H-NMR spectrum on 150 μg o f xenognosin A shown above the normal ID-spectrum (downfield region o n l y ) .


60


conditions gave a difference i n T-^'s of 0.5 sec between the doublets of the A 2 B 2 system. This was deemed s u f f i c i e n t support to assign the styrene chromophore as i n 1. Later work by David Graden with a synthetic sample of jL allowed for a quantitation of both the p a r t i a l l y relaxed data (Figure 5) and nOe difference data (Figure 6). The second structural problem involved the regiochemical as signment of the single methoxyl group. This assignment could not be v e r i f i e d by differences i n T^ times, so advantage was taken of spe c i f i c i t y of the association between pyridine and the a c i d i c phenolic protons. T i t r a t i o n of the acetone-d solution to 20% p y r i d i n e - d (v:v) gave clear s h i f t s of the h i g h f i e l d and lowfield doublets of the A 2 B 2 of 3 Hz and 1 Hz respectively. Two additional aromatic signals, δ 6.35 and δ 6.44, were shifted downfield by 4 Hz and 3 Hz respectively, suggesting that the free hydroxyl group on the other aromatic ring was flanked by two ortho protons as shown i n 1. Mass spectral confirmation of that assignment was obtained from CAD ana lyses of the m/z 137 fragment ion generated from chemical ionization ( C H 4 ) of 1^ (Figure 7) (.10) . The m/z 137 ion comes from α-cleavage of the o l e f i n to generate the benzyl cation shown i n Figure 7. I t was reasoned that the further fragmentation of that ion would be de pendent on the regiochemistry of the methoxy group. The regioisomer i c ions needed for mass spectral comparisons were accessible through Dr. V. Kamat's synthesis of the isomeric benzyl alcohols. Under chemical i o n i z a t i o n conditions, these alcohols protonate and lose H 2 O to generate the same ions at m/z 137. CAD analyses con firm the suspected difference i n fragmentation and establish the me thoxy ortho to the a l k y l substituent i n 1 (Figure 8) . The s k e l e t a l assignment of xenognosin Β proved to be more straightforward than did xenognosin A. The UV (MeOH, Xmax 248 nm, sh 300) and the c h a r a c t e r i s t i c one proton singlet at δ 8.19 (100 MHz, acetone-d^) are very diagnostic of isoflavanoids. Two trisubstituted aromatic rings and an aromatic methoxy group (δ 3.78) completed the %-NMR assignment and revealed the basic structure _2. The remaining regiochemical assignment involved the placement of the methoxy methyl group on one of three possible oxygen atoms - very s i m i l a r to the problem faced with xenognosin A. The chemical i o n i z a t i o n MS(CH^) gave l i t t l e fragmentation, but CAD (N ) analyses of the Mfl molecu l a r ion gave two ions at m/z 136 and m/z 148 (Figure 9) a r i s i n g from a retro-Diels-Alder fragmentation of the heterocyclic r i n g . The masses of these ions established the association of the methoxy group with the m/z 148 fragment, but did not locate i t at either the 2* or 4 position of the r i n g . C a p i t a l i z i n g on the higher energy fragmen tation afforded by electron impact mass spectrometry (70 ev, 150°C), the retro-Diels-Alder fragmentation was completely suppressed and the major fragment ion appeared at (M-17) . Through comparison with other 2'-substituted isoflavanes (11), the M-17 fragment can be as signed to result from the loss of an hydroxyl r a d i c a l to give the s t a b i l i z e d oxonium ion (Figure 10). These data established the methoxy group at 4 and the struc ture of xenognosin Β as _2. This sample also proved to be i d e n t i c a l by NMR, MS, and b i o l o g i c a l a c t i v i t y to a synthetic sample of 2', 7dihydroxy-4 -methoxy isoflavone (generously provided by Professor Paul Dewick, University of Nottingham, U.K.). Two d i f f e r e n t uses of mass spectrometry have proven c r i t i c a l to


6

5

2

f

+

f

1


LYNN


5 0/ m

ml

. d -ACETONE ο


360 M H z . 4 PULSES

2.2 S e c 3.0 S e c

I ' ' ' I ' 7.4 7.2

Figure

5.

*H-NMR

1

u

' I ' • ' I ' ' ' I ' ' • ι • • • ι 7.0 6.8 6.6 6.4 6.2

relaxation

'

6.0

PPM

study o f x e n o g n o s i n A a t 360 MHz.

H . He He Ho H 6

H (1Q5 /o)

2

Hy

e

3

Η (14.8 Λ»)

XENOGNOSIN A

β

0

H (220 /o) e

b

CH,

4

CH,

">

!

ill l

JJJk -pr-r-^r-p, .0

6.5

6. C

5.S

S.O

-r. 5

-t.C

3.5

FFr

F i g u r e 6. Xenognosin A nOe d i f f e r e n c e spectrum. (360 MHZ, acetone-d^). I r r a d i a t i o n o f H 2 g i v e s the enhancements shown i n the upper t r a c e .


62



100

M/Z +

F i g u r e 7. CI MS ( C H , 150 °C) g e n e r a t e d ( M + l ) i o n was quadrap o l e s e l e c t e d and s u b j e c t e d to c o l l i s i o n a c t i v a t e d d e c o m p o s i t i o n C ^ ) to g i v e the mass spectrum ( 6 ) . 4

F i g u r e 8. The m/z 137 i o n was s e l e c t e d from the s o u r c e and c o l l i s i o n a l l y decomposed ( ^ ) to g i v e the CAD s p e c t r a . The top spectrum i s the fragment a r i s i n g from the i s o l a t e d x e n o g n o s i n and the bottom two s p e c t r a a r i s e from the i s o m e r i c b e n z y l a l c o h o l s ( 6 ) .



5.

LYNN

63


—I

I

100

200

M/Z F i g u r e 9. Xenognosin Β was c h e m i c a l l y i o n i z e d (CH^, 150 °C) and the ( M + l ) i o n quadrapole s e l e c t e d and s u b j e c t e d t o c o l l i s i o n a c t i v a t i o n d e c o m p o s i t i o n (N2) t o g i v e the mass spectrum. +

M-17 F i g u r e 1Q P o s t u l a t e d s t r u c t u r e f o r the major fragment xenognosin Β under e l e c t r o n impact mass s p e c t r o m e t r y .

i o n of



64


the microgram scale structure assignment of regiochemistry of methoxyl substituents on these haustorial inducing factors. In fact, the s i m i l a r i t y of the dihydroxy and methoxy substituents on these phenylpropenoids suggested that the b i o l o g i c a l a c t i v i t y may be l i n k ed to t h i s f u n c t i o n a l i t y . Formononetin (7-hydroxy-4'-methoxy i s o flavane), _3, isolated from the same source, was completely devoid of b i o l o g i c a l a c t i v i t y and added further support for the necessity of the f u n c t i o n a l i t y . Dr. Kamat developed an e f f i c i e n t t o t a l synthesis of xenognosin A (Scheme 1) which possessed s u f f i c i e n t f l e x i b i l i t y for structure/ a c t i v i t y relationships to be explored (12). The several analogues which he was able to prepare uncovered a s t r i c t s t r u c t u r a l s p e c i f i c i t y associated with c e r t a i n parts of the xenognosin A skeleton. Modification to the styrene system either through removing (4f ) or methylating (4g) the para-hydroxyl group had l i t t l e effect on the a c t i v i t y , but a simple change i n the regiochemistry of the methoxy substituent on the t r i s u b s t i t u t e d ring (4h) severely reduced the a c t i v i t y (Figure 11). Further reduction (6) or oxidation (7) of the propene bridge greatly reduced the b i o l o g i c a l activity, and removal of either the methoxyl or hydroxyl groups from the t r i s u b s t i t u t e d ring leaves the compound completely i n a c t i v e . These s t r u c t u r e / a c t i v i t y studies point toward a precise struc t u r a l dependence on certain f u n c t i o n a l i t y of the xenognosin A mole cule (13). The meta-methoxyphenol f u n c t i o n a l i t y seems most s i g n i f i cant since shows s l i g h t a c t i v i t y i n the agar assay. Even though these studies are hampered by the d i f f e r i n g s o l u b i l i t y of the ana logues and the stress i m p l i c i t i n removing the organisms from agar and placing them i n depression s l i d e s with d i s t i l l e d (the half maximal response to xenognosin A i n depression s l i d e s i s 10"^M and i n the agar assay i s at least two orders of magnitude more sensi tive) , the development of some insight into the s p e c i f i c i t y of this response has been gained. This s p e c i f i c i t y i s remarkable i n l i g h t of the rather broad host range that Agalinis i s capable of p a r a s i t i zing. Nonetheless, flavanoids are well known as stable taxonomic characters and several flavanoids and phenolics are often involved i n host allelopathy (14). Xenognosin Β was suspected of being a biosynthetic precursor of the red clover phytoalexin ± medicarpin, and i n fact was synthesized and shown to be biosynthetically incor porated into the phytoalexin before i t was ever found to occur na t u r a l l y (15). Xenognosin A has now been found i n v a r i e t i e s of Pisum as a stress metabolite associated with host defense (16). I t seems perfectly reasonable that these taxonomically c h a r a c t e r i s t i c and p h y s i o l o g i c a l l y active components which are produced by root tissue would serve as cues for host selection i n a root parasite. Haustorial Inducers i n a Natural Host While the a c t i v i t y and the s t r u c t u r a l s p e c i f i c i t y of the xenognosins were very encouraging, Astragalus gummifer i s native to the Middle East and would not be expected to have co-evolved as a host for the southeastern U.S. native, A g a l i n i s purpurea. The questions of whe ther molecules of this type were actually exuded from host roots i n s u f f i c i e n t quantities to constitute host selection s t i l l remained. For that reason, John Steffens switched h i s attention to Lespedeza



5. L Y N N


xenognosin Β

± medicarpin

formononetin

* maackiain

1

-

H

i

a) H , Pd/C (10%), EtOH, AO PSI, 24 h r , 95%. 2

b) TBDMS-C1, ( E t ) N , DMAP, C H C 1 , 25*C, 10 h r , q u a n t i t a t i v e . 3

2

2

c) 1 eq. DIBAL, t o l u e n e , -78'C, 2 h r , 95%. d) £ - T B D M S - 0 - C H B r , Mg, THF, 25*C, 1 h r , 85%. 6

4

e) C H N , MeOH, 4*C, 8 h r , 95%. 2

2

f ) CH S0 C1, 3

2

( E t ) N , THF, 25*C, 2 h r , 60%. 3

g) ( C H ) N F , THF, 25°C, 0.5 h r , 90%. 4

Scheme

9

4

1. S y n t h e s i s

of xenognosin A ( 1 2 ) .


65



XENOGNOSIN

A

4f

4g

4h

XENOGNOSIN Β

F i g u r e 11. R e l a t i v e a c t i v i t i e s of h a u s t o r i a l i n d u c e r s when p r e s e n t e d i n s o l u t i o n t o 2-to-3 week-old p l a n t s o f A g a l i n i s purpurea. P l a n t s d e v e l o p e d an average gf two h a u s t o r i a each when p r e s e n t e d w i t h x e n o g n o s i n A a t 10~ M.(13).

4 R, a) O C H

R OCH3 2

3

R OCH3 3

b)

OH

OH

OH

Ο

Η

OCH3

OCH3

d)

Η

OH

OCH3

c)

OH

OH

OCH3

f)

OH

OCH3

Η

g)

OH

OCH3

OCH

OH

OH

h) OCH3

3

Analogues o f

7 sin A


5.


LYNN

sericea (Leguminosae), a forage legume which could be grown i n the laboratory f o r the production of exudate and was readily parasitized by Agalinis (17). Four hundred grams (fresh weight) of 3-month old vermiculite-grown Lespedeza roots were extracted with 50% aqueous MeOH, dried i n vacuo and l y o p h i l i z e d . Twelve grams of this material were extracted with acetone and concentrated to y i e l d 2.5 g of a dark brown o i l . This o i l was applied d i r e c t l y to droplet countercurrent chromatography (CHC^/MeOH/^O, 7:13:8, descending mode) and the early eluting active f r a c t i o n was repeatedly f l a s h chromatographed (SiOo, CH Cl /Me0H) and f i n a l l y p u r i f i e d by HPLC (reverse phase C , 83% Me0H/H 0) giving 2 mg of a c r y s t a l l i n e s o l i d , 8. Mass spectrometry analyses (18) (EI, 70 eV) gave a molecular ion at m/z 458.3757, C Q H 0 3 (calcd 458.3760) and major fragment ions at m/z 234 and 224, suggesting a retro-Diels-Alder fragmenta tion of an olean-12-ene triterpene bearing two oxygen atoms on the A-B ring fragment (19). Seven methyl singlets i n the h i g h f i e l d r e gion of the iH-NMR (360 MHz, acetone-d^) and a single o l e f i n i c pro ton (6 5.5, t, J=7.5 Hz) supported the oleanene assignment (Figure 12). Three hydroxyl substituents were i d e n t i f i e d with deuteriumexchange negative ion CI MS (EtOD, Ν~0) (20) by the appearance of a molecular ion at m/z 459 [(M-H)~ with 2 exchanges]. The 3β- and 24-hydroxyl f u n c t i o n a l i t i e s were suggested by iH-NMR ( δ 3.45(H-3, dd, J=5.4, 11 Hz), δ 4.2 (H-24, d, J = l l Hz), δ 3.5 (H'-24, bd, J = l l Hz) (21). Disruption of the intramolecular hydrogen bonding with Me S0-d6 and by acetylation, taken together with a 2.9% nOe of the C-25 methyl upon i r r a d i a t i o n of H -24 (22), confirmed this assign ment . Placement of the third hydroxyl group was more d i f f i c u l t . Elec tron impact fragmentation had r e s t r i c t e d i t s location to the D-E ring fragment, and the proton residing on the same carbon (δ 3.42, t, J=4.5 Hz) was best assigned as an e q u i t o r i a l proton flanked by a single methylene. This r e s t r i c t e d the hydroxyl group to an a x i a l position at C-15, C-16, C-21 or C-22. CAD-MS proved invaluable i n establishing substituent regio chemistry i n the xenognosins and had proved useful i n triterpenes (23). Comparison of the mass spectrum of the triacetate of J$ with 3-oxo-15-acetoxyolean-12-ene showed that both compounds gave an m/z 276 fragement from the retro-Diels-Alder rearrangement and both of these ions lost acetic acid to an m/z 216 ion. CAD analyses (1 μΤ Ar as c o l l i s i o n gas) of these two fragment ions gave considerably d i f f e r e n t spectra, r u l i n g out the C-15 hydroxylation of 8. NOe difference experiments (22) to a i d i n methyl group assign ment were conducted with the 33,24-phenylborate of (phenylboronic acid, benzene, reflux w/Dean Stark trap) since the methyl signals were more e a s i l y resolved following this modification. Key enhance ments obtained from the nOe difference experiments and their assign ments are l i s t e d i n Table I. Additionally, CH -27 and CH..-30 could be assigned from correlation with other triterpenoids (24; . The remaining two methyl groups, C H 3 - 2 9 and C H 3 - 2 6 , were tentatively assigned to the signals at δ 0.87 and δ 0.97 respectively. I t be came clear during the course of the work that d e r i v a t i z a t i o n of the 1,3-diol of introduced small unexpected chemical s h i f t changes i n methyl signals distant from the Α-ring, therefore making the predic tive value of the empirically derived correlation tables unreliable. 2

l g


67

2

2

3

5 o

2

f

3


68


Table I.

Nuclear Overhauser Enhancement Difference Value for Soyaspogenol Β Signal enhanced assignment δ

Signal i r r a d i a t e d δ assignment H-18

f

H-24

CH -25


3

CH -28

1.05

1.3

3.17(bd)

CH -23

1.02

0.6

4.1(d)

CH -25

0.93

1.9

4.1(d)

4.9

2.2(dd)

H-24

%

3

3

3

1

H-24

0.93(s)

P r e i r r a d i a t i o n (selective 180°) of each signal was followed by a 90° observed pulse delayed by 0.7s. This spectrum (550-1000 tran sients) was acquired simultaneously with a spectrum i n which one selective pulse was 3 ppm u p f i e l d of tetramethylsilane, and the two spectra were computer subtracted to observe the enhancements. Nevertheless, the use of the methyl groups as diagnostic s i g nals for the l o c a l i z a t i o n of ring f u n c t i o n a l i t y i s invaluable. T i t r a t i o n of the phenylborate ester of 8 with E u ( f o d ) 3 gave the data shown i n Figure 13. The invariance of the methyls CH -23 and C H 3 - 2 5 v e r i f i e d phenyl borate protection of the d i o l against s h i f t reagent complexation and r e s t r i c t s complexation to the t h i r d hydroxyl group (Figure 13). Three methyl groups, CH -29, C H 3 - 2 8 , and CH -30, d i s played the greatest induced chemical s h i f t with added s h i f t reagent, and this l o c a l i z e d the hydroxyl group to the Ε-ring. Closer exami nation suggested that C-21 hydroxylation (3, a x i a l ) would predict C H 3 - 2 9 and 30 to be most affected by the s h i f t reagent, and that placement at C-22 (a, axial) would predict C H 3 - 2 8 and 30 to expe rience the greatest downfield s h i f t . Neither of these expectations were r e a l i z e d , probably due to the angular dependence of the s h i f t reagent induced chemical s h i f t s (25). For that reason, attention was turned to experiments that would increase the r e s o l u t i o n obtainable i n the -^H-NMR spectrum. Professor James Roark, v i s i t i n g from Kearney State College, was experimenting with methods for s e l e c t i v e l y observing the transmission of coupling information over large numbers of bonds. The monoterpene dl-camphor best represents what he found. Figure 14 shows a contour plot (26) of the homonuclear proton c o r r e l a t i o n map of camphor. The corres ponding ID spectrum i s shown along the ordinate. Connection of the off-diagonal cross peaks with t h e i r corresponding diagonal partners establishes the spin coupling between adjacent protons and allows for v i r t u a l l y the complete proton assignment of camphor. Close i n spection of the camphor map shows that the "W-type" 4-bond coupling between H-2x and H-4x v i s i b l e i n ID spectrum (J=3.8 Hz) has no cross peak i n the 2D spectrum of Figure 14. Protons with J couplins of 4 Hz or less have proven to give cross peaks that are weak or not present i n low r e s o l u t i o n COSY experiments (27), but the r e s o l u t i o n of smaller couplings i s possible by the i n s e r t i o n of fixed delays following each of the two pulses (28). For example, an 0.2 sec delay (Δ = 0.2 sec) following each of the two 90° pulses results i n the appearance of intense cross peaks between H-2x and H-4x. Extending the delay to 400 msec r e s u l t s i n the appearance of 3

3

3



LYNN


F i g u r e 12. H-NMR spectrum Lespedeza s e r i c e a .

o f the h a u s t o r i a l

OQ

e

i n d u c e r from

^ *. -

*

- • #

*

*-* ' '

.4.

»

' i - V - »'

»-·"""

ao

θ!θ7

0.14

a 21

0.28

uM EiKfod) - uM Phenylborate 3

F i g u r e 13. E f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f the NMR s h i f t reagent E u ( f o d ) ~ on methyl resonances o f soyasapogenol Β p h e n y l borate. Eu(fod;3 was d i s s o l v e d i n a minimum amount o f a c e t o n e - d and added t o a 2 ml s o l u t i o n o f soyasapogenol Β p h e n y l b o r a t e i n the same s o l v e n t . S p e c t r a were o b t a i n e d a t 360 MHz.


(

70



2.0

1.5

10

PPM

F i g u r e 14. Contour p l o t of the 360 MHz H-NMR c o r r e l a t i o n spectrum of dl-camphor. A 64 x256 data s e t was accumulated w i t h q u a d r a t u r e phase d e t e c t i o n i n both dimensions and the data s e t was z e r o f i l l e d once i n the dimension and symmetrized. T^ was 5 sec and t was incremented by 1.63 msec. T o t a l a c c u m u l a t i o n time was 24 minutes and data workup and p l o t t i n g took 15 min. 1



5.

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Parasitic Angiosperms: Allelochemicals and

71

Host Selection

several additional cross peaks. The higher contour s l i c e i n Figure 15a shows only the intense quaternary methyl diagonal signals, but with the addition of the delay (Figure 15b), cross peaks are obser ved between the high and the low f i e l d methyl s i n g l e t s . The obser vation of these cross peaks a r i s i n g from the ^ J coupling between the gem-dimethyls of camphor allows for the assignment of the bridgehead methyl to the central singlet at δ 0.89. This assignment i s further supported by a lower contour s l i c e of the same data, Figure 16, where cross peaks appear for the coupling between H-3 and the central methyl. D i f f e r e n t i a t i o n of the gem-dimethyls i s readily apparent from the cross peaks appearing between H-4d and 5d and the high f i e l d methyl (δ = 0.81). The analogous coupling i s also obser ved between H-2d and the downfield methyl (δ 0.94). Thus, i n just two experiments that f a c i l i t a t e d the observation of proximities over two to f i v e bonds, a l l the proton assignments of camphor have been made. The assignments made here with delayed COSY agree with pre vious assignments of camphor made by rigorous comparisons with model systems (29). Since the use of the ring protons of camphor had proven so ef fective i n assigning the three methyl singlets through long range coupling interactions, the reverse usage of the methyl groups to assign f u n c t i o n a l i t y along the triterpene skeleton should be equally e f f e c t i v e . Protection of the Α-ring of 8 as an acetonide (2,2 dimethoxypropane, acetone, pTsOH) and oxidation (PDC, pyridinium t r i f l u o r o a c e t a t e , r t , 4 h) generated the ketoacetonide j). This o x i dation shifted the protons cx to the carbonyl downfield from the bulk of the backbone methylenes and greatly simplified their assignment (Figure 17). The cross peaks connecting H-18 (δ 2.3) to H-19e (δ 1 . 3 5 ) and H-19a (δ 2.07) are also pointed out i n Figure 17. The i n s e r t i o n of a 0.25 s delay i n the COSY sequence s i g n i f i c a n t l y changes the o v e r a l l appearance of the 2D-map and the observed cross peaks (Figure 18). Α Λ Ι coupling cross peak connects H-19a with the h i g h f i e l d methyl singlet ( C H 3 3 O ) . This same methyl singlet shows coupling to one of the protons adjacent to the carbonyl allowing i t s assignment as H-21a. Another cross peak v e r i f y i n g a \J coupling between H-19e and the other methylene proton adjacent to the carbo nyl, H-21e, further confirms the s i t e of oxidation as C-22 and so assigns the structure of the h a u s t o r i a l inducer as 33,223,24-trihydroxyolean-12-ene. During the course of t h i s work Kitagawa et a l . (30) revised the structures of soyasapogenol Β to this structure on the basis of extensive chemical and X-ray analyses. Our work u t i l i zing small scale NMR and MS spectra data i s i n agreement with that of Kitagawa. f

Quantitation of Haustorial Inducers i n Root Exudate Now that an h a u s t o r i a l inducing factor has been characterized i n a host plant that could be grown i n the laboratory, the levels of the compounds actually exuded could be analyzed. John Steffens and Rody Spivey focused on developing methods that would allow for suitable quantitation. E f f o r t s were made to quantitate not only the terpenoid components, but also the flavanoid, genistein (4 ,5,7-trihydroxyisoflavone) , which was found to be a major isoflavone of Lespedeza. Genistein was analyzed to gain an estimate of l e v e l s of phenylpro1


72


Me"

JO

•


^

(Tw-90 A-tl-90*A-t2) e

°

n

δ= 0 sec.

I '

1

1

I

1

1

I 2.0

1

1

1

1

ι ' •' ι ' I

| 1.5 1

1

1

I

1

' ' I

1

1

1

' I

10

1

1

08

PPM

B. δ=0.4 sec.

Me'

I "

' I "

Ί

2.0

1

"

ι

1

"

I Ί

' I 1.5

F i g u r e 15. H i g h e r contour s l i c e and (B) the data i n F i g u r e 17.

1

1

1

I

1

of (A)

"

I ' 1.0 1

'

I '

the data

'

PPM

i n Figure


14


5.

LYNN


2.0

15

1.0

73

PPM

F i g u r e 16. Same experiment as performed i n F i g u r e 14 w i t h Δ = 0.4 sec. S i x t e e n t r a n s i e n t s were c o l l e c t e d r a t h e r than the 4 t r a n s i e n t s i n F i g u r e 14.



74


F i g u r e 17. Contour p l o t of the 360MHz homonuclear s p i n c o r r e l a t i o n mpa o f 10 (2 mg, CDCL^, h i g h - f i e l d e x p a n s i o n ) w i t h no d e l a y i n s e r t e d i n the p u l s e sequence shown a t the top of the f i g u r e . Assignments o f c r o s s peaks i n d i c a t i n g c o u p l e d s p i n s i n the E - r i n g are shown w i t h tljie d o t t e d l i n e s . The c o r r e s p o n d i n g r e g i o n o f the one-dimensional H NMR s p e c t r a i s p r o v i d e d on the a b s c i s s a . The 2-D c o r r e l a t i o n map i s composed o f 128 χ 512 data p o i n t s p e c t r a , each composed o f 16 t r a n s i e n t s . A 4-s d e l a y was a l l o w e d between each p u l s e sequence ( T ^ ) and t ^ was incremented by 554s. Data was a c q u i r e d w i t h q u a d r a t u r e phase d e t e c t i o n i n both dimensions, z e r o f i l l e d i n the t ^ dimension, and the f i n a l 256 χ 256 data was symmetrized. T o t a l time of the experiment was 2.31 h ( 1 7 ) .


LYNN


(Tw-90 A-tl-90 A-t2) e

n


e

F i g u r e 18. A 0.2-s d e l a y e d COSY spectrum o f the a l i p h a t i c r e g i o n of 10 (2mg, C D C L ) . Long-range W - t y p e c o u p l i n g o f 19 and 21 a x i a l p r o t o n s t o 30-CH and c o u p l i n g a c r o s s the gem d i m e t h y l s from 19eq t o 21 eq e s t a b l i s h the p o s i t i o n o f o x i d a t i o n a t C-22. The spectrum was o b t a i n e d under c o n d i t i o n s s i m i l a r t o those i n F i g u r e 1, except t h a t 32 t r a n s i e n t s were a c q u i r e d f o r each o f 128 χ 512 data p o i n t s p e c t r a ( 1 7 ) . M

M

3

3



76


panoids such as xenognosin A and Β which might be exuded by plants biosynthesizing molecules of this type. Seeds of Lespedeza sericea were surface s t e r i l i z e d by soaking i n Captan (1 g/1) 1 h, 35% Clorox for 20 min, and washing with IN HCI and water. For quantitation of components i n Lespedeza roots, seeds were sown on moistened f i l t e r paper i n p l a s t i c P e t r i dishes. At 15d post-germination, roots (70 for isoflavone analysis; 210 for triterpenes) were dissected o f f , frozen and l y o p h i l i z e d . The dried tissue was then homogenized with 4 ml methanol and further extracted with 3 additional volumes of the same solvent. After f i l t r a t i o n through glass wool and concentration i n vacuo at 30°C, the extracts underwent droplet counter-current chromatography i n 7:13:8 chloroform/methanol/water (7:13:8, v/v) run i n the descending mode with the organic phase mobile. Fractions corresponding i n retention time to the soyasapogenols and genistein were collected, pooled and concentrated i n vacuo. Each f r a c t i o n was then passed over a s i l i c a gel column 0.5 χ 2 cm eluted i s o c r a t i c a l l y with 8% methanol d i c h l o romethane (v/v). The eluate was then concentrated iji vacuo and f i l tered p r i o r to HPLC. HPLC analyses for genistein (65% Me0H:H20 (v/v), 260 nm detection) and for the terpenoids (84% Me0H:H 0 (v/v), 214 nm detection) were carried out on a Waters 5V C±q reverse phase column eluted at 1 ml/min. For quantitation of root exudate components, surface s t e r i l i z e d Lespedeza seeds were allowed to germinate and the seed coats were separated from the young plants. At 3d post germination the root of each plant was inserted through Nitex mesh suspended over water i n a s t e r i l e P e t r i dish. In this way, plants were supported by the mesh so that only the roots were allowed to come into contact with the water. Every 3d for the next 9d, exudate was removed and repla ced with s t e r i l e d i s t i l l e d water; plates were discarded when signs of contamination appeared. Exudate collected i n t h i s way from 3200 plants, f i l t e r e d through an 0.8 M i l l i p o r e f i l t e r and lyophi l i z e d , yielded 185 mg dry exudate. The l y o p h i l i z e d exudate was extracted with methanol and the r e s u l t i n g extract, after f i l t r a t i o n and concentration in vacuo, was chromatographed under conditions i d e n t i c a l to those described above for root extracts. Thin layer chromatograms and HPLC analyses showed a major ter penoid component that eluted with the synthesized 22-keto d i o l , 10. This compound, now known as soyasapogenol E, was present as a minor component i n the root extract, but i n exudate appears to be present i n l e v e l s equal to or exceeding soyasapogenol Β (Table II). Genis tein i s found i n the roots at levels comparable to that of soyasapo genol Β but i s exuded at levels about two orders of magnitude lower than the soyasapogenols. Also, the r a t i o of soyasapogenol Β to Ε i n the root i s 6 to 1, whereas greater l e v e l s of soyasapogenol Ε are exuded than soyasapogenol Β. This apparent selective exudation may either represent an active aspect of root metabolism or r e f l e c t c e l l u l a r compartmentalization of secondary metabolites and their passive leakage from the root. B e l l and co-workers (31-32) have shown a compartmentalization of plant chemical defenses i n the cot ton root, with triterpenoid synthesis occurring i n the epidermis, proanthocyanidins i n the hypodermis, sesquiterpenoids i n the cortex, and proanthocycanidins i n the endodermis. I f a s p a t i a l d i s t r i b u t i o n of this kind i s maintained i n Lespedeza sericea, this could account 2



5. LYNN

Table I I .

Root Exudate and Extract Quantitation Lespedeza sericea Soyasapogenol Ε

Genistein

291 ± 1 (0.06)

50.0 ± 0.2 (0.01)

304 ± 6 (0.03)

42.8 ± 0.1 (0.34)

56.7 ± 0.1 (0.45)

0.422 ± 0.01 (0.002)

Soyasapogenol Β Root Extract pmol/root (% dry wt root)


77

Root Exudate (3245 plants) pmol exuded/root/day (% dry wt exudate)

Each figure represents the mean and standard error of three r e p e t i tions. The exudation pattern from the f i r s t 5 days of c o l l e c t i o n does not appear to d i f f e r from the second 5 days of c o l l e c t i o n . The r e l a t i v e levels of soyasapogenols and genistein within the root are not d i f f e r e n t at 15 days after germination, nor do they d i f f e r at 3 months of age. Soyasapogenols were not detected i n shoot portions of seedlings. Variance i n root size of 5-day old plants was not taken into account i n either the c a l c u l a t i o n of pmol/root or pmold exuded/root. The weight of f i l t e r e d and l y o p h i l i z e d exudate of 375 plants was used to extrapolate to the figure of 3245 i n d i v i d u a l s used for exu date c o l l e c t i o n i n the analysis of exudation/root. for the d i f f e r e n t i a l exudation of r e l a t i v e l y large quantities of epidermally l o c a l i z e d soyasapogenol Β and E, while the isoflavone genistein, occurring i n large quantities i n c e l l s d i s t a l to the e p i dermis, would be represented at much lower l e v e l s i n the root exu date. A l t e r n a t i v e l y , the exudation of the soyasapogenols, which are probably not d i f f e r e n t i a l l y l o c a l i z e d i n the root, could represent a more active process. According to the data i n Table II, by day 15 approximately 94% of the soyasapogenol Ε synthesized i n the root has been released into the root exudate. In contrast, only 69% of the soyasapogenol Β synthesized i n the root i s found i n root exu date. Thus, the d i f f e r e n t i a l secretion of soyasapogenol Β and Ε may account for the observed r a t i o of soyasapogenol Β to Ε within the root. Conclusion Several naturally occurring compounds have now been characterized which are capable of inducing the d i f f e r e n t i a t i o n of the haustorium i n Agalinis purpurea. They f a l l into two s t r u c t u r a l classes; the phenylpropenoids, xenognosin A and B, and a terpenoid, soyasapoge nol B. Both the biosynthetic connection of the xenognosins with the taxononically useful flavanoids and the s t r u c t u r a l s p e c i f i c i t y of their b i o l o g i c a l a c t i v i t y suggest that these compounds are i d e a l l y suited as host recognition cues for p a r a s i t i c plants. The con nection between flavanoids and root allelopathy (33) and the speci f i c connections established by Dewick (15) and Carlson and Dolphin (16) tying the xenognosins i n with plant stress metabolites again



78


connects these compounds to s p e c i f i c , stable metabolites and there fore are l i k e l y cues of a suitable host. The strengths of the arguments i n favor of phenylpropenoid r e cognition cues i d e n t i f i e d i n the nonhost gum tragacanth made the i s o l a t i o n of the terpenoid from Lespedeza quite unexpected. However, many of the arguments supporting the phenolics also support the terpenoid. The haustorial inducing a c t i v i t y of soyasapogenol Β i s dependent on certain s t r u c t u r a l features. Neither the 22-keto d e r i vative, soyasapogenol E, nor a series of olean-12-ene triterpenes from cactus (generously supplied by Professor Kirchner, University of Arizona) possessed any detectable a c t i v i t y . The soyasapogenols have only been found i n the Leguminosae and therefore may have re s t r i c t e d occurrence, but their taxonomic usefulness i s not as well established as the flavanoids . The soyasapogenols have also been attributed with defensive r o l e s . In their glycosylated form, they are toxic and reduce herbivory i n leguminous seeds (34). A group of more highly oxidized oleanene triterpenes, the averacins, are found i n oat roots and are potent resistence factors to "take a l l " disease caused by the fungus Gaeumannomyces graminis (35). I f Lespedeza and genistein exudation can be taken as representative, then i t seems unlikely that quantities of these phenolics s u f f i c i e n t for the induction of haustoria would be exuded. Therefore the s t r u c t u r a l s p e c i f i c i t y , the stable and s p e c i f i c metabolic produc tion, and Lespedeza s metabolic control over the exudation of the soyasapogenols suggest that the terpenoids are more appropriate recognition cues for these parasites. The haustorial inducing a c t i v i t y of soyasapogenol Β i s , how ever, much weaker than that of xenognosin A. In the f i l t e r paper disk method, 20 nmol of soyasapogenol Β i s the lowest quantity which w i l l induce haustoria, whereas xenognosin A w i l l induce large numbers of haustoria at 1 nmol. Haustoria are not induced when soyasapogenol Β i s presented as a 10 uM solution but xenognosin A i s quite active at the same concentration. Unlike the xenognosins which constitute v i r t u a l l y a l l of the a c t i v i t y of gum tragacanth, the soyasapogenol Β a c t i v i t y represents only a portion of the a c t i v i t y of Lespedeza. The continued reduction i n b i o l o g i c a l a c t i v i t y during the fractionation greatly complicated the work with Lespedeza u n t i l John Steffens was able to demonstrate a dramatic increase i n the a c t i v i t y of soyasapogenol Β when i t was combined with another weakly active f r a c t i o n . No such s y n e r g i s t i c stimu l a t i o n of a c t i v i t y has ever been detected with the xenognosins and this demonstration with the terpenoids may indicate a considerably more sophisticated, multi-component system of host recognition than previously expected. At present, there i s no data to suggest that any of the s y n e r g i s t i c a l l y acting substances are phenolic although very l i t t l e about the structure of these compounds i s known. The remarkably rapid process of c e l l d i v i s i o n and d i f f e r e n t i a tion which leads to the complete formation of the haustorium f o l lowing stimulation suggests that a parasite c e l l or group of c e l l s are poised and ready for immediate response. I t therefore may be reasonable for a b i o l o g i c a l l y active allelochemical, through some fundamentally d i f f e r e n t pathway, to t r i p the i n i t i a l physiological events i n the induction of the haustorium. In fact, xenognosin A can induce haustorial development across a broad range of p a r a s i t i c 1

1



5.

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Parasitic Angiosperms: Allelochemicals and

Host Selection

79

angiosperms, including A g a l i n i s , Striga a s i a t i c a (unpublished re s u l t s ) , and Sopubia d e l p h i n i t o l i a (Sahai and Shiranna, pers. comm.). Soyasapogenol Β only shows a c t i v i t y i n the A g a l i n i s system although the synergistically active components have not been vigorously tested on other parasites. Only through more work on t h i s system and the investigation of other p a r a s i t i c plants w i l l the role of the haustorium i n host se l e c t i o n be understood. The p a r a s i t i c plants have provided a b i o l o g i c a l system to direct the i s o l a t i o n and i d e n t i f i c a t i o n of natural l y occurring allelochemicals which have profound physiological ef fects on these plants. The c e l l s that ultimately lead to the haus torium can then be chemically manipulated with the very r e a l hope of c o n t r o l l i n g haustorial expression. The s p e c i f i c chemical control of t h i s organ, an organ found only i n the p a r a s i t i c plants, provides a fundamentally new and very s p e c i f i c way of combatting the a g r i c u l t u r a l l y devastating parasites such as S t r i g a and Orobanche. The technologies for the further study of these systems and other a l l e 1ochemical based systems are now i n place i n several laboratories and the next ten years o f f e r to provide rewarding insights into the chemical basis of b i o l o g i c a l recognition phenomena. Acknowledgments The progress i n these studies would never have been possible without the collaborative e f f o r t between this labora tory and that of Professor James Riopel at the University of V i r g i n i a . The funding provided by USDA Competitive Research Grant 5901-0410-9-0257 and USDA Cooperation Agreement 58-7B30-0-196 made t h i s collaboration possible. We also g r a t e f u l l y acknowledge the funding from Research Corporation and the Frasch Foundation. I applaud the e f f o r t s of John Steffens, who has contributed i n a major way to every aspect of t h i s work; Dr. Vinayak Kamat and David Graden, whose synthetic and spectroscopic talents proved invaluable; and Professor Jim Roark, who i n a r e l a t i v e l y short time made a l a s t i n g contribution to the work. L i t e r a t u r e Cited 1.

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Lynn, D. G.; Steffens, J . C.; Kamat, V. S.; Graden, D. W.; Shabanowitz, J . ; Riopel, J . L. J . Am. Chem. Soc. 1981, 103, 1868-70. 7. Graden, D. W. Ph.D. Thesis, University of V i r g i n i a , V i r g i n i a , 1984. 8. Simulations were performed on an NTC 1280 computer with a program similar to the LAOCOON program. Castellano, S. Α.; Bothner-By, A. A. J . Chem. Phys. 1964, 41, 3863. 9. Bachers, G. E.; Schaefer, T. Chem. Rev. 1971, 71, 617-26. Noggle, J . H.; Schirmer, R. E. "The Overhauser E f f e c t : Chem ical Applications"; Academic Press: New York, 1971. 10. A l l MS/MS data was obtained on a Finnigan 3200 spectrometer modified f o r triple quadrapole work. 11. Egushi, S.; Haze, M.; Nakayama, S.; Hayashi, S. Org. Mass Spec. 1977, 12, 51-2. 12. Kamat, V. S.; Graden, D. W.; Lynn, D. G.; Steffens, J . C.; Riopel, J . L. Tetrahedron L e t t . 1982, 1541-44. 13. Steffens, J . C.; Lynn, D. G.; Kamat, V. S.; Riopel, J . L. Ann. Bot. 1982, 50, 1-7. 14. B e l l , A. A. Ann. Rev. Pl. Physiol. 1981, 32, 21-81. 15. Dewick, P. M. J. Chem. Soc. Chem. Comm. 1975, 656-8. 16. Carlson, R. E.; Dolphin, D. H. Phytochem. 1982, 21, 1733-36. Carlson, R. E.; Dolphin, D. H. Phytochem. 1981, 20, 2281-84. 17. Steffens, J . C.; Roark, J . L.; Lynn, D. C.; Riopel, J . L. J . Am. Chem. Soc. 1983, 105, 1669-71. 18. High resolution EI MS was obtained through Harvey Laboratories, Inc. on a VG 7070. 19. Budzikiewicz, H.; Wilson, J . M.; Djerassi, C. J . Am. Chem. Soc. 1963, 85, 3688-3699. 20. Hunt, D. R.; Sethi, S. K. J. Am. Chem. Soc. 1980, 102, 6953-63. 21. Isuda, Y.; Sano, T.; Isobe, K.; Miyauchi, M. Chem. Pharm. B u l l . 1974, 22, 2396-2401. 22. P r e i r r a d i a t i o n (selective 180°) of 24-H' was followed after a delay (0.7 s) by a 90° observed pulse. This spectrum (550-1000 transients) was acquired simultaneously with a spectrum i n which one selective pulse was 3 ppm upfield of Me Si, and the two spectra were computer subtracted to observe the enhance ments. Solomon, I. S. Phys. Rev. 1955, 99, 559-565. H a l l , L. D.; Saunders, J . K. M. J . Chem. Soc., Chem. Commun. 1980, 368-370. 23. Chen, M. T.; Barbalas, M. P.; Pegues, R. F.; McLafferty, F. W. J . Am. Chem. Soc. 1983, 105, 1510-1513. 24. Itô, S. in "Natural Products Chemistry"; Nakanishi, K.; Goto, T., Itô, S., Natori, S., Nozoe, S., Eds.; Kodansha L t s . : Tokyo, 1974, pp. 365-366. 25. McConnell, H. M.; Robertson, R. E. J. Chem. Phys. 1958, 29, 1361-65. 26. Benn, R.; Günther, H. Angew Chem. Int. Ed. Engl. 1983, 22, 350-380. 27. Lynn, D. G.; P h i l l i p s , N. J . ; Hutton, W. C.; Shabanowitz, J . ; Fennel, D. I.; Cole, R. J. J . Am. Chem. Soc. 1982, 104, 731922. 28. Bax, Α.; Freeman, R.; Morris, G. J . Magn. Reson. 1981, 42, 1648. Bax, Α.; Freeman, R. J . Magn. Reson. 1981, 41, 542-61. 4


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R E C E I V E D August 16, 1984


The Chemistry of Allelopathy - American Chemical Society

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