Naturally Occurring Pest Bioregulators - ACS Publications - American

Chapter 15

Arthropod-Resistant and -Susceptible Geraniums Comparison of Chemistry D. Hesk , L. Collins , R. Craig , and R. O. Mumma Downloaded by NORTH CAROLINA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch015

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Department of Entomology, Department of Chemistry, and Department of Horticulture, Pesticide Research Laboratory and Graduate Study Center, Pennsylvania State University, University Park, PA 16802

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Glandular trichome exudate from insect resistant and susceptible geraniums (Pelargonium xhortorum) was chemically analyzed to d e t e r m i n e differences between the two plant lines. Hplc analysis of t h e exudate from resistant plants s h o w e d that it w a s p r e d o m i n a n t l y made up of u n s a t u r a t e d a n a c a r d i c acids, with the C ω5 and C ω5 anacardic acids contributing nearly 8 0 % of the total. By contrast, the e x u d a t e from t h e s u s c e p t i b l e plants w a s chiefly saturated, with the C and C saturated anacardic acids contributing nearly 5 0 % of the total. The C ω5 and C ω 5 anacardic acids were only present in trace a m o u n t s . In addition a number of other significant peaks o b s e r v e d in the c h r o m a t o g r a m s of extracts f r o m s u s c e p t i b l e plants, a n d also seen in small amounts in the resistant profile, were isolated a n d characterized by m a s s spectrometry a n d nmr spectroscopy. Two unsaturated anacardic acids, which contributed nearly 3 0 % of the total exudate in the susceptible plant, and only seen in trace amounts in t h e resistant plant, were identified as C ω6,9 and C ω9 anacardic acids. A number of o d d chain length and branched chain anacardic acids, were also isolated a n d identified. 22

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0097-6156/91/0449-0224$07.75/0 © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Downloaded by NORTH CAROLINA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch015

15.

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Arthropod-Resistant and -Susceptible Geraniums 225

The common garden geranium (Pelargonium xhortorum) has been shown to possess a potent chemical defense consisting of glandular trichomes which secrete a viscous sticky exudate.(Iz5) T h i s s t r a t e g y , w h i c h is p r i m a r i l y a p h y s i c a l entrapment mechanism, rather than a toxic o n e , is effective against small a r t h r o p o d species, a n d may also provide resistance against larger herbivores. The exudate from the trichomes w a s found to be comprised of a mixture of anacardic acids, which in the mite resistant plants, w a s s h o w n to be mainly C22 a n d C24 ω 5 unsaturated anacardic acids. Small amounts of t h e saturated a n a l o g u e s w e r e also f o u n d in t h e e x u d a t e f r o m resistant p l a n t s ( l j i 4 - ) . A n a l y s i s of e x u d a t e f r o m mite susceptible geraniums, showed that it contained the same four compounds, but with the saturated materials predominating (5. 6 ) . A n a l y s e s w e r e performed on a number of resistant a n d susceptible plants in order to determine whether the chemical composition w a s constant within each plant line. While it w a s found that the chemical profile of the resistant plants w a s fairly constant, the exudate from susceptible plants exhibited v a r i a b i l i t y . F u r t h e r m o r e , on c l o s e e x a m i n a t i o n of t h e g a s chromatographic trace of the exudate from susceptible plants, it was determined that in addition to the four previously identified c o m p o u n d s , there were a number of other significant peaks, w h i c h were unidentified. In addition, as all t h e peaks eluted close together, it w a s possible that the apparent variability w a s due to unresolved peaks (5. 6). Hence, t h e objectives of this study w e r e to d e v e l o p an improved separation of the anacardic acids, to characterize the unknown compounds in order to fully compare the resistant and s u s c e p t i b l e plants a n d to d e t e r m i n e w h e t h e r t h e e x u d a t e composition w a s indeed constant within both t h e resistant and susceptible lines.

Experimental Plant S o u r c e . Geraniums were maintained in a g r e e n h o u s e environment using standard cultural practices. The plant line 7 1 17-7 and its corresponding self-pollinated progeny, family 8 7 - 5 , had been previously determined to be mite a n d aphid resistant, a n d plant line 71-10-1 a n d its c o r r e s p o n d i n g self-pollinated progeny, families 85-26, 87-13 and 87-14, had previously been determined to be mite and aphid susceptible (Z). Newly opened

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

226

NATURALLY OCCURRING PEST BIOREGULATORS


inflorescences, from 12 plants, 6 resistant and 6 susceptible, w e r e s e l e c t e d from r a n d o m locations in the g r e e n h o u s e for chemical analysis. Isolation and purification of exudate. The exudate from both the resistant and susceptible plants was collected by immersing the inflorescences (minus the petals) in methylene chloride for one hour. The extract was then dried over anhydrous sodium sulfate, filtered and evaporated to leave the crude anacardic acid residue. This p r o c e d u r e had p r e v i o u s l y been s h o w n to p r o d u c e a representative extract of anacardic acids (5. 6 ) . The carboxyl group in the anacardic acids w a s methylated ( d i a z o m e t h a n e - d i e t h y l ether, 5 minute t r e a t m e n t ) before the acids were separated from the other extracted material by thin layer c h r o m a t o g r a p h y (tic). Merck 2 0 x 2 0 c m silica gel F254 plates with a pre-concentration zone were used. The plates were developed in a two step solvent system consisting of benzene: diethyl ether: ethanol: acetic acid (50: 4 0 : 1: 0.5, by volume) followed by hexane: diethyl ether: acetic acid (84: 15: 1, by volume). Development was allowed to proceed to about half the length of the plate in the first step and the entire length of the plate in the second. The monomethylated anacardic acid band was located by viewing at 254nm and comparing the Rf value with that of a monomethyl anacardic acid standard, before it w a s s c r a p e d from the plate a n d eluted with m e t h y l e n e c h l o r i d e (3x2mL). The methylene chloride extract was filtered through a pasteur pipette containing a glass wool plug, and then evaporated to leave the purified anacardic acids. Finally the phenolic group on the anacardic acids was methylated (diazomethane-diethyl ether plus a few drops of methanol, 30 minute treatment (3.)) to give the dimethylated anacardic acids. Bulk Extraction of Anacardic Acids for NMR. Separate batches (2.5Kg each) of resistant and susceptible inflorescences w e r e s o a k e d in m e t h y l e n e chloride for one hour. The m e t h y l e n e chloride extracts were dried, filtered and evaporated as d e s c r i b e d a b o v e to leave c r u d e resistant a n d s u s c e p t i b l e exudates. A preliminary clean up of each was performed using c o l u m n c h r o m a t o g r a p h y . Each crude extract w a s dissolved in hexane (15mL) and 5mL of that w a s applied to a silica gel chromatography column (18cm χ 2cm) and eluted successively with (i) hexane (100mL), (ii) 4 % diethyl ether, 1 % acetic acid in



15. HESK ET AL.

Arthropod-Resistant and -Susceptible Geraniums

227

hexane (200mL), (iii) 10% diethyl ether, 1 % acetic acid in hexane ( 2 0 0 m L ) , (iv) 5 0 % diethyl ether, 1 % acetic acid in hexane (200mL) and (v) 2 5 % methanol in diethyl ether (100mL). The c o l u m n eluent w a s collected in 10mL t u b e s and each w a s analyzed by tic in order to determine the elution point of the anacardic acids. The tubes containing the anacardic acids were combined and subsequently pooled with the acids collected from the other two column runs. T h e p a r t i a l l y p u r i f i e d a n a c a r d i c a c i d s , f r o m b o t h the resistant and susceptible plants were further cleaned up prior to hplc separation using Merck 20x20cm, 2mm thickness preparative tic plates. The compounds were applied to several plates as the dimethylated compounds (diazomethane, 30 minute t r e a t m e n t (fi.)), and run in the same two step solvent system described above. The plates were v i e w e d at 2 5 4 n m and the anacardic acid bands were cut and eluted from the silica using methylene chloride (3x30mL). The purified anacardic acids were then ready for separation by preparative hplc. HPLC Analysis of Anacardic Acids. The anacardic acids collected from both the resistant and susceptible plants were analyzed by high performance liquid chromatography (hplc) using a 25cm χ 4 . 6 m m Supelcosil 5μ LC8 DB column. The solvent system used consisted of isopropanol, acetonitrile, 0.01 M acetic acid (49: 14: 37, by volume) and was run isocratically at 1.2mL/min. Detection was achieved using a Waters 490 uv detector at 212nm, with the trace being recorded on a Shimadzu 3A recorder-integrator. In preparative chromatography of the the bulk extract, a 2 5 c m χ 10mm Supelcosil 5μ LC8 DB semi-preparative column was used. In this case the mobile phase was modified to isopropanol, acetonitrile, 0.01 M acetic acid (49: 15: 30, by volume) and run i s o c r a t i c a l l y at 3 m L / m i n . T h e e l u t i n g s o l v e n t f r o m each a b s o r p t i o n peak w a s collected into s e p a r a t e vials, a n d the solvent was removed by rotary evaporation. Each anacardic acid was further purified by hplc, to achieve a purity of greater than 95%. GC-MS Analysis. The d i m e t h y l a t e d a n a c a r d i c acids w e r e analyzed by gas chromatography-mass spectrometry (gc-ms). All analyses were performed in the El mode, using a Kratos model MS-25 mass spectrometer, with a 30m χ 0.53mm RTX-5 (Restek Corp., cross bonded 9 5 % d i m e t h y l - 5 % diphenyl polysiloxane) capillary column with a temperature program of 2 5 0 - 2 8 0 ° C at 5°C/minute.


228



NMR Analysis. The dimethylated anacardic acids were analyzed by proton nmr s p e c t r o s c o p y . All s a m p l e s w e r e d i s s o l v e d in deuterochloroform and were run at 360Mz on a Bruker AM 360. D i m e t h y l Disulfide D e r i v a t i z a t i o n . Dimethyl disulfide (DMDS) derivatization was performed on the unsaturated anacardic acids in order to locate the position of the double bond (2.)· A solution of the anacardic acid (0.05-0.1 mg) w a s d i s s o l v e d in hexane (1.75mL). DMDS (2.5mL) and iodine solution (0.25mL of a 60mg I2/1T1L ether) were added to the reaction vial, and the reaction w a s allowed to run overnight at 40°C. After the reaction w a s complete, 5mL of a 5% (w/v) solution of sodium thiosulfate was added to the reaction mixture to remove the excess iodine. The organic layer was removed, and evaporated to leave the crude D M D S d e r i v a t i v e , which w a s purified by hplc, before being submitted for mass spectrometric analysis.

Results. HPLC Analysis. Hplc analysis of the resistant exudate (Figure 1) showed that there are two major compounds which are known to be the C22 ω5 (compound B) and C24 ω5 (compound I) unsaturated a n a c a r d i c acids from earlier work, and a n u m b e r of minor compounds of which only the C22 saturated (compound G) and C24 saturated (compound M) anacardic acids have previously been identified ( 2 - 6 ) . Of the uncharacterized compounds, Ε and J are of particular interest, as they are present in similar quantities to the C22 and C24 saturated anacardic acids. This analysis is in good agreement with the capillary gc method used by Walters, who identified the four major anacardic acids and found them in similar quantities to those seen in the hplc trace. Hplc analysis of the susceptible exudate by contrast revealed some differences in the composition to that reported by Walters (5. 6). A very complicated profile w a s seen, with at least ten peaks each contributing over 1 % to the total exudate composition (Figure 2). Of those, only the C22 saturated (compound G) and C24 saturated (compound M) anacardic acids were known and hence the majority of the anacardic acids making up the exudate were u n k n o w n s t r u c t u r e s . T h u s t h e r e w a s c l e a r l y a n e e d to characterize the unknown c o m p o u n d s to obtain a full chemical profile of the susceptible exudate. Of particular interest was the almost complete lack of the C22 ω 5 (compound B) and C24 ω 5




15. HESK ET AL.


229


Figure 2: HPLC Profile of Glandular Trichome Exudate From Susceptible Geraniums


15.

HESK ET AL.



(compound I) anacardic acids, contrasting with the analysis by Walters, who observed that they were present in amounts varying from 3 0 % to 6 0 % of the total susceptible exudate composition ( i L fi). In o r d e r to resolve this a m b i g u i t y , the major p u r i f i e d anacardic acids from the hplc were analyzed under the same gc conditions used by Walters (Table I).

Table I: HPLC and GC Retention Times of Anacardic Acids Code A Β C D Ε F G H I J Κ L M N 0

Name C20 sat C22 ω5 C21 anteiso C24 ω6,9 C23 ω6 C22 iso C22 sat. C24 ω9 C24 co5 C23 anteiso C23 s a t C24 iso C24 sat C25 anteiso C25 sat

HPLC Rt(min) 19.0 20.5 21.9 22.8 25.2

GC Rt(Min) nd 7.74

27.8 29.0 30.7

nd 7.02 9.50

31.5

9.82

33.6 36.1 43.2 45.2 52.5 56.7

7.81 8.11 8.73 9.29 9.94 nd

nd 10.11 nd

It was found that compound J, a major peak in the susceptible co-chromatographed with the C22 ω5 (compound B) peak on gc and c o m p o u n d s Η and Ν closely chromatographed with the C24 ω 5 (compound I) peak. Thus it appears that the difference in results can be accounted for by the improved separation achieved on the hplc over the capillary gc method, and hence what were believed to be ω 5 anacardic acids in the susceptible exudate were really other compounds which happened to co-chromatograph with them. Such a problem is not encountered in the resistant exudate, as the co5 anacardic acids are by far the major components.


232


Mass Spectral and NMR Analysis.


The mass spectral and nmr d a t a from each of the unknown anacardic acids are given in Tables II and III respectively, and the results for each compound are discussed in detail below. The spectra were also obtained for the previously known anacardic acids (Compounds B, G, I, and M) and are listed in Tables II and III for comparative purposes. However because their structures are already known (5. 6). they are not discussed below. C o m p o u n d A. The mass spectrum of dimethylated A showed a molecular ion at m/z 348 and a significant peak at m/z 3 1 7 , indicating a loss of - O C H 3 (Table II). The region from 100 to 200 m/z is practically identical in all the d i m e t h y l a t e d a n a c a r d i c acids under investigation, because as can be seen from Figure 3, none of the ions in this region contain the side c h a i n , which differentiates each anacardic acid. The base peak ion occurs at m/z 161 produced by the loss of water from the ion produced by α-cleavage of the side chain from the aromatic ring (m/z 1 7 9 ) . T h e r e are also substantial peaks at m/z 148 and m/z 121, indicating losses of - O C H 3 and - ( 0 = C ) - O C H 3 , respectively from the m/z 179 fragment. The principal resonances seen in the nmr spectrum of A, together with all the other anacardic acids isolated, are shown in T a b l e III. For the p u r p o s e s of clarity, the two s i g n a l s c o r r e s p o n d i n g to m e t h y l a t e d carboxyl ( 8 3 . 8 8 p p m singlet) and phenol (53.79ppm singlet) groups have been omitted, as have the aromatic signals. In all cases the aromatic region consisted of two pairs of doublets at 66.73 and 56.79ppm, and a double doublet at 57.23ppm, fully consistent with an ABC type system. T h e nmr s p e c t r u m of A s h o w s a triplet at 5 2 . 5 1 p p m corresponding to the methylene group adjacent to the ring (Table III). The methylene group beta to the ring can also be seen as a multiplet at 8 1 . 5 4 p p m , with remainder of the methylene groups present in an intense broad resonance at 61.23ppm. No resonances in the vinylic region of the spectrum are seen, thus confirming the result from mass s p e c t r o m e t r y that A is s a t u r a t e d . The remaining signal in the spectrum is the terminal methyl group at 5 0 . 8 7 p p m , which in this case is a simple triplet, integrating for three protons. Thus as there is only one methyl group present it can be concluded that the side chain in A is unbranched, and hence is identified as the C o straight chain saturated anacardic acid. 2



N 0

L M m

J Κ k

I

F G H

anteiso

C25 sat.

C25

C26 ω5

C24 sat.

C24 iso

C2sœ6

C24 ω5 C23 anteiso C23 sat.

C24 ω9

C22 iso C22 sat.

2

43 41 34 41

388

418 418

390 390 416 404 404 430

402

39 61

31 36 19

40 51 31

20

73 29

362 400

376 376 402

M 46 20

Mwt 348 374

18 29

16 20 19

16 22 30 31

24 23 31

27

44 62

52 59

51 49 32

50 55 41

51 55 98 37

91

52 64

100 100

100 100 100

100 100 100 100

100 100 100

100

100 100

INTENSITY (% BASE PEAK ION) M-31(2) M/Z 161 M/Z 180 31 100 51 15. 47 100

15 14

33 15 14 20 14 56 21

15 14 30

33

15 35

M/Z 148 15 79

22 19

23 17 31 22 24 34

32

23 15 45

36

18 38

M/Z 1 19 34

Mass Spectrometric Analysis of Anacardic Acids in Glandular Trichome Exudate

Name Code A C20 sat. Β C22 ω5 C C21 anteiso D C 4 ω6,9 Ε C23 co6

Table II:



0.87f

(Straight)

0.86f

(Anteiso)

0.83m

(Straight)

0.90Γ

(Straight)

0.86f

(Iso)

0.844

(Straight)

0.89f

(Straight)

0.89f

(Anteiso)

0.83m

(Straight)

0.87f

(Straight)

0.85f

(Straight)

0.86f

(Iso)

0.83d

(Straight)

0.86f

(Straight)

0.89f

(Anteiso)

0.85m

(Straight)

1.23m

1.23m

1.29m

1.23m

1.22m

1.29m

1.29m

1.22m

1.23m

1.26m

1.23m

1.24m

1.27m

1.30m

1.24m

1.24m

1.23m

2

CH C/fCH 2

1.53m

1.55m

1.56m

1.55m

1.52m

1.57m

1.56m

1.54m

1.55m

1.55m

1.55m

1.54m

1.53m

1.57m

1.54m

1.55m

1.54m

2

CH -CH-Ar

* m=*multiplet, Mriplet, ^ d o u b l e t , ctef-double doublet

0

N

m

M

L

k

Κ

J

I

H

G

F

Ε

D

C

Β

0.87r

(Straight)

A

3

CH

Compound

2.03m

2.01m

2.00m

1.98m

1.99m

2.7Qdd

2.04m

1.99m

2

CH -CH«

2.51 f

2.52f

2.54f

2.51 f

2.51 f

2.54f

2.54r

2.51 f

2.51 f

2.51 f

2.51 f

2.51/

2.50f

2.54f

2.52f

2.51 f

2.51/

2

CH -Ar

Table III: N M R Analysis of Anacardic Acids in Glandular Trichome Exudate*

5.0Hz

3.8Hz

5.36m J-4.5, 4.7Hz

5.36m J-4.5, 4.6Hz

5.33m J-4.5, 4.6Hz

J-3.6,

5.32m

J~5.2,

5.32m

5.38m

4.7Hz

5.32m

CH-CH

J-4.6,


15.

HESK ET AL.


OCH

3

COOCH3

M (-OCH or HOCH3)


v

(-R)

3

\ M-31.M-32

(-H2O)

(-OCH )>y 3

m/z148

m/z149

m/z 121

Figure 3: Mass Spectral Fragmentation of Dimethylated Anacardic Acids C o m p o u n d C. The mass spectrum again s h o w e d the c o m m o n anacardic acid ions, and a molecular ion of m/z 362, which suggested that Compound C was a C21 saturated anacardic acid. The nmr spectrum also indicated that C was saturated, and the t e r m i n a l methyl signal at 5 0 . 8 5 p p m was complexed and represented 6 protons. Hence C was identified as a C21 branched chain anacardic acid, and by comparison with the nmr spectra of iso and anteiso branched chain fatty acid standards, the terminal methyl signal was identical to the terminal methyl resonance of an anteiso group, thus C is in all probability the C21 anteiso saturated anacardic acid.


236



It should be noted t h o u g h , that although the nmr spectrum will eliminate the iso and straight chain isomers as possible structures for C, it is theoretically possible for the branch to be located in a position other than the anteiso. Such c o m p o u n d s would also give an nmr spectrum very similar to the anteiso, but as such c o m p o u n d s are relatively rare in plants, C can be classified as the C21 anteiso saturated anacardic acid, with a high degree of confidence. C o m p o u n d D. The mass spectrum of D gave a molecular ion of m/z 4 0 0 , and the expected ion at m/z 368 from the loss of C H 3 O H . Compound D was therefore identified as a C24 unsaturated anacardic acid, but containing two double bonds. Nmr a n a l y s i s of D c o n f i r m e d that the c o m p o u n d w a s u n b r a n c h e d (60.89ppm, triplet, 3 protons) and unsaturated, and furthermore indicated the presence of more than one double bond, due to the extra signal at 52.78ppm, and a highly complex vinyl signal at 55.38ppm, in addition to the resonance at 5 2 . 0 4 p p m . As the latter is due to a methylene group adjacent to a vinyl group, it was postulated that the resonance at 62.78ppm could be due to a methylene group located between two double bonds. However in order to establish the position of unsaturation, it is necessary to prepare the dimethyl disulfide (DMDS) derivative. DMDS simply adds across a double bond to leave a C H 3 S - group attached both carbons. The presence of the methyl thioether g r o u p s on adjacent c a r b o n s renders the bond between t h e m susceptible to cleavage during mass spectrometry, and hence it is possible to determine the position of the original double bond (a).

DMDS has previously been used to establish the location of double bonds in mono-unsaturated c o m p o u n d s (SL); however it appeared to work very well in the case of compound D, giving one major peak on the hplc. The mass spectrum turned out to be much more complicated than expected, because of partial decomposition of the derivative under the conditions employed. From Figure 4, the ion at m/z 2 9 1 , which is derived from the loss of C H 3 O H from m/z 323 places one double bond 9 carbons in from the terminal methyl group. The position of the second double bond was localized from the ion m/z 347, which arises from the loss of a C H S C H u n i t from the 9,10 carbon bond to leave a thioepoxide group, a break across the 6,7 carbon bond and a loss of C H S H . The ion at m/z 315 arises from the loss of C H O H from m/z 347. Hence the second double bond is located 6 carbons in from the terminal methyl group and therefore c o m p o u n d D is 3

3

3

3



m/2 478

\

•MeSH

m/2 399

- MeOH

m/2 431

^SMe

m/2 291 (B)

m/2 323

Figure 4: Mass Spectral Fragmentation of the DMDS Derivative of D

m/2 315

• MeSH

12

• C6H SMe

MeSMe

m/2 588


SMe

238


identified as being a C24 ω 6 , 9 unsaturated anacardic acid. The other major ions in the spectrum can be accounted for by loss of C H 3 S C H 3 from the molecular ion (not seen) to leave m/z 526. That ion itself loses CH3SH to give m/z 478, and further losses of S C H and CH3OH from m/z 478 account for the ions at m/z 431 and m/z 399. A small ion at m/z 464 is formed from the loss of two CH3SCH3 groups to leave the double thio-epoxide.


3

C o m p o u n d Ε. Ε was found to have a molecular ion at m/z 388 i n d i c a t i n g t h a t the s t r u c t u r e w a s c o n s i s t e n t w i t h a C 2 3 unsaturated anacardic acid. The mass spectrum of the DMDS derivative of Ε showed the expected molecular ion at m/z 482, and two significant fragments at m/z 319, and m/z 1 3 1 . The ion at m/z 131 is the fragment expected from the cleavage of the bond between adjacent methyl thioether groups, when the bond occurs six c a r b o n s from the methyl end of the side c h a i n . However, as anacardic acids containing branched chains had a l r e a d y b e e n i d e n t i f i e d , m/z 131 c o u l d also represent a fragment, with an iso or anteiso structure and hence the double bond would then occur 5 carbon atoms from the terminal methyl group. This ambiguity was easily solved by the nmr spectrum, which showed that the terminal methyl signal (80.86ppm) was a simple 3 proton triplet, which hence proved that the double bond was located at the ω 6 position. In addition coupling constants of 5.2 and 5.0Hz indicated that the the double bond was cis. Hence Ε is identified as the cis C23CÛ6 unsaturated anacardic acid. C o m p o u n d F. A molecular ion at m/z 376 and the knowledge that the retention time of F was slightly earlier than G, which was already known as the C22 straight chain saturated anacardic acid, strongly suggested that Compound F was a C22 branched chain saturated anacardic acid. The nmr of the terminal methyl signal (80.83ppm) showed it contained 6 protons, and was split into a simple doublet, consistent with an '/so type' end chain. Hence F is the C22 iso saturated anacardic acid. C o m p o u n d H . The mass spectrum shown in Figure 5 has a molecular ion at m/z 402 and the usual ions at m/z 180, 1 6 1 , 148 and 121 indicating that H is a C24 u n s a t u r a t e d a n a c a r d i c acid. The ion at m/z 370 is formed from the loss of C H O H from the molecular ion, The DMDS derivative gave the spectrum shown in Figure 6. The expected molecular ion at m/z 496 was seen and the fragment at m/z 173 located the position of unsaturation as 3




15. HESK ET A L


In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991. 350

ur* M/Z

4

0

0

450

Figure 6: El Mass Spectrum of the Dimethyl Disulphide Derivative of Dimethylated Compound H from Geranium

300


500

15.

HESKETAL.

Arthropod-Resistant and Susceptible Geraniums


being 9 carbon atoms from the end of the terminal methyl group. The fragment at m/z 2 9 1 , formed from the loss of CH3OH from m/z 323, supported this conclusion. In addition the nmr spectrum showed that the side chain was unbranched (50.85ppm, triplet), and also that the geometry of the double bond was c/s, due to J values of only 3.6 and 3.8Hz. Hence compound H is identified as the c/s C24 ω9 unsaturated anacardic acid. C o m p o u n d J . J was found to have a molecular ion at m/z 390, together with the characteristic ions in the 100 to 200 m/z r e g i o n , s u g g e s t i n g that the structure w a s a C23 saturated anacardic acid. As can be seen from Figure 7 a nmr analysis confirmed that J was saturated, and examination of the terminal methyl group signal (50.83ppm multiplet), s h o w e d it integrated for 6 protons and contained a splitting pattern consistent with anteiso branching. Hence J is more than likely the C23 anteiso saturated anacardic acid. C o m p o u n d K. The mass spectrum w a s practically identical to that of compound J , with the same molecular ion at m/z 390, but with a slightly later hplc retention t i m e , a n d hence it w a s thought likely that Κ was a C23 s t r a i g h t chained saturated anacardic acid. Confirmation of this was provided from the nmr spectrum which showed a simple three proton terminal methyl triplet at 50.89ppm, as well as the absence of any signals in the vinyl region. Hence Κ is the C23 straight chain saturated anacardic acid. C o m p o u n d k. This minor peak in resistant exudate, and not seen in the susceptible exudate, gave a molecular ion at m/z 416 suggesting that the structure was a C25 u n s a t u r a t e d a n a c a r d i c a c i d . The mass spectrum of the D M D S derivative g a v e the expected molecular ion at m/z 510, a fragment at m/z 131 and an ion at m/z 347, indicating that a 6 carbon fragment had been lost. However as in the case of Compound E, it was also possible that this 6 carbon fragment was branched, thus meaning that the double bond would be effectively located in the ω 5 position. This ambiguity w a s easily cleared up by the nmr s p e c t r u m which s h o w e d a s i m p l e triplet which i n t e g r a t e d for 3 protons at 50.89ppm, and also because of the small J values (4.5, 4.6Hz), the geometry of the double bond was shown to be cis. Hence k is the cis C25 ω6 unsaturated anacardic acid.


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C o m p o u n d L. The mass spectrum gave a molecular ion at m/z 4 0 4 t o g e t h e r with the other e x p e c t e d a n a c a r d i c acid ions. Furthermore as compound L had an earlier retention time on the hplc than compound M, the known C24 saturated straight chained anacardic acid, it was thought likely that L was a C24 b r a n c h e d chain saturated anacardic acid. The exact structure was confirmed by nmr spectroscopy (Figure 7b), which showed the terminal methyl group (50.84ppm) to be a 6 proton doublet, a splitting pattern consistent with iso branching. Thus L is the C24 iso saturated anacardic acid. C o m p o u n d m. This was another c o m p o u n d only seen in the resistant exudate, and from a molecular ion of m/z 430 it was identified as a C26 unsaturated anacardic acid. The spectrum of the DMDS derivative gave a molecular ion of m/z 524 and the fragment at m/z 117 placed the double bond 5 carbons in from the t e r m i n a l methyl g r o u p The ion at m/z 3 7 5 , w a s also c o n s i s t e n t with a 5 c a r b o n f r a g m e n t being lost. The nmr spectrum showed a simple triplet, integrating for 3 protons, at the t e r m i n a l methyl p o s i t i o n , s h o w i n g that the c h a i n w a s unbranched, and thus confirming the ω5 double bond position. In addition the small J values of 4.5 and 4.7 Hz placed the double bond geometry as c/s, and hence m is identified as the cis C26 ω5 unsaturated anacardic acid. Compound N. The spectrum showed the compound had a molecular weight of 418, consistent with a C25 saturated anacardic acid. Nmr analysis s h o w e d that the splitting of the terminal methyl group (80.83ppm multiplet, 6 protons) was complex and identical to the terminal methyl group splitting in the nmr spectrum of the anteiso standard. Hence Ν is in all probability the C25 anteiso saturated anacardic acid. C o m p o u n d O. This gave a practically identical spectrum to N, with the same molecular weight, and because of the slightly later retention time on the hplc it was strongly suspected that Ο was the C25 straight chain isomer. This was readily confirmed by nmr as the terminal methyl group at 50.86ppm was a simple 3 proton triplet, meaning that Ο is the C25 straight chain saturated anacardic acid.




15. HESK ET A L




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DigcUSSion. Having identified the majority of the anacardic acids in both resistant and susceptible exudate, it is possible to more fully highlight the differences between the two, than w a s previously the case.


Table IV: Percentage Composition of Anacardic Acids in Geranium Glandular Trichome Exudate. Code A Β C D Ε F G H I J Κ k L M m N 0

Name C20 sat C22 ω5 C21 anteiso C24 ω6,9 C23 (û6 C22 iso C22 sat. C24

Naturally Occurring Pest Bioregulators - ACS Publications - American

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