Harnessing Insect-Specific Enzymes to Activate Novel Proinsecticides

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 24, 2016 | http://pubs.acs.org Publication Date: June 26, 1984 | doi: 10.1021/bk-1984-0255.ch008

Harnessing Insect-Specific Enzymes to Activate Novel Proinsecticides GLENN D. PRESTWICH, RYOHEI YAMAOKA, SELOKA PHIRWA, ANGELO DEPALMA, MICHAEL ANGELASTRO, and APURBA K. GAYEN Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794 The 29-fluorophytosterols, members of a new class of selective pro-insecticides, have been synthesized and examined in vivo in tobacco hornworms. Dealkylation at C-24 of the steroid side chain by insects releases the latent poison fluoroacetate, resulting in dose-dependent reductions in growth rate, maximum weight, and survival when fed at 1 to 100 ppm to Manduca sexta. Larval steroid composition is unaffected. The effects of six related 29-monofluorosterols show that both the 22,23-and 24,28-double bonds increase toxicity dramatically. Inhibitors of sterol dealkylation suppress the toxicity of 29-fluorostigmasterol, whereas a cholesterol supplement does not alleviate the toxic effects. Metabolism of [29- H]-29-fluorostigmasterol releases tritiofluoroacetate, as demonstrated by the isolation and identification of [2- H]-erythro-2-fluorocitrate from in vivo experiments with 5th instar larvae. Rates of steroid dealkylation measured with [29- H]-phytosterols are higher in younger larvae, with sitosterol being more efficiently dealkylated than stigmasterol in a l l stages. 3

3

3

The b i o r a t i o n a l design of p e s t i c i d e s i n the agrochemical industry lags over a decade behind the analogous b i o r a t i o n a l design of drugs i n the pharmaceutical industry. This lag r e f l e c t s two primary problems: (1) poor understanding of i n s e c t biochemistry v i s JT v i s mammalian biochemistry, and (2) d i f f i c u l t y i n implementing the d e s i r a b l e switch away from cheap, broad-spectrum b i o c i d e s . R a t i o n a l l y - d e s i g n e d compounds which are substrate analogs f o r insect-unique enzymes s a t i s f y the prime c r i t e r i a f o r the development of future insect c o n t r o l agentsQ,2). 0097-6156/ 84/ 0 2 5 5 - 0 1 2 7 $ 0 6 . 0 0 / 0 © 1984 A m e r i c a n C h e m i c a l S o c i e t y

Magee et al.; Pesticide Synthesis Through Rational Approaches ACS Symposium Series; American Chemical Society: Washington, DC, 1984.


128

PESTICIDE SYNTHESIS THROUGH RATIONAL APPROACHES

In analogy to the use of prodrugs i n chemotherapyC3), enzyme-activated p r o - i n s e c t i c i d e s are p o s s i b l e , i n which the target enzyme would convert a less t o x i c precursor into an i n a c t i v a t o r of a s p e c i f i c p r o t e i n (see, f o r example, the chapter by T. Fukuto i n t h i s volume). There are two forms such p r o i n s e c t i c i d e s might take. Suicide substrates(4) can be designed such that a l a t e n t r e a c t i v e f u n c t i o n a l i t y i s both unmasked by and then i r r e v e r s i b l y binds to the enzyme a c t i v e s i t e . A l t e r n a t i v e l y , the enzyme may cause the release of a l a t e n t or l e s s r e a c t i v e toxicant from a nontoxic precursor, such that the enzyme system involved i n the l i b e r a t i o n of the toxicant i s d i f f e r e n t from that a c t u a l l y s u f f e r i n g the biochemically adverse e f f e c t s of the unmasked p o i s o n . Vfe have r e c e n t l y reported the s u c c e s s f u l harnessing of the insect phytosterol d e a l k y l a t i o n pathway to release f l u o r o a c e t a t e from 29-fluorophytosterols in Manduca sexta larvae jln v i v o ( 5 , 6 ) . In t h i s paper we present f u r t h e r evidence f o r (a) the dealkyation rates of f l u o r i n a t e d and n o n f l u o r i n a t e d phytosterols by hornworms, (b) the suppression of 29-fluorostigmasterol t o x i c i t y by chemical i n h i b i t i o n of d e a l k y l a t i o n , and (c) the i s o l a t i o n of t r i t i a t e d f l u o r o c i t r a t e as the ultimate biochemical l e s i o n r e s u l t i n g from 29-fluoro stigmasterol dealkylation. Insect s t e r o i d metabolism has two b i o c h e m i c a l l y d i s t i n c t i v e components: d e a l k y l a t i o n of p h y t o s t e r o l s to c h o l e s t e r o l and p o l y h y d r o x y l a t i o n of c h o l e s t e r o l to ecdysone. We w i l l focus on the f i r s t of these. Lacking the a b i l i t y to synthesize s t e r o l s de novo, i n s e c t s instead have evolved a d e a l k y l a t i o n pathway to convert plant s t e r o l s to c h o l e s t e r o l ( 7 - 1 0 ) . The d e a l k y l a t i o n pathways are apparently absent in most other higher and lower organisms, which can convert mevalonate to squalene and thence i n t o sterols(Γ1). S p e c i f i c i n s e c t i c i d e s are p o s s i b l e based on these biochemical d i f f e r e n c e s . S p e c i f i c a l l y , many phytophagous i n s e c t s degrade s i t o s t e r o l (l^X^H) v i a f u c o s t e r o l (2), f u c o s t e r o l epoxide (3>), and desmosterol (£) to c h o l e s t e r o l (^). Stigmasterol (6) i s degraded analogously v i a the s t i g j u a s t a t r i e n o l 7. ( F i g . 1 ) . The evidence for the steps i n d i c a t e d (dehydrogenation, epoxidation, fragmentation, and Δ^-reduction) has been obtained using a z a s t e r o l s as i n h i b i t o r s of Δ^Α- reductase, or using a l l e n i c and imino f u c o s t e r o l analogs as i n h i b i t o r s of epoxidation and dealkylation(7-10). Although d e a l k y l a t i o n i s r e s t r i c t e d to arthropods, not a l l i n s e c t s possess t h i s a b i l i t y , nor do a l l insects employ the same s t e r o i d nucleus. In general, phytophagous i n s e c t s are capable of d e a l k y l a t i o n w h i l e zoophagous i n s e c t s lack t h i s a b i l i t y . Although about twelve d i f f e r e n t insects(7,8,12) have been examined, the d i s t r i b u t i o n of d e a l k y l a t i v e enzymes both w i t h i n and outside the c l a s s I n s e c t a i s poorly known. This i s l a r g e l y due to the cumbersome task of f o l l o w i n g the conversion of a



F i g u r e 1.

6. STIGMASTEROL

I, SITOSTEROL(X=H)

N

Steroid

OH N

OH

dealkylation

2, FUCOSTEROL

OH

pathway i n i n s e c t s .

N

4, DESMOSTEROL

*0Η

5, CHOLESTEROL

*0Η


130 14


C

- r i n g l a b e l l e d p h y t o s t e r o l to the dealkylated form. We have attempted to remedy t h i s s i t u a t i o n by developing r a p i d , more s e n s i t i v e assay u s i n g [29-^H] p h y t o s t e r o l s and d o u b l y - l a b e l l e d [29- H], [ 2 6 - ^ C ] - s t e r o l s . P a r t i t i o n assay f o r d e a l k y l a t i o n


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A r a p i d and s e n s i t i v e method f o r determining the extent and rate of p h y t o s t e r o l d e a l k y l a t i o n i n vivo o r i n v i t r o has been developed r e c e n t l y i n our l a b o r a t o r i e s ( 1_3). The method i s based on the use o f [29-^H]-phytosterols ( F i g . 2) as substrates which then release [ H ] - a c e t a t e (or i t s metabolic equivalent) upon d e a l k y l a t i o n . Following i n vivo or iri v i t r o i n c u b a t i o n with the [29-^H] s t e r o l substrate, the i n s e c t or tissue i s homogenized and p a r t i t i o n e d between water and e t h y l a c e t a t e . L i q u i d s c i n t i l l a t i o n counting of the two layers gives a d i r e c t measure of d e a l k y l a t i o n , since the net change due to d e a l k y l a t i o n i s transformation of l i p i d - s o l u b l e [^Hj-sterol to aqueous [ H ] acetate (and i t s subsequent entry into other pathways). With d o u b l y - l a b e l l e d s t e r o l s containing l^C £ the s t e r o i d nucleus, the change i n 3H/14Ç r a t i o can define the degree of d e a l k y l a t i o n even more p r e c i s e l y . To test t h i s method, we f i r s t employed Manduca sexta t h i r d and f i f t h - i n s t a r larvae and pupae, using both [29-**H]-sitosterol ( s p e c i f i c a c t i v i t y 0.55 mCi/mmole or 2.91 χ 10^ dpm/mg) and [29-^H]-stigmasterol ( s p e c i f i c a c t i v i t y 0.55 mCi/mmole o r 2.90 χ 1θ6 dpm/mg) synthesized i n our l a b o r a t o r i e s ( 1 4 ) . These r e l a t i v e l y low s p e c i f i c a c t i v i t i e s were chosen to optimize t o t a l recovery of r a d i o a c t i v i t y , which was poor at 100-fold higher specific activities. Pupae were i n j e c t e d abdominally and l a r v a e were i n j e c t e d p e r o r a l l y with 5 HI of a dimethylformamide (DMF) s o l u t i o n of the [29- H] s t e r o l (100,000 dpm per i n d i v i d u a l f o r pupae and 5th i n s t a r s , 20,000 dpm X 5 larvae for t h i r d i n s t a r s ) and then frozen at the times i n d i c a t e d . D e a l k y l a t i o n was c a l c u l a t e d on a per gram fresh weight b a s i s from the aqueous dpm at each time point c o r r e c t e d f o r the aqueous dpm a t zero time. "Percent d e a l k y l a t i o n " was a l s o c a l c u l a t e d by d i v i d i n g the c o r r e c t e d aqueous dpm by the t o t a l recovered dpm. Recovery o f a p p l i e d r a d i o a c t i v i t y was i n i t i a l l y >95% and declined i n a roughly l i n e a r manner t o 80% a f t e r 8 h r , r e f l e c t i n g e x c r e t i o n and r e d i s t r i b u t i o n . Less than 1% o f i n j e c t e d [3H]-acetate was converted i n t o l i p i d s during an 8 hr i n c u b a t i o n . High aqueous dpm at zero time (up to 4% o f applied dpm for pupae) were a t t r i b u t e d to the presence o f DMF and p r o t e i n s i n the aqueous l a y e r which could s o l u b i l i z e the l a b e l l e d s t e r o l . Table 1 shows the p r e l i m i n a r y r e s u l t s at two time points f o r the nanomoles of two [ 2 9 - H ] - s t e r o l s dealkylated per gram fresh weight (mean +1 S.D.) f o r four r e p l i c a t e s . For t h i r d i n s t a r s , approximately 23% d e a l k y l a t i o n of s i t o s t e r o l and 11% d e a l k y l a t i o n of s t i g m a s t e r o l was observed a t 4 hours, which i n d i c a t e s r a p i d 3

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3

3



PRESTWICH ET A L .

Insect-Specific Enzymes and Proinsecticides

3

Figure 2. [29- H]-Fhytosterol p a r t i t i o n assay f o r rapid measurement of d e a l k y l a t i o n i n v i v o .


Magee et al.; Pesticide Synthesis Through Rational Approaches ACS Symposium Series; American Chemical Society: Washington, DC, 1984. 0.57 0.18

5th I n s t a r

Pupa

(0.03)

(0.03)

7.54 (0.37)

1 hr

(1.1)

1.59

(0.27)

3.85 (0.86)

19.7

4 hr

[29- H]-sitosterol

3

1 hr

0.11

(0.01)

(0.04)

1.74 (0.27) 0.19

mean (S.D.)

0.28

0.58

6.37

(0.02)

(0.09)

(0.47)

4 hr

[29- H]-stigmasterol

3

f r e s h weight,

i n v i v o by Manduca s e x t a .

D e a l k y l a t i o n , nmole sterol/gram

J

D e a l k y l a t i o n of [29- H]-phytosterols

3rd I n s t a r

Stage

Table 1.


S

X

H

m w

α

H

m

T3

Κ)

8.

PRESTWICH ET AL.


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and e f f i c i e n t turnover of the i n j e c t e d [ 2 9 - H ] - p h y t o s t e r o l s . Pupal and f i f t h i n s t a r values are 3-4% f o r s i t o s t e r o l and 2-3% for s t i g m a s t e r o l (these numbers i n d i c a t e aqueous dpm 2-3 f o l d higher than the zero time v a l u e ) . For both [ 2 9 - H ] - s i t o s t e r o l and [ 2 9 - H ] - s t i g m a s t e r o l , the turnover per gram insect i s approximately ten f o l d higher i n t h i r d i n s t a r s than i n f i f t h i n s t a r s or pupae, suggesting a greater l e v e l of d e a l k y l a t i o n a c t i v i t y i n the more r a p i d l y growing l i f e stages. A l s o , an increased rate of d e a l k y l a t i o n for s i t o s t e r o l r e l a t i v e to stigmasterol i s apparent, c o n s i s t e n t with the greater abundance of s i t o s t e r o l i n the host plants of Manduca l a r v a e . We are now r e f i n i n g t h i s technique to coadminister the [ ^ C ] - r i n g l a b e l l e d s t e r o l s with t h e i r [29- H] counterparts in order to more c l e a r l y define the f a t e s of both fragments of the d e a l k y l a t i o n pathway, and to separate true d e a l k y l a t i o n from conjugation and passive transport processes. A f u l l report on these methods as a p p l i e d to a l l l i f e stages of Manduca and Tenebrio i s forthcoming(13). We f e e l that t h i s technique can be adapted to give q u a n t i f i a b l e r e s u l t s on the u t i l i z a t i o n of s t e r o l s with d i f f e r e n t A/B r i n g and s i d e chain f u n c t i o n a l i t i e s by a wide range of i n s e c t s . 3


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T o x i c i t y of 29-Fluorophytosterols S u b s t i t u t i o n of f l u o r i n e for hydrogen at C-25 and C-26 o f p h y t o s t e r o l s and at C-20, C-22, C-24 or C-25 of c h o l e s t e r o l provided compounds which d i d not a f f e c t Manduca sexta growth or development s i g n i f i c a n t l y a t 50 ppm i n the d i e t ( 1 5 ) . In c o n t r a s t , we predicted that the C-29 f l u o r o p h y t o s t e r o l s ( F i g . 1, X=F) would release the metabolic equivalent of f l u o r o a c e t a t e as a r e s u l t of d e a l k y l a t i o n O ,6) . The 2 9 - f l u o r o p h y t o s t e r o l s ( F i g . 3) a l l showed s i g n i f i c a n t impairment of growth and development of l a r v a l tobacco hornworms when fed at 1 t o 100 ppm to Manduca s e x t a ^ . I t i s c l e a r that the Δ22 s t e r o l s 8. and 1^ were more t o x i c and caused more severe s t u n t i n g than the 22,23-dihydro analogs 2, and 1^ ( F i g . 4 ) . Moreover, C-24 stereochemistry was not as important i n determining r e l a t i v e t o x i c i t y as the absence or presence of the Δ22 o l e f i n i c bond. The 2 9 - f l u o r o f u c o s t e r o l 1J3 was found to be intermediate in i t s e f f e c t s between the 2 9 - f l u o r o s i t o s t e r o l £ and 2 9 - f l u o r o s t i g m a s t e r o l 8. Each of the three 2 9 - f l u o r o p h y t o s t e r o l s examined i n d e t a i l (^, 13) showed a c l e a r dose dependency f o r the r e d u c t i o n of the l a r v a l growth r a t e , maximum l a r v a l weight reached, l a r v a l m o r t a l i t y , and percent pupation(f>). As expected, the 2 9 , 2 9 - d i f l u o r o s t i g m a s t e r o l 10 was s i g n i f i c a n t l y l e s s toxic than 29-f l u o r o s t i g m a s t e r o l 8.. Larvae fed 2 9 - f l u o r o s t e r o l s matured slowly but progressed through the normal i n s t a r molts, as determined by d a i l y observations to detect apolysis. Severely poisoned larvae had trouble shedding s k i n s and developed abnormally large 5 t h - i n s t a r heads on only 2nd or 3rd i n s t a r - s i z e d bodies. The a b i l i t y o f 2 9 - f l u o r o s t e r o l s to




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PRESTWICH ET A L .


MAXIMUM WEIGHT GAIN

ι

I

I

I

I

I

I

I

I

I

0

10

20

30

40

50

60

70

80

90

1

100

PERCENT OF CONTROL

Figure 4. E f f e c t s o f 2 9 - f l u o r o p h y t o s t e r o l s on growth and pupation of Manduca sexta, fed from second i n s t a r on 50 ppm 29F-sterol i n d i e t .



136


overcome the usual gating phenomenon i n Manduca i s noteworthy. We reasoned(_5,6^) that the slow steps i n s t e r o l d e a l k y l a t i o n were (a) 24,28-desaturation to give 2 9 - f l u o r o f u c o s t e r o l or the corresponding 2 9 - f l u o r o s t i g m a s t a t r i e n o l , and (b) epoxidation of the 24,28 double bond. The s u b s t i t u t i o n of the weakly e l e c t r o n withdrawing fluorome thy1 f o r methyl causes d e s a t u r a t i o n and epoxidation reactions to proceed s l u g g i s h l y on the 2 9 - f l u o r o analogs, while proceeding unimpaired with nonfluorinated s t e r o l s . With the Δ22 bond present, e l e c t r o n release into an i n c i p i e n t conjugated diene can compensate f o r the e l e c t r o n withdrawing e f f e c t of the fluoromethyl group. Preparation and bioassay of the unsaturated f l u o r i d e s and t h e i r epoxy d e r i v a t i v e s i s i n progress to v e r i f y t h e i r intermediacy in the the d e a l k y l a t i o n pathway. Importantly, l a r v a l s t e r o i d composition i s unaffected by the monofluorophytosterols and monofluorocholesterols(5,15). That i s , none of these m a t e r i a l s i n t e r f e r e with the d e a l k y l a t i o n process. In f a c t , of the f i f t e e n f l u o r i n a t e d s t e r o l s t e s t e d , the 29-fluoro d e r i v a t i v e s were unique in e x h i b i t i n g t o x i c i t y . For these explanations o f 2 9 - f l u o r o s t e r o l t o x i c i t y to be v a l i d , we needed to demonstrate that an i n h i b i t o r of d e a l k y l a t i o n would protect larvae from the l a t e n t t o x i c i t y of an ingested 2 9 - f l u o r o s t e r o l . As the i n h i b i t o r , we chose the 24,28-allene 14. p r e v i o u s l y prepared(_16) and tested i n vivo i n Bombyx mori(17) by Ikekawa and h i s a s s o c i a t e s . This m a t e r i a l was shown to i n t e r f e r e with the s i t o s t e r o l to f u c o s t e r o l and the f u c o s t e r o l to f u c o s t e r o l epoxide conversions, but not to a f f e c t silkworm larvae fed on f u c o s t e r o l epoxide, desmosterol, or c h o l e s t e r o l . Our experimental r e s u l t s are summarized i n F i g . 5. Hornworm larvae fed diet c o n t a i n i n g 300 ppm c h o l e s t e r o l developed normally, i n c o n t r a s t to those fed 2 9 - f l u o r o s t i g m a s t e r o l (30 ppm) and 300 ppm c h o l e s t e r o l . A p r o t e c t i v e " d i l u t i o n " e f f e c t was found when 300 ppm s t i g m a s t e r o l was fed w i t h the 2 9 - f l u o r o s t i g m a s t e r o l , but m o r t a l i t y was s t i l l h i g h . Most d r a m a t i c a l l y , a d d i t i o n o f 100 ppm of aliène L4 to the 2 9 - f l u o r o s t i g m a s t e r o l plus c h o l e s t e r o l treatment completely reversed the e f f e c t s of the 29-fluorostigmasterol. With d e a l k y l a t i o n blocked, no a c t i v a t i o n of the l a t e n t poison occurred. T o x i c i t y and

i s o l a t i o n of f l u o r o c i t r a t e

F l u o r o a c e t a t e undergoes a " l e t h a l synthesis"(18) to 2 - f l u o r o c i t r a t e which may r e v e r s i b l y i n h i b i t aconitase and which i r r e v e r s i b l y binds to a membrane-associated c i t r a t e transport protein(19,20). I n s e c t i c i d a l and other b i o c i d a l uses of f l u o r o a c e t a t e (or i t s metabolic precursors) r e c e i v e d considerable a t t e n t i o n twenty-five years a g o ( 2 0 but most uses have been abandoned due to high n o n s p e c i f i c vertebrate t o x i c i t y of these compounds. Vfe have reported the use of o)~fluoro f a t t y acids and t h e i r d e r i v a t i v e s as delayed-action toxicants for targeted


8.

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MAXIMUM WEIGHT GAIN

CHOLESTEROL, 300ppm

29F - STIGMASTEROL, 30 ppm CHOLESTEROL, 300 ppm

29F - STIGMASTEROL, 30ppm STIGMASTEROL, 300ppm

29F-STIGMASTEROL, 30ppm CHOLESTEROL, 300ppm 24, 28

ALLENE, 100ppm

I

I

I

I

I

I

I

I

I

I

I

0

10

20

30

40

50

60

70

80

90

100

PERCENT OF C O N T R O L

Figure 5. P r o t e c t i o n from t o x i c i t y of 2 9 - f l u o r o s t i g m a s t e r o l (8) by 24,28-allene 14. Data show growth and pupation f o r Manduca s e x t a .



138


termite c o n t r o l using the b a i t - b l o c k method(22). Selected fluorοlipids give acceptably long delay times, a t t r a c t a n c y , and high k i l l at low o r a l doses. S e l e c t i v i t y f o r termites i s achieved i n t h i s scheme by a targeted d e l i v e r y of the poisons in a food source. In the case of the 2 9 - f l u o r o p h y t o s t e r o l s , the r e l e a s e of f l u o r o a c e t a t e from a masked poison which could not be a c t i v a t e d by v e r t e b r a t e s would seem to be a superior strategy f o r achieving selective t o x i c i t y . F l u o r o c i t r a t e and several metabolic precursors (e.g., f l u o r o a c e t a t e , (E)-16-fluorohexadec-9-enoic a c i d ) produced e f f e c t s i n hornworms analogous to those seen f o r 2 9 - f l u o r o s t i g m a s t e r o l , a l b e i t at lower doses(5). The a c t i v e (2R, 3R) steroisomer(23), ( - ) - e r y t h r o - 2 - f l u o r o c i t r a t e was 100-1000 f o l d more t o x i c than 2 9 - f l u o r o s t i g m a s t e r o l and 10-100 f o l d potent than f l u o r o a c e t a t e or (E)-16-fluorohexadec-9-enoic a c i d . The conversion of f l u o r o a c e t a t e to the coenzyme A t h i o e s t e r and then condensation with oxaloacetate ( F i g . 6) gives only a s i n g l e f l u o r o c i t r a t e isomer i n mammalian systems(19). Both enzymic r e a c t i o n s have s i g n i f i c a n t l y lower v e l o c i t i e s r e l a t i v e to the normal acetate to a c e t y l CoA to c i t r a t e c o n v e r s i o n . For example, pig heart c i t r a t e synthase has the same K f o r a c e t y l CoA and f l u o r o a c e t y l CoA, but the V f o r f l u o r o a c e t y l CoA i s only l/300th of the V f o r a c e t y l CoA(19). A l s o , we expected the ω-fluorofatty a c i d to be more e f f i c i e n t l y converted t o f l u o r o c i t r a t e than e i t h e r f l u o r o a c e t a t e or 2 9 - f l u o r o s t i g m a s t e r o l , since the 3-oxidation pathway provides the CoA d e r i v a t i v e d i r e c t l y , whereas the d e a l k y l a t i o n pathway formally gives fluoroacetaldehyde as the released fragment. In a d d i t i o n to the c i r c u m s t a n t i a l i n vivo evidence f o r f l u o r o c i t r a t e as the ultimate biochemical l e s i o n we desired to demonstrate unambiguously that i t was produced as a metabolite of 29-fluorostigmasterol. The t o x i c i t y of f l u o r o c i t r a t e and the r e s u l t i n g l e t h a l accumulation of c i t r a t e i n mouse, f l y and cockroach t i s s u e s have been shown i n early experiments with fluoroacetamide and f l u o r o a c e t a t e ( 2 4 ) . However, to our knowledge, complete c h a r a c t e r i z a t i o n of ( 2 R , 3 R ) - 2 - f l u o r o c i t r a t e as the l e t h a l metabolite i n vivo has not p r e v i o u s l y been r e p o r t e d . We thus prepared(25) [29-^H]-29-fluorostigmasterol, [ 2 9 - H ] - 2 9 - f l u o r o s i t o s t e r o l and [16- H]-16-fluorohexadec-9-enoic a c i d to enable i s o l a t i o n of [ 2 - 3 H ] - 2 - f l u o r o c i t r a t e from _in v i v o incubations using Manduca s e x t a . Four e a r l y f i f t h i n s t a r larvae (1-2 g each) were starved f o r 24 hr and then each was fed on a 20 mg slab of diet t r e a t e d with 5 μΐ of a DMF s o l u t i o n containing ca. 3 χ 1 0 dpm of [29- H]-29f l u o r o s t i g m a s t e r o l ( s p e c i f i c a c t i v i t y 150 mCi/mmol), [29- H]-29f l u o r o s i t o s t e r o l ( s p e c i f i c a c t i v i t y 240 mCi/mmol), o r [16- H]-16-fluorohexadec-9-enoic acid ( s p e c i f i c a c t i v i t y 33 mCi/mmol). A f t e r 8 h r s , the larvae were frozen and l a t e r homogenized i n d i v i d u a l l y i n ethyl acetate-water (1:1). U n l a b e l l e d ( - ) - e r y t h r o - 2 - f l u o r o c i t r a t e as the t r i s ( c y c l o h e x y l m

m a x

m a x

3

3

6

3

3

3



PRESTWICH ET AL.


Γ

29FT stigmasterol

dealkylation

V j ^ ^ V ^ —

^

^

^

^

cholesterol

T-CHCOOH

VΤ

FÇH C-SCoA

Ô ÇOOH

CoASH

^ citrate synthase

c=o ι

ÇOOH T-C-F HO-C-COOH

[2-H]3

(-) erythro

-

2-fluorocitrate

CH COOH 2

2

Ç^ oxaloacetate COOH Malic acid

I

TCA CYCLE 3

Figure 6. Metabolic conversion of [29- H]-29-fluoros t i g m a s t e r o l to [ 2 - H ] - 2 - f l u o r o c i t r a t e i n v i v o . 3



140

PESTICIDE SYNTHESIS T H R O U G H RATIONAL APPROACHES

ammonium) s a l t (1 mg) was added to the aqueous l a y e r , s i n c e we estimated that only s e v e r a l nanograms of l a b e l l e d f l u o r o c i t r a t e per i n s e c t would be produced. The lengthy p u r i f i c a t i o n scheme(25) c o n s i s t s of the f o l l o w i n g key f e a t u r e s : (1) e l u t i o n of i s o c i t r a t e , c i t r a t e , and f l u o r o c i t r a t e from ion exchange column w i t h 2 Ν ammonium formate; (2) a c i d i f i c a t i o n , l y o p h i l i z a t i o n and diazomethane methylation followed by s i l i c a g e l chromatography to give the t r i m e t h y l esters of c i t r a t e , i s o c i t r a t e , and f l u o r o c i t r a t e ( f r a c t i o n s checked by c a p i l l a r y GC a n a l y s i s ) ; (3) benzoylation and s i l i c a g e l chromatography of the benzoates; (4) s e p a r a t i o n of the diastereomeric trimethyl ester benzoates by reverse phase HPLC (Ce~PXS 10/25, 34% C H 3 C N - H 2 O ) . At each stage, the l o c a t i o n of the l a b e l l e d m a t e r i a l was followed by LSC of chromatographic f r a c t i o n s , with the dpm i n steps (2) and (3) moving only with the f l u o r o c i t r a t e - c o n t a i n i n g f r a c t i o n s . In a d d i t i o n , c a p i l l a r y GC (30 m DB-5) was used to q u a n t i f y c i t r a t e , i s o c i t r a t e and f l u o r o c i t r a t e trimethyl esters and benzoates i n each f r a c t i o n . F i n a l l y , dpm were observed only i n the erythro isomer of t r i m e t h y l 2 - f l u o r o c i t r a t e benzoate. The progress of the p u r i f i c a t i o n i s given i n Table 2. Full details w i l l be published s e p a r a t e l y ( 2 5 ) . We observed that the ω-fluorofatty a c i d was much more e f f i c i e n t l y converted to f l u o r o c i t r a t e (0.57% of applied dpm) than was the 2 9 - f l u o r o s t i g m a s t e r o l (0.005% of a p p l i e d dpm). Conversion of [ 2 9 - H ] - 2 9 - f l u o r o s i t o s t e r o l to f l u o r o c i t r a t e was examined, but i n s u f f i c i e n t l a b e l remained a f t e r step (2) f o r further p u r i f i c a t i o n s ^ , ^ ) . This suggests much lower d e a l k y l a t i o n e f f i c i e n c y f o r t h i s analog (

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