Conformationally Rigid Peptides as Models for Selective Herbicides

Chapter 14 Conformationally Rigid Peptides as Models for Selective Herbicides 1

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J. V. Edwards , H. G. Cutler , P. S. Zorner , and C. B. Coffman 1

Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, LA 70179 Richard B. Russel Research Center, U.S. Department of Agriculture, Athens, GA 30163 Agricultural Research Center, BASF Chemicals Division, Research Triangle Park, NC 27709-3528 Northeastern Region Beltsville Agricultural Research Center, U.S. Department of Agriculture, Beltsville, MD 20705

Downloaded by UNIV LAVAL on May 9, 2016 | http://pubs.acs.org Publication Date: November 3, 1987 | doi: 10.1021/bk-1987-0355.ch014

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Synthetic peptide analogs derived from the phytotoxin tentoxin, have been examined for their phototoxicity and herbicidal potential to a variety of plants and agronomically significant weeds. Structurally modified analogs included both cyclic and acyclic derivatives. Acyclic tripeptides were modified from the synthetic intermediate tert-butyloxycarbonyl-Leucyl-N(methyl) dehydrophenylalanyl-glycine methyl ester. Modifications at the leucine side chain and dehydrophenylalanine nitrogen were introduced by substituting different alkyl groups. Selective alkylation at the amide bond nitrogen of dehydrophenylalanine provided analogs which showed herbicidal potential. The cyclic peptides induced chlorosis in both barnyardgrass and morningglory. The tripeptide analog tert-butyloxycarbonyl-Valyl-N(ethyl) dehydrophenylalanyl-glycine methyl ester exhibited root growth inhibition in barnyardgrass and inhibited bolting in mustard seed. Similar analogs containing an N-ethylated amide nitrogen and dehydrophenylalanine exhibited some growth regulating activity in wheat coleoptiles. The effects of stereochemical and rigid conformation on biological activity are discussed. The u s e o f b i o l o g i c a l l y d e r i v e d c h e m i c a l s as h e r b i c i d e s has m e t w i t h l i m i t e d success. Despite t h i s , biotechnology o f f e r s potential f o r t h e e x p l o r a t i o n o f n a t u r a l l y o c c u r r i n g compounds as h e r b i c i d e s , w h i c h may s e r v e as i m p e t u s f o r new s y n t h e t i c a n d s t r u c t u r e / f u n c t i o n approaches i n h e r b i c i d e developement U ) . I n t h i s r e g a r d we h a v e implemented a program t o study b i o l o g i c a l l y a c t i v e peptides from p l a n t s and f u n g i . S p e c i f i c a l l y , we f o c u s e d on p h y t o t o x i c c y c l i c

0097-6156/87/0355-0151$06.00/0 © 1987 American Chemical Society

Baker et al.; Synthesis and Chemistry of Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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SYNTHESIS AND CHEMISTRY OF AGROCHEMICALS

t e t r a p e p t i d e s s e c r e t e d by f u n g i . Cyclic peptides having a diverse r a n g e o f b i o l o g i c a l a c t i v i t i e s i n p l a n t s h a v e been i s o l a t e d and s t r u c t u r a l l y c h a r a c t e r i z e d ( F i g u r e 1) ( 2 - 6 ) . These a c t i v i t i e s v a r y i n b o t h t h e i n d u c e d p a t h o l o g i c a l and p l a n t g r o w t h r e g u l a t i n g r e s p o n s e as w e l l as s e l e c t i v i t y . F o r e x a m p l e AM t o x i n , a h o s t s e l e c t i v e t o x i n s e c r e t e d by A . m a l i , i n d u c e s n e c r o s i s on o n l y c e r t a i n a p p l e l e a f c u l t i v a r s ~ T 2 ) , w h e r e a s t e n t o x i n (5) (Figure 2 ) , a n o n - s e l e c t i v e t o x i n , induces cïïlorosis (a y e l l o w i n g of p l a n t t i s s u e ) i n some p l a n t s s u c h as l e t t u c e and mung b e a n b u t n o t i n o t h e r s s u c h as c o r n Tentoxin induced c h l o r o s i s r e s u l t s from i n t e r f e r e n c e


with

c h l o r o p l a s t development ( 9 - 1 1 ) . I n t e r e s t i n g l y , t h e s e compounds ( F i g u r e 1 ) s h a r e s i m i l a r s t r u c t u r a l f e a t u r e s , n o t a b l y t h e 12-atom peptide r i n g s , which are r i c h i n a l k y l amino a c i d s i d e c h a i n s . Some o f t h e p e p t i d e s c o n t a i n u n u s u a l a m i n o a c i d s i d e c h a i n s p o s s e s s i n g o l e f i n i c (AM t o x i n , t e n t o x i n ) and e p o x i d e f u n c t i o n a l i t i e s (HC t o x i n ) . In view of these b i o l o g i c a l and s t r u c t u r a l c h a r a c t e r i s t i c s i t i s t i m e l y t o e x p l o r e t h e r o l e o f c o n f o r m a t i o n , s i d e c h a i n s , and p e p t i d e b a c k b o n e modifications in the phytotoxic p r o f i l e s of these peptides. We s e l e c t e d t e n t o x i n as a model f o r e x a m i n i n g some a s p e c t s o f s t r u c t u r e versus f u n c t i o n . The c o n f o r m a t i o n a l r i g i d i t y r e s u l t i n g f r o m t h e 1 2 - a t o m p e p t i d e r i n g has a l l o w e d p r e v i o u s w o r k e r s t o d e t e r m i n e bond a n g l e s by u s i n g NMR t e c h n i q u e s t o i d e n t i f y p r e f e r r e d c o n f o r m e r s a s s o c i a t e d w i t h b i o l o g i c a l a c t i v i t y ( 1 2 and 1 3 ) . Our p r e v i o u s a n a l o g s t u d i e s h a v e e x p l o r e d t h e r o l e oT~"the ï î F a t o m r i n g i n b i o l o g i c a l a c t i v i t y and compared b i o c h e m i c a l p r o f i l e s f o r t h e mode o f a c t i o n o f t h e t w o c o n f o r m a t i o n a l ^ s i m i l a r a n a l o g s t e n t o x i n and [ P r o ] t e n t o x i n ( 2 4 ) . Of p r a c t i c a l i n t e r e s t i s t e n t o x i n ' s p o t e n t i a l as a s e l e c t i v e h e r b i c i d e s i n c e i t i s a c t i v e i n s o r g h u m s p e c i e s s i m i l a r t o j o h n s o n g r a s s b u t n o t i n c o r n (.7,8.). R e c e n t l y we r e p o r t e d t h e t o t a l s y n t h e s i s o f t e n t o x i n and a conformational!y similar peptide [ P r o ] tentoxin (24). Synthesis o f t e n t o x i n r e q u i r e s s y n t h e t i c s t e p s b o t h common t o a n d d i f f e r e n t f r o m n o r m a l s o l u t i o n p h a s e p e p t i d e s y n t h e s e s due t o t h e p r e s e n c e o f u n u s u a l m o d i f i c a t i o n s i n c l u d i n g N - m e t h y l a t e d a m i d e b o n d s and a dehydrophenylalany1 residue. An a d v a n t a g e i n p e r f o r m i n g t h e t o t a l s y n t h e s i s o f a p e p t i d e s u c h as t e n t o x i n i s t h e c o n v e n i e n t a p p r o a c h a f f o r d i n g piecemeal assessment o f unique f u n c t i o n a l groups f o r their role in biological a c t i v i t y . I n o r d e r t o assess t h e c o n t r i b u t i o n o f these unusual m o d i f i c a t i o n s t o c h l o r o s i s i n d u c t i o n , s y n t h e t i c i n t e r m e d i a t e s f r o m t h e s y n t h e s i s were assayed f o r chlorosis induction in lettuce seedlings. A l t h o u g h o n l y one p e p t i d e demonstrated i n d u c t i o n o f s i g n i f i c a n t l y low c h l o r o p h y l l l e v e l s i n l e t t u c e i t was f o u n d t h a t t h i s a n a l o g a l s o i n h i b i t e d r o o t growth. F u r t h e r i n v e s t i g a t i o n s i n t o sequence r e q u i r e m e n t s f o r t h e r o o t i n h i b i t i o n revealed t h a t the t r i peptide d e r i v a t i v e B o c - L e u - N ( C H 3 ) A P h e - G l y - 0 M e (3) was s u f f i c i e n t f o r f u l l b i o l o g i c a l response ( r o o t growth i n h i b i t i o n ) . The s t r u c t u r e o f a n a l o g 3 ( R ^ i s i s o b u t y l and R2 i s m e t h y l ) w h i c h d e m o n s t r a t e s r o o t g r o w t h i n h i b i t i n g e f f e c t s i s shown i n F i g u r e 3 . T h o u g h t h i s p e p t i d e has c o n s i d e r a b l y i n c r e a s e d c o n f o r m a t i o n a l f l e x i b i l i t y over t h e c y c l i c p e p t i d e t e n t o x i n , i t c o n t a i n s two b a c k b o n e and s i d e c h a i n m o d i f i c a t i o n s w h i c h c o n f e r i n c r e a s e d c o n f o r m a t i o n a l r i g i d i t y t o t h e m o l e c u l e when c o m p a r e d w i t h b a c k b o n e non-modified oligopeptides. I t i s helpful to r e c a l l t h a t the c o n f o r m a t i o n o f a p e p t i d e i s d e t e r m i n e d by i t s o v e r a l l three-dimensional structure (14). I f t h e bond a n g l e s and b o n d 1

1

z


14. EDWARDS ET AL.

Peptides as Models for Selective Herbicides 153

Peptide Tentoxin cyclo[-N(CH )-Ala-Leu-N(CH3)A Phe-Gly-] z

3


HC-toxin cy clo[-Aoe-D-Pro-Ala-D-Ala-]

Induces chlorosis in some plants (lettuce, mung bean) but not others (corn, tomato) (5,7,8). Inhibits growth of susceptible corn roots (3).

Cyl-2 cyclo-[Aoe-D-O-methy1Tyr-11e-P i p-]

cyclo[-Pro-Val-Pro-Val-]

Inhibits lettuce root growth elongation and rice seedling growth (6). Retards stem growth of rice seedlings. Promotes stem growth (4).

(D-Val isomer) Malformin cyclo[D-Cys-D-Cys-Val-D-Leu-Ile] AM-toxin cyclo[-Ala-Hmb-Amp-ΔΑΙa-]

Figure 1.

Biological Activity

Induces malformations in corn roots (16-18). Causes veinal necrosis on apple leaf cultivars (2).

Abbreviated structures and biological activities of phyto-active, cyclic peptides. Abbreviations according to IUPAC-IUB Commission (1972) Biochemistry 11,17261732 are used. Δ denotes a dehydroamino acid of the Ζ configuration. ζ

^ C H

Figure 2.

3

Structure and preferred conformation of tentoxin

(A. 12).




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lengths are held constant the conformation is describable. Important dihedral angles for defining conformation in peptides are the Ψ, Φ and ω angles (Figure 3). Because of the considerable conformational freedom present in peptides i t is desirable to fix and rigidify the conformation in order to probe the relation of conformation to biological activity (15). In the field of mammalian hormone and neurotransmitter peptides this approach has been investigated over the last 10-15 years. For this reason we decided to study the biological effects of structural features which would predictably rigidify and/or change conformation in the tripeptide sequence found to have root growth inhibiting properties. Some structural changes incorporated in this study which either reduce conformational flexibility (15) or change the spatial orientation of the amino acid side chain include: N-alkylation (substitution of a methyl, ethyl, or propyl for a hydrogen at the nitrogen of an amide bond), conversion of an sp center at an α-carbon to a sp center, substitution of a D amino acid for an L amino acid and substitution of a hydrogen at the α carbon with a methyl group. Previously conformational studies by Yitoux et al. (25) on dipeptides containing N-methylation have shown the conformational specificity induced by N-methylation. Conformations of tri peptides containing dehydrophenylalanine have been recently discussed by Chauhan et al. (26). In initial structure activity relationshTp studies we have held constant the olefinic moiety at position 2 and varied alkyl groups at the 2 position amide bond. This was made possible through selective N-alkylation. The selective N-alkylation step shown in Figure 4 is pivotal to the synthesis of analogs described here, and preliminary profiles of the structure activity relation for the root growth inhibitors revealed that alkylation at the 2 position of the synthetic intermediate was necessary for biological activity at the 100-500 micromolar level. Utilizing this reaction we have thus far been successful in N-alkylating at dehydrophenylalanine with methyl, ethyl, and propyl groups in good yield. We focus here on an approach in which synthetic fragments of a naturally occurring cyclic peptide such as tentoxin can be studied as a model for conformational^ rigid peptide phytotoxins and for their potential by selective herbicidal activity. Our initial studies include the following steps: 1) Screen synthetic intermediates for phytotoxicity in plants sensitive to tentoxin. 2) Appropriately derivatize synthetic intermediates showing activity at 100 micromolar or less. 3) Test these analogs in a plant assay sensitive to a broad range of biologically active compounds. 4) Test analogs in crop plants and agronomically important weeds both in excised and intact plant tissue. This report constitutes the results of four different plant assays. The modified peptides were i n i t i a l l y screened in lettuce and cress seedlings and subsequently tested on agronomically important weeds. 3

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Synthesis Cyclic peptides of this study were prepared utilizing synthetic routes previously reported. The full details of the synthesis of tentoxin and [Pro ] tentoxin have been reported elsewhere (24). 1


14. EDWARDS ET AL.

Peptides as Models for Selective Herbicides

155

Tripeptides utilized in this study were synthesized employing synthetic steps identical to those in the cyclic peptide syntheses. Variations of the N-terminal amino acid and in the alkyl group at the nitrogen of the 2-position dehydrophenylalanine were accomplished through substitution of the appropriate tert-butyloxycarbonyl amino acid and alkyl iodide at the appropriate synthetic step, respectively. The details and physical constants of these synthetic tri peptide analogs will be reported elsewhere (Edwards J . Y. and Cutler H. G . , unpublished results).


Root growth bioassay of germinating lettuce and curly cress seedlings Aliquots of oligopeptide compounds to be tested were dissolved in ethyl acetate. One m i l l i l i t e r aliquots of the samples were pipetted onto filter paper and 15 lettuce or cress seeds were uniformly distributed on the the filter paper surface and allowed to imbibe in the dark at 20 to 25°C for 24h. The samples were subsequently placed in a growth chamber in continuous light at 28°C for 72h, and the root lengths were measured. Wheat coleoptile assay An assay previously utilized for detecting growth inhibition and promotion and cited for its validity in screening for phytotoxicity was employed. The techniques utilized were those of Cutler et. al. (18). Petri dish assay for herbicidal activity. Compounds were applied to sterile filter paper in a petri dish at a concentration of 500 micromolar in a solution of 5% acetone and 95% water. Surface sterilized seeds of lettuce, barnyardgrass, morningglory, and mustard seed were placed on filter paper. A visual assessment of chlorosis was made. Radical and coleoptile lengths were measured seven days after seed germination. Two replications per compound were performed. Discussion of structure function relationships The peptides discussed in this structure/activity relation study were i n i t i a l l y tested in the lettuce and cress seedling bioassay where the root growth inhibition activity was originally discovered. The results of this study are shown in Table I. It is interesting that replacement of the methyl group at the amide nitrogen of dehydrophenylalanine in 3 with an ethyl group, resulting in 1 gives a shift from inhibition to growth promotion. On the other hand when the stereogenic center at leucine in the N-ethylated analog is converted from the R to S configuration a shift from growth inhibition to growth promotion is observed. Further derivatization in the form of various combinations of R^ and R2 group subsitutions gave the root growth responses seen in Table 1. Compound 8 is the most rigid of the analogs. A gem dimethyl functionality at the α carbon of the 1 position amino acid


156


in combination with R2 as a methyl group and a dehydrophenylalanine residue in compound 8 resulted in promoted growth of both lettuce and cress seedlings. Previously an extended structure function relation analysis was performed to assess the role of varying degrees of conformational rigidity present in the tri peptide sequence (H-Leu-Phe-Gly-OH) on Table. I

Results of in vitro lettuce and cress root growth assay for seven tnpeptide analogs. Lettuce/Cress Seedling Assays Promotion


Compound 1 2 3 4 5 6 8

*%Root Growth at 10" M Inhibition 6

z

Boc-Leu-N(C H )A Phe-Gly-OMe 40-50%(L),19%(C) Boc-D-Leu-N(C Hc)APhe-Gly-0Me Boc-Leu-N(CH 7A*Phe-Gly-0Me Boc-D-Ala-A Phe-Gly-OMe Boc-Val-N(C H )A Phe-Gly-OMe 40%(C) Boc-Val-N(CH M Phe-Gly-0Me Boc-Aib-N(CH )A Phe-Gly-0Me 40%(L),90%(C) 2

5

2

3

z

50*(L),80%(C) 60%(L),50%(C) 40*(L),20%(C)

z

2

5

z

3

85%(C)

z

3

plant growth (Edwards J . V. and Cutler G. G . , unpublished results). This approach was the subject of a study done in a wheat coleoptile assay. The wheat coleoptile assay is a primary plant bioassay sensitive to a broad range of biologically active compounds (18). The synthetic tentoxin fragments had activities comparable to other phytotoxic compounds in this assay. These studies showed trends of increased activity as the peptide backbone was modified both structurally and conformationally. For example the order of growth inhibition with wheat coleoptiles was Boc-Leu-Phe-Gly-OMe < Boc-Leu-A Phe-Gly-OMe < Boc-Leu-N(CH3)A Phe-Gly-0Me. This parallels an increase in backbone rigidity in the tripeptide sequence Leucyl-phenylalanyl-glycine. Interestingly the growth promotion observed from analogs in this assay structurally paralleled analogs found to promote growth in the lettuce seedling assay. The results of a wheat coleoptile study are shown for analogs 1, 3 and 5. Peptides which were N-ethylated at the two position nitrogen gave significant promotion when compared with the N-methylated sequence (Figure 5). Promising a c t i v i t é s observed against barnyardgrass, morningglory, and mustard seed were found in the herbicidal assays. The highest levels of root growth inhibition were observed in treatments with analogs 1, 2, and 5 (Figure 5). The same compounds inhibited bolting of mustard seed. It should be noted that these assays (lettuce/cress, wheat coleoptile, and weed) were done in different laboratories under double blind conditions. It is interesting that those analogs which gave promotion in the lettuce and wheat coleoptile assays were the same ones demonstrating promising herbicidal activity in the in vitro weed assays. Compound 5 showed the most herbicidal promise" of any compound tested. In the whole plant assays phytotoxic responses observed seemed to be tolerated by the plant. Only in the case of analog 2 z

z


14. EDWARDS ET AL.

CH3 H3C-

Peptides as Models for Selective Herbicides

ο Art!

k \>

H

0

—Η—Α—CH2—ê—Ο—CH3

Scô Si

2

General formula for tripeptides of this study. Arrows with accompanying Greek letters indicate dihedral bond angles.

CHj


r

-ί—Ν—CH—H—Ν -f—C -f—ζ

CH3 Figure 3.

f^Sp

ο

H3C

0

Η

0

- J1 — 0O-Ê-Hjh-Cf^-H^^ — ϋ—γ—C —C—Ν—C—^—N-

CH3 iC-CH H3C—CH CH3 R-I/^C^/ie-crown-o

Ο ® /

CH3 HC 3

—ë—

Conformationally Rigid Peptides as Models for Selective Herbicides

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