Biologically Active Natural Products - American Chemical Society

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Chapter 3

Approaches to Structure—Function Relationships for Naturally Occurring Cyclic Peptides A

Study of Tentoxin

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Judson V. Edwards , Oliver D. Dailey, Jr. , John M . Bland , and Horace G. Cutler 2

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Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, LA 70179 Richard B. Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, GA 30613

Approaches to studying structure/activity relationships in naturally occurring peptides with agronomic relevance are discussed. The cyclic tetrapeptide tentoxin has been examined for the relation of its conformational properties to biological activity and its use as a template for derivation of synthetic analogs possessing divergent phyto-active properties. The cyclic tetrapeptide HC toxin is discussed for similar properties. Approaches to amide bond and amino acid modifications are discussed; these include the incorporation of thiomethylene ether, retro-inverso, alkyl spacer, N-alkyl, and "cyclopropylog" modifications in tentoxin and biologically active synthetic fragments. M i c r o b i a l and P l a n t P e p t i d e s The r o l e o f p e p t i d e s i n r e g u l a t i n g fundamental b i o l o g i c a l f u n c t i o n s i n both animal {I) and m i c r o b i a l c e l l s (2J i s w e l l documented. S t r u c t u r e - f u n c t i o n s t u d i e s employing s y n t h e t i c a l l y m o d i f i e d analogs have proven an i n d i s p e n s i b l e t o o l second o n l y t o t h e b i o l o g i c a l assay i n e v a l u a t i o n o f b i n d i n g and t r a n s d u c t i o n o f p e p t i d e s . In c o n t r a s t , l e s s a t t e n t i o n has been g i v e n t o s t r u c t u r e and f u n c t i o n o f indigenous and m i c r o b i a l l y f i x e d p e p t i d e s i n h i g h e r p l a n t s ( f o r a review see r e f e r e n c e 3 J • Evidence f o r p l a n t p e p t i d i c hormones ( o r f r a g m e n t s ) , analogous t o mammalian hormones, which r e g u l a t e v e g e t a t i v e growth and development i s a b s e n t , but morphogenetic s i g n a l f u n c t i o n s have been h y p o t h e s i z e d f o r o l i g o p e p t i d e s (1,5»). Reports o f b i o l o g i c a l l y a c t i v e p e p t i d e s i n p l a n t s and microbes have r e c e n t l y appeared {3,6-9). Of t h e p e p t i d e s c h a r a c t e r i z e d , both c y c l i c and l i n e a r s t r u c t u r e s have been f o u n d , and i n many cases unusual amino a c i d s have been noted (10,11). M i c r o b i a l p e p t i d e s t h a t possess s e l e c t i v e b i o l o g i c a l a c t i v i t y i n r o o t s o r a e r i a l t i s s u e s o f p l a n t s have been i s o l a t e d . The p l a n t r e g u l a t o r y e f f e c t s o f t h e b i c y c l i c p e n t a p e p t i d e m a l f o r m i n , a fungal m e t a b o l i t e , have been known f o r some t i m e (2,9,12,13) and t h e s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s e x p l o r e d ( 1 4 7 . R h i z o b a c t e r i a have This chapter not subject to U.S. copyright Published 1988 American Chemical Society

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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been found t o enhance p l a n t growth by p r o d u c t i o n o f p e p t i d e s i d e r o p h o r e s ( 1 5 ^ 1 6 ) , and recent a t t e n t i o n has been g i v e n t o a group of p e p t i d e s ( R h i z o b i n s ) exuded by soybean nodules t h a t may be i n v o l v e d i n n i t r o g e n f i x a t i o n (9j_. A v a r i e t y of c y c l i c t e t r a p e p t i d e s have demonstrated very potent and h i g h l y s e l e c t i v e p h y t o t o x i c and p l a n t growth r e g u l a t i n g p r o p e r t i e s ( 1 1 ) .

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A g r i c u l t u r a l P o t e n t i a l o f Some P h y t o - a c t i v e N a t u r a l l y Peptides

Occurring

The p o t e n t i a l use of b i o l o g i c a l l y - a c t i v e n a t u r a l products as t e m p l a t e s f o r t h e development of a g r i c u l t u r a l products has r e c e i v e d i n c r e a s e d a t t e n t i o n i n recent y e a r s . Our i n t e r e s t has been t o study t h e s t r u c t u r a l f e a t u r e s of f u n g a l l y - s e c r e t e d , c y c l i c t e t r a p e p t i d e s r e s p o n s i b l e f o r b i o l o g i c a l a c t i v i t y i n p l a n t s . The s t u d i e s d e s c r i b e d here deal p r i n c i p a l l y with two t y p e s o f approaches: a n a l y s i s of p r e d i c t a b l e conformational features relevant to b i o l o g i c a l a c t i v i t y , and t h e use of c y c l i c p e p t i d e s as a t e m p l a t e f o r d e r i v i n g b i o a c t i v e fragments. The fungal m e t a b o l i t e t e n t o x i n has been employed as a model compound. An u n d e r s t a n d i n g of t h e e f f e c t of p r e d i c t a b l e a l t e r a t i o n s i n secondary s t r u c t u r e r e s u l t i n g from primary s t r u c t u r a l changes p r o v i d e s a b e t t e r understanding o f c o n f o r m a t i o n a l requirements f o r b i o l o g i c a l a c t i v i t y . A thorough knowledge o f the c o n f o r m a t i o n a l requirements f o r a c t i v i t y i n t h e p l a n t i n c r e a s e s u n d e r s t a n d i n g of t h e mode of a c t i o n and f u r t h e r s t h e p o t e n t i a l f o r m a n i p u l a t i n g f u n g a l l y s e c r e t e d p e p t i d e s o f a g r i c u l t u r a l i n t e r e s t . For example t h e developments of a g o n i s t s and a n t a g o n i s t s from c o n f o r m a t i o n a l s t u d i e s on n e u r o p e p t i d e hormones have l e d t o an i n c r e a s e d understanding of s i t e s o f a c t i o n and development of t h e r a p e u t i c drugs ( 1 7 ) . With the d i s c l o s u r e o f a b i o l o g i c a l l y a c t i v e n a t u r a l product t h e q u e s t i o n o f t e n a r i s e s as t o how t h e compound may be employed as a t e m p l a t e f o r d e r i v a t i o n of s i m p l e r b i o a c t i v e f r a g m e n t s . Several examples o f b i o l o g i c a l a c t i v i t y of s u b t l y m o d i f i e d n a t u r a l products or s y n t h e t i c fragments have proven f r u i t f u l i n t h i s r e g a r d . The s y n t h e t i c d i p e p t i d e c a r b o b e n z o x y p r o l y l v a l i n o l i s a good example ( 1 8 ) . The p e p t i d e i s a s y n t h e t i c fragment of HC t o x i n (a c y c l i c t e t r a p e p t i d e which i s a potent i n h i b i t o r of a genotype of c o r n r o o t s ) y e t the fragment possesses an a l t o g e t h e r d i f f e r e n t b i o l o g i c a l response. Treatment of c o r n p l a n t s w i t h t h e s y n t h e t i c fragment provides f o r increased y i e l d s i n u n f e r t i l i z e d corn f i e l d s . We r e p o r t here approaches t o examining t h e p o t e n t i a l use of t h e c y c l i c t e t r a p e p t i d e t e n t o x i n as a t e m p l a t e f o r development of compounds of p o t e n t i a l h e r b i c i d a l and p l a n t growth r e g u l a t i n g activity. Both amide bond s u b s t i t u t i o n s and novel amino a c i d s have been used t o a s s e s s t h e n a t i v e c o n f o r m a t i o n of t e n t o x i n and f o r d e r i v a t i o n of novel b i o l o g i c a l l y - a c t i v e p e p t i d e s which are s y n t h e t i c fragments o f t e n t o x i n . The c y c l i c t e t r a p e p t i d e t e n t o x i n cyclo[N(Me)Ala-Leu-N(Me)A P h e - G l y ] ( F i g u r e 5) i s a secondary m e t a b o l i t e of A l t e r n a r i a a l t e r n a t a . T e n t o x i n i s of i n t e r e s t a g r i c u l t u r a l l y f o r i t s s e l e c t i v e h e r b i c i d a l a c t i o n . It induces c h l o r o s i s (a y e l l o w i n g i n p l a n t s due t o d e p l e t e d c h l o r o p h y l l l e v e l s ) i n s u s c e p t i b l e weed s p e c i e s w h i l e not e f f e c t i n g major crop p l a n t s such as corn and soybean ( 1 9 ) . z

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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3. EDWARDS ET AL.

Naturally Occurring Cyclic Peptides

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As a c y c l i c t e t r a p e p t i d e , t e n t o x i n i s among a c l a s s of f u n g a l l y s e c r e t e d p e p t i d e s c o n t a i n i n g f o u r amino a c i d s . W i t h i n t h e past 10-15 y e a r s many of t h e s e analogs have been d i s c o v e r e d and t h e i r p h y t o t o x i c and growth r e g u l a t i n g e f f e c t s e l u c i d a t e d ( 1 6 ) . The s t r u c t u r a l and c o n f o r m a t i o n a l c h a r a c t e r i z a t i o n o f a v a r i e t y o f t h e s e analogs has been a c h i e v e d by e x t e n s i v e NMR, c r y s t a l l o g r a p h i c , and t h e o r e t i c a l c a l c u l a t i o n s (21). S t r u c t u r a l s i m i l a r i t i e s among t h e p e r t i n e n t p e p t i d e s i n c l u d e aromatic g r o u p s , a l k y l s i d e c h a i n s , and a t w e l v e atom p e p t i d e r i n g . V a r i o u s novel amino a c i d s are f o u n d , such as as dehydro amino a c i d s ( i n t e n t o x i n and t h e AM t o x i n s ) and t h e 8 - o x o - 9 , 10-epoxydecanoic a c i d [ i n HC t o x i n and C y l - 2 ( 1 1 ) ] . F i v e combinations of backbone c o n f o r m a t i o n are p o s s i b l e along t h e r i n g of c y c l i c t e t r a p e p t i d e s i n c l u d i n g : a l l c i s ; a l l t r a n s , 3 c i s and 1 t r a n s ; 3 t r a n s and 1 c i s ; and 2 c i s and 2 t r a n s . As a r e s u l t o f t h e s e backbone v a r i a t i o n s , d i s t i n c t i v e c o n f o r m a t i o n a l f e a t u r e s are present which may p r o v i d e u s e f u l leads i n d e s i g n i n g mimetic compounds. For example, t h e presence of a 7 - t u r n i n HC t o x i n , and c y c l o - ( L - P r o - D - V a l - ) 2 (found t o promote stem growth of r i c e s e e d l i n g s ) , r e s u l t i n g from a sequence s p e c i f i c r e v e r s e t u r n , suggests t h e p o s s i b l e use of r e v e r s e t u r n mimics t o develop s m a l l e r n o n - p e p t i d i c m o l e c u l e s w i t h analogous a c t i v i t y . A seven-membered r i n g compound of t h e t y p e r e c e n t l y proposed by Huffman and C a l l a h a n (20) may be u s e f u l i n t h e development of s i m p l i f i e d non-peptide analogs. P r e v i o u s work suggests t h a t t h e c y c l i c p e p t i d e backbone s e r v e s as a c a r r i e r which induces b i n d i n g , w h i l e a r e a c t i v e s i t e at a s p e c i f i c amino a c i d p a r t i c i p a t e s as t h e r e c e p t o r l i g a n d . Thus, t h e attachment of r e a c t i v e l i g a n d s t o c a r r i e r s m i m i c k i n g t h e p e p t i d e c o n f o r m a t i o n may p r o v i d e u s e f u l l e a d compounds. T e n t o x i n - an E x c e p t i o n t o Other N a t u r a l l y O c c u r r i n g Tetrapeptides.

Cyclic

S i n c e t h e a c t i v i t i e s of many of t h e c y c l i c t e t r a p e p t i d e s depend on t h e i r c o n f o r m a t i o n s , the r e l a t i o n s h i p between primary s t r u c t u r e and c o n f o r m a t i o n i s important t o u n d e r s t a n d . R e c e n t l y a u s e f u l set of e m p i r i c a l r u l e s has been r e p o r t e d t o p r e d i c t t h e c o n f o r m a t i o n of c y c l i c t e t r a p e p t i d e s on t h e b a s i s of p r i m a r y s t r u c t u r e ( 2 1 ) . The r u l e s are based on t h e many c o n f o r m a t i o n s r e p o r t e d f o r c y c l i c t e t r a p e p t i d e s ; t h e s e r u l e s have proven u s e f u l i n f i n d i n g a s t a r t i n g c o n f o r m a t i o n i n energy c a l c u l a t i o n s , and they t a k e i n t o c o n s i d e r a t i o n 7 - t u r n s , and t h e e f f e c t o f D-amino a c i d s on c o n f o r m a t i o n . Tentoxin r e p r e s e n t s an i n t e r e s t i n g e x c e p t i o n t o t h e r u l e g o v e r n i n g D-amino a c i d s i n t h a t t h e e f f e c t of t h e N - m e t h y l d e h y d r o p h e n y l a l a n i n e i s t o adopt an a l l L c o n f i g u r a t i o n a l sequence. The s h i f t t o an L form would not be a n t i c i p a t e d w i t h t e n t o x i n s i n c e g l y c i n e i s t r e a t e d as a D-amino a c i d a c c o r d i n g t o t h e r u l e s o f Kato et a l . I t has been proposed t h a t N-methyl-a, 3 - d e h y d r o a l a n i n e has a deep energy minimum (-7 k c a l w i t h = -80 and ¥ = - 10) ( 2 2 ) . S i m i l a r t o r s i o n a l angles have been found w i t h a l l p e p t i d e s having a Zo symmetry c o n f o r m a t i o n (21) . In a s s e s s i n g t h e r e l a t i v e c o n f o r m a t i o n a l f e a t u r e s of t e n t o x i n we have employed r u l e 2 o f Kato et al_. ( 2 1 ) : " i n a r i g h t - h a n d e d p e p t i d e r i n g , t h e c a r b o n y l group a c y l a t i n g a D r e s i d u e i s o r i e n t e d t o t h e upperside". The e f f e c t of t h e N-methylated amide bonds and

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s u b s t i t u t e d D-amino a c i d s on b i o l o g i c a l a c t i v i t y was t e s t e d . In a d d i t i o n t h e r e l a t i v e r o l e of t h e 12-membered r i n g w i t h r e s p e c t t o t h e f u n c t i o n a l groups was c o n s i d e r e d . T e n t o x i n analogs were designed and s y n t h e s i z e d t o e v a l u a t e s t e r e o c h e m i c a l and s t r u c t u r a l p r o p e r t i e s . B i o l o g i c a l t e s t s of t h e analogs demonstrated an i n t e r e s t i n g e f f e c t of D-amino a c i d - c o n t a i n i n g analogs on b i o l o g i c a l a c t i v i t y ( 2 3 ) , Since t e n t o x i n e x h i b i t e d d e v i a t i o n s from the e m p i r i c a l r u l e s , only t h o s e analogs where d e h y d r o p h e n y l a l a n i n e had not been N-methylated were s u b j e c t t o c o n f o r m a t i o n a l p r e d i c t i o n when s u b s t i t u t i n g D-amino a c i d s . S u b s t i t u t i o n of D-amino a c i d s f o r L-amino a c i d s produced an i n c r e a s e i n a c t i v i t y i n one analog and a decrease i n a n o t h e r . The i n c r e a s e d a c t i v i t y was observed i n t h e analog where a s u b s t i t u t i o n of D - A l a n i n e f o r L - A l a n i n e was made w i t h no N - m e t h y l a t i o n . On the o t h e r hand, a s u b s t i t u t i o n o f D-Leu f o r L-Leu i n an N-methylated analog produced a decrease i n a c t i v i t y . The p r e d i c t e d p r e f e r r e d c o n f o r m a t i o n a l s t a t e s f o r some c h l o r o s i s - i n d u c i n g t e n t o x i n analogs i n o r d e r o f t h e i r b i o l o g i c a l a c t i v i t y were c a l c u l a t e d ( F i g u r e 1 ) . Approaches t o S t u d y i n g Amide Bond and Amino A c i d M o d i f i c a t i o n s Tentoxi n

in

The use of amide bond m o d i f i c a t i o n s i n s t u d y i n g b i o a c t i v i t y p r o f i l e s has been very u s e f u l i n a s s e s s i n g b i o l o g i c a l l y a c t i v e p e p t i d e s and t h e s t a t u s of p e p t i d e backbone s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s . The f o l l o w i n g q u e s t i o n s were asked: 1) To what e x t e n t are t h e alignment and s t e r e o c h e m i s t r y of the p e p t i d e backbone c r i t i c a l ? 2) How does the m o d i f i c a t i o n of t h e backbone a f f e c t t h e r e s i s t a n c e toward enzymatic d e g r a d a t i o n ? 3) To what e x t e n t are r i g i d i t y and f l e x i b i l i t y o f p e p t i d e s m a n i f e s t e d i n t h e backbone? 4) Can a g o n i s t s and a n t a g o n i s t s be prepared by i n t r o d u c i n g p e p t i d e backbone modifications? These q u e s t i o n s have been asked about animal p e p t i d e hormones, and they are e q u a l l y a p p l i c a b l e t o p l a n t - a c t i v e c y c l i c t e t r a p e p t i d e s . We have s t u d i e d t h e i n c o r p o r a t i o n of a number of amide bond and amino a c i d m o d i f i c a t i o n s i n both t e n t o x i n and fragments of t e n t o x i n . A number of amide bond m o d i f i c a t i o n s were considered (Figure 2 ) . A Thiomethylene E t h e r - C o n t a i n i n g

Pseudo-dipeptide

The t h i o m e t h y l e n e e t h e r s u b s t i t u t i o n f o r an amide bond i s c h a r a c t e r i z e d by i t s enzymatic r e s i s t a n c e , c o m p a t i b i l i t y w i t h common p e p t i d e c o n f o r m a t i o n a l f e a t u r e s and s y n t h e t i c a c c e s s i b i l i t y from c h i r a l amino a c i d p r e c u r s o r s ( 2 4 ) . T h i s s u b s t i t u t i o n has been e x p l o r e d i n e n k e p h a l i n a n a l o g s , and i t has been found t o both c o n f e r i n c r e a s e d r e s i s t a n c e t o p r o t e o l y s i s as w e l l as n o n s e l e c t i v i t y i n c y c l i c enkephalin analogs. The p r e p a r a t i o n o f p e p t i d e s c o n t a i n i n g a t h i o m e t h y l e n e e t h e r s u b s t i t u t i o n f o r an amide bond has p r e v i o u s l y been a c c o m p l i s h e d w i t h a d i p e p t i d e s u r r o g a t e ( 2 4 ) . For t h e purposes o f our s t u d i e s we t a r g e t e d t h e d i p e p t i d e s u r r o g a t e Boc-Val¥ [CH2S]Phe-0H (1), s y n t h e s i z e d from t h e c h i r a l p r e c u r s o r s S - v a l i n o l and R - p h e n y l a l a n i n e (Scheme 1 ) . Bromination of phenylalanine r e s u l t s i n r e t e n t i o n of c o n f i g u r a t i o n . P r e p a r a t i o n of t h e a-mercapto a c i d of p h e n y l a l a n i n e from t h e bromo a c i d r e s u l t s i n i n v e r s i o n o f t h e c o n f i g u r a t i o n . Using t h e B o c - v a l i n y l t o s y l a t e and t h e disodium s a l t

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Figure 1

Analogs of t e n t o x i n i n t h e i r p r e d i c t e d c o n f o r m a t i o n . R e l a t i v e potency and t h e s t e r e o g e n i c c o n f i g u r a t i o n at a l a n i n e and l e u c i n e r e s i d u e s are n o t e d . Data from r e f .

23.

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Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Figure 2

CONR

R^H

CSNH

y

Many enhanced and equipotent analogs known.

Conformational R i g i d i t y X, angle 0° ( i n Z), 120° ( i n E)

Conservative replacement.

Potential inhibitor for metal loproteases.

S i m p l i f i c a t i o n , maintain receptor a f f i n i t y

Maintenance of side chain topology.

flexibility

Effect

Increased

[Degree of protease resistance = Xs]

X

X

X

XXX

XX

XXX

Proteolytic Resistance

Amide bond and amino a c i d s u b s t i t u t i o n s which c o n f e r p r o t e o l y t i c r e s i s t a n c e and v a r y i n g c o n f o r m a t i o n a l and p h y s i c a l p r o p e r t i e s on p e p t i d e s .

NH of amide

aC-R

CO of amide

n

[Xxx] Y y

/fC^ix H2C CH2

n

CONH

NHCO

2

CONH

Replaced Substituent

CH S

Replacing Substituent

Peptide Mimetics

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o S3

^ £j g el

w

^ ^

>

o jS £

o 2

P

w

o

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3.

EDWARDS ETAL.

1.

NH CHCH OH • 2

2

( B 0 C )

2°

>Boc-NHCHCH OHI CH(CH ) 2

L

CH(CH )

3 2

NOBr

2

6

2

2

(R)-phenylalanine

2

2

3 2

•HSCHC0 H CH C H

5

2

CH

2

6

5

(S)-2-mercapto3-phenyl propanoic acid

(R)-2-bromo-3phenyl-propanoic acid

Boc~NHCHCH OTs + HSCH-C0 H CH(CH )

6

2

2

L

5

->Boc~NHCHCH OTs I CH(CH ) 3 2

Na CS3

-> BrCHCOoH I CH C H

2

CH C H

TsCI pyridine

3 2

NH CHC0 H 2

41

Naturally Occurring Cyclic Peptides

-> Boc-NH-CHCHoSCHC0 H 2

CH(CH ) C H 3 2

2

B o c - L - V a l ^ t C H S ] L-Phe-OH I 2

Scheme 1

S y n t h e s i s o f t h e p s e u d o d i p e p t i d e Boc-Val[CH2S]Phe-0H.

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

42

BIOLOGICALLY ACTIVE NATURAL PRODUCTS

o f a - m e r c a p t o p h e n y l a l a n i n e , a c o u p l i n g r e a c t i o n was performed i n d i m e t h y l s u l f o x i d e . The r e a c t i o n gave Boc-Val¥[CH2S]Phe-0H i n 46% y i e l d ; t h i s analog i s a potent i n h i b i t o r of l e t t u c e r o o t s ( J . V . Edwards, u n p u b l i s h e d r e s u l t s ) . T h i s a n a l o g , however f a i l e d t o e x e r t a h e r b i c i d a l e f f e c t i o n on m o r n i n g g l o r y and b a r n y a r d g r a s s , although an a u x i n - l i k e response was observed with m o r n i n g g l o r y ( J . V . Edwards and D. C a r l s o n , u n p u b l i s h e d r e s u l t s ) .

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Retro-Inverso

Modifications

In r e t r o - i n v e r s o m o d i f i e d analogs of p e p t i d e s , t h e d i r e c t i o n of t h e amide bonds i n t h e backbone has been r e v e r s e d but t h e t h r e e d i m e n s i o n a l o r i e n t a t i o n of t h e s i d e c h a i n s i s m a i n t a i n e d ( 2 5 , 2 6 ) . The R u s s i a n group headed by Shemyakin u t i l i z e d r e t r o - i n v e r s o analogs i n t h e development of a topochemical approach t o t h e study of s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s i n p e p t i d e systems ( 2 7 ) . Their s t u d i e s d e a l t w i t h c y c l i c p e p t i d e s and d e p s i p e p t i d e s , n o t a b l y G l y , Glyl-O-gramicidin j e n n i a t i n a n t i b i o t i c s . I n v e r s i o n of each c h i r a l c e n t e r (and concomitant r e v e r s a l o f the p e p t i d e backbone) r e s u l t e d i n enantiomers e x h i b i t i n g e x a c t l y t h e same b i o l o g i c a l activity. S i m i l a r s t u d i e s have d e a l t w i t h l i n e a r b i o a c t i v e p e p t i d e s . F o r example, a l l D - r e t r o - b r a d y k i n i n (28) and an a l l D - r e t r o analog of a m e l a n o c y t e - s t i m u l a t i n g (MSH) p e p t i d e (29) have been s y n t h e s i z e d . However, t h e o r i g i n a l s p a t i a l o r i e n t a t i o n of a l l s i d e chains i n t h e analogs was not r e t a i n e d s i n c e t h e C- and N - t e r m i n i were r e v e r s e d . The end-group problem can be circumvented by replacement of t h e C - t e r m i n a l amino a c i d r e s i d u e by a 2 - a l k y l m a l o n y l r e s i d u e and t r a n s f o r m a t i o n of t h e N - t e r m i n a l r e s i d u e i n t o a gem-diaminoalkyl residue (26,29). Goodman and coworkers have conducted a number of s t u d i e s on p a r t i a l l y - m o d i f i e d , r e t r o - i n v e r s o analogs o f b i o l o g i c a l l y a c t i v e p e p t i d e s i n which a s e l e c t e d p e p t i d e bond i s r e v e r s e d through t h e i n c o r p o r a t i o n of gem-diaminoalkyl and 2 - a l k y l m a l o n y l d e r i v a t i v e s . (26). Much of t h e research has d e a l t w i t h p a r t i a l l y m o d i f i e d r e t r o - i n v e r s o analogs of t h e l u t e i n i z i n g h o r m o n e - r e l e a s i n g hormone (LH-RH) (30) and e n k e p h a l i n s ( 3 1 - 3 3 ) . In many i n s t a n c e s , t h e analogs have e x h i b i t e d enhanced b i o l o g i c a l a c t i v i t y and i n c r e a s e d s t a b i l i t y toward enzymatic d e g r a d a t i o n ( 3 1 , 3 2 ) . The h e r b i c i d a l l y a c t i v e p e p t i d e t e n t o x i n i s an i n t e r e s t i n g model f o r i n c o r p o r a t i o n of t h e r e t r o - i n v e r s o m o d i f i c a t i o n . C o n f o r m a t i o n a l p r o p e r t i e s are a s s o c i a t e d with c h l o r o s i s i n d u c t i o n and a p r o t e o l y t i c a l l y r e s i s t a n t p e p t i d e bond may enhance t h e b i o l o g i c a l a c t i v i t y of the n a t u r a l l y occurring t o x i n . 5

s

a n (

S y n t h e s i s of a R e t r o - i n v e r s o M o d i f i e d Analog of T e n t o x i n S t u d i e s were c a r r i e d out on a s y n t h e s i s of d i a s t e r e o m e r s of c y c l o [R,S-mLeu-N(CH3)A Phe-Gly-gAla] (compound 2 i n Scheme 2) wherein t h e amide bond of t h e d i p e p t i d e u n i t A l a - L e u i s r e v e r s e d . The p r e f i x m denotes t h e malonic a c i d d e r i v a t i v e of t h e analogous amino a c i d and t h e p r e f i x g denotes t h e gem-di ami n o - a l k y 1 d e r i v a t i v e of t h e c o r r e s p o n d i n g amino a c i d ( 3 0 - 3 3 ) . The t - b u t y l monoester of i s o b u t y l m a l o n i c a c i d , ( 3 ) , m a l o n i c a c i d analog of l e u c i n e was prepared (Scheme 2 ) . D i e t h y l i s o b u t y l m a l o n a t e z

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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3.

EDWARDS ET AL.

Naturally Occurring Cyclic Peptides

43

was prepared i n 74% y i e l d i n accordance w i t h t h e l i t e r a t u r e method (35). Treatment w i t h IN e t h a n o l i c KOH i n acetone a f f o r d e d t h e monoester ( 3 2 ) . R e a c t i o n with t - b u t a n o l and DCC i n t h e presence of 4 - d i m e t h y l ami nopyri d i ne (DMAP) a f f o r d e d t h e t - b u t y l e t h y l d i e s t e r i n 76% y i e l d which was s e l e c t i v e l y h y d r o l y z e d t o 3 w i t h IN NaOH (aqueous) i n m e t h a n o l . There was no r e a c t i o n upon treatment o f t h e d i e s t e r with IN e t h a n o l i c KOH i n a c e t o n e . R e a c t i o n of 3 w i t h N - h y d r o x y s u c c i n i m i d e (HOSu) i n t h e presence of DCC (32) p r o v i d e d t h e N - h y d r o x y s u c c i n i m i d e e s t e r 4 which was o b t a i n e d i n 56% y i e l d upon r e c r y s t a l l i z a t i o n from i s o p r o p a n o l . R e a c t i o n of 4 w i t h d , l - 3 p h e n y l s e r i n e e t h y l e s t e r (5, o b t a i n e d from t h e £ - t o l u e n e s u l f o n a t e s a l t ) (35) i n t h e presence of 1 - h y d r o x y b e n z o t r i a z o l e (HOBt) p r o v i d e d 6 i n 69% y i e l d . Attempts t o prepare 6 from 3 and 5 by t h e mixed a n h y d r i d e method (35) gave poor (25% o r l e s s ) . S e l e c t i v e s a p o n i f i c a t i o n of 6 a f f o r d e d t h e a c i d 7 i n 94% y i e l d . Treatment of 7 w i t h excess a c e t i c anhydride i n t h e presence of sodium a c e t a t e (35) e f f e c t e d s t e r e o s e l e c t i v e d e h y d r a t i o n forming t h e a z l a c t o n e of t-BuO-R,S-mLeu-A Phe (8) i s o l a t e d i n 96% y i e l d as a yellow o i l . R e a c t i o n o f 8 w i t h a 20% excess of t h e d i p e p t i d e H-Gly-Ala-OMe (prepared as shown i n Scheme 3) i n r e f l u x i n g e t h y l a c e t a t e f u r n i s h e d t h e t e t r a p e p t i d e t-BuO-R,S-mLeu-A Phe-Gly-Ala-OMe 9. Flash chromatography of t h e crude product a f f o r d e d unreacted 8 (23% r e c o v e r y ) and 9 i n 54% y i e l d (71% based upon recovered s t a r t i n g material). Compound 9 was s e l e c t i v e l y N-methylated at d e h y d r o p h e n y l a l a n i n e by t r e a t m e n t w i t h excess K2CO3 and iodomethane i n t h e presence of a c a t a l y t i c amount of 18-crown-6 (35) t o p r o v i d e 10 i n 98% y i e l d . T e n t o x i n i t s e l f a l s o c o n t a i n s an N - m e t h y l a l a n i n e m o i e t y . However, t h e second N-methyl group has l i t t l e or no e f f e c t on b i o l o g i c a l acti v i t y . The c o n v e r s i o n of the a l a n i n e r e s i d u e of 10 t o i t s gem-diamino d e r i v a t i v e was e f f e c t e d by treatment of 10 w i t h l i q u i d ammonia i n methanol with s e l e c t i v e f o r m a t i o n of t h e amide 11 i n 93% y i e l d . Treatment of 11 w i t h [ b i s ( t r i f 1 u o r o a c e t o x y ) i o d o ] benzene (TIB) i n 75% a c e t o n i t r i l e / w a t e r (36,32,32,33) a f f o r d e d t h e amine s a l t 12 i n 43% y i e l d f o l l o w i n g work-up. The use of TIB i n t h e c o n v e r s i o n of amides t o amines o f f e r s a s i g n i f i c a n t improvement over e a r l i e r methods i n v o l v i n g t h e Hofmann rearrangement, C u r t i u s rearrangement, or Schmidt r e a c t i o n . In p r e p a r a t i o n of the f i n a l c y c l i z a t i o n s t e p , t h e t - b u t y l e s t e r 12 was d e p r o t e c t e d with 40% t r i f l u o r o a c e t i c a c i d / d i c h l o r o r n e t h a n e , p r o v i d i n g t h e a c i d 13 i n q u a n t i t a t i v e y i e l d . Compound 13 was t r e a t e d w i t h t r i e t h y l amine i n DMF at -20°C u n t i l t h e pH reached 8 . Thereupon, 1.2 e q u i v a l e n t s of d i p h e n y l p h o s p h o r y l a z i d e (DPPA), 1 e q u i v . HOBt, and 0.1 e q u i v . DMAP were added and the m i x t u r e was s t o r e d at - 5 ° f o r f o u r days ( 3 5 ) . I s o l a t i o n of t h e n e u t r a l product a f f o r d e d t h e r e t r o - i n v e r s o m o d i f i e d analog 2 i n 41% y i e l d . Treatment of l e t t u c e seedlings with 2 r e s u l t e d i n nearly equivalent b i o l o g i c a l a c t i v i t y t o t h e parent analog (23) at c o n c e n t r a t i o n s of 100 yM and 10 yM. F u l l c h l o r o s i s i s m a i n t a i n e d at 100 yM (O.D. D a i l e y and J . V . Edwards, u n p u b l i s h e d r e s u l t s ) . z

z

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

BIOLOGICALLY ACTIVE NATURAL PRODUCTS

CH2 (C02 C2H5 )2

(CH3)2 CHCH2 B r

C2HsONa C2HS0H.A 74* 1

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( C H 3 )2CHCH2CH(C02C2H5 ) 2

(CH3 )2 CHCH2CH(CO2 C2 Hs)(CO2 H)

t

j^-BuOH,

DCC OoC-HRT* 76%

CH2CI2, 1

(CH3 )2CHCH2CH(C02C2H5 )(C02t-Bu)

(CH3 )2CHCH2CH(C02t-Bu)(C02H)

N KOH (EtOH) Acetone *" 96%

N NaOH MeOH 87%

HOSu,

DCC.THF

3

O

H

O

56%