Tremorgenic Mycotoxins - ACS Publications

9 Tremorgenic Mycotoxins

Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch009

ALEX CIEGLER and RONALD F. VESONDER Northern Regional Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Peoria, Ill. 61604 RICHARD J. COLE National Peanut Research Laboratory, Dawson, Ga. 31742

Ten tremorgenic mycotoxins have been reported in the literature, but structures have been determined for only five of these. The toxins can be separated into three group ings based on their nitrogen content: one, three, or four atoms per molecule. Structural investigations are underway on one of the major unidentified tremorgens, penitrem A; current data indicate the presence of an indole nucleus and an isoprene unit.

Most described mycotoxins function as hepatotoxins, nephrotoxins, or dermal toxins, but two classes of substances have been isolated that appear to act at the level of the central nervous system. The first of these, citreoviridin, causes paralysis in the extremities of laboratory animals sometimes followed by convulsions and respiratory arrest (1); tremoring has not been ascribed to this substance. The second class encompasses several compounds which cause sustained trembling in host animals. This review covers only the second set of substances, the tremorgens. It is unusual for secondary fungal metabolites to elicit a sustained tremoring response in animals. Only 10 tremorgenic compounds have been reported in the literature, and structures have been determined for five of these, verruculogens TR-1 and TR-2, fumitremorgen B, tryptoquivaline, and tryptoquivalone. The tremorgens can be divided into three groupings based on their nitrogen content: Group A composed of penitrems A, B, and C (PA, PB, and PC) contain only one nitrogen per molecule; Group Β composed of fumitremorgens A and Β ( F T A and FTB) and verruculogens (TR-1 and TR-2) contain three nitrogen atoms per molecule (2); Group C composed of tryptoquivaline and trypto163 Rodricks; Mycotoxins and Other Fungal Related Food Problems Advances in Chemistry; American Chemical Society: Washington, DC, 1976.

164

MYCOTOXINS

quivalone contain four nitrogens per molecule. a v a i l a b l e o n the t e n t h c o m p o u n d W i l s o n and W i l s o n (3) published Group

N o e l e m e n t a l analysis is

i s o l a t e d f r o m Aspergillus

flavus

(4).

A

P e n i t r e m A w a s o r i g i n a l l y e x t r a c t e d f r o m t w o strains of


by

a l t h o u g h a m o l e c u l a r w e i g h t of 501 has b e e n

cyclopium

( l a t e r i d e n t i f i e d as P . crustosum)

Pénicillium

that were the principal

c o n t a m i n a n t s of feedstuffs c a u s i n g disease o u t b r e a k s a m o n g sheep a n d horses ( 4 ) .

A t h i r d p r o d u c i n g s t r a i n c a m e f r o m peanuts n o t i n v o l v e d i n

food intoxication. Subsequently the same toxin was isolated f r o m P . p a l i t a n s N R R L 3468, a c u l t u r e f o u n d i n a l m o s t m o n o t y p i c g r o w t h o n a s a m p l e of m o l d y c o m m e r c i a l f e e d s u s p e c t e d of b e i n g i m p l i c a t e d i n deaths of d a i r y c o w s ( 5 ) . tremortin A .

T h e tremorgen found b y Ciegler (5)

T h i s n a m e is n o w w i t h d r a w n i n d e f e r e n c e

was

named

to t h e t r i v i a l

name penitrem A advanced b y W i l s o n (4) w h o h a d initially discovered this class of t r e m o r g e n i c substances. B e c a u s e the t w o k n o w n Ρ Α - p r o d u c i n g P e n i c i l l i a w e r e c l o s e l y r e l a t e d , C i e g l e r a n d P i t t ( 6 ) s c r e e n e d a d d i t i o n a l Pénicillium

species to d e t e r m i n e

i f t r e m o r g e n p r o d u c t i o n h a d t a x o n o m i c s i g n i f i c a n c e a n d to w h a t extent the o c c u r r e n c e of v a r i o u s P e n i c i l l i a i n foods a n d feeds m i g h t represent a potential health hazard. was

confined

Asymetrica.

Tremorgen

to s e v e r a l species O t h e r subsections

production among

i n the subsection

the

Penicillia

Fasiculata,

i n A s y m e t r i c a o r sections

section

other

than

A s y m e t r i c a tested s h o w n o p r o d u c t i o n of t r e m o r g e n . S o m e of the species t h a t p r o d u c e P A are c o m m o n c o n t a m i n a n t s of grains or specific foodstuffs.

P . crustosum,

a good tremorgen producer,

is a c o n t a m i n a n t of v a r i o u s r e f r i g e r a t e d foods, g r a i n s , a n d c e r e a l p r o d u c t s a n d causes a soft b r o w n r o t i n a p p l e s .

W h e t h e r or n o t t r e m o r g e n is

p r o d u c e d d u r i n g t h e a p p l e - r o t t i n g process is n o t k n o w n . F o u r c u l t u r e s ( P . cyclopium,

P . palitans,

P . crustosum,

P . puberulum)

isolated from

m o l d y c o m m e r c i a l feedstuffs w e r e c a p a b l e of p r o d u c i n g P A o n a v a r i e t y of a g r i c u l t u r a l c o m m o d i t i e s w i t h l o w t e m p e r a t u r e s f a v o r i n g t o x i n a c c u m u lation (7).

I t w a s s u b s e q u e n t l y f o u n d t h a t m o s t of the P A p r o d u c e r s

also s y n t h e s i z e d t w o a d d i t i o n a l c l o s e l y r e l a t e d t r e m o r g e n s , P B a n d P C (8). P A c a u s e d p e r c e p t i b l e t r e m o r s i n m i c e at 250 /xg/kg; P B r e q u i r e d 1.3 m g / k g f o r a s i m i l a r r e a c t i o n . T h e single d o s e L D c a l c u l a t e d a c c o r d i n g to W e i l s f o r m u l a ( 9 ) fidence

5 0

of P A f o r m i c e

w a s 1.05 w i t h a 9 5 %

con

i n t e r v a l of 0.51-2.17 m g / k g ; t h e P B d o s e w a s 5.84 m g / k g w i t h

a 9 5 % c o n f i d e n c e i n t e r v a l of 4 . 1 3 - 8 . 2 6 m g / k g ( 8 ) .

P C is f a r less t o x i c

t h a n P A or P B , b u t studies o n its p r o p e r t i e s a n d t o x i c i t y w e r e l i m i t e d b y l o w y i e l d s . F u r t h e r t o x i c o l o g i c a l effects of p e n i t r e m h a v e b e e n d e s c r i b e d

Rodricks; Mycotoxins and Other Fungal Related Food Problems Advances in Chemistry; American Chemical Society: Washington, DC, 1976.

9.

C I E G L E R

E T

Tremorgenic

A L .

165

Mycotoxins


Applied Microbiology

Figure 1. Thin-layer chromatogram (silica gel G-HR, developed in chloroform.acetone, 93:7; sprayed with FeCl ) of a crude solvent extract of Pénicillium palitans. 1: erogsterol; 2: penitrum C; 3: penitrum B; 4: crude solution; 5: penitrum A; 6: viridicatin. 3

b y W i l s o n et a l . (4, 10) P A produces

and Cysewski ( I I ) .

S t e r n (12)

speculated that

tremor b y i n h i b i t i n g the interneurons w h i c h i n h i b i t the

α - m o t o r cells of t h e a n t e r i o r h o r n of the s p i n a l c o l u m n . T h e p e n i t r e m s c a n b e r e a d i l y e x t r a c t e d f r o m m o l d m y c e l i a or c o n taminated agricultural commodities

b y maceration i n a blendor

with

chloroform-methanol; they can be detected using thin-layer chromatog raphy ( T L C ) on silica gel G - H R ( M e r c k )

developed

acetone (93:7, v / v )

3

and sprayed with F e C l .

i n chloroform-

T h e p a t t e r n of

FeCl 3

p o s i t i v e c o m p o u n d s o n t h e T L C p l a t e appears to b e t h e s a m e f o r most p e n i t r e m p r o d u c e r s a n d is i l l u s t r a t e d i n F i g u r e 1. A s i m p l e a n d specific c o l o r i m e t r i c assay d e p e n d s o n the f o r m a t i o n of a blue color v i a methanolysis ( 1 3 ) ;

absorbance

at 630 n m is a l i n e a r

f u n c t i o n of p e n i t r e m c o n c e n t r a t i o n b e t w e e n 2.5 a n d 3 0 / t g / m l . P e n i t r e m s c a n b e c r y s t a l l i z e d f r o m e t h a n o l - w a t e r i n the f o r m of fine n e e d l e s : P A , 237°-239°C (decomposition,

turning reddish b r o w n ) ; P B

poses s i m i l a r l y b e t w e e n 185° a n d 195 ° C . d i e t h y l ether, c h l o r o f o r m ,

acetone,

mp

decom

T h e p e n i t r e m s are s o l u b l e i n

and methanol; they have

limited

solubility i n water and i n both hydrochloric acid and sodium hydroxide (5%

concentrations).

B o t h p e n i t r e m s are r a p i d l y d e g r a d e d to f o r m a

y e l l o w i s h s o l u t i o n , p r o g r e s s i n g t h r o u g h g r e e n a n d t h e n to b l u e , w h e n d i s s o l v e d i n alcohols o r acetone a n d t r e a t e d w i t h v a r i o u s d i l u t e a c i d s . T h e s e changes are c o n c u r r e n t w i t h loss of t o x i c i t y . P u r i f i e d p e n i t r e m s are u n s t a b l e i n c h l o r o f o r m w h e n e x p o s e d d i r e c t l y to l i g h t . T h i s c h a n g e is p r e s u m a b l y a r e s u l t of a c i d f o r m a t i o n i n the solvent. T h e structures of t h e p e n i t r e m s h a v e n o t b e e n d e t e r m i n e d .

The

e l e m e n t a l analysis a n d the h e a v i e s t d e t e c t a b l e i o n i n t h e mass s p e c t r a of both

toxins

correspond

to

a formula

of

C H 4 4 N O e C l for 3 7


PA

and

166

MYCOTOXINS

Table I.


PPM,

δ

N M R " D a t a of P A i n P y r i d i n e - * ^ Proton Count

Assignment

3 3 3 3 3 12 1 1 1 1 3 2 1 1 1 1 1 1 1 43

quarternary m e t h y l CH -C—0 olefinic m e t h y l olefinic m e t h y l aromatic m e t h y l

1.35 s 1.5 s 1.7 s 1.8 s 2.15 s 3.6-2.0 3.75 S 3.85 d ( J , H z 20) 4.3 d 4.4 s 4.7 t,s 5.0 b,s 5.15 b,s 5.4 d 5.45 s 6.0 s 6.1 d 6.9 s 11.7 Total β

C

3 7

3

acidic O H

N M R = nuclear magnetic resonance; P A = penitrem A .

H45N0

5

for P B ( 8 ) .

B o t h tremorgens g i v e a p o s i t i v e L i e b e r m a n -

B u r c h a r d r e a c t i o n , b u t n e i t h e r is p r e c i p i t a t e d w i t h d i g i t o n i n . N o a m i n o acids

or sugars c a n b e

detected

i n the

acid hydrolyzate

of

either

compound. T h e ultraviolet (uv) at 295 (€ =

s p e c t r u m of P A i n m e t h a n o l shows a b s o r p t i o n

11,600) a n d 2 3 3 n m (c = 3 7 , 0 0 0 ) ; P B has peaks at 286 (t

13,100) a n d 2 2 7 n m (c =

38,450) a n d a s h o u l d e r at 297 n m ( = €

I n 0.1ÎV H C 1 P A has n e w p e a k s at 272 ( =

15,400) a n d 2 5 4 n m

c

=

11,000). (

€

=

2 3 , 7 0 0 ) ; the p e a k at 295 n m d i s a p p e a r e d , a n d t h e p e a k at 233 n m s h i f t e d to 2 2 7 n m (c = 25,500).

T h e s e s p e c t r a suggest a n i n d o l e n u c l e u s .

The

o n l y change i n s p e c t r u m for P B i n a c i d s o l u t i o n is a n e w b r o a d a b s o r p t i o n b e t w e e n 230 a n d 2 4 5 n m (c = 28,200). t r u m changes i n a l k a l i n e s o l u t i o n

B o t h tremorgens s h o w n o spec

(8).

I n u n p u b l i s h e d d a t a w e h a v e s h o w n some s t r u c t u r a l features b y i r , N M R , a n d c h e m i c a l transformations. T h e i r s p e c t r u m of P A i n c h l o r o f o r m s h o w e d s t r o n g O H a b s o r p t i o n at 3580 c m " ; a b a n d at 3475 c m " 1

1

i n d i c a t e d either a n N - H s t r e t c h as i n p y r r o l e s ( i n d o l e s ) o r a h y d r o g e n bonded

O H ; a m e d i u m b a n d at 1650 c m "

1

could result f r o m

C=C

s t r e t c h i n g a t t r i b u t e d to a l k y l C H , C H ; m e t h y l a b s o r p t i o n at 1370 a n d 3

2

1360 c m ; h y d r o x y l at 1055 c m " . 1

1

T h e N M R s p e c t r u m of P A i n d e u t e r a t e d p y r i d i n e ( T M S as r e f e r e n c e ) e x h i b i t e d signals w h i c h a c c o u n t f o r 43 of its 44 protons.


Assign-

9.

Tremorgenic

CIEGLER E T A L .

167

Mycotoxins

merits w e r e m a d e o n l y to the m e t h y l signals ( T a b l e I ) . T h e H p r o t o n of t h e N H c o u l d n o t b e seen as i t occurs as a b r o a d s i g n a l at δ 7.4 w h e r e p y r i d i n e also exhibits signals. A c e t y l a t i o n of P A gave a p r o d u c t w i t h a m p 2 0 3 ° - 2 0 5 ° C ( d e c o m poses, turns d u l l r e d ) .

H i g h - r e s o l u t i o n mass s p e c t r o s c o p y w a s i n a c c o r d

for a m o l e c u l a r f o r m u l a

C39H46NO7CI

incorporating one acetyl group.

T h e i r of the a c e t y l a t e d m a t e r i a l s t i l l s h o w e d O H ( p o s s i b l y a h i n d e r e d Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch009

OH

w h i c h does n o t acetylate w i t h p y r i d i n e - a c e t i c a n h y d r i d e a t r o o m

t e m p e r a t u r e ) , a n d a p e a k at 657 ( M , 18) i n the mass s p e c t r u m w a s i n a g r e e m e n t for a h y d r o x y l g r o u p . T h e b a n d at 3480 c m " , s t r o n g e v i d e n c e 1

f o r a n N H a b s o r p t i o n , w a s s t i l l present i n the i r . T h e N M R signals of a c e t y l a t e d P A are i n T a b l e I I . T h e N M R s p e c t r u m is i n a g r e e m e n t w i t h t h e mass s p e c t r a l analysis i n t h a t one h y d r o x y l g r o u p w a s a c t y l a t e d

(one

n e w m e t h y l p e a k at δ 2.05). T h e p e a k at δ 6.9 i n the N M R s p e c t r u m of P A disappears o n a c e t y l a t i o n , a n i n d i c a t i o n that this p e a k c o u l d r e s u l t f r o m t h e p r o t o n of a h y d r o x y l g r o u p . Table II. PPM,

δ

1.39 s 1.42 s 1.5 s 1.7 s 1.72 s 1.85 s 2.05 s 2.15 s 3.5-2.0

5.6 d

6.1 d 11.77

N M R D a t a of Acetylated P A in P y r i d i n e - ^ Proton

Assignment

Count

methyl methyl isolated C H methyl methyl isolated C H acetate m e t h y l aromatic m e t h y l

3 3 2 3 3 2 3 3

2

2

appearance of a new peak e q u i v a l e n t t o one p r o t o n a t δ 3.6 (b,s) t h i s is a p p a r e n t l y a shift of the 5.45 s peak i n the P A spectrum s t i l l present, c o u l d i n d i c a t e an acidic O H

N M R Decoupling Experiments. I r r a d i a t i o n of t h e p r o t o n f o r P A at δ 6.1 collapses the d o u b l e t at δ 4.3 to a s m a l l e r d o u b l e t , i n d i c a t i n g a l l y l i c c o u p l i n g to a m e t h i n e p r o t o n of the f o l l o w i n g t y p e :

N

R

1

H

.R

1/

c=c—c


168

MYCOTOXINS

Ηδ3.2 G e m i n a l c o u p l i n g of the t y p e

G

C

R -c

Ηδ3.8

-R is i n d i -

R

c a t e d b y i r r a d i a t i n g the s i g n a l at δ 3.0 w h i c h causes the d o u b l e t at δ 3.85 to c o l l a p s e t o a b r o a d singlet.

H y d r o g é n a t i o n of a c e t y l a t e d P A u s i n g


A d a m s c a t a l y s t a p p e a r e d to cause d e c o m p o s i t i o n .

H o w e v e r the m e t h y l

protons at δ 2.15 r e m a i n e d ; this is e v i d e n c e of a n a r o m a t i c m e t h y l g r o u p . B e c a u s e H m i t e d m a t e r i a l h i n d e r e d f u r t h e r c h e m i c a l analysis, o n l y t e n t a t i v e s p e c u l a t i o n c a n b e m a d e o n a structure f o r P A . B a s e d o n s p e c t r o s c o p i c d a t a t h e presence of a n i n d o l e n u c l e u s c a n b e p r o p o s e d w i t h a f u l l y s u b s t i t u t e d a r o m a t i c m o i e t y , e.g.,

In

a d d i t i o n one O H is r e a d i l y a c e t y l a t e d , a n d a p p a r e n t l y o n e is n o t

acetylated.

T h i s w o u l d a c c o u n t for t w o of t h e o x y g e n f u n c t i o n s .

The

p r o t o n at δ 11.77 i n the N M R s p e c t r u m i n d i c a t e s a n a c i d i c O H or b o n d e d OH

(e.g., h y d r o x y l g r o u p b o n d e d to c a r b o n y l f u n c t i o n ) .

I f t h e latter is

t h e case w e c o u l d i n t e r p r e t the b a n d at 1650 c m " i n t h e I R to b e a b o n d e d 1

c a r b o n y l ; this w o u l d a c c o u n t for t w o m o r e oxygens. T h e r e m a i n i n g t w o o x y g e n s c o u l d b e a c c o u n t e d for b y ether l i n k a g e s . H o w e v e r t h e N M R does n o t e x c l u d e a n e p o x i d e l i n k a g e . M o r e d e t a i l e d s t r u c t u r a l analyses w i l l d e p e n d o n t h e a v a i l a b i l i t y of a d d i t i o n a l t o x i n . Group

Β

Verruculogen. V e r r u c u l o g e n ( T R - 1 ) , a t r e m o r g e n i c m y c o t o x i n , w a s p r o d u c e d b y Pénicillium

verruculosum

( A T C C #24640; N R R L

i s o l a t e d f r o m peanuts t h a t w e r e m o l d e d as a r e s u l t of i m p r o p e r conditions (14).

5881) storage

I n a d d i t i o n to c a u s i n g severe tremors i n m i c e a n d one-

d a y o l d cockerels v e r r u c u l o g e n has a n L D

5 0

of 2.4 m g / k g i n m i c e a n d

15.2 m g / k g i n cockerels, i . p . W h e n i t w a s a d m i n i s t e r e d o r a l l y , t h e L D w a s 126.7 m g / k g i n m i c e a n d 365.5 m g / k g i n cockerels.


5 0

9.

CIEGLER ET AL.

Tremorgenic

169

Mycotoxins

H o t u j a c et a l . ( 1 5 ) s t u d i e d the m o d e of a c t i o n of v e r r u c u l o g e n u s i n g b o t h m i c e a n d t h r e e - to s e v e n - d a y o l d c h i c k e n s .

O n t h e basis o f t h e i r

results u s i n g a s u b s t a n c e w h i c h i n c r e a s e d y - a m i n o b u t y r i c a c i d l e v e l s i n the central nervous system of m i c e a n d a y - a m i n o b u t y r i c a c i d derivative w h i c h w a s c a p a b l e of p a s s i n g the b l o o d - b r a i n b a r r i e r , t h e y

concluded

t h a t v e r r u c u l o g e n - i n d u c e d tremors i n m i c e w e r e c a u s e d b y a d e c r e a s e of g a m m a - a m i n o b u t y r i c a c i d levels i n the c e n t r a l n e r v o u s system.


I n a subsequent study using three- to seven-day-old chickens i n w h i c h y-aminobutyric

a c i d itself e a s i l y p e n e t r a t e d

the b l o o d - b r a i n barrier,

t h e y f o u n d a d i r e c t a n t a g o n i s t i c effect of γ - a m i n o b u t y r i c a c i d o n v e r r u c u logen-induced tremors. A l t h o u g h the biosynthesis of v e r r u c u l o g e n has n o t b e e n s t u d i e d , i t m a y b e s p e c u l a t e d o n t h e basis of its c h e m i c a l s t r u c t u r e ( S t r u c t u r e I )

CH

3

CH

3

Structure

I

t h a t i t is f o r m e d f r o m the a m i n o a c i d s p r o l i n e a n d t r y p t o p h a n a n d t w o isoprene units. T h e close s t r u c t u r a l a n d s p e c t r a l s i m i l a r i t i e s of T R - 1 a n d a c l o s e l y r e l a t e d substance T R - 2 w i t h those o f f u m i t r e m o r g e n A a n d Β ( S t r u c t u r e II)

( F T A a n d F T B ) s t r o n g l y suggest a c o m m o n b i o s y n t h e t i c p a t h w a y .

P e r h a p s T R - 1 comes f r o m F T B v i a h y d r o p e r o x i d e . tain an additional isoprene moiety. gens w a s d e t e c t e d

F T A a p p e a r s to c o n

A l t h o u g h n e i t h e r of t h e f u m i t r e m o r -

i n extracts of the v e r r u c u l o g e n - p r o d u c i n g m o l d ,

m e t a b o l i t e e x h i b t i n g the s a m e R

f

a

o n T L C a n d c o l o r reactions as those

Structure

II


170

MYCOTOXINS

o b s e r v e d f o r T R - 2 w a s present.

S i n c e t h e c o n v e r s i o n of T R - 1 t o T R - 2

w a s o b s e r v e d o n l y u n d e r the specific c o n d i t i o n s r e p o r t e d ( 2 )

a n d since

T R - 2 w a s n o t a p r o d u c t f r o m p h o t o d e g r a d a t i o n , i t is p l a u s i b l e t h a t T R - 2 is also o f n a t u r a l o r i g i n a n d m a y b e a b i o s y n t h e t i c p r e c u r s o r to T R - 1 . Verruculogen

(mp

233°-235°C)

moderately soluble i n benzene,

was very soluble i n chloroform,

e t h y l acetate, acetone,

and dimethyl-

s u l f o x i d e , s p a r i n g l y s o l u b l e i n e t h a n o l , m e t h a n o l , a n d toluene, a n d i n s o l u


b l e i n hexane a n d water. T h e tremorgen r a p i d l y degraded into several p h o t o p r o d u c t s w h e n s u b j e c t e d to n o r m a l l a b o r a t o r y l i g h t c o n d i t i o n s . B e c a u s e of its size a n d c o m p l e x i t y , t h e c h e m i c a l s t r u c t u r e o f v e r r u culogen (Structure I ) was elucidated b y single crystal x-ray diffraction studies i n l i e u o f c h e m i c a l m e t h o d s .

T h e tremorgen was neutral a n d

c r y s t a l l i z e d w i t h one m o l e c u l e of b e n z e n e

(C27H32O7N3

· C H ). e

It was

6

c h a r a c t e r i z e d b y 6 - O - m e t h o x y i n d o l e , d i k e t o p i p e r a z i n e , β-methylcrotonyl m o i e t i e s , a n d a n o v e l e i g h t - m e m b e r e d p e r o x i d e r i n g system T h e u v s p e c t r u m of T R - 1 s h o w e d A =

11,000), a n d 2 9 5 n m (

indole compounds

(2).

€

m a x

E t 0 H

(2).

226 ( e — 47,500), 277 (c

= 9 7 0 0 ) w h i c h is t y p i c a l of 2,3,6-substituted

T h e C D s p e c t r u m of T R - 1 i n e t h a n o l s h o w e d

t w o C o t t o n effects c o r r e s p o n d i n g to t h e first t w o u v b a n d s ; t h e t h i r d c o t t o n effect w a s n o t o b s e r v e d . + 0 . 1 6 ; at 265 n m i t w a s Ac =

T h e C o t t o n effect at 290 n m w a s Ac

=

+ 0 . 5 6 . M a j o r a b s o r p t i o n s i n the i r spec

t r u m w e r e 3520 a n d 3480 c m "

1

( O H i n d o l e , or b o t h ) , 1660 c m

p i p e r a z i n e ) , a n d d o u b l e t at 1 3 6 5 - 1 3 5 5 cm"*

1

(diketo

(gem-dimethyl).

T h e p r o t o n N M R s p e c t r u m of v e r r u c u l o g e n ( F i g u r e 2 ) w a s c h a r a c t e r i z e d b y e x t r e m e l y w e l l - d e f i n e d c h e m i c a l shifts t h a t w e r e a s s i g n e d as f o l l o w s : T h e t h r e e a r o m a t i c protons h a d t h e t y p i c a l p a t t e r n f o r a n o r t h o d o u b l e t at δ 7.85 ( / = 9.0 H z ) ( C 1 6 ) , o r t h o - m e t a d o u b l e t of d o u b l e t s at δ 6.74 ( / = 2.0 a n d 9.0 H z ) ( C 1 7 ) , a n d a m e t a d o u b l e t at δ 6.52 ( / — 2.0 Hz)

(C19).

T h e l a t t e r s i g n a l w a s p a r t i a l l y o b s c u r e d b y another p r o t o n

s i g n a l ( δ 6.57) a s s i g n e d to t h e p r o t o n at ( C 2 1 ) ( d o u b l e t ; / =

8.0 H z ) . A

v i n y l p r o t o n ( C 2 2 ) c o u p l e d to ( C 2 1 ) w a s o b s e r v e d as a d o u b l e t ( / = Hz)

a t δ 4.98.

methine protons located on ( C 1 3 ) . as a d o u b l e t (7 = doublet ( / =

8.0

V i n c i n a l coupling was observed between the O H a n d T h e O H p r o t o n a p p e a r e d at δ 4.74

3.0 H z ) , a n d t h e m e t h i n e p r o t o n w a s at δ 5.57 as a

3.0 H z ) . T h e a d d i t i o n of D 0 to t h e N M R s a m p l e r e s u l t e d 2

i n t h e d i s a p p e a r a n c e of the s i g n a l at δ 4.74 a n d a c o n c o m i t a n t c o l l a p s e i n t h e c o u p l i n g of t h e s i g n a l at δ 5.57. T h e O H p r o t o n ( D 0 2

exchangeable)

l o c a t e d o n ( C 1 2 ) r e s o n a t e d at δ 4.25 as a s h a r p singlet. T h e h y d r o x y l s on (C12)

a n d ( C 1 3 ) w e r e i n the cis c o n f i g u r a t i o n . T h e g e m - d i m e t h y l

protons o n the i s o v a l e r y l m o i e t y ( C 2 4 a n d C 2 5 ) w e r e e q u i v a l e n t w h e n a n a l y z e d i n c h l o r o f o r m - d s o l u t i o n a n d r e s o n a t e d at δ 1.66 as a six p r o t o n singlet;

i n d i m e t h y l sulfoxide-de

singlets at δ 1.58 a n d δ 1.70.

they appeared

as t w o

three-proton

T h e t w o g e m i n a l m e t h y l groups ( C 2 9 a n d


CIEGLER ET AL.

9.


ι

ι

Figure

C30)

I'

'

i

2.

Tremorgenic

r

ι

171

Mycotoxins

ι

ι

ι

ι

ι

ι

ι

ι

ι

ι

ι

Proton magnetic resonance spectrum of verruculogen taken in chloroform-d solution

a t t a c h e d to ( C 2 8 )

ι

ι

ι

(Structure

I)

w e r e o b s e r v e d at δ 1.95 a n d δ 0.97 as t h r e e -

p r o t o n singlets. T h e t w o protons a d j a c e n t to t h e p r o l i n e n i t r o g e n a t p o s i t i o n 9 r e s o n a t e d as a n i l l - d e f i n e d t r i p l e t a t δ 3.50; t h e other f o u r p r o t o n s c o m p r i s i n g the p r o l i n e r i n g a n d the m e t h y l e n e protons at p o s i t i o n 3 1 g a v e r i s e to a n u n s t r u c t u r e d m u l t i p l e t b e t w e e n δ 1.5 a n d 2.5. T h e p r o t o n at p o s i t i o n 6 w a s a s s i g n e d to t h e c h e m i c a l s h i f t at δ 4.40

(ill-defined

t r i p l e t ) . T h e ( C 3 ) m e t h i n e p r o t o n w a s s t r o n g l y c o u p l e d to o n l y one o f t h e m e t h y l e n e protons o n ( C 3 1 )

a n d a p p e a r e d as a d o u b l e t at δ 5.98

( / — 10.0 H z ) . T h e m e t h o x y p r o t o n s r e s o n a t e d as a t h r e e - p r o t o n singlet at δ 3.75. D o u b l e t resonance e x p e r i m e n t s i n b o t h d e c o u p l i n g a n d I N D O R modes verified the coupling between the protons on ( C 2 1 ) and between

(C13)

and ( O H 1 3 ) .

and

T h e a p p e a r a n c e of t h e C 3 )

(C22)

methine

p r o t o n as a d o u b l e t w a s consistent w i t h t h e g e o m e t r y o f t h e m o l e c u l e as d e t e r m i n e d b y x - r a y c r y s t a l l o g r a p h y . D a t a f r o m studies to d e t e r m i n e t h e m o l e c u l a r f o r m u l a w e r e i n i t i a l l y somewhat conflicting.

H o w e v e r h i g h r e s o l u t i o n , mass s p e c t r a l analysis

effecting i o n i z a t i o n v i a e l e c t r o n - i m p a c t a t 70 e V p r o v i d e d t h e mass (m/e

511.2360) w h i c h a n a l y z e d f o r

C27H33N3O7

(2).

correct

T h e mass

s p e c t r u m s h o w e d , i n a d d i t i o n t o t h e m o l e c u l a r i o n at m/e 511, p r o m i n e n t f r a g m e n t ions at m/e 496 (loss of C H ) , m/e 4 9 3 (loss o f H 0 ) , a n d 3

2

m/e

427 (loss o f C H 0 ) . T h e l a t t e r f r a g m e n t a t i o n also o c c u r r e d c h e m i c a l l y 5

8


172

MYCOTOXINS

w h e n verruculogen was hydrogenated using palladium on carbon ( 5 % ) i n an ethanol solution ( 2 ) . This chemical reaction yielded two products: one p r o d u c t w a s a s m a l l m o l e c u l e i d e n t i f i e d as i s o v a l e r a l d e h y d e ( C H i O ) 5

0

w h i c h o r i g i n a t e d v i a c l e a v a g e of t h e b o n d s b e t w e e n ( C 2 1 ) a n d t h e i n d o l e n i t r o g e n a n d b e t w e e n t h e p e r o x i d e oxygens.

Concomitantly the double

b o n d b e t w e e n ( C 2 2 ) a n d ( C 2 3 ) w a s s a t u r a t e d to f o r m i s o v a l e r a l d e h y d e . T h e p r o d u c t ( T R - 2 ) w a s assigned S t r u c t u r e I I after c o m p a r i n g its Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch009

s p e c t r a l d a t a w i t h t h a t o f v e r r u c u l o g e n ( 2 ) . H i g h r e s o l u t i o n , mass spec t r a l analysis o f T R - 2 s h o w e d t h e largest detectable mass at m/e w i t h a computer-calculated formula of

C22H27N3O6.

showed

(c

A

m

a

x

E

t

0

H

224

(e —

37,400), 268

429.1898

Its U V s p e c t r u m

= 6 8 3 0 ) , a n d 294 n m (

7 5 4 0 ) w h i c h is t y p i c a l f o r 2,3,6-substituted i n d o l e c o m p o u n d s

c

—

(2). The

I R s p e c t r u m of T R - 2 s h o w e d m a j o r absorptions at 3450 ( O H a n d / o r i n d o l e ) , 1660 ( d i k e t o p i p e r a z i n e ) , a n d 1380 c m " ( C H ) . 1

3

P r i n c i p a l considerations w e r e o b t a i n e d f r o m t h e p r o t o n a n d spectra ( 2 ) .

1 3

C-NMR

T h e p r o t o n N M R of T R - 2 ( F i g u r e 3 ) h a d t h e t y p i c a l

c h e m i c a l shifts f o r ortho d o u b l e t

( δ 6.60, 7 = 3.0, a n d 9 . 0 H z ) , a n d a

m e t a d o u b l e t at δ 6.87 (7 = 3.0 H z ) i n a c c o r d a n c e w i t h t h e protons p o s i t i o n e d at ( C 1 6 ) ,

(C17),

a n d ( C 1 9 ) of the indole ring.

T h e proton

assigned to p o s i t i o n 3 w a s at δ 5.37 ( m u l t i p l e t ) . A c h e m i c a l shift f o r one D 0 exchangeable p r o t o n a p p e a r i n g at δ 5.94 w a s assigned to t h e O H 2



9.

C I E G L E R

E T

Tremorgenic

A L .

173

Mycotoxins

Figurée. The C-NMR spectrum of verruculogen (Structure I). ( )— Assignments may be reversed. *—Assignment uncertain. All values in ppm downfield from Τ MS; solvent CDCl . 13

3

g r o u p f o r m e d o n ( C 2 8 ) . T h e s i x - p r o t o n s i g n a l i n the s p e c t r u m of v e r r u c u l o g e n r e s o n a t i n g at δ 1.66 a n d assigned to t h e g e m - d i m e t h y l g r o u p a t t a c h e d to ( C 2 3 )

w a s absent i n the s p e c t r u m of T R - 2 . A l s o a b s e n t

w e r e the t w o c o u p l e d m e t h i n e p r o t o n s r e s o n a t i n g i n t h e T R - 1 s p e c t r u m at δ 6.57 ( C 2 1 ) a n d 4.98 ( C 2 2 ) ( d o u b l e t s ; / — 8.0 H z ) . F u r t h e r evidence supporting the (Structure I I ) proposed for T R - 2 was p r o v i d e d w h e n the

1 3

C - N M R s p e c t r a of T R - 1 a n d T R - 2 w e r e c o m

p a r e d ( F i g u r e s 4 a n d 5 ) . T h o s e five carbons are p r e s e n t i n t h e s p e c t r u m o f T R - 1 b u t are absent f r o m t h e

1 3

1 3

C-NMR

C - N M R spectrum of T R - 2

c o r r e s p o n d e d to ( C 2 3 ) , ( C 2 4 ) , ( C 2 5 ) , ( C 2 6 ) , a n d ( C 2 7 ) .

These data

are consistent w i t h t h a t e x p e c t e d f o r t h e p r o p o s e d c o n v e r s i o n o f v e r r u c u l o g e n T R - 1 to T R - 2 . Fumitremorgens. O n l y t w o b r i e f m a n u s c r i p t s h a v e b e e n p u b l i s h e d on fumitremorgens A a n d Β ( F T A a n d F T B ) , two closely related toxins p r o d u c e d b y A . fumigatus

(16, 17).

T h e s t r u c t u r e o f F T B w a s estab

l i s h e d i n the l a t t e r p u b l i c a t i o n a n d w a s s h o w n to c o n t a i n p r o l i n e a n d 6-methoxyindole

groups

(Structure I I I ) .

F T B appears

identical

Structure 111


to


Figure S. The C-NMR spectrum of TR-2 (Structure II). ( )—Assignments may be reversed. *—Assignment uncertain. AU values in ppm downfield from TMS; solvent CDCl . 13

s

l a n o s u l i n , t h e m a j o r m e t a b o l i t e o f Pénicillium lanosum (18).

T h e struc

t u r e o f F T B appears a l m o s t i d e n t i c a l t o t h a t o f v e r r u c u l o g e n

(TR-1)

except f o r t h e presence o f t h e p e r o x i d e m o i e t y l i n k i n g t h e t w o i s o p r e n e fragments.

B o t h F T A a n d F T B are soluble i n chloroform a n d ethyl

acetate, b u t o n l y s l i g h t l y s o l u b l e i n m e t h y l a n d e t h y l alcohols. I n t h e u v s p e c t r a there a r e t h r e e m a x i m a : A a x m

6 6 , 9 0 0 ) , 275.5 ( c — 1 2 , 0 0 0 ) ,

a n d 2 9 5 n m ( = 8500). €

E t 0 H

225.5

(e =

F T A has the fol

l o w i n g properties: Colorless prisms f r o m methanol; m p 202.5°-203.5°C; elemental composition

C32H41N3O7;

m/e 5 7 9 ; i r s p e c t r a ( K B r ) : 3420,

2940, 1670, 1565, 1440, 1370, 1300, 1160, 1070, a n d 1035 c m " . 1

FTB

c r y s t a l l i z e s f r o m m e t h a n o l as colorless needles w i t h t h e f o l l o w i n g c h a r a c teristics: m p 2 1 1 ° - 2 1 2 ° C ; e l e m e n t a l c o m p o s i t i o n

C27H33N3O5; m/e 4 7 9 ;

t h e I R s p e c t r a w a s s i m i l a r to t h a t o f F T A . Toxicity data indicated that 1 m g F T A / k g mice caused sustained t r e m b l i n g w h i c h w a s m o r e severe w h e n F T B w a s i n j e c t e d i . p . a t a s i m i l a r dose.

The L D

(i.p.)

c a u s e d 7%

5 0

was not determined, b u t 5 m g of either F T A or F T B d e a t h w i t h i n 96 h r . T h e f u m i t r e m o r g e n s h a v e n o t

been indicated i n a n y natural occurring mycotoxicosis.



9.

C I E G L E R

E T

Tremorgenic

A L .

175

Mycotoxins

Structure IV

Structure V

Group C I n a p r i v i l e g e d c o m m u n i c a t i o n f r o m G . Bùchi, M a s s a c h u s e t t s I n s t i tute of Technology

(19),

w e l e a r n e d t h a t h e a n d h i s colleagues

i s o l a t e d t w o n e w t r e m o r g e n i c toxins f r o m Aspergillus

had

clavatus—trypto-

quivaline (Structure I V ) a n d tryptoquivalone (Structure V ) .

The pro

d u c i n g f u n g u s h a d b e e n i s o l a t e d o r i g i n a l l y f r o m a s a m p l e of m o l d y r i c e collected i n a T h a i household w h e r e a y o u n g b o y h a d d i e d of an u n i d e n t i fied toxicosis. T h e i s o l a t e d s t r a i n p r o d u c e d t w o n e w n o n t o x i c m e t a b o l i t e s , kotanin and desmethylkotanin (20), h i g h l y toxic cytochalasin Ε

as w e l l as s m a l l a m o u n t s

of

the

(21).

T h e m o r e p o l a r of the t w o n e w t r e m o r g e n s , t r y p t o q u i v a l i n e , h a d t h e following properties: m p 153°-155°, [ a ] composition C cm" ; uv A 1

m a x

3 1 7 n m (c =

2 9

2 5

D 1 4 2 ° ; m/e

546.2; e l e m e n t a l

H 3 o N 0 ; I R ( C H C 1 ) 3520, 1790, 1735, 1680, a n d 1615 4

228 (

E t 0 H

7

c

3

— 3 7 , 0 0 0 ) , 275 (e — 8 5 5 0 ) , 305 (e —

3 0 4 0 ) ; N M R ( C D C 1 ) δ 1.03 ( d , 3, 7 = 3

3800),

7 H z ) , 1.17 ( d , 3,

7 =

9 H z ) , 1.50 (s, 3 ) , 1.52 (s, 3 ) , 2.19 (s, 3 ) , 2.63 ( m , 1 ) , 3.10 ( d , 1,

J =

10Hz),

5.61

3.15 ( d , 1, J =

10Hz),

3.63 ( b , 1 ) , 4.04 ( b , 1 ) , 5.00 (s, 1 ) ,

( d , 1, 7 = 9 H z ) , 5.70 (t, 1, 7 — 1 0 H z ) , 7 . 1 2 - 7 . 9 0 ( m , 7 ) ,

8.22

( d , 1, 7 — 8 H z ) . C h e m i c a l a n d s p e c t r a l d a t a i n d i c a t e d t h a t t r y p t o q u i v a l i n e is a s p i r o - y - l a c t o n e rather

than

a δ-lactone. F i g u r e 6 shows a c o m

p u t e r g e n e r a t e d d r a w i n g of t h e x - r a y m o d e l o f the

p-bromophenylure-

thane derivative of tryptoquivaline. T h e less p o l a r t r e m o r g e n , t r y p t o q u i v a l o n e , h a d t h e f o l l o w i n g c h a r a c teristics: m p 2 0 2 ° - 2 0 4 ° ; [ « ] C

2 e

H 4N 04; U V A 2

4

m a x

E t 0 H

2 5

D 2 5 4 ° ; m/e 488.17; e l e m e n t a l c o m p o s i t i o n

234 ( c — 3 4 , 9 5 0 ) , 292 (

6300); I R ( C H C 1 ) 2525,1790,1735,1715, 3

δ 1.24 ( d , 3, 7 — 7 H z ) , 1.31 ( d , 3, J =

c

— 9550), 3 2 0 n m ( 1

7 H z ) , 1.59 ( d , 3, J =

( d o f d , 1, 7 — 1 0 , 1 4 H z ) , 347 ( d o f d , 1, 7 — 1 1 , 1 4 H z ) , 5.12 1, 7 =

7 H z ) , 4.36 ( q u a r t e t , 1, 7 =

— 3

7 H z ) , 3.09 (quintent,

7 H z ) , 5.24 (s, 1 ) , 5.51 (t, 1, 7 =

H z ) , 7.12 ~ 7.94 ( m , 7 ) , 8.28 ( d , 1, J =8

€

a n d 1680 c m " ; N M R ( C D C 1 )

Hz).


10


176

MYCOTOXINS

Figure 6. Computer generated drawing of the x-ray model of the p-bromophenyl-

Tremorgenic Mycotoxins - ACS Publications

Recommend Documents