5 Aspergillus Toxins Other Than Aflatoxin RICHARD J. COLE National Peanut Research Laboratory, U.S. Department of Agriculture, P.O. Box 637, Dawson, Ga. 31742
Available physical, chemical, and biological data are presented for all known Aspergillus mycotoxins other than the aflatoxins with emphasis on the chemistry of those that are of major interest. Included in this latter category are the ochratoxins, the sterigmatocystins, and the aspergillic acid group. Other Aspergillus mycotoxins presented in less detail are kojic acid, austamide, ascladiol, terreic acid, viriditoxin, cytochalasin E, maltoryzine, 3-nitropropanoic acid, oxalic acid, helvolic acid, gliotoxin, fumigatin, fumagillin, terrein, spinulosin, and butenolide.
Members of the genus Aspergillus represent some of the most prevalent mycotoxin-producing fungi associated with feed and food materials. The known mycotoxins of Aspergillus spp., other than aflatoxins, are pre sented in this review. Major emphasis is on the chemistry of those Aspergillus toxins currently recognized to be of major interest, including the ochratoxins, the sterigmatocystins, and aspergillic acid. A l l Aspergillus toxins, until proved otherwise, are considered potentially hazardous to animal health. Ochratoxins
The ochratoxins comprise a group of chemically related metabolites isolated originally from culture extracts of Aspergillus ochraceus (1, 2) and subsequently from other Aspergillus spp. (3) and from Pénicillium viridicatum (4, 5). The achratoxins contain a 3,4-dihydro-3-methylisocoumarin moiety linked through a carboxyl group to L-β-phenylalanine by a secondary amide bond. The most toxic derivatives, ochratoxins A (Structure Ia) and C (Structure Ic) contain a chlorine atom at position 5 (6,7,8). Ochratoxin Β (Structure Ib), which differs from ochratoxin A 68
5.
69
Aspergillus Toxins
COLE
Structure I.
=
Ochratoxins
=
=
la: R H, IL Cl, R, H lb: R = H, Ri = H, Ri = H
H
le: R R= = H,R CiHs, = Rï = Rs = Id: Cl,RCl, = OHH 1
s
b y t h e absence o f c h l o r i n e at p o s i t i o n 5, w a s c o n s i d e r a b l y less t o x i c (6, 8, C h u et a l . ( J O ) p o s t u l a t e d t h a t t h e c h l o r i n e atoms o n o c h r a t o x i n s A
9).
a n d C p l a y an indirect role i n toxicity. T h e y presented a direct correlation b e t w e e n t h e d i s s o c i a t i o n constants f o r t h e p h e n o l i c h y d r o x y l g r o u p s o n the ochratoxins a n d t h e i r a c u t e t o x i c i t y . T h e y suggested t h a t t h e p h e n o l i c h y d r o x y l g r o u p i n t h e d i s s o c i a t e d f o r m w a s necessary f o r t o x i c i t y a n d t h a t the c h l o r i n e a t o m m a y h a v e a d i r e c t effect o n t h e d i s s o c i a t i o n o f the p h e n o l i c h y d r o x y l groups i n ochratoxins A a n d C , r e n d e r i n g t h e m toxic. T h e y f u r t h e r n o t e d t h a t the a c i d d i s s o c i a t i o n constant o f o c h r a t o x i n Β w a s o n e - t e n t h as large as o c h r a t o x i n A , a n d t h e t o x i c i t y o f o c h r a t o x i n Β w a s c o r r e s p o n d i n g l y a b o u t o n e - t e n t h t h a t of o c h r a t o x i n A (9). D a t a o n t h e t o x i c i t y of t h e r e c e n t l y r e p o r t e d 4 - h y d r o x y o c h r a t o x i n A ( S t r u c t u r e I d ) w e r e n o t presented i n d e t a i l , b u t t h e t o x i n w a s r e p o r t e d to b e n o n - l e t h a l to rats at 40 m g / k g ( i n t r a p e r i t o n e a l ) (5). O c h r a t o x i n A w a s l e t h a l to a l l rats tested a t this dosage l e v e l . T h e c h e m i c a l structures o f t h e ochratoxins w e r e e l u c i d a t e d b y S o u t h A f r i c a n scientists ( J , 2, 6) a n d s u b s e q u e n t l y p r o v e d b y synthesis ( I I ) . A c i d h y d r o l y s i s o f o c h r a t o x i n A g a v e L - / ? - p h e n y l a l a n i n e a n d 7-carboxy-5chloro-3,4-dihydro-8-hydroxy-3-methyhsocoumarin.
Support for a second
ary a m i d e was presented b y the I R spectrum w h i c h showed a t y p i c a l a m i d e I b a n d a t 1678 c m " ( C = 0 s t r e t c h i n ) a n d a m i d e I I b a n d a t 1
1535 c m
1
( N - H bending)
boxyl group
absorptions
a n d a t 3380 c m " ( N - H s t r e t c h i n g ) . C a r 1
appeared
at 1723 c m "
1
(C=0
stretching),
a n d a b r o a d b a n d a p p e a r e d b e t w e e n 2500 a n d 3000 c m " ( O - H s t r e t c h 1
i n g ) . T h e lactone f u n c t i o n w a s o b s e r v e d a t 1678 c m " ( s h i f t e d t o l o w e r 1
frequency
because of intramolecular Η b o n d i n g w i t h t h e 8 h y d r o x y l
g r o u p ) a n d at 1132 c m "
1
( C - O - C stretching).
T h e u v spectrum of ochratoxin A showed A
m a x
E t 0 H
215 (c — 36,800)
a n d 333 n m (e = 6400); o c h r a t o x i n Β h a d t h e s a m e u v s p e c t r u m w i t h t h e exception (A
m a x
E t 0 H
of a hypsochromatic
shift o f t h e l o n g w a v e
length
band
218, c — 37,200 a n d 318 n m , = 6900) (2). €
T h e h i g h r e s o l u t i o n mass s p e c t r u m o f o c h r a t o x i n A s h o w e d
m/e
403.08187 w i t h a c a l c u l a t e d e l e m e n t a l c o m p o s i t i o n o f C o H i C l N O e a n d , 2
8
i n a c c o r d a n c e w i t h t h e c a l c u l a t e d f o r m u l a , a n isotope p e a k a t m/e 405 (2).
A p r o t o n a t e d m o l e c u l a r i o n p e a k a t n o m i n a l mass 404 w a s o b s e r v e d
i n t h e l o w r e s o l u t i o n mass s p e c t r u m o f o c h r a t o x i n A after i o n i z a t i o n w i t h isobutane.
chemical
70
MYCOTOXINS
I ι ι ' • ι
1 , ι ι, ι, I ! ι ι ι, I ι, ι ι ι I ι ι ι ι I , ι ι ι I ι ι ι ι I ι, ι !, ι I ι ι ι ι 1 ι ι ; ι I
' Figure
8 7 1. Sixty MHz
^-τ
ι ι ι I ι
6 5 4 3 2 1 proton magnet resonance spectrum of ochratoxin chloroform-ά solution
i^lJ^J
ί~~ in
T h e p r o t o n m a g n e t i c resonance s p e c t r u m of o c h r a t o x i n A ( F i g u r e 1 ) consisted of a n A B X system i n t h e 3 , 4 - d i h y d r o i s o c o u m a r i n m o i e t y .
The
signals for t h e s e c o n d a r y m e t h y l at p o s i t i o n 3 r e s o n a t e d at δ 1.54 as a doublet ( / =
7.0 H z ) . I n c l u d e d i n this system w e r e t h e m e t h y l e n e p r o
tons at p o s i t i o n 4 w h i c h r e s o n a t e d at δ 3.2 ( c o m p l e x s i g n a l ) a n d t h e m e t h i n e p r o t o n at p o s i t i o n 3 w h i c h r e s o n a t e d at a p p r o x i m a t e l y δ 4.75 ( c o m p l e x s i g n a l ) . T h e s e signals are s i m i l a r to those o b s e r v e d f o r t h e same 3 , 4 - d i h y d r o i s o c o u m a r i n system i n m e l l e i n ( S t r u c t u r e I I ) ( 1 2 ) :
the
m e t h i n e p r o t o n ( p o s i t i o n 3 ) a p p e a r e d as a sextet at δ 4.50 (7 = 7.0 H z ) . S t r o n g l y c o u p l e d t o the m e t h i n e p r o t o n w e r e
the secondary
methyl
( p o s i t i o n 3 ) w h i c h r e s o n a t e d at δ 1.50 as a d o u b l e t (7 — 7.0 H z ) a n d the m e t h y l e n e protons ( p o s i t i o n 4 ) w h i c h r e s o n a t e d at δ 2.83 as a d o u b l e t (7 = 7.0 H z ) .
( T h e s e three v a l u e s w e r e i n c o r r e c t l y r e p o r t e d as 3.0 H z . )
N e a r l y s u p e r i m p o s e d o n the m e t h y l e n e protons ( δ 3.20 ) of o c h r a t o x i n A w a s a c o m p l e x t w o - p r o t o n s i g n a l a s s i g n e d to the b e n z y l i c m e t h y l e n e protons, a n d n e a r l y s u p e r i m p o s e d o n the m e t h i n e s i g n a l (δ 4.55) w a s a
Structure II. Mellein
5.
71
Aspergillus Toxins
C O L E
c o m p l e x s i g n a l ( δ 5.02)
assigned to the m e t h i n e p r o t o n p o s i t i o n e d next
to t h e a m i d e n i t r o g e n . T h e s i n g l e a r o m a t i c p r o t o n of the d i h y d r o i s o c o u m a r i n m o i e t y r e s o n a t e d at δ 8.60 as a singlet; the five a r o m a t i c protons of p h e n y l a l a n i n e r e s o n a t e d at δ 7.40 as a singlet. T h e c a r b o x y l p r o t o n w a s o b s e r v e d at δ 13.00 a n d the h y d r o x y l p r o t e i n at δ 11.61. T h e e x t r e m e d o w n field p o s i t i o n of t h e latter w a s t y p i c a l of a n Η - b o n d e d O H g r o u p .
This
same extreme d o w n f i e l d p o s i t i o n w a s r e p o r t e d for the Η - b o n d e d d i h y d r o i s o c o u m a r i n O H protons of m e l l e i n a n d 4 - h y d r o x y m e l l e i n (δ — 11.03 a n d 11.03) (12).
T h e a m i d e p r o t o n o n o c h r a t o x i n A r e s o n a t e d at δ 8.75 as a
doublet ( / =
8.0 H z ) .
T h e N M R s p e c t r u m of o c h r a t o x i n Β c o n t a i n e d
s i m i l a r c h e m i c a l shifts b u t d i f f e r e d f r o m o c h r a t o x i n A b y the presence of t w o o r t h o - c o u p l e d a r o m a t i c protons ( l o c a t e d o n positions 5 a n d 6 ) reso n a t i n g at δ 8.22 a n d 7.05 (7 — 8.0 H z ) The
1 3
C-NMR
(off
(2).
center resonance d e c o u p l i n g s p e c t r u m )
of d i -
m e t h y l i s o c o u m a r i n c a r b o x y l a t e as p r e s e n t e d b y M a e b a y a s h i et a l . ( J 3 ) is s h o w n i n F i g u r e 2. T h e t w o signals o c c u r r i n g at l o w e s t field, 31.56 a n d 27.65 p p m d o w n f i e l d f r o m C S , w e r e a s s i g n e d to c a r b o n y l carbons 1 a n d 2
11 r e s p e c t i v e l y . T h e c h e m i c a l shifts b e t w e e n 71.08 a n d 49.12 p p m w e r e assigned to the a r o m a t i c carbons w i t h the s i g n a l for t h e a r o m a t i c c a r b o n C - 6 e a s i l y r e c o g n i z e d f r o m t h e off resonance d e c o u p l i n g s p e c t r u m .
The
t w o m e t h o x y c a r b o n s w e r e l o c a t e d at 128.19 a n d 139.80 p p m . T h e m e t h y l ene c a r b o n at C - 4 w a s o b s e r v e d at 158.66 p p m ; t h e t e r t i a r y m e t h y l ( C - 1 0 ) o n p o s i t i o n 3 of the d i h y d r o i s o c o u m a r i n m o i e t y w a s at 171.95 p p m .
The
m e t h i n e c a r b o n ( C - 3 ) w a s a s s i g n e d to the c h e m i c a l shift at 118.79 p p m . T h e most r e c e n t o c h r a t o x i n - t y p e c o m p o u n d r e p o r t e d w a s 4 - h y d r o x y ochratoxin A (Structure Id)
( 5 ) f r o m Pénicillium
viridicatum.
Charac
teristic differences b e t w e e n the N M R s p e c t r a of o c h r a t o x i n A a n d 4hydroxyochratoxin A were i n the dihydroisocoumarin moiety. A n Ο C H O C12
J
AMY3
OCH Ο 113 " 3
J
11
'CHo 10
J
aromatic C
CH ÎC3) nOCH OCH3
(C6)
3
c=o ici) C=Ol iCll)!
Figure 2.
C—off
13
(C12)
CDCI
J
mi
(C13)
rm
center resonance decoupling spectrum of marin carborylate in chloroform-d solution
CH
2
C«3
(cio) rm
dimethylisocou-
72
MYCOTOXINS
system i n the 3,4 p o s i t i o n e d a r e a w a s e v i d e n t i n t h e N M R s p e c t r u m of 4 - h y d r o x y o c h r a t o x i n A w h e r e a s o c h r a t o x i n A h a d a n A B X system i n t h i s region. T h e A M Y
3
system c o n s i s t e d of t h e f o l l o w i n g : a s e c o n d a r y m e t h y l
at p o s i t i o n 3 w h i c h a p p e a r e d as a d o u b l e t at δ 1.68
(7 = 7 . 0 H z ) ,
the
m e t h i n e p r o t o n at p o s i t i o n 3 w h i c h r e s o n a t e d as a q u a r e t e t of d o u b l e t s at δ 4.80 (7 = 2 , 7 H z ) , a n d the m e t h i n e p r o t o n o n p o s i t i o n 4 w h i c h r e s o n a t e d as a d o u b l e t at δ 5.11 (7 = 2 H z ) . T h e biosynthesis of t h e ochratoxins has b e e n s t u d i e d w i t h t h e a i d of 1 4
C - and
1 3
C - l a b e l e d precursors.
I t has b e e n d e m o n s t r a t e d t h a t p h e n y l
a l a n i n e w a s i n c o r p o r a t e d d i r e c t l y i n t o t h e o c h r a t o x i n s ( 1 3 , 14, 1 5 ) , a n d i t w a s p r e s u m e d t h a t the biosynthesis of p h e n y l a l a n i n e o c c u r r e d i n t h e usual manner—i.e., v i a the shikimic a c i d pathway.
S e a r c y et a l .
(14)
r e p o r t e d t h a t t h e i r d a t a w e r e consistent w i t h t h e h y p o t h e s i s t h a t t h e m a j o r p o r t i o n of t h e i s o c o u m a r i n m o i e t y of o c h r a t o x i n A w a s s y n t h e s i z e d v i a acetate c o n d e n s a t i o n w i t h most of t h e
1 4
C - l a b e l from
supplemented
[ 2 - C ] s o d i u m acetate l o c a t e d i n c a r b o n s 2, 4, a n d 6 ( S t r u c t u r e I I I ) . 1 4
o
Structure
HI.
Ochratoxin
A
( T h e n u m b e r i n g system of t h e i s o c o u m a r i n n u c l e u s i n t r o d u c e d b y S e a r c y et a l . (14)
is r e t a i n e d to f a c i l i t a t e d i s c u s s i o n . )
T h e y o b s e r v e d l i t t l e or
n o r a d i o a c t i v i t y i n carbons 1, 3, 5. 9, 10, or 11 of t h e i s o c o u m a r i n m o i e t y . T h e y c o n c l u d e d t h a t the absence of r a d i o a c t i v i t y i n c a r b o n 10 suggested t h a t it was not d e r i v e d f r o m acetate. a p f e l (15)
I n similar studies, Steyn a n d H o l z -
reported that the isocoumarin a c i d moiety was derived f r o m
five acetate units b y h e a d - t o - t a i l c o n d e n s a t i o n , a n d t h e y c o n c l u d e d , t h e r e fore, t h a t carbons 9 a n d 10 w e r e also d e r i v e d f r o m acetate.
T h e y sug
gested that the absence of a p p r e c i a b l e r a d i o a c t i v i t y o b s e r v e d b y S e a r c y et a l . ( J 4 ) i n carbons 9 a n d 10 m a y h a v e a r i s e n f r o m a l o w y i e l d of acetate f r o m d e g r a d a t i o n experiments a n d f r o m t h e r e l i a n c e o n t o t a l r a d i o a c t i v i t y r a t h e r t h a n o n specific r a d i o a c t i v i t y . T h e y also e s t a b l i s h e d w i t h the a i d of m e t h i o n i n e - S - C H 3 that t h e source of t h e c a r b o x y l c a r b o n at p o s i t i o n 4 14
was v i a transmethylation probably from r e c e n t l y M a e b a y a s h i et a l . ( 1 3 )
using
1 3
S-adenysylmethionine.
C - N M R studies c o n f i r m e d the
p a r t i c i p a t i o n of s o d i u m f o r m a t e - C i n t h e f o r m a t i o n of the 1 3
More
carboxyl
f u n c t i o n at p o s i t i o n 4. T h e s e studies s t r o n g l y suggested t h a t t h e i s o c o u m a r i n a c i d w a s d e rived
v i a t h e a c e t a t e - m a l o n a t e p a t h w a y w i t h t h e e x c e p t i o n of the c a r
b o x y l f u n c t i o n at C
4
w h i c h w a s d e r i v e d f r o m the C i p o o l . T h e p o i n t i n
5.
COLE
Aspergillus
73
Toxins
the biosynthesis of o c h r a t o x i n A at w h i c h the c h l o r i n e a t o m w a s i n c o r p o rated was not determined. It was assumed that phenylalanine was formed via the shikimic acid pathway. Sterigmatocystins T h e s t e r i g m a t o c y s t i n s are a g r o u p of c l o s e l y r e l a t e d f u n g a l m e t a b o lites c h a r a c t e r i z e d b y a x a n t h o n e n u c l e u s f u s e d to a d i h y d r o d i f u r a n o or a tetrahydrodifurano moiety.
T h e most e c o n o m i c a l l y i m p o r t a n t m e m
b e r of t h e g r o u p is s t e r i g m a t o c y s t i n ( S t r u c t u r e I V a ) f r o m A . nidulans,
A . rugulosus,
a n d Bipoloris
Aspergillus
(16),
members
include aspertoxin
(3-hydroxy-6,7-dimethoxydifuroxanthone)
( S t r u c t u r e I V b ) , ( I S , 19, 20),
O-methylsterigmatocystin (Structure I V c )
(21)
sp. (17),
other
versicolor
and dihydro-O-methylsterigmatocystin (Structure V a )
Aspergillus
flavus;
5-methoxysterigmatocystin
(Structure
(22),
from
IVd)
(23),
Structure
9
IVa:
IV. Sterigmatocystins R = H,R = CHs, Rs = H, Rs H R = OH, Ri = CHs, Ri = CHs, Rs — H R = H, Ri = CHs, R* = CHs, Rs H R = H, R, = CHs, Rs = H,R = OCHs R = H,R = H,R*=z H, Rs = H 1
=
IVb: IVc:
=
r=
IVd:
S
IVe:
6-demethylsterigmatosystin (Structure I V e )
1
dihydrosterigmatocys-
(24),
tin (Structure V b ) (25), a n d dihydrodemethylsterigmatocystin (Structure Vc)
(25)
f r o m Aspergillus
versicolor.
T h e m a j o r differences a m o n g the
v a r i o u s sterigmatocystins are t h e p r e s e n c e o r absence of u n s a t u r a t i o n i n the d i f u r a n o r i n g s y s t e m ( s i m i l a r to aflatoxins B i a n d B ) 2
a n d i n the
s u b s t i t u t i o n p a t t e r n o n p o s i t i o n s 6, 7, a n d 10 of the xanthone r i n g system a n d / o r p o s i t i o n 3 of t h e d i f u r a n o system. E n g l e b r e c h t a n d A l t e n k i r k (26)
s t u d i e d t h e t o x i c i t y of s t e r i g m a t o
c y s t i n analogs o n p r i m a r y c e l l c u l t u r e s . T h e y c o n c l u d e d t h a t c o m p o u n d s c o n t a i n i n g the A - f u r o b e n z o f u r a n - r i n g s y s t e m ( S t r u c t u r e I V ) w e r e m o r e 1 2
t o x i c t h a n those c o n t a i n i n g a s a t u r a t e d f u r o b e n z o f u r a n - r i n g system ( S t r u c ture V ) .
T h e c a r c i n o g e n i c i t y of s t e r i g m a t o c y s t i n has b e e n w e l l
Structure
V. Sterigmatocystins Va: R = CH R* = CHs Vb: R = CHs, Ri == H Vc: R = H, Ri = H 3y
docu-
74
M Y C O T O X I N S
m e r i t e d (27, 28, 29).
E n g e l b r e c h t a n d A l t e n k i r k (26)
further suggested
t h a t a c a r b o n y l g r o u p u n s a t u r a t e d i n the α,β p o s i t i o n a n d a n u n s a t u r a t e d b o n d i n the A - - p o s i t i o n are r e q u i r e d f o r c a r c i n o g e n i c i t y . A l s o a m e t h o x y 1
2
g r o u p at p o s i t i o n 6 e n h a n c e d t o x i c i t y of these c o m p o u n d s , a n d a m e t h o x y g r o u p at p o s i t i o n 7 d e c r e a s e d t o x i c i t y . H o l z a p f e l et a l . ( 1 7 ) , i n studies o n t h e acute t o x i c i t y of s t e r i g m a t o c y s t i n to a l b i n o rats, r e p o r t e d v a l u e s of 1 2 0 - 1 6 6 m g / k g (per B u l l o c k et a l . (16) cystin.
o$) a n d 6 0 - 6 5 m g / k g
LD
5
0
(IP).
e l u c i d a t e d the c h e m i c a l s t r u c t u r e of s t e r i g m a t o
S t e r i g m a t o c y s t i n is a p a l e y e l l o w c r y s t a l l i n e c o m p o u n d
m e l t i n g p o i n t of 2 4 6 ° C ( d e c ) 208, 235, 249, a n d 329 n m ( l o g
4.28, 4.39, 4.44, a n d 4.12,
€
with a
Its u v s p e c t r u m s h o w e d
(30).
A
E t 0 H
m a x
respectively)
T h e u v s p e c t r u m a g r e e d w i t h s p e c t r a of m a n y o t h e r h y d r o x y l a t e d
(16).
a n d / o r m e t h o x y l a t e d xanthones
T h e most c h a r a c t e r i s t i c features
(30).
of t h e i n f r a r e d ( i r ) s p e c t r u m of s t e r i g m a t o c y s t i n w e r e 3450 c m " 1650 c m "
1
( y - p y r o n e ) , 1627 c m " , 1 6 1 0 c m " , a n d 1590 c m " 1
1
(OH),
1
(phenyl)
1
(16).
T h e h i g h r e s o l u t i o n mass s p e c t r u m of s t e r i g m a t o c y s t i n s h o w e d 324.0627 w h i c h a n a l y z e d for C i H n 0 . 8
m/e
T h e most p r o m i n e n t p e a k i n t h e
6
c h e m i c a l - i o n i z a t i o n mass s p e c t r u m w a s at n o m i n a l mass m/e 325 w i t h n o p r o m i n e n t f r a g m e n t ions. T h e p r o t o n N M R s p e c t r u m of s t e r i g m a t o c y s t i n consisted of c h e m i c a l shifts f o r t w o
different systems:
x a n t h o n e system.
the d i h y d r o d i f u r a n o
system a n d
the
C o u p l i n g b e t w e e n the three n o n e q u i v a l e n t p r o t o n s
of
t h e x a n t h o n e s y s t e m , H , H , a n d H i , g a v e rise to a n A B X s p e c t r u m i n 8
9
0
w h i c h J A X — 7BX — 8.1 H z ( F i g u r e 3 ) . c o n s i s t e d of a t r i p l e t at δ 7.64 ( / = t r u m was complicated
b y the H
T h e X p o r t i o n of this s p e c t r u m
8.1 H z ) . T h e A B p o r t i o n of the s p e c p r o t o n of t h e d i h y d r o d i f u r a n o
4
s y s t e m w h i c h r e s o n a t e d i n the same a r e a ( s u p e r i m p o s e d shifts f o r H et a l . (16)
8
a n d H i between δ 6.7-7.0) ( F i g u r e 3 ) . 0
observed
8
ring
chemical
However, Bullock
a c o u p l i n g constant of 2 H z i n a c o m p l e x
c o r r e s p o n d i n g to the c h e m i c a l shifts of t h e H 6.8; 7AB =
on
group
a n d H i protons ( a b o u t δ 0
2 H z ) of s t e r i g m a t o c y s t i n . R o d r i c k s et a l . (19)
reported that
t h e c o r r e s p o n d i n g A B p o r t i o n of the A B X system ( x a n t h o n e p r o t o n s ) i n a s p e r t o x i n acetate was n o t e n t i r e l y d i s c e r n i b l e , b u t t h r e e d o u b l e t s
were
o b s e r v e d w i t h a c o u p l i n g constant of 1 H z (δ 6.98 a n d 6.78; J B — 1 H Z ) A
i n t h e r e g i o n of the H
8
and H i
0
protons.
superimposed on the acetal proton ( H ) 4
T h e remaining doublet
was
of t h e d i h y d r o d i f u r a n o system.
T h e r e f o r e , analysis of t h e A B X s y s t e m of a s p e r t o x i n acetate w a s Jax JBX =
8.0HZ
(ortho substituted)
and / B = A
1HZ
(meta
=
substituted)
(19).
N M R a n a l y s i s of the c o r r e s p o n d i n g protons i n t h e s p e c t r u m of d i h y d r o - o - m e t h y l s t e r i g m a t o c y s t i n (22)
p r o v i d e d a m o r e d i s c e r n i b l e v i e w of
t h e x a n t h o n e protons since t h e a c e t a l p r o t o n ( d o u b l e t δ 6.5, / = w a s n o t s u p e r i m p o s e d o n the c h e m i c a l shifts of protons H
8
6.0Hz)
a n d Η χ of t h e 0
5.
C O L E
ι ι ι
I
Aspergillus Toxins
ι ι ι ι I ι ι ι ι
75 1
I
I
1080
1
i: f
,° 3 C H
Figure
3.
Proton NMR
spectrum of sterigmatocystin
in cloroform-d
solution
x a n t h o n e system. I t c a n be r e a d i l y o b s e r v e d f r o m this s p e c t r u m ( F i g u r e 4 ) t h a t t h e X p o r t i o n of the s p e c t r u m ( H d i - o r t h o t r i p l e t at δ 7.57
(7 =
9
p r o t o n ) a g a i n a p p e a r e d as a
8.0 H z ) ; the A B p o r t i o n ( H
8
and
Ηχο)
resonated as t w o 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 7.0 a n d 6.8, r e s p e c t i v e l y (7 = 8.0 H z a n d 1.0 H z ) . T h e values for 7 A X — 7 B X — 8 . 0 H z a n d 7AB =
1.0 H z also agree w i t h 7-ortho a n d 7-meta for b e n z e n o i d systems.
T h e N M R s p e c t r u m of s t e r i g m a t o c y s t i n also c o n t a i n e d the t y p i c a l c h e m i c a l shifts for protons of a d i h y d r o d i f u r a n o s y s t e m s i m i l a r to those o b s e r v e d for the c o r r e s p o n d i n g protons i n aflatoxin Βχ (31, 3 ) : Ηχ = t r i p l e t , δ 6.62 (7 = 2 . 5 H z ) ; H H
3
=
t r i p l e t s of d o u b l e t , δ 4.81 (7 =
6.85 (7 = 7 H z ) .
The noncoupled
2
33)
(Figure
= t r i p l e t , δ 5.50 (7 =
2.5Hz);
2.5 a n d 7.0 H z ) ; H
4
— doublet, ca. δ
a r o m a t i c p r o t o n , H , r e s o n a t e d at δ 5
6.50 as a singlet, a n d t h e m e t h o x y protons at p o s i t i o n 6 w e r e o b s e r v e d at δ 4.00 ( F i g u r e 3 )
(16).
T h e s t r u c t u r a l analysis of the p - b r o m o b e n z o a t e d e r i v a t i v e of s t e r i g matocystin b y x-ray diffraction (33)
a g r e e d w i t h t h e s t r u c t u r e of s t e r i g
m a t o c y s t i n p r o p o s e d b y B u l l o c k et a l . (16).
T h e s t r u c t u r a l assignment
of s t e r i g m a t o c y s t i n w a s v e r i f i e d f u r t h e r t h r o u g h t o t a l synthesis m e t h y l s t e r i g m a t o c y s t i n (34)
(zb)-O-
a n d b y the c o n v e r s i o n of O - m e t h y l d i h y d r o -
sterigmatocystin into dihydroaspertoxin b y treating it w i t h methanolic a l k a l i a n d t h e n w i t h l e a d tetraacetate a n d d i l u t e a l k a l i n e h y d r o l y s i s
(1).
β!ο
Figure
j
0
4.
Proton
6
|
0
. . . . . .
NMR spectrum of cystin in chloroform-d
j
q
.
^
ppj
'
(5)
4 0
dihydro-O-methyhterigmatosolution
H o l k e r a n d M u l h e i r n ( 3 5 ) s t u d i e d t h e biosynthesis of sterigmatocys t i n b y d e g r a d a t i o n of
1 4
C - l a b e l e d t o x i n p r o d u c e d b y Aspergillus
versicolor
f r o m [ 1 - C ] acetate. T h e y r e p o r t e d t h a t t h e d i s t r i b u t i o n of r a d i o a c t i v i t y 1 4
i n d i c a t e d t h a t t h e x a n t h o n e r i n g system i n s t e r i g m a t o c y s t i n
probably
o r i g i n a t e d v i a the a c e t a t e - m a l o n a t e p a t h w a y a n d t h a t t h e 4 - c a r b o n b i s f u r a n m o i e t y also s e e m e d to arise f r o m h e a d - t o - t a i l c o n d e n s a t i o n of t w o acetate u n i t s w i t h t h e C — C b o n d j o i n i n g the x a n t h o n e a n d b i s f u r a n m o i e t i e s d e r i v e d f r o m acetate m e t h y l groups.
T h e y also o b s e r v e d that
t h e l e v e l of r a d i o a c t i v i t y i n the b i s f u r a n m o i e t y w a s s i g n i f i c a n t l y l o w e r t h a n t h a t i n the xanthone system. F r o m the a b o v e observations H o l k e r a n d M u l h e i r n ( 3 5 ) suggested t h a t s t e r i g m a t o c y s t i n w a s d e r i v e d f r o m t w o separate k e t i d e u n i t s c o m b i n e d i n a n u n k n o w n f a s h i o n . S i n c e sterigmatocystins, v e r s i c o l o r i n s , a n d aflatoxins a l l c o n t a i n the furobenzofuran
r i n g system, i t has b e e n s p e c u l a t e d t h a t t h e y h a v e
a
c o m m o n b i o g e n e t i c p a t h w a y or that the aflatoxins m a y b e d e r i v e d f r o m s t e r i g m a t o c y s t i n a n d / o r v e r s i c o l o r i n t y p e precursors
( 3 2 , 3 5 , 36,
R e c e n t e v i d e n c e p a r t i a l l y s u p p o r t e d these hypotheses.
H s i e h et a l . ( 3 3 )
demonstrated that
14
37).
C - s t e r i g m a t o c y s t i n w a s efficiently c o n v e r t e d to afla
t o x i n B i b y t h e r e s t i n g m y c e l i u m of Aspergillus
parasiticus.
T h e i r results
5.
C O L E
77
Aspergillus Toxins
indicated a biosynthetic p a t h w a y leading from 5-hydroxysterigmatocystin to s t e r i g m a t o c y s t i n a n d t h e n to aflatoxin B . x
S c h r o e d e r et a l . ( 3 9 )
re
p o r t e d t h a t a n o r a n g e v e r s i c o l o r i n - t y p e p i g m e n t , t e n t a t i v e l y i d e n t i f i e d as v e r s i c o n a l acetate, a c c u m u l a t e d i n c u l t u r e s of Aspergillus
flavus w i t h a
c o n c o m i t a n t r e d u c t i o n i n aflatoxin p r o d u c t i o n as a r e s u l t of t h e i n h i b i t o r y a c t i o n of the i n s e c t i c i d e d i c h l o r v o s . Aspergillic
Acid
A s p e r g i l l i c a c i d ( S t r u c t u r e V I ) , first of a n u m b e r of closely r e l a t e d p y r a z i n e metabolites reported, was discovered a n d n a m e d b y W h i t e a n d W h i t e a n d H i l l (41).
(40)
A s w i t h m a n y other m y c o t o x i n s , a s p e r g i l l i c
a c i d w a s o r i g i n a l l y d i s c o v e r e d b e c a u s e of its a n t i b i o t i c p r o p e r t i e s . A s p e r g i l l i c a c i d a n d its analogs are m a j o r m e t a b o l i t e s A . flavus a n d o t h e r Aspergillus
of c e r t a i n strains of
spp.
A s p e r g i l l i c a c i d is a c u t e l y toxic to m i c e ( 1 0 0 - 1 5 0 m g / k g , i p ) b u t has n o c h r o n i c effects at s u b l e t h a l dosages (41).
T h e analogs of a s p e r g i l l i c
a c i d s h o w e d ranges of t o x i c i t y f r o m n e a r z e r o to t o x i c i t y e q u a l i n g t h a t f o r a s p e r g i l l i c a c i d (42,
43, 44).
T o x i c i t y a p p e a r e d to b e r e l a t e d to the
h y d r o x a m i c a c i d f u n c t i o n a l i t y , a n d little effect o n t o x i c i t y w a s o b s e r v e d f o r differences i n t h e 3 a n d 6 p o s i t i o n e d s i d e - c h a i n substituents. M a c D o n a l d (45, 46) 1 4
i n studies w i t h D L - l e u c i n e - C a n d L - i s o l e u c i n e -
C s h o w e d t h a t Aspergillus
1 4
flavus
synthesized aspergillic acid a n d h y -
d r o x y a s p e r g i l l i c a c i d ( S t r u c t u r e V I I ) f r o m one m o l e c u l e of l e u c i n e p l u s one m o l e c u l e of i s o l e u c i n e .
T h i s c o n c l u s i o n w a s b a s e d o n the f a c t that
aspergillic acid from m e d i u m supplemented
with L-isoleucine- C had 1 4
m o s t of the r a d i o a c t i v i t y i n t h e i s o l e u c i n e m o i e t y a n d o n l y a s m a l l a m o u n t i n the l e u c i n e m o i e t y . T h e o p p o s i t e was f o u n d w h e n a s p e r g i l l i c a c i d w a s produced i n m e d i u m supplemented w i t h DL-leucine- C. D a t a were simi 1 4
l a r f r o m studies o n t h e biosynthesis of h y d r o x y a s p e r g i l l i c a c i d . I n another s t u d y a s p e r g i l l i c a c i d - C w a s c o n v e r t e d to h y d r o x y a s p e r g i l l i c a c i d - C , 1 4
1 4
b u t the reverse w a s not true. A l s o , i n t h e e a r l y stages of g r o w t h of A . flavus,
m o r e a s p e r g i l l i c a c i d t h a n h y d r o x y a s p e r g i l l i c a c i d w a s present i n
the m e d i u m , b u t i n the later stages, h y d r o x y a s p e r g i l l i c a c i d p r e d o m i n a t e d . T h e a b o v e findings s u p p o r t t h e hypothesis t h a t h y d r o x y a s p e r g i l l i c a c i d is produced irreversibly from aspergillic acid.
CH
'3
Structure VI.
ό
Aspergillic
Acid
Structure VII. Hydroxyasper gillic Acid
78
MYCOTOXINS
Structure VIII. Νeoaspergillie Acid I n l a t e r studies u s i n g radioisotopes, M i c e t i c h a n d M a c D o n a l d
(47)
showed that neoaspergillic a c i d (Structure V I I I ) was biosynthesized from two molecules of leucine.
R e s u l t s also s t r o n g l y suggested t h a t t h e se
q u e n c e i n t h e biosynthesis o f n e o a s p e r g i l l i c a c i d w a s (2) flavacol
( S t r u c t u r e I X ) —» n e o a s p e r g i l l i c a c i d - >
leucine-»
neohydroxyaspergillic
acid (Structure X ) .
, C M - C H C
H
2
^ N ^ O H
^ C H - C H ^ N ^ O H
3
Stucture IX.
C
Ffovacol
H
3
O H
£
Structure X. Neohydroxyas pergillic Acid
E l u c i d a t i o n o f t h e c h e m i c a l structure o f a s p e r g i l l i c a c i d ( C12H20N2O2 ) ( S t r u c t u r e V I ) p r i m a r i l y arose f r o m a n d D u t c h e r a n d W i n t e r s t e i n e r (50)
t h e w o r k of D u t c h e r
(48,
49)
a l o n g w i t h s u b s e q u e n t studies b y
ether investigators w h i c h e v e n t u a l l y r e v i s e d t h e n a t u r e o f t h e side c h a i n s a n d e s t a b l i s h e d t h e i r l o c a t i o n o n t h e p y r a z i n e r i n g (51, 52, 53, 54). P r i n c i p l e c h e m i c a l features
of aspergillic acid are pyrazine ring,
c y c l i c h y d r o x a m i c a c i d , s e c - b u t y l a n d i s o b u t y l moieties.
T h e various
analogs o f a s p e r g i l l i c a c i d differ f r o m e a c h other p r i m a r i l y i n t h e n a t u r e of t h e s i d e - c h a i n substituents o n positions 3 a n d 6. A s p e r g i l l i c a c i d a n d m o s t analogs c a n exist i n e i t h e r t h e h y d r o x a m i c a c i d f o r m
(2-hydroxy-
pyrazine-l-oxide) or the l-hydroxy-2-pyrazinone form (Structure X I ) . T h e u v spectrum of aspergillic a c i d was A a n d 235 n m (c = 10,500) a n d A
m a x
m a x
E t 0 H
328 (e — 8500)
336 n m ( c = 10,800) i n 0.05M p h o s
p h a t e buffer, p H 7.3. T h e i r s p e c t r u m s h o w e d absorptions at 3120, 2940,
RAN^OH O
R f ^ N ^ O O H
Structure XI. Ατ±Β Aspergillic Acid Nu cleus
5.
79
Aspergillus Toxins
C O L E
2850, 2 8 0 0 - 2 2 5 0 ( b r o a d ) , 2040 ( a b s e n t i n c h l o r o f o r m s o l u t i o n ) , 1 5 8 5 , 1 1 5 0 , a n d 710 c m "
1
1640,
(48).
T h e N M R s p e c t r u m of a s p e r g i l l i c a c i d t a k e n i n t r i f l u o r o a c e t i c a c i d s o l u t i o n w i t h t e t r a m e t h y l s i l a n e as i n t e r n a l reference s h o w e d t h e f o l l o w i n g r e c o g n i z a b l e features: a c h e m i c a l shift r e s o n a t i n g at δ 7.83 f o r a single p r o t o n w a s assigned to t h e a r o m a t i c p r o t o n o n p o s i t i o n 5 ( S t r u c t u r e V I ) ; the m e t h y l e n e protons a t t a c h e d to C - 3 r e s o n a t e d as a d o u b l e t (7 =
8.0
H z ) at δ 3.12; the m e t h i n e p r o t o n i n the s e c - b u t y l side c h a i n a t t a c h e d to C - 6 w a s o b s e r v e d at δ 3.73 ( m u l t i p l e t ) , a n d the f o u r m e t h y l g r o u p s o n the sec-
a n d i s o b u t y l side c h a i n s w e r e at δ 1.02 a n d δ 1.51 ( β - m e t h y l o n
t h e s e c - b u t y l side c h a i n w a s p r e s u m a b l y s u p e r i m p o s e d o n g e m - d i m e t h y l T h e 3 p r o t o n d o u b l e t at δ 1.51 (7 = 6 H z ) w a s a s s i g n e d
doublet) (47).
to the m e t h y l g r o u p o n the s e e - b u t y l s i d e c h a i n ( p o s i t i o n 6 ) a n d a p p a r e n t l y s h o w e d v i r t u a l c o u p l i n g to t h e adjacent m e t h i n e p r o t o n w h i c h w a s i n t u r n c o u p l e d to the adjacent m e t h y l e n e protons. Kojic
Acid
Kojic acid [5-hydroxy-2-(hydroxymethyl)-4ii-pyran-4-one] t u r e X I I ) , a r e l a t i v e l y c o m m o n m e t a b o l i t e of Aspergillus
(Struc
spp. a n d i n
o
Structure XII. Kojic Acid p a r t i c u l a r of A . flavus,
w a s first d i s c o v e r e d b y S a i t o ( 5 5 ) .
Yabuta
(56)
s t u d i e d t h e c h e m i s t r y of k o j i c a c i d a n d w a s m a i n l y r e s p o n s i b l e f o r e l u c i d a t i n g its c h e m i c a l structure. E a r l y w o r k o n k o j i c a c i d w a s n o d o u b t r e l a t e d to its a n t i m i c r o b i a l properties.
C o n s i d e r a b l e e m p h a s i s r e m a i n e d o n the p o t e n t i a l usefulness
of k o j i c a c i d i n s p i t e of reports of its t o x i c i t y to a n i m a l s . T h u s , k o j i c a c i d has b e e n the target of extensive c h e m i c a l r e s e a r c h ( D a t a sheet N o . 502, Charles Pfizer and C o . ) . A l t h o u g h k o j i c a c i d has n o t b e e n d i r e c t l y i m p l i c a t e d i n n a t u r a l o u t breaks of m y c o t o x i c o s i s , i t r e m a i n s a p o t e n t i a l p r o b l e m i n v i e w of the l a r g e n u m b e r of m i c r o o r g a n i s m s c a p a b l e of i t . T h e L D
5 0
of p r o d u c i n g l a r g e
amounts
of k o j i c a c i d i n 17 g m i c e w a s 30 m g i p i n j e c t i o n
(57).
K o j i c a c i d also s h o w e d t o x i c i t y i n p l a n t cells at 1 0 M ( 5 8 ) . _ 1
K o j i c a c i d is c h a r a c t e r i z e d b y a γ - p y r o n e n u c l e u s s u b s t i t u t e d o n p o s i ations 2 a n d 5 w i t h a h y d r o x y m e t h y l a n d a h y d r o x y g r o u p . trum showed A
m a
x
E t 0 H
268 n m (e = 8000) a n d 216 n m ( = €
Its u v spec 11,000).
80
MYCOTOXINS
T h e i n f r a r e d s p e c t r u m of k o j i c a c i d shows t y p i c a l γ - p y r o n e a b s r o p tions—i.e., C = 0 frequencies
s t r e t c h i n g f r e q u e n c y at 1765 c m " a n d v c = c s t r e t c h i n g 1
at 1620 c m "
1
a n d 1588 c m " .
O t h e r significant absorptions
1
o c c u r r e d a t 3 2 8 5 ( O H ) , 1350, 1285, 1230, 1142, 1085, 990, 944, a n d 865 cm' . 1
T h e N M R spectrum of kojic acid, taken i n D 0 solution, exhibited 2
c h e m i c a l shifts f o r a t w o - p r o t o n s i g n a l at δ 4.54 ( s i n g l e t , 2 - h y d r o x y m e t h y l g r o u p ) a n d o n e - p r o t o n singlets at δ 6.59 a n d δ 8.10 for the protons p o s i t i o n s 3 a n d 6.
S i g n a l s f o r the t w o O H protons w e r e n o t
on
observed
b e c a u s e of c h e m i c a l e x c h a n g e w i t h D 0 . A c h e m i c a l shift r e s o n a t i n g at 2
δ 4.69 w a s a s s i g n e d t o H D O
(59).
I n s p i t e of extensive r e s e a r c h o n t h e biosynthesis of k o j i c a c i d , its m o d e of f o r m a t i o n w a s d u b i o u s . T h e w o r k of A r n s t e i n a n d B e n t l e y ( 6 0 , 61, 62, 63, 64)
y
i n a series of elegant experiments u s i n g
cursors w i t h s u b s e q u e n t
1 4
C-labeled pre
d e g r a d a t i o n of t h e p r o d u c t s , p r o v i d e d
strong
evidence that kojic a c i d was formed directly f r o m the oxidation of D g l u c o s e . T h e y s u g g e s t e d t h a t D-glucose c o u l d b e o x i d i z e d to 3 - k e t o g l u c o n i c a c i d l a c t o n e w h i c h c o u l d i n t u r n take t w o possible p a t h w a y s to kojic acid. B o t h pathways involve enzymatic dehydration and reduction f r o m 3 - k e t o g l u c o n i c a c i d l a c t o n e to f o r m k o j i c a c i d . F u r t h e r s u p p o r t f o r these p a t h w a y s w a s p r o v i d e d w h e n i t w a s e x p e r i m e n t a l l y s h o w n t h a t g l u c o n i c a c i d a n d g l u c o n o l a c t o n e b o t h serve as precursors for k o j i c a c i d b i o s y n t h e s i s (64).
A n excellent c o m p r e h e n s i v e r e v i e w of k o j i c a c i d has
been prepared b y Beelik
(65).
Aust amide S t e y n (66,
67)
r e c e n t l y r e p o r t e d o n t h e c h e m i c a l structures of
n e w d i k e t o p i p e r a z i n e c o m p o u n d s i s o l a t e d f r o m cultures of ustus.
five
Aspergillus
A u s t a m i d e ( S t r u c t u r e X I I I ) a n d 1 2 , 1 3 - d i h y d r o a u s t a m i d e are c h a r
a c t e r i z e d b y a b a s i c Ψ-indoxyl m o i e t y s u b s t i t u t e d o n p o s i t i o n t w o w i t h a seven-membered
s p i r a n r i n g system a n d c o n t a i n i n g i n a d d i t i o n d i k e t o
piperazine a n d proline moieties.
T h e u v spectra of b o t h
s h o w e d t y p i c a l Φ-indoxyl c h r o m o p h o r e s ( A
m a x
E t 0 H
15
Structure XIII. mide
Austa
compounds
234, 256, a n d 392 n m ) .
5.
C O L E
81
Aspergillus Toxins
A u s t a m i d e contained additional U V absorptions ( A n m ) a t t r i b u t e d to the e n a m i d e c h r o m o p h o r e cm"
(Ψ-indoxyl C = 0 ) ,
C = 0
groups).
a n d 1680 c m '
1
E t 0 H
268 a n d 282
(66,67).
T h e i r s p e c t r u m of a u s t a m i d e s h o w e d 3420 c m " 1
x
m a
( N H g r o u p ) , 1700
1
a n d 1650 c m "
1
(diketopiperazine
Dihydroaustamide h a d similar ir absorption for
m a j o r f u n c t i o n a l groups.
the
M a s s spectral analysis of austamide s h o w e d a
molecular ion peak ( m * )
at m/e
363 w h i c h a n a l y z e d f o r C21H21N3O3.
T h e b a s e p e a k a p p e a r e d at m/e 203 ( C i i H i i N 0 ) w h i c h r e s u l t e d f r o m 2
2
cleavage of the s p i r a n r i n g to f o r m a n a l i c y c l i c f r a g m e n t at m/e
218
(C12H14N2O2) f o l l o w e d b y a loss of a m e t h y l g r o u p . D i h y d r o a u s t a m i d e s h o w e d a m * p e a k at m/e 365 w i t h a c o r r e s p o n d i n g f r a g m e n t r e p r e s e n t i n g the a l i c y c l i c p a r t o f t h e m o l e c u l e at m/e 220
(67).
C h a r a c t e r i s t i c features of the N M R s p e c t r u m o f a u s t a m i d e
were
c h e m i c a l shifts for t w o n o n e q u i v a l e n t g e m i n a l m e t h y l g r o u p s r e s o n a t i n g at δ 1.38 ( s i n g l e t ) a n d δ 0.88 ( s i n g l e t ) ; t h e o l e f i n i c p r o t o n s l o c a t e d o n t h e other p a r t of the isoprene u n i t a p p e a r e d at δ 4.89 ( d o u b l e t ) a n d at δ 6.82 ( d o u b l e t ) ( J A B — 10 H z ) . T h e n o n e q u i v a l e n t m e t h y l e n e p r o t o n s at p o s i t i o n 3 resonated at δ 3.06 ( e q u i t o r i a l q u a r t e t ) a n d δ 2.10 ( a x i a l q u a r t e t ) as p a r t of a n A B X system w i t h J B — 14, / A X — 5, a n d J x — 12 H z . T h e A
B
X p o r t i o n consisted o f the m e t h i n e p r o t o n o n t h e d i k e t o p i p e r a z i n e m o i e t y r e s o n a t i n g at δ 4.99 as a p a i r of d o u b l e t s ( J x — 5; J x = A
12 H z ) .
B
T h e protons i n the p r o l i n e r i n g c o m p r i s e d a n A M X system. 2
2
The
m e t h y l e n e protons adjacent to t h e p r o l i n e n i t r o g e n a p p e a r e d as t w o o v e r l a p p i n g triplets at δ 3.85 (7 = 9, 9 H z ) ; the c h e m i c a l shifts f o r the t w o protons at p o s i t i o n 19 r e s o n a t e d as a sextet at δ 2.40 ( / = 3, 9, 9 H z ) . T h e a r o m a t i c protons o n the i n d o x y l n u c l e u s w e r e o b s e r v e d b e t w e e n δ 7.7 a n d δ 6.6, a n d the N H p r o t o n ( D 0 e x c h a n g e a b l e ) w a s o b s e r v e d at δ 4.73 as a 2
b r o a d s i g n a l . T h e N M R s p e c t r u m of d i h y d r o a u s t a m i d e w a s s i m i l a r except that the olefinic t r i p l e t at δ 6.26 w a s absent a n d a p r o t o n ( p o s i t i o n 12) a p p e a r e d at δ 4.18; c h e m i c a l shifts f o r the p r o l i n e p r o t o n s b e c a m e m o r e complex. T h e other three d i k e t o p i p e r a z i n e s c o n s i s t e d of c l o s e l y r e l a t e d 2,3d i s u b s t i t u t e d indoles. T h e m a j o r c o m p o u n d of t h i s g r o u p , p r o l y l - 2 - ( l ' , l ' dimethylallyltryptophyldiketopiperazine
[C21H25N3O2]
(Structure X I V )
w i l l serve as a m o d e l f o r d i s c u s s i o n . I t h a d t y p i c a l U V a b s o r p t i o n f o r 2,3-disubstituted indole ( A 3.85, 3.91, a n d 3.85).
m a x
E t 0 H
225, 275, 283, a n d 291 n m ; l o g c 4.51,
T h e i r s h o w e d c h a r a c t e r i s t i c N H a b s o r p t i o n at
3480, 3460, a n d 3365 c m " . T h e a m i d e I b a n d s o c c u r r e d at 1685 1
(weak
s h ) a n d 1670 c m ' . A b s e n c e of a n a m i d e I I b a n d s u p p o r t e d t h e p r e s e n c e 1
of a d i k e t o p i p e r a z i n e system. T h e mass s p e c t r u m of S t r u c t u r e X I V h a d a m * p e a k at m/e 351 a n d one p r o m i n e n t p e a k at m/e
198 ( b a s e p e a k ) r e s u l t i n g f r o m c l e a v a g e of
the b o n d b e t w e e n carbons 8 a n d 9. T h e N M R s p e c t r u m s h o w e d c h e m i c a l
82
M Y C O T O X I N S
shifts f o r t w o D 0 e x c h a n g e a b l e p r o t o n singlets at δ 8.75 a n d δ 5.72 a r i s i n g 2
f r o m the N H protons. T h e f o u r a r o m a t i c protons a p p e a r e d as a m u l t i p l e t b e t w e e n δ 7.52-6.95; the g e m - d i m e t h y l protons w e r e l o c a t e d at δ 1.50 as a s i x - p r o t o n singlet. T h e t h r e e e x o c y c l i c protons o n positions 19 a n d 20 c o m p r i s e d a A A X system w i t h the X p a r t ( H o n p o s i t i o n 19) at δ 6.10 2
(/
_
A X
18.0 H z ; 7 1 χ — 9 H z ) a n d the A A p a r t at δ 5.08 ( 7 A X — 18.0 H z ; 1
Δ
7A1A1 =
9HZ).
T h e 3 protons at positions 8 a n d 9 r e s o n a t e d as a n A B X system at δ 4.44 ( H o n p o s i t i o n 9 — X p a r t ; 7 A X — 4; 7 B X — 11.0 H z ) , δ 3.75 (7AB -
15.5; 7 A X -
4 H z ) , a n d δ 3.17 ( H „ )
(7AB -
15.5; 7 B X -
(H ) A
11.0Hz).
A s i g n a l a r i s i n g f r o m the m e t h i n e p r o t o n at p o s i t i o n 12 w a s o b s e r v e d as a t r i p l e t at δ 4.05 (7 = 7 H z ) . T h e protons o n p o s i t i o n 15 w e r e l o c a t e d at δ 3.66, a n d the other f o u r protons of the p r o l i n e r i n g w e r e
between
δ 2.4 a n d 1.8 as a m u l t i p l e t . A u s t a m i d e ( S t r u c t u r e X I I I ) w a s r e p o r t e d as t o x i c to d u c k l i n g s , b u t n o specific t o x i c o l o g i c a l d a t a h a v e b e e n r e p o r t e d (66). concerning
biosynthesis of
A l t h o u g h no data
the diketopiperazines were
a v a i l a b l e , the
amino acids, tryptophan a n d proline, m i g h t be involved. Ascladiol A
p a t u l i n - p r o d u c i n g s t r a i n of Aspergillus
w h e a t flour (68) XV).
clavatus
isolated
from
produced a new mycotoxin named ascladiol (Structure
A s c l a d i o l ( C H 0 ) , a m e t a b o l i t e closely r e l a t e d to p a t u l i n , w a s 7
8
4
Structure XV. Ascladiol
Structure XIV. Diketo piperazine
o n l y o n e - f o u r t h as a c u t e l y t o x i c to m i c e as p a t u l i n . b a n d s i n the i r s p e c t r u m w e r e 1735 c m " five-membered
1
l a c t o n e r i n g s y s t e m ) a n d 3300 c m "
spectrum showed A
m a
x
E t 0 H
M a j o r absorption
a n d 1750 c m ' 1
(OH)
1
(supporting
(68).
The uv
271 n m .
T h e p r o t o n m a g n e t i c resonance s p e c t r u m t a k e n i n a c e t o n e - d
e
solu
t i o n s h o w e d resonances f o r t w o m e t h i n e protons at δ 6.29 ( m u l t i p l e t ; C - 2 ) a n d δ 5.87 ( q u a r t e t ; C - 5 ) . T h e t w o p a i r of m e t h y l e n e protons l o c a t e d o n C - 6 a n d C - 7 w e r e p o s i t i o n e d at δ 4.74 a n d δ 4.30.
Superimposed on the
m e t h y l e n e protons at δ 4.74 w e r e t w o D 0 e x c h a n g e a b l e protons assigned 2
to t h e O H protons o n C - 6 a n d C - 7
(68).
5.
Aspergillus
C O L E
Terreic
83
Toxins
Acid
T h e a n t i b i o t i c t e r r e i c a c i d ( S t r u c t u r e X V I ) w a s first d i s c o v e r e d W i l k i n s a n d H a r r i s (69).
by
Its u t i l i t y w a s n e g a t e d because i n v i v o tests
s h o w e d that i t w a s h i g h l y t o x i c to m a m m a l s (70). of t e r r e i c a c i d to m i c e s h o w e d a n L D
5 0
Intravenous i n j e c t i o n
of 7 1 - 1 1 9 m g / k g
(70).
T h e c h e m i c a l s t r u c t u r e of t e r r e i c a c i d w a s p r o p o s e d b y S h e e h a n et a l . (71)
as 2 , 3 - e p o x y - 6 - h y d r o x y t o l u q u i n o n e
(Structure X V I ) . T h e structure
a s s i g n m e n t w a s b a s e d o n c o m p a r i s o n s of the N M R s p e c t r a of t e r r e i c a c i d and 2,3-epoxy-l,4-naphthoquinone
( S t r u c t u r e X V I I ) together w i t h p h y s i
cal data and chemical transformation products.
Structure XVI. Terreic Acid
Structure XVII. Naphthoquinone
T h e i r s p e c t r u m of t e r r e i c a c i d h a d s h a r p absorptions at 3300, 1655, a n d 1629 c m ' c o m p a t i b l e w i t h the presence of a n e n o f i z e d
1,2,4-triketone
system.
(CH ),
1
A d d i t i o n a l s t r o n g absorptions w e r e
1350, 1305, 1200, 1135, 1035, a n d 760 c m "
1
1690, 1380
(71).
1370,
3
T h e u v spectrum h a d
m a x i m a at 214 ( l o g c 4.03) a n d 316 n m ( l o g e 3 . 8 8 ) ; the l a t t e r a b s o r p t i o n s h i f t e d to 304 n m i n a n a c i d s o l u t i o n . A s i m i l a r shift to 304 n m w a s o b s e r v e d w h e n i t w a s c o n v e r t e d to the m e t h y l e t h e r d e r i v a t i v e . T h e c h e m i c a l shifts f o r t e r r e i c a c i d 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 w e r e r e p o r t e d r e l a t i v e to t h e O H a b s o r p t i o n of w a t e r . T h e N M R s p e c t r u m h a d t h r e e d i s t i n g u i s h a b l e resonances
a t t r i b u t e d to t h e m e t h y l
( + 1 0 7 H z ) , the t w o e p o x i d e protons
( + 3 0 H z ) , and the O H proton
( — 93 H z ) .
The
epoxide
protons
of
group
5,6-epoxy-3-hydroxytoluquinone
( + 32 H z ) a n d t e r r e i c a c i d w e r e s i m i l a r l y p o s i t i o n e d
(71).
Viriditoxin V i r i d i t o x i n w a s i s o l a t e d f r o m m y c e l i a of a t o x i g e n i c strain of gillus veri-nudans 73).
Asper
f o u n d d u r i n g routine screening for toxigenic f u n g i
(72,
V i r i d i t o x i n w a s s h o w n to b e a s y m m e t r i c a l d i m e r w i t h S t r u c t u r e
XVIII.
E l e m e n t a l a n d mass s p e c t r a l analyses e s t a b l i s h e d t h e m o l e c u l a r
Ο
OH
OH
Structure
XVIII.
Viriditoxin
84
MYCOTOXINS
w e i g h t as 662 w i t h a m o l e c u l a r f o r m u l a of C34H30O14. e t h a n o l s o l u t i o n s h o w e d u v a b s o r p t i o n at A
T h e toxin in
266 a n d 380 n m
m a x
(73).
Significant c a r b o n y l absorptions i n t h e i r s p e c t r u m w e r e 1740 c m "
1
( e s t e r ) a n d 1635 c m ' . T h e latter a b s o r p t i o n s h i f t e d to 1720 after a c e t y l a 1
t i o n w h i c h suggested a h y d r o g e n - b o n d e d lactone. F u r t h e r s u p p o r t arose f r o m t h e N M R s p e c t r u m w h i c h s h o w e d a D 0 e x c h a n g e a b l e p r o t o n at 2
δ 13.70. T h i s e x t r e m e d o w n f i e l d p o s i t i o n is t y p i c a l of a h y d r o g e n - b o n d e d OH
group.
A c h e m i c a l shift f o r a n a d d i t i o n a l O H p r o t o n a p p e a r e d at
δ 9.72. T w o m e t h o x y resonances w e r e at δ 3.66 a n d δ 3.74. C h e m i c a l shifts i n the a r o m a t i c r e g i o n (singlets at δ 6.24 a n d 6.78) w e r e a s s i g n e d to t h e t w o m e t a - p o s i t i o n e d a r o m a t i c protons o n the n a p t h a l e n e r i n g system. T h e m e t h y l e n e protons i n the l a c t o n e r i n g a n d t h e e x o c y c l i c m e t h y l e n e p r o t o n s w e r e c o u p l e d w i t h the adjacent m e t h i n e p r o t o n .
T h e methine proton
resonated as a m u l t i p l e t at δ 4.96, a n d the m e t h y l e n e protons p a r t i a l l y o v e r l a p p e d at δ 2.76 ( m u l t i p l e t ) a n d δ 2.81 ( d o u b l e t ) . an L D
5 0
Viriditoxin had
of 2.8 m g / k g ( i p ) i n 20 g m i c e . N o b i o s y n t h e t i c d a t a w e r e g i v e n
for viriditoxin. Cytochalasin
Ε
A recent r e p o r t i m p l i c a t e d c y t o c h a l a s i n Ε f r o m Aspergillus
clavatus
to h u m a n m o r t a l i t y f r o m i n g e s t i o n of m o l d - d a m a g e d r i c e (74, 7 5 ) .
Cyto
c h a l a s i n Ε ( S t r u c t u r e X I X ) c o n t a i n e d m o n o - s u b s t i t u t e d a r o m a t i c , sec o n d a r y a m i d e , e p o x i d e , k e t o n e , a n d a l k y l v i n y l c a r b o n a t e moieties Its i r s p e c t r u m s h o w e d m a j o r absorptions at 3475 c m "
1
(75).
( O H , N H ) , 1765
c m " , 1 6 6 0 c m " , a n d 1720 c m " . 1
1
1
P r o t o n c h e m i c a l shifts f o r c y t o c h a l a s i n Ε w e r e a s s i g n e d as f o l l o w s : t w o exchangeable protons a p p e a r e d at δ 6.93 ( N H p r o t o n o n C - 2 )
and
δ 4.1 ( O H p r o t o n o n C - 1 5 ) ; t h e a r o m a t i c protons o c c u r r e d as a m u l t i p l e t at δ 7.1. Resonances for 4 m e t h y l groups w e r e o b s e r v e d at δ 1.0
(doublet,
/ = 6 H z ; C - 5 ) , δ 1.2 ( s i n g l e t ; C - 6 ) , δ 1.13 ( d o u b l e t , 7 = 6 H z ; C - 1 3 ) , and
δ 1.4 ( s i n g l e t ; C - 1 5 ) .
T w o s t r o n g l y c o u p l e d p r o t o n s r e s o n a t e d at
δ 5.45 ( d o u b l e t ) a n d δ 6.25 ( d o u b l e t , 7 =
11 H z ) . T h e s e w e r e assigned
t o the protons l o c a t e d o n C - 1 6 a n d C - 1 7
(75).
T h e c o r r e c t e d s t r u c t u r e a n d stereochemistry w e r e o b t a i n e d v i a s i n g l e c r y s t a l x - r a y d i f f r a c t i o n analysis ( 7 5 ) .
Cytochalasin Ε reportedly killed
rats w i t h i n a f e w h o u r s after d o s i n g . T h e L D ( i p ) a n d 9.1 m g / k g ( o r a l )
5 0
v a l u e s w e r e 2.6 m g / k g
(75).
Maltoryzine T w o cases of f e e d p o i s o n i n g i n d a i r y cattle w e r e t r a c e d to m a l t sprout c o n t a m i n a t e d w i t h a t o x i g e n i c s t r a i n of Aspergillus
oryzae v a r .
microsporia
5.
Aspergillus
COLE
Toxins
o:
OH
Structure XIX. Cytochalasin Ε
Ο
Structure XX. Maltoryzine
(76) . T h e t o x i n w a s n a m e d m a l t o r y z i n e (C11H14O4) ( S t r u c t u r e X X ) (77) . M a l t o r y z i n e h a d a n L D
5 0
of 3 m g / k g ( i p ) i n m i c e .
T h e u v s p e c t r u m of this t o x i n w a s A 3.1), a n d 320 n m ( l o g c 2.1).
220 ( l o g c 4 . 1 ) , 280 ( l o g €
m a x
T h e I R s p e c t r u m of m a l t o r y z i n e s u p p o r t e d
O H ( 3 3 0 0 c m " ) , ketone (1700 c m " ) , a n d a r o m a t i c moieties (1600 a n d 1
1500 c m " ) . 1
1
T h e c h e m i c a l s t r u c t u r e of m a l t o r y z i n e w a s d e d u c e d
from
c h e m i c a l d e g r a d a t i o n studies of the t r i m e t h o x y d e r i v a t i v e of m a l t o r y z i n e (77). Other Toxins of A s p e r g i l l u s Spp. T h e f o l l o w i n g is a b r i e f s u r v e y of o t h e r t o x i c Aspergillus B u s h et a l . (78)
metabolites.
isolated a n d identified 3-nitropropanoic a c i d (Structure
X X I ) f r o m toxic extracts of A . flavus c u l t u r e s . 3 - N i t r o p r o p a n o i c a c i d has also b e e n r e p o r t e d as a m e t a b o l i t e of A . oryzae. together w i t h p y r a z i n e c o m p o u n d s
I n b o t h cases, i t o c c u r r e d
w h i c h m a y suggest a r o l e i n the
nitrification pathway. O x a l i c a c i d ( S t r u c t u r e X X I I ) is a m e t a b o l i c p r o d u c t of s e v e r a l f u n g i i n c l u d i n g A . flavus, A. glaucus,
A. luchuensis,
a n d A . niger.
T h e toxicologi-
c a l properties of o x a l i c a c i d m a y r e l y o n t h e presence of l a r g e q u a n t i t i e s i n c o n t a m i n a t e d f e e d s u p p l i e s or o n s y n e r g i s t i c effects w i t h o t h e r m e t a b o lites f u n c t i o n i n g i n concert.
Unfortunately, fundamental information
I
Structure XXI. β-Nitropropanoic Acid
Structure XXII. Oxalic Acid
86
MYCOTOXINS
r e l a t i v e to t h e s y n e r g i s t i c effects of f u n g a l metabolites o c c u r i n g n a t u r a l l y is m i n i m a l or l a c k i n g a l t h o u g h i t is r e c o g n i z e d t h a t s y n e r g i s m occurs naturally. Helvolic acid (fumigacin)
(Structure X X I I I ) , a toxic antibiotic pro
d u c e d b y some isolates of A . fumigatus,
w a s r e p o r t e d almost s i m u l t a n e
o u s l y b y W a k s m a n et a l . ( 7 9 ) a n d C h a i n et a l . ( 8 0 ) . T h e correct c h e m i c a l
CH OH
Ο
2
Structure Helvolic
Structure XXIV. Gliotoxin
XXIII. Acid
s t r u c t u r e of h e l v o l i c a c i d w a s d e t e r m i n e d b y c h e m i c a l a n d p h y s i c a l c o n s i d e r a t i o n s a n d p r o t o n m a g n e t i c resonance studies
(81).
G l i o t o x i n ( C ^ H ^ N a C ^ ) ( S t r u c t u r e X X I V ) , a n a n t i b i o t i c first r e p o r t e d f r o m Gliocladium
frimbriatum
s e v e r a l f u n g i i n c l u d i n g Aspergillus a n d Α . Τ err eus (85, 8 6 ) .
(82),
is a m e t a b o l i c p r o d u c t
fumigatus
(83),
A . chevalieri
of (84),
G l i o t o x i n is c h a r a c t e r i z e d b y a d i s u l f i d e b r i d g e
across a d i k e t o p i p e r a z i n e r i n g system. T h e b a s i c s t r u c t u r e is a 3 , 6 - e p i d i thio-2,5-dioxopiperazine moiety. oxopiperazines
A l t h o u g h several other e p i p o l y t h i o d i -
occur naturally (87),
( S t r u c t u r e X X V ) (86)
only gliotoxin and acetylaranatin
h a v e b e e n r e p o r t e d as metabolites of
Aspergillus
sp. I n a d d i t i o n to potent a n t i b i o t i c p r o p e r t i e s , g l i o t o x i n w a s t o x i c to r a b b i t s ( L D
5 0
— 45 m g / k g ) , m i c e ( L D
( L D o — 5 0 - 6 5 m g / k g ) (88); 5
5 0
acutely
— 5 0 m g / k g ) , a n d rats
at s u b l e t h a l doses the a n i m a l s h a d k i d n e y
lesions. A c o m p r e h e n s i v e r e v i e w of t h e biosynthesis of e p i p o l y t h i o d i o x o piperazines was presented b y T a y l o r (87). O t h e r t o x i g e n i c metabolites of A . fumigatus XXVI)
(89),
fumagillin (Structure X X V I I )
X X V I I I ) (91, 92, 93, 94),
are f u m i g a t i n ( S t r u c t u r e (90),
Terrein (Structure
and spinulosin (Structure X X I X ) (88).
Ο
Ο Structure XXV. Acetylaranatin
Structure XXVI. Fumigatin
Ojima
5.
HOOC
87
Aspergillus Toxins
COLE
(CH=CH)
C=0
4
Structure XXVIII. Terrein
Structure XXVII. Fumagillin
•OH
δ
OH
Structure XXIX. Spinuhsin
Structure XXX. Butenolide
et al. ( 9 5 ) r e c e n t l y r e p o r t e d t h e i d e n t i t y of a n e w b u t e n o l i d e ( S t r u c t u r e XXX)
f r o m c u l t u r e filtrates o f A . terreus.
T h e y also f o u n d s i x other
closely r e l a t e d metabolites associated w i t h this c o m p o u n d .
Information
n o t p r e s e n t e d i n this r e v i e w is p r o v i d e d i n R e f . 96. Literature
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