Isolation and identification of the toxic principle from Tetradymia glabrata
by Samuel Kenneth Reeder
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in Chemistry
Montana State University
© Copyright by Samuel Kenneth Reeder (1971)
Abstract:
Tetradymol, 3,4a(R),5(S)-trimethyl-8a(S)- hydroxy-4,4a 5,6,7,8,8a ,-9-octahydro-naphtho [2,3-b] furan
(II), has been isolated from Tetradymia glabrata, a desert plant of the great Salt Lake Basin, which is
the cause of extensive range losses of sheep. This compound was shown to be an active principle of the
plant by sheep and mouse feeding experiments.
The structure of the compound was established on the basis of chemical and spectral data, and
confirmed by an X-ray crystallographic structure of the 2-chloromercury derivative.
(Formula not captured by OCR) ISOLATION AND IDENTIFICATION OF THE TOXIC PRINCIPLE
FROM TETRADYMIA GLABRATA
by
Samuel K enneth Reeder.
A t h e s i s su b m itte d t o th e G raduate F a c u lty in p a r t i a l
f u l f i l l m e n t o f th e re q u ire m e n ts f o r th e d egree
of
DOCTOR OF PHILOSOPHY
in
C hem istry
MONTANA STATE UNIVERSITY
.Bozeman, Montana
March, 197.1
ACKNOWLEDGMENT
The a u th o r would l i k e t o e x p re ss h is g r a titu d e to th e fo llo w in g
in d iv id u a ls f o r t h e i r g u id a n c e , a s s i s t a n c e , and encouragem ent in th e
p u rs u it of th is re sea rc h p ro je c t.
D r. P . W.-J e n n in g s , G raduate A d v iso r
D r. M. W. H u ll, A s s is ta n c e in sheep t e s t i n g ex p erim en ts
D r. D. E . W orley, A s s is ta n c e in sm a ll an im al t e s t i n g ex p erim en ts
D r. J . W. J u t i l a P ro v id in g mice f o r t e s t i n g p u rposes ■
Dr'. K. A nderson, P erform ing h is to p a th o lo g y s tu d ie s
D r. W. Herz and Mr. A . H a ll,. P erform ing 90 MHz nmr stu d y
D r9 W. L . W aters, Perform ing 60 MHz nmr s tu d ie s and s p in
d eco u p lin g experim ents
D r. C. N. Caughlan and D r. G. D. S m ith , D eterm ining th e X -ray
c r y s ta llo g r a p h ic s t r u c t u r e o f te tra d y m o l
He would f u r t h e r l i k e to g iv e s p e c ia l re c o g n itio n t o h is w if e ,
C am ille , who l i k e Aaron o f old h eld up h is hands t h a t th e b a t t l e m ight
be won.
Acknowledgement i s a l s o due th e N a tio n a l Defence E d u catio n A ct f o r
th e a s s is ta n c e o f a f e llo w s h ip , th e Montana H e art A s s o c ia tio n f o r
re s e a rc h f u n d s , and th e Endowment R esearch F oundation o f Montana S ta te
U n iv e rs ity f o r o th e r r e s e a r c h f u n d s .
iv
TABLE OF CONTENTS
Page
LIST OF FIGURES .............................................................
LIST OF TABLES
..............................................................................
v
vi i
LIST OF SCHEMES..............................................................................................................v i i i
ABSTRACT
. . . . . . .
......................................................... . . . . . . .
ix
INTRODUCTION............................................................................................ .... . . .
I
D ISCU SSIO N ..................................................................................................................
17
I.
P h y s io lo g ic a l A c t i v i t y ..............................
I I . I s o l a t i o n o f th e T o x i n .............................................
I I I . S p e c tr a l and Chem ical E lu c id a tio n o f S tr u c tu r e . . . .
IV.
X-Ray S t r u c t u r a l A n a ly sis . . . . .
...................................
17
34
48
90
EXPERIMENTAL S E C T IO N ..........................
I.
II.
III.
IV .
V.
V I.
R e a g e n t s .................................................................................................
I n s t r u m e n t s ...........................
..........................................
P la n t M a t e r i a l ..........................................................................
Animal T e s t i n g ...................................................................................
I s o l a t i o n Techniques f o r Tetradym ol ...................................
R e a c t i o n s . ...........................................................................................
LITERATURE CITED
95
95
96
97
99
103
107
115
V
LIST OF FIGURES
Page
1.
A Diagram o f H ep atic F u n c t i o n .............................................................
2.
A Diagram o f H ep atic Lobules
3.
The Urea C y c l e ........................................................................................................... 7
4.
Range o f th e O ccurrence o f T e tr a d y m ia ....................................................... 13
5.
The P la n t, Tetradym ia g la b r a t a
6.
E le c tro c a rd io g ra p h o f Sheep S-230 ............................................................. 20
7.
IR Spectrum o f T e tr a d y m o l.....................
8.
-2
......................................................................
3
.................................................................. 14
50
UV Spectrum o f T e tr a d y m o l.........................................................
50
9.
A 90 MHz Spectrum o f Tetradym pl . . ............................................................ 51
10.
A 60 MHz' Spectrum o f ( I I A ) .............................................................................. 54
1 1.
IR S p e c tra o f (I IA ), (V I), and ( V I l ) ........................................................57
1 2.
A 60 MHz Spectrum o f ( V I ) ..........................
59
13.
A 60 MHz Spectrum o f ( V I l ) .................................................................
60
1 4.
IR S p e c tra o f (XIX), (X V Il), and ( X V I I A ) ............................................... 64
15.
A 60 MHz Spectrum of ( X I X ) ................................... ■......................................65
1 6.
P ro b ab le P o s itio n s f o r th e A d d itio n o f one Mole o f
Hydrogen to T e t r a d y m o l .................................................................................. 66
17.
A 60 MHz Spectrum o f ( X V I I l ) ..........................................................................67
1 8.
A 60 MHz Spectrum o f ( X V I l A ) ...................................
69
19.
P o s s ib le T r ic y c lic Ring Systems f o r Tetradym ol
............................... 73
20.
IR S p e c tra o f (XVIIB), (XXV), and (XXVl) .....................
76
VX
Page
21.
A 60 MHz Spectrum o f ( X X V ) .............................................................................. 77
22.
A 60 MHz Spectrum o f (XVIIB)
23.
A 60 MHz Spectrum o f (X X V l).............................................................................. 82
24.
P y rid in e Induced S h i f t in th e NMR Spectrum
o f (XVIIB) ..............................................................................
........................................................................... 79
84
25.
P y rid in e Induced S h i f t in th e NMR Spectrum
o f ( I I A ) ......................................................................................................... ; . 85
26.
Tetradym ol I s o m e r s .............................................. .............................................87
27.
C onform ation f o r Easy D ehyd ratio n in th e MS
o f T e tr a d y m o l............................................................................................................ 89
28.
T etradym ol M ercury C h lo rid e D e riv a tiv e Bond
D ista n c e s from X-Ray S t r u c t u r e ................................... ■ ...........................92
29. ,C r y s ta l Packing P a tte r n f o r T etradym ol
Mercury C h lo rid e ....................................................................................................94
30.
A pparatus f o r Sheep F eedings
..................................................................
100
V ii
LIST OF TABLES
Page
I.
Sheep Feeding E xperim ents Using th e
2
Sheep Feeding E xperim ents Using th e P la n t E x t r a c t ..................................26
3.
Sm all Animal Feeding E xperim ents Using th e P la n t
E x t r a c t ........................................................................................................................... 28
4.
Mouse Feeding E xperim ents ...............................................................................
5.
E f f e c t o f G rinding on P la n t E x tra c tio n
6.
Y ie ld s o f H ydrogenated P ro d u cts U sing D if f e r e n t
C a ta ly s ts ........................................................
7.
E lu tio n o f C hrom atographic Columns
Whole P la n t ................................
18
31
...................................................... 40
63
.................................................... 105
viii
LIST OF SCHEMES
Page
1.
I s o l a t i o n o f th e Toxin Followed by T o x i c i t y ............................... .... . 3 5
2.
I s o l a t i o n of th e Toxin Followed by T o x ic ity ( I l ) .................. . . „ 3 7
3.
I s o l a t i o n Procedure f o r T etradym ol ............................................ ....
4.
R ea c tio n s of P e p e ric A c i d .............................................................................. 56
5.
H ydrogenation o f T e t r a d y m o l ...................... ................................................... .63
42
V
ix
ABSTRACT
T etradym ol, 3 ,4 a (R ), 5 (S ) - tr im e th y l- 8 a ( S ) - h y d r o x y - 4 ,4 a 5 ,6 ,7 ,8 ,8 a ,9 -o c ta h y d ro -n a p h th o [ 2 ,3 - b J fu ra n ( I I ) , has been is o la te d from
T etradym ia g la b r a t a , a d e s e r t p la n t o f th e g r e a t S a l t Lake B asin ,
which i s th e cause o f e x te n s iv e ran g e lo s s e s o f sh e e p . T his compound
was shown to be an a c ti v e p r in c ip le o f th e p la n t by sheep and mouse
fe e d in g e x p e rim e n ts.
The s t r u c t u r e o f th e compound was e s ta b lis h e d on th e b a s is o f
chem ical and s p e c t r a l d a ta , and confirm ed by an X -ray c r y s ta llo g r a p h ic
s t r u c t u r e o f th e 2 -ch lo ro m ercu ry d e r i v a t i v e .
OH
(II)
INTRODUCTION
The l i v i n g organism i s a v ery complex u n i t p erfo rm in g a g r e a t
number o f in te g r a te d f u n c tio n s , b u t i t could be c o n sid e re d a s an
in te g r a te d chem ical p la n t ta k in g in raw m a te r ia ls and c o n v e rtin g them
in to energy and p ro d u c ts t h a t i t needs f o r b u ild in g and o th e r f u n c tio n s .
However, a s i s th e case in m ost chem ical p l a n t s , th e e n te r in g raw
m a te r ia ls and th e p ro d u c ts m ust be, p u r if ie d o f th e nonusuable m a te r ia ls
and b y -p r o d u c ts , and th e s e m a te r ia ls m ust be e lim in a te d .
In mammals th e r e a r e a number o f organs t h a t a re re s p o n s ib le f o r
th e above ty p e o f p u r i f i c a t i o n and e lim in a tio n ; th e lu n g s , i n t e s t i n e s ,
k id n e y s , and l i v e r to name a few .
One o f th e s e , th e l i v e r , i s
im p o rta n t f o r p u r i f i c a t i o n o f th e raw m a te r ia ls coming from th e
d ig e s tiv e t r a c t , rem oval o f some d e le te r io u s m a te r ia ls o f norm al
m etabolism from th e b lo o d , and e x c r e tio n o f th e s e m a te r ia ls or t h e i r
c o n ju g a te s by means o f th e b i l e d u c t.
These m a t e r i a l s , t o be e x c r e te d ,
a re o f many d i f f e r e n t ty p e s and r e q u ir e d iv e rs e enzym atic p ro c e sse s f o r
r e a c tio n a n d /o r e x c r e tio n .
The l i v e r , th u s , p lay s a v e ry im p o rta n t
r o le in th e f u n c tio n o f th e t o t a l system and because o f t h i s d iv e r s ity
o f fu n c tio n can be in ju r e d in many w ay s.
A b r i e f o u tlin e o f h e p a tic f u n c tio n w ith r e s p e c t t o su b sta n c es
e n te r in g th e body from th e i n t e s t i n e i s shown in F ig u re I .
Food
su b sta n c e s and o th e r m olecules p ass th ro u g h th e i n t e s t i n a l w a lls in to
th e blood c a p i l l a r i e s and a re th e n tr a n s p o r te d in to th e p o r t a l v e in
2
B mfieo j i m a
F ig u re I .
A Diagram o f H ep atic F u n ctio n
which t r a n s p o r ts them in to th e l i v e r .
Here th e blood p a sse s in to
s m a lle r v e s s e ls u n t i l i t re a ch e s th e s in u s o id s o f th e lo b u le s .
C la s s i­
c a l l y th e s e lo b u le s a re th e sm all u n its o f th e l i v e r bounded by p lan es
drawn betw een th e l i n e s o f th e h e p a tic t r i a d ; h e p a tic a r t e r y , p o r ta l
v e in , and b i l e d u c t surrounded by G lis s o n ’s c a p s u le ; c e n te re d a b o u t th e
c e n t r a l v e in a s shown in F ig u re 2 .
The p u r i f i c a t i o n o f incoming blood
The c l a s s i c h e p a tic lo b u le
i s o u tlin e d w ith s o lid
l i n e s , th e p o r t a l lo b u le
w ith an in te r r u p te d l i n e ,
and th e l i v e r a c in u s o r
f u n c tio n a l u n i t w ith a
d o tte d l i n e . The b ran ch es
o f a p o r t a l v e in and a
h e p a tic a r t e r y ( s o l i d )
from one p o r t a l a re a a re
shown a t low er r i g h t .
P o r ta l a re a s a re la b e le d
"Pn , c e n t r a l v e in s "C".
F ig u re 2 .
A Diagram o f H e p atic Lobules^
ta k e s p la c e in th e s in u s o id s by means o f th e parenchym al and K u p ffer
c e l l s w ith th e m a te r ia ls t o be e x c re te d going in to th e b i l e d u c ts and
th e p u r if ie d blood th e n p a ss in g in to th e r a m if ic a tio n s o f th e h e p a tic
venous system and f i n a l l y in to th e i n f e r i o r vena c a v a , a p a r t o f th e
more g e n e r a l c i r c u l a t o r y sy stem .
In humans th e t o t a l h e p a tic blood
flow i s a p p ro x im a te ly one and one h a l f l i t e r s p e r m inute w ith s ix ty to
e ig h ty p e r c e n t o f t h i s flow coming from th e p o r t a l v e in t o th e h e p a tic
v e in .
The o th e r tw enty t o f o r t y p e r c e n t comes from th e h e p a tic a r t e r y
2
to th e h e p a tic v e in .
About tw enty p e r c e n t o f th e t o t a l blood o f th e
4
human body i s to be found in th e l i v e r and s p le e n .
3
Harmful s u b sta n c e s e n te r in g th e l i v e r from th e p o r t a l v e in a f f e c t
th e l i v e r in two b a s ic w ays.
They can s e n s i t i z e th e l i v e r and le a d to
an a l l e r g i c 'r e a c t i o n , induced h e p a t i t u s , or th e y can d em o n strate a tr u e
h e p a to to x ic a c t i v i t y , to x ic h e p a t i t u s .
4
tlH ep ato to x in s a r e a h etero g en eo u s group o f n a t u r a l l y o c c u rrin g and
s y n th e t ic chem ical compounds’1^ w ith d iv e rs e a c t i o n s .
e x h i b i t a number o f common c h a r a c t e r i s t i c s .
They d o , how ever,
They " ( I ) e x h i b i t a
d i s t i n c t i v e h i s t o l o g i c a l p a tte r n f o r any g iv e n h e p a to to x in , (2 ) v ary in
s e v e r ity in d i r e c t r e l a t i o n t o th e d o se , (3 ) can be e l i c i t e d in a l l
i n d iv id u a ls , (4 ) a r e re p ro d u c a b le in la b o r a to r y a n im a ls , and (5 ) a p p ea r
a f t e r a p r e d ic ta b le and u s u a lly b r i e f l a t e n t p e rio d fo llo w in g
e x p o s u re .
„6
Numerous t e s t s o f l i v e r f u n c tio n have been d e v ise d t o determ ine
b o th th e e x te n t and th e mode o f a t t a c k o f h e p a to to x in s .
Among th e s e
a re t e s t s o f th e rem oval and e x c r e tio n o f in je c te d dyes from th e b lo o d ,
and le v e l s o f p a r t i c u l a r enzymes o r m e ta b o lite s in th e b lo o d .
In
g e n e r a l, th e s e t e s t s m easure th e l i v e r ’s a b i l i t y to ’’s o r t ” m olecules
and e x c r e te them d i r e c t l y o r in an a l t e r e d form,, and i t s a b i l i t y to
e n z y m a tic a lly perform ch em ical r e a c t i o n s .
S p e c if ic a l ly one o f th e m ost w id ely used m easures o f l i v e r
f u n c tio n i s th e BSP ( s u lfo b ro m p h th a le i n ) e x c r e tio n t e s t .
N orm ally,
t h i s o rg a n ic dye i s in je c te d in tra v e n o u s ly and i t s d isa p p e a ra n c e from
__I!
th e blood m onitored by th e use o f a c o lo r im e te r .
The t e s t i s an
ex trem ely s e n s i t i v e measure o f g e n e r a l l i v e r f u n c tio n .
The dye is
known t o be removed from th e blood a lm o st e x c lu s iv e ly by th e l i v e r and
t o be e x c re te d th ro u g h th e b i l e d u c t a f t e r c o n ju g a tio n w ith g lu ta th io n .
Thus, t h i s t e s t g iv e s th e sum o f th e u p ta k e , c o n ju g a tiv e , and e x c r e tiv e
a b i l i t i e s o f th e l i v e r .
The th r e e m ost common t e s t s o f s p e c i f i c enzyme a c t i v i t y r e la te d t o
th e l i v e r a re th e serum a lk a l in e p h o sp h atase (SAP), g lu ta m ic o x alo ­
a c e t i c tra n sa m in a se (GOT), and g lu ta m ic p y ru v ic tra n sa m in a se (GPT)
t e s t s which a re ru n on blood seru m .8
These t e s t s have been shown to be
d ia g n o s tic f o r d i f f e r e n t ty p e s o f h e p a to d y s fu n c tio n .
The SAP a c t i v i t y
i s g r e a t e r w ith b i l i a r y o b s tr u c tio n th a n h e p a to c e llu la r damage w h ile
th e GOT and GPT a c t i v i t i e s a re much g r e a te r in h e p a to c e llu la r damage
/
th a n b i l i a r y o b s tr u c tio n .
P o s s ib le re a so n s f o r th e changes in enzyme
le v e ls t h a t have been s u g g e s te d , b u t n o t p ro v en , a re an in c re a s e d p ro ­
d u c tio n o f th e enzymes o r a d ecreased e x c r e tio n o f th e enzymes by th e
liv e r.
Though th e s e p o s s i b i l i t i e s a re l o g i c a l th e y a re n e ith e r
o rig in a l nor d e f in itiv e .
Much work i s l e f t to be done i n o rd e r to
o b ta in an u n d e rsta n d in g o f th e im portance o f th e s e enzymes t o l i v e r
f u n c tio n .
A n a ly sis f o r th e amount o f ammonia t h a t can be fr e e d from th e
blood upon th e a d d itio n o f b ase has a ls o been used a s a m easure o f
l i v e r f u n c tio n .
The form in which t h i s ammonia is c a r r ie d in th e blood
6
i s n o t known" b u t i t o b v io u sly could n o t be f r e e ammonia a t p h y sio lo g ­
i c a l pH.
Some a u t h o r i t i e s ^ have su g g este d t h a t t h i s e a s i l y a v a ila b le
ammonia i s c a r r ie d in th e blood as sim ply ammonium h y d ro x id e w hile
others'*"^ fa v o r a p r o te in bound 'fo rm .
R eg a rd le ss o f i t s form in th e
b lo o d , t h i s ammonia, which p rim a rily comes from th e g a s t r o - i n t e s t i n a l
t r a c t , i s n o rm ally co n v erted in to u rea by th e l i v e r c e l l s th ro u g h th e
b io c h em ica l pathway known a s th e u re a c y c le (F ig u re 3 ) .
This u rea i s
th e n allo w ed t o e n te r th e h e p a tic v e in and t o c i r c u l a t e th ro u g h o u t th e
body a s a u s e f u l reduced form o f n itr o g e n .
In c re a se d le v e l s o f blood
ammonia have been a t t r i b u t e d t o th e sh u n tin g o f p o r t a l blood around th e
l i v e r o f th e d ecreased a b i l i t y o f th e parenchym al c e l l s t o s y n th e s iz e
u r e a „ A bnorm ally h ig h c o n c e n tra tio n s o f blood ammonia can le a d t o
pulmonary edema, which has been a t t r i b u t e d to membrane i r r i t a t i o n or
in c re a s e d venous p re s s u re a s a r e s u l t o f damage to th e sy m p ath etic
nervous sy stem .
12
Ammonia i s a ls o re p o rte d to le a d to n e u r a l damage
r e s u l t i n g in coma and u ltim a te ly d e a th .
13
The above t e s t s and o t h e r s , a lo n g w ith h i s t o l o g i c a l s t u d i e s , a re
o fte n used t o g iv e a p ic tu r e o f th e g ro ss changes in th e l i v e r caused
by h e p a to to x in s .
But th e s e methods do n o t m o n ito r fu n c tio n s t h a t a re
s p e c i f i c enough t o e lu c i d a te th e a c t u a l mechanism o f th e a t t a c k of th e '
h e p a to to x in .
In a l l in s ta n c e s th e y sim ply show t h a t th e l i v e r i s n o t
f u n c tio n in g p ro p e rly o r has been damaged.
"C o n sid erin g t h a t h e p a to to x in s d i f f e r m arkedly in ch em ical
7
(CH9
)
2 '3
I
I
(CH2 ) 0
C itru llin e
A rg in in e
co7
I 2
J
CO"
NH9 CH9
I : I
C=N-C-H
I
I _
NH CO2
Fum erate
H-c-Sn
to ;
A sp a rta te
ATP
(CH2 )g
H-C-Shq
I
_
COn
3
A rg in in o s u c c in a te
F ig u re 3„
The Urea Cycle
s t r u c t u r e , i t i s h ig h ly im probable t h a t th e y a l l a tta c k th e l i v e r in
th e same way.
The u n ifo rm ity o f th e b io c h em ica l fin d s in th e l i v e r
a f t e r exposure to h e p a to to x in s o f d iv e rs e c h a r a c te r s u g g e s ts t h a t ,
a lth o u g h th e s e a g e n ts p ro b ab ly i n i t i a t e c e l l u l a r in ju r y in d i f f e r e n t
w ays, th e su b se q u e n t c h a in o f b io c h em ica l e v e n ts le a d in g t o n e c ro s is
Jl
11 Il
8
fo llo w s a common p a t h w a y T h e problem seems t o be t h a t only a few
h e p a to to x in s have been s tu d ie d i n te n s iv e ly a t th e b io c h e m ic a l l e v e l .
The m ost work seems t o have been 'done on th e 'h alo g e n a ted hydrocarbons
w ith numerous compounds b e in g r e l a t e d t o them r a t h e r th a n b ein g
in d iv id u a lly s t u d i e d .
A number o f mechanisms f o r th e i n i t i a l a t t a c k o f a h e p a to to x in
have been p ro p o se d .
Some o f th e s e mechanisms have r a t h e r good e x p e r i­
m ental evidence w h ile o th e rs a re much more p o o rly fo u n d e d .
a re :
Among th e m •
7,(1 ) im pairm ent o f h e p a tic blood f l o w , . . . ; (2 ) a l t e r a t i o n s in
l i p i d m etab o lism ; and (3 ) d is tu rb a n c e s in p r o te in s y n th e s is and energy
p ro d u c tio n in th e l i v e r r e s u ltin g , from d i r e c t in ju r y t o h e p a tic c e l l
membranes, o r m e ta b o lic a l t e r a t i o n s in e s s e n t i a l c o n s ti tu e n ts o f th e
I c.
h e p a tic c e l l . "
A b r i e f e x am in atio n o f th e s e mechanisms r e v e a ls t h a t th e most
w eakly su p p o rte d one i s t h a t su g g e s tin g im pairm ent o f h e p a tic blood
flow a s a prim ary l e s i o n .
The e v id en c e "^ shows t h a t th e r e i s i n t r a ­
lo b u la r v a s o c o n s tr ic tio n in carbon t e t r a c h l o r i d e p o iso n in g t h a t can be
blocked by cordotom y, a pro ced u re t h a t b lo c k s sy m p ath etic s tim u li which
c o n tr o l th e s p h in c te r s in th e blo o d v e s s e ls which in tu r n c o n tr o l th e
blood flo w .
T his o p e ra tio n f u l l y p r o te c ts a g a in s t h e p a tic n e c r o s is and
a l t e r a t i o n s in m ito c h o n d ria l enzym atic a c t i v i t y u s u a lly seen fo llo w in g
carbon t e t r a c h l o r i d e , p o is o n in g .
However, th e ev id en ce from th e
cordotomy i s r e f u te d by o th e r w orkers
17
who have found t h a t th e
o p e ra tiv e p ro ced u re w ith o u t a c t u a l cordotomy a ls o p r o t e c t s .
They
suspect that hypothermia is the real factor here, not the neural change„
There a re n e u r a l changes in. to x ic h e p a ti tu s h u t th e s e a r e u s u a lly
attributed to ammonia to x ic ity or other factors after the i n i t ia l
a t t a c k on th e l i v e r .
R e g a rd le s s , th e p rim ary le s i o n h e re i s in th e
sympathetic nervous system not in the liv e r blood flow , i f the above
mechanism i s c o r r e c t .
The blood flow does o b v io u sly change in to x ic
hepatitus, but th is does not n ecessarily point to the sympathetic
nervous system n o r indeed t o blood flow changes a s a p rim ary l e s i o n .
I t could w e ll be- t h a t s w e llin g in parenchym al c e l l s due t o some ty p e o f
c e l l u l a r damage i s th e cause o f th e change in blood f lo w .
A lte r a tio n s in l i p i d m etabolism and r e s u l t a n t f a t t y l i v e r i s
certain ly an important factor in liv e r disease but there seems to be
l i t t l e evidence t h a t i t i s a prim ary l e s i o n .
Many to x in s cause f a t t y
liv e r but a few that do cause th is symptom have been shown to decidedly
cause o th e r changes in th e l i v e r f i r s t .
rev iew
18
None m entioned in K la ts k in !s
have been shown t o cause f a t t y i n f i l t r a t i o n f i r s t .
I t seems
more l i k e l y t h a t f a t t y i n f i l t r a t i o n i s an ev id en ce o f some change in th e
liv e r not the change i t s e l f , i . e „, something has happened to an enzyme
f
system o r a membrane t h a t a llo w s th e f a t t y b u ild - u p .
The third suggested mechanism involves the more basic components
of the C e lltS function—membrane permeability, energy u tiliz a tio n , and
enzymatic catabolism and anabolism.
Changes in liv e r membrane
10
p e rm e a b ility have been n o ted a s th e r e s u l t , o f th e a t t a c k o f numerous
to x in s .
But a g a in , a t l e a s t in carb o n t e t r a c h l o r id e .a n d e th io n in e
p o is o n in g , th e s e changes a p p ea r to be a secondary e f f e c t T h e
s u g g e s tio n t h a t a n o n -p o la r s o lv e n t such a s carbon t e t r a c h l o r i d e
d is s o lv e s a segm ent o f th e membrane i s much le s s th a n p le a s in g as th e
c o n c e n tra tio n s t h a t a re to x ic a re o f such a low l e v e l t h a t i t i s d i f ­
f i c u l t t o conceive o f t h i s o c c u rrin g in many c e l l s o f th e l i v e r .
A more
u s e f u l p o s tu la te would be t h a t th e p a r t i c u l a r to x ic m olecule in te r a c te d
d i r e c t l y w ith a s p e c i f i c m olecule in th e membrane, some enzyme, o r
a n o th e r s p e c i f i c m olecule o f im portance in th e c y to p la sm .
The b e s t
example o f t h i s l a t t e r i n t e r a c t i o n i s th e proven e f f e c t o f e t h i o n i n e .
T his m olecule has been shown
20
to s p e c i f i c a l l y form a compound w ith
a d e n o s in e , S -ad e n o sy l e th io n in e , t h a t can n o t be used by th e c e l l .
This
bonding o f ad en o sin e o b v io u sly c u ts o f f th e su p p ly o f ATP, th e main
energy c u rre n cy o f th e c e l l , and t h u s , s e t s o f f a sequence o f e v en ts
t h a t cause th e c e l l ’s d e s tr u c tio n in c lu d in g in th e p ro c e ss detachm ent
of ribosom es from th e endoplasm ic re tic u lu m and f a t t y i n f i l t r a t i o n .
One
has d i f f i c u l t y o b serv in g ATP s u p p lie s by e i t h e r e le c tr o n m icroscopy o r
l i g h t m icroscopy— in f a c t i t i s im p o ssib le to do so a t p r e s e n t—so th e s e
means a g a in a re shown only t o g iv e g ro ss ch an g es, i . e . , th e r e s u l t s o f
m inute b io c h e m ic a l change n o t th e change i t s e l f .
P erhaps th e m ost i n te n s iv e ly s tu d ie d h e p a to tb x in i s carbon t e t r a ­
c h lo rid e which has been found t o f i r s t a t t a c k th e endoplasm ic re tic u lu m '
11
d is lo c a tin g , th e ribosom es and su b se q u e n tly i n h i b iti n g p r o te in s y n th e s is
and plasma l i p o p r o te in s y n t h e s i s .
21
a re e f f e c te d i s n o t known, how ever.
The means by which th e s e changes
A f te r th e i n i t i a l a t t a c k o f a
h e p a to to x in "there a re sometimes r a t h e r d r a s t i c changes in membrane
p e rm e a b ility le a d in g t o m ito c h o n d ria l d e s tr u c tio n and c e l l u l a r s w e llin g .
T his h y p e rte n s io n can c u t o f f th e c e l l ’s oxygen su p p ly from th e b lo o d ,
a s has a lre a d y been n o te d .
T his can th e n le a d t o a sto p p ag e o f th e
a n a b o lic p ro c e ss e s coupled w ith th e r e le a s e o f a u t o l y t i c enzymes w hich
r e s u l t in th e c e l l ’s d e s tr u c ti o n .
th e r e seem to be l i t t l e
n e c ro s is .
W ith o th e r ty p e s o f h e p a to to x in s
o r no in fla m a tio n b u t r a t h e r s im ply a c u te
I t i s a l s o im p o rta n t t o n o te t h a t n e c r o s is can be lo c a liz e d
w ith in th e lo b u le a s c e n t r e - , m id z o n a l, p e r ip h e r a l, o r p a n lo b u la r
depending on b o th th e to x in and i t s c o n c e n tr a tio n .
22
I t i s n o t known,
how ever, w hether th e lo c a t io n o f th e le s i o n in th e l i v e r i s due t o th e
r a t i o o f blood flow t o c e l l m ass, th e l o c a l i z a t i o n o f enzym atic a c t i v i t y
o r o th e r f a c t o r s .
What i s needed i s s tu d ie s o f a c tu a l b io c h em ica l
in te r a c t io n s on th e m o le c u la r l e v e l w ith th e s p e c i f i c to x in t o f in d
where and how i t i s i n t e r a c t i n g .
When an u n d e rsta n d in g o f t h i s occurs
on th e m o le c u la r l e v e l i t w i l l be p o s s ib le to b e g in to d e sig n m olecules
t h a t w i l l r e v e rs e th e to x ic e f f e c t o r r e p la c e th e bound m e ta b o lite s .
One d e m o n stra tio n o f l i v e r d y s fu n c tio n i s p h o to s e n s itiz a tio n , ty p e
I I I o f C la r e .
23
In t h i s syndrome th e in ju r e d l i v e r i s u n ab le to perform
i t s norm al f u n c tio n o f rem oving th e pigm ent p h y llo e r y th r in ( I ) from th e
12
p o r t a l blood and e x c r e tin g i t in th e b i l e .
In t h i s manner th e pigm ent
i s a b le to re a c h th e p e r ip h e r a l c i r c u l a t i o n a n d , th e n c e , th e s k in
t i s s u e where p h o to s e n s itiz a t ion o c c u rs .
An o u ts ta n d in g example o f t h i s
syndrome i s found in th e work o f R im ington and Quinn
24
w ith g e e ld ik k o p ,
"b ig y ello w -h e ad " o f sh e e p , in South A f r i c a .
C la re
25
s u g g e s ts t h a t th e "b ig head" problem o f sheep in th e i n t e r ­
m ountain a re a o f th e w e ste rn U nited S ta te s o f America i s a n o th e r example
o f ty p e I I I p h o t o s e n s i t i z a t i o n .
H is s u g g e s tio n is based on two s tu d ie s
o f th e d e s e r t p l a n t , Tetradym ia g l a b r a t a , t h a t were com pleted p re v io u s
t o 1940.
T . gl a b r a t a is a s tr o n g ly sc e n te d sh ru b o f th e Compositae fa m ily ,
S enecio t r i b e , t h a t grows over th e r a t h e r broad re g io n t h a t i s shown in
F ig u re 4 .
The p la n t ( f ig u r e 5) resem b les sage b ru sh and i s found in th e
same g e n e r a l re g io n a s s a g e .
I t can be a s much a s th r e e f e e t t a l l .
The
c u r r e n t y e a r 's grow th (A ), or new g ro w th , has t h i n l i g h t c o lo re d le a v e s
t h a t a re p o in te d and a b o u t one h a l f in c h lo n g .
The e n t i r e new growth
13
( le a v e s , stem , and flo w e r b u d s) a re tom entose (covered w ith f in e w h ite
h a irs ).
The o ld e r grow th (B) i s woody and h ig h ly branched w ith few er
le a v e s .
These le a v e s a re more ro u n d ed , s h i n i e r , s m a lle r and th ic k e r th a n
th o s e o f th e new g ro w th .
F ig u re
The flo w e r heads t h a t a p p e a r from A p ril t o m id-
Range o f th e O ccurrence o f Tetradym ia
June a re y ello w b u t soon tu r n w h ite from th e s il k y h a i r s on th e s e e d s .
14
A- New Growth
Mounted p la n t specimen
in th e Montana S ta te
U n iv e rs ity Herbarium
B- O lder
Growth
F ig u re 5.
The P la n t, T etradym ia g la b r a t a .
15
Flem ing
f i r s t in d ic a te d t h a t T. g la b r a t a was re s p o n s ib le f o r n o t
only th e "b ig head" symptoms o f f a c i a l and e a r t i s s u e s w e llin g , b u t
a ls o th e d e a th o f many sheep t h a t o ccu rred d u rin g th e s e " b ig head" o u t­
b re a k s on th e ran g es o f N evada.
and h is colleagues
28
In th e y e a rs up th ro u g h 1922, Fleming
conducted many feeding experiments and a b rief
chemical study in an attempt to ascertain (I ) the conditions required
for poisoning, (2) the amount of the plant required for poisoning, and
(3) the nature of the toxic p rin cip le.
They succeeded in showing that
a feeding of approximately two and one h a lf per cent of the SheeptS body
weight of the green tops and buds was s u ffic ie n t to k i l l the animal but
there is no mention of the sw elling of.th e head in th eir la te r paper.
They a t t r i b u t e d th e d e a th s o f th e an im als to h e p a to d y s fu n c tio n and
c a r d ia c f a i l u r e caused by a s u b sta n c e c o n ta in e d in th e p l a n t .
T h e ir
work on chem ical c h a r a c te r iz a ti o n was s c a n ty b u t th e y d id succeed in
d e m o n stratin g th e t o x i c i t y o f th e p etro leu m e th e r and a c e to n e e x tr a c ts
o f th e g re e n p la n t by a s in g le r a b b i t fe e d in g e x p erim en t w ith each
e x tra c t.
Clawson and Huffman
29
w orking on th e "b ig head" problem in Utah
o b ta in e d much th e same r e s u l t s a s Flem ing
30
had e a r l i e r r e p o r te d .
These
la te r workers did not attempt any chemical characterization of the toxin .
They d id , how ever, p ursue th e problem o f p h o to s e n s itiz a tio n a s th e re , was
no external edema observed resulting from th eir early feeding experim e n ts .
They l a t e r re p o rte d
31 n in e sheep had developed " b ig head" when
16
e x p e rim e n ta lly fe d Tetradym ia g la b r a t a w h ile b ein g herded on th e d e s e r t
ra n g e .
Huffman th e n t r i e d th e c o -fe e d in g o f T . g la b r a t a w ith many
o th e r ran g e p la n ts t o f in d i f th e p h o to s e n s itiz a tio n was due t o th e
i n t e r a c t i o n o f two p l a n t s .
In h is u n p u b lish ed d a ta
32
i s found t h a t , of
th e a p p ro x im a te ly f o r t y p la n ts c o - f e d , only by fe e d in g A rte m isia
t r i d e n t a t a or nova w ith T . g la b r a t a o r c an e scens could any "b ig head"
s w e llin g symptoms be n o te d .
This d o e sn ’t fo llo w from th e C lare ty p e
I I I p h o to s e n s itiz a tio n a s p h y llo e r y th r in ( I ) i s th e p ro d u c t o f th e
m etabolism o f c h lo ro p h y ll which can come from any h ig h e r p la n t so u rce
n o t j u s t A rte m is ia .
However, in a l l whole p l a n t , T e tra d y m ia , fe e d in g
ex p erim en ts th e m ost c h a r a c t e r i s t i c le s i o n was l i v e r n e c r o s is so th e
problem i s s t i l l p ro b a b ly r e l a t e d to C lare ty p e I I I p h o to s e n s itiz a tio n
though th e s e n s i t i z i n g pigm ent may be d i f f e r e n t .
The fo llo w in g in v e s t ig a ti o n was u n d e rta k e n to a s c e r t a i n th e
chem ical s t r u c t u r e o f th e compound o r compounds re s p o n s ib le f o r th e
t o x i c i t y o f T. g la b r a t a and to f u r t h e r s tu d y i t s e f f e c t on th e liv in g
sy stem .
DISCUSSION
T his d is c u s s io n i s d iv id e d in to fo u r m ajor s e c tio n s to f a c i l i t a t e
p r e s e n ta tio n and o rg a n iz e th e m a t e r i a l s .
The s e c tio n s d e a l w ith :
I . p h y s io lo g ic a l a c t i v i t y , ' I I . i s o l a t i o n o f th e t o x i n , I I I . chem ical
and s p e c t r a l e lu c i d a tio n o f s t r u c t u r e , and IV . X -ray s t r u c t u r a l
a n a ly s is .
I.
P h y s io lo g ic a l A c tiv ity
■In agreem ent w ith th e fin d in g s o f Clawson and Huffman,
33
th e to x ic
p r in c ip le o f T . g la b r a t a was lo c a liz e d in th e new grow th and flo w e r buds
o f th e p l a n t .
When th e whole to p o f th e p l a n t ; which in c lu d e d c o n s id e r­
a b le o ld e r , d ry , woody m a t e r i a l ; was fed a p p ro x im a te ly f iv e tim es as
much p la n t m a te r ia l was re q u ire d to produce d e ath (sheep S-553) as w ith
j u s t buds and flo w e rs (sheep P-311) a s i s shown in Table I .
Since th e s e
two sam ples o f p la n t m a te r ia l were pick ed a t d i f f e r e n t tim es i t m ight be
argued t h a t th e la c k o f t o x i c i t y d em o n strated by th e whole p la n t was due
to d i f f e r e n t grow th s ta g e s o f th e p l a n t .
T his does n o t a p p ea r to be
t r u e , how ever, s in c e Tg I was picked e a r l i e r th a n Tg I I and Fleming
and Huffman
35
34
in d ic a te t h a t t o x i c i t y d ecreased a s th e sea so n p ro g re s s e d .
I t should a ls o be n o ted t h a t due t o l a t e r a in s in 1968 th e p la n t was in
a q u ite s im ila r grow th s ta g e a t b o th p ic k in g s .
be l i t t l e to x in in th e o ld e r g row th .
Thus, th e r e appeared to
The hexane e x t r a c t o f th e r o o t
s to c k o f th e p la n t showed no evid en ce o f th e a c tiv e p r in c ip le o f
T . g la b r a t a when i t was an aly zed by t i c .
n
D
i
f t
f ' 'i
/
1____d - i .
Table I
Sheep Feeding E xperim ents Using th e Whole P la n t
**
Sheep
number
W eight
pounds
Age
y e a rs
Days
fed
P la n t
fed
T o ta l
pounds
fed
% p la n t
o f body
w eig h t
S-553
115
2
11
Tg I
16
13.8
S-536
108
2
7
Tg I
6
5.5
S-230
108
2
11
Tg I
16. 6
1 5 .4
5/35
7/70
9/26
d e ath
S-563
HO
2
9
Tg I
1 3 .5
12.3
4 /8
6/13
8/7
r e le a s e
2.5
2. 3
Tg H
3 .0
2.6
At
3 .0
2.6
At*
P-380
114 '
2
•3
-
NH4 I e v e l^
BSP c le a ra n c e
8/11
10/16
R e s u lt
d e ath
r e le a s e
d e ath
P-311
110
2
4
Tg H
4.0
3. 6
4 /2 4
H-641
102
,1
3
Tg I I I
4 .1
4 .0
2/12
H -52 2
120
I
0
0 /4
c o n tr o l
H-552
132
I
0
0/4
c o n tr o l
3/13
4 /2 6
d e ath
2/36
d e ath
Ewe-I
87
0
I
0/3
*
A rte m is ia t r i d e n t a t a picked w h ile v ery lu s h n e a r Bozeman5 Montana
c o n tr o l
^Recorded a s a f r a c t i o n w ith th e day o f th e fe e d in g ex p erim en t when th e Nht le v e l t e s t was >
ru n in th e num erator and th e jug/ml o f NH4 found in th e serum reco rd ed a s th e
d e n o m in a to r.
.
Recorded as above w ith th e day in th e num erator and Ti_ in m inutes reco rd ed as th e
d en o m in ato r.
^
(
:
19
The r e s u l t s o f th e o r a l fe e d in g ex p erim en ts w ith T . g la b r a t a ,
re c o rd ed in T able I , g iv e an id e a o f th e mode o f a c ti o n o f th e to x ic
p rin c ip le .
The BSP t e s t c l e a r l y re v e a le d t h a t th e l i v e r was undergoing
s e v e re damage.
The norm al sh e e p , a s i l l u s t r a t e d by Ewe-1, had a one-
h a l f tim e (tim e re q u ire d f o r one h a l f o f th e dye to be e lim in a te d ) o f
from th r e e t o seven m inutes w hile a sheep re c e iv in g T . g la b r a ta demon­
s t r a t e d a o n e -h a lf tim e ( T i) o f from s ix te e n m inutes in sheep S-553 to
2,
a s h ig h a s sev e n ty m inutes in sheep S -2 3 0 .
A nother t e s t o f l i v e r f u n c tio n , blood ammonia, a ls o re v e a le d t h a t
th e r e was se v e re h e p a tic d y s fu n c tio n .
The norm al sh e e p , a s i l l u s t r a t e d
by H-522 and H -552, had a blood ammonia l e v e l o f a b o u t 4 jug/ml.
When a
sheep was fed T . g la b ra t a t h i s l e v e l ro s e to 13 jug/ml in H-641 and 24
jUg/ml in P -311.
Both th e s e t e s t s in d ic a te d t h a t th e l i v e r was mal­
fu n c tio n in g p re v io u s t o th e d e ath o f th e sheep b u t d id n o t show how th e
to x in a tta c k e d th e l i v e r n o r d id th e y r e v e a l i f th e l i v e r was a tta c k e d
firs t.
S ince Flem ing
36
had s ta t e d t h a t th e a c e to n e e x t r a c t was p o s s ib ly
c a r d ia c - a c tiv e a l l sheep were m onitored by EKG f o r p o s s ib le h e a r t damage
37
b u t no s i g n i f i c a n t change was n o te d .
An example o f an EKG ta k en
b e fo re any p la n t fe e d in g and one ta k e n a f t e r f i f t e e n days had e lap se d in
th e fe e d in g o f S-230 (T able I ) i s shown in F ig u re 6 .
The only change
noted i s a s l i g h t s lo p e in th e S-T segm ent which in d ic a te d some damage
b u t n o t o f a marked n a t u r e .
20
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L ead 4
A f te r f i f t e e n d a y s o f th e f e e d in g e x p e r im e n t had e l a p s e d ,
two d a y s b e f o r e d e a th
Lead 4
F ig u re 6.
E le c tro c a rd io g ra p h o f Sheep S-230
21
Sheep S-536 was r e le a s e d a f t e r fe e d in g i t a s u b le t h a l dosage o f
To g la b r a t a t o f in d i f th e r e m ight be a delayed a c t i v i t y o f th e to x in
o f i f p h o to s e n s itiz a tio n m ight occur „ The sheep was v i s u a l l y examined
on a number o f o c ca sio n s up to two months a f t e r r e le a s e and found t o be
n o rm a l.
Sheep S-563 was r e le a s e d a f t e r th e fe e d in g o f a s u b le th a l
dosage s in c e c o -fe e d in g o f A . t r i d e n t a t a had caused no p h o to s e n s itiz a ­
t i o n n o r in c re a s e in t o x i c i t y o f T . g l a b r a t a .
This sh eep was a ls o
observed a f t e r r e le a s e and dem on strated no v i s i b l e a d v e rse e f f e c t s from
th e f e e d in g .
A u to p sie s were perform ed on a l l sheep t h a t were s a c r i f i c e d o r d ied
a s a r e s u l t o f T . . g la b r a t a f e e d in g s .
s e v e re l i v e r n e c r o s is was e v id e n t.
from t a n . t o c la y in c o l o r .
w ith w atery g re e n b i l e .
In a l l c ase s m a c ro sc o p ic a lly
The l i v e r t i s s u e was f r i a b l e and
The. g a l l b la d d e r was extended and f i l l e d
M ost, 5 o u t o f 6 , o f th e sheep d em onstrated
c o n s id e ra b le lung c o n g e s tio n w h ile one h a l f dem o n strated some macro­
sc o p ic changes in th e k id n e y in c lu d in g roughness o f some o f th e c o r t i c a l
p o r ti o n , a m arbled ap p earan ce o f th e o u ts id e o f th e c o r te x , and a y ello w
d is c o l o r a t io n (one example o f e a c h ) .
Samples o f t i s s u e were ta k e n from th e l i v e r , lu n g , h e a r t , k id n e y ,
a d r e n a l g la n d , b r o n c h ia l lymph n o d e , p a n c re a s , abomasum, s p le e n , and
th y ro id g lan d f o r m ic ro sco p ic e x a m in a tio n .
D r. K. A nderson in th e
D ia g n o stic L a b o ra to ry lo c a te d a t Montana S ta te U n iv e rs ity examined th e
t i s s u e specim ens and made th e fo llo w in g o b s e r v a tio n s , some o f which a re
22
sum m arized.
The l i v e r t i s s u e t y p i c a l l y d em o n strated ’’marked p a n lo b u la r
n e c r o s is o f l i v e r parenchym a.
N e c ro sis was c e n tro 'lo b u la r in lo c a tio n
and b o rd e re d on more p e r ip h e r a l p a r ts o f th e lo b u le s in which f a t t y
change was e v id e n t in d e g e n e ra te d , t>ut s t i l l v i a b l e , c e l l s a d ja c e n t t o
th e p o r t a l t r i a d s .
...T h e le s io n s d e sc rib e d a re n o n - in f lam m atory.
N e cro sis ten d ed t o ex ten d from ope c e n t r a l v e in to a n o th e r and r e s u lte d
in an ap p earan ce o f p s e u d o - lo b u la tio n .
a re a s appeared v i a b l e .
K u p ffe r c e l l s w ith in n e c r o tic
In p o r t a l a r e a s , th e r e ap p eared t o be an e a r l y ,
s l i g h t p r o l i f e r a t i o n of b i l e d u c t s .”
38
The k id n ey t i s s u e showed hyperem ia ( in c re a s e in th e amount o f
b lo o d ) e s p e c ia ll y in th e m e d u lla ry p o r tio n and some g e n e r a l c o n g e stio n
and s w e llin g .
The lu n g s d is p la y e d a l l th e way from s l i g h t t o extrem e c o n g e stio n
and from a lv e o l a r emphysema to i n t r a ^ a Iv e o la r and in tr a - b r o n c h io l a r
hemmorhage.
Some b ro n c h io le s showed ’’pronounced e x f o l i a t i o n o f l i n in g
e p ith e liu m s u g g e s tiv e o f th e p re sen c e o f an i r r i t a n t . ”
39
The c a r d ia c t i s s u e re v e a le d some c o n g e s tio n and a few sub­
e p i c a r d i a l hemmorhages b u t th e r e was no g r e a t change from norm al h e a r t
tis s u e .
In th e only sampfe o f abomasum t i s s u e examined hyperem ia was
e v id e n t and th e r e was ”a s l i g h t m ononuclear inflam m atory re sp o n se in th e
d e ep e r a s p e c t o f th e mucosa and in some r e l a t e d a re a s o f th e submuco sa.”
40
The o th e r t i s s u e s examined seemed t o be q u ite norm al
23
m ic r o s c o p ic a lly .
From th e s e s tu d ie s i t was concluded t h a t th e a c ti v e p r in c ip le o f
T . g la b r a t a was h e p a to to x ic t o a l l a n im als t e s t e d .
These fin d in g s
in d ic a te d t h a t any damage t o o th e r body t i s s u e s was m inor and p ro b ab ly
o f a secondary n a t u r e .
The cause o f d e a th was p ro b ab ly h e a r t f a i l u r e
induced by a c u te to x e m ia .
T hat a problem e x is te d in r e l a t i n g p h o to s e n s itiz a tio n to p o iso n in g
by T . g la b r a t a was a llu d e d t o in th e in tr o d u c tio n .
t h a t ,rb ig head" was an example o f w hat C lare
p h o to s e n s itiz a tio n .
42
Huffman
41
su g g ested
l a t e r c a l l s ty p e I I I .
However, t h i s was m erely a su rm ise a s he p re se n te d
no e x p erim en ts in h is a r t i c l e t o s u p p o rt the; s u g g e s tio n .
C lare
su p p o rte d t h i s view in h is a r t i c l e b u t a g a in w ith o u t e v id e n c e .
43
a ls o
In th e
ex p erim en ts d e sc rib e d in t h i s stu d y no s w e llin g o f e x t e r i o r t i s s u e was
observed in any a n im a l.
The sheep were on g reen p a s tu r e w ith much sun­
l i g h t d u rin g th e tim e o f th e e x p e rim e n ta l fe e d in g s so t h a t i f c h lo ro p h y ll
were re q u ire d in th e d i e t f o r th e accu m u latio n o f p h y llo r e th r in le a d in g
t o a s u n lig h t induced p h o to s e n s itiz a tio n th e c o n d itio n s should s u re ly
have been m e t, e s p e c ia ll y s in c e even a d i e t o f hay and c h a f f p ro v id e s
enopgh s e n s i t i z i n g pig m en t.
44
As th e fe e d in g s were o v er v a rie d le n g th s
o f tim e i t does n o t fo llo w t h a t a s m a ll amount o f l i v e r damage, caused
by T . g la b ra t a ., fo llo w ed by g reen fe e d le a d s t o p h o to s e n s itiz a tio n as
was e a r l i e r su g g este d by Huffman.
45
S in ce th e s w e llin g o f e x te r n a l t i s s u e was n o t observed in th e
24
p r e s e n t stu d y i t le n d s credence t o H uffm an's l a t e r o b s e rv a tio n s ^ ^ t h a t
have a lre a d y been n o te d .
I t would th u s seem v ery u n lik e ly t h a t "b ig
head" i s a ty p e I I I syndrome b u t r a t h e r t h a t i t i s a new ty p e t h a t
r e q u ir e s v e ry s p e c ia l c o n d itio n s and p erh ap s a p a r t i c u l a r pigm ent from
a n o th e r p l a n t t o s e rv e as s e n s i t i z e r a f t e r l i v e r d e s tr u c ti o n .
If i t
was a sim ple r e l a t i o n s h i p betw een A. t r i d e n t a t a and T . g la b ra t a , p h o to ­
s e n s i t i z a t i o n should have been observed in sheep P-380 b u t t h i s was n o t
th e c a s e .
D r. M. Madsen o f Ufah S ta te U n iv e rs ity re p o rte d
th a t i f a
sheep was f a s te d th e n fe d a s u b le t h a l dose o f T. g la b r a t a fo llo w ed by
w a te r and A. t r i d e n t a t a p h o to s e n s itiz a tio n r e g u la r ly o c c u rre d .
above fin d in g has n o t been checked in t h i s la b o r a to r y .
The
T h e re fo re , th e
way p h o to s e n s itiz a tio n o c c u rs , i f indeed i t does from T . g la b ra ta
p o is o n in g , i s s t i l l u n c e r ta in .
When th e hexane and a c e to n e e x t r a c t s o f T . g la b ra t a were s e p a r a te ly
g iv en t o sheep by means o f c a p s u le s , th e same t o x i c i t y dem onstrated w ith
th e whole p la n t was e x h ib ite d a s shown in Table 2 .
S in ce i t was n o t
c e r t a i n t h a t hexane o r a c e to n e would e x t r a c t a l l th e to x in from th e
p la n t m a t e r i a l , th e e x t r a c t o f a sample e q u iv a le n t t o tw ice th e to x ic
dose was fe d in each in s t a n c e .
The r e s u l t s o f th e BSP t e s t s and th e
blood ammonia t e s t s on H-636 and H-665 reco rd ed in T able 2 in d ic a te d th e
same l i v e r d y s fu n c tio n a s had been found p re v io u s ly .
The fin d in g s of
h is to p a th o lo g ic a l s tu d ie s a ls o su p p o rte d t h i s c o n c lu s io n .
When th e
p la n t t h a t had been e x tr a c te d w ith a c e to n e and hexane was fed in a
25
q u a n tity , t h a t had been s u f f i c i e n t t o k i l l in p re v io u s ex p erim en ts (w ith
u n e x tra c te d p la n t) no t o x i c i t y was observed (sheep H-641 and H -6 7 1 ).
Thus, i t was re v e a le d t h a t th e to x ic p r in c ip le o f T. g la b r a ta had been
q u ite e f f e c t i v e l y e x tr a c te d in to hexane and a c e to n e .
The f r e s h p la n t was a ls o e x tr a c te d w ith e th a n o l.
T h is e th a n o l
e x t r a c t was ev ap o rate d t o a v is c o u s o i l th e n d ilu te d s l i g h t l y w ith
aqueous e th a n o l.
The e x t r a c t was p a r t i t i o n e d in to two f r a c t i o n s by
e x tr a c ti o n w ith p e n ta n e .
These two f r a c t i o n s , th e p en tan e e x t r a c t o f
th e e th a n o l e x t r a c t and th e rem ain in g p a r t o f th e e th a n o l e x t r a c t a f t e r
pentane e x t r a c t i o n , were e v ap o rate d and s e p a r a te ly fe d t o sheep H-628
and H-634 r e s p e c t i v e l y .
N e ith e r o f th e e x tr a c ts d em o n strated t o x i c i t y .
The e th a n o l e x tr a c te d p la n t was a l s o fe d t o sheep H-653 and showed no
to x ic ity .
The re a so n s f o r th e la c k o f t o x i c i t y d em onstrated in th e s e
e x p erim en ts w ith th e e th a n o l e x t r a c t was n o t c e r ta in s in c e th e e x tr a c ts
were n o t an aly zed t o determ in e w hat m olecules were p r e s e n t e x c e p t in th e
case o f th e p en tan e e x t r a c t o f th e e th a n o l e x t r a c t .
T his d id show a
h ig h c o n c e n tra tio n o f th e compounds l a t e r shown t o be to x ic from T .
g la b r a t a .
I t could w e ll be t h a t p o t a l l th e to x in was e x tr a c te d from
th e aqueous e th a n o l la y e r o r t h a t e th a n o l was n o t as e f f e c t i v e in
e x tr a c ti n g th e to x in from th e p l a n t .
Thus, th e la c k o f t o x i c i t y could
p o s s ib ly be a t t r i b u t e d t o p a r t i t i o n o f th e to x in in to to o many f r a c tio n s
re d u c in g th e a c t u a l dosage t o a l e v e l below th e e f f e c t i v e d o se .
However, th e r e a re o th e r p o s s i b i l i t i e s in c lu d in g th e d e s tr u c tio n o f th e
Table 2
Sheep Feeding Experiments Using the Plant Extracts
Sheep
number
W t.
lb s ,
H-636
+
*'
NH^ l e v e l
BSP c le a ra n c e I
R e s u lt
4 .5
3/12
5/18
3/36
5 /7 9
Death
. 5 .0
4 .8
3/14
5/13
7/14 5 / 4 4
7 /3 9
V. i l l ^
s a c r if ic e d
Pentane
of
e th a n o l
2 .5
2 .5
3/6
R elease
E th an o l
rem ains
2 .5
2 .5
3/6
R elease
Age
y rs ,
E x tr a c t
fe d
E x tr a c t
from
pounds
% p la n t
o f body
w eig h t
98
I
A cetone
4 .4
H-665
104
I
Hexane
H-634
103
H-r628
104
P la n t
Pounds
e x tr a c te d fed
w ith
H-651
102
I
A cetone
2.2
2.2
3 /5
Re-use
H-671
90
I
Hexane
2 .5
2.8
3 /8
R elease
H-653
100
I
E th an o l
2 .5
2 .5
3 /6
R elease
Recorded a s a f r a c t i o n w ith th e day o f th ^ fe e d in g ex p erim en t when t h e zNH^ le v e l t e s t was
ru n in th e num erator and th e jug/ml o f NH4 found in th e serum re c o rd ed a s^ th e denom inator.
^Recorded a s above w ith th e day in th e n u m erator and th e Ti_ in m inutes reco rd ed as th e
d e n o m in ato r.
^
f i V. i l l meaning v e ry s i c k , a c t u a l l y down and on th e v erg e o f d e a th .
N>
Ox
27
to x in in th e crude e x t r a c t by th e h ig h e r te m p e ra tu re s re q u ire d in
s tr ip p in g o f f th e e th a n o l o r by some o th e r f a c t o r .
The experim ents w ith th e hexane and a c e to n e e x t r a c t s , n e v e r th e
l e s s , d id confirm F le m in g 's f i n d i n g ^ t h a t th e l i p i d e x t r a c t was t o x i c .
Since sheep re q u ire d such la r g e q u a n t i t i e s o f t o x i n , due to t h e i r
s i z e , and were q u i t e ' ex p en siv e a s a la b o r a to r y an im al i t was d ecid ed to
a tte m p t u s in g s m a ll an im als f o r t e s t i n g p u rp o s e s.
A f te r tr y in g f iv e
d i f f e r e n t an im al s p e c ie s (T able 3) and f in d in g t h a t th e a c e to n e and
hexane e x t r a c t s were b o th h e p a to - to x ic , in a l l b u t one c a s e , mice were
chosen a s a t e s t a n im al b ecause th e y were in e x p e n s iv e , were g e n e r a lly
e a s i e r t o h a n d le , and d i d n 't b i t e a s h a r d .
A cetone e x tr a c te d a p p ro x im a te ly 3 .7 tim es a s much t o t a l m a te r ia l
from th e f r e s h p l a n t as d id h e x a n e .
T hus, t o feed th e e x t r a c t o f th e
same w eig h t o f p la n t i t was n e c e s s a ry t o fe e d 3 .7 tim e s a s much o f th e
crude a c e to n e e x t r a c t a s o f th e hexane e x t r a c t .
For t h i s re a so n th e
p e rc e n ta g e s o f body w eig h t d i f f e r betw een th e two e x t r a c t s in Table 3 .
Each fe e d in g r e p r e s e n ts th e e x t r a c t o f f r e s h p la n t e q u iv a le n t t o
a p p ro x im a te ly 5 p e r c e n t o f th e a n im a l's body w e ig h t.
When th e s e an im als were opened a f t e r d e ath a marked change had
ta k e n p la c e in th e l i v e r .
In s te a d o f th e norm al d ark mahogany c o lo r o f
th e l i v e r th e r e were observed w h ite p a tc h e s on a l i g h t p in k b ack g ro u n d .
M icroscopic ex am in atio n re v e a le d f o r th e g r e a t e r p a r t th e same ty p e
f in d in g s a s were observed in sheep t i s s u e .
The lu n g s were c o n g e ste d ,
28
Table 3
Small Animal Feeding Experiments Using the Plant Extract
Animal
R ab b it
R ab b it
Guinea p ig
Guinea pig
G e rb il
White r a t
B lack r a t
Mouse
Mouse
Mouse
Mouse
W eight (g )
3220
2680
780
620
77
336
445
22
22
22
22
E x tr a c tio n
S o lv e n t
% E x tr a c t
o f body w t.
Hours
t o d e ath
Hexane
A cetone
Hexane
A cetone
A cetone
Hexane
A cetone
Hexane
Hexane
A cetone
A cetone
0.25
0.95
0.25
0.95
0 .9 5 .
0.25
0.95
0.25
0.25
0.95
0.95
1 4 .5
2 0 .5
6
12
33
3
not i l l
3
5
4
4
*
A ll e x tr a c ts ta k e n from an amount o f p la n t e q u iv a le n t t o 5% o f
th e sm a ll a n im a l’s body w e ig h t.
th e h e a r t and b r a in t i s s u e appeared t o be norm al w h ile th e l i v e r t i s s u e
d is p la y e d th e m ost prom inent c h a n g e s.
Cloudy s w e llin g o f th e parenchym al c e l l s w ith o u t n e c r o s is was
a p p a re n t in th e g e r b i l , w h ile th e g u in ea p ig showed s c a tte r e d a re a s o f
b la n c h in g o f th e parenchym al c e l l s s u g g e s tiv e o f e a r ly d e g e n e ra tiv e
change b u t w ith o u t n e c r o s i s .
A cute n e c r o s is s im ila r t o t h a t found in
sheep was a p p a re n t in th e r a b b i t w ith one obvious d i f f e r e n c e .
In sheep
th e n e c r o s is was c e n tr o - lo b u la r w h ile in th e r a b b i t i t was p e r i p o r t a l in
d is trib u tio n .
th e r a b b i t .
There was only a b e g in n in g o f b i l e d u c t p r o l i f e r a t i o n in
P a n lo b u la r d e g e n e ra tio n and n e c r o s is o f h e p a tic parenchym al
29
c e l l s was a p p a re n t in th e m ouse.
N e cro sis a f f e c te d numerous c e l l s b u t
was preceeded by d e g e n e ra tiv e changes and was n o t p r e s e n t in c le a r ly
d e fin e d f o c i a s was n o ted in th e l i v e r s o f s h e e p T h e r e appeared t o
be no d if f e r e n c e s in th e m ic ro sco p ic f in d in g s betw een any two anim als
o f th e same s p e c ie s fe d th e hexane o r a c e to n e e x t r a c t in d ic a tin g th e
to x ic m a te r ia l was p r e s e n t in b o th e x t r a c t s „
F lem ing^^ in d ic a te d t h a t th e one r a b b i t t h a t d ied a s a r e s u l t o f
th e t o x i c i t y o f th e a c e to n e e x t r a c t had s u ffe re d Jiear-f; f a i l u r e .
He th e n
su g g este d t h a t th e r e were two d i f f e r e n t to x in s in th e p l a n t , a c a r d ia c a c ti v e and a h e p a to - a c tiv e s u b s ta n c e .
S ince th e amount o f p la n t
e x tr a c te d f o r fe e d in g and th e r e s u l t s o f th e n ecro p sy f in d in g s were th e
same f o r b o th e x t r a c t s in a l l a n im als o f th e same s p e c ie s te s t e d in t h i s
la b o r a to r y , th e p o s tu la tio n o f d i f f e r e n t a c tiv e p r in c ip le s in th e two
e x t r a c t s seems t o la c k fo u n d a tio n .
To t h i s can be added t h a t th e
r e s u l t s o f blood ammonia and BSP t e s t s perform ed on sheep H-636 and
H-665 (T able 2 ) in d ic a te d th e two e x t r a c t s were a ls o e f f e c t in g th e sheep
in th e same way.
A p o s s ib le re a so n f o r th e s e r e s u l t s d i f f e r i n g from th o s e o f
Flem ing
51
a llu d e d to a b o v e , i s found in h is use o f only one r a b b i t f o r
t e s t i n g p u rp o s e s .
He su g g ested h e a r t f a i l u r e as th e cau se o f d e ath b u t
w ith o u t s u f f i c i e n t e x p e rim e n ta l ev id en ce t o w a rra n t t h i s c o n c lu s io n .
From th e p ap er
52
i t a p p ea rs t h a t th e cause o f d e ath was n o t obvious and
was t h e r e f o r e a t t r i b u t e d t o h e a r t f a i l u r e .
The o th e r p o s s ib le cau ses o f
30
d e a th f o r t h i s one r a b b i t could have been s t r e s s from th e fe e d in g s , a
tr a c e co n tam in ate in th e a c e to n e , o r even some ty p e o f in f e c tio n t h a t
was overlooked in th e n e c ro p sy .
Had Flem ing used more e x p e rim e n ta l
an im als h is r e s u l t s would p ro b a b ly ' have been q u ite d i f f e r e n t and in
l i n e w ith .th e f in d in g s re c o rd ed in t h i s c u r r e n t s tu d y .
A f te r th e pure to x i n , te tra d y m o l' ( I I , page 4 9 ), had been is o la te d
53
i t was im p o rta n t t o .e s t a b l i s h a n ap p ro x im ate LD^q
f o r mice .
As th e
to x in i s a s o l i d , i t was n e c e s s a ry t o u se a c a r r i e r f o r th e o r a l
f e e d in g s .
P ropylene g ly c o l, 50 p e r c e n t e th a n o l, in n -h e x a n e , and n -
hexane were a l l t r i e d a s c a r r i e r .
As i s i l l u s t r a t e d "in T able 4 th e
t o x i c i t y o f th e e x t r a c t was g r e a tly low ered o r removed when i t was fe d
in p ro p y len e g ly c o l (group 8) w h ile th e p u r if ie d to x in d is s o lv e d in 50
p e r c e n t e th a n o l in n-hexane (groups 1 -5 ) d em o n strated th e same t o x i c i t y
a s th e c a r r i e r a lo n e (groups 6 and 7 ) .
When th e crude e x t r a c t (groups
9 and 10) o r p u r if ie d compound (groups 13-20) was fed in n-hexane a lo n e
a much more c o n s is te n t p ic tu r e was r e v e a le d .
F u r th e r i t was shown t h a t
n-hexane a lo n e (groups 11 and 12) was n o n -to x ic when fe d o r a lly in doses
a b o u t t h r e e .tim e s t h a t re q u ire d f o r i t a s a c a r r i e r .
T able 4 i l l u s t r a t e s t h a t th e LD^q (m ice) o f te tra d y m o l l i e s some­
where betw een 170 and 330 mg/kg (groups 15 and 1 6 ).
A u to p sie s were perform ed on a number o f mice t h a t were fe d p u r if ie d
to x in .
Two o f th e m ice t h a t did n o t d ie from group 16 (T able 4) were
a u to p s ie d r e v e a lin g s e v e re ly n e c r o tic l i v e r s „
One l i v e r was a lm o st
31
T able 4
Mouse Feeding Experiments
m l/kg t o t a l
volume
I . Pure ( I I ) ''
50% e th a n o l in
n-hexane
2.
«
3.
" .
4.
"
5.
"
460
360
280
Number o f
an im als
% dying in
one week
CO
OO
mgA g o f
to x in
9
25
3 .6
190
160
3 .5
3 .1
4 .0
8
8
8
8
50
63
75
50
0 .0
3 .3
8
50
0 .0
5.0
9
45
8 . Crude e x t r a c t
in pro p y len e
g ly c o l
2000-3000
3 .0 - 4 .5
. 5
00
9 . Crude e x t r a c t
in n-hexane
1 0.
"
2100-3300
4 .4 - 5 .8
12
100
1100-1200
1 . 9 -2 .0
4
25
1 1. N-hexane
12.
"
0.0'
4 .0 - 4 .9
0 .0
7 .0 -8 .6
8
7
00
00
1 3. Sublimed ( I I )
in n-hexane
14.
"
750-580
4 .8 - 6 .2
7
100
330
3.3
9
100
1 5. Pure ( I I ) in
n-hexane
16.
"
17.
"
18.
"
19.
”
20.
"
330
CO
CO
M a te ria ls
a n d /o r c a r r i e r
9
100
6 . 50% e th a n o l in
n-hexane
7.
"
170
3.3
282
3 .9
200
140
100
3 .9
3 .9
3 .9
'
11
5
5
5
5
.
Pure ( I l ) r e f e r s to su b lim e d ? b ase washed te tra d y m o l
45
40
00
00
00
32
t o t a l l y w h ite .
A lso th r e e mice t h a t did n o t d ie from each o f th e groups
17 th ro u g h 20 (T able 4) were a u to p s ie d .
Those in group 17 showed- v e ry
marked le s io n s w h ile th e le s io n s were m a c ro sc o p ic a lly m odest in group
1 8.
Groups 19 and 20 had no m a c ro sc o p ic a lly v i s i b l e l e s i o n s .
A ll o f th e observed le s io n s in th e mice shown in T able 4 were q u ite
s im ila r to th o s e seen in th e mice fe d th e crude hexane e x t r a c t (T able 3 ,
p a g e .2 8 ) , b u t no h is to p a th o lo g y s tu d ie s have been made on t h i s t i s s u e .
These g ro ss f in d in g s d o, how ever, s u p p o rt th e c o n c lu s io n t h a t te tra d y m o l
i s th e a c ti v e p r in c ip le o f T . g la b r a t a .
F u rth e r ev id en ce f o r t h i s •
c o n c lu sio n i s p re s e n te d in th e fo llo w in g s e c tio n .
In g e n e r a l i t could be concluded from a l l th e s e p h y s io lo g ic a l
s tu d ie s t h a t th e a c ti v e p r in c ip le o f T . g la b r a t a was a h e p a to to x in o f
m oderate t o x i c i t y .
The e f f e c t s on th e l i v e r in a l l in s ta n c e s were
dem onstrated as c e l l u l a r damage b u t th e method by which t h i s damage was
m ediated was n o t d e term in e d .
One item o f u n u su a l i n t e r e s t was th e d if f e r e n c e in lo c a tio n o f th e
le s i o n in r a b b i t s and sh e e p , p e r ip h e r a l lo b u la r and c e n tro -rlo b u la r
re s p e c tiv e ly .
D r. A nderson o f Montana S ta te U n iv e rs ity s t a t e d ^ t h a t he
knew o f no o th e r in s ta n c e o f t h i s ty p e o f in te r s p e c ie s d if f e r e n c e in th e
m a n if e s ta tio n o f th e h e p a to to x ic ity o f a p a r t i c u l a r s u b s ta n c e .
This
fin d in g s u g g e s ts , b u t does n o t p ro v e , t h a t some c e l l s a r e a tta c k e d more
r e a d i l y th a n o th e rs in th e l i v e r by te tra d y m o l.
S ince blood flow i s
s im ila r in th e l i v e r s o f b o th sheep and r a b b i t s , i f th e p o in t o f a tta c k
33
o f th e h e p a to to x in was in t h i s flo w , s i m i l a r le s io n s would be e x p e c te d .
The a u th o r s u g g e s ts , w ith o u t p r o o f , t h a t te tra d y m o l i s r e a l l y a tta c k in g
a p a r t i c u l a r enzyme system t h a t i s lo c a liz e d in s p e c i f i c c e l l s o f th e
liv e r.
T hat th e s e c e l l s a r e lo c a liz e d in d i f f e r e n t a re a s o f th e l i v e r s
o f d i f f e r e n t anim als seems t o be a l o g i c a l p o s t u l a t e .
lo c a tio n s o f th e le s io n s th e n l o g i c a l l y f o llo w s .
)
The d i f f e r e n t
34
II.
Isolation of the Toxin
T his s e c tio n o f th e d is c u s s io n is p re se n te d in th r e e p a r t s .
The
f i r s t p a r t (A) w i l l d e a l w ith th e a ssig n m en t o f g r e a t e s t t o x i c i t y t o a
p a r t i c u l a r m olecule by a p ro c e ss o f e lim in a tio n u t i l i z i n g mice a s a t e s t
a n im a l.
The second p a r t (B) w i l l d e a l w ith th e methods used in i s o l a ­
tin g s u f f i c i e n t p u r if ie d compound f o r s t r u c t u r a l e lu c id a tio n and p a r t i a l
d e te rm in a tio n o f th e LD^q o f th e to x in f o r m ice.
The t h i r d p a r t (C)
w i l l d e a l w ith some o f th e c h a r a c t e r i s t i c s o f te tra d y m o l, th e to x ic
p r in c ip le o f T= g la b r a t a , e x c lu d in g th e ch em ical s t r u c t u r e .
A.
When e i t h e r th e crude n-hexane o r a ce to n e e x t r a c t was s p o tte d on
A n a s il S t i c p la te s and developed w ith 40 p e r c e n t n-hexane in d ie th y l
e th e r , i t s e p a ra te d in to a number o f s p o ts w hich were made v i s i b l e by
s p ra y in g th e p la f e w ith s u lfu ric -d ic h ro m a te s o l u t i o n .
These sp o ts were
g iv en th e numbers in d ic a te d in Scheme I .
The t o t a l n-hexane e x t r a c t was f i r s t d iv id e d by s i l i c a g e l chroma­
to g ra p h y in to 9 n o n -p o la r f r a c t i o n (A ), m a te r ia l e lu te d o f f th e column
w ith 50 p e r c e n t n-hexane. in d i e th y l e t h e r , and a p o la r f r a c t i o n (B ),
m a te r ia l e lu te d o f f th e column w ith 10 p e r c e n t m ethanol in d ie th y l
e th e r.
I.
The m a te r ia ls e lu te d and q u a n t i t i e s in v o lv ed a re shown in Scheme
The n o n -p o la r f r a c t i o n dem o n strated t o x i c i t y when a d m in is te re d t o a
m o u se.in th e amount in d ic a te d which i s r e l a t i v e l y e q u iv a le n t to th e
to x ic dosage o f th e t o t a l e x t r a c t .
35
Scheme I
Isolation of the Toxin Followed by Toxicity
S o lv e n t f r o n t '
Fed .23% body w t. , Death
24% crude e x t r a c t
Fed .20% body w t.
A liv e
OO o
0
9Q_o '1% body w t.
Death 7 h r s .
24% cru d e e x t r a c t
Fed .20% body w t.
Death
90_4
14% crude e x t r a c t
Fed .10% body w t.
A liv e
- 1 2 % b o d y wt
Death 7 h r s .
26% crude e x t r a c t
Fed .23% body w t.
A liv e
F ed, A liv e
0
S p o ttin g l in e
Mice were used a s t e s t an im als f o r th e above fe e d in g e x p e rim e n ts .
^ A n a sil S t i c , developed in 40:60 n -h e x a n e : d ie th y l e t h e r .
36
Next th e n o n r-p o lar. f r a c t i o n o f th e e x t r a c t was d iv id e d in to f o u r
f r a c t i o n s (C-F) shown in Scheme I by s i l i c a g e l chrom atography.
These
f r a c t i o n s were th e # a d m in is te re d t o mice in a c o n c e n tra tio n a b o u t f o u r
tim es a s g r e a t a s th e y o ccu rred in th e to x ic dosage o f th e o r ig i n a l
. crude e x t r a c t .
Only th e mouse re c e iv in g f r a c t i o n ( P ) 5 th e m a te ria ls
la b e le d 29-3A .th ro u g h 2 9 -4 , dem o n strated to x ic e f f e c t s .
Since a t th e s e
h ig h dosages only f r a c t i o n (D) d is p la y e d t o x i c i t y th e o th e r f r a c tio n s
were p la ce d a s id e and f r a c t i o n (D) was f u r t h e r s e p a ra te d by s i l i c a g e l
chrom atography in to two p o rtio n s c o rre sp o n d in g t o 29 t-3 and 29-4 a s shown
in th e schem e.
These f r a c t i o n s were th e n a d m in is te re d to mice in th e
h ig h dosage r a t e and b o th dem on strated t o x i c i t y .
The m a te r ia l la b e le d 29-3 was now f u r t h e r p u r if ie d by r e c r y s t a l l i z a ­
t i o n and fe d a s shown in Scheme 2 .
There were n o t enough fe e d in g s h e re
to e s t a b l i s h w ith any c e r t a i n t y th e e x a c t to x ic dose b u t i t must l i e
betw een 790 mg/kg and 270 m g/kg.
A t t h i s p o in t i f was found t h a t 29-3
was r e a l l y two compounds by changing from A n a s il t o alum ina t i c .
Thus,
29-3 was f r a c tio n a te d by alum ina chrom atography in to 29-3A and 29-3B.
These m a te r ia ls were th e n fe d in th e c o n c e n tra tio n s shown in Scheme 2
w ith th e r e s u l t t h a t th e mouse r e c e iv in g 29-3A d ied w h ile th e mouse
r e c e iv in g 29-3B rem ained a l i v e .
As th e c o n c e n tra tio n in th e p la n t o f
29-3B i s l e s s th a n one h a l f t h a t o f 29-3A , t h i s amount o f 29-3B is a
much l a r g e r dosage based on y ie ld from th e p la n t th a n th e amount o f
29-3A fe d t o th e m ouse.
37
Scheme 2
*
Isolation of the Toxin Followed by Toxicity
(II)
.079% body w t.
/Death
.079% body w t.
'D eath
29-3_______
R e c ry s ta lliz e ^
29-3A
.15% body w t . , Death
.044% body w t.
"A live
Alumina chrom atography
.027% body s t .
A liv e
29-3B
.10% body w t . , A liv e
007% body w t.
A liv e
,Mother liq u o r s
A liv e (2 m ice)
C ry s ta ls
S ublim ate
'.034% body wt
D eath
Sublime
R e c r y s ta lliz e
Base wash
Residue
R esidue
\ .013% body w t.
A liv e
*
Mice were used a s t e s t an im als f o r th e above fe e d in g e x p e rim e n ts.
38
A t t h i s p o in t 29-3A was s e t a s id e and f u r t h e r work was done on
29-4 which was g iv e n th e t r i v i a l name te tra d y m o l.
The te tra d y m o l c o n ta in in g y e llo w is h s o lid from th e chrom atographic
s e p a r a tio n o f 29-3 from 29-4 was sublim ed and fe d t o mice r e s u l t i n g in ■
t h e i r d e a th s a s shown in Scheme 2 .
n o t d em o n strate t o x i c i t y .
The re s id u e from th e s u b lim a tio n d id
The w h ite c r y s t a l l i n e su b lim a te was f u r t h e r
p u r if ie d by b ase e x tr a c ti o n and r e c r y s t a l l i z a t i o n to y ie ld a pure com­
pound whose LDgQ f o r mice l i e s betw een 170 and 330 mg/kg (se e Table 4 ,
page 3 1 ).
As was m entioned on page 2 7 , th e hexane e x t r a c t from p la n t e q u iv a ­
l e n t t o 5 p e r p e n t o f th e body w e ig h t o f a mouse i s l e t h a l to t h a t m ouse.
I t i s q u ite p o s s ib le t h a t t h i s dosage i s g r e a t e r th a n th e minimum
re q u ire d f o r d e a th , a s th e minimum to x ic dosage o f th e cru d e e x t r a c t was
n o t p u rsu e d .
However, when th e q u a n t i t i e s o f th e v a rio u s f r a c tio n s o f
th e crude hexane e x t r a c t a re c o n s id e r e d ; a s shown in Scheme I , page 35;
t h i s dosage could n o t be tq o f a r o u t o f l i n e .
I t was a ls o shown; as
re c o rd ed in Table .4, page 31; t h a t p u r if ie d te tra d y m o l ( I I ) was l e t h a l
t o a l l mice t e s t e d in a c o n c e n tra tio n o f ap p ro x im a te ly 300 m g/kg.
By
th e p u r i f i c a t i o n pro ced u re o u tlin e d in Scheme 3 , page 42 i t was observed
t h a t sublim ed te tra d y m o l could be is o la te d ( l a t e r shown t o be a t l e a s t
90 p e r c e n t p u re ) in a q u a n tity e q u a l t o a b o u t 0.3 p e r c e n t o f th e w et
buds w e ig h t.
A c o n s id e r a tio n o f th e s e numbers showed t h a t i f th e
i s o l a t i o n p ro ced u re were q u a n t i t a t i v e , which i t o b v io u sly was n o t , th e
I
39
to x in from p la n t e q u iv a le n t to 10 p e r c e n t
le th a l.
of
th e mouse’s w eig h t was
When th e lo s s e s in i s o l a t i o n and e x tr a c ti o n were c o n sid e re d
alo n g w ith th e t o x i c i t y o f 29-3A, a n o th e r to x in is o la te d from T.
g la b r a t a (se e page 3 6 ), th e le v e l s o f t o x i c i t y p o in te d t o th e c o n c lu sio n
t h a t te tra d y m o l was m ainly re s p o n s ib le f o r th e t o x i c i t y o f th e hexane
e x t r a c t in m ic e .
Though t h i s stu d y d id n o t d i r e c t l y show t h a t te tra d y m o l was th e
m olecule re s p o n s ib le f o r th e e x te n s iv e ran g e lo s s e s o f sheep in th e
G re at B a s in , i t did s u g g e s t t h a t th e r e was a good p o s s i b i l i t y o f t h a t
c o n c lu s io n o S up p o rt f o r th e c o n c lu sio n was found in s im ila r necro p sy
f in d in g s and s im ila r le v e l s o f t o x i c i t y in mice and sh e e p .
B.
A f te r i t was determ ined t h a t te tra d y m o l, o r 2 9 -4 , was th e to x ic
p r in c ip le o f T . g la b r a t a i t was e s s e n t i a l t o i s o l a t e l a r g e r q u a n titie s
fo r s tru c tu ra l s tu d ie s ,
In o rd e r t o d eterm in e how b e s t to e x t r a c t th e
to x in from th e p l a n t , two sam ples (1165 g ) o f buds and new growth were
random ly ta k e n .
One o f th e s e sam ples was th e n ground w ith a meat
g r in d e r in n-hexane w h ile th e p la n t was s t i l l f r o z e n .
I t was th e n
e x tr a c te d th r e e tim e s , f o r 20-24 h o u rs each tim e , w ith a t o t a l o f 6
l i t e r s o f n-hexane each tim e .
The o th e r sample was l e f t unground and
e x tr a c te d in th e same m anner.
A f te r t h i s was com pleted th e unground,
e x tr a c te d m a te r ia l was ground and e x tr a c te d two tim es w ith n -h e x a n e .
The r e s u l t s o f th e s e e x tr a c tio n s a re summarized in T able 5.
40
T able 5
E f f e c t o f G rindin g on P la n t E x tra c tio n
Ground
Unground
W eight o f p la n t m a te r ia l
1165 g
1165 g
*
ND
W eight o f crude e x t r a c t
2 0 .7 g
9 .3 g
1 4 .1 g
P e r c e n t o f p la n t w eig h t
1.78%
0.8%
1.2%
Crude e x t r a c t c o n c e n tra tio n
o f te tra d y m o l ( I l ) 0
**
Amount o f te tra d y m o l ( I I )
0 .6
1 .0
0 .2
1 .4
1 .0
0.3
Ungroundground
Amount of 29-300
*
Not determ ined
“ ^ R e la tiv e t o a c o n c e n tra tio n o f 1 .0 in th e unground crude e x t r a c t
R e la tiv e to an amount o f 1 .0 in th e unground crude e x t r a c t
00R e la tiv e t o an amount o f 1 .0 in th e ground crude e x t r a c t
A ll c o n c e n tra tio n s were determ ined by s p o t m atching on alum ina t i c
v is u a liz e d by c h a r rin g w ith s u lfu ric -d ic h ro m a te s o l u t i o n .
S ince m ost o f th e te tra d y m o l was e x tr a c te d from th e p la n t w ith o u t
g r in d in g , and th e unground e x t r a c t had l e s s co n tam in atin g s u b s ta n c e s ,
v
\
■t
C
C
r
n
Ol
b u lk e x tr a c tio n s o f th e p la n t m a te r ia l were perform ed on.unground
m a te ria l.
In o rd e r to determ in e th e ap p ro x im ate y ie ld o f to x in from th e p la n t
a 100 g sample o f buds and flo w e rs and a 100 g sample o f new growth were
e x tr a c te d s e p a r a te ly w ith o u t g rin d in g and p u r if ie d a s o u tlin e d in Scheme
3 , page 4 2 .
The y ie ld a f t e r alum ina chrom atography o f th e two e x tr a c ts
41
re v e a le d t h a t th e r e was l e s s to x in in th e new grow th th a n th e r e was in
th e flo w e rs and b u d s .
Keeping in mind th e m oderate i n s t a b i l i t y o f th e t o x i n , which i s
d is c u s s e d on pages 4 5 -4 7 , te tra d y m o l was p u r if ie d f o r chem ical and
p h y s io lo g ic a l s tu d ie s u sin g a number o f methods as, shown in Scheme 3 .
Chromatography removed m ost o f th e co n tam in atin g m a te r ia ls le a v in g a
y e llo w is h s o lid t h a t gave-one s l i g h t l y e lo n g a te d s p o t on t i c .
These
y e llo w is h im p u r itie s could be removed e i t h e r by m u ltip le r e c r y s t a l l i z a ­
tio n s o r by s u b lim a tio n .
When th e r e c r y s t a l l i z e d m a te r ia l was su b lim ed ,
a re s id u e rem ained behind t h a t d is p la y e d an i r c a rb o n y l band t h a t had
been p r e s e n t, b u t v e ry s m a ll, in th e m a te r ia l b e fo re s u b lim a tio n .
Thus,
s in c e s u b lim a tio n removed b o th th e y ello w and th e c a rb o n y l c o n ta in in g
im p u rity , th e column chrom atographed m a te r ia l was sublim ed d i r e c t l y to
y ie ld w h ite c r y s t a l s .
These c r y s t a l s were s t i l l n o t a pure compound by
e le m e n ta l a n a l y s i s , how ever, so th e y were d is s o lv e d in c o n c e n tra te d
e th a n o lic potassiu m hydro x id e and allow ed to sta n d o v e rn ig h t in a c lo s e d ,
l i g h t p r o te c te d , v i a l a t a b o u t 25°C.
When t h i s m ix tu re was worked up and
th e compound r e c r y s t a l l i z e d i t gave a s a t i s f a c t o r y a n a l y s i s .
W hether th e
im p u rity r e a c te d o r was e x tr a c te d w ith b ase was n o t c e r t a i n .
There w as,
how ever, q u ite a n o tic e a b le y e llo w in g o f th e b a s ic e th a n o lic s o lu tio n
when i t was observed a f t e r s ta n d in g o v e rn ig h t.
This c o lo r a tio n did
in d ic a te some deco m p o sitio n b u t n o t n e c e s s a r ily o f th e im p u rity .
m ight be su g g este d t h a t te tra d y m o l was m odified d u rin g t h i s r a t h e r
It
42
Scheme 3
Isolation Procedure for Tetradymol
P la n t to p s
S o rt
Buds and flo w e rs
Hexane
e x tr a c ti o n
SiOg
chrom atography
0.75%
0.30%
AlgO3
chrom atography
0.50%
0.21%
S u b lim atio n
0.31%
P e rc e n tag e s based on is o la te d m a te r ia l a t each s te p r e l a t i v e
t o th e whole g re e n p la n t m a te r ia l.
v ig o ro u s p u r i f i c a t i o n s t e p .
However, when th e p u r if ie d m a te r ia l was
checked by i r , nm r, u v , and ms th e r e seemed t o be no s i g n i f i c a n t d i f ­
fe re n c e s r e s u l t i n g from th e b ase e x t r a c t i o n .
A lso th e p o s itio n and
shape o f th e s p o t on alum ina t i c and th e c o lo r v i s u a l i z a t i o n r e a c tio n s
43
o f th e compound were i d e n t i c a l b e fo re and a f t e r th e e x t r a c t i o n .
F u r th e r
th e o p t i c a l r o t a t i o n o f th e compound in c re a s e d from +44° to +56° a s a
r e s u l t o f th e e x t r a c t i o n .
These p h y s ic a l fin d in g s coupled w ith th e
fin d in g s o f p h y s io lo g ic a l a c t i v i t i e s o f a b o u t th e same m agnitude w ith
m a te r ia l b e fo re and a f t e r e x tr a c ti o n le d t o th e c o n c lu s io n t h a t
te tra d y m o i was unchanged by t h i s b ase w ash.
Co
A. number o f c h a r a c t e r i s t i c s o f te tra d y m o i, th e to x in from t e t r a -
dymia g l a b r a t a , a re g iv e n below .
T etradym oi c r y s t a l l i z e d a s w h ite n e e d le s from n -h e x a n e 'and had a
m e ltin g p o in t o f 9 2 -9 2 .5°C.
When te tra d y m o i (2 9 -4 ) was mixed w ith E h r lic h ’s r e a g e n t^^ th e r e
developed an im m ediate la v e n d e r c o lo r t h a t changed t o b lu e in one h a l f
hour and f i n a l l y g re e n i n two h o u r s .
I f in s te a d te tra d y m o i was added to
91 p e r c e n t fq rm ic a c id and th e p -d im eth y l-am in o benzaldehyde added an
im m ediate in te n s e ,dark b lu e c o lo r r e s u lte d t h a t was s t a b l e f o r a t l e a s t
th e h o u r u n t i l th e s o lu tio n was d is c a rd e d .
Tetradym oi showed an Rf = .34 in 50 p e r c e n t hexane in d ie th y l
e th e r on alum ina t i c .
I t gave a b r i g h t ro s e -la v e n d e r c o lo r when th e t i c
s p o t was sprayed w ith e i t h e r v a n i l l i n - s u l f u r i c o r s u lfu ric -d ic h ro m a te
re a g e n ts .
The compound 29-3A showed an Rf = .60 in th e same system b u t
d is p la y e d a b r i g h t b lu e - b la c k c o lo r w ith th e s u lfu ric -d ic h ro m a te sp ra y
re a g e n t and a b r i g h t v i o l e t c o lo r w ith th e v a n i l l i n - s u l f u r i c sp ray
re a g e n t.
44
T etradym ol d is p la y e d one w e ll d e fin e d peak by g lc u sin g a 6 f o o t ,
I p e r c e n t OV-I liq u id phase on GC-Q s u p p o rt in a l / 8 - i n c h copper
colum n; a 6 f o o t , 2 p e r c e n t SE-30 l i q u i d phase on GC-Q su p p o rt in a
1 /8 - in c h g la s s colum n; and a 6 f o o t , I p e r c e n t OV-17 l i q u id phase on
GG-Q in a l / 8 - i n c h aluminum column.
When th e SE-30 column o r th e OV-17
column were u t i l i z e d , 29-3A would n o t d em o n strate any peak in th e gas
chrom atograph.
I t w ould, how ever, come th ro u g h th e OV-I column.
M icro­
gram q u a n t i t i e s o f th e s e compounds w e re ■c a p tu re d from th e gas chrom ato­
g ra p h ic d e te c to r p o r t in c a p i l l a r y tu b e s w h ile th e flam e was e x tin ­
guish ed .
When th e s e sam ples were s p o tte d on t i c , th e y re v e a le d th e same
Rf and c o lo r r e a c tio n s a s a u th e n tic te tra d y m o l and 29-3A.
A c o n s id e r a tio n o f th e r e s u l t s re c o rd ed in Table 5, page 40,
su g g este d t h a t te tra d y m o l was m ain ly a s u rfa c e l i p i d a s i t was e a s ily
e x tr a c te d in hexane from th e whole g re e n p l a n t .
The o th e r p o s s ib le
to x ic c o n s t i t u e n t , 29-SA, was only s l i g h t l y e x tr a c te d by hexane u n d er
th e s e c o n d itio n s b u t was e a s i l y e x tr a c te d a f t e r th e p la n t i s ground u p .
S ince b o th o f th e s e compounds were s o lu b le in hexane and seemed t o
e x t r a c t in a manner e q u a l t o each o th e r from th e ground p la n t th e ■
d if f e r e n c e in e x t r a ct a b i l i t y from th e unground p la n t was p o s s ib ly due to
th e lo c a t io n o f th e m olecules in th e p l a n t .
There was a ls o th e p o s s i b i l ­
i t y t h a t te tra d y m o l was c o n c e n tra te d in th e i n t e r c e l l u l a r sp aces a s one
o f th e sap components r a t h e r th a n a p p e a rin g on th e p l a n t Ts s u r f a c e .
above e x tr a c ti o n ex p erim en t would n o t d i f f e r e n t i a t e betw een th e two
The
45
p o s s ib ilitie s .
The i n s t a b i l i t y o f te tra d y m o l was n o ted on numerous o ccasio n s
d u rin g i s o l a t i o n and c h a r a c te r iz a ti o n s t u d i e s .
Flem ing^^ showed t h a t
th e p l a n t m a te r ia l l o s t t o x i c i t y on d ry in g and s t o r a g e .
Though th e
m olecule does sublim e i t does so t o an a p p re c ia b le e x te n t only under
vacuum, o r w ith h e a t n e a r i t s m e ltin g p o i n t .
I f th e m olecule was n o t
l o s t i n t h i s manner i t p ro b a b ly was decomposed o r a l t e r e d on s to r a g e ,
T his problem was avoided in t h i s la b o r a to r y by s to ra g e o f a l l . p l a n t i
m a te r ia l and e x t r a c t s a t -20°C in s e a le d c o n ta i n e r s .
A nother d e m o n stra tio n o f i n s t a b i l i t y was in th e b e h a v io r o f th e
compound on alum ina chrom atography.
E lu tio n chrom atography, on h ig h ly
a c ti v a te d alum ina could n o t be e f f e c te d w ith te tra d y m o l a s th e compound
decomposed o r became bound so t i g h t l y to th e su p p o rt t h a t i t could n o t
be e lu te d w ith 5 p e r c e n t m ethanol in d ie th y l e t h e r .
The t o t a l y ie ld
from th e column amounted t o only 55 p e r c e n t o f th e m a te r ia l p laced on
th e colum n.
F u r th e r th e upper s e c tio n o f th e column to o k on a y ello w
c o lo r s u g g e s tiv e o f deco m p o sitio n on th e column b u t no a tte m p t was made
t o f in d th e d ecom position p ro d u c ts .
s id e r a b le number o f r e a c tio n s
57
Alumina i s known t o m ediate a co n -
a s c o n d e n s a tio n s , d e h y d ra tio n s , o x id a­
t i o n s , and re a rra n g em e n ts so t h i s proposed d eco m p o sitio n was n o t to o
s u rp ris in g .
To av o id th e above p roblem , th e column s u p p o rt m a te r ia l
was f i r s t d e a c tiv a te d w ith m eth an o l.
L e d e re r and L e d e r e r ^ re fe re n c e methods o f d e a c tiv a tin g alum ina by
46
th e a d d itio n o f w a te r , b u t t h i s r e q u ir e s a f a i r l y lo n g m ixing tim e in
th e a tte m p t t o a v o id th e problem s o f inhom ogeneity due t o th e sm a ll
volume o f w a te r b e in g added t o a v e ry h y d ro sc o p ic a d s o r b e n t.
problem was avoided by th e use o f m ethanol mixed w ith b e n ze n e .
This
When
t h i s s o lv e n t m ix tu re was s t i r r e d w ith alum ina f o r a s h o r t p e rio d and
th e n allo w ed t o co o l, ( to remove th e h e a t o f a d s o r p tio n ) i t y ie ld e d a
homogeneous a d s o rb e n t o f a s u ita b le a c t i v i t y f o r th e s e compounds a s.w a s
dem onstrated by f a i r l y sh a rp bands and l i t t l e t a i l i n g T h i s s lu r r y
could be poured d i r e c t l y in to th e colum n, e lu te d w ith a sm all amount o f
h e x an e , and used w ith o u t f u r t h e r p r e p a r a t io n .
When t h i s column was used
th e r e was s t i l l a sm a ll amount o f m a te r ia l t h a t could n o t be e lu te d from
i t (a p p ro x im a te ly 15 p e r c e n t) b u t t h i s was n o t deemed t o be s i g n i f i c a n t .
F l o r i s i l was t r i e d as an a d s p fb e n t b u t was n o t e f f e c t i v e in s e p a r­
a tin g te tra d y m o l from 2 9-3A.
I t a l s o le d t o some decom position a s was
dem o n strated by a p in k c o lo r t h a t developed on th e colum n.
S i l i c a g e l chrom atography was q u ite u s e f u l f o r rem oving th e c o lo re d
p o la r m a te r ia ls and th e v e ry n o n -p o la r compounds from th e crude g re e n brown hexane e x t r a c t b u t d id n o t g iv e as good a s e p a r a tio n o f 29-3A and
B from te tra d y m o l a s d id a lu m in a .
There seemed t o be no lo s s o f compound
on th e s i l i c a g e l column a s th e y ie ld s were g r e a te r th a n 98 p er c e n t from
th e chrom atography.
B esid es i n s t a b i l i t y t o chrom atography, te tra d y m o l e x h ib ite d l i g h t
s e n s itiv ity .
When i t was d is s o lv e d in hexane and l e f t in a capped,
47
c leiar g la s s v i a l on th e desk to p n e a r a n o rth fa c in g window a w h ite
s o lid c o lle c te d on th e g la s s n e a r e s t th e l i g h t .
T his s o lid was in s o l ­
u b le in d i e t h y l e th e r and was much more p o la r th a n te tra d y m o l by t i c .
In one in s ta n c e te tra d y m o l a ls o decomposed w h ile s to r e d in s o lu tio n in
a s e a le d v i a l under r e f r i g e r a t i o n .
T his deco m p o sitio n h as n o t been
o b se rv e d , how ever, when th e compound was s to re d in a c r y s t a l l i n e form a t
- 2 O0C under a n itr o g e n a tm o sp h ere .
The p ro d u c t(s ) o f th e proposed
d ecom position have p o t been in v e s t ig a te d .
48
III.
S p e c tr a l and Chem ical E lu c id a tio n o f S tr u c tu r e
The s t r u c t u r a l e lu c i d a tio n o f te tra d y m o l ( I I ) , 8 a (S )-h y d ro x y -3 ,
4 a (R) , 5 (S ) - t r imet h y l - 4 , 4 a , 5 , 6 , 7 , 8 , 8 a ,9 -o c ta h y d rp -n a p h th o [ 2 , 3 - b ] f u r a n ,
has proved t o be an i n t e r e s t i n g problem .
The compound p re s e n te d a num­
b e r o f u n u su al f e a tu r e s in c lu d in g a f u r a n r i n g ; th e co m p arativ ely r a r e ,
n o n -is o p re n o id erem ophilane s k e l e t o n ; and an a n g u la r h y d ro x y l f u n c tio n ,
which has been found in only one o th e r e re m o p h ila n e .
59
T his e lu c id a tio n
was co m p licated by th e m oderate i n s t a b i l i t y o f th e compound which was
n o ted in p re v io u s s e c tio n s o f t h i s t h e s i s .
Beyond th e s e d em o n stratio n s
o f i n s t a b i l i t y th e compound was v e ry l a b i l e t o c o n c e n tra te d form ic o r
h y d ro c h lo ric a c id g iv in g a s p ro d u c ts b la c k t a r s which were a ls o th e
e v e n tu a l p ro d u c ts when te tra d y m o l was d is s o lv e d in d e u tero ch lo ro fo rm o r
carbon t e t r a c h l o r i d e f o r nmr s t u d i e s .
The i n s t a b i l i t y o f te tra d y m o l t o
a c id s could be r a t i o n a l i z e d a s due t o th e r e a c t i v i t y o f th e fu ra n
n u c l e u s b u t th e d ecom position i n th e c h lo r in a te d s o lv e n ts i s a s y e t
u n e x p la in e d .
By com bining th e e le m e n ta l a n a ly s is (B a led , f o r
76.88%; H, 9.04%.
Found:
C,
C, 76.84%; H, 9.25% .) and th e ms p a re n t peak
(M+234 m/e) th e m o le c u la r form ula o f ^25^22^2 was a s c e r t a i n e d .
A c u rs o ry
in s p e c tio n o f t h i s form ula re v e a le d t h a t any s t r u c t u r e proposed f o r
te tra d y m o l ( I I ) m ust in c o rp o ra te f iv e u n s a tu r a tio n s , two oxygens, and
f i f t e e n c a rb o n s .
Three o f th e s e u n s a tu r a ti o n s , one o f„th e oxygens, and
f iv e carbons a re in c lu d e d in th e m ethyl fu ra n r in g a s w i l l p r e s e n tly be
49
shown.
The o th e r oxygen m ust be p a r t o f a h y d ro x y l m oiety as in th e i r
spectrum o f t h i s compound shown in F ig u re 7 d em o n strated th e ty p i c a l
s tro n g O-H s tr e tc h i n g freq u e n cy a t 3400 Cm- 1 . 61
From th e nmr sp ectru m ,
OH
(II)
R=H
(IIA ) R = HgCl
shown in F ig u re 8 , two more o f th e carbon atoms could be a ssig n e d t o a
secondary m ethyl g ro u p , reso n an ce a t 0 .66 ppm (3H d , J=6 c p s ) , and a
t e r t i a r y m ethyl g ro u p , reso n an ce a t 0 .9 0 ppm (3H s ) .
These assig n m en ts
l e f t e ig h t carbons and two u n s a tu r a tio n s unaccounted f o r in th e
m o le c u la r fo rm u la .
They w i l l be shown below to be in c o rp o ra te d in a
[ 4 .4 .0 ] b i c y c l i c r in g system .
The p resen ce o f a fu ra n r in g in th e m olecule was in d ic a te d by
s e v e r a l m eans.
The compound gave th e u s u a l c o lo r r e a c tio n s f o r a t r i -
s u b s tit u te d fu ra n r in g a s la v e n d e r w ith th e E h rlic h r e a c t i o n , red w ith
th e L ieberm an-B urchard r e a c t i o n , and p in k w ith v a n i l l i n in d i l u t e ,
a lc o h o lic s u l f u r i c a c i d . ^
Beyond th e s e g e n e r a l r e a c t i o n s , te tra d y m o l d is p la y e d s p e c tr a l
ev id en ce c h a r a c t e r i s t i c o f a t r i s u b s t i t u t e d f u r a n .
The uv max 222 mp
(lo g c 3 . 83) (F ig u re 8) i s in e x c e lle n t agreem ent w ith t h a t o f m enthofu ra n ( I I I ) which d e m o n strates uv max 220 m/l ( l o g E 3 . 7 8 ) . ^
fu r a n has th e same proposed m ethyl fu ra n chrom ophore.
Mentho-
The nmr spectrum
50
F ig u re 7 .
F ig u re 8.
IR Spectrum o f T etradym ol ( I l ) (M icro KBr P e l l e t )
UV Spectrum o f T etradym ol ( I I ) (M ethanol, 1 .3 5 x 10 4 M olar)
F ig u re 9 . A 90 MHz Spectrum o f Tetradym ol ( I l ) run in D, Benzene w ith TMS as an I n te r n a l
S ta n d a rd .
52
o f ( I I ) , F ig u re 9 , showed a s in g le p ro to n a t 7 .04 ppm w eakly coupled
(shown by s p in d e co u p lin g ) t o a th r e e p ro to n reso n an ce d o u b le t a t 1 .7 7
(III)
CH3
R=H
(IIIA ) R = HgCl
Sir
z
a ,
\
XR
ppm (J= 1 .2 c p s) which a g re ed v e ry w e ll w ith th e s h i f t p o s itio n and
c o u p lin g o f th e a ( o r 2 ) p ro to n and th e P (o r 3) m ethyl group r e s p e c t­
iv e ly o f th e t r i s u b s t i t u t e d fu r a n n u c le u s o f such compounds a s e u ry p o s a l 64 (IV ) (7 .1 2 and 2 .0 4 ppm J = I c p s ) , p e ta s a lb in e 65 (V) (7 .0 5 and 2 .0 5
ppm J = l . l c p s ) , and m e n th o fu ra n ^ ( I I I ) (6 .8 4 and 1 .8 5 ppm J= 1 .0 c p s ) .
The i r spectrum o f ( I l ) F ig u re 7 , d is p la y e d two bands a t 1655 and
—1
1560 cm
t h a t a re c h a r a c t e r i s t i c o f th e t r i s u b s t i t u t e d fu ra n
n u c le u s 67’ 68 a lo n g w ith th e s tro n g r in g b re a th in g band a t 1005 cm ^ 169
OH
(IV)
(V)
53
In a d d itio n t o th e s e s p e c t r a l c h a r a c t e r i s t i c s te tra d y m o l gave th e
t y p i c a l fu r a n d e r iv a tiv e s when t r e a t e d w ith m ercury ( I I ) c h lo r id e ,
chromium t r i o x i d e , h y d ro g en , and m a le ic a n h y d rid e .
Each o f th e s e
d e r iv a tiv e s i s d is c u s s e d in d e t a i l below .
A fu ra n n u c le u s w ith an a, (o r 2 ) p o s itio n u n s u b s titu te d i s v e ry
e a s i l y m e rcu rated a t t h a t p o s i t i o n .
70
As an exam ple, sim p ly adding
3 , 4 , 5 - tr im e th y l fu r a n to a b u ffe re d s o lu tio n o f m ercury ( I l ) c h lo rid e
a t room te m p e ra tu re im m ediately gave h ig h y ie ld s o f th e 2 -ch lo ro m ercu ry
d e riv a tiv e .
71
When te tra d y m o l was added t o th e m ercury ( I l ) c h lo rid e
s o lu tio n th e r e was an im m ediate p r e c i p i t a t i o n o f th e m ercury c h lo rid e
d e r iv a tiv e (IIA ) w hich was r e c r y s t a l l i z e d from e th a n o l t o y ie ld b e a u ti­
f u l w h ite n e e d le s .
A good e le m e n ta l a n a ly s is was n o t o b ta in e d on th e
compound from th e one sample su b m itte d b u t mass s p e c t r a l d a ta (M+469 m/e
p lu s a l l th e o th e r peaks c lu s te r e d a b o u t th e p a re n t io n peak f o r th e
v a rio u s is o to p e s o f c h lo rin e and m ercury) was o b ta in e d t h a t agreed w ith
th e a ss ig n e d s t r u c t u r a l fo rm u la .
The nmr spectrum (F ig u re 10) o f t h i s
a d d u c t d is p la y e d th e d isa p p e a ra n c e o f th e reso n an ce o f th e fu ra n p ro to n
from th e re g io n o f 7 ppm when ru n in DCGlg (n o t show n), w h ile th e i r
spectrum (F ig u re 11) s t i l l d is p la y e d th e th r e e fu ra n bands n e a r 1650,
1550, and 1000 cm
—1
3400 cm .
a lo n g w ith th e h y d ro x y l s tr e tc h i n g freq u en cy a t
When t h i s a d d u c t was d is s o lv e d in aqueous e th a n o l, and
hydrogen s u l f i d e bubbled th ro u g h th e s o l u t i o n , te tra d y m o l ( I I ) was
re g e n e ra te d
a s d e m o n strated by t i c .
Thus, th e compound was n o t
2.0
F ig u re 1 0.
4.0
s!o
P P M (T )
6.0
7.0
8.0
9 .0
A 60 MHz Spectrum o f (IIA ) ru n in P y rid in e w ith TMS a s an I n te r n a l S ta n d a rd .
ib
55
changed by th e s u b s t i t u t i o n r e a c t i o n .
o f Eastman
73
T his was an alo g o u s t o th e work
who made th e 2-m ercury c h lo rid e d e r iv a tiv e (IIIA ) o f
m en th o fu ran .
F i n a l l y , t h i s chlo ro m ercu ry s u b s tit u te d compound from
te tra d y m o l was su b m itte d t o X -ray c r y s ta llo g r a p h ic a n a ly s is to y ie ld
th e s t r u c t u r e shown as (XXVIl) on page 90.
When ( I I ) was r e a c te d w ith th e S a r e t t r e a g e n t,
t r i o x id e - p y r id in e com plex, i t y ie ld e d a compound
74
a chromium
mp 161-
1 6 2 .5°C, M+266 m /e) which had added two oxygen atoms to th e m o lecu lar
form ula o f ( I I ) .
To t h i s compound was a ssig n e d th e s tr u c t u r e (V I), a
h e m ik e ta l la c to n e o r p s e u d o -a c id .
T his s tr u c t u r e was analogous t o th e
chromium t r i o x i d e o x id a tio n p ro d u c t, p e p e ric a c i d , d e riv e d from m enthofu r a n ( I I I ) whose s tr u c t u r e was proved by Woodward and Eastm an.
75
These w orkers a ss ig n e d th e s t r u c t u r e (V Il) on th e fo llo w in g b ases (shown
in Scheme 4 ) :
(a ) O x id a tiv e c lea v a g e w ith p o tassiu m perm anganate
y ie ld e d P-m ethyl a d ip ic a c id ( V I I I ) .
(b) D eh y d ratio n w ith sodium
hydrogen s u l f a t e y ie ld e d th e anhydro compound (IX ) which re v e rte d t o
(V II) on tr e a tm e n t w ith a l k a l i ,
(c ) C a ta ly tic h y d ro g e n a tio n was
56
d i f f i c u l t , b u t under d r a s t i c c o n d itio n s y ie ld e d ( X) .
sodium amalgum y ie ld e d th e y -k e to a c id ( X l ) .
R eduction w ith
(d) T i t r a t i o n t o p h en o l-
p h th a lie n did n o t open th e la c to n e r in g b u t tre a tm e n t w ith sodium
hydro x id e in e th a n o l re v e a le d uv max 265 mjU, which is a ss ig n e d t o an
a ,p - u n s a tu r a te d y -k e to a c id ( X I I ) .
O th er w orkers have found th e same
Scheme 4
R ea c tio n s o f P e p e ric Acid (V II)
ty p e o f compounds in th e o x id a tio n o f o th e r h ig h ly s u b s tit u te d fu ra n
d e r iv a tiv e s a s (X III) from a t r o l a c t y l o n 76 and (XIV) from i s o s e r i c e n in e .
The o x id a tio n p ro d u c t (V I) from te tra d y m o l was a w hite
c r y s t a l l i n e h ig h m e ltin g s o lid t h a t d em o n strated uv max 221 mji
57
HIl 3 1 1
WAVtNUMBlR CM'
WAVtltNCTH IN
F ig u re 1 1 . IR S p e c tra o f ( I I A ) , (V I), and (V II ).
( a l l s p e c tr a run in m icro KBr p e l l e t s )
58
(X III)
(lo g E 4 .0 5 ) which compared fa v o ra b ly w ith th e uv a b s o r p tio n o f th e
o x id a tio n p ro d u c t o f m enthofuran ( V I l ) , uv max 216 mjn (lo g E 4 .0 7 ) ,
made in t h i s la b o r a to r y .
A lso th e i r s p e c tr a o f th e s e two compounds
showed th e same ty p e o f u n s a tu ra te d c arb o n y l band (F ig u re 11) b u t
d if f e r e d in th e oxygen hydrogen s t r e t c h i n g freq u en cy a re a p o s s ib ly due
t o th e two h y d ro x y ls in (V l) and only th e one in ( V I l ) .
The nmr s p e c tra
(F ig u re s 12 and 13) o f th e s e two compounds a ls o d is p la y e d d i s t i n c t
s i m i l a r i t i e s in th e lo c a t io n o f th e reso n an ce o f th e m ethyl group on
carbon 3 a t 2 .7 5 and 2 .8 5 ppm f o r (V I) and (V Il) r e s p e c tiv e ly and th e
re so n an c es around 2 .5 ppm t h a t were a ss ig n e d t o th e p ro to n s on carbons
4 and 9 in (V I) t o carbons 4 and 7 in ( V I I ) .
When te tra d y m o l ( I I ) was tr e a t e d w ith m a leic a n h y d rid e a m ix tu re o f
th r e e compounds (by t i c ) r e s u l t e d .
A mass spectrum o f t h i s m ix tu re was
ta k e n which showed a p a re n t peak (M+332 m /e) which had th e c o r r e c t
m o le c u lar w eig h t f o r a D ie ls -A ld e r ty p e a d d u ct s im ila r t o th o se w ith
fu ra n (XV) and (XVI) .
T his m ix tu re was n o t f u r t h e r c h a r a c te r iz e d a s i t tu rn e d pink th e n
F ig u re 1 2.
A 60 MHz Spectrum o f (V I) ru n in DCC1„ w ith TMS a s an I n te r n a l Standard
F ig u re 13. A 60 MHz Spectrum o f (V II) run in P y rid in e w ith th e P y rid in e P ro to n s as an
I n t e r n a l S ta n d a rd .
61
O
brown on exposure t o a i r in d ic a tin g i n s t a b i l i t y and d e co m p o sitio n .
This was n o t s u r p r is in g a s m a le ic a n h y d rid e a d d u cts o f fu ra n compounds
a re many tim es u n s ta b le
79
and b e s id e s te tra d y m o l i t s e l f was n o t v ery
s ta b l e u n d er a e r o b ic c o n d itio n s .
Perhaps th e m ost u s e f u l o f th e fu ra n d e r i v a t i v e s , f o r c h a r a c te r iz a ­
t i o n p u rp o se s, were th e h y d ro g e n a tio n p ro d u c ts .
Due t o th e e s s e n t i a l l y
p la n a r c h a r a c te r o f th e f u r a n r in g and s u b s tit u e n ts su rro u n d in g i t ,
c a t a l y t i c h y d ro g e n a tio n y ie ld e d a number o f iso m e ric p ro d u c ts .
These
in c lu d e d two m ajor compounds (XVIIA) and (XVIIB) which had added two
moles o f hydrogen t o ( I I ) , a s dem o n strated by th e ms, and one m ajor
compound (X V IIl) which had added one mole o f hydrogen, by ms.
In
a d d itio n t o th e s e p ro d u c ts a m ix tu re o f iso m e ric d eh y d rated -h y d ro g en ated
p ro d u c ts a s (XIX) were a l s o o b ta in e d , a s re v e a le d by ms.
These l a t t e r
compounds a re shown below to have l o s t th e h y d ro x y l and be f u l l y hy d ro ­
g en ated .
A ll o f th e s e h y d ro g e n a tio n p ro d u c ts could be s e p a ra te d in to
fo u r f r a c t i o n s ; (XVIIA), (XVIIB), (X V IIl), and (XIX); by e lu tio n
chrom atography on a lu m in a .
The r e a c tio n and p ro d u c ts a re g iv en in
62
Scheme 5.
The r a t i o s o f th e p ro d u c ts co u ld be changed by th e use o f d i f f e r e n t
c a t a l y s t s and s o lv e n ts a s i s i l l u s t r a t e d i n Table 6.
Note e s p e c ia lly
t h a t th e y i e l d o f th e d e h y d rated -h y d ro g en ated p ro d u c ts was d ecreased
from 18 p e r c e n t t o 6 p e r c e n t by changing from Pd on c h a rc o a l in
e th a n o l to Rh on alum ina in m e th a n o l.
S ince th e s e p ro d u c ts (XIX) were
shown t o be a d i f f i c u l t l y s e p a ra b le m ix tu re , Rh on alum ina was used a s
th e c a t a l y s t in m e th an o l.
T his in c re a s e d ease o f d e h y d ra tio n w ith Pd
had been observed by o th e r w o rk e rs .
80
The d e h y d ra ted -h y d ro g en a te d compounds (XIX) were shown to be a
m ix tu re o f a t l e a s t s ix compounds by g l c .
A ll o f th e compounds y ie ld e d
a p a re n t peak (M+222 m /e) by g lc/m s which corresponded t o th e lo s s o f '
w a te r and .the a d d itio n o f th r e e moles o f hydrogen t o ( I l ) .
One o f
th e s e isom ers was p a r t i a l l y p u r i f i e d f o r s p e c t r a l use by re p e a te d
alum ina chrom atography.
The i r spectrum (F ig u re 14) o f t h i s p a r t i a l l y
-I
p u r if ie d compound no lo n g e r d e m o n strated th e 3400 cm band o f th e
h y d ro x y l, n o r th e 1550 and 1650 cm
bands o f th e fu r a n r in g (th e m ix tu re
o f XIX lik e w is e d id n o t d is p la y th e s e b a n d s ).
The nmr spectrum (F ig u re
15) d is p la y e d re so n an c es f o r th r e e p ro to n s in th e re g io n betw een 3 .5
and 4 .5 ppm which could be a ssig n e d t o th o s e p ro to n s on th e carbons
b e a rin g th e e th e r linlcage in th e te tr a h y d r o f u r a n n u c le u s .
81
This
spectrum a ls o showed re so n an c es f o r th r e e m ethyl groups a tta c h e d to
carbons 3 , 4 a , and 5.
These- m ethyl groups were now a l l on s a tu r a te d
63
Scheme 5
H ydrogenation o f Tetradym ol ( I I )
Ch
( XVIIA ( B)
( XVI I I )
T able 6
Y ie ld s
o f H ydrogenated P ro d u cts Using D if f e r e n t C a ta ly s ts
^ X Q a ta ly s t
P ro d u ct
Rh on alum ina in m ethanol
Pd on c h a rc o a l in e th a n o l
6%
18%
(XVIIA)
22%
52%
(XVIIB)
52%
18%
(X V III)
19%
14%
(XIX)
*
Y ie ld s based on is o la te d p ro d u c ts
64
16
IB
20
25
30
40
F ig u re 15.
A 60 HHz Spectrum o f (XIX) run in DCCl3 w ith TMS as an I n te r n a l S ta n d a rd .
66
carbon atoms and were ex p ected t o re s o n a te a t h ig h f i e l d .
Compound (X V III) ( Ci5 H2 4 °2 ’ mP 145-146°C , M+236 m /e) re v e a le d by
i t s p h y s ic a l c o n s ta n ts t h a t i t re p re s e n te d th e a d d itio n o f one mole o f
hydrogen t o ( I I ) .
These two hydrogen atoms could m ost l o g i c a l l y be added
in one o f th r e e p o s itio n s in th e fu ra n r i n g ; 9 a -3 a , 9 a -2 , o r 3-2 (F ig u re
1 6 ).
They were m ost l i k e l y added to th e 2-9a p o s itio n (n e x t to th e
e th e r lin k a g e ) s in c e th r e e p ro to n s ap p eared down f i e l d betw een 4 .5 and
5 .5 ppm in th e nmr spectrum o f (X V III) shown in F ig u re 1 7 .
This s h i f t
p o s itio n was only s l i g h t l y below th e ex p ected s h i f t p o s itio n o f th e
oH
OH
OH
F ig u re 1 6 . P ro b ab le P o s itio n s f o r th e A d d itio n o f one Mole o f Hydrogen
to T etradym ol ( I I ) .
reso n an ce o f a p ro to n a l l y l i c to a double bond and lo c a te d on a carbon
82
b e a rin g an e th e r lin k a g e . "
The reso n an ce f o r th e m ethyl a tta c h e d to
carbon 3 was n o t a ss ig n e d in th e nmr spectrum o f (X V III), b u t i f t h a t
carbon were s a tu r a te d th e m ethyl a tta c h e d t o i t would be expected to
re s o n a te above 1 .3 ppm a s a m ethyl group lo c a te d on a s a tu r a te d carbon
n e x t t o a double bond is n o t u s u a lly s h i f t e d much below 1 .0 ppm.
83
S ince th e r e were only two m ethyl reso n an c es above 1 .3 ppm in th e nmr
F ig u re 1 7.
A 60 MHz Spectrum o f (X V IIl) ru n in
B enzene.
68
spectrum o f (X V III) (which can e a s i l y be a ss ig n e d t o th e m ethyls on
carbons 4a and 5 ) , th e m ethyl on carb o n 3 seemed m ost l i k e l y t o be on an
u n s a tu r a te d p o s i t i o n .
In a d d itio n t o t h i s nmr s p e c t r a l d a ta th e r e was
a d d i t i o n a l ev id en ce a g a in s t th e a d d itio n o f th e two hydrogen atoms in
th e 3a-9a o r 2-3 p o s itio n s from th e i r spectrum o f (X V IIl) (F ig u re 1 4 ).
I f e i t h e r o f th e s e s t r u c t u r e s was c o r r e c t th e compound would c o n ta in an
enol e th e r.
The observed i r spectrum di<3 n o t c o n ta in th e s tro n g bands
around 1250 cm ^ t h a t a re c h a r a c t e r i s t i c o f an e n o l e t h e r ^ and th e
a lk e n e s t r e t c h i n g v i b r a t i o n a t 1700 cm*"1 was sq low i n i n t e n s i t y t h a t i t
a ls o seemed u n lik e ly t o have a r i s e n f^om an e n o l ^ t h e r . ^
T his compound
was c o n v e rte d v e ry slow ly i n t o (XVIlB) on f u r t h e r c a t a l y t i c h y d ro g e n a tio n
w ith Rh on alum ina in m ethanol which su g g este d t h a t t h i s compound d id n o t
a r i s e from an im p u rity in th e o r i g i n a l compound ( I I ) and th u s , m ust have
had th e same g e n e r a l s t r u c t u r e a s te tra d y m o l and th e o th e r h y d ro g e n a tio n
p ro d u c ts o f t h a t compound. .
The o th e r two m ajor components o f t h i s h y d ro g e n a tio n m ix tu re (XVIIA9
C15H2 6 °2 ? mp 75-77°C , M+238 m /e; XVIIB? P15H2602 ? mp 104-105°C 9 M+238 m /e)
were e v id e n tly isom ers o f th e compound which has th e c o m p letely s a tu r a te d
fu ran r in g .
These two compounds were d i f f e r e n t by t i c and g l c , and
showed some d if f e r e n c e in t h e i r s p e c t r a , b u t d is p la y e d th e same molecu­
l a r w e ig h t by ms and had th e same e le m e n ta l a n a l y s i s .
T h e ir i r s p e c tr a
(F ig u re s 14 and 20) b o th dem onstrated ,a r a t h e r sh arp h y d ro x y l band in
I
th e 3400 cm
re g io n and th e lo s s o f th e f u r a n bands a t 1650 and 1550
F ig u re 1 8.
A 60 MHz Spectrum o f (XVIIA) run in Benzene.
70
-I
cm
which su p p o rte d th e a ssig n e d s t r u c t u r e (XVIIA and B ).
The nmr s p e c tr a o f th e s e two te tr a h y d r o compounds (F ig u re s 18 and
22) re v e a le d th e same th r e e p r o to n s 1 reso n an ces betw een 3 .0 and 4 .1 ppm
which had been a ss ig n e d t o th o s e p ro to n s on carbons 2 and 9a in a l l th e
hydrogenated compounds, and re so n an c es f o r th r e e m ethyl groups above 1 .3
ppm f o r th o s e m ethyls on carbons 3 , 4 a , and 5, which were a l l now s a t u r ­
a te d p o s i t i o n s .
One o f th e s e compounds (XVIIB) w i l l be d isc u sse d in more
d e t a i l in a l a t e r p o rtio n o f t h i s s e c tio n d e a lin g w ith i t s d e h y d ra tio n .
F u r th e r s tu d ie s were n o t c a r r ie d o u t t o d eterm in e which o f th e e ig h t
p o s s ib le isom ers th e s e two compounds re p re s e n te d and d e ta i le d a n a ly s is
t o c o n firm t h a t b o th (XVIIA) and (XVIIB) were a s in g le isom er was n o t
ta k e n beyond th e g lc and t i c c h a r a c te r iz a ti o n on two s u p p o r ts , in each
c a s e , which showed b o th compounds t o be homogeneous.
W ith th e in c lu s io n o f a m ethyl fu r a n in th e m o le c u lar s tr u c tu r e t o
a c c o u n t f o r th r e e u n s a tu r a ti o n s , one oxygen atom , and f iv e carbon atoms
o f th e m o le c u lar form ula th e r e were rem ain in g two u n s a tu r a tio n s , one
oxygen atom and te n carbon atoms t o be accounted f o r .
As was p re v io u s ly
n o ted two more o f th e carbon atoms co u ld be a ssig n e d to a secondary
71
m ethyl group and a t e r t i a r y m ethyl g ro u p , w h ile th e o th e r oxygen was
accounted f o r in a h y d ro x y l m o ie ty ,
t e r t i a r y a s th e nmr spectrum o f ( I l )
an ces in th e re g io n betw een 3 .3 and
T his h y d ro x y l m oiety was pro b ab ly
F ig u re 8, showed no p ro to n re s o n :. 5 ppm where th o se p ro to n s on th e
OH
(XXIV)
(X X II)
(XXI)
oh
I
(X X III)
OH
(IV )
(N um bering o f t h e s e compounds f o llo w s th e o r i g i n a l l i t e r a t u r e .)
72
p r e s e n t.
F u r th e r th e h y d ro x y l could n o t be a c y la te d w ith p y rid in e and
a c e t i c a n h y d rid e , which i s t y p i c a l l y used a s a t e s t to confirm t h a t a
h y droxyl i s t e r t i a r y _^ ^
ES>t?^
The p o s itio n o f two more carbon atoms could be p o s tu la te d from th e
nmr spectrum o f ( I l ) .
T his spectrum (F ig u re 8) d is p la y e d two AB q u a r te ts
due to gem inal c o u p lin g a t 2 .1 and 3 .0 ppm (J=17 c p s) and a t 2 .2 and 2 .4
ppm (J=9 c p s) which could be a ssig n e d t o p ro to n s on th e carbon atoms 4
and 9a betw een th e fu r a n n u c le u s and t e r t i a r y carbon a to m s.
The a s s ig n -
ment o f th e s e p o s itio n s and s p l i t t i n g s had an alo g y in n e o lin d e ra n e
90
(XX) (C-^q AB spectrum 2 .3 and 3 .2 5 ppm, J=15 c p s ) , 9-hydroxyfu ro erem o p h ila n e
91
(XXIV) (C , AB spectrum 1 .9 0 and 2.5 7 ppm, J= 1 6 .5 c p s ) , c i s 92
furo erem o p h ilo n e
(XXl) (C^ AB spectrum 2 .2 0 ppm, J = 1 7 .5 c p s ) , t r a n s fu ro erem o p h ilan e
93
(XXIl) (C^ AB spectrum 2 .4 0 ppm, J= 1 6 .0 c p s ) ,
I in d e r e n e 9^r (X X IIl) (Cg AB spectrum 2.6 3 and 2 .8 0 ppm, J=15 c p s ) , and
e u ry o p s o l9^ (IV ) (C^ AB spectrum 3.0 0 and 2.78 ppm, J n o t g iv en b u t
s ta te m e n t made t h a t carbon 9 p ro to n s g iv e a q u a r t e t ) .
T his assig n m en t
f o r th e p ro to n s in te tra d y m o l ( I l ) gave th e p a r t i a l s t r u c t u r e (A ).
(A)
R / H
73
Since te tra d y m o l ( I l ) would ta k e up only two moles o f hydrogen on
c a t a l y t i c h y d ro g e n a tio n th e o th e r two u n s a tu r a tio n s o f th e m o lecu lar f o r ­
mula were accounted f o r a s rin g s w ith th e hydroxyl group a t one r in g
ju n c tu r e and a m ethyl group a t th e o th e r .
The h y d ro x y l group and t e r t i ­
a ry m ethyl could n o t be on th e same carbon s in c e th e t e r t i a r y m ethyl was
to o f a r u p f i e l d .
I f th e y were b o th on th e same carbon atom th e m ethyl
would n o rm ally be below 1 .0 ppm.
96
The rem aining carbons and one s id e o f th e fu ra n r in g could be
imagined a s combined in a t l e a s t s ix b a s ic r in g s y s te m s ; 4 ,4 ,0 ; 5 ,3 ,0 ;
6 ,2 ,0 ; 7 ,1 ,0 ; 4 ,3 ,1 ; and 4 ,2 ,2 a s i l l u s t r a t e d in F ig u re 1 9 .
The o th e r
isom ers w ith th e fu r a n r in g on th e fo u r membered r in g in th e 6 ,2 ,0 system
o r on th e f iv e membered r in g in th e 5 ,3 ,0 system could be r e je c te d s in c e
th e y d id n o t have th e two a H y l i c seco n d ary carbons t h a t have been shown
t o be p r e s e n t n e x t t o th e fu r a n r in g in th e above p a ra g ra p h s .
F ig u re 1 9 .
P o s s ib le T r ic y c lic Ring Systems f o r T etrad y m o l.
74
By th e fo llo w in g p ro c e ss o f e lim in a tio n i t was p o s s ib le to p o s tu ­
l a t e th e 4 ,4 ,0 r in g system a s th e m ost l i k e l y c a n d id a te f o r th e s tr u c t u r e
o f te tra d y m o l.
The 7 ,1 ,0 system was u n lik e ly s in c e th e r e were no p ro to n
re so n a n c e s f o r th e cy clo -p ro p a n e r in g in th e re g io n from 0 .0 to 0 .5 ppm
in th e nmr spectrum o f ( I l ) which i s th e norm al s h i f t p o s itio n f o r th o se
p ro to n re s o n a n c e s .
97
Second, th e 6 ,2 ,0 system was a l s o u n lik e ly by th e
same ty p e re a so n in g s in c e th e nmr spectrum d id n o t r e v e a l p ro to n re s o n ances around 3 .5 ppm f o r th o s e p ro to n s on th e cy clo b u tan e r i n g .
98
T h ird , a 5 ,3,0. system appeared u n lik e ly s in c e a d eh y d ro g en atio n (arom at i z a t i o n ) a tte m p t w ith e i t h e r S in b o ilin g t r i g Iyme o r Pd on c h a rc o a l a t
320°G y ie ld e d e i t h e r s t a r t i n g m a te r ia l or a m ix tu re o f l i g h t yellow non­
p o la r compounds (by t i c ) w ith no h i n t o f th e b r i g h t b lu e o f an a z u le n e .
S ince a z u le n e s have such an in te n s e c o lo r
99
and would be ex p ected t o be
formed in a t l e a s t s m a ll y ie ld s from th e s e r e a c tio n s on a 5 ,3 ,0 system "^^
t h a t system could be q u ite s a f e ly ru le d o u t.
F o u rth , i f th e 4 ,3 ,1 system
was c o r r e c t , a r a t h e r sh arp s ig n a l in th e nmr spectrum f o r th e b rid g e
p ro to n s would be expected s in c e t e r t i a r y carbons a d jo in th e b rid g e carbon
a t b o th b rid g e h e a d s .
T his sh a rp s ig n a l was n o t o b s e rv e d .
Now i f p a r t i a l
s t r u c t u r e (A) was c o r r e c t i t e lim in a te d a l l e x c e p t th e 4 , 4 , 0 ; 4 ,3 ,1 ; and
4 ,2 ,2 system s s in c e th e s e a lo n e o f th e su g g ested r in g system s had two
and only two carbons betw een th e f u r a n and th e b rid g e h e ad c a rb o n s .
F i f t h , none o f th e s e system s e x c e p t th e 4 ,4 ,0 and 3 ,5 ,0 had v ery good
p re c e d e n t in known s e s q u ite rp e n e s y s te m s .
By th e above re a so n in g i t
75
was p o s s ib le to p o s tu la te th e p a r t i a l s t r u c t u r e (B) in c o rp o ra tin g th e
4 ,4 ,0 r in g sy stem .
There a re two b a s ic ty p e s o f known s e s q u ite rp e n e system s having th e
p a r t i a l s tr u c t u r e (B)
102
, th e erem o p h ilan es (B I) and th e eudesmanes (B 2).
Both o f th e s e system s have th e secondary m ethyl group a t carbon 5 w ith
v a ry in g s te r e o c h e m is tr ie s o f th e r in g ju n c tu r e and m ethyl g ro u p s .
In
o rd e r t o determ in e which o f th e s e two was re p re s e n te d in th e s tr u c tu r e
o f te tra d y m o l ( I l ) , an a tte m p t was made to d eh y d rate ( I l ) u sin g th e m ild
(B)
^ Rgi
R19R2 = CH3 or
(B I) R2 = CH3
(B2) R1 = CH3
c o n d itio n s o f F l o r i s i l in r e f lu x in g benzene ."LUU T his did produce a
c o lo r le s s l i q u id p ro d u c t (XXV) which was p u r if ie d by chrom atography on
d e a c tiv a te d a lu m in a .
The i r spectrum o f t h i s compound (F ig u re 20) i n d i ­
c a te d d e h y d ra tio n had o ccu rred by th e absence o f th e h y d ro x y l b a n d .
The
s h i f t in th e uv a b s o r p tio n from Xmax= 222 m^l in te tra d y m o l ( I l ) to 288
m /j
in t h i s d eh y d rated p ro d u c t (XXV) su g g ested t h a t th e new double bond
was co n ju g ated to th e fu ra n r i n g .
The nmr spectrum (F ig u re 21) showed a
one p ro to n reso n an ce a t 6.8 ppm which could be a ssig n e d t o th e a o r 2
p ro to n on th e f u r a n r in g and a one p ro to n reso n an ce a t 5.8 ppm which
could be a ssig n e d t o th e p ro to n on th e new double bond in (XXV)
76
25
3
3.S
4
4.5
WAVtlENCTH IN MICRONS
S
5.5
6
65
7
7.3
8
9
IO
Il
12
14
16
18
20
25
30
40
WAVEltNCTH IN MICRONS
WAVENUMBER CM1
F ig u re 2 0 . IR S p e c tra o f (XVIIB) (sp ectru m ru n in a m icro KBr p e l l e t ) ,
(XXV) and (XXVI) ( s p e c tr a run n e a t on s a l t p l a t e s ) .
0
F ig u re 2 1.
A 60 MHz Spectrum o f (XXV) ru n in S ila n o r C (DCClq w ith 1% IMS).
fVM (I)
78
in d ic a tin g i t was a t r ! s u b s t i t u t e d double bond.
Compound (XXV) p ro b ab ly
had th e s t r u c t u r e shown b u t i t was n o t f u r t h e r c h a r a c te r iz e d as i t was
decomposing q u ite r a p id ly w h ile th e s p e c tr a were b ein g r u n .
This de­
co m position was dem onstrated by a change in th e ap p earan ce o f s o lu tio n
from c o lo r le s s t o orange and th e ap p earan ce o f a new more p o la r compound
when th e s o lu tio n was checked by t i c .
Because o f t h i s problem o f i n s t a ­
b i l i t y , th e te tr a h y d r o p ro d u c t (XVIIB) was d eh y d rated t o f in d i f i t was
more s ta b l e and p o s s ib ly e a s i e r to c h a r a c te r iz e .
(XXV)
T his te tr a h y d r o p ro d u c t (XVIIB) was s u c c e s s f u lly d eh y d rated t o form
a h y d ro g e n a ted -d eh y d ra te d p ro d u c t (XXVl).
With t h i s m olecule th e lo c a ­
t i o n o f th e double bond could be a s c e r ta in e d , a s i s shown below , and a
ch o ice be made betw een th e two s e s q u ite rp e n e t y p e s .
When te tra d y m o l ( I I ) was c a t a l y t i c a I l y hydrogenated i t y ie ld e d , a s
has been a lre a d y n o te d , a m ix tu re o f p ro d u c ts which had added two o r fo u r
hydrogen atoms o r had l o s t w a ter and added s ix hydrogen a to m s.
The p ro ­
d u c t in h ig h e s t c o n c e n tr a tio n , u t i l i z i n g Rh on alum ina in m eth an o l, had
in c o rp o ra te d fo u r hydrogen atoms to s a tu r a te th e fu ra n r in g (XVIIB).
F u r th e r a ssig n m en ts could be made, how ever, to t h i s s tr u c t u r e from th e
nmr sp ec tru m .
P PM (T )
F ig u re 2 2.
A 60 MHz Spectrum o f (XVIIB) ru n in S ila n o r C (DGCl3 w ith
1%
IMS).
80
(XVIIB)
In t h i s nmr spectrum (F ig u re 22) were d isp la y e d reso n an ces f o r
th r e e p ro to n s a t 4.07 ppm (1H m ), 3.80 ppm (IR d J=8 c p s ) , and 3.38 ppm
(IR dd J= 8 ,9 c p s) which could be a ss ig n e d to th e p ro to n s on th e carbons
2 and 9a b e a rin g th e e th e r lin k a g e o f th e te tr a h y d r o f u r a n (3 .8 ppm
t y p i c a l f o r p ro to n s on th e
fu r a n )
a
p o s itio n o f th e u n s u b s titu te d te tr a h y d r o ­
The one p ro to n reso n an ce a t 4.07 ppm was a ss ig n e d to th e
p ro to n on carbon 9a which was shown to be coupled to a two p ro to n r e s ­
onance a t 1 .8 0 ppm by a s p in d eco u p lin g e x p e rim e n t.
ance was a ss ig n e d t o th e p ro to n s on carbon 9 .
T his l a t t e r re s o n ­
The ap p earan ce o f th e
re so n an c es o f th e two p ro to n s a t 3.38 and 3.80 ppm was p ro b ab ly due to
gem inal c o u p lin g betw een th e p ro to n s on carbon 2 (J=8 c p s ) , w h ile th e one
p ro to n on carbon 3 was coupled w ith only one o f th e carbon 2 p ro to n s
(J=9 c p s ) .
T his was e v id e n tly due to a r i g i d c o n fo rm atio n t h a t h e ld
th e s e p ro to n s a t such an a n g le a s t o a llo w e f f i c i e n t c o u p lin g betw een
only one o f them and th e carbon 3 p ro to n .
This c o u p lin g c o n s ta n t a n g le
dependence i s m a th e m a tic a lly e x p re ssed in th e K arp lu s e q u a tio n .105
From
th e g rap h determ ined by t h i s e q u a tio n th e d ih e d r a l a n g le betw een th e
p ro to n s on carbon 2 and t h a t on carbon 3 m ust be ap p ro x im a te ly 90° and
81
When t h i s hydrogenated p ro d u c t (XVIIB) was d eh y d rated w ith I 9 in
b o ilin g h e p tan e i t y ie ld e d a c o lo r le s s liq u id h aving th e p ro p e r raolecuI a r w eig h t (M+220 m /3) f o r (XVIIB) minus HgO.
T his l i q u id d is p la y e d an
i r spectrum (F ig u re 20) t h a t was devoid o f th e O-H s tr e tc h i n g freq u en cy
in th e re g io n around 3400 cm ^ and a f t e r p u r i f i c a t i o n by chrom atography
on alum ina and c a r e f u l vacuum d i s t i l l a t i o n gave (XXVI) in 50 p er c e n t
y ie ld based on s t a r t i n g m a t e r i a l .
T h is compound was homogeneous by g lc
(XXVI)
on th e one column u s e d .
The nmr spectrum o f (XXVI), re c o rd ed in F ig u re
23 d is p la y e d a one p ro to n reso n an ce a t 5.47 ppm (m) which could be
a ss ig n e d t o th e p ro to n on a t r i s u b s t i t u t e d double bond a tta c h e d to carbon
8.
When t h i s nmr spectrum was compared to t h a t o f (XVIIB) (F ig u re 2 2 ) ,
a two p ro to n reso n an ce was noted a t 2 .5 2 ppm which was dow nfield from th e
reso n an ce p o s itio n o f th e s e p ro to n s p re v io u s to th e d e h y d ra tio n .
T his
reso n an ce could be a ss ig n e d t o p ro to n s a l l y l i c t o th e new double b o n d .
A lso i t was no ted in t h i s com parison t h a t th e two p ro to n q u a r te t a t 1 .8 0
ppm (th e two p ro to n s on carbon 9 in XVIIB, F ig u re 22) was now e i t h e r
a b s e n t from or moved in th e sp e c tru m .
L a s tly i t was observed t h a t th e
re so n a n c e s a ss ig n e d t o th e p ro to n s on carb o n s 2 and 9a were only s l i g h t l y
O
O
IO
F ig u re 23.
A 60 MHz Spectrum o f (XXVl) ru n in D, Benzene.
83
a f f e c t e d by th e d e h y d ra tio n .
K eeping th e above f a c t s in m ind, i t was p o s s ib le to a s s ig n th e
double bond t o th e 8-8a p o s itio n by th e fo llo w in g p ro c e ss o f e lim in a tio n .
F i r s t , th e double bond could n o t be 4 a-5 b ecau se i t would be t e t r a s u b s t i tu te d and th e r e would be no p ro to n reso n an ce a t 5.47 ppm.
Second, i f th e
double bond was lo c a te d in th e 8a-9 p o s itio n th e p ro to n on carbon 9a
should be g r e a tly a f f e c t e d s in c e i t would be b o th a l l y l i e t o a double
bond and gem inal t o an e th e r lin k a g e .
T his s h i f t was n o t observed .
T h ird , i f th e double bond was lo c a te d 4-4a th e two a H y l i c p ro to n s ( to
th e double bond) would be q u ite d i f f e r e n t and would n o t be ex p ected t o ■
re s o n a te a t th e same s h i f t p o s i t i o n .
There was only one p eak , though
somewhat b ro a d , f o r th e two p ro to n s w hich could be seen t o be a f f e c te d
by th e new double b o n d .
A lso th e reso n an ce f o r th e t e r t i a r y p ro to n on
carbon 5 in t h i s in s ta n c e would n o t be ex p ected to be s h i f t e d below 2
ppm s in c e th e e f f e c t o f a double bond i s n o t s u f f i c i e n t t o move an a l l y l i e p ro to n below t h a t s h i f t p o s i t i o n .
107
F u rth e r a 4~4a double bond
would n o t be e x p ec te d to so m arkedly a f f e c t th e carbon 9 p ro to n s as was
e v id e n c e d .
T his l e f t only th e 8-8a p o s itio n as in d ic a te d in th e proposed s t r u c ­
tu r e (KXVI).
The nmr spectrum matched t h i s s t r u c t u r e q u ite w e ll.
The
re so n an c e o f th e two p ro to n s on carb o n 9 would be ex p ected t o be s h if te d
from 1 .8 0 ppm dow nfield to a p o s itio n below 2 .0 ppm due to th e a d d itiv e
e f f e c t o f b o th th e double bond and e th e r oxygen; and th u s t h i s was a t r i -
60 MHz Spectrum ru n in DCCl
F ig u re 2 4 .
60 MHz Spectrum run in P y rid in e
S h if ts a re in d ic a te d in ppm from i n t e r n a l TMS
P y rid in e Induced S h i f t in th e NMR Spectrum o f (XVIIB)
60 MHz Spectrum ru n in p y rid in e
F ig u re 2 5 .
60 MHz Spectrum ru n in DCCI1
S h if ts a re in d ic a te d in ppm from in t e r n a l TMS
P y rid in e Induced S h i f t in th e NMR Spectrum o f (IIA )
86
s u b s t i t u t e d double b o n d .
The p ro to n s on th e 7 p o s itio n were n o t
a s s ig n e d a s th e y were s t i l l n o t s e p a ra te d s u f f i c i e n t l y t o be o b serv ed ,
-I Q O
a s was expected s in c e th e y were a f f e c t e d by only a sim ple double bond.
The lo c a t io n o f th e double bond in t h i s 8-8a p o s itio n o b v io u sly
r e q u ir e s th e erem ophilane r a t h e r th a n th e eudesmane s k e le to n .
The c i s ty p e r in g ju n c tu r e a t 8a and 4a was e s ta b lis h e d on th e
b a s is o f th e s o lv e n t induced s h i f t o f th e t e r t i a r y m ethyl group re s o n ­
ance i n th e nmr spectrum o f (XVIIB) a s shown in F ig u re, 24 ;
A .m ethyl
group which i s c i s t o a h y d ro x y l m oiety i s c o n s id e ra b ly more d e sh ie ld e d
when th e spectrum o f th e compound i s ru n in p y rid in e th a n when i t i s ru n
109
in d e u te ro c h lo ro fo rm .
From th e a v a il a b le t a b l e s , th e p y rid in e -in d u c e d
CDCI3
s h i f t (&G H ^ = - .1 8 ppm) o f th e carbon 4a m ethyl reso n an ce ag reed w ith
a d ih e d r a l bond a n g le betw een th e h y d ro x y l group and m ethyl group o f
j u s t over s i x t y d e g r e e s .
HO
T his th e n was a c i s r in g j u n c t u r e . The
CDCl3 ■
p y rid in e -in d u c e d s h i f t (A „ M = - .2 2 ppm) o f th e m ethyl on carbon 4a in
U5 5
th e m ercury c h lo r id e d e r iv a tiv e (IIA ) (F ig u re 25) a ls o su p p o rted th e c i s
r in g ju n c tu r e a ssig n m e n t a s t h i s s h i f t was t h a t g iv en f o r a d ih e d ra l
a n g le o f 6 0°.
T his ty p e r in g ju n c tu r e i s t y p i c a l o f th e known erem ophi-
la n e s .H l
The secondary m ethyl o f an erem ophilane i s t y p i c a l l y c is w ith
r e s p e c t t o th e t e r t i a r y m ethyl g ro u p .
112
I f t h i s was th e case in t e t - '
radym ol two conform ers (IIB ) and (H G ) a re p o s s ib le (F ig u re 2 6 ).
When
th e s t a b i l i t i e s o f th e two conform ers were compared i t was observed t h a t
87
F ig u re 2 6 .
Tetradym ol Iso m ers.
88
(H G ) h as one l e s s m e th y l, rin g - c a rb o n gauche i n t e r a c t i o n and did n o t
have th e 1 ,3 d i a x i a l h y d ro x y l, m ethyl i n t e r a c t i o n .
T hus, i t would be
ex p ected t h a t conform er (H G ) would p re d o m in a te . The observed s o lv e n t
DGCl3
induced s h i f t (Afi H ^ = - .0 3 ppm) o f th e m ethyl group on carbon 5 o f th e
m ercury c h lo rid e d e r iv a tiv e was so s m a ll t h a t s t r u c t u r e ( H E ) , w ith th e
h y d ro x y l and m ethyl groups d i a x i a l , seemed u n l i k e l y .
T his conform a­
t i o n a l a ssig n m en t was f u r t h e r su p p o rte d by th e i n t e n s i t y (23 p er c e n t o f
b a s e ) o f th e p a re n t peak (M+234) in th e mass spectrum o f ( I l ) which
in d ic a te d t h a t th e fo rm a tio n o f th e s ix membered r in g f o r th e m ost
f a c i l e d e h y d ra tio n
114
was n o t f a v o r a b le .
As could be r e a d i l y seen co n -
form er (IIB ) was i d e a l l y a rra n g ed f o r t h i s d e h y d ra tio n a s shown in F ig ­
u re 2 7.
A lso i t was n o ted t h a t th e h y d ro x y l m oiety in (H E ) was more
h ig h ly h in d e re d by th e 1 ,3 h y d ro x y l, m ethyl i n t e r a c t i o n th a n i t was in
(H G ).
T his h in d e ra n c e should have speeded th e e lim in a tio n o f th e
115
h y d ro x y l group i n th e ms in l i n e w ith Biemannt S
w ork.
I t m ight be
e x p e c te d , how ever, t h a t th e ra p id in v e rs io n o f t h i s c is r in g ju n c tu re
would e lim in a te th e above argum en t, i . £ . , th e ju n c tu r e co u ld i n v e r t in
tim e f o r th e h y d ro x y l group to e lim in a te in th e ms, b u t i t was n o ted
t h a t th e c o n fo rm a tio n a l change r e q u ir e s th e e c lip s in g o f two m ethyl
groups and a h y d ro x y l group in th e t r a n s i t i o n p lu s th e in te rc o n v e r s io n
o f two c h a ir forms o f c y c lo h e x a n e .
T his p ro c e ss o b v io u sly r e q u ir e s a
c o n s id e ra b le amount o f e n e rg y .
The above argum ents s u p p o rtin g th e s t r u c t u r e (H G ) gave j u s t as
89
F ig u re 2 7 .
C onform ation f o r Easy D eh y d ratio n in MS.
good s u p p o rt t o b o th conform ers (IID ) and (H E ) which a re iso m e ric w ith
(H E and C) a b o u t carbon 5.
On th e b a s is o f th e s e s tu d ie s i t was n o t
p o s s ib le to choose betw een th e two iso m e rs, n o r was i t p o s s ib le to
a s c e r t a i n th e a b s o lu te c o n f ig u r a tio n o f th e m olecule though th e c o n fig ­
u r a tio n shown i s t h a t o f th e t y p i c a l e re m o p h ila n e .
In o rd e r to determ in e th e c o n fig u ra tio n a b o u t carbon 5 in
te tra d y m o l ( I l ) , co n firm th e r e s t o f th e proposed s t r u c t u r e , and d e t e r ­
mine th e a b s o lu te c o n f ig u r a tio n , th e m ercury c h lo rid e d e r iv a tiv e (IIA )
o f te tra d y m o l was su b m itte d t o X -ray c r y s ta llo g r a p h ic a n a ly s is — th e
r e s u l t s o f which a re o u tlin e d in th e n e x t s e c tio n o f t h i s d is c u s s io n .
IV .
X-Ray S t r u c t u r a l A n a ly sis
The X -ray c r y s t a l s t r u c t u r e o f th e m ercury c h lo r id e d e r iv a tiv e o f
te tra d y m o l (XXVIl) a u th e n tic a te d th e proposed s tr u c t u r e and f u r t h e r
re v e a le d t h a t th e carb o n 5 m ethyl group was c is w ith r e s p e c t to th e
h y d ro x y l group and carbon 4a m ethyl g ro u p .
a b s o lu te c o n f ig u r a tio n was t h a t shown b elo w .
(XXVII)
I t a ls o re v e a le d t h a t th e
91
D rc D. S m ith, a p o s t- d o c to r a l s tu d e n t o f D r. C. Caughlan who p e r­
formed th e X -ray c r y s t a l s tu d y , re p o rte d t h a t th e c r y s t a l d isp la y e d
orthorhom bic symmetry w ith u n i t c e l l dim ensions
1 0 .3 0 4 (9 ) X , and c = 1 9 .7 5 9 (1 7 ) X as o b ta in e d from a GE XRD-5 d i f f r a c t ­
om eter u sin g Mo ICa r a d i a t i o n .
While d a ta was b ein g c o lle c te d on th e
c r y s t a l , i t p a r t i a l l y decomposed in th e X -ray beam.
T his was made
e v id e n t by a d e c re a se i n th e s ta n d a rd s p o ts o f a p p ro x im a te ly 30 p e r c e n t
and a g ra d u a l d ark en in g o f th e c r y s t a l .
Of th e 1159 p ie c e d a ta s e t , 787 p ie c e s were found t o be more th a n
tw ice th e s ta n d a rd d e v ia tio n o f th e i n t e n s i t i e s and were c o n sid e re d
o b s e rv e d o The o th e r 372 r e f l e c t i o n s were c o n sid e re d unobserved and were
n o t in c lu d e d in th e re fin e m e n t.
A f te r v a rio u s re fin e m e n t p ro c e d u res th e f i n a l R was found to be 5 .4
p e r c e n t and th e w eighted R 5.9 p e r c e n t f o r th e s t r u c t u r e shown.
The p o s i t i o n a l and th e rm a l p a ra m ete rs o f th e enantiom orph were a ls o
r e f in e d b u t th e re fin e m e n t stopped a t an R o f 6 .5 p e r c e n t.
This s i g n i ­
f i c a n t d if f e r e n c e in R proves th e a b s o lu te c o n fig u ra tio n o f (XXVIl).
The c r y s t a l s tr u c t u r e re v e a le d a number o f o th e r i n t e r e s t i n g
fe a tu re s .
When a l e a s t sq u a re s p la n e was drawn th ro u g h th e atoms o f th e
fu r a n r in g and th e m ethyl group on carbon 3, th e maximum d e v ia tio n from
t h i s p lan e was 0.07 X by carbon 2 which was p ro b ab ly v e ry n e a r ly w ith in
th e e x p e rim e n ta l e r r o r .
Thus, th e f u r a n r in g was e s s e n t i a l l y p la n a r .
The bond d is ta n c e s (F ig u re 28) in d ic a te d t h a t th e r e was a c o n s id e ra b le
92
d e lo c a liz a tio n o f e le c tr o n s in th e fu ra n r in g s in c e th e bonds were
s h o r t (e x c e p t th e oxygen I t o carbon 2 bond) and th e bond betw een c a r ­
bons 3 and 3a was v e ry n e a r ly th e same le n g th a s t h a t betw een carbons
3a and 9 a .
I f t h i s was s imply a b u ta d ie n e system h e ld by an oxygen
b r id g e , a s has sometimes been su g g este d f o r f u r a n ,116 th e above two
bond le n g th s sh ould d i f f e r a p p r e c ia b ly .
These fin d in g s o f p la n a r ity
and s h o r t bond le n g th s l e n t s u p p o rt t o th e assig n m en t o f a ro m a tic
c h a r a c te r to t h i s fu r a n r i n g .
O H
F ig u re 2 8 . Tetradym ol Mercury C h lo rid e D e riv a tiv e Bond D istan c e s from
X-Ray S t r u c t u r e .
From th e c r y s t a l packing d a ta , a u n i t c e l l i s shown in F ig u re 2 9 ,
i t was determ ined t h a t th e compound e x i s t s in th e l a t t i c e as a dim er
h e ld by hydrogen bonding betw een th e h y d ro x y l group o f one m olecule and
th e m ercury o f th e o th e r .
T his could have accounted f o r th e 6 degree
In
Li
93
v a ria n c e from l i n e a r i t y o f th e c arb o n , m ercury, c h lo r in e b o n d s.
The d ih e d r a l a n g le betw een th e carbon 8a h y d ro x y l group and th e
t e r t i a r y m ethyl group on carbon 4a was 5 7 ,3 ° which was v e ry c lo se to
th e a n g le su g g ested from th e nmr s p e c t r a l d a ta on page 86 o f t h i s t h e s i s .
W ith th e heavy atoms p r e s e n t, th e X -ray s t r u c t u r a l a n a ly s is o f t h i s
compound proved t o be a r a t h e r s tr a ig h t- f o r w a r d and sim p le problem ,
Since th e compound was so e a s i l y p re p a re d a n d , a c c o rd in g t o th e
lite ra tu re
117
fu ra n s w ith th e a o r 2 p o s itio n u n s u b s titu te d c h a r a c te r ­
i s t i c a l l y form th e ch lo ro -m ercu ry d e r iv a tiv e so r e a d i l y , th e a u th o r
s u g g e s ts t h a t f o r new furano-com pounds o f t h a t ty p e t h i s p rocedure i s
th e method o f c h o ice f o r th e e lu c id a tio n o f s t r u c t u r e .
r .\.
94
F ig u re 2 9 .
C r y s ta l Packing P a tte r n f o r Tetradym ol Mercury C h lo rid e
EXPERIMENTAL SECTION
I.
Reagents
B Io -S il A, 100-200 mesh, o b ta in e d from Bio-Rad L a b o ra to rie s and
n e u t r a l a lu m in a , 100 m esh, o b ta in e d from V entron C o rp o ra tio n , were used
d i r e c t l y , w ith o u t a c t i v a t i o n , f o r e l u t i o n column chrom atography in a l l
b u t one e x p e rim e n t.
n ig h t a t 12O0C.
In t h i s in s ta n c e th e alum ina was a c ti v a te d o v er­
F l o r i s i l , '60-100 mesh, o b ta in e d from F lo r id in Company,
was a c ti v a te d o v e rn ig h t a t 12O0C b e fo re use in chrom atography„
For t h i n la y e r chrom atography A n a s il S , o b ta in e d from A nalabs I n c . ,
and aluminum oxide G (ty p e E ) , o b ta in e d from E. M. R e a g e n ts, were u s e d .
The t h i n la y e r chrom atographic p la te s were p o u red , a s a w a te r s lu r r y
w ith a Desaga s p re a d e r g iv in g a 250 m icron th ic k n e s s , and th e n a c tiv a te d
a t 12O0C f o r one h o u r.
The fo llo w in g s o lv e n ts were found t o be s a t i s f a c t o r y f o r use in
e x t r a c t i o n , column e l u t i o n , and t h i n la y e r developm ent d i r e c t l y from th e
c o n ta in e r w ith o u t f u r t h e r p u r i f i c a t i o n :
n -h e x a n e , P h i l l i p s P etro leu m ;
anhydrous d i e th y l e t h e r , anhydrous m e th an o l, e th y l a c e t a t e , p y r id in e ,
and b e n ze n e , B aker r e a g e n t.
For making th e m ic r o - p e lle ts used in o b ta in in g in f r a r e d s p e c tr a ,
.anhydrous KBr, SPEX I n d u s tr ie s I n c . , was found to be q u ite s a t i s f a c t o r y
d i r e c t l y from th e s u p p l i e r 's sm a ll c o n ta i n e r s .
The s o lv e n ts S ila n o r C,
Merk; Dfi b e n ze n e , D iaprep I n c . ; p y r id in e , carbon t e t r a c h l o r i d e , and
a b s o lu te m eth an o l, Baker an aly zed r e a g e n t were used as s o lv e n ts in
96
s p e c t r a l a n a ly s e s .
The v a n i l l i n - s u l f u r i c sp ra y re a g e n t was made up by adding 1 .5 g
v a n i l l i n , USP Merk, to 50 ml a b s o lu te e th a n o l and th e n adding 0.25 ml o f
c o n c e n tra te d s u l f u r i c a c i d .
E h r lic h ’s
118
re a g e n t was made up by adding 50 mg o f p -d im e th y l-
amino b e n za ld e h y d e, Eastman r e a g e n t, to I ml -95 p e r c e n t e th a n o l and
th e n adding 2 drops o f c o n c e n tra te d h y d ro c h lo ric a c id .
A f r e s h Chromerge, M anostat C o rp ., s o lu tio n in s u l f u r i c a c id was
used d i r e c t l y a s th e s u lfu ric -d ic h ro m a te sp ra y re a g e n t f o r t i c .
The o th e r re a g e n ts and s o lv e n ts were used a s re c e iv e d w ith th e
e x c e p tio n o f n -h e p ta n e which was washed w ith s u l f u r i c a c i d , d rie d and
d is tille d .
II.
In stru m e n ts
I n f r a r e d s p e c tr a were o b ta in e d u s in g e i t h e r a Beckman IR-4 or
IR-20 s p e c tro p h o to m e te r.
S p e c tra were ru n in m icro KBr p e l l e t s , a s a
I
n e a t , t h i n f ilm betw een sodium c h lo rid e p l a t e s , o r in s o lu tio n in carbon
t e t r a c h l o r i d e u sin g sodium c h lo rid e in f r a r e d c e l l s .
N u c lea r m agnetic reso n an ce s p e c tr a were o b ta in e d u sin g a V arian
model A-60 f o r th e 60 MHz s p e c tr a and a B roker in s tru m e n t f o r th e 90
MHz s p e c t r a .
Spin d eco u p lin g ex p erim en ts were perform ed on compound ( I I )
w ith th e 90 MHz in s tru m e n t w h ile th e s e ex p erim en ts were perform ed on ( I I )
and (XVIIB) w ith a 60 MHz in s tru m e n t.
97
U l t r a v i o l e t s p e c tr a were o b ta in e d in anhydrous m ethanol s o lu tio n
u sin g matched q u a rtz c e l l s on a Cary Model 14.
A nalyses o f dye c o n c e n tra tio n s o b ta in e d in an im al fe e d in g e x p e r i­
ments were determ ined u s in g a Beckman DU.
Mass s p e c t r a l d a ta were o b ta in e d u sin g a V arian Mat CH 5 w ith b o th
a s o lid probe and a g lc i n t e r f a c e .
O p tic a l r o t a t i o n s were determ in ed on a Rudolph Model 180 in
anhydrous m eth an o l.
For d e te rm in a tio n o f p u r ity a Beckman Model GC 4 gas chrom atograph
was u t i l i z e d .
I t was equipped w ith v a rio u s colum ns, a flam e d e te c to r ,
and te m p e ra tu re programmed oven.
E lem en tal a n a ly s e s were perform ed by C h em an aly tics, Tempe, A riz o n a .
X-Ray c r y s ta llo g r a p h ic d a ta were ta k e n on a GE XRD-5 d if f r a c to m e te r .
III.
P l a n t ,M a te ria l
Four p ic k in g s o f p la n t m a te r ia l were made which a re d e sig n a te d Tg
I-IV .
The Tg^ I p ic k in g was made a p p ro x im a te ly f i f t e e n m ile s so u th o f
Luci n , U tah, which i s in th e n o rth w e s te rn p a r t o f th e s t a t e , on May 4 -5 ,
1968.
The p l a n t had flo w e rs , b u d s , and new grow th p r e s e n t when p ic k e d .
The to p fo u r t o e i g h t in c h es o f th e p la n t was broken o f f and s tu f f e d in to
"gunny" sack s which were cooled w ith d ry ic e and p la ce d in an en clo sed
t r a i l e r f o r t r a n s p o r t a t i o n to Bozeman.
Upon a r r i v a l a t Bozeman, th e
p l a n t m a te r ia l was p la ce d in a r e f r i g e r a t o r a t 40°C f o r one day .
I t was
98
th e n chopped and fro z e n f o r s to ra g e a t - 2 O0C.
The Tg TI p ic k in g was made a t th e same lo c a t io n a s Tg I on J u n e '
1 8 -1 9 , 1968.
The p l a n t was in th e f u l l bloom or f r u i t s ta g e o f g ro w th .
T his tim e only new g row th, flo w e rs , and buds were p ic k e d .
This p la n t
m a te r ia l was p la ce d in p l a s t i c b a g s, a lo n g w ith dry i c e , and th e s e bags
were s to re d i n an in s u la te d c h e s t f o r t r a n s p o r ta tio n t o Bozeman.
Upon
a r r i v i n g a t Bozeman, th e p la n t m a te r ia l was s to re d in th e cold room a t
- 2 O0C.
The Tg I I I p ic k in g was made J u ly 1 -4 , 1968, a p p ro x im a te ly f i f t y
m ile s n o r th o f th e a re a where. Tg I and I I had been o b ta in e d .
By t h i s
tim e th e p la n t was b e g in n in g to d ry o u t, b u t a few o f th e bushes were
found t h a t were s t i l l q u ite s u c c u le n t w ith b u d s, f lo w e r s , and new grow th
p re s e n t.
A gain th e o ld e r grow th was d is c a rd e d and th e new er growth
packed in p l a s t i c bags w ith d ry ic e and ta k e n to Bozeman.
The Tg IV p ic k in g was made on June 15, 1969, a lo n g th e highway
a b o u t 20 m ile s s o u th o f P ark V a lle y , U tah .
T his was in th e same g e n e r a l
re g io n where th e o th e r p ic k in g s were made.
The p la n t was v ery lu s h w ith
many buds and flo w e rs a lo n g w ith c o n s id e ra b le new g ro w th .
new grow th were pick ed and p laced in p l a s t i c b a g s .
The buds and
These were ta k e n to
a cold room (O0C) a t th e U n iv e rs ity o f Utah and s to re d f o r fo u r d a y s,
th e n tr a n s p o r te d to Bozeman and s to r e d a t - 2 O0C.
Tg I was i d e n t i f i e d by D r. W. E . B ooth, c u r a to r o f th e herb ariu m a t
Montana S ta te U n iv e r s ity , a s a u th e n tic Tetradym ia g la b r a t a .
Tg I I was
99
confirm ed a s T . g la b r a t a by D r. Holmgren, c u r a to r o f th e h erbarium a t .
Utah S ta te U n iv e r s ity .
Samples o f b o th o f th e s e p ic k in g s were f i l e d f o r
r e f e r e n c e a t th e Montana S ta te U n iv e rs ity Herbarium a s numbers 63708
and 63737.
The re g io n where t h i s p la n t m a te r ia l was picked was v ery sandy and
a r i d w ith a s a l t f l a t j u s t a few m ile s away.
The e le v a tio n was a b o u t
4000 f e e t above sea l e v e l .
When th e hexane e x t r a c t s o f each o f th e f o u r p ic k in g s were an aly zed
by t i c th e y were a l l found to c o n ta in th e compound t h a t was l a t e r shown
to be th e a c ti v e p r in c ip le o f T . g la b r a t a .
A lso i t was p o s s ib le to
e x t r a c t t h i s p r i n c i p l e from p l a n t .m a t e r i a l t h a t had been s to re d fro z e n
f o r two y e a r s .
IV.
Animal T e s tin g
A.
F eeding T echniques:
Sheep fe e d in g s o f th e whole p l a n t were f i r s t
a tte m p te d w ith a b a l l i n g gun u t i l i z i n g la r g e g e l a t i n c a p su le s c o n ta in in g
ground p la n t m a t e r i a l .
This method was found to be v e ry slow f o r b o th
fe e d in g and p r e p a r a tio n o f th e c a p s u le s .
Forced fe e d in g th ro u g h a
stom ach tu b e was th e n a tte m p ted and found to be q u ite s a t i s f a c t o r y .
The p la n t m a te r ia l was p re p a red f o r d i r e c t stom ach fe e d in g by f i r s t
g rin d in g i t , w h ile f r o z e n , w ith a m eat g r in d e r .
The r e s u l t a n t fib e ro u s
mass was f u r t h e r p u lv e riz e d in a Waring b le n d e r to y ie ld a t h i c k s l u r r y .
This s l u r r y was th e n fo rc e d by m oderate p o s itiv e a i r p re s s u re d i r e c t l y
100
in to th e stomach o f th e sheep th ro u g h a 10 mm Tygon t u b e .
The a p p a ra tu s
i s i l l u s t r a t e d in F ig u re 30.
P la n t e x t r a c t s were p re p a red f o r sheep fe e d in g in th e fo llo w in g
m anner.
The s t i l l fro z e n p la n t m a te r ia l was f i r s t ground in th e cold
s o lv e n t in which i t was to be e x t r a c t e d .
Then more co ld s o lv e n t was
added and th e r e s u l t a n t m ix tu re allo w ed to sta n d in a c o o l room (10°C)
f o r 24 h o u rs .
A t th e end o f t h i s tim e th e liq u id was f i l t e r e d o f f , th e
s o lv e n t e v ap o rate d and re s u b m itte d t o th e p la n t m a te r ia l, and th e
e x tr a c te d m a te r ia ls s to r e d a t IO0C in p r e p a r a tio n f o r f e e d in g .
c y c le was perform ed th r e e tim e s .
A.
B.
C.
D.
F ig u re 30.
Hand a I r pump
2 l i t e r g la s s j a r
S te e l b it e - t u b e
Tygon stom ach tube
A pparatus f o r Sheep F e e d in g s.
This
101
The e th a n o l e x t r a c t was f u r t h e r t r e a t e d by e x tr a c ti n g th e p en tan e
to g iv e a " p o la r" and a n o n -p o la r" f r a c t i o n which were fe d s e p a r a te ly .
These p la n t e x tr a c ts were a d m in is te re d to sheep w ith a b a llin g gun
and c a p s u le s .
The p la n t t h a t had been e x tr a c te d was fe d a s a s lu r r y
w ith th e stomach tu b e .
F eedings o f th e whole p la n t were n o t a tte m p ted w ith mice o r o th e r
la b o r a to r y a n im a ls .
They were fed only e x tr a c ts and p u r if ie d compounds
which were a l l a d m in is te re d o r a l l y .
The e a r ly fe e d in g s were accom plished
u s in g a sy rin g e w ith a b lu n t n e e d le covered w ith p l a s t i c .
This method o f
fe e d in g had th e problem s o f lo s in g p a r t o f th e dosage in th e a n im a l’ s
mouth and s u f f o c a tio n o f th e an im al by in je c tin g m a te r ia l in to th e
t h r o a t , n o t th e e so p h a g u s.
I t was l a t e r found t h a t mouse fe e d in g s could
be b e t t e r accom plished by i n je c tin g th e dosage d i r e c t l y in to th e stom ach.
T his was done by means o f a 7 cm FE 2 0 , " In tra m e d ic " , p o ly e th y le n e .tu b e
(C lay Adams) a tta c h e d to a 26 gauge n e e d le on a ^ cc tu b e r c u lin s y r in g e .
A lm ost th e e n t i r e le n g th o f th e tu b e could be passed down th e esophagus
o f th e mouse and in to th e stom ach w h ile th e mouse was h e ld s e c u re ly in a
su p in e p o s i t i o n .
T his method o f fe e d in g was used f o r th e mouse fe e d in g s
numbered 8-20 in Table 4 .
A ll mice used were Swiss Manor m ales from th e colony o p erated by
D r. J . J u t i l a a t Montana S ta te U n iv e r s ity .
m ately 6 weeks a t th e tim e o f fe e d in g .
The u s u a l age was a p p ro x i­
102
B.
T e sts o f F u n c tio n :
The fo llo w in g t e s t s , BSP c le a ra n c e and ammonia
l e v e l , were ru n only on s h e e p .
To perform th e BSP c le a ra n c e t e s t
,
5 ml o f a s ta n d a rd s o l u t i o n , 50 mg/ml, o f su lfo b ro m o p h th ale in (XXVIl)
(Hynson, W estc o tt and Dunning) was in je c te d in to th e ju g u la r v e in in th e
s h e e p 's n e ck .
A liq u o t p o rtio n s o f blood were w ithdraw n from th e ju g u la r
v e in on th e o th e r s id e o f th e s h e e p 's neck a t a p p ro p ria te i n t e r v a l s .
t y p i c a l tim e sequence f o r blood sam ples w o u ld :
A
0 m inutes (b e fo re
in j e c t i o n f o r a b la n k r e a d in g ) , 5, 1 5 , 30, and 60 m in u te s .
Each blood
sample was mixed im m ediately a f t e r draw ing w ith an EDTA s o lu tio n p re ­
pared from EDTAP t a b l e t s (Cambridge Chem ical P ro d u c ts ) to p re v e n t
c lo ttin g .
The blood was th e n c e n tr if u g e d .
One ml o f th e serum was
p ip e tte d in to 10 ml o f w ater to which was added 3 ml o f a 0 .IN sodium
hydro x id e s o lu tio n t o develop th e dye c o lo r .
s o lu tio n was re a d a t 580 mju.
The ab so rb an ce o f th e
Log A was th e n p lo tte d a g a in s t tim e to
o b ta in th e u s u a l f i r s t o rd e r r a t e c o n s ta n t.
From t h i s was c a lc u la te d
th e h a lf - tim e f o r th e dye r e t e n t i o n .
To perform th e ammonia l e v e l t e s t
120
an a l iq u o t p o r tio n o f blood was
drawn and im m ediately mixed w ith a 10% TCA s o lu tio n and s to re d in i c e . '
T his blood sample was th e n c e n tr ifu g e d and 0 .5 ml o f th e r e s u l t a n t serum
p laced in a t e s t t u b e .
To t h i s was added 2 .5 ml o f a s o lu tio n o f 11 .2 g
l i q u id p henol and 0.050 g sodium n f tr o f e r r ic y a n i d e made up to one l i t e r
w ith w a te r .
To t h i s s o lu tio n was added 2 .5 ml o f a s o lu tio n o f 5.0 g
sodium hydro x id e and 7 .0 g sodium h y p o c h lo rite (P urex) made up to one
103
l i t e r w ith w a te r .
This serum m ix tu re was' a g ita te d in a therm ost a te d
(37°C) w a ter b a th f o r t h i r t y m in u tes t o develop th e c o lo r r e a c tio n and
th e n re a d a t 625 m^ a g a in s t w a te r.
■
This re a d in g was th e n co n v erted to
jug/ml u s in g a s ta n d a rd curve fo rm u lated u sin g a range o f ammonium c h lo r ­
id e s o l u t i o n s .
During th e tim e o f th e sheep fe e d in g ex p erim en ts t h e i r h e a r t a c tio n
was m onitored by th e e le c tro c a rd io g ra p h (EKG) u t i l i z i n g fo u r l e a d s .
These le a d s were th e s ta n d a rd le a d s I , I I , and I I I
122
, and a back le a d
IV w hich extended from th e back a t th e sh o u ld e r t o th e l e f t f r o n t l e g .
V.
I s o l a t i o n T echniques f o r T etradym ol ( I I )
A.
E x tr a c tio n P ro c e d u re :
The p la n t m a te r ia l was t r a n s f e r r e d w h ile s t i l l
fro z e n in to a la r g e s t a i n l e s s cone in a room k e p t a t a b o u t IO0G.
T his
cone was f i t t e d w ith an a l l m e ta l ( b r a s s ) g a te v a lv e a t th e bottom
su p p o rtin g a wad o f g la s s w ool.
Cold n-hexane was added in s u f f i c i e n t
q u a n tity to co m p letely cover th e p la n t m a te r ia l.
The cone was covered
w ith aluminum f o i l and allow ed to sta n d f o r from 8 to 20 h o u r s .
The
s o lv e n t and e x tr a c te d m a te r ia ls were th e n d ra in e d o f f th ro u g h th e bottom
v a lv e and th e s o lv e n t removed from th e e x t r a c t under w a te r a s p i r a t o r
vacuum in a steam h e a te d s t i l l .
The n-h ex an e was th e n re s u b m itte d to
th e e x tr a c ti o n cone and th e dark green-brow n re s id u e f u r t h e r c o n c e n tra te d
under w a ter a s p i r a t o r vacuum w ith th e r o tq r y e v a p o ra to r a t a te m p e ra tu re
o f SO0G.
T his crude e x t r a c t was th e n s to re d in th e co ld ( - 2 0°G) u n t i l
104
chrom atographed.
The p la n t m a te r ia l was e x tra c te d th r e e tim es, in t h i s
m anner.
B.
S i l i c a Gel Chrom atography:
The s i l i c a g e l , B io - S il A, was made up a s
as a s l u r r y in 10% d i e t h y l e th e r in n -h e x a n e ,
in to a t y p i c a l g la s s chrom atography column.
T his s l u r r y was poured
A fte r d ra in in g th e s o lv e n t
t o th e to p o f th e a d so rb e n t th e column was charged w ith crude e x t r a c t a t
th e r a t i o o f I g crude e x t r a c t t o 50 g d ry s i l i c a g e l .
The column was
th e n e lu te d in th e manner shown in T able 7 .
The f r a c t i o n s from th e column were m onitored by alum ina t i c and
th o se c o n ta in in g te tra d y m o l were c o n c e n tra te d and s to re d a t - 2 0°G in
p r e p a r a tio n f o r alum ina e l u tio n chrom atography.
The column and a d s o rb e n t could be re -u s e d e f f e c t i v e l y a t l e a s t two
more tim es by re v e rs in g th e e lu o tr o p ic s e r i e s to n-hexane a f t e r each ru n ,
and th e n re c h a rg in g th e column and e l u tin g as u s u a l.
C.
Alumina Chrom atography:
The alum ina was made up a s a s lu r r y in 50%
benzene in n-hexane t o which had been added anhydrous m ethanol in a r a t i o
o f 3 ml m ethanol t o each 100 g o f alum ina to be s l u r r i e d .
T his s lu r r y
was poured in to a t y p i c a l g la s s column and th e s o lv e n t d ra in e d t o th e
a d s o rb e n t s u r f a c e .
The column was n e x t e lu te d w ith a 50% benzene in n -
hexane s o lu tio n a t a r a t i o o f I ml f o r each I g o f d ry a lu m in a .
When
t h i s s o lu tio n had d ra in e d to th e to p o f th e a d so rb e n t th e column was
charged a t th e r a t e o f I g te tra d y m o l m ix tu re , from th e s i l i c a g e l
105
column, to 200 g alum ina and e lu te d a s shown in Table 7 .
T able 7
E lu tio n o f C hrom atographic Columns
S o lv e n t q u a n tity l/k g
. S o lv e n t m ix tu re used
n-hexane
d ie th y l e th e r
m ethanol
S i l i c a g e l e lu tio n
I
2 .
3
4 .
5 '
6
90%
85%
70%
40%
10%
15%
30%
60% .
100%
97%
3%
6
2
2
2
2
4
3%
I
I
I
Alumina e l u tio n
50%
I
2
3
50%
100%
97%
The f r a c t i o n s from th e column were m onitored by alum ina t i c and
th o se f r a c t i o n s c o n ta in in g only te tra d y m o l were c o n c e n tra te d and s to re d
a t - 2 0°C in p r e p a r a tio n f o r s u b lim a tio n .
The column and a d s o rb e n t could be re fu s e d e f f e c t i v e l y two more
tim es
b y
r e v e r s in g th e e lu o tr o p ic s e r i e s t o n-hexane a f t e r each ru n , and
th e n re c h a rg in g th e column and e lu tin g a s u s u a l .
106
In one in s ta n c e th e alum ina was a c tiv a te d b e fo re u se and packed
u s in g 30 p e r c e n t d i e th y l e th e r i n n - h e x a n e .
In t h i s in s ta n c e t e t r a -
dymol and 29-3 did n o t come o f f th e column u n t i l i t was washed w ith 2
p e r c e n t m ethanol in d i e th y l e t h e r .
to g e th e r .
D.
When th e y did come o f f th e y did so
T o ta l y ie ld from th e column was a b o u t 55 p e r c e n t.
F l o r i s i l Chrom atography:
F re s h ly a c tiv a te d F l o r i s i l (27 g ) was made
up a s a s lu r r y u sin g I p e r c e n t d i e t h y l e th e r in n -h e x a n e.
This s l u r r y
was poured in to a t y p i c a l g la s s chrom atography column and th e s o lv e n t
d ra in e d t o th e s u rfa c e o f th e a d s o r b e n t. .The column was th e n charged
w ith 350 mg o f th e m ix tu re o f te tra d y m o l ( I I ) and 29-3 from th e s i l i c a
g e l colum n.
T his column was th e n e lu te d u sin g a g r a d ie n t from n-hexane
d ie th y l e th e r m ix tu re s to d i e th y l e th e r m ethanol m ix tu re s .
As th e
e lu tio n proceeded th e column tu rn e d a b r i g h t p in k , and rem ained t h a t
c o lo r .
F.
The t o t a l y ie ld from th e column was ab o u t 90 p e r c e n t.
S u b lim atio n : ' The y ello w s o lid from th e alum ina colum n, t h a t was
shown t o c o n ta in te tra d y m o l ( I l ) by t i c , was r e c r y s t a l l i z e d fo u r tim es
from n-hexane t o g iv e w h ite c r y s t a l s .
These w h ite c r y s t a l s (173 mg) were
th e n sublim ed tw ice a t . 0.15 mm Hg w ith an o i l b a th te m p e ra tu re o f 55 to
60°C w h ile th e co ld f in g e r was m ain ta in ed a t ta p w a ter te m p e ratu re o f
from 5 t o 9°C.
The s u b lim a tio n gave 135 mg (80%) o f w h ite c r y s t a l s ; mp
9 1 .5 -9 2 .5 °C , [ a ] ^ + 44° (C 0 .9 0 , MeOH): which were s t i l l im pure.
A n a l. C a lc d . f o r C]_5H22^2:
0,76.88% ; H,9.4% .
Found:
C, 75.29%;
107
H5 8.99%; N3 l e s s th a n 0.1%.
L a te r s u b lim a tio n s were c a r r ie d o u t a s above on th e y ellow s o lid
d i r e c t l y from th e alum ina column which gave th e w h ite c r y s t a l s .
This
sublim ed m a te r ia l was used f o r th e v a rio u s d e g re d a tiv e r e a c tio n s d e s­
c rib e d h e r e in and f o r th e base e x tr a c ti o n t h a t f i n a l l y gave th e pure
compound .
F.
Base E x t r a c tio n :"■The sublim ed w h ite c r y s t a l s (0 .3 6 0 g ) were
d is s o lv e d in a 0.6M e th a n o lic p o tassiu m hydroxide s o lu tio n and k e p t in
a capped, l i g h t - p r o t e c t e d v i a l o v e rn ig h t a t a p p ro x im a te ly 25 C.
m a te r ia l was th e n e x tr a c te d th r e e tim es w ith n -h e x a n e .
T h is
The n-hexane
e x t r a c t was washed w ith s a l t s o l u t i o n , d rie d w ith NagSO^, and e v a p o ra te d .
The r e s u l t a n t c r y s t a l l i n e m a te r ia l (0 .2 8 2 g , 78%) was r e c r y s t a l l i z e d
tw ice from n-hexane t o y ie ld t i n y tr a n s p a r e n t w h ite n e e d le s ; mp 9 2 .S0C,
[ a ] ^ 5+ 56° (C 4 .3 , MeOH).
The mass spectrum showed m/e 234 (M+, 24%),
216 (5 % ), 126 (2 8 % ), 1 1 1 (3 8 % ), 109 (1 0 0 % ), 108 (4 5 % ).
A n a l. C a lc d . f o r C^gH22O2 :
C, 76.88%; H, 9.40%.
Found:
C, 76.84%;
H,. 9.25% .
V I. R eactio n s
A.
H ydrogenation o f T etrad y m o l: In a t y p i c a l r e a c tio n 1 .9 g o f 5% Rh
on alum ina was p la ce d in 300 ml o f a b s o lu te m ethanol and shaken under 35
pounds sq u are in c h p re s s u re o f hydrogen f o r one h a l f hour in a P a rr
s h a k e r.
The r e a c tio n b o t t l e was th e n opened and 2.57 g (0 .0 1 1 m ole) o f
108
sublim ed te trad y rjio l ( I l ) added „ The b o t t l e was th e n r e c lo s e d , hydrogen ■
p re s s u re o f 35 poupds/s q u a re in c h r e - e s t a b l i s h e d , and sh ak in g co n tin u ed
f o r 18 hours ( s h o r te r tim es o f from I to 5 h o u rs were i n s u f f i c i e n t f o r
com plete r e a c t i o n ) .
A t th e end o f t h i s tim e th e s o lu tio n was f i l t e r e d
th ro u g h a s in te r e d g la s s fu n n e l to remove th e c a t a l y s t .
(C au tio n I
If
th e fu n n e l i s allow ed to go co m p letely dry d u rin g f i l t r a t i o n i t spon­
ta n e o u s ly i g n i t e s .)
The s o lv e n t was th e n ev ap o rated from t h i s m ix tu re
on a r o ta r y e v a p o ra to r under w ater a s p i r a t o r vacuum.
The y ie ld of. th e
p ro d u c ts was 2 .6 0 g .
The r e s u l t a n t m ix tu re o f compounds was. s e p a ra te d by re p e a te d column
chrom atography on alum ina u sin g an e lu o tr o p ic s e r i e s o f n -h ex an e, b en zen e,
d ie th y l e t h e r , and e th y l a c e t a t e .
The in d iv id u a l compounds, w ith th e
e x c e p tio n of (XIX), were f u r t h e r p u r if ie d by s u b lim a tio n ( a t 60°C and
0.05 mm Hg) and re p e a te d r e c r y s t a l l i z a t i o n from n -h e x a n e .
The p u r i f i c a ­
tio n was m onitored by g lc u sin g a 6 f o o t , I p er c e n t OV-I liq u id phase
on GC-Q su p p o rt in a l / 8 -in c h copper colum n.
P u r ity was confirm ed by t i c
and e le m e n ta l a n a l y s i s .
The p ro d u c ts were a s fo llo w s w ith t h e i r is o la te d form , m e ltin g
p o in t, Rf (o n .alu m in a t i c u sin g 40% n-hexane in d i e th y l e t h e r ) , e le m e n ta l
fo rm u la , mass s p e c t r a l p a re n t p eak , and a n a ly s is g iv en in t h a t o r d e r .
The y ie ld s o f in d iv id u a l p ro d u c ts a re g iv e n in Table 6, page 63.
109
(XIX)
C o lo rle s s liq u id ( a t l e a s t 7 components by g l c ) , .9 0 ,
C i5H26O, 222 m /e, no a n a ly s is o b ta in e d .'
(XVIIA)
C o lo rle s s p la te s from n -h e x a n e , 75 .5 -7 7 °C , .4 7 ,
cI 5H26O2 ? 238 m /e. C a lc d .: C, 7 5 .6 3 ; H, 1 0 .9 2 .
Found:
C, 75.88 ; H, 1 0 .9 2 .
(XVIIB) . C o lo rle s s b lo c k s from n -h e x a n e , 104-105°C, .3 6 ,
Ci s h26O2 , 238 m /e. C a lc d .: C, 7 5 .6 3 ; H, 1 0 .9 2 .
Found:
(X V III)
B.
C, 7 5 .4 5 ; H, 1 1 .0 0 .
C o lo rle s s n e e d le s from n -h e x a n e , 145-1460C, .1 0 ,
^15^24^2?
m/ e • C a lc d .: C, 7 6 .2 7 ; H, 1 0 .1 7 .
Found: C, 7 6 .1 8 ; H, 1 0 .1 8 .
M ercury C h lo rid e D e riv a tiv e o f T etrad y m o l:
123
To 2.7 2 g (0 .0 1 m ole)
HgCl2 in 25 ml h o t H2Q was added 5.44 g (0 .0 4 m ole) NaOAc in 10 ml HgO,
th e n 24 ml e th a n o l.
The s o lu tio n was allow ed t o sta n d a t room tem pera­
tu r e o v e rn ig h t, when i t was f i l t e r e d and u se d .
When 600 mg o f sublim ed tetu ad y m o l ( I l ) (0.0026 m ole) was d is s o lv e d
in 8 ml o f 95% e th a n o l and slow ly added to 17 ml (0.0028 moles Hg) o f
th e above s o lu tio n th e r e was an immediate fo rm atio n o f fh e f lo c c u le n f
p r e c i p i t a t e o f th e m ercury c h lo rid e d e r i v a t i v e .
f i l t e r e d to g iv e 1 .4 9 g (c ru d e ) o f a w h ite s o l i d .
The s o lu tio n was
T his p ro d u c t was r e ­
c r y s t a l l i z e d from h o t e th a n o l to y ie ld tr a n s p a r e n t, w h ite n e e d le s , mp
£ 0 5 -2 0 6 .5°C (w ith some d e c o m p o sitio n ).
When a few m illig ra m s o f th e m ercury c h lo rid e d e r iv a tiv e o f t e t radym ol was d is s o lv e d in an aqueous e th a n o l s o lu tio n and HgS bubbled
th ro u g h th e s o lu tio n f o r a few m in u te s , te tra d y m o l was re g e n e ra te d as
HO
dem onstrated by i t s m o b ility and c o lo r r e a c tio n on t i e .
C.
M aleic A nhydride Adduct o f T etrad y m o l:
To a s o lu tio n o f 140 g
m aleic a n h y d rid e in 40 ml o f d ie th y l e th e r was added 50 mg o f
(II).
te tra d y m o l
This s o lu tio n was capped and allow ed to sta n d in th e d ark a t room
te m p e ra tu re o v e rn ig h t.
The r e s u l t a n t m ix tu re was s p o tte d on A n a sil t i c
and developed w ith 50% a c e to n e in m ethanol to r e v e a l th r e e s p o ts made
v i s i b l e by sp ra y in g w ith s u lfu ric -d ic h ro m a te s o l u t i o n .
an Rf .8 2 J1 .3 5 , and .1 8 .
s y ste m .)
Tfyese s p o ts fyad
(M aleic an h y d rid e had an Rf .27 in fyhis
The s o lv e n t was e v ap o rate d u n d er vacuum and th e w hite s o lid
su b m itte d to mass s p e c t r a l a n a ly s is to r e v e a l a p a re n t peak M+332 m /e.
B efore th e m a te r ia l could be p u r if ie d and f u r t h e r c h a r a c te r iz e d i t
tu rn e d p in k and th e n brown o v e r n ig h t.
No a tte m p t was made to p r o te c t
from a i r or l i g h t a f t e r th e s o lv e n t was e v a p o ra te d .
D.
S a r e t t O x id atio n o f Tetradym ol:"1"2^
To an ic e cooled f l a s k c o n ta in in g
200 ml of p y rid in e was slow ly ad d ed ^ w ith m agnetic s t i r r i n g , 20 g o f
CrOg.
A f te r t h i s m ix tu re had s t i r r e d f o r 10 m in u te s, 2 .0 2 g o f t e t r a d y ­
mol ( I I ) in 200 ml o f p y rid in e was ad d ed .
The c o o lin g was now rem oved,
a condenser f i t t e d , and th e su sp e n sio n allow ed to s t i r in th e dark f o r
8 hours a t which tim e no te tra d y m o l was d e te c ta b le by t i c .
e th e r was added
125
D ie th y l
to th e su sp e n sio n to p r e c i p i t a t e th e r e s t of th e
chromium s a l t s and th e su sp e n sio n f i l t e r e d .
The s o lu tio n was a c i d i f i e d
w ith 6N HCl and re p e a te d ly e x tr a c te d w ith e th y l a c e ta te and d ie th y l
Ill
e th e r.
The e x t r a c t was washed w ith an a c i d i f i e d s a l t s o lu t i o n , d rie d
and e v a p o r a te d .
The gummy p ro d u c t c r y s t a l l i z e d w ith d i f f i c u l t y a f t e r
th e a d d itio n o f n-hexane and r e c r y s t a l l i z e d from d i e th y l e th e r t o y ie ld
0.92 g (40%) o f th e p ro d u c t (V l) a s w h ite c r y s t a l s , mp 1 6 1 -1 6 2 .5°C, uv
max 221 mjli (lo g E 4 .0 5 ) .
The mass spectrum showed m/e 266 (M+, < 0.1% ),
123 (100%).
A n a l. C a lc d . f o r C1 5H2 2 °4 :
6 7 .6 5 ; H, 8 .3 3 .
Found:
C, 6 7 .5 0 ;
' H, 8 .4 1 .
E.
S a r e t t O x id a tio n o f M enthofuran:
In a manner an alogous to th e above
p ro ced u re 10 g o f m enthofuran was o x id iz e d .
Ip t h i s in s ta n c e th e r e a c ­
t i o n was worked up by adding water"*"^ and e x tr a c tin g w ith n-hexane and
d i e th y l e t h e r , to remove th e u n re a c te d m en th o fu ran , th e n a c id i f in g w ith
HCl and e x tr a c ti n g w ith d ie th y l e t h e r .
A m onstrous em ulsion r e s u lte d
and i t was only w ith d i f f i c u l t y t h a t 1 .9 g (16%) o f th e o x id a tio n
p ro d u c t (V II) was o b ta in e d a s w h ite c r y s t a l s from m ethyl a c e ta te and n h exane, mp 190-191°C (re p o rte d 188°C)"*"^^, uv max 216
F.
A ttem pted A c e ty la tio n o f T etrad y m o l:
mfJ,
(lo g 8 4 .0 7 ) .
Tetradym ol ( I I ) (4 .8 mg) was
d is s o lv e d in I ml p y rid in e t o which was added I ml a c e t i c a n h y d rid e .
128
The system was flu s h e d w ith n itr o g e n , s e a le d and allow ed to stan d in th e
dark a t room te m p e ra tu re o v e rn ig h t.
When t h i s s o lu tio n was worked up in
th e u s u a l manner s t a r t i n g m a te r ia l was is o la te d in a b o u t 50 p e r c e n t
y ie ld and no a c e ta te could be d e te c te d by t i c .
112
When a c e ty la tio n was a tte m p ted w ith a c e t y l ' c h lo rid e in n -h e x a n e,
th e s o lu tio n tu rn e d b lu e and gave an u n s ta b le n o n -p o la r compound t h a t
behaved on t i c in much th e same manner a s th e d e h y d ra tio n p ro d u c t o f
te tra d y m o l u sin g F l o r i s i l .
A c e ty la tio n was a ls o a tte m p te d u sin g a c e tic - f o r m ic a n h y d rid e .
129
To 51 g o f a c e t i c an h y d rid e (0 .5 m ole) was added 23 g o f 91 p e r c e n t
fo rm ic a c id a t O0C.
T his s o lu tio n was th e n h e ate d to 45°C f o r I hour
■and used d i r e c t l y when c o o l.
When 37 mg o f te tra d y m o l was added to 0 .5
ml o f t h i s s o lu tio n i t im m ediately tu rn e d y e llo w .
A t th e end o f 2
m inutes th e s o lu tio n was re d tu rn in g la v e n d e r in a few more m in u te s .
The r e a c tio n m ix tu re tu rn e d g re e n when i t was quenched w ith aqueous
NaHCOg a f t e r 2 h o u rs.'
I t was e x tr a c te d w ith d ie th y l e th e r and s p o tte d
on A n a s il S t i c and developed in d i e th y l e t h e r .
The s p o tte d m a te r ia l
tu rn e d b lu e a s soon a s i t touched th e a d so rb e n t and t h i s b lu e s p o t d id
n o t move on developm ent.
There w as3 how ever, one c o lo r le s s su b sta n c e in
th e r e a c tio n m ix tu re t h a t did. move t o Rf ,8 and was made v i s i b l e by
sp ra y in g w ith s u lfu ric -d ic h ro m a te s o lu t i o n .
This compound was n o t f u r ­
th e r c h a r a c te r iz e d due t o low y i e l d .
G.
D ehy d ratio n o f T etradym ol:
Sublimed te tra d y m o l (130 mg) was d is s o lv e d
in 10 ml o f benzene and re flu x e d over 2 g o f F l o r i s i l .
130
The F l o r i s i l
a lm o st im m ediately tu rn e d la v e n d e r and in 20 m inutes was b lu e - b la c k .
90 m inutes a lm o st a l l th e te tra d y m o l was gone, a s evidenced by t i c , so
th e benzene s o lu tio n was d ecanted and th e F l o r i s i l e x tr a c te d w ith
In
113
m e th a n o l.
The m ethanol e x t r a c t was orange le a v in g th e F l o r i s i l a sky
b lu e c o l o r .
(T h is c o lo r was v e ry s ta b l e on th e F l o r i s i l a s i t rem ained
w ith l i t t l e v i s i b l e change f o r weeks w h ile open in th e la b o r a to r y .)
The
benzene s o lu tio n y ie ld e d a n o n -p o la r component which could be is o la te d by
e lu tio n chrom atography.
However, t h i s compound r a p id ly decomposed to a
more p o la r compound w hile s p e c tra were b ein g ru n .
H.
A ttem pted A ro m a tiz a tio n :
Sublimed te tra d y m o l (85 mg) was d is s o lv e d
in 15 ml of trig ly m e t o which was th e n added 27 mg o f s u l f u r .
s o lu tio n was re flu x e d f o r I h o u r, th e n steam d i s t i l l e d .
This
'
'
The r e s u l t a n t
l i g h t y ello w s o lid showed i t s e l f by t i c and ms t o be i d e n t i c a l w ith
te tra d y m o l ( I l ) p lu s a l i t t l e e le m e n ta l s u l f u r .
When a n o th e r sample was
re flu x e d under th e same c o n d itio n s f o r 12 ho u rs much o f th e te tra d y m o l
d isa p p e a re d le a v in g a re d d is h s o l u t i o n .
When t h i s s o lu tio n was an aly zed
by t i c th e r e was s t i l l te tra d y m o l rem ain in g along w ith some more p o la r
compounds in v e ry low c o n c e n tra tio n .
An a tte m p t was a ls o made to a ro m a tiz e th e te tra d y m o l by th e p ro ced u re
o f M in ato .
132
An in tim a te m ix tu re o f 50 mg o f te tra d y m o l and 200 mg o f 5
p e r c e n t Pd on carbon was h e ate d u n d er a n itr o g e n atm osphere to 320°C f o r
5 m in u te s .
The r e s u l t a n t y ellow o i l was chrom atographed on s i l i c a g e l t o
y ie ld two f r a c t i o n s t h a t were much l e s s p o la r th a n te tra d y m o l.
The
m a te r ia ls were an aly zed by g lc on a 15 f o o t , I p e r c e n t OV-I liq u id
phase on GC-Q s u p p o rt in a 1 /8 -in c h copper column t o r e v e a l a t l e a s t 7
components t h a t were n o t f u r t h e r c h a r a c te r iz e d .
114
I-.
D ehydration o f (XVIIB):
To 50 ml o f a c id w ashed, r e d i s t i l l e d
heptane was added 20 pig o f io d in e th e n 680 mg o f (XVIIB),,133
The f l a s k
was f i t t e d w ith a condenser and m agnetic s t i r r e r and th e s p lu tio n r e ­
flu x e d f o r 2 .5 h o u r s .
The r e s u l t a n t m ix tu re o f p ro d u c ts wps chrom ato­
graphed on alum ina u sin g hexane and d ie th y l e th e r t o y ie ld fo u r f r a c tio n s
w ith a t l e a s t e ig h t com ponents, by g l c .
The o v e r a ll y ie ld was 430 mg.
The m ajor component (XXVI) was p r e s e n t in a y ie ld o f 50 p e r c e n t from
s t a r t i n g m a te r ia l, and o f is o la te d m a te r ia l 75 p e r c e n t.
The compound
(XXVI) was c a r e f u l ly vacuum d i s t i l l e d from a sm a ll s e a le d tube t o a d ry
ic e a c e to n e t r a p a t 0.05 mm Hg a s a f u r t h e r p u r i f i c a t i o n f o r s p e c t r a l
a n a l y s i s ,■
t
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MONTANA STATE UNIVERSITY LIBRARIES
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R eed er, Samuel
I s o l a t i o n and i d e n t i f i ­
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p r in c ip le from
T etradym ia g la b r a ta