Isolation, Separation, And Structural Identification Of Streptomyces Quinones

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Content ISOLATION, SEPARATION, AND STRUCTURAL IDENTIFICATION
OF STREPTOMYCES QUINONES
by
PHILIP G. PHILLIPS
A D i s s e r t a t i o n P r e s e n te d to th e
FACULTY OF THE GRADUATE SCHOOL,
UNIVERSITY OF SOUTHERN CALIFORNIA
In P a r t i a l F u lf il lm e n t o f th e
R eq u irem en ts f o r t h e D egree
DOCTOR OF PHILOSOPHY
( H ic ro b io lo g y )
Ju n e 1971
71-27 ,94-6
I
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f University Microfilms, A XEROX C om pany, Ann Arbor, Michigan
f e ' . .
PH ILLIPS, P h i l i p G. , 1941-
ISOLATION, SEPARATION, A N D STRUCTURAL
IDENTIFICATION OF STREPTOMYCES QUINONES.
U n i v e r s i t y o f S o u th e rn C a l i f o r n i a , P h .D ., 1971
B io c h e m is tr y
T H IS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED
UNIVERSITY O F S O U T H E R N CALIFORNIA
TH E G R A D U A T E S C H O O L
U N IV E R S IT Y PA R K
LOS A N G E L E S , C A L IF O R N IA 9 0 0 0 7
T h is dissertation, w ritte n by
.......................PHILIP. G . PHILLIPS.................................
under the direction o f h.i.S ... Dissertation Com
mittee, and approved by all its members, has
been presented to a n d accepted by The Gradu
ate School, in p a rtia l fulfillment of require
ments of the degree o f
DOCTOR O F PHILOSOPHY
- ............................................. I f
D a te J«ReJL971
DISSERTATION COMMITTEE
Chairman
TABLE OF CONTENTS
D e d ic a tio n iv
Acknowledgments v
L i s t o f T ab les v i
L is t o f F ig u re s v i i
I n tr o d u c tio n 1
P la s to q u in o n e s 1
R hodoquinones 2
U biquinones 3
N aphthoquinones 6
Q uinone F u n c tio n s 12
Q uinone B io s y n th e s is 13
M a te r ia ls and M ethods 16
O rganism s Used 16
C hem icals and S u p p lie s 16
L ip id E x tr a c tio n o f Whole C e lls 17
Column C hrom atography 18
T hin L ayer C hrom atography 19
S y n th e s is and P u r i f i c a t i o n o f S y n th e tic Compounds 21
P h y s ic a l M ethods 26
R e s u lts 29
S e p a ra tio n o f Q uinones 29
I n f r a r e d S p e c tro sc o p y 40
Mass S p e c tro m e try 40
N u c le a r M agnetic R esonance S p ectro sco p y 45
• •
l i
TABLE OF CONTENTS (CONTINUED)
O z o n o ly sis S tu d ie s 55
Q uinone R e s to r a tio n o f O x id a tiv e P h o s p h o ry la tio n 56
D is c u s sio n 63
R e fe re n c e s 70
• 3 •
111
DEDICATION
I w ould l i k e to d e d i c a te t h i s t h e s i s to my w ife P h y l l i s a n d to
o u r c h i ld r e n D avid and B eth w hose p a t ie n c e has made t h i s work p o s s i b l e .
a l s o
I w ould l i k e t o d e d i c a te t h i s t h e s i s to a l l o f t h e p eople o f
t h i s w orld who a r e l e s s f o r t u n a t e t h a n I . I hope my s c i e n t i f i c
a c h ie v e m e n ts w i l l in some way make u p f o r p a r t o f t h i s i n j u s t i c e .
ACKNOWLEDGMENTS
I w ould l i k e t o e x p r e s s my d e e p a p p r e c i a t i o n t o D r. A rn o ld F .
B ro d ie f o r h i s c o n tin u e d g u id a n c e d u r in g t h e c o u r s e o f my s t u d i e s .
A lso I w ish t o th a n k D rs. P a t r i c k J . Dunphy, K en n eth L. S e r v i s , and
N ic h o la s N ic o la id e s f o r t h e i r a s s i s t a n c e . I am a l s o in d e b te d t o
D r. I r v i n g G ordon who h a s c o n s t a n t l y d is p la y e d an i n t e r e s t in my
p r o g r e s s .
v
LIST OF TABLES
Number T i t l e Page
1 The O ccu rren ce o f U biquinones 5
2 The O ccu rren ce o f M enaquinones 8 , 9
3 Quinone R e s to r a tio n o f O x id a tiv e P h o s p h o ry la tio n 57
4 Quinone R e s to r a tio n o f O x id a tiv e P h o s p h o ry la tio n 58
5 Quinone R e s to r a tio n o f O x id a tiv e P h o sp h o ry la tio n 59
vi
LIST OF FIGURES
Number T i t l e Page
1 S e p a r a tio n o f M enaquinones by A d so rp tio n C h rom atography 30
2 U l t r a v i o l e t S p e c tra o f M K g in I s o o c ta n e 32
3 O x id iz ed and Reduced U l t r a v i o l e t S p e c tra o f M K g in E th a n o l 33
4 S e p a r a tio n o f M enaquinones by S i l v e r Io n Im p reg n a ted
A d so rp tio n C hrom atography 34
5 S e p a r a tio n o f S tre p to m y c e s Q uinones by S i l v e r Io n
Im p re g n a te d A d s o rp tio n C hrom atography 35
6 S e p a r a tio n o f M enaquinones by R ev ersed P h ase
C hrom atography 36
7 S e p a r a tio n o f S tre p to m y c e s Q uinones by R ev ersed P h ase
C hrom atography 39
8 I n f r a r e d S p e c tra o f M K g 41
9 N u c le a r M agnetic R esonance S p e c tra o f M K g ( I I - H ) i n CCl^ 46
10 N u c le a r M agnetic R esonance S p e c tra o f Band 2 in C C ^ 47
11 N u c le a r M ag n etic R esonance S p e c tra o f Band 1 in B enzene 50
12 N c u le a r M ag n etic R esonance S p e c tra o f Band 2 in B enzene 51
13 N u c le a r M ag n etic R esonance S e p c tra o f Band 3 in B enzene 52
14 N u c le a r M agnetic R esonance S p e c tra o f Band 5 in B enzene 53
15 N u c le a r M agnetic R esonance S p e c tra o f Band 6 in B enzene 54
vii
INTRODUCTION
The S tre p to m y c e s q u in o n e s w h ich form th e s u b j e c t m a tte r o f t h i s
t h e s i s w ere d is c o v e r e d w h ile s u rv e y in g s e v e r a l m ic ro o rg a n is m s f o r
q u in o n e c o n t e n t . When i t w as4fo u n d t h a t t h i s o rg a n is m c o n ta in e d a
m u l t i q u in o n e s y s te m , t h a t i s m ore th a n o ne q u in o n e s p e c i e s , i t was
o f i n t e r e s t t o d e te rm in e t h e e x t e n t and ty p e o f q u in o n e v a r i a t i o n .
H aving d e v e lo p e d s e v e r a l new te c h n iq u e s f o r t h e s e p a r a t i o n o f
q u in o n e s i t was f e a s i b l e t o e x p e c t t h a t a l l o f t h e q u in o n e s p e c ie s
c o u ld b e i s o l a t e d w ith s u f f i c i e n t p u r i t y an d in am o u n ts w h ich w ould
a llo w f o r a c c u r a t e s t r u c t u r a l i d e n t i f i c a t i o n s .
As t h e s tu d y p r o g r e s s e d ad d e d i n t e r e s t was g e n e r a te d b e c a u s e
th e ty p e o f v a r i a t i o n d is p la y e d b etw ee n t h e v a r io u s m o le c u le s had
n o t p r e v io u s l y b e e n d e s c r ib e d . I n g e n e r a l q u in o n e s a r e fo u n d u b iq
u i t o u s l y i n b o th t h e p l a n t and a n im a l k in g d o m s. T h ese q u in o n e s
d i s p l a y a w ide v a r i e t y o f s t r u c t u r a l v a r i a t i o n s w hich form a b a s i s
f o r d i v i d i n g them i n t o s e v e r a l m a jo r g r o u p s . S t r u c t u r a l v a r i a t i o n
w ith i n g ro u p s i s a l s o q u i t e common. W h ile i t i s beyond t h e sco p e
o f t h i s i n t r o d u c t i o n t o re v ie w c o m p le te ly t h e w ide ra n g e o f e x i s t i n g
n a t u r a l q u in o n e s , b r i e f m e n tio n w i l l b e made o f some o f th o s e com
pounds n o t t o b e d is c u s s e d in d e t a i l .
P la s to q u in o n e C h lo r o p la s ts o f h ig h e r p l a n t s c o n t a in a b e n z o q u in o n e
w h ich was f i r s t i s o l a t e d from a l f a l f a by K o fle r i n l ^ B ( 1 ) . When
t h i s compound was s u b s e q u e n tly i s o l a t e d fro m c h l o r o p l a s t s o f g re e n
1
p la n ts by C rane ( 2 ) , i t was g iv e n th e name p la s to q u in o n e (PQ ). The
p la s to q u in o n e i s o l a t e d by C rane i s a 5 ,6 -d im e th y l benzoquinone su b
s t i t u t e d a t th e C-3 p o s iti o n w ith a C 1 + 5 is o p re n o id s id e c h a in , ( I ) .
The p la s to q u in o n e s id e c h a in i s r e l a t e d to a n a t u r a l p ro d u c t
a lc o h o l, s o la n e s o l, f i r s t is o l a t e d from to b a c c o p la n ts ( 3 ) , and
su b se q u e n tly c h e m ic a lly i d e n t i f i e d a s a 0 ^ 5 is o p re n o id a lc o h o l (4 -,5 ).
S in ce i t s p re s e n c e in h ig h e r p l a n t s was d e m o n stra te d , p la s to q u in o n e
h as a ls o been shown t o o c c u r in brown a lg a e , b lu e -g re e n a l g a e , re d
a lg a e ( 6 ) and in g re e n a lg a e ( 6 ,7 ) . Q uinones r e l a t e d to PQg have
a ls o b een i d e n t i f i e d ( 8 ) . These compounds d is p la y e d u l t r a fc io le t
a b s o rp tio n s p e c tr a s im i la r to th e o r i g i n a l p la sto q u in o n e ( c a l le d
p la sto q u in o n e A) b u t d i f f e r e d in t h e i r ch ro m a to g rap h ic b e h a v io r.
These p la s to q u in o n e s a r e c a l le d p la s to q u in o n e B, C, D e t c . A p l a s t o
quinone o f s h o r te r c h a in le n g th th a n PQg h as a ls o been is o la te d and
was shown to be PQ^ ( 9 ) .
Rhodoquinone A u n iq u e benzoquinone was is o l a t e d from th e p h o to
s y n th e tic b a c te riu m R h o d o sp iriliu m rubrum (1 0 ) w hich was g iv e n th e
name rh o d o q u in o n e. The s t r u c t u r e o f t h i s compound ( I I ) was id e n
t i f i e d a s a r in g s u b s t i t u t e d amino b e n zo q u in o n e.
O
3
O
NH2
II
U b iq u in o n e The m a jo r p a r e n t b en zo q u in o n e fo u n d in a n im a ls , p l a n t s ,
and m ic ro o rg a n ism s i s a 2 - m e th y l- 3 - p o ly is o p r e n o id , 5 ,6 - d im e th o x y - l,4 -
b en z o q u in o n e named u b iq u in o n e (Q) o r coenzym e Q ( I I I ) .
CH CH
The m a jo r d i f f e r e n c e s s e e n i n t h i s compound a r e v a r i a t i o n s i n t h e
le n g th o f t h e C -3 is o p r e n o id s i d e c h a i n . A p a r t i a l l i s t o f th e
o c c u rre n c e o f u b iq u in o n e s in n a t u r e i s g iv e n i n T a b le 1 . Two p o i n t s
sh o u ld b e made c l e a r a t t h i s tim e . F i r s t , t h a t t h i s t a b l e i s g r e a t l y
a b b r e v ia te d due t o t h e tre m e n d o u s l i s t o f p u b l i c a t i o n s c o n c e rn in g
th e n a t u r a l o c c u r r e n c e o f u b iq u in o n e s .
More im p o r ta n t, h o w ev er, i s th e f a c t t h a t t h e hom ologues id e n
t i f i e d fro m t h e s e v a r io u s s o u r c e s a e e , in many c a s e s , o n ly t h e m a jo r
u b iq u in o n e fo u n d . T h is i s b e c a u se u n t i l r e c e n t l y p u r i f i c a t i o n
te c h n iq u e s w ere o n ly d e v e lo p e d w hich a llo w e d f o r r a t h e r u n s o p h is
t i c a t e d s e p a r a t i o n s . F o r i n s t a n c e , w h ile E s c h e r ic h ia c o l i i s r e
p o r te d i n m o st p u b l i c a t i o n s t o c o n t a in Qg» i t h a s r e c e n t l y b een
4
shown t h a t , in f a c t , t h e u b iq u in o n e fro m t h i s o rg a n is m c a n b e s e p
a r a t e d i n t o Q^q th r o u g h an d p e r h a p s , ev e n t o show t h e p r e s e n c e
o f Q3 th r o u g h Q i ( 3 4 ) .
As i s t h e c a s e w ith o t h e r t y p e s o f q u in o n e s , s t r u c t u r a l v a r -
i s t i o n s o t h e r th a n c h a in l e n g t h d i f f e r e n c e s do o c c u r in t h e u b i
q u in o n e s . P a r t i c u l a r m e n tio n o f o n ly o n e ty p e o f v a r i a t i o n w i l l be
made h e r e b e c a u s e i t i s s i m i l a r t o t h a t s e e n w ith t h e S tre p to m y c e s
m e n aq u in o n es w h ich a r e t h e s u b j e c t o f t h i s t h e s i s . I n 1962 L a v a te
e t a l . (3 5 ) i s o l a t e d a u b iq u in o n e fro m P e n i c i l l i u m s t i p i t a t u m w h ich
d i f f e r e d fro m t h e Q ^ 0 fo u n d i n m o st P e n i c i l l i u m s p e c i e s (2 3 ) i n t h a t
i t p o s s e s s e d o n e s a t u r a t e d d o u b le bond i n i t s i s o p r e n o i d s i d e c h a i n .
The f o llo w in g y e a r , a u b iq u in o n e w as i s o l a t e d fro m G ib b e r e l l a
f u j i k u r o i w h ich a l s o had o n e s a t u r a t e d d o u b le bond in i t s s i d e
c h a in ( 2 6 ) . I n t h i s c a s e t h e u s e o f n u c l e a r m a g n e tic r e s o n a n c e
s p e c tr o s c o p y d e m o n s tr a te d t h a t t h e s a t u r a t e d is o p r e n o i d u n i t was
t h e o ne a t t h e - t e r m i n a l en d o f t h e c h a in ( i . e . f u r t h e s t fro m t h e
r i n g ) . O th e r e v id e n c e show ed t h a t t h i s q u in o n e h ad t e n is o p r e n o i d
u n i t s m ak in g t h e name o f t h i s com pound Q iq (X -H ) w h ich i d e n t i f i e s
t h e t e n t h (X ) i s o p r e n o i d u n i t a s t h e s a t u r a t e d o n e . I t s h o u ld b e
m e n tio n e d t h a t t h i s o rg a n is m a l s o c o n t a i n s Q 1 q . I n 1964 L a v a te
an d B e n tly (3 6 ) ex am in ed 18 m o ld s an d fo u n d t h a t 10 o f them c o n
t a i n e d m o n o - s a tu r a te d h o m o lo g u es o f Q io« t h e s e c a s e s , h o w e v e r,
no f u r t h e r i d e n t i f i c a t i o n c o n c e r n in g t h e e x a c t l o c a t i o n o f s a t u r a t i o n
w as m ade. T h ese q u in o n e s a r e r e f e r r e d t o a s Q 1 0 (2 -H ) i n d i c a t i n g
t h a t t h e r e a r e tw o h y d ro g e n s p r e s e n t i n t h e s i d e c h a in w h ich a r e
i n a d d i t i o n t o t h e n o rm a l is o p r e n o i d c o m p lem e n t.
5
TABLE 1
The O ccu rren ce o f U biquinones
S o u rce Homologue R e fe re n c e
P ro to z o a
Qe. Q9a
1 1 , 1 2
Sea u rc h in
Qio
13
I n s e c ta
^9* Qioa
1 4 ,1 5
A n n elid a
Qio
14
C ru c ta c e a
Qio
14
V a rio u s f i s h e s
Q 1 0
16
A m phibians
Q 1 0
14
V a rio u s b i r d s
Qio
1 4 ,1 7
Mammalian s o u rc e s
Q9 » Qioa
1 4 ,1 6 -2 2
Fungi
Q6 th r u Q ioab
1 4 ,2 3 -2 6
B a c te r ia Q4 th r u Qgab 7 ,1 0 ,1 4 ,2 3 ,2 7 -2 9
P la n ts Qg th r u Q 10a 7 ,1 4 ,2 5 ,3 0 -3 3
a ) Some in d i v id u a l s o f th e s e so u rc e g ro u p s c o n ta in more th a n one
s i n g l e u b iq u in o n e hom ologue b u t a l l do n o t c o n ta in a l l hom ologues
l i s t e d .
b ) In some c a s e s th e s e o rg a n ism s a l s o c o n ta in p a r t i a l l y h y d ro
g e n a te d hom ologues.
c ) Some e v id e n c e s u g g e s ts t h i s sh o u ld re a d th ro u g h Q 1 0 .
N ap h th o q u in o n e s The o t h e r m a jo r g ro u p o f q u in o n e s t o be d is c u s s e d
w h ich a r e p r e s e n t in l i v i n g o rg a n is m s a r e th e n a p h th o q u in o n e s . T hese
com pounds a r e b u i l t o n a b a s ic s t r u c t u r e o f a n a p h th a le n e r i n g sy ste m .
One n a p h th o q u in o n e w h ich h a s b e e n shown t o o c c u r i n p l a n t s (3 7 ) i s
V ita m in K j_ a l s o c a l l e d p h y llo q u in o n e . The s t r u c t u r a l i d e n t i f i c a t i o n
o f t h i s com pound, o r i g i n a l l y d is c o v e r e d by Dam ( 3 8 ) , was w orked o u t
in s e v e r a l l a b s (3 9 -4 3 ) and was v e r i f i e d by s y n t h e s i s a s b e in g 2 -
m e t h y l - 3 - p h y t y l - l , 4 -n a p h th o q u in o n e ( I V ) , ( 4 4 ) .
T h ere a r e o n ly two known n a t u r a l l y o c c u r r in g v a r i a t i o n s o f t h i s com
pound a lth o u g h many s y n t h e t i c v a r i a t i o n s have b e e n d e s c r i b e d , some
o f w h ich w i l l be r e f e r r e d t o l a t e r in a d is c u s s io n o f t h e i r u s e in
t h e s tu d y o f o x i d a t i v e p h o s p h o r y la tio n .
A m o d ifie d V ita m in was i s o l a t e d from s p in a c h w hich la c k e d a
m e th y l g ro u p a t t h e C-2 p o s i t i o n ( 4 5 ) . T h is compound i s named
d e s m e th y l V ita m in K -^ an d i s s i m i l a r t o d e s m e th y l m en aq u in o n es t o be
d is c u s s e d i n t h e f o llo w in g s e c t i o n . A seco n d m o d ifie d V ita m in was
i s o l a t e d by A lle n e t a l . (4 6 ) an d i t s s t r u c t u r e , a lth o u g h n o t f u l l y
i d e n t i f i e d , was r e p o r t e d t o c o n t a in a h y d ro x y l g ro u p i n t h e s i d e c h a in
d i s t a l t o t h e c h a in te r m i n a l is o p r e n o id u n i t .
By f a r t h e m a jo r g ro u p o f n a p h th o q u in o n e s fo u n d i n n a t u r e a r e th e
m e n aq u in o n es (MKS) (V ).
O
IV
7
O
n= 1 - 9
V
I t can be seen t h a t V itam in i s a s t r u c t u r a l l y m o d ifie d MK^. The
s t r u c t u r e co u ld b e named M K ^ ( I I , I I I , IV -H ). B ecause t h i s i s th e
g ro u p o f quinones o f m ajor i n t e r e s t t o t h i s t h e s i s , an e x ten d ed l i s t
o f m enaquinone o c c u re n c e i s p re s e n te d in T ab le 2.
I t sh o u ld be n o te d t h a t in th e c a s e o f th e m enaquinones a
s i t u a t i o n an alo g o u s t o t h a t seen w ith th e u b iq u in o n e s can u n d o u b te d ly
b e e x p e c te d . T hat i s , t h a t th e r e a r e c e r t a i n l y many hom ologues
p r e s e n t in some o rg an ism s th e p re s e n c e o f w hich mould be d e m o n stra te d
by a more in te n s iv e s e a r c h . An exam ple o f t h i s i s found in
S tre p to m y c e s. In 1959 L e s te r and C rane (1 4 ) r e p o r te d t h a t th e s e
o rg an ism s c o n ta in e d a m enaquinone, how ever, t h i s compound was n o t
c h a r a c te r iz e d f u r t h e r . As w i l l be d e m o n stra te d in t h i s t h e s i s , th e
o rg an ism s tu d ie d h a s a t l e a s t e ig h t d i f f e r e n t m enaquinones. A lso
S trep to m y ces o liv a c e u s h a s b een shown to c o n ta in a t l e a s t th e f i v e
m enaquinone-9 hom ologues found in th e org an ism s tu d ie d h e re ( 4 7 ) . I t
may w e ll be t h a t t h e org an ism u sed in t h i s t h e s i s stu d y was S.
o liv a c e u s s in c e d ue t o com m ercial re a s o n s no e x a c t i d e n t i f i c a t i o n o f
t h e S treptom yces o rg an ism in t h i s stu d y c o u ld be made.
As in d ic a te d in T ab le 2 c a u tio n m ust b e ta k e n in assum ing t h a t
a n im a ls p o s se ss a m enaquinone. The e v id e n c e p re s e n te d f o r i t s ( t h e i r )
8
T A B L E 2
The O cc u rre n c e o f M enaquinones
S o u rce
S tre p to m y c e s s p e c ie s
M ycobacterium p h l e i
M ycobacterium t u b e r c u l o s i s
E s c h e r ic h ia c o l i
S tr e p to c o c c u s f a e c a l i s
C hlorobium th i o s u l f a to p h i lu m
C hloropseudom onas e th y lic u m
C o ry n e b a c te riu m d i p t h e r i a e
C o ry n e b a c te riu m c r e a t en o v o ra n s
B a c i ll u s s u b t i l i s
B a c i ll u s m eg ateriu m
B a c i ll u s c e re u s
S a r c in a f l a r a
S ta p h y lo c o c c u s a lb u s
S a r c in a l u t e a
P ro te u s v u l g a r i s
Chrom atium vinosum
S ta p h y lo c o c c u s a u re u s
Homologue R e fe re n c e
MKg, MKq(II-H)MKg(4-H)
MKg (6-H), MKg(8-H)
MK8 (2-H), MKg(4 - H )MKg(6-H) 47
MKg ( I I - H )
MKg (2-H)
M K g ( 2 - H ) M K g ( 2-H)
M K ? , MKg, M K g
M K g , d e sm e th y l M K g 3
d e sm e th y l MIC
d e sm e th y l M K g
d e sm e th y l M K g
mk7
c h lo ro b iu m q u in o n e
M K g ( 2 - H )
M K g ( 2 - H ) , M K q ( 2-H)
M K i 0 (2-H) 9
M K y
MKy
MICy
MK_
MK.
M K
"8
MK,
MKC
M K g t h r u M K C
48
49
5 0 , 5 1
52
53
5 5 , 5 6
57
51
58
58
59
59
58
58
58
60
61
9
TABLE 2 c o n tin u e d
The O c c u rre n c e o f M enaquinones
S o u rc e
B a c te r o id e s m e la n in o g e n ic u s
H a lo b a c te riu m c u tiru b ru m
B a c i l l u s s te a r o th e r m o p h ilu s
R h o d o s p iriliu m v i r d i s
R h o d o s p in llu m ru b ru m
R h o d o s p in llu m fu lv u m
S a r r a t i a m a rc e sc e n s
F la v o b a c te riu m s p e c ie s
M ic ro c o c c u s l y s o d e i k t i c u s
Most p l a n t s
A nim als
Human
B o v in e
a ) T h ese o rg a n ism s a l s o c o n t a in
b ) S ee t e x t f o r s t r u c t u r e .
c ) S ee t e x t f o r a d i s c u s s i o n o f
p r e s e n c e o f th e s e q u in o n e s .
H omologue R e fe re n c e
MK7 t h r u MKg 62
M K q 63
M IG j 51
M K ga 6 U
M Kg 65
M K 5 , M K 6 51
M K 3 t h r u M K 5 , M K
M K 8 (2 -H ) B 51
V ita m in K^ 6 6
M K 4 67
M K ? t h r u M K . c
V ita m in K^ 6 8
MK9-MK12# MKg(2-H)C 6 9 ,7 0
o t h e r q u in o n e t y p e s .
t h e e v id e n c e d e m o n s tra tin g th e
10
e x is te n c e (6 7 -7 0 ) i s in t h e o p in io n o f many p e o p le n o t s u f f i c i e n t to
draw any c o n c r e te c o n c lu s io n s . T here have b een p u b lic a tio n s p r e
s e n tin g e v id e n c e f o r and a g a i n s t m enaquinone e x is te n c e b u t in any
c a s e , th e l e v e l s r e p o r te d c o u ld be a c c o u n te d f o r by b a c t e r i a l contam
i n a t i o n . E vid en ce o b ta in e d by l i p i d e x t r a c t i n g l a r g e q u a n t i t i e s o f
b e e f h e a r t showed a c o m p le te a b se n c e o f any ty p e o f n ap h th o q u in o n e
( 6 5 ) . The o n ly o th e r m enaquinone b e s id e s M K^ i d e n t i f i e d by c h e m ic a l
means i s one w hich was i d e n t i f i e d a s b e in g a compound more l i p o p h i l i c
th a n M K j_ q ( 7 1 ) . No f u r t h e r s t r u c t u r a l i d e n t i f i c a t i o n o f t h i s com
pound h as b een m ade.
As can be s e e n from T a b le 2 m ost o f th e n a t u r a l m enaquinones
fo u n d t o d a te v a ry l i t t l e from th e b a s ic f u l l y s u b s t i t u t e d mena
q uinone s t r u c t u r e V. The m a jo r v a r i a t i o n s s e e n can b e d iv id e d in to
t h r e e g ro u p s: th o s e w hich a r e m ethyl s u b s t i t u t e d in th e C-2 p o s iti o n
and v a ry in th e le n g th o f th e is o p re n o id s id e c h a in (MKn ) (V ), th o s e
w hich a r e m e th y l s u b s t i t u t e d in th e C-2 p o s i t i o n and have p a r t i a l l y
s a tu r a t e d C-3 is o p r e n o id s i d e c h a in s (MKjj ( X-H) ) , f o r exam ple MKg
( I I - H ) , ( V I ),
CH
VI
and th o s e w hich la c k C-2 m e th y l s u b s t i t u t i o n (d e sm e th y l M K jj) (V II)
11
o
VII
n = l-9
To th e M K jj s e r i e s m ust b e added M K Q (m en ad io n e) r e p o r te d t o b e p r e
s e n t i n S_. a u re u s ( 6 1 ) . T h ere seem s to be no g e n e r a l r u l e w hich
g o v e rn s th e m ixed a p p e a ra n c e o f h om ologues. T h at i s , some o rg a n ism s
have o n ly p a r t i a l l y s a t u r a t e d h o m o lo g u es, f o r exam ple M. p h l e i ; some
o rg a n ism s h ave b o th p a r t i a l l y s a t u r a t e d and f u l l y u n s a tu r a t e d
h o m o lo g u es, f o r exam ple S tre p to m y c e s ; w h ile o t h e r s have f u l l y u n
s a t u r a t e d and d e sm e th y l h o m o lo g u es, f o r exam ple E. C o l i . T h ere i s t o
d a te no exam ple o f an o rg a n ism h a v in g a p a r t i a l l y s a t u r a t e d compound
and a d e sm e th y l d e r i v a t i v e . Some o rg a n is m s , su ch a s E _ . c o l i , a r e o f
i n t e r e s t in t h a t th e y p o s s e s s b o th u b iq u in o n e s an d m en aq u in o n es, o r
a s in t h e c a s e o f £ . ru b ru m , b o th rh o d o q u in o n e and a m enaq u in o n e.
I t i s a l s o o f i n t e r e s t t h a t w h ile t h e u b iq u in o n e c o n te n t o f E . c o l i
v a r i e s from t o Q g , o n ly M K g ad d d e sm e th y l M K g a r e p r e s e n t .
T h e re a r e o t h e r ty p e s o f m o d if ic a tio n w hich o c c u r , b u t th e s e
seem t o be r a r e and i s o l a t e d t o one o r s e v e r a l c l o s e l y r e l a t e d
o rg a n is m s . F o r ex am p le, C. t h i o s u l f a to p h i lu m , a s se e n in T ab le 2 ,
c o n ta in s a m enaquinone r e f e r r e d t o a s cholobium q u in o n e V I I I .
O
VIII
1 H
->6
12
T h is and c l o s e l y r e l a t e d o rg a n ism s ( s e e T a b le 2 ) a r e t h e o n ly known
s o u r c e s o f t h i s q u in o n e , a n d no f u n c t i o n a l p r o p e r t i e s f o r i t h a v e
b een d e s c r ib e d . O th e r ex am p les o f u n d e te rm in e d s t r u c t u r a l m o d if ic a
t i o n s i n m e n aq u in o n es a r e c a s e s s u c h a s th o s e in a n im a l t i s s u e s w here
v e ry p o l a r m en aq u in o n es h a v e b e e n t e n t a t i v e l y i d e n t i f i e d ( 7 7 ) . T h e re
have a l s o b een r e p o r t s o f c i s hom ologues b e in g p r e s e n t in b a c t e r i a in
a d d i t i o n t o t h e i r t r a n s iso h o m o lo g u e s ( 9 0 ) . To t h i s l i s t s h o u ld be
ad d ed t h e m o d i f ic a t io n s o f V ita m in a l r e a d y d is c u s s e d .
Q uinone F u n c tio n s To d a t e , t h e r e i s no known f u n c t i o n a l d i f f e r e n c e
b etw ee n v a r io u s h o m o lo g u es, h o w ev er, a d e q u a te s t u d i e s a d d r e s s e d t o
t h i s p ro b lem have n o t b e e n c a r r i e d o u t . T h a t t h e s h o r t e r hom ologues
a r e m o st p ro b a b ly n o t b i o s y n t h e t i c in t e r m e d i a te s w i l l b e d is c u s s e d
u n d e r t h e s e c t i o n o n t h e b i o s y n t h e s i s o f q u in o n e s . I t ca n b e se e n
t h a t a s t h e is o p r e n o id s i d e c h a in becom es l o n g e r , t h e compound b e
com es m ore l i p o p h i l i c and t h i s may b e o f some im p o rta n c e t o membrane
s t r u c t u r e , i . e . some q u in o n e s w ould h a v e m ore a f f i n i t y f o r h ig h l y
l i p i d p o r t i o n s o f m em branes.
I n g e n e r a l q u in o n e s a r e fo u n d i n membrane f r a c t i o n s . A g e n e r a l
r o l e f o r m e n aq u in o n es an d c e r t a i n u b iq u in o n e s i n b a c t e r i a l e l e c t r o n
t r a n s p o r t h a s b een w e ll e s t a b l i s h e d ( 7 2 - 7 5 ) . L ik e w ise a r o l e f o r
u b iq u in o n e i n m ito c h o n d r ia l e l e c t r o n t r a n s p o r t seem s w e ll e s t a b l i s h e d
( 9 1 ) . I t i s o f i n t e r e s t t h a t i n c e r t a i n o rg a n is m s (M. p h l e i f o r
e x a m p le ), t h e r e l a t i v e c o n c e n t r a t i o n o f m enaquinone t o o th e r e l e c t r o n
c a r r i e r s i s v e r y h ig h . T h is i s a l s o t h e c a s e w ith u b iq u in o n e in
a n im a l m ito c h o n d r ia . I t may w e ll b e , t h e r e f o r e , t h a t t h e q u in o n e 's
13
f u n c ti o n i s n o t s o l e l y r e l a t e d t o o x id a tiv e p h o s p h o r y la tio n . Be
c a u se o f t h e i r s o l u b i l i t y p r o p e r t i e s , q u in o n e s may a l s o s e rv e
s t r u c t u r a l f u n c tio n s in membrane sy ste m s a lth o u g h d i r e c t e v id en ce
f o r t h i s i s in d e e d d i f f i c u l t t o o b ta in . Some l i g h t may have been
sh ed on t h i s problem by Cox and G ibson ( 7 6 ) , who i s o l a t e d m u tan ts o f
E. c o l i w hich la c k e d e i t h e r m enaquinones o r u b iq u in o n e s . W hile th e s e
m u ta n ts a r e n e g a tiv e f o r one o f th e q u in o n e s p e c ie s , th e y a p p e a r to
be no rm al in a l l r e s p e c t s o th e r th a n e l e c t r o n t r a n s p o r t . T h is may
s u g g e s t t h a t b o th q u in o n e s a r e n o t n e c e s s a ry f o r membrane s t r u c t u r e ,
Q uinones may a l s o b e in v o lv e d in m echanism s t o t r a p f r e e r a d i c a l s ,
a w e ll known c h e m ic a l p r o p e r ty o f q u in o n e s.
B io s y n th e s is W hile th e b io s y n th e tic r o u te f o r t h e u b iq u in o n e r in g
i s w e ll u n d e rs to o d from work w ith b o th R. rubrum and a n im a ls , l i t t l e
i s known a b o u t th e b io s y n th e s i s o f th e m enaquinone r i n g . The s te p s
in v o lv e d in u b iq u in o n e b io s y n th e s is h ave b een o u t l i n e d in d e t a i l ,
and t h e in te r m e d ia te s in v o lv e d in th e s e s te p s have b e e n i s o la te d
( 7 7 ,7 8 ) . An e a r l y s t e p in t h i s s y n th e s is in v o lv e s a c o n d e n s a tio n o f
a p o ly is o p r e n o id c h a in p ro b a b ly a s an a l c o h o l, w ith p -h y d ro x y b e n z o ic
a c i d . S u b seq u en t t o t h i s th e m ethoxyl and m eth y l g ro u p s a r e added
and t h e compound i s o x id iz e d t o y i e l d th e f u l l y s y n th e s iz e d p ro d u c t.
The s o u rc e o f b o th m eth o x y l and m e th y l g ro u p s h a s been shown t o
be m e th io n in e in r a t ( 7 9 ) , E , c o l i ( 8 0 ) , A s p e r g ill iu s fu m ig a tu s ( 8 1 ) ,
and in R. rubrum ( 8 2 ) . I t sh o u ld be n o te d t h a t in t h e c a s e o f R.
rubrum d e f i n i t e e v id e n c e e x i s t s o n ly f o r t h e o r i g i n o f th e r in g
m ethyl*
14
The a d d i t i o n o f th e is o p r e n o id s id e c h a in h a s b een shown n o t to
ta k e p la c e by s e q u e n t i a l a d d i t i o n s o f s i n g l e is o p r e n o id u n i t s . W hile
t h i s h a s n o t b e e n shown in a w id e v a r i e t y o f s y s te m s , F r i i s e t a l .
(7 7 ) h av e d e m o n stra te d i t in R. ru b ru m . A lso Hammond and W hite (6 1 )
c o n c lu d e d t h a t w ith a u re u s t h e s h o r t e r m enaquinone hom ologues
( i . e . M K q, mki » e t c . ) d id n o t s e r v e a s p r e c u r s o r s f o r t h e lo n g e r
members o f t h e s e r i e s . L ik e w ise w ith E. c o l i , p r e lim in a r y e v id e n c e
h a s s u g g e s te d t h a t t h e u b iq u in o n e s a r e n o t s y n th e s iz e d in t h i s m anner
( 8 3 ) . The b io s y n th e s i s o f is o p r e n o id u n i t s in a n im a ls and f u n g i
p ro c e e d s v ia t h e w e ll c h a r a c t e r i z e d m e v a lo n ic a c i d (MVA) pathw ay ( 8 4 ) .
W ith b a c t e r i a t h e s i t u a t i o n seem s t o b e e n t i r e l y d i f f e r e n t a s t h e
in c o r p o r a tio n o f M V A i n t o q u in o n e s id e c h a in s h a s o n ly b e e n demon
s t r a t e d in one o rg a n ism ( 6 5 ) . T h is one c a s e was in a r a t h e r odd
b a c te riu m in t h a t i t c o n ta in s s q u a le n e and i t s h y d ro g e n a te d d e r i v
a t i v e s ( 6 3 ) , com pounds n o t fo u n d in o t h e r b a c t e r i a . The b a c t e r i a l
b io s y n t h e t i c r o u t e h a s b een v e r y d i f f i c u l t t o i n v e s t i g a t e d ue t o th e
f a c t t h a t no c e l l f r e e sy stem f o r is o p r e n o id b io s y n th e s i s h a s b een
d e m o n s tra te d , e x c e p t in th e a f fo re m e n tio n e d b a c te riu m w hich u s e s
M V A ( 6 5 ) .
The b i o s y n t h e t i c s te p s le a d in g t o t h e m enaquinone r i n g sy stem
h av e n o t b e e n f u l l y w orked o u t . The r i n g m e th y l g ro u p h a s b een
shown t o b e d e r iv e d from m e th io n in e a s i n th e c a s e o f u b iq u in o n e
( 8 5 ,8 6 ) . The p r e c u r s o r o f t h e a ro m a tic p o r ti o n o f t h e r i n g sy stem
h a s b e e n shown t o b e s h ik ira ic a c i d ( 8 1 - 8 3 ) . T h is was d e m o n stra te d
m ost c o m p le te ly by C am pbell e t a l . (8 9 ) who showed t h a t c a rb o n s 1 and
5 - 1 0 o f t h e n a p h th o q u in o n e r i n g sy stem w ere d e r iv e d i n t a c t from
15
u n ifo rm ly la b e l e d s h ik im a te . C arbons 2 a n d 3 o f t h e r i n g sy ste m
a p p e a r t o b e d e r iv e d from a c e t a t e ( 8 8 ) .
The s t e p s in v o lv e d in t h e p a r t i a l h y d ro g e n a tio n o f is o p r e n o id
s id e c h a in s h a v e o n ly b e e n s tu d ie d t o a l i m i t e d d e g re e ( 9 2 ) . T h is
s tu d y s u g g e s te d t h a t h y d ro g e n a te d t a k e s p la c e a f t e r t h e c o m p le tio n o f
th e f u l l y u n s a tu r a t e d m enaquinone and i s m e d ia te d by NA DH an d o r
NADPH.
Beyond t h i s l i t t l e i s known o f t h e s t e p s o r in t e r m e d i a te s i n
v o lv e d i n m enaquinone b i o s y n t h e s i s . I t i s t h e p u rp o s e o f t h i s t h e s i s
t o d e s c r ib e t h e i s o l a t i o n , s e p a r a t io n an d s t r u c t u r a l i d e n t i f i c a t i o n
o f a s e r i e s o f m en aq u in o n es from a member o f t h e S tre p to m y c e s g ro u p .
16
MATERIALS A N D M ETH O D S
O rganism s Used The S trep to m y ces s p e c ie s u sed was th e k in d g i f t o f
C albiochem , Los A n g eles, C a l if o r n ia . The m a te r ia l was s u p p lie d a s
a c h lo ro fo rm su sp e n sio n o f w hole c e l l s .
14. p h le i was u sed in e x p e rim e n ts in v o lv in g quinone r e s t o r a t i o n
o f o x id a tiv e p h o s p h o ry la tio n . S tr a i n ATCC 354 was grown and h a r v e s t
ed by th e m ethod o f B ro d ie and G ray ( 7 2 ) . The e le c tr o n tr a n s p o r t
p a r t i c l e s w ere p re p a re d a c o o rd in g to th e m ethod o f B ro d ie ( 7 3 ) , and
th e s u p e rn a ta n t from th e p a r t i c l e s was f r a c t io n a t e d w ith ammonium
s u l f a t e . The f r a c t i o n w hich p r e c i p i t a t e d betw een 35 and 60% was
d is s o lv e d in 0 .0 1 M MgC^ and d i a l i z e d a g a in s t la r g e volum es o f 0 .005
M T ris-H C l b u f f e r , pH 7 .5 , c o n ta in in g 0 .0 1 M MgClg. The d i a l i z e d
m a te r ia l was r e f e r r e d to a s A S II.
P h ie i was a l s o u sed a s a so u rc e o f M K g ( I I - H ) . The e x t r a c t
io n and p u r i f i c a t i o n o f t h i s compound was c a r r i e d o u t in th e same
m anner a s th e S tre p to m y c e s 1 q u in o n e s t o be d e s c r ib e d .
C hem icals and S u p p lie s A cid washed s i l i c i c a c i d , 100-200 m esh, was
o b ta in e d from th e C la rk so n C hem ical Company, W illia m s p o rt, P e n n a .,
and P e r m u tit, F o lin , was o b ta in e d from t h e F is h e r C hem ical Company.
The t h i n la y e r a d s o rb a n ts u s e d , S i l i c a g el-G and X ieselg u h r-G w ere
o b ta in e d from Brinkm ann S c i e n t i f i c , a s was m ost o f th e o th e r t h i n
la y e r equipm ent and s u p p lie s .
The s o lv e n ts u se d f o r s p e c tro s c o p y w ere a l l o f s p e c tr o q u a lity ,
17
Those u sed f o r ch ro m ato g rap h y w ere e i t h e r s p e c tr o - o r c h ro m a to g ra p h ic
q u a l i t y , o r in th e c a s e o f d i e t h y l e t h e r and p e tro le u m e t h e r (3 0 -6 0 °
b p .) th e y w ere r e d i s t i l l e d from lo w e r g ra d e s o lv e n t s .
The m enaquinone s ta n d a r d s , w hich w ere u se d a s m a rk e rs , w ere a
g i f t o f D r. O tto I s l e r , H offm ann-LaRoche Company, B a s a l, S w itz e rla n d .
V itam in was o b ta in e d c o m m e rc ia lly and was f u r t h e r p u r i f i e d a s
d e s c rib e d in th e m ethods s e c t i o n . M enadione, 1 , 4 -n a p h th o q u in o n e , and
la p a c h o l w ere p u rc h a se d a s c r y s t a l l i n e compounds and u sed w ith o u t
f u r t h e r p u r i f i c a t i o n . P h y to l was o b ta in e d from C albiochem i n a
t r i p l e d i s t i l l e d form and u sed w ith o u t f u r t h e r p u r i f i c a t i o n . G eren y l
a c e to n e and m eth y l h ep ta n o n e w ere p u rc h a se d from K and K C hem ical
Company. The o th e r v ita m in K hom ologues u sed w ere s y n th e s iz e d a s
d e s c rib e d in th e m ethods s e c t i o n . O th e r c h e m ic a ls u se d a r e commonly
a v a i l a b l e .
L ip id E x tr a c tio n o f Whole C e lls To th e c h lo ro fo rm s u s p e n s io n o f
S tre p to m y c e s c e l l s was added enough m e th an o l t o b r in g th e r a t i o o f
c h lo ro fo rm :m e th a n o l t o 2 :1 . The m ix tu re was e x t r a c te d by a g i t a t i o n
in a W earing B le n d e r and th e r e s u l t i n g su sp e n sio n was f i l t e r e d th ro u g h
p a p e r t o c o l l e c t t h e s o l i d c e l l fra g m e n ts . The e x t r a c t i o n p ro c e d u re
(9 3 ) was th e n r e p e a te d on th e c e l l fra g m e n ts tw ic e m o re, and th e
l i q u i d from th e e x t r a c t i o n was p o o le d and p la c e d in s e p e r a to r y fu n
n e l s . C a re fu l a d d i tio n o f w a te r in volum es e x c e s s t o th e am ount o f
m eth an o l u se d r e s u l t e d in t h e s e p a r a tio n o f a c h lo ro fo rm lo w e r la y e r
from a m e th a n o l-w a te r u p p e r l a y e r . The lo w er l a y e r , c o n ta in in g th e
q u in o n e s, was rem oved and ta k e n t o d ry n e s s in vacuo o r u n d e r a stre a m
18
o f n i t r o g e n . The m a t e r i a l re m a in in g a f t e r re m o v a l o f t h e c h lo ro fo rm
was a d a rk y e llo w o i l w hich was t h e s t a r t i n g m a t e r i a l f o r a l l f u r t h e r
p u r i f i c a t i o n s t e p s . The r e l a t i v e am ount o f m a t e r i a l o b ta in e d by t h i s
p ro c e d u re w as a p p ro x im a te ly 1 g o f y e llo w l i p i d f o r e v e ry 84 g o f
w et w e ig h t c e l l s u s e d . M. p h l e i l i p i d c o n ta in in g M K g ( I I - H ) was e x
t r a c t e d by i d e n t i c a l p r o c e d u r e s .
C h ro m a to g ra p h ic S e p a r a tio n s T h ere w ere tw o m ain p ro b le m s in v o lv e d
in t h e f i n a l s e p a r a t io n o f in d i v i d u a l s p e c ie s o f q u in o n e s . The f i r s t
was t o s e p a r a t e t h e q u in o n e c o n t e n t from o th e r l i p i d s and th e seco n d
was t h e s e p a r a t i o n o f q u in o n e hom ologues from e a c h o t h e r . W hile t h e
l a t t e r ty p e o f s e p a r a t i o n in v o lv e d o n ly t h i n l a y e r c h ro m a to g ra p h y t h e
fo rm e r in v o lv e d b o th t h i n l a y e r an d colum n c h ro m a to g ra p h y .
Column C h rom atography G la s s colum ns w ere u se d p a c k e d e i t h e r w ith
P e r m u t it, F o l i n , o r w ith a c i d w ashed s i l i c i c a c i d . D e a c tiv a te d
P e r m u tit was p r e p a r e d by s h a k in g a 2% (v /w ) aq u e o u s s l u r r y o f P e r m u tit
f o r 12 h o u r s . F o r e v e ry gram o f l i p i d lo a d e d o n t h e co lu m n s a p p ro x
im a te ly 20 gram s o f a d s o r b a n t w ere u s e d . F o r exam ple o n e m a jo r s e p
a r a t i o n in v o lv e d t h e e x t r a c t i o n o f 2 .6 Kg w et w e ig h t o f c e l l s y i e l d i n g
31 g o f y e llo w l i p i d . F o r th e s e p a r a t i o n o f t h i s 31 g o f m a t e r i a l
500 g o f P e r m u tit was p ack ed in a 4 .5 x 20 cm co lu m n . B e fo re t h e
a d d i t i o n o f t h e l i p i d , t h e colum n was w ashed w ith 1 . 1 o f p e tro le u m
e t h e r . The 31 g o f l i p i d , i n p e tro le u m e t h e r , was th e n lo a d e d in t o
t h e colum n and t h e f o llo w in g f r a c t i o n s c o l l e c t e d : 1 .1 o f 3% d i e t h y l
e t h e r in p e tro le u m e t h e r ( f r a c t i o n I ) , th e n f o u r f r a c t i o n s o f 500 ml
19
e a c h o f 4% d i e t h y l e t h e r i n p e tro le u m e t h e r ( f r a c t i o n s X I, I I I , IV
and V ). The l a t t e r f o u r f r a c t i o n s c o n ta in e d 558 mg o f m a t e r i a l o r
a b o u t 1 g f o r e v e ry 56 g o f c ru d e l i p i d s t a r t i n g m a t e r i a l . F r a c ti o n
I I I , 373 mg, c o n ta in e d a h ig h p r o p o r ti o n o f q u in o n e w h ile f r a c t i o n s
I I , IV an d V c o n ta in e d m in im a l am ounts a s ju d g e d by t h i n l a y e r
c h ro m a to g ra p h y . F or t h i s r e a s o n f r a c t i o n I I I was u se d f o r a l l
f u r t h e r p u r i f i c a t i o n w ork.
W hile m ore th a n o n e b a tc h o f c e l l s was e x t r a c t e d d u r in g t h e
c o u rs e o f t h e s e s t u d i e s , t h e exam ple c i t e d was by f a r t h e l a r g e s t .
The m eth o d o lo g y u s e d f o r s m a lle r s c a l e p u r i f i c a t i o n s w as e x a c t l y
th e same o n ly on a p r o p o r t i o n a l l y s m a l le r s c a l e . The u s e o f P e r m u tit
h a s an a d v a n ta g e o v e r s i l i c i c a c id in t h a t s i l i c i c a c i d te n d s t o
h e a t up on a d d i t i o n o f l i p i d and t h i s c a u s e s b u b b le s an d colum n
d i s r u p t i o n . W hile th e s e p a r a t i o n s a c h ie v e d w ith s i l i c i c a c id w ere
f a r m ore c o m p le te , P e rm u tit w as fo u n d t o be a d e q u a te t o o b t a i n th e
d e g re e o f s e p a r a t i o n r e q u ir e d a t t h i s s t a g e o f p u r i f i c a t i o n .
T h in L ay e r C hrom atography S i l i c a g e l-G was u se d a s t h e p r i n c i p a l
r e s i n f o r a d s o r p ti o n c h ro m a to g ra p h y . When u se d w ith o u t t h e a d d i t i o n
o f s i l v e r i o n s , t h e s i l i c a g e l (50% aq u e o u s s l u r r y ) was p o u re d o n to
20 x 20 g l a s s p l a t e s a t a t h i c k n e s s o f e i t h e r 250 o r 500 )i. The
p l a t e s w ere a llo w e d t o d ry o v e r n ig h t w ith no f u r t h e r a c t i v a t i o n
n e c e s s a r y . When s i l v e r io n s w ere in c o r p o r a te d i n t o t h e l a y e r s , a
9% s i l v e r n i t r a t e s o l u t i o n w as u se d t o make th e 50% s l u r r y o f s i l i c a
g e l ( 9 4 ) . The s i l v e r io n im p re g n a te d s l u r r y was p o u re d o n to 20 x 20
cm g l a s s p l a t e s a t a t h i c k n e s s o f 375 j i , w hich w ere a c t i v a t e d a t 110°
20
f o r 45 m in . These p l a t e s i f s to r e d in a d a r k , m o is tu re f r e e e n v iro n
m ent, w ere s t a b l e f o r up to one m onth. Im p ro p er h a n d lin g , su ch a s
ex p o su re t o l i g h t , in a c t i v a t e d th e s e p l a t e s r a p i d l y .
R ev ersed p h a se ch ro m ato g rap h y was c a r r i e d o u t u s in g K ie se lg u h r-G
powder a s t h e a d s o r b a n t. A 50% aqueous s l u r r y o f th e r e s i n was p o u r
ed o n to 20 x 20 cm g la s s p l a t e s a t a th ic k n e s s o f 250 p . The p l a t e s
w ere a llo w e d t o d ry o v e rn ig h t and r e q u ir e d no f u r t h e r a c t i v a t i o n .
B efo re u se t h e p l a t e s w ere im m ersed in a 5% ( v /v ) l i q u i d p a r a f f i n in
p etro le u m e t h e r s o lu ti o n and rem oved to a llo w e v a p o ra tio n o f th e
p e tro le u m e t h e r .
The q u in o n e s w ere v i s u a l iz e d on a d s o r p tio n p l a t e s w ith th e a id
o f rhodam ine 6 G ( 9 5 ) , T h ere a r e two m ethods f o r th e u s e o f rh o d a -
m ine. D eveloped p l a t e s c a n be sp ra y e d w ith a s o lu ti o n o f rhodam ine
o r th e dye c a n be in c o rp o r a te d i n t o th e la y e r a t th e tim e th e p l a t e
i s p o u red . I n th e c a s e w here s i l v e r io n s w ere n o t used th e aqueous
s l u r r y o f s i l i c a g e l c o n ta in e d 0,1% rh o d am in e. When s i l v e r io n s w ere
u sed i t was im p o rta n t to ddd th e rhodam ine ( f i n a l c o n c e n tr a tio n 0.05%)
to th e s i l v e r n i t r a t e s o l u t i o n and th e n t o add q u ic k ly and p o u r th e
s i l i c a g e l . T h is av o id e d t h e a p p e a ra n c e o f a p r e c i p i t a t e w hich to o k
p la c e i f t h e p ro c e d u re was n o t c a r r i e d o u t r a p i d l y . In e i t h e r th e
ab sen ce o r p re s e n c e o f s i l v e r io n s rhodam ine ca u se d th e q u in o n e s to
a p p e a r a s d a r k r e d a r e a s on a p in k b ackground in no rm al l i g h t o r a s
p u rp le a r e a s on a y e llo w -g re e n b ackground u n d er u l t r a v i o l e t l i g h t .
W ith r e v e r s e d p h a se ch ro m ato g rap h y t h e q u in o n e s w ere v i s u a l i z e d
by th e b o ro h y d rid e Emmerie E n g el r e a g e n t ( 9 6 ,9 7 ) . The q u in o n e s
ap p e a re d a s y e llo w t o brown s p o ts u n d e r no rm al l i g h t . W hile th e
21
b o ro h y d rid e m ethod i s d e s t r u c t i v e , th e rh o d am in e m ethod i s com
p l e t e l y n o n - d e s t r u c t i v e . The re c o v e ry o f q u in o n e s from t h i n l a y e r s
was a c c o m p lish e d by s c r a p in g t h e a d s o rb a n t from t h e d e s ir e d a r e a o f
t h e p l a t e and e l u t i n g t h e q u in o n e from t h e pow der w ith d i e t h y l e t h e r .
W ith t h e u s e o f s i l v e r im p re g n a te d p l a t e s a h ig h e r p e rc e n ta g e o f r e
c o v e ry w as a c h ie v e d i f th e q u in o n e s w ere e l u t e d from t h e r e s i n w ith
a c e to n e r a t h e r th a n d i e t h y l e t h e r . W hile t h e a c e to n e a l s o e l u te d
rh o d a m in e , p a r t i t i o n o f th e a c e to n e b etw een d i e t h y l e t h e r and w a te r
rem oved t h e dye t o th e aq u eo u s p h a se and t h e q u in o n e t o th e e t h e r
p h a s e .
B ecau se su c h a w ide ra n g e o f s o lv e n t sy ste m s w ere u se d w ith
a d s o r p tio n ch ro m a to g rap h y r e f e r e n c e t o s p e c i f i c sy ste m s w i l l be made
a t th e a p p r o p r ia te tim e s . R ev ersed p h a se sy ste m s u se d a c e to n e - w a te r
m ix tu re s s a t u r a t e d w ith l i q u i d p a r a f f i n . The e x a c t p e r c e n ta g e s w i l l
be i n d i c a te d w here n e c e s s a r y . In a l l c a s e s t h e ch ro m a to g rap h y ta n k s
w ere l i n e d w ith f i l t e r p a p e r t o f a c i l i t a t e r a p i d s a t u r a t i o n o f th e
a tm o s p h e re . The lo a d in g l i m i t s f o r p l a t e s w ere a s f o llo w s : f o r 500 p
a d s o r p tio n p l a t e s up t o 30 mg, and one h a l f o f t h a t f o r 250 p p l a t e s ,
f o r s i l v e r im p re g n a te d p l a t e s up t o 8 mg, and f o r r e v e r s e d p h a se
p l a t e s up t o 3 mg.
S y n th e s is and p u r i f i c a t i o n o f S y n th e tic Compounds C om m ercially
o b ta in e d V itam in was p u r i f i e d by colum n c h ro m a to g rap h y on P e rm u tit
o r s i l i c i c a c i d . The V itam in was e l u t e d from t h e s e colum ns by 3%
d i e t h y l e t h e r in p e tro le u m e t h e r . The colum n p u r i f i e d V ita m in was
th e n f u r t h e r p u r i f i e d on 500 p s i l i c a g e l p l a t e s in 12% b u ty l e t h e r /
22
h e x a n e . T h is sy ste m r e s o l v e s t h e v ita m in i n t o i t s C-3 c i s and C-3
t r a n s is o m e rs ( 9 8 ) . R e p e a te d p u r i f i c a t i o n w ith t h i s sy ste m a llo w s f o r
t h e i s o l a t i o n o f p u re is o m e re s w hich c a n b e m a in ta in e d a t f r e e z e r
te m p e r a tu r e s i n t h e d a r k . The f o r e g o in g p ro c e d u re was a l s o t h e one
u se d t o s e p a r a t e an d p u r i f y M K g ( I I - H ) from M. p h l e i l i p i d .
Gamma-hydroxy v ita m in K -^ was s y n th e s iz e d by a m o d ifie d p ro c e d u re
o f W agner e t a l . ( 9 9 ) . To 2 g o f v ita m in (colum n p u r i f i e d ) was
ad d ed d ro p w is e 20 ml o f c o n c e n tr a te d s u l f u r i c a c i d . T h is r e a c t i o n
was c a r r i e d o u t in a n i c e b a th w ith c o n s ta n t s t i r r i n g . The r e a c t i o n
was a llo w e d t o c o n tin u e f o r t h i r t y m in u te s , th e n th e m ix tu re was
s lo w ly p o u re d o n to a n ic e / w a t e r m ix tu r e w ith c o n s ta n t s t i r r i n g . The
c ru d e p ro d u c t was th e n e x t r a c t e d w ith d i e t h y l e t h e r fro m th e i c e /w a te r
m ix tu re and t h e d i e t h y l e t h e r w as th e n w ashed w ith a NaHCOg s o l u t i o n
t h r e e tim e s and th e n w ith w a te r t h r e e ti m e s . The e t h e r l a y e r was th e n
d r i e d o v e r a n h y d ro u s m agnesium s u l f a t e and c o n c e n tr a te d in v a c u o . The
r e s u l t i n g d e e p r e d o i l was p la c e d on a p e r m u tit colum n and was e l u t e d
a f t e r a 10% d i e t h y l e t h e r in p e tro le u m e t h e r w ash , w ith a 25%, m ix tu re
o f t h e same s o l v e n t s . The 25% f r a c t i o n was ta k e n t o d r y n e s s and
c h ro m a to g ra p h e d on 500 ji l a y e r s o f s i l i c a g e l i n 15% e t h y l a c e t a t e /
b e n z e n e . The Rp o f t h e V -h y d ro x y v ita m in i s 0 .6 2 . The y i e l d o f
p ro d u c t (IX ) i s a p p ro x im a te ly 40% and i t s u l t r a v i o l e t s p e c t r a in i s o
o c ta n e shows s h a rp a b s o r p ti o n p e a k s a t 2 4 4 , 2 4 9 , 2 6 4 , an d 273 ji w ith
a n o th e r b ro a d p eak a t 325 p .
D ih y d ro p h y ty l v ita m in was p r e p a r e d a s f o llo w s from colum n
p u r i f i e d v ita m in t ^ . To 2 g o f v ita m in in d i e t h y l e t h e r was added
an e x c e s s o f p a lla d iu m c h a r c h o l. T h is was p la c e d i n a h y d ro g e n a tio n
23
CH
IX
a p p a ra tu s w hich was s u b s e q u e n tly e v a c u a te d . The m ix tu re was th e n
s u b je c te d t o 50 p s i o f hydrogen gas an d a llo w e d t o r e a c t w ith c o n s ta n t
sh a k in g f o r t h i r t y m in u te s . The p ro d u c t was f i l t e r e d t o rem ove th e
c h a rc h o l and th e e t h e r was washed t h r e e tim e s w ith w a te r . The e t h e r
la y e r was d r ie d o v e r an h y d ro u s magnesium s u l f a t e and t h e e t h e r was
th e n rem oved in v a c u o . The o i l y p ro d u c t was p la c e d on a p e rm u tit
colum n and e lu te d w ith 3% d i e t h y l e t h e r in p e tro le u m e t h e r . The
colum n p ro d u c t was th e n p u r i f i e d w ith 375 p s i l v e r im p reg n ate d l a y e r s
in 12% 2 -b u tan o n e ( m e th y l- e th y l k e to n e )/h e x a n e . T h is system s e p a r a te s
th e d ih y d ro p h y ty l v ita m in (Rp 0 .7 5 ) from u n re a c te d v ita m in
(Rp 0 .6 6 ) . The y i e l d o f p ro d u c t i s a b o u t 80% i f c a u tio n i s o b se rv e d
n o t t o l e t th e r e a c t i o n go f o r to o lo n g a tim e . The u l t r a v i o l e t
s p e c tr a o f d ih y d ro p h y ty l v ita m in (X ) shows s h a rp a b s o r p tio n p eak s
a t 242, 248, 263 and 270 p w ith a b ro a d p eak a t 325 mp.
O
X
D esm ethyl v ita m in K -^ was p r e p a r e d by a m o d ifie d p ro c e d u re o f
F i e s e r e t a l . ( 1 0 0 ) . 6 g o f l,i+ -n a p h th o q u in o n e in 250 ml o f d i e t h y l
e t h e r was sh a k e n w ith 75 ml o f 30% aq u eo u s (w /v ) Na2 S 2 0 i|. The e t h e r
s o l u t i o n was w ashed w ith a s a t u r a t e d NaCl s o l u t i o n and d r ie d o v e r
a n h y d ro u s m agnesium s u l f a t e . To t h e r e s u l t i n g s o l u t i o n was ad d ed 20
m l o f p e tro le u m e t h e r , and t h e r e s u l t i n g p r e c i p i t a t e d p ro d u c t was
c o l l e c t e d on a B uchner f u n n e l a s a g ra y p o w der. T h is p ro d u c t in 15
ml o f p -d io x a n e c o n t a in in g 1 .5 ml o f B F g - e th e ra te was ad d ed d ro p w is e
t o 1 g o f p h y to l i n 10 ml o f p - d io x a n e . The m ix tu re was sh a k e n f o r
tw e n ty m in u te s and c o o le d . 1 0 0 ml o f e t h e r was ad d ed and t h e m ix tu re
was w ashed w ith 2 x 50 ml o f w a te r . The r e s u l t i n g e t h e r s o l u t i o n was
e x t r a c t e d f i v e tim e s w ith a m ix tu r e o f 30 ml o f 2% aq u e o u s (w /v ) NaOH
p lu s 10 m l o f 30% Na2 S 2 0 ^ p lu s 5 ml o f s a t u r a t e d N aC l. The r e s u l t i n g
e t h e r s o l u t i o n was d r i e d o v e r a n h y d ro u s m agnesium s u l f a t e and c o n
c e n t r a t e d t o a s y ru p v a c u o . To t h i s was ad d ed 20 ml o f p e tro le u m
e t h e r . T h is m ix tu re was th e n c e n t r i f u g e d an d t h e p e l l e t was d is s o lv e d
in 25 m l o f d i e t h y l e t h e r c o n ta in in g 1 g s i l v e r o x id e an d 1 g a n
h y d ro u s m agnesium s u l f a t e . The m ix tu re was sh a k e n and a llo w e d t o
s ta n d f o r 60 m in u te s . The l i q u i d was th e n f i l t e r e d and c o n c e n tr a te d
t o a s y ru p w h ich was d is s o lv e d in p e tro le u m e t h e r an d p la c e d on a
colum n o f d e a c t i v a t e d p e r m u t i t . F o llo w in g a w ash o f 200 m l o f
p e tro le u m e t h e r t h e d e s m e th y l v ita m in was e l u t e d w ith 2 0 0 ml o f a
6 % d i e t h y l e t h e r i n p e tro le u m e t h e r s o l u t i o n . The colum n p u r i f i e d
p ro d u c t was p u r i f i e d f u r t h e r o n 500 p s i l i c a g e l p l a t e s w ith e i t h e r
15% 2-b u ta n o n e /h e x a n e o r 5% m e th a n o l/b e n z e n e . The y i e l d i s a p p ro x
im a te ly 5% an d t h e u l t r a v i o l e t s p e c t r a o f d e s m e th y l v ita m in (X I)
25
show s s h a r p a b s o r p t i o n p e a k s a t 2 4 2 , 2 4 9 , 255 an d 265 mp w ith a b ro a d
p e a k a t 325 mp.
The p r e p a r a t i o n o f q u in o n e d i a c e t a t e s w as a c c o m p lish e d a s f o l
lo w s . The q u in o n e (lO m g) was d i s s o l v e d i n 3 ml o f a c e t i c a n h y d r id e .
To t h i s w as ad d ed 50 mg o f z in c d u s t an d 3 m ic ro d ro p s o f t r i e t h y l -
a m in e . The m ix tu r e was a llo w e d t o r e a c t f o r t h r e e m in u te s on a s te a m
b a t h . To t h e f l a s k w as th e n ad d e d 40 m l 1^0 an d NaHCOg ( i n s m a ll
p o r t i o n s ) u n t i l no f u r t h e r r e a c t i o n o c c u r r e d . The m ix tu r e w as f i l t e r
ed an d e x t r a c t e d f i v e tim e s w ith d i e t h y l e t h e r . The p o o le d e t h e r
f r a c t i o n s w ere t h e n w ashed w ith HjO, 1 N HC1, an d a n d d r i e d o v e r
a n h y d ro u s m agnesium s u l f a t e . The e t h e r s o l u t i o n w as th e n ta k e n t o
d r y n e s s an d t h e r e s u l t i n g m a t e r i a l , in p e tro le u m e t h e r , w as p la c e d
on a p e r m u t it co lu m n . A f te r w ash in g t h e colum n w ith 100 m l 10%
d i e t h y l e t h e r , in p e tro le u m e t h e r , t h e d i s c e t a t e p r o d u c t w as e l u t e d
w ith a 50% s o l u t i o n o f t h e same s o l v e n t s . The y i e l d o f p r o d u c t i s
v e r y h ig h a n d a p p ro a c h e s 100%. The u l t r a v i o l e t s p e c t r a o f 2 , 3 -
d i s u b s t i t u t e d - 1 , 4 - d i a c e t y l n a p h th o q u in o n e s ( X I I ) shows o n e m a jo r
s h a r p a b s o r p t i o n p e a k a t 232 mp.
O z o n o ly s is o f t h e m en aq u in o n e d i a c e t a t e s was c a r r i e d o u t a s
f o l l o w s . To 2 mg o f q u in o n e d i a c e t a t e was ad d ed ( a t - 7 8 ° ) 15 ml o f
O
XI
O - C - C h U
ii
o
XII
e t h y l c h l o r i d e . The e n t i r e r e a c t i o n B u st be ru n a t low te m p e ra tu re
a s e t h y l c h lo r id e b o i l s a t 1 2 ° . O zone, d e l iv e r e d from an Ozone Re
s e a r c h and Equipm ent Company m odel #03V2 o z o n a to r , was b u b b led th ro u g h
th e r e a c t i o n m ix tu re a t a r a t e o f 0 .2 5 g 0 3 / h o u r . A fte r th e a p p e a r
an ce o f a d eep b lu e c o l o r , th e r e a c t i o n was a llo w e d to c o n tin u e f o r
t h i r t y m in u te s . The o z o n a to r was th e n tu r n e d o f f and oxygen was
a llo w e d to b u b b le th ro u g h th e r e a c t i o n f o r t h i r t y m in u te s. The f l a s k
was th e n rem oved from t h e d ry ic e a c e to n e m ix tu re and th e e t h y l c h lo
r i d e was a llo w e d t o e v a p o ra te o f f t o a s m a ll volum e (a p p ro x im a te ly 1
m l). To t h i s was added 20 ml o f d i e t h y l e t h e r c o n ta in in g s u f f i c i e n t
tr i p h e n y l phosphene to re d u c e t h e o z o n id e . T h is was r e f lu x e d a t *+0°
f o r 30 m in u te s and th e m ix tu re was ta k e n t o d ry n e s s a t 7 0 °. The
o z o n id e s th u s o b ta in e d w ere a n a ly z e d d i r e c t l y by g a s ch ro m a to g rap h y .
P h y s ic a l M ethods
Gas ch ro m ato g rap h y was c a r r i e d o u t on a Beckman m odel GC-4 g a s
ch ro m a to g ra p h . The colum n u sed was 1 /4 " x 6 ' packed w ith 3% 0V101
(d im e th y l s i l i c o n e ) on an i n e r t s u p p o r t. T em p eratu re o f th e ru n was
programmed from 10 0 -2 6 0 ° in 16 m in u te s . The s o lv e n t used was d i e t h y l
27
e t h e r .
U l t r a v i o l e t s p e c tr a w ere re c o rd e d on e i t h e r a Cary m odel 1 1 o r 14
re c o r d in g s p e c tro p h o to m e te r. S p e c tra from 360-225 mji w ere re c o rd e d
in is o o c ta n c e ( 2 ,2 ,4 - tr im e th y l p e n ta n e ) w ith th e e x c e p tio n o f red u ce d
quinone s p e c t r a , w hich w ere ta k e n a s f o llo w s . The q uinone was d i s
so lv e d in 3 ml o f e th a n o l c o n ta in in g 0 .0 1 volum e o f 1 M ammonium
a c e t a t e aqueous b u f f e r (pH 5 . 0 ) . F o llo w in g th e re c o rd in g o f an
o x id iz e d s p e c t r a , th e quinone was red u ce d by th e a d d itio n o f a s u f f i
c i e n t amount o f a f r e s h l y p re p a re d aqueous s o lu tio n o f sodium b o ro -
h y d rid e . A fte r a llo w in g a b o u t 1 m in u te f o r co m p lete r e d u c tio n a r e
duced s p e c tr a was ta k e n .
I n f r a r e d s p e c tr a w ere re c o rd e d on a P e rk in Elm er m odel 21 i n f r a
re d s p e c tro p h o to m e te r. The sa m p le s, in th e form o f o il s ,w e r e a p p lie d
d i r e c t l y to a p o ta ssiu m brom ide d is c t o w hich a second d is c was p la c e d
so t h a t th e o i l s p re a d betw een them in a t h i n l a y e r . The m achine was
f i t t e d w ith a P e rk in Elm er beam co n d en sin g d e v ic e w hich allo w e d e x c e l
l e n t s p e c tr a t o b e ta k e n o f a s l i t t l e a s 1 mg o f m a t e r i a l .
N u clea r M agnetic R esonance S p e c tra w ere re c o rd e d on a V arian
Model HA-100 s p e c tro p h o to m e te r o p e r a tin g a t 100 Me. The s o lv e n ts used
w ere CDCI3 o r b e n z e n e . In b o th c a s e s th e s ig n a l f o r lo c k in g th e
m ag n etic f i e l d was s u p p lie d by te tr a m e th y l s x le n e w hich was in c o r
p o ra te d a s an i n t e r n a l s o u rc e .
Mass s p e c tr a w ere re c o rd e d on one o f tw o m a ch in es. Some work was
28
done on a V a ria n m odel M 6 6 mass s p e c tro p h o to m e te r o p e r a tin g a t an
io n iz in g p o t e n t i a l o f 70 V and a p r e s s u r e o f 1 x 10 " 7 T o rr. The
sam p les w ere v a p o riz e d a t 250° and i n j e c t e d by a d i r e c t i n l e t sy ste m .
The seco n d m achine u se d was a P e rk in s Elm er H a ta c h i m odel RMU-6 E m ass
s p e c tro p h o to m e te r o p e r a tin g a t an io n iz in g p o t e n t i a l o f 70 v and a
p r e s s u r e o f 6 x 10 - 7 T o r r . The te m p e ra tu re o f io n i z a ti o n was 285°
and a g a in th e sam ple was i n j e c te d by a d i r e c t i n l e t sy stem .
W arburg e x p e rim e n ts w ere ru n on a G ilso n r e s p ir o m e te r a t 30° by
s ta n d a rd m anom etric te c h n iq u e s . The r e a c t i o n s w ere sto p p e d by th e
a d d i tio n o f 1 ml o f 10% TCA and th e p r e c i p i t a t e d p r o te i n was c e n
t r i f u g e d o u t. The r e s u l t i n g s u p e r n a ta n t m a te r ia l was a s sa y e d f o r
in o rg a n ic p h o sp h a te by th e m ethod o f F is k e and SubbaRow (1 0 1 ). P ro
t e i n c o n c e n tr a tio n was m easured by th e m ethod o f S tadtm an e t a l . (102)
29
RESULTS
S e p a r a tio n o f Q uinones The g e n e r a l u s e f u ln e s s o f t h i n l a y e r
c h ro m a to g rap h y (TLC) in t h e s e p a r a t io n and p u r i f i c a t i o n o f a w ide
v a r i e t y o f compounds h a s b e e n w e ll e x p l o i t e d . G e n e ra lly s p e a k in g ,
p l a t e s can b e d e v e lo p e d in l e s s th a n a n h o u r, o r w ith m icro te c h n iq u e ,
in a s l i t t l e a s f i v e m in u te s . The am ount o f m a t e r i a l w hich can be
s e p a r a te d i s v e ry l a r g e com pared w ith o th e r te c h n iq u e s , and th e c o s t
o f t h e m ethod i s v e ry lo w . The u s e o f TLC f o r q u in o n e w ork h a s b een
e x te n s iv e and s o lv e n t sy ste m s can b e fo u n d t o e f f e c t th e s e p a r a t io n
o f compounds w ith a lm o s t a n y ty p e o f v a r i a t i o n .
The s e p a r a t io n o f t h e S tre p to m y c e s * q u in o n e s beg an w ith colum n
f r a c t i o n I I I (373 m g). T h is m a t e r i a l was c h ro m a to g rap h ed on 500
s i l i c a g e l l a y e r s (3 0 m g /la y e r) in 12% b u ty l e th e r /h e x a n e . M arker
s p o ts o f c i s and t r a n s v ita m in w ere a l s o r u n . I t i s known t h a t
t h i s sy stem i s c a p a b le o f s e p a r a t in g c i s and t r a n s iso m e rs v e ry w e ll
( 9 8 ) , b u t o th e r ty p e s o f hom ologues o n ly p o o rly ( 6 5 ) . An e x c e p tio n
t o t h e l a t t e r p o i n t i s t h e s e p a r a tio n o f m e th y la te d com pounds and
t h e i r d e sm e th y l hom o lo g u es. F ig u re 1 shows th e ty p e o f s e p a r a t io n s
w hich c a n b e e x p e c te d w ith t h i s sy ste m . The S tre p to m y c e s * q u in o n e
was fo u n d t o b e s e p a r a te d i n t o two b a n d s , a m ajo r one m ig r a tin g w ith
t h e c i s v ita m in m a rk e r (Rp 0 .3 5 ) and a m in o r o n e a h e a d o f i t
(Rp 0 .* fl) . The b an d s w ere c o l l e c t e d i n d i v i d u a l l y and re c h ro m a to
g ra p h e d u n t i l th e y w ere f r e e o f m u tu a l c o n ta m in a tio n . U l t r a v i o l e t
s p e c tr a o f th e tw o i s o l a t e d com ponents w ere ta k e n and p ro v ed to b e
30
Figure 1 Separation of Menaquinones by Adsorption Chromatography
i d e n t i c a l . They w ere t y p i c a l o f a 2 ,3 - d i s u b s t i t u t e d - 1 ,4 - n a p h th o
q u in o n e (1 0 3 ) show ing a b s o r p tio n peak s a t 242, 248, 26 0 , 268 and
326 mp w ith a s h o u ld e r a t 238 mp ( F ig . 2 ) . Reduced u l t r a v i o l e t
s p e c t r a o f b o th com ponents w ere a l s o ta k e n and a l s o p ro v ed t o be
i d e n t i c a l . B oth showed th e p re s e n c e o f a m ajo r a b s o r p tio n p eak a t
243 mp and m inor p eak s a t 290 and 340 mp ( F ig . 3 ) . On th e b a s is o f
t h i s e v id e n c e , i t was co n c lu d e d t h a t th e o rg an ism c o n ta in e d c i s and
t r a n s iso m e rs o f a 2 , 3 - d i s u b s t i t u t e d m e n a q u in o n e (s). No a tte m p t was
made t o d i s t i n g u i s h betw een tw o p o s s i b i l i t i e s f o r th e o c c u rre n c e o f
th e s e is o m e rs . T h at i s , w h eth er th e y a r o s e n a t u r a l l y o r w ere form ed
by non en zy m atic r e a c t i o n s o c c u rrin g d u rin g i s o l a t i o n and f r a c t i o n
a t i o n p ro c e d u re s . Such non en zy m atic is o m e ra tio n r e a c t i o n s a r e
known to o c c u r w ith v ita m in K ty p e compounds ( 6 5 ,1 0 4 ) .
B ecause c i s and t r a n s iso m e rs o f m enaquinones m ig ra te t o id e n
t i c a l p o s iti o n s on th e o th e r TLC sy stem s u sed in t h i s w o rk , th e two
ban d s w ere reco m b in ed and a l l f u r t h e r s e p a r a tio n s w ere done w ith t h i s
p o o le d m a t e r i a l . The p o o le d m a te r ia l was ch ro m ato g rap h ed on s i l v e r
n i t r a t e im p reg n ate d l a y e r s (3 7 5 u ) in 12% 2 -b u ta n e /h e x a n e . T h is
sy stem i s p a r t i c u l a r l y u s e f u l f o r th e s e p a r a tio n o f m enaquinone homo
lo g u e s d i f f e r i n g in e i t h e r C-3 c h a in le n g th o r in th e d e g re e o f
s a t u r a t i o n o f th e C-3 s id e c h a in . F ig u re 4 shows a t y p i c a l s e p a r a tio n
o f m enaquinones u s in g t h i s sy ste m . The b a s i s f o r d i f f e r e n t i a l m ig r a t
io n o f compounds w ith s i l v e r io n s i s th e com plexing betw een v bonds
and th e io n s ( 1 0 5 ) . T h e re fo re , th e s h o r t e r th e C-3 s id e c h a in o r t h e
h ig h e r th e d e g re e o f s a t u r a t i o n i n th e s id e c h a in , t h e h ig h e r t h e Rp.
v a lu e w i l l b e . As can be se e n in F ig u re 4 , a d i f f e r e n c e o f one
20
EmM
2 4 0 2 8 0 320 3 6 0
{ WAVELENGTH (rnji)
T
i
i
i
i
j
i
F ig u re 2 U l t r a v i o l e t S p e c tra o f M K g in I s o o c ta n e
33
5 0
OXIDIZED
REDUCED
4 0
3 0
20
3 2 0 2 8 0 2 0 0 2 4 0 3 6 0
WAVELENGTH (my)
F ig u r e 3 O x id iz e d and R educed U l t r a v i o l e t S p e c tr a o f M Kg in E th a n o l
3*1
Figure * t Separation of Menaquinones by Silver Ion Impregnated
Adsorption Chromatography
NOUVUniVS dO 33U930
Hi9N3~l NIVH9
i 8 8 8 8 8 S 8 8 9 o
- 1 i i i i i i i I i"
0 1
1
o >
I
< 0
tO
♦
in
o
ro
C J
Figure 5 Separation of Streptomyces Quinones by Silver Ion
Impregnated Adsorption Chromatography
(h - h)
36
Figure 6 Separation of Menaquinones by Reversed Phase Chromatography
methyl (|]-H)
M K o
37
is o p re n o id u n i t r e s u l t s in an Rp ch an g e o f a p p ro x im a te ly 0 .0 8 , f o r
exam ple M K g and MKg, w h ile th e Rp d if f e r e n c e betw een a f u l l y un
s a tu r a t e d and mono s a tu r a te d compound i s a p p ro x im a te ly 0 . 1 a s f o r
exam ple w ith MKg and MKg ( I I - H ) .
When th e p o o led quinone f r a c t i o n from S tre p to m y c e s was chrom
ato g ra p h e d u s in g t h i s sy stem , e ig h t bands w ere seen (F ig u re 5 ) . The
bands w ere a s s ig n e d num bers a s shown in th e f i g u r e . I t can be seen
t h a t band 6 c o m ig ra te d w ith M K g w h ile band 5 c o m ig ra te d w ith MKg
( I I - H ) . The bands w ere e lu te d s e p a r a te ly from th e a d s o rb a n t and
u l t r a v i o l e t s p e c tr a o f each was ta k e n . A ll o f th e s p e c tr a w ere id e n
t i c a l t o th o s e shown in F ig u re s 2 and 3. T hat i s , th e y w ere a l l id e n
t i c a l t o t h a t o f a 2 , 3 - d is u b s titu te d - l,4 - n a p h th o q u in o n e . Bands l a ,
3 a, and 4 w ere p r e s e n t in t r a c e am ounts w h ile bands 1 , 2 , 3, 5 and 6 ,
p r e s e n t in g r e a t e r am ounts, a p p e a re d in m olar r a t i o s o f 4 :2 0 :4 :2 :1
r e s p e c t i v e l y , a s ju d g ed by s p e c tro p h o to m e tric m easurem ent. U sing
t h i s sy stem t h e r e was a n in th band w hich m ig ra te d w ith a lo w er Rp
v a lu e th a n band 6 b u t b ecau se i t s u l t r a v i o l e t s p e c tr a in no way r e
sem bled a q u in o n e , i t was o f no f u r t h e r i n t e r e s t .
S m all am ounts o f th e e ig h t is o l a t e d q u in o n es w ere mixed to g e th e r
f o r ch rom atography on a re v e rs e d p h ase sy stem . F ig u re 6 shows th e
ty p e o f m enaquinone s e p a r a tio n o b ta in e d w ith r e v e r s e d ph ase chrom
a to g ra p h y on 250 p. K ie s e lg u h r G la y e r s in 97% a c e to n e /w a te r. I t can
be se e n t h a t a s w ith th e s i l v e r n i t r a t e sy ste m , a s h o rte n in g o f th e
C-3 s id e c h a in r e s u l t s in h ig h e r R p v a lu e s , a s f o r exam ple M K g and
MKg. W ith t h i s sy stem , th e a b s o lu te change o f Rp v a lu e seen w ith
d e c re a s e o f one is o p re n o id u n i t (a p p ro x im a te ly 0 . 1 0 u n i t s ) i s g r e a t e r
38
th a n t h a t o b se rv e d w ith th e s i l v e r n i t r a t e sy ste m . U n lik e th e s i l v e r
n i t r a t e sy ste m , t h i s sy stem show s n e g a tiv e ch an g e s o f Rp v a lu e w ith an
in c r e a s in g d e g re e o f s a t u r a t i o n . As can be s e e n in t h i s f i g u r e M K g
( I I - H ) m ig ra te s a p p ro x im a te ly 0 .0 3 Rp u n i t s b e h in d M K g .
When th e p o o le d S tre p to m y c e s bands from s i l v e r n i t r a t e chrom
a to g ra p h y w ere ru n w ith t h i s s y s te m , th e r e s u l t s shown in f i g u r e 7
w ere o b ta in e d . As c a n b e seen t h e r e l a t i v e m ig ra tio n s w ere o p p o s ite
to th o s e se e n in f i g u r e 5 ( s i l v e r n i t r a t e s y s te m ). H ere a g a in band
5 m ig ra te d w ith M K g ( I I - H ) and band 6 m ig ra te d w ith MKg,
On th e b a s is o f th e s i l v e r n i t r a t e ch ro m ato g rap h y ( f i g u r e 5 ) , i t
w ould be p r e d ic te d t h a t th e i n d i v id u a l q u in o n e s v a r ie d in th e le n g th
o f t h e C-3 s id e c h a in . T hat i s , band 1 would b e one is o p r e n o id u n i t
s h o r t e r th a n band 2 , e t c . H ow ever, when th e r e v e r s e d p h a se system
r e s u l t s ( f i g u r e 7 ) a r e ta k e n i n t o a c c o u n t, t h i s e x p la n a tio n would no
lo n g e r be te n a b le . The o n ly e x p la n a tio n f o r t h e o b se rv e d r e s u l t s o f
b o th sy stem s was t h a t t h e compounds v a r ie d in t h e d e g re e o f u n
s a t u r a t i o n o f th e C-3 is o p r e n o id s id e c h a in . I f i t i s assum ed t h a t
band 6 i s M K g by i t s co m m ig rato ry b e h a v io r on b o th s y ste m s , and on
s i m i l a r g ro u n d s t h a t band 5 i s M K g (2-H), th e n by m easurem ent o f Rp
v a lu e ch an g es band 3 , 2 , and 1 c a n be assum ed t o be M Kg(^-H), M K g
(6-H) and M K g (8-H) r e s p e c t i v e l y . By th e same re a s o n in g and b e c a u se
band 4 m ig ra te d w ith M K & (2-H) ( n o t shown) i t c o u ld a l s o be assum ed
t h a t ban d s 4 , 3a and l a were M K g ( 2 - H ) , M K g ( 4 - H ) and M K g ( 6 - H ) r e s p e c t
i v e l y . I t sh o u ld be n o te d t h a t ch ro m ato g rap h y o f l i p i d s from
S tre p to m y c e s o liv a c e u s u s in g t h e s e sy stem s showed q u a l i t a t i v e l y
s i m i l a r r e s u l t s .
39
N O iivum vs jo aauoaa
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Figure 7 Separation of Streptomyces Quinones by Reversed Phase
Chromatography
<31-H)
I n f r a r e d S p e c tro s c o p y S p e c tr a o f t h e f i v e m a jo r b an d s ( 1 , 2 , 3 , 5
and 6 ) w ere ta k e n . A ll o f t h e s p e c t r a w ere q u a l i t a t i v e l y s i m i l a r and
w ere t y p i c a l o f t h o s e e x p e c te d o f a n a p h th o q u in o n e w ith an is o p r e n o id
s i d e c h a in ( f i g u r e 8 ) . The p ro m in e n t band a t 2900 cm" 1 and t h e
a s s o c i a t e d s h o u ld e r a t 2860 cm" 1 a r e a s s o c i a t e d w ith CH s t r e t c h i n g
w h ile t h e b an d s a p p e a r in g a t 1445 and 1379 cm"1 , a r e a s s o c i a t e d w ith
CCH2 an d CCHg s t r e t c h i n g r e s p e c t i v e l y . The b a n d s a p p e a r in g in th e
1 2 5 0 -1 3 5 0 and 1 5 5 0 -1 7 0 0 -cm"1 r e g io n s a r e a s s o c i a t e d w ith t h e a r o m a tic
q u in o n e r i n g . The m ost i n t e n s e p eak a t 1660 cm” 1 was d u e t o C=0
s t r e t c h i n g o f t h e q u in o n e and t h e l e s s i n t e n s e 1620 cm” 1 p e a k was due
t o th e C=C s t r e t c h i n g o f t h e r i n g . The band a t 1598 cm” 1 was d u e t o
t h e C=C s k e l e t a l i n p la n e v i b r a t i o n s o f t h e c o n ju g a te d a r o m a tic r i n g
(1 0 6 ) an d t h e b an d a t 712 cm” 1 was a s s o c i a t e d w ith t h e CH o u t o f p la n e
d e fo rm a tio n s o f t h e f o u r a d j a c e n t r i n g h y d ro g e n s o f t h e n a p h th o
q u in o n e n u c le u s . The b a n d s a t 1 2 5 0 -1 3 5 0 cm” 1 w hich a r e a s s o c i a t e d
w ith a l l V ita m in K ty p e com pounds h av e n o t b e e n g iv e n d e f i n i t e a s s i g n
m en ts ( 1 0 7 ) . The m ain b an d in t h e 840 cm” 1 r e g i o n w hich a p p e a r s a t
838 cm"1 , th e s h o u ld e r a p p e a r in g a t 812 cm" 1 a n d t h e s m a ll band a t
8 8 8 cm" 1 a r e a l l r e p r e s e n t a t i v e o f t h e is o p r e n o id c h a in o f t h e n a p h th
o q u in o n e . The weak b an d a t 1740 cm" 1 was b e l ie v e d to t h e p r e s e n c e
o f s l i g h t c a rb o n y l c o n ta m in a tio n .
Mass S p e c tro m e try The sa m p le s w ere p r e p a r e d f o r b o th m ass s p e c t r o
sco p y an d n u c l e a r m a g n e tic re s o n a n c e s p e c tr o s c o p y in t h e f o llo w in g
m a n n er. F o llo w in g s e p a r a t i o n by s i l v e r n i t r a t e c h ro m a to g ra p h y , t h e
s e p a r a te d com pounds w ere c h ro m a to g ra p h e d on t h e m e n tio n e d r e v e r s e d
Figure 8 In frared Spectra o f M K (
FREQUENCY (CM4 )
1200 1100
5000 4000 3000 2500 2000 1800 1600 1400 1 000 950 900 850 800 750
WAVELENGTH (MICRONS)
p h a se s y ste m , th e n w ere r u n tw ic e on 250 s i l i c a g e l G p l a t e s , o n ce
in 12% b u ty l e th e r /h e x a n e and o n c e in 5% d i e t h y l e th e r /b e n z e n e . T hese
l a s t tw o s te p s e lim in a te d a l l o f t h e p a r a f f i n w hich was e l u t e d fro m
th e r e v e r s e d p h a se sy stem a lo n g w ith th e q u in o n e s . At t h i s p o i n t ,
th e sam p les w ere s to r e d u n t i l u s e a t - 1 0 ° u n d e r a n a tm o sp h e re o f n i t r o
g e n .
Mass s p e c tr a o f t h e f i v e s a m p le s , (b a n d s 1 , 2 ,3 , 5 and 6 ) and o f
a u t h e n tic MKg an d MKg ( I I - H ) w ere th e n r e c o rd e d on a V a ria n m odel M 6 6
s p e c tro p h o to m e te r. The m o le c u la r io n p eak o f MKg ( I I - H ) a p p e a re d a t
m /e 786 in ag ree m en t w ith Beau e t a l . ( 9 4 ) , and t h a t o f MKg a p p e a re d
a t m /e 78 4 . The m o le c u la r io n s o f ban d s 1 , 2 ,3 , 5 and 6 a p p e a re d a t m/e
7 9 2 , 7 9 0 , 7 8 8 , 786 and 784 r e s p e c t i v e l y . A ll o f th e com pounds, b o th
unknowns and s ta n d a r d s a l s o showed in t e n s e p eak s a t m/e 225 w hich i s
c h a r a c t e r i s t i c o f a fra g m e n t o f 2 , 3 - d i s u b s tit u te d - l ,4 - n a p h t h o q u in o n e s
w hich h a s b een a s s ig n e d o n e o r b o th o f t h e fo llo w in g s t r u c t u r e s b y
D iM ari e t a l . (1 0 8 ) .
The a p p e a ra n c e o f th e p e a k s a t m /e 225 s u g g e s ts t h a t th e s e com pounds
a r e a l l 1 ,4 -n a p h th o q u in o n e s w ith a t l e a s t one u n s a tu r a te d is o p r e n o id
u n i t a d ja c e n t t o t h e r i n g sy ste m . The m o le c u la r io n s a t m /e 7 9 2 , 790,
7 8 8 , 7 8 6 , and 784 s u g g e s ts , a s d o e s t h e i r c h ro m a to g ra p h ic b e h a v io r ,
O O
t h a t bands 1 ,2 ,3 ,5 and 6 a r e M K g( 8 -H ), M K g( 6 -H ), M K g(4— H ), M K g(2-H ) and
M K g r e s p e c t iv e l y . B ecause o f th e r e l a t i v e l y low s e n s i t i v i t y o f th e
m achine no f u r t h e r a n a l y s i s a s to th e e x a c t lo c a tio n o f th e s a tu r a te d
d o u b le bonds c o u ld be made. Such a n a ly s is h a s been shown t o be f e a
s i b l e w ith c e r t a i n q u in o n e s by L ed e rer (1 0 9 ) and Dunphy e t a l . (9 0 ).
T hese w orkers w ere a b le t o d e m o n stra te s e q u e n tia l lo s s e s o f is o p re n o id
u n i t s in th e c ra c k in g p a t t e r n s o f th e M K g( I I - H ) , F o llo w in g a lo s s o f
15 mass u n i t s due t o th e l o s s o f m ethyl g ro u p s, a l o s s o f 69 mass
u n i t s due to an u n s a tu r a te d to- te r m in a l is o p re n o id u n i t was seen
fo llo w e d by th e l o s s o f s i x 6 8 mass u n it fra g m e n ts c o rre sp o n d in g t o
u n s a tu r a te d i n t e r n a l is o p re n o id u n i t s . B ecause th e peak a p p e a rs a t
225 t h i s m eant th e re m a in in g u n it m ust be s a tu r a t e d .
B ecause i t was o f i n t e r e s t t o d is c o v e r th e e x a c t lo c a tio n s o f th e
s a tu r a t e d is o p re n o id u n i t ( s ) , a h ig h e r r e s o lu t io n m achine, P e rk in
Elm er H ita c h i RMU-6 E, was em ployed. D e s p ite many a tte m p ts o n ly band
5 was s u c c e s s f u lly a n a ly z e d by t h i s m ethod, th e o th e r compounds (b an d s
1 , 2 , and 3) g iv in g o n ly t e n t a t i v e a n sw e rs. Band 5 showed a m o le c u la r
io n a t m /e 786, fo llo w e d by peaks a t m/e 7 7 1 , lo s s o f m eth y l groups
m /e 717, lo s s o f a c o -te rm in a l u n s a tu r a te d u n i t , m /e 6*^9, 5 81, 513,
*♦45, 377 and 309 l o s s o f 6 i n t e r n a l u n s a tu r a te d u n i t s . T h is c ra c k in g
p a t t e r n i s s im i la r t o th o s e seen by L e d e re r (109) and Dunphy e t a l .
( 9 0 ) , and c o n firm s t h a t t h e second is o p re n o id u n i t from th e r in g i s
th e s a tu r a te d o n e . T h e re fo re , band 5 was a s s ig n e d a s t r u c t u r e id e n
t i c a l t o M K g ( I I - H ) .
Band 3 showed a m o le c u la r io n a t m /e 788 and a peak a t m/e 719
c o rre sp o n d in g t o th e l o s s o f m ethyl g ro u p s . F ollow ing t h i s a peak
44
was se e n a t m /e 7X9 c o rre s p o n d in g to l o s s o f a to - te r m in a l u n s a tu r a te d
u n i t . F o llo w in g t h i s , peaks w ere seen a t m/e 6 5 1 , 583, 5 1 5 , 447 and
379 c o rre s p o n d in g to l o s s o f 5 i n t e r n a l u n s a tu r a te d u n i t s . B ecause
th e l a r g e m /e p eak was s e e n , an d from n u c le a r m a g n e tic re s o n a n c e
s p e c tro s c o p y d a ta ( t o be d is c u s s e d in t h e fo llo w in g s e c t i o n ) , i t can
be assum ed t h a t th e r in g a d ja c e n t is o p re n o id u n i t i s u n s a tu r a te d so
t h a t m ost l i k e l y u n i t s number I I and I I I ac c o u n t f o r th e f o u r e x tr a
h y d ro g en s m aking band 3 M K g (II,III-H ). I t sh o u ld b e s t r e s s e d t h a t
due t o a p o o r c e r i b r a t i o n sy stem and m u l tip l e m in o r io n s t h e peaks
s e e n h e re may p o s s ib ly be o f f by one m ass u n it in some c a s e s so t h a t
th e r e s u l t s a r e t e n t a t i v e .
Band 2 showed a m o le c u la r io n a t m /e 790 fo llo w e d by a peak a t
m /e 775 due t o th e l o s s o f m e th y l g ro u p s . F o llo w in g t h i s a peak was
s e e n a t m /e 719 due t o th e l o s s o f an O J-te rm in a l s a tu r a t e d u n i t .
T h is was fo llo w e d by p eak s a t m /e 651, 58 3 , and 515 due t o t h e l o s s
o f t h r e e i n t e r n a l u n s a tu r a te d u n i t s . Beyond t h i s no d e f i n i t e id e n
t i f i c a t i o n o f s t r u c t u r e co u ld b e made b y th e c r a c k in g p a t t e r n e x c e p t
t h a t a l a r g e p eak a t m /e 225 in d ic a te d t h a t th e r i n g te r m in a l is o
p re n o id u n i t was u n s a tu r a te d . T his th e n means t h a t th e r e a r e two
s a tu r a t e d u n i t s lo c a te d in some c o m b in a tio n in u n i t s I I , I I I , IV o r
V, B io c h em ical e v id e n c e ( t o b e d is c u s s e d in a s e p a r a te s e c t i o n )
s u g g e s te d t h a t a t l e a s t one o f th e s a t u r a t e d bonds was in u n i t I I .
Band I showed a m o le c u la r io n p eak a t m/e 7 9 2 , and a l o s s o f
m e th y l g ro u p s se e n a s a peak a t m/e 7 7 7 . F o llo w in g t h i s , a p eak a t
m /e 721 d e m o n stra te d th e lo s s o f an w - te r m in a l s a tu r a t e d u n i t . Be
tw een t h i s p o in t and a m ajor m /e 225 p e a k no m eaning c o u ld b e made
45
o u t o f t h e c r a c k in g p a t t e r n . T h is m eans t h a t t h e s i x re m a in in g h y d ro
g en s (2 a r e i n u n i t IX ) a r e lo c a te d i n t h r e e is o p r e n o id u n i t s in some
p a t t e r n i n u n i t s I I - X I I I .
P a r t o f th e p ro b le m in v o lv e d i n t h e m ass s p e c tr o p h o to m e tr ic
a n a l y s i s o f th e s e com pounds u n d o u b te d ly a r i s e s from t h e f a c t t h a t a s
h y d ro g e n a tio n o f is o p r e n o i d u n i t s i n c r e a s e s , t h e r e i s n o s e l e c t i v i t y
o f c r a c k in g o f f is o p r e n o id u n i t s . T h is i s b e c a u s e w ith o u t d o u b le
bonds t h e r e i s l i t t l e s e l e c t i v e a d v a n ta g e f o r c e r t a i n io n s o v e r o t h e r s
and c o n s e q u e n tly many m inor io n s a p p e a r . I f enough s u i t a b l e s ta n d a r d s
w ere a v a i l a b l e w ith v a r io u s known p a t t e r n s o f h y d r o g e n a tio n , th a n
h ig h r e s o l u t i o n m ass s p e c tro m e try m ig h t y i e l d d e f i n i t e a n s w e rs a s t o
th e e x a c t l o c a t i o n o f t h e s a tu r a t e d is o p r e n o i d u n i t s .
N u c le a r M ag n etic R eso n an ce S p e c tro s c o p y N u c le a r M ag n etic R esonance
(NMR) s p e c t r a o f b a n d s 2 and 5 w ere ta k e n a s w e ll a s t h a t o f M K g( I I - H ) .
The s p e c t r a a r e r e p o r t e d a s V v a l u e s . The s p e c t r a w ere ta k e n in CCl^
and w ere b o th t y p i c a l o f m e n aq u in o n es. The s p e c t r a o f M K g( I I - H ) i s
shown i n f i g u r e 9 , a n d t h a t o f band 2 i s show n in f i g u r e 1 0 . A ss ig n
m en ts f o r V v a lu e s i n f i g u r e 10 w ere made a s f o ll o w s . The com plex
p a t t e r n fro m 2 .0 t o 2 .5 i s due to t h e f o u r p r o to n s a d j a c e n t in t h e
b e n z o id r i n g . The p e a k s a t 4 .9 5 , 8 .1 8 and 8 .2 8 a l l r e s u l t from t h e
d o u b le bond in t h e C-3 is o p r e n o id s id e c h a i n . The t r i p l e t a t
4195 i s d u e t o th e C -2 m e th y l g ro u p . The d o u b le t a t 6 .6 9 i s due t o
th e r i n g a d j a c e n t is o p r e n o id u n i t a n d r e f l e c t s th e p r e s e n c e o f t h e
m e th y le n e g ro u p a d j a c e n t to th e r i n g . The f a c t t h a t t h e r e i s a
d o u b le t a t 6 .6 9 i n d i c a t e s t h a t t h e r i n g - t e r m i n a l is o p r e n o id u n i t i s
46
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F ig u re 10 N u c le a r M ag n e tic .R e so n a n c e S p e c tra o f Band 2 in CCl^
48
u n s a t u r a t e d . I f t h e r i n g - t e r m i n a l is o p r e n o id w ere s a t u r a t e d , i t w ould
b e r e f l e c t e d by a s h i f t i n th e 6 .6 9 d o u b le t t o an u p f i e l d t r i p l e t
a p p e a rin g a ro u n d 6 .9 5 ( 1 1 0 ) . The s i n g l e t a t 7 .8 8 i s d u e t o t h e r i n g
m e th y l g ro u p a t t h e C-2 p o s i t i o n o f t h e q u in o n e r i n g .
The l a r g e p e a k a t 8 . 0 4 i s a t t r i b u t e d t o t h e m e th y le n e g ro u p s in
t h e C - 3 s i d e c h a i n . The l a r g e p e a k a t 8 . 4 6 i s d u e to t h e t r a n s m e th y l
g ro u p s on i n t e r n a l and c o -te rm in a l is o p r e n o id u n i t s . Of p a r t i c u l a r
i n t e r e s t i s t h e a b s e n c e o f a p eak a t 8 . 3 4 — 8 . 4 0 w hich i s s e e n in M K g
( I I - H ) ( f i g u r e 9) a t 8 . 3 4 and i s a t t r i b u t e d t o t h e c o n t r i b u t i o n o f
e i t h e r c i s o r t r a n s C o -te rm in a l is o p r e n o id u n i t s . T h is te n d e d t o
i n d i c a t e , a s d id m ass s p e c t r a d a t a , t h a t t h e c o - te r m in a l is o p r e n o id
u n i t was s a t u r a t e d . The re m a in in g tw o m a jo r p e a k s , th e s i n g l e t a t
8 . 8 and t h e d o u b le t a t 9 . 1 8 a r e d u e t o t h e C - 3 s i d e c h a in C H 2 a n d C Hg
g ro u p s r e s p e c t i v e l y . I t s h o u ld b e n o te d t h a t t h e s e p e a k s a t 8 . 8 and
9 . 1 8 o n ly a p p e a r when t h e r e i s s a t u r a t i o n o f o n e o r m ore d o u b le bonds
i n t h e s i d e c h a in ( 36) an d t h a t t h e i r s i z e i s d i r e c t l y p r o p o r ti o n a l
t o t h e d e g re e o f s a t u r a t i o n . T h is can b e s e e n i n f i g u r e s 9 an d 1 0
w here b an d 2 ( f i g u r e 1 0 ) w ith s i x e x t r a s id e c h a in h y d ro g e n s d i s
p la y s l a r g e r 8 . 8 an d 9 . 1 8 p e a k s th a n M K g ( I I - H ) ( f i g u r e 9) w ith tw o
e x t r a s i d e c h a in h y d ro g e n s .
I t i s p o s s i b l e t o s e p a r a t e o u t t h e e f f e c t s o f i n t e r n a l and
te r m i n a l s a t u r a t i o n m ore c o m p le te ly by u s in g b e n z e n e a s a s o lv e n t
f o r N M R s p e c t r a ( 1 1 0 ) . T h is i s p o s s i b l e f o r t h e f o llo w in g r e a s o n . In
CClj^ i n t e r n a l a n d u ) - te r m in a l c i s m e th y ls a s w e ll a s r i n g te r m in a l
t r a n s m e th y l g ro u p s a p p e a r a s p e a k s a t a b o u t 8 .3 4 . A lso b o th i n t e r n a l
and U > -te rm in al t r a n s m e th y ls a r e r e f l e c t e d a s a p eak a t 8 .4 2 . In
49
b en zen e how ever, to - te r m in a l u n i t s r e f l e c t c i s m e th y ls a t ab o u t 8 .3 2
and tr a n s m ethyls a t a b o u t 8 .4 3 , w h ile i n t e r n a l u n i t s r e f l e c t c i s
m e th y ls a t about 8 .2 5 and t r a n s m e th y ls a t a b o u t 8 .3 8 . T h e re fo re th e
f i e l d s a t about 8 .3 2 and 8 .4 3 a r e c l e a r o f o b s tr u c tio n from s ig n a ls
p ro d u ced by i n t e r n a l u n i t s . I f s ig n a l s a p p e a r a t th e s e p o in ts i t
r e f l e c t s th e f a c t t h a t w - te r m in a l s a t u r a t i o n i s n o t p r e s e n t.
For t h i s re a s o n s p e c tr a o f bands 1 ,2 ,3 , and 5 and M K g (II-H ) were
ta k e n in b enzene. B ecause th e benzene w i l l g iv e r i s e to p e a k s in th e
2 .0 to 2 .5 re g io n t h e s p e c tr a can o n ly be re c o rd e d u p f ie ld from 3 .0 .
F ig u re s 1 1 ,1 2 ,1 3 ,1 4 and 15 show th e r e s u l t s o f N M R s p e c tr a o f bands
1 , 2 ,3 , 5 and MKg ( I I - H ) r e s p e c t i v e l y . B ecause th e a r e a o f i n t e r e s t
l i e s u p f ie ld o f 7 .0 o n ly t h a t p o r tio n o f th e s p e c tr a i s shown in th e
f i g u r e s . Of i n t e r e s t i s th e f a c t t h a t s p e c tr a o f band 3 , 5 and M K g
( I I - H ) ( f ig u r e s 1 3 ,1 4 , and 15 r e s p e c t iv e l y ) show p ro m in en t peaks a t
8 .3 4 and 8.43 in d i c a ti n g t h a t t h e r e a r e c i s and t r a n s m ethyl g ro u p s in
t h e to - te r m in a l is o p re n o id u n i t showing t h a t t h i s u n i t i s u n s a tu r a te d .
The ab sen ce o f th e s e p eak s in th e s p e c tr a o f bands 1 and 2 ( f ig u r e s 11
an d 1 2 r e s p e c t iv e l y ) d e m o n stra te a s d id m ass s p e c t r a l d a ta t h a t th e s e
compounds p o sse ss to - te r m in a l s a t u r a t i o n . A lso o f i n t e r e s t i s th e
f a c t t h a t s p e c tra o f M K g (II - H ) and band 5 ( f i g u r e s 15 and 14 r e s p e c t
i v e l y ) show e q u iv a le n t s iz e d p eak s a t 8 . 8 and 9.18 due t o t h e two
e x t r a s id e c h a in p ro to n s w h ile s p e c tr a o f bands 3 ,2 , and 1 ( f ig u r e s 1 3 ,
12 and 11 r e s p e c t iv e l y ) show p eak s a t 8 . 8 and 9.18 w hich in c re a s e in
s i z e p r o p o r tio n a lly t o th e number o f s a t u r a t i n g p ro to n s in t h e i r s id e
c h a i n s .
50
7 . 0 8.0 9 . 0 10, 0
Figure 11 Nuclear Magnetic Resonance Spectra of Band 1 in Benzene
7 . 0 8.0
9 . 0 10.0
Figure 12 Nuclear Magnetic Resonance Spectra of Band 2 in Benzene
52
1
s
1
1
I
i
1
t
!
1
7. 0
8. 0 1Q0 9. 0
Figure 13 Nuclear Magnetic Resonance Spectra of Band 3 in Benzene
53
i /
i ■■ H '
i
i
i
i
t
!
I
i
i
!
i
|
i
i
\
i
>
I
I
7. 0
9. 0 10,0
Figure 1^ Nuclear Magnetic Resonance Spectra of Band 5 in Benzene
54
— I * ---* ---i ---1 ---1 ---1 __i__i __i __I __i__i— t
7 , 0 8,0
9 . 0 1 0 . 0
Figure 15 Nuclear Magnetic Resonance Spectra of MKQ (II-H) in Benzene
O z o n o lsis S tu d ie s Ozone i s known to add t o d o u b le b o n d s. The r e
s u l t i n g o z o n id e p ro d u c t ca n th e n be s p l i t b y r e d u c t io n t o y ie ld f r a g
m ents o f compounds b ro k en a t p o in t s o f u n s a tu r a t io n . I t w as, t h e r e
f o r e , o f i n t e r e s t t o p erfo rm o z o n o ly s is on th e S tre p to m y c e s q uinones
t o s e e i f fra g m e n ts c o u ld r e v e a l t h e r e l a t i v e l o c a t i o n o f s a tu r a te d
is o p re n o id u n i t s . T h at i s i f two s a tu r a t e d u n i t s w ere a d ja c e n t th e
re c o v e re d frag m e n t w ould b e f i v e c a rb o n s lo n g e r th a n i f s a tu r a te d
u n i t s w ere n o t a d j a c e n t . B ecause i t would b e e x tre m e ly d i f f i c u l t t o
o b ta in s ta n d a r d s f o r a l l p o s s i b le c o m b in a tio n s th e r e s u l t s o f t h i s
stu d y w ere r a t h e r d i f f i c u l t t o i n t e r p r e t an d w ere s u g g e s tiv e a t b e s t .
The s id e c h a in p ro d u c ts o f M Kg ( I I - H ) sh o u ld b e a f i v e carb o n
k e to -a ld e h y d e , r e s u l t i n g from a d ja c e n t u n s a tu r a te d is o p re n o id u n i t s
and a te n c a rb o n k e to -a ld e h y d e r e s u l t i n g from th e one s a tu r a te d u n i t .
P ro d u c ts o f o z o n o ly s is o f M K g ( I I - H ) w ere a n a ly z e d by g as chrom ato
g rap h y and showed tw o m ajo r p eak s n o t a s s o c ia te d w ith th e re d u c ta n t
u sed w hich g av e s e v e r a l p e a k s . Those p eak s due to M K g ( I I - H ) p ro d
u c ts w ere lo c a te d a t 9 .1 0 and 2 .7 5 m in u te s . The f i v e c a rb o n k e to -
a ld e h y d e p ro b a b ly came o f f t h e colum n in th e s o lv e n t p eak so th e
2 .7 5 p eak was th o u g h t t o b e e l i c i t e d by th e te n c a rb o n k e to -a ld e h y d e .
The 9 .1 0 peak was a lm o s t i d e n t i c a l t o a 9 .1 2 p eak s e e n w ith MK^, w hich
sh o u ld y i e l d o n ly a r i n g a ld e h y d e p r o d u c t. Band 5 showed two m ajor
p e a k s ; one a t 9.0 8 and one a t 2 .6 7 m in u te s . T hese w ere th o u g h t to
r e p r e s e n t i d e n t i c a l p ro d u c ts t o th o s e o f M K g ( I I - H ) . Band 3 showed
p eak s a t 9 .0 9 ( r i n g p e a k ) and 7 .6 1 , w ith a v e ry weak s ig n a l a t 2 .6 9 .
The 7 .6 1 p eak may b e due t o two a d ja c e n t s a tu r a t e d is o p r e n o id u n it s
y ie ld in g a f i f t e e n ca rb o n k e to -a ld e h y d e . T h is w ould a g re e w ith th e
56
m ass s p e c t r a l d a ta w hich s u g g e s te d t h a t is o p re n o id u n i t s I I and I I I
w ere s a t u r a t e d . Band 2 showed m ajo r p eak s a t 9 .0 5 ( r in g p e a k ) and
7 .5 6 (2 a d ja c e n t s a tu r a t e d u n i t s ) . The p ro d u c t from one te r m in a l
s a tu r a t e d is o p re n o id u n i t w hich i s known to o c c u r in band 2 would
have come o u t in th e s o lv e n t p eak a s ju d g e d from a n a l y s i s o f a
s ta n d a r d f o r t h i s fra g m e n t. A t h i r d p eak in th e a n a l y s i s o f band 2
a t 8 .1 4 c o u ld n o t be a s s ig n e d a s t r u c t u r e . Band 1 showed p eak s a t
9 .0 7 ( r i n g p e a k ), 7 .6 0 (2 a d ja c e n t s a tu r a t e d u n i t s ) and 3 .8 1 . The
3 .8 1 peak c o rre s p o n d s t o a s ta n d a r d f o r tw o te r m in a l s a tu r a te d u n i t s
w hich g iv e s r i s e t o a peak a t t h e same tim e o r s l i g h t l y s o o n e r. A lso
p r e s e n t in t h i s sam ple was a p eak a t 3 .3 1 w hich i s u n e x p la in e d and
a p eak a t 8 .1 6 w hich i s s i m i l a r to th e u n e x p la in e d peak se e n w ith
band 2 .
Q uinone R e s to r a tio n o f O x id a tiv e P h o s p h o ry la tio n The n a t u r a l
q u in o n e found in M. p h l e i ca n b e s e l e c t i v e l y d e s tro y e d by l i g h t a t
360 mu ( 7 4 ) . T h is tr e a tm e n t r e s u l t s in th e d e s tr u c t io n o f NAD+ lin k e d
o x id a tio n and a s s o c ia te d p h o s p h o ry la tio n . O x id a tio n can be r e s to r e d
by t h e a d d i tio n o f many 1 ,4 -n a p h th o q u in o n e s . H owever, th e re c o v e ry
o f a s s o c ia t e d p h o s p h o ry la tio n i s d ep en d en t on v e ry s p e c i f i c q uinone
s t r u c t u r e (1 1 1 ,1 1 2 ). T h is phenomenon i s d e m o n stra te d by th e d a ta in
T a b le s 3 and 4 . The d a ta in th e s e t a b l e s was o b ta in e d u s in g a system
w hich o n ly a llo w s p h o s p h o ry la tio n t o o c c u r a t s i t e 2 .
As can be se e n from th e t a b l e s , p h o s p h o ry la tio n was r e s t o r e d t o
th e i n a c t i v a t e d sy stem by o n ly t r a n s V itam in o r th e n a t u r a l q u in o n e ,
M K g ( I I - H ) . T hese two m o le c u le s have in common th e 1 ,4 -n a p h th o q u in o n e
TABLE 3
U n irra d ia te d
I r r a d i a t e d m inus Quinone
I r r a d i a t e d
I r r a d i a t e d
I r r a d i a t e d
I r r a d i a t e d
I r r a d i a t e d
I r r a d i a t e d
I r r a d i a t e d
I r r a d i a t e d
I r r a d i a t e d
+ K x
+ tr a n s K
+
+
+
+ M K g (II-H )
+
+
+
c i s K1
mk3
M K C
la p a c h o l*
m enadione*
p h o sp h o lip id *
Quinone R e s to ra tio n o f O x id a tiv e P h o sp h o ry la tio n
Oxygen u p ta k e P h o sp h ate e s t e r i f i e d Oxygen u p ta k e P h o sp h ate e s t e r i f i e d P /0
uatom s um oles uatom s/um um oles/um
T o ta l T o ta l Q uinone added Quinone added
1 0 .7 2 .5 0 .2 3
2 .3 0 . 0 0 . 0
1 0 .5 2 . 6 2 . 0 0 .5 0 .2 5
1 7 .1 5 .4 2 . 1 0.7 0.3 2
5 .4 0 . 0 2 . 6 0 . 0 0 . 0
1 9 .5 0 . 0 4 .0 0 . 0 0 . 0
3 .4 0 . 0 1 .7 0 . 0 0 . 0
1 1 .4 3 .2 2 . 1 0 . 6 0.28
1 8 .9 0 . 0 0 . 0
2 4 .0 0 . 0 0 . 0
3 .0 0 . 0 0 . 0
The r e a c tio n m ix tu re f o r th e s e e x p e rim e n ts c o n ta in e d 15 um oles DPNH, 10 um oles AMP, 50 um oles KF,
30 um oles MgCl2 , 20 um oles g lu c o s e , 3 mg y e a s t h e x o k in a se , 100 um oles HEPES-K0H pH 7 .4 , 12 um oles K H2 P01 | ,
7 .5 rag p a r t i c u l a t e p r o te i n , 0 .3 ml AS I I (32 mg p r o te in /m l) , 0 .2 ml quinone s o lu tio n ; and s u f f i c i e n t w a te r
to b rin g th e t o t a l volume t o 1 .5 m l. The r e a c tio n was ru n f o r 10 min a t 30° and sto p p ed by th e a d d itio n o f
1 ml 10% TCA. Where in d ic a te d th e p a r t i c l e s w ere i r r a d i a t e d f o r 30 m in.
C o n c e n tr a tio n unknown
Cn
TABLE 4
Quinone R e s to ra tio n o f O x id a tiv e P h o s p h o ry la tio n
Oxygen u p ta k e P h o sp h ate e s t e r i f i e d Oxygen u p ta k e P h o sp h ate e s t e r i f i e d P /0
uatom s um oles uatom s/um um oles/um
T o ta l T o ta l Quinone added Q uinone added
U n irra d ia te d 1 0 . 6 4 .8 0 .4 5
I r r a d i a t e d m inus Quinone 3 .3 0 . 0 0 . 0
I r r a d i a t e d + K ^ 1 3 .7 5 .5 3 .3 1 .3 0 .4 0
I r r a d i a t e d + DHP 6 .3 0 . 0 1 , 1 0 . 0 0 . 0
I r r a d i a t e d + D M K 1 0 . 0 0 . 0 4 .2 0 . 0 0 . 0
I r r a d i a t e d + T 5 -OH 7 .1 0 . 0 3 .9 0 . 0 0 . 0
C o n d itio n s s im i la r t o th o s e in T able 3 , e x c e p t t h a t 1 2 .6 mg o f p a r t i c u l a t e p r o te in was u se d .
w
C O
59
TABLE 5
Q uinone R e s to r a tio n o f O x id a tiv e P h o s p h o ry la tio n
Oxygen u p ta k e P h o sp h a te e s t e r i f i e d P /0
U n ir r a d ia te d
(u a to m s)
9 .5
(u m o les)
5 .3 0 .5 6
I r r a d ia te d - m in u s q u in o n e 5 .3 0 .7 0 .0 3
I r r a d i a t e d t M K g ( I I - H ) 9 .4 3 .4 0 .3 6
I r r a d i a t e d + Band 2 8 . 6 3 .0 0 .3 5
I r r a d i a t e d + V itam in 1 2 . 1 2 .9 0 .2 4
C o n d itio n s w ere s i m i l a r t o T ab le 3 , e x c e p t t h a t a l l f l a s k s
c o n ta in e d 5 um oles o f q u in o n e .
60
n u c le u s w ith a C-2 m e th y l g ro u p a n d a C-3 is o p r e n o id s i d e c h a in w ith a
r i n g te r m i n a l u n s a t u r a t e d is o p r e n o id u n i t and a d j a c e n t s a t u r a t e d i s o
p r e n o id u n i t . I t w ould a p p e a r , t h e r e f o r e , t h a t t h e a b i l i t y o f an y
q u in o n e t o r e s t o r e c o u p le d p h o s p h o r y la tio n t o l i g h t i n a c t i v a t e d ex
t r a c t s w ould be e v id e n c e o f t h i s f i r s t tw o u n i t s a t u r a t i o n p a t t e r n .
B ecau se o f t h e l i m i t e d am ount o f S tre p to m y c e s q u in o n e , i t was o n ly
p o s s i b l e t o t e s t b an d 2 w ith t h i s sy ste m . T a b le 5 shows t h a t , in f a c t ,
b and 2 d o e s r e s t o r e c o u p le d a c t i v i t y w ith t h i s a s s a y sy ste m w hich
s u p p o r ts o t h e r d a t a s u g g e s tin g t h a t tw o o f t h e s a t u r a t i n g h y d ro g e n s
o f band 2 a r e in t h e seco n d is o p r e n o id u n i t from t h e r i n g .
B eca u se o f a g e n e r a l i n t e r e s t i n is o p r e n o id b io s y n t h e s i s much tim e
was s p e n t lo o k in g f o r a e e l l f r e e sy stem f o r q u in o n e b i o s y n t h e s i s .
W hile t h e pathw ay o f n o n - b a c t e r i a l is o p r e n o id b i o s y n t h e s i s , in v o lv in g
m e v a lo n ic a c i d , h a s b e e n w e ll w orked o u t , no e v id e n c e h a s e v e r b e e n
g a th e r e d t h a t b a c t e r i a u s e t h i s b i o s y n t h e t i c r o u t e . I n a d d i t i o n no
o t h e r p ath w ay h a s b e e n fo u n d in b a c t e r i a so t h a t t h e e n t i r e i s s u e i s
v e ry c lo u d e d . I t was hoped t h a t i f an e f f i c i e n t c e l l f r e e sy ste m c o u ld
b e fo u n d some l i g h t m ig h t b e sh ed on t h e s te p s in v o lv e d i n t h e h y d ro
g e n a tio n o f q u in o n e s su c h a s t h o s e fo u n d in th e S tre p to m y c e s o rg a n is m .
B eca u se o f t h e l i m i t e d a v a i l a b i l i t y o f S tre p to m y c e s c e l l s s e v e r a l
o t h e r o rg a n is m s w ere u s e d . T hese in c lu d e d , M. p h l e i , _E, c o l i an d M.
l y s o d e i k t i c u s . When any o f t h e s e o rg a n is m s was grow n in t h e p r e s e n c e
o f la b e l e d m e v a lo n ic a c i d , s e v e r a l am ino a c i d s o r a c e t a t e n o r a d i o
a c t i v i t y w as in c o r p o r a te d i n t o t h e q u in o n e s . G row ing M. p h l e i i n t h e
p r e s e n c e o f u n if o r m ly l a b e l e d l a c t a t e r e s u l t e d i n th e in c o r p o r a tio n o f
l a b e l i n t o t h e q u in o n e w hich was shown by c h e m ic a l d e g r a d a tio n t o b e in
61
b o th t h e s i d e c h a in and t h e r i n g sy ste m .
F u r th e r e x a m in a tio n o f t h i s in v i t r o sy ste m showed t h a t o n ly when
t h e l a b e l was i n e i t h e r c a rb o n num ber 2 o r 3 was l a b e l in c o r p o r a te d i n
t o t h e q u in o n e . When c a rb o x y la b e l e d l a c t a t e was u se d i n t h e g ro w th
medium t h e q u in o n e s w ere u n la b e le d and t h e CO2 w hich was g iv e n o f f by
t h e c u l t u r e was l a b e l e d . T h is i n d i c a t e d t h a t t h i s t h r e e c a rb o n p r e
c u r s o r was d e c a r b o x y la te d p e rh a p s t o form a n a c t i v e tw o c a rb o n f r a g
m ent w hich c o u ld th e n b e u s e d f o r q u in o n e b i o s y n t h e s i s .
H aving fo u n d a known p r e c u r s o r m o le c u le f o r t h e q u in o n e a s tu d y
w as u n d e rta k e n t o f i n d a n in v i t r o sy ste m f o r t h e b i o s y n t h e s i s o f t h e
M Kg ( I I - H ) i n M. p h l e i . A ll a tte m p ts a t t h i s w ere n e g a t iv e a lth o u g h
a g r e a t num ber o f v a r i a t i o n s w ere t r i e d . The fo llo w in g i s a p a r t i a l
l i s t o f t h o s e v a r i a t i o n s .
1 . A d d itio n o f many c o f a c t o r s su c h a s ATP, GTP, ADP, DPNH, DPN,
TPNH, TPN, FAD, l i p o a t e , g l u t a t h i o n e , p y r id o x a l p h o s p h a te ,
an d a c e ty l-C o A .
2 . A d d itio n o f many o x i d i z a b l e s u b s t r a t e s su c h a s - k a t o -
g l u t a r a t e , i s o c i t r a t e , m a la te , £ - h y d r o x y b u ty r a te , s u c c in a te
a n d p y r u v a te .
3 . R unning t h e e x p e rim e n ts a e r o b i c a l l y o r a n a e r o b i c a l l y .
4 . I r r a d i a t i o n o f c e l l f r e e e x t r a c t s a t 360 mji t o d e s tr o y
en d o g en o u s q u in o n e s .
5 . R unning t h e e x p e rim e n ts a t v a r io u s te m p e r a tu r e s and io n i c
s t r e n g t h s .
6 . P r e p a r in g e x t r a c t s fro m c e l l s a t 35° t o a v o id d e s t r u c t i o n
o f a p o s s i b l y c o ld s e n s i t i v e enzym e.
62
7 . R e a d d itio n o f c e l l membrane m a te r ia l a f t e r s e p a r a tio n from
a s o n ic a te d p r e p a r a tio n .
8 . A d d itio n o f c e l l f r e e mammalian e x t r a c t s .
9. A d d itio n o f c e l l f r e e e x t r a c t s from M. l y s o d e i k t i c u s .
10. A d d itio n o f m enadione o r 2 - m e t h y l - l , U -n ap h th o q u in o n e a s
p o s s i b l e r i n g sy stem p r e c u r s o r s .
1 1 . A d d itio n o f M K g and NADT, T2 0 , o r re d u c e d t r i t a t e d l i p o a t e
t o lo o k f o r r a d i o a c t i v e M K g ( I I - H ) , i . e . th e h y d ro g e n a tio n
s t e p .
1 2 . A d d itio n o f d e sm e th y l v ita m in K ^ o r d e sm e th y l M K g and
S -a d e n o s y l m e th io n in e to lo o k f o r t h e m e th y la te d d e r i v a t i v e s
V ita m in o r M K g r e s p e c t i v e l y , i . e . th e m e th y la tio n s t e p .
13. A d d itio n o f 2-m ethyl-1,M — n ap h th o q u in o n e and p h y to l t o lo o k
f o r v ita m in K^, i . e . th e p r e n y la tio n s t e p .
The ab o v e l i s t i s an o v e r s im p lif ie d summary and many v a r i a t i o n s o f
some o f t h e s e m a jo r e x p e rim e n ts w ere t r i e d .
The o n ly c e l l f r e e sy stem f o r q u in o n e b io s y n th e s is w hich was found
was in a H a lo b a c te riu m s p . h o w ev er, t h i s b a c t e r i a was fou n d t o in c o r
p o ra te m e v alo n ic a c id and th u s m ost l i k e l y h a s a b io s y n th e tic pathw ay
an alo g o u s t o t h a t o f n o n - b a c t e r ia l s y ste m s.
63
DISCUSSION
T h is t h e s i s h a s d e s c r ib e d th e i s o l a t i o n , s e p a r a t io n and id e n
t i f i c a t i o n o f a s e r i e s o f m en aquinones from a member o f th e S tre p to m y c e s
g ro u p . F iv e m a jo r q u in o n e s h av e b een i d e n t i f i e d and t h r e e m in o r s p e c ie s
t e n t a t i v e l y i d e n t i f i e d . W hile NMR, Mass S p e c t r a l , u l t r a v i o l e t , and
i n f r a r e d d a ta h ave a l l c o n firm e d t h e b a s i c s t r u c t u r a l i d e n t i f i c a t i o n
o f th e f i v e m a jo r com pounds, i t s h o u ld b e n o te d t h a t t h e TLC d a ta was
s u f f i c i e n t t o i d e n t i f y th em , a t l e a s t a s M K g , M K g(2 -H ), M K g(4 -H ),
M K g ( 6 -H ) , and M K g ( 8 -H ).
The l a t t e r p o in t i s v e ry im p o rta n t b e c a u se i t o u t l i n e s a r a p id
m ethod f o r th e i d e n t i f i c a t i o n o f many unknown q u in o n e s . I t h a s b een
shown t h a t sim p le a d s o r p tio n c h ro m a to g ra p h y can s e p a r a t e th e n a p h th o
q u in o n e c o n te n t from m ost c o n ta m in a tin g l i p i d s . An e x c e p tio n t o t h i s
i s t h e f in d i n g t h a t some f a t t y a c i d s m ig ra te w ith Rp v a lu e s s i m i l a r t o
n a p h th o q u in o n e s w ith many s o lv e n t s y s te m s . T h a t i s n o t a m a jo r p ro b
lem b e c a u s e t h e r e a r e s o lv e n t sy ste m s (B enzene f o r in s ta n c e ) w hich
a l l b u t e lim in a te t h i s , and b e c a u se s i l v e r io n a d s o r p tio n c h ro m a to
g ra p h y e lim in a te s f a t t y a c i d s w hich g e n e r a lly ru n w ith v e ry h ig h Rp
v a l u e s . When ex am in in g o rg a n ism s w ith b o th b e n z o q u in o n e s and n a p h th o
q u in o n e s (JE. c o l i f o r e x a m p le ), a d s o r p tio n ch ro m a to g rap h y c a n s e p a r a te
t h e two s p e c ie s p r o v id in g t h e p r o p e r s o lv e n t sy stem i s u sed (B enzene
o r d i e t h y l e th e r /b e n z e n e m i x tu r e ) . Once s e p a r a te d t h e two s p e c ie s o f
q u in o n e s r e a c t s i m i l a r l y a s t o i n t e r s p e c i e s s e p a r a t io n on t h e o th e r
s o lv e n t sy ste m s u s e d .
I t h a s b een shown t h a t s i l v e r io n a d s o r p tio n ch ro m a to g rap h y i s a
p o w e rfu l t o o l f o r t h e s e p a r a t io n o f com pounds w hich v a ry o n ly s l i g h t l y
6*t
fro m e a c h o t h e r . The p r e s e n c e o f n a n d n t l d o u b le b o n d s in tw o com
p ounds w h e th e r by a d d i t i o n o f a n is o p r e n o id u n i t o f b y e l i m i n a t i o n by
h y d ro g e n o f a d o u b le bond fro m a n is o p r e n o i d u n i t r e s u l t s i n Rp v a lu e
c h a n g e s s u f f i c i e n t t o a llo w q u a n t i t a t i v e s e p a r a t i o n . T h is i s d u e t o
t h e d i f f e r e n t i a l b in d in g o f t h e com pounds w ith s i l v e r io n s . S u i t a b l e
s o lv e n t s y ste m s f o r t h i s ty p e o f s e p a r a t i o n in c lu d e d 2 -b u ta n o n e /h e x a n e
o r m e th a n o l/b e n z e n e m i x tu r e s . W hile t h e i d e a l p e r c e n ta g e s o f th e s e
m ix tu r e s f o r ra e n a q u in o n e s, su c h a s th o s e s e p a r a t e d i n t h i s r e p o r t a r e
a p p r o x im a te ly 12 an d 5% r e s p e c t i v e l y , i t h a s b e e n fo u n d t h a t f o r t h e
s e p a r a t i o n o f a n a lo g o u s u b iq u in o n e s m ore p o l a r c o m b in a tio n s , i n th e
n e ig h b o rh o o d o f 30 an d 12% r e s p e c t i v e l y m u st b e u se d ( 6 5 ) . T h is
m ethod r e s u l t s in h ig h e r Rp v a l u e s f o r com pounds o f s h o r t e r c h a in le n g
t h o f lo w e r d e g r e e s o f s a t u r a t i o n , i . e . fe w e r d o u b le bon d s p r e s e n t*
A m ethod w hich c o m p lim e n ts t h e s i l v e r io n sy ste m i s th e r e v e r s e d
p h a s e s y s te m . T h is i s b e c a u s e w h ile t h e Rp v a lu e d i f f e r e n c e s f o r
c h a in l e n g t h v a r i a t i o n a r e q u a l i t a t i v e l y t h e same a s t h e s i l v e r io n
s y s te m , t h e Rp v a lu e s f o r d i f f e r e n t i a l s a t u r a t i o n a r e q u a l i t a t i v e l y
r e v e r s e d . H ere a g a in t h e u b iq u in o n e s a c t s i m i l a r l y a n d r e q u i r e o n ly
v a r i e d m ix tu r e s o f s o l v e n t s , i n t h i s c a s e l e s s p o la r* An ex am p le o f
t h i s i s f o r i n s t a n c e 97% a c e to n e / w a t e r f o r m e n aq u in o n e s e p a r a t i o n an d
91% a c e to n e / w a t e r m ix tu r e f o r u b iq u in o n e s e p a r a t i o n ( 6 5 ) .
T h e r e f o r e , by u s in g b o th s y ste m s i t i s r e l a t i v e l y e a s y t o d i s t i n
g u is h b e tw e e n d i f f e r e n t i a l c h a in l e n g t h an d o r d i f f e r e n t i a l s a t u r a t i o n .
The g e n e r a l u s e f u l n e s s o f t h e s e tw o m e th o d s i n i d e n t i f y i n g b a c t e r i a l
m e n aq u in o n es h a s b e e n shown e ls e w h e re ( 5 1 ) , an d w i l l u n d o u b te d ly be
u s e d i n c r e a s i n g l y in t h e f u t u r e . C o n firm a tio n o f g e n e r a l s t r u c t u r e
65
a f t e r th e s e TLC m e th o d s i s d ep en d en t o n ly on u l t r a v i o l e t s p e c tr a and o r
mass s p e c t r a l d a ta * The mass s p e c tr a p ro v id e s m o le c u la r w e ig h ts and in
c e r t a i n c a s e s p a r t i a l o r com plete s t r u c t u r a l d a t a . W ith t h e c a s e o f
th e compounds s e p a r a te d h e re , m o le c u la r io n s a t m/e 7 92, 7 9 0 , 7 88, 786,
and 7 8 4 s u g g e s te d a s d id th e TLC d a t a t h a t t h e compounds w ere M K g (8-H),
MKg(6 -H ), M K g ( 4 - H ) , MKg(2-H) and MK g . The f in d in g o f a l a r g e peak a t
m/e 225 i s a l s o v a l u a b le in t h a t i t i n d i c a t e s t h a t in f a c t t h e com
pound i s a n a p h th o q u in o n e s ( a ls o o b ta in a b le from u l t r a v i o l e t s p e c tr a )
w ith a t l e a s t a r i n g a d ja c e n t u n s a tu r a te d is o p re n o id u n i t ( n o t known
from u l t r a v i o l e t s p e c t r a ) . The u b iq u in o n e s can a l o s be a n a ly z e d by
mass s p e c tro s c o p y w h ich y ie ld s s i m i l a r in fo rm a tio n . In th e c a s e o f
M K g o r any f u l l y u n s a tu r a te d q u in o n e m o le c u la r w eig h t d a ta i s c o n n e c t
io n w ith TLC and UV d a t a i s s u f f i c i e n t to make p o s i t i v e s t r u c t u r a l id e n
t i f i c a t i o n . In t h e c a s e o f p a r t i a l l y s a tu r a t e d com pounds, how ever,
more in fo rm a tio n i s r e q u ir e d . In some c a s e s , h ig h r e s o l u t i o n mass
s p e c t r a l c ra c k in g p a t t e r n s can be s u f f i c i e n t f o r s t r u c t u r a l id e n
t i f i c a t i o n . T h is w as seen h e re f o r M K g ( 2 - H ) w hich by i t s c ra c k in g
p a t t e r n was i d e n t i f i e d a s M K g ( I I - H ) . I t sH ould a l s o be p o in te d o u t
t h a t m ass s p e c t r a l in fo rm a tio n was s u f f i c i e n t in p a r t b e c a u se t h i s
p a r t i c u l a r problem h a d been worked o u t b e f o r e w ith s u p p o rtin g c h e m ic a l
d e g ra d a tio n d a ta ( 1 0 8 ) . A lso , in t h e p r e s e n t s tu d y , M K g ( 4 - H ) was
t e n t a t i v e l y i d e n t i f i e d by i t s mass s p e c t r a l c ra c k in g p a t t e r n a s M K g ( I I ,
I I I - H ) .
W hile i n f r a r e d d a t a i s in no way q u a n t i t a t i v e , i t i s a l s o a good
q u a l i t a t i v e m ethod f o r b a s ic s t r u c t u r a l i d e n t i f i c a t i o n . In th e p r e s e n t
c a s e , IR d a ta i d e n t i f i e d th e q u in o n e s a s m enaquinones w ith is o p re n o id
66
s u b s t i t u t i o n . When h y d r o x y la tio n o c c u rs in q u in o n e s i d e c h a i n s , a s
f o r ex am p le, * -h y d ro x y v ita m in an a d d i t i o n a l p eak due to 0-H
s t r e t c h i n g can b e s e e n a t a b o u t 3500 cm“^ . T h is th e n p ro v id e s a
v a lu a b le t o o l f o r t h e e l im i n a tio n o r c o n fo rm a tio n o f s id e c h a in h y d ro x y
l a t i o n a s s ig n m e n t. In t h i s c o n n e c tio n , c a r e m ust be ta k e n to e l im i n a te
th e p o s s i b i l i t y o f w a te r b e in g p r e s e n t in t h e h ig h ly h y d ro s c o p ic KBr
d i s c s u se d f o r sam ple p r e p a r a t i o n , a s su ch c o n ta m in a tio n a l s o g iv e s
r i s e t o a s i m i l a r 3500 cm“ l a d s o r p tio n p e a k .
N u c le a r m a g n e tic re s o n a n c e s p e c tro s c o p y h a s b een a p o w e rfu l t o o l
in th e i d e n t i f i c a t i o n o f c o u n t le s s c h e m ic a l s t r u c t u r e s . L ik e m ass
s p e c t r a l d a ta i t can b e b o th q u a l i t a t i v e and q u a n t i t a t i v e . The o n ly
d is a d v a n ta g e o f N M R com pared t o m ass s p e c tr a i s t h a t N M R r e q u i r e s mg
am ounts o f sam ple com pared t o ug am ounts f o r m ass s p e c t r a . Q uinones
a r e p a r t i c u l a r l y a m e n ia b le t o s tu d y by N M R b e c a u se th e y p o s s e s s r e
l a t i v e l y few ty p e s o f h y d ro g e n s .
In t h e p r e s e n t s tu d y , a l l o f th e m a jo r q u in o n e s w ere i d e n t i f i e d
by N M R a s h a v in g s p e c t r a s i m i l a r t o a u t h e n t i c m enaquinone com pounds.
As w ith U V s p e c t r a , N M R p o s i t i v e l y i d e n t i f i e d s u b s t i t u t i o n a t t h e C-2
p o s i t i o n and showed th e s u b s t i t u t i o n t o be by a m e th y l g ro u p . N M R
a l s o s u b s t a n t i a t e d th e m ass s p e c t r a l f in d in g t h a t th e r i n g a d ja c e n t
is o p r e n o id u n i t was u n s a t u r a t e d . The p re s e n c e o f s a t u r a t e d is o p r e n o id
u n i t s was a l s o shown by t h e a p p e a ra n c e o f d i s t i n c t i v e s i g n a l s f o r t h e
new s p e c ie s o f h y d ro g e n . N M R s p e c tr a ta k e n in b en zen e a l s o showed t h a t
two o f t h e f i v e m a jo r b an d s (MKg(6 -H ) and MKg(8 -H )) p o s s e s s e d
te r m in a l s a t u r a t e d is o p r e n o id u n i t s , a f in d i n g w hich was a l s o d e r iv e d
from m ass s p e c t r a l d a t a .
67
W hile ch e m ic a l d e g ra d a tio n o f th e compounds by o z o n o ly s is and
su b se q u e n t a n a l y s i s o f th e p ro d u c ts d id n o t r e v e a l d e f i n i t e an sw ers
a s t o th e s t r u c t u r e o f th e compounds i t d id p ro v id e i n t e r e s t i n g c l u e s .
T h is m ethod i s by d e f i n i t i o n o f li m it e d v a lu e b e c a u se i t can o n ly show
th e p re s e n c e o f a d ja c e n t s a tu r a te d is o p re n o id u n i t s and n o t t h e i r
a b s o lu te p o s i t i o n . An e x c e p tio n t o t h i s p o in t i s when th e s a t u r a t i o n
o c c u rs a t th e W -te rm in a l o r 6> -te rm in a l and a d ja c e n t is o p re n o id u n i t s .
Gas c h ro m a to g ra p h ic e x a m in a tio n o f band 1 (MKg( 8 -H )) in d ic a te d t h a t
th e O -te rm in a l and i t s a d ja c e n t is o p re n o id u n i t w ere s a tu r a te d w h ile
band 2 (MKg(6 -H )) had o n ly i t s W -te rm in a l is o p re n o id u n it s a tu r a t e d .
W hile q u in o n e s o f known s t r u c t u r e have b een u sed to i d e n t i f y
s t r u c t u r a l re q u ire m e n ts f o r th e r e s t o r a t i o n o f o x id a tiv e p h o s p h o ry la t
io n , th e r e v e r s e h a s , t o th e b e s t o f my know ledge, n e v e r been u se d .
T hat i s , t e s t i n g unknown s t r u c t u r e s and d e r iv in g s t r u c t u r a l id e n
t i f i c a t i o n on th e b a s is o f t h e i r a b i l i t y to r e s t o r e co u p led a c t i v i t y
to in a c tiv a te d b a c t e r i a l e x t r a c t s . Such b io c h e m ic a l e v id e n c e f o r th e
p a r t i a l s t r u c t u r e o f band 2 (MKg( 6 -H )) was o b ta in e d . From i t s a b i l i t y
to r e s t o r e co u p le d p h o s p h o ry la tio n to in a c tiv a te d e x t r a c t s , i t can be
assum ed t h a t band 2 h a s a s a tu r a te d is o p re n o id u n i t in th e second
p o s iti o n from th e r i n g .
W hile i t was im p o s s ib le t o make d e f i n i t e s t r u c t u r a l a ssig n m e n ts
f o r a l l o f th e m ajo r com pounds, th e fo llo w in g s t r u c t u r a l in fo rm a tio n
h as b een d e r iv e d .
T here i s , o f c o u rs e , no e v id e n c e which can g iv e c lu e s a s to why
such a v a r i e t y o f s im i la r m o le c u le s i s p r e s e n t in t h i s o rg an ism . The
compounds exam ined h e re w h ile v a ry in g in p o l a r i t y do n o t r e p r e s e n t a s
B a n d 5
B a n d 1
w id e a r a n g e a s f o r exam ple t h e u b iq u in o n e c o n te n t o f E . c o l i . I t i s
d i f f i c u l t t o s e e w here su c h m in o r c h a n g e s in l i p o p h i l i c i t y w ould be
o b l i g a t o r y t o c e l l f u n c t i o n b u t t h i s may b e t h e c a s e . I t may a l s o be
t h a t some o f t h e com pounds a r e e i t h e r b i o s n y t h e t i c o r d e g r a d a tiv e
i n t e r m e d i a t e s . In t h i s c o n n e c tio n , when c e l l f r e e e x t r a c t s o f th e
o rg a n ism w ere in c u b a te d w ith s u f f i c i e n t e n e rg y s o u r c e s , and NADT o r
T2 O no i n c o r p o r a tio n o f t r i t i u m i n t o q u in o n e s was se e n ( 6 5 ) . T h is
r e s u l t may b e m e a n in g le s s i n l i g h t o f t h e f a c t t h a t no c e l l f r e e
sy ste m f o r q u in o n e s y n t h e s i s h a s b e e n fo u n d . As m e n tio n e d in t h e
i n t r o d u c t i o n , th e o ne e x c e p tio n t o t h i s l a t t e r p o i n t i s an o rg a n ism
w hich i n c o r p o r a te s MVA. H ow ever, t h e S tre p to m y c e s o rg a n ism when
grow n in t h e p r e s e n c e o f M V A shows no l a b e l e d q u in o n e ( 6 5 ) .
From an y e x a m in a tio n o f l i v i n g m a tte r one c a n s e e t h a t b i o l o g i c a l
m is ta k e s a r e r a r e l y fo u n d . T h a t i s , i t i s d i f f i c u l t t o b e l i e v e t h a t
o n ly one o f t h e e i g h t q u in o n e s fo u n d h e r e h a s a b i o l o g i c a l f u n c t i o n ,
t h e r e s t b e in g e r r o r s o f b i o s y n t h e s i s . Any f i n a l an sw er t o t h i s
p ro b lem w ould in d e e d b e q u i t e d i f f i c u l t t o r e a c h .
70
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Creator Phillips, Philip G. (author)

Core Title Isolation, Separation, And Structural Identification Of Streptomyces Quinones

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Degree Program Molecular Microbiology and Immunology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,oai:digitallibrary.usc.edu:usctheses,OAI-PMH Harvest

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Language English

Advisor Brodie, Arnold F. (committee chair), Burman, Pradip Roy (committee member), Katze, Jon R. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-523649

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Rights Phillips, Philip G.

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