STUDIES

m THE

BXOSIHTHESlS OP NICCTIHE AHD LIGNUi

By
L o v e ll J . Dewey

A THESIS

Submitted to th e School o f Graduate S tu d ies o f M ichigan
S ta te C ollege o f A gricu ltu re and A pplied Science
In p a r t i a l f u lf illm e n t o f th e requirem ents
f o r th e degree o f
DOCTOR OF PHILOSOPHY
Department o f Chemistry

195U

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ACKNOWLBDCMENT

The author w ish es t o e g r e s s h is s in c e r e a p p re c ia tio n
to Dr, Richard D. Byerrum whose a s s is t a n c e , guidance and
Gounsel have g r e a tly f a c i l i t a t e d th e com p letion o f t h i s
problem . The author i s a lso in d eb ted to o th er members o f
th e Department o f Chemistry who have given h e lp fu l advice
from tim e to tim e and e s p e c ia lly to D r. Robert M. R erbst
who k in d ly made th e arrangements fo r th e independent
deuterium a n a ly s e s. S p e c ia l g r a titu d e i s a ls o due t o Dr.
E. H„ L u cas, P ro fe sso r o f H o r tic u ltu r e , fo r h is in v a lu ­
able a s s is ta n c e and advice in th e c u lt iv a t io n o f th e
p la n ts u sed in th e in v e s t ig a t io n s .
F i n a l l y , th e w r ite r w ishes to thank th e Atomic Energy
Commission and th e Department o f Chemistry o f M ichigan
S ta te C o lleg e fo r p rovid in g funds in support o f t h i s work.

A* WA? ,W W W %
i. JU.

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360184

VITA

The author was born February 1 ? , 1927 In Kalamazoo, M ichigan.
He r e c e iv e d h is secondary ed u cation a t th e W, K. K ellogg High School
near A ugusta, M ichigan.

A fte r graduation he served in th e U n ited

S t a t e s Navy fo r s ix t e e n m onths.

In September 19U6 he en tered

Kalamazoo C o lle g e and was graduated i n dune 1990 w ith a Bachelor o f
A rts Degree*

During th e summers between th e sch o o l y ea rs a t Kalamazoo

C o lle g e , he worked in th e p la n t breed in g resea rch program a t th e A . M.
Todd Company i n Kalamazoo.

In th e f a l l o f 199® he e n r o lle d in th e

Graduate School o f M ichigan S ta te C o lle g e as a Teaching A s s is ta n t in
Chem istry and remained a t th a t p o s itio n u n t i l duly 1991 when he was
made a S p e c ia l Graduate R esearch A s s is ta n t under an Atomic Energy
Commission g r a n t.

He h eld t h i s p o s itio n during the remainder o f h is

graduate program a t M ichigan S ta te C o lle g e .

ABSTRACT

U sing m eth ion in e, doubly la b e le d in the m ethyl group w ith C14
and deuterium , i t has been shown th a t th e m ethyl group o f m ethionine
can g iv e r i s e t o the m ethyl group o f n ic o tin e through tra n sm eth y la tio n .
A lso in s im ila r experim ents th e m ethoxyl groups o f the l i g n i n o f both
tobacco and b a r le y have been shown t o a r is e from th e m ethyl group o f
m ethionine through tra n sm eth y la tio n .

The l a t t e r tran sm eth ylation

in v o lv e s the tr a n sfe r o f m ethyl groups from s u lfu r to oxygen, a r e ­
a c tio n which i s not known to oocur in animal m etabolism .

In a d d itio n

i t has been in d ic a te d th a t th e d ir e c t tr a n sfe r o f th e m ethyl group o f
m ethionine to form th e m ethosyl group o f lig n in r ep re sen ts a sy n th e sis
o f p art o f th e lig n in m o le c u le .

From the fin d in g s o f th e se and other

s tu d ie s i t i s concluded th a t tran sm eth ylation r e a c tio n s are o f general
importance in th e metabolism o f h igh er p la n t s .
Calcium g ly c o la te -2 -C 14 has been shown to g iv e r i s e to r a d io a c tiv e
n ic o tin e when adm inistered to tobaeco p la n t s .

H ost i f n ot a l l o f the

r a d io a c t iv it y o f th e n ic o tin e was lo c a liz e d in th e m ethyl group.
A comparison o f th e r a te o f in co rp o ra tio n o f the alpha carbon o f g ly c o la t e in to th e m ethyl group o f n ic o tin e w ith r a te s ob tain ed in p rev io u s
m eth y la tio n s t u d i e s , where m eth ion in e, fo rm a te, b ic a rb o n a te , c h o lin e
and g ly c in e were ad m in istered , has been made.
fo llo w in g c o n c lu sio n s were dr awns

From th e comparison th e

l ) the alpha carbon o f g ly c o la te i s

n ot converted to e ith e r formate or bicarb onate b efo re i t s in co rp o ra tio n

-1 -

in t o the m ethyl group o f n ic o t in e , 2) g ly c in e i s n o t converted to
g ly c o la te b efo re th e in co rp o ra tio n o f th e alpha carbon o f g ly c in e in to
th e n ic o tin e m ethyl group and 3) g ly c o la te may f i r s t be converted to
g ly o x y la te or th e alpha carbon may f i r s t be converted to formaldehyde
b e fo re e n te r in g the n ic o tin e m ethyl group.
By ad m in isterin g o r n ith in e -2 -C 14 hydrochloride to tobacco p la n ts
i t has been shown th a t p art o f th e n ic o tin e m olecule may a r is e from
o r n it h in e .

H alf o f th e radiocarbon o f th e is o la t e d n ic o tin e was shown

to be p re sen t i n th e 2 p o s it io n o f th e p y r r o lid in e r in g and the oth er
h a lf some^&ere in th e 3 S h and $ p o s itio n s o f the p y r r o lid in e r in g .
I t was p o stu la te d th a t th e second h a lf o f th e radiocarbon was lo c a te d
in th e 5 p o s itio n o f th e p y r r o lid in e r in g .

T h erefore, th e co n clu sio n

was drawn th a t o r n ith in e i s converted to a sym m etrical in term ed iate
b efo re I t s in co rp o ra tio n in to th e p y r r o lid in e r in g o f n ic o t in e .

L ess

than two per c en t o f th e r a d io a c t iv it y o f th e n ic o tin e was found i n th e
m ethyl group and none was found i n th e p y r id in e r in g .
When ly s in e -2 -G 14 hydrochloride was adm inistered to tobacco p la n ts
under th e same c o n d itio n s as were employed in the o r n ith in e stu d y , the
n ic o t in e i s o l a t e d was co n sid era b ly l e s s r a d io a c t iv e .

I t was p o stu la te d

th a t ly s in e i s not incorp orated d ir e c t ly in to the n ic o tin e m olecule but
i s f i r s t converted to some precursor o f p r o lin e or o r n ith in e such as
glutam ic a c id .

TABLE OF CONTENTS
Pag©
INTRODUCTION AND HISTORICAL............................... , . , .......................................

1

EXPERIMENTAL AND RESULTS.......................................................................................
S y n th e sis o f Labeled P recu rso rs, . . , , ......................................................
D eterm ination o f th e I s o to p e s
. . . . . . . . . . ...............
P rep aration o f the P l a n t s , . . . . ...............
I s o la t io n and P u r ific a tio n o f N i c o t i n e .. ...............
T ransm ethylation Experiments w ith Doubly Labeled M eth ion in e.
A . N ic o tin e Study.........................................
A d m in istration o f m eth ion in e.................................................
Exchange s tu d y ........................
R e s u lt s ......................................................
B . L ignin S tu d y .
...................
A dm inistration o f m eth ion in e.................................................
I s o la t io n o f l i g n i n ....................................................................
.......................................
D em ethylation o f l i g n i n
R e s u lt s
..................................................................................
M ethylation Experiments w ith Calcium G ly c o la te ............................
Uptake o f Calcium g ly c o la t e ................................................................
A d m in istration o f th e r a d io a c tiv e g l y c o l a t e , . . . , , . ,
D em ethylation o f the n i c o t in e ................................................
R e s u lt s
..........
B io sy n th e tic S tu d ies w ith O rn ith in e-2-C 14,
...............
Uptake o f o r n ith in e h yd roch lorid e................................................
A d m in istration o f the r a d io a c tiv e o r n ith in e .............................
D egradation o f th e r a d io a c tiv e n ic o t in e ...................
1) O xidation w ith potassium permanganate
2) D ecarboxylation o f th e r a d io a c tiv e n ic o t in ic a c i d , , .
3) D em ethylation o f th e r a d io a c tiv e n ic o t in e ....................
R e s u lt s
.........................................
B io sy n th e tic S tu d ies w ith Lysine-2-C ...............
DISCUSSION...................................................................................................................
T ransm ethylation s t u d i e s
M ethylation stu d y ..........................
S tu d ie s w ith o r n ith in e -2 -C 1 4

...............
............................................................

SUMMARY............................................................................................................................
REFERENCES
APPENDIX

7
7
8
12
lit
15
15
15
16
17
19
19
20
21
22
21425
26
27
28
29
29
31
33
33
37
38
39
Ii3
U7
hi
50
52
60

......................................................................
67

LIST OF TABLES

TABLE
I
XI
III
IV

Deuterium and C14 in the Fed M ethionine and th e N ico tin e
D ip ic r a te ................................................
Deuterium and G14 in th e Fed M ethionine and in th e M ethyltrietbylammonium Iod id e Obtained from th e I s o la t e d L ig n in .

PAGE

10
23

L ocation o f R a d io a c tiv ity in the N ic o tin e M olecule A fter
th e A d m in istration o f Calcium CELyeolate-2-C14..........................

29

L ocation o f R a d io a c tiv ity in th e N ic o tin e M olecule A fter
th e A d m in istration o f O rnithine-2-C 14 h y d ro ch lo rid e...............

ho

DffRQDUCTIQN AIR) HISTORICAt

1

INTRODUCTION AND HISTORICAL
In th e I n v e s tig a tio n d escrib ed here two d i s t i n c t m etab olic p r o c esses
i n h igh er p la n ts have been s tu d ie d .

F i r s t , i t was d e sir ed to e s t a b lis h

whether or n ot tra n sm eth y la tio n 3 th e tr a n s fe r o f an in t a c t m ethyl group
from one compound to an oth er, i s a gen eral m etabolic r e a c tio n i n h igh er
p la n t s .

Secondly^ variou s compounds have been te s t e d as p o s s ib le

p recu rso rs o f th e n ic o tin e m olecule in an attem pt t o e s t a b lis h a pathway
f o r th e b io s y n th e s is o f n ic o t in e .
T ransm ethylation as an im portant m etab olic p rocess in th e higher
animal has been w e ll e s ta b lis h e d by th e u se o f is o to p ic a l l y la b e le d
compounds (1 ) and w ith is o la t e d enzyme system s ( 2 ) ,

Although th e im port*

ance o f th e tran sm eth ylation p ro c ess has been recogn ized f o r some tim e
in animal m etabolism ; no d ir e c t attem pt to demonstrate t h i s m etab olic
p ro cess i n high er p la n ts has been p u b lish ed up to th e p r e se n t tim e.
In d ir e c t evid en ce 9 however 9 has been p resen ted by s e v e r a l i n v e s t i g a t o r s .
Brown and Byerrum ( 3) have shown th a t th e m ethyl group o f m ethionine i s
more r a p id ly in corp orated in to th e n ic o tin e m ethyl group than i s the
carbon o f form ate and have su g g ested tran sm eth ylation as a p rocess to
account fo r th e se r e s u l t s .

Byerrum and Wing (L) u sin g c h o lin e , another

im portant m ethyl group donor in animal m etabolism ; have shown th a t t h i s
compound i s e q u a lly as e f f e c t i v e i n g iv in g r i s e to the m ethyl group o f
n ic o t in e as m eth io n in e.

S tu d ies reported by Marion and co-w orkers (5 )

show th a t th e m ethionine m ethyl carbon e n te r s more r a p id ly in to the

2

m ethyl groups o f th e b a r le y a lk a lo id s , H-methyl ty r a n in e , horde nine and
gramine, and o f th e chO line o f b a r le y than does the carbon o f f o r n a te .
However, c h o lin e la b e le d in th e m ethyl group w ith carb on -lh did not g iv e
r i s e to r a d io a c tiv e hordenine in d ic a tin g th a t c h o lin e does not serv e as
a m ethyl donor i n hordenine form ation,

Dubeck and Kirkwood (6 ) have

found th a t the m ethionine m ethyl group se rv e s as a precu rsor o f th e 0 and N-m ethyl groups o f th e c a sto r bean a lk a lo id , r i c i n i n e , but th a t
n e ith e r th e m ethyl groups o f c h o lin e nor th e carbon o f form ate g iv e r i s e
t o th e m ethyl groups o f t h i s a lk a lo id .

The f a ilu r e o f c h o lin e to donate

m ethyl groups i n th e p la n ts stu d ied was a ttr ib u te d t o th e la c k o f an
enzymic system , which would o x id iz e c h o lin e t o bet& ine.

The hetaim e

appears t o be th e a c tu a l m ethyl donor a t l e a s t i n animal m etabolism (7 ) *
Another stu d y by F lo k str a (8 ) has shown th a t t h e m ethyl group o f
m ethionine i s in corp orated more r a p id ly in to th e m ethoxyl carbon in th e
l ig n i n o f b a r le y p la n ts than i s th e carbon o f form ate.

Barrenscheen and

von V&lyi-Nagy (9 ) have found th a t m ethionine and glycocyaraine admin­
is t e r e d t o ground wheat gem * in c r e a se s i x - t o e ig h t - f o ld th e s y n th e s is
o f c r e a t in e .

F in a lly , Ahmad and Karim (10) have demonstrated th a t

m ethionine and c r e a tin in e stim u la te th e b io s y n th e s is o f c h o lin e in th e
s e e d lin g o f th e ch ick -p ea by about 30 per c e n t.

I t has been p o s tu la te d

th a t th e form ation o f m o m ico tin e in th e le a v e s o f N lc o tla n a g lu tin o s a
a t th e expense o f n ic o t in e in v o lv e s a tran sm eth ylation p r o c ess ( l l ) .
H ost o f th e in v e s tig a to r s o f p la n t m a te r ia l m entioned above a ls o have
su g g ested th a t tran sm eth ylation i s a m etab olic r e a c tio n in h igh er p la n ts

3

b u t f i n a l p ro o f has boon la c k in g p rev io u s t o t h i s stu d y ,

Xt th e r e fo r e

seemed o f i n t e r e s t to adm inister m eth ionine doubly la b e le d i n th e m ethyl
group w ith earb on -lh and deuterium t o in t a c t p la n ts and determ ine whether
or n ot th e m ethyl group was tr a n sfe r r e d as an e n t i t y to form th e m ethyl
group o f n ic o t in e and th e m ethoxyl group o f l i g n i n .
I n a d d itio n t o the tran sm eth ylation s tu d ie s th ere has been con sid er*
a b le I n t e r e s t i n th e o r ig in o f m ethyl groups from sou rces oth er than the
known m ethyl donor compounds.
m entioned ab ove.

The form ate s t u d ie s have alread y been

Another compound which has been stu d ie d in both anim als

(12) and p la n ts (13) i n t h i s connect io n i s g ly c in e .

Haraill dem onstrated

th a t th e alpha carbon o f g ly c in e i s incorp orated in to th e m ethyl group
o f n ic o t in e a t about th e same r a te as th e m ethyl group o f m eth ion in e.
S in ce th e g ly c in e alpha carbon was in corp orated in t o n ic o tin e more r a p id ly
than fo rm a ts, one p rop osal advanced to e x p la in th e m eth y la tin g a c tio n o f
th e alpha carbon o f g ly o in e was th a t th e g ly c in e may be h y d r o ly tic a l l y
deansinated t o give g ly c o lic a c id , the alpha carbon o f which could then
be reduced to g iv e m ethyl groups w ithou t going to form ate.

In stu d y in g

th e m eth y la tio n o f guanidine ace t i c so ld Barrenseheen and Gigante found
th a t g ly c o lic a c id had n e ith e r an a c tiv a tin g nor in h ib it in g e f f e c t when
g ly c o lic a c id and guanidine ace t i c so ld were Incubated w ith an enzyme
p rep a ra tio n from e t i o l a t e d wheat se e d lin g s ( l i t ) .

S im ila r ly , S te en a h o lt

has found th a t g ly c o lic a cid d id n ot m eth ylate guanidine ace t i c a c id to
c r e a tin e when th e y were Incubated to g eth er w ith su sp en sio n s o f v a rio u s
animal t i s s u e s or when p erfu sed through i s o l a t e d r a b b it h e a r t ( 1 5 ) .

k

In v iew o f th e se n eg a tiv e fin d in g s in i s o l a t e d system s from both p la n ts
and anim als on th e a b i l i t y o f g ly c o lic a c id to serv e as a m eth y la tin g
agen t i t s e a le d o f i n t e r e s t t o in v e s tig a te th e r a te o f in co rp o ra tio n o f
th e alpha carbon o f g ly c o lic a c id in to th e n ic o tin e m ethyl group In
I n t a c t p la n t s .

In a d d itio n i t would be p o s s ib le to compare th e r a id s

o f in c o rp o ra tio n o f th e alpha carbons o f g ly c in e and g ly c o lic acid in t o
th e m ethyl group o f n ic o t in e ,

The m eth ylatin g a b i l i t y o f g ly c o lic acid

has been stu d ie d i n the p re sen t work u sin g calcium g ly c o la te la b e le d
w ith earbon~lU in th e alpha p o s it io n .
An oven more e x te n s iv e l it e r a t u r e than th a t in th e m eth ylation f i e l d
has accumulated i n th e search f o r precu rsors o f th e p y rid in e and
p y r r o lid in e r in g s o f n ic o t in e .

Before th e general a v a i l a b i l i t y o f

carbon*!!* th e s tu d ie s on th e b io s y n th e s is o f a lk a lo id s in v o lv ed m ainly
th e fe e d in g o f supposed p recu rsors to p la n ts and then m easuring th e
in c r e a se In a lk a lo id con ten t i n th e tr e a te d p la n ts over c o n tr o l p l a n t s .
The r e s u l t s o f th e s e s tu d ie s were o fte n open to q u estio n e it h e r because
the a lk a lo id in c r e a se was sm all or in more r ec e n t y ea rs because a e r ia l
p o r tio n s o f p la n ts were employed in th e o r ig in a l s t u d ie s and Dawson (16)
has shown th a t In the ca se o f th e tobacco a lk a lo id , n i c o t in e , sy n th e sis
occurs in th e r o o t s .

H e i n and Linear (17) were ab le to show an in c re a se

i n n ic o tin e c o n ten t o f tobacco p la n ts when s o lu tio n s o f p r o lin e and
o r n ith in e were in je c te d in to th e stem s o f th e p la n t s .

Dawson (18) has

rep orted th a t 1 - p r o lin e , n ic o t in ic a c id , l-p y r r o lid o n e c a r b o x y lic acid
and d-glu tam ic a c id in c r e a se d the n ic o tin e c o n ten t o f th e le a v e s o f

5

tobacco s h o o t s , which were c u ltu red In aqueous s o lu tio n s o f th e se a c id s .
However , Dawson (19) has sin c e employed i n i t i a l l y n ic o t in e - f r e e tobacco
le a v e s and has f a i l e d to o b ta in a sy n th e s is o f n ic o tin e from p r o lin e and
n ic o t in ic a c id .

More r e c e n tly P e tr o s in l ( 2 0) has reported th a t p r o lin e ,

g ly c in e , 3- in d o le a c e t ic a c id , tryp toph an, a la n in e , a sp a r tic a c id ,
glutam ic a c id and n ic o t in ic acid caused th e appearance o f n ic o tin e i n
germ inating seed s o f tobacco c u ltu red in the dark.
Other s tu d ie s have taken the form o f sp e c u la tiv e p rop osals f o r
a lk a lo id b io g e n e s is u sin g a n a lo g ie s from organic r e a c tio n s which were
known to take p la c e i n th e la b o r a to ry .

Robinson*a (2 1 ) scheme fo r

a lk a lo id b io g e n e s is i s a good example o f t h i s approach.

In h is scheme

f o r th e b io s y n th e s is o f n ic o tin e Robinson proposed th a t 2-hydroxy-N-m ethyl
p y r r o lid in e condensed w ith acetone d ie a r b o x y lic a c id , which supposedly
would a r is e from c i t r i c a c id .

The condensation product would be d e-

carb oxylated and then rea c te d w ith two m oles o f formaldehyde and one
mole o f ammonia t o form it k e to -3“( 2

-m ethyl p y r r o lid in e ) p ip e r id in e .

The l a t t e r then would be converted to n ic o tin e by th e l o s s o f th e k eto
group and 2 m oles o f hydrogen.
L ater T r ie r (22) proposed a pathway fo r th e o r ig in o f both the
p y rid in e and p y r r o lid in e r in g s o f n ic o tin e from a s in g le amino a c id ,
p r o lin e .

More r e c e n tly Mortimer ( 23) has su g g ested tryptophan as a l i k e l y

p recu rsor o f n ic o tin e and has p resen ted a mechanism fo r th e in co rp o ra tio n
o f a t l e a s t part o f th e tryptophan

m olecule in to n ic o t in e .

James (2b ) and

Dawson (1 9 ) have prepared review s which p re sen t a more e x te n s iv e survey
o f th e s tu d ie s on a lk a lo id b io g e n e sis than can be given h e r e .

6

F in a lly th e r ec e n t s tu d ie s by Dawson and co-w orkers (25) end Bowden
(2 6 ) in which compounds la b e le d w ith carbon-lij. were t e s t e d as p o s s ib le
p recu rso rs o f n ic o tin e should be m entioned.

When th e s y n th e s is o f

n ic o tin e in r o o t c u ltu r e s was stu d ie d w ith n ic o tin ic a cid -ca rb o x y l-C 14
as a p recu rsor * Dawson e t a l . were n ot ab le to i s o l a t e r a d io a c tiv e
n ic o tin e in d ic a tin g th a t th e carboxyl group o f n ic o t in ic acid i s u n a v a il­
ab le fo r n ic o tin e s y n th e s is in tobacco r o o t c u lt u r e s .

S im ila r ly when

DL-tryptophan la b e le d in th e b e ta p o s itio n o f th e s id e ch ain w ith carbon-ll*
was adm inistered t o tobacco p la n ts by Bowden no r a d io a c t iv it y was found
in the is o la t e d n ic o t in e .

N eith er o f th e se s t u d i e s 9 how ever, r u le s out

th e p o s s i b i l i t y th a t th e p y r id in e r in g o f n ic o tin e may a r is e from n ic o ­
t i n i c a c id or tryptophan.
In view o f t h i s la c k o f d e f in it e knowledge concerning the b iosyn ­
t h e s i s o f n ic o tin e and c o n sid erin g th e f a c t th a t o r n ith in e o fte n has been
su g g ested as a precursor o f n ic o tin e i t was decided to adm inister both
o r n ith in e and l y s in e la b e le d w ith carb on-lii to in t a c t tobacco p la n ts and
determ ine whether or not th ey are incorp orated in to th e n ic o tin e m o lec u le.

EXPatZMSmi. ABO RESOWS

7

EXPERIMENTAL AND RESULTS
S y n th esis o f Labeled Precursors
DL-methionine i s o to p ic a l l y la b e le d i n th e m ethyl group was sy n th e­
s is e d by red ucin g DL-homocys t in e w ith sodium i n liq u id ammonia and
t r e a t in g th e red u ctio n product w ith la b e le d m ethyl io d id e e s s e n t i a l l y
according t o th e method o f du Vigneaud, Eyer and Haraon (2 7 ) w ith th e
ex c e p tio n th a t th e m od ified apparatus designed by Brown (2 8 ) was used
i n the s y n t h e s is , and th e p u r if ic a t io n procedure o u tlin e d by M e lv ille
and co-w orkers (29) was employed.

In th e ca se o f th e DL-methionine

la b e le d w ith deuterium in th e m ethyl group d eu terated m ethyl Iod ide was
used w h ile fo r th e p rep aration o f DL-methionlne la b e le d w ith carb on-lk
i n th e m ethyl group G ^ -m e th y l io d id e was employed.

Both la b e le d samples

o f m ethyl Io d id e were purchased from T ra cerla b , I n c . , B oston ,

The doubly

la b e le d m ethionine was th en obtained by m ixing th e C14 la b e le d m ethionine
w ith the d eu terated m ethionine in th e r a t i o o f 10 to 90 per c e n t (by
w eigh t) r e s p e c t iv e ly .
Calcium g ly c o la te -2 -G *4 was sy n th e sise d from brom oacetic a c id ,
which was ob tain ed front N uclear Instrument and Chemical C orporation ,
C h icago, according to th e procedure o u tlin e d by Hughes, Ostwald and
T o lb ert ( 30) fo r th e p rep aration o f calcium g ly c o la te from e h lo r o a c e tic
a c id .

About 30 mg. o f bromo&cetlc acid -2 -C 14, U70 a g . o f in a c t iv e

brom oacetic a c id , 2 g . o f calcium carbonate and 10 m l. o f water were

8

p la c e d In a round bottom f la s k and heated on the steam bath fo r 2 1 /2
d a y s.

At th e end o f t h i s tim e th e r e a c tio n m ixture was f i l t e r e d w h ile

h o t u sin g no wash water and th e f i l t r a t e p la ced i n th e r e fr ig e r a to r *
The calcium carbonate was then washed thoroughly and th e wash saved fo r
a second crop*

At th e end o f 2k hours th e f i r s t crop o f c r y s t a ls was

f i l t e r e d o f f , washed w ith a b so lu te a lc o h o l and d ried in a vacuum d e s ic ­
ca to r*

The calcium g ly c o la te was shown to co n ta in o n ly one r a d io a c tiv e

compound by paper chromatography and rad ioau tograp h s.

The paper chromato­

graphs were developed w ith phenol sa tu ra ted w ith an aqueous s o lu tio n
co n ta in in g 6*3 per c e n t sodium c it r a t e and 3*? per c e n t sodium dihydrogen
phosphate in a chamber sa tu ra te d w ith a c e t ic a c id vapor ( 3 1 ) .
O rnithine-2-G i4 h yd rochlorid e and ly s in e -2 -C 14 hydrochloride were
purchased from Tracer l a b , I n c . , Boston and were used as such i n th e
experim ents d escrib ed l a t e r .
D eterm ination o f th e Iso to p e s
A ll r a d io a c t iv it y measurements ob tain ed in th e se s tu d ie s were made
i n a window-less flo w counter (T ra eerla b , I n c .) u sin g a N uclear Instrum ent
and Chemical C orporation sc a le r *

The gas used i n th e flo w counter was a

m ixture o f helium (9 9 .0 5 per c e n t) and isobutane ( 0 .9 5 per c e n t) and was
purchased from The Matheson Co. , I n c , , East R u therford, New J e r s e y .

The

o v e r - a l l e f f i c ie n c y o f th e counter was about 19 per c e n t as determ ined
u s in g a N a tio n a l Bureau o f Standards sodium bicarb onate sam ple.

A ll

sam ples were p la te d in ^ i n f in it e ly th ic k n la y e r s on aluminum d is h e s w ith

9

an area o f 2 ,8 3 square cm. fo r c o u n tin g .

The counts were c o r r e c te d fo r

s e lf -a b s o r p t io n (s e e Appendix I ) e x cep t where noted t© th e co n tra ry and
th en converted t o counts per minute ( c .p .m ,) per mM.
For th e d eterm ination o f th e r a d io a c t iv it y in th e experim ents where
m ethionine doubly la b e le d w ith deuterium and carbon-lU was fe d th e
m eth io n in e, th e n ic o tin e d ip ic r a te and th e quaternary io d id e obtained
from th e l ig n i n raethoxyl group as d escrib ed l a t e r were burned a t 70Q°C.
i n a m icro-com bustion tu b e ,

The combustion tube was packed w ith th e

"com bination f i l l i n g 9 ©f copper o x id e , le a d p e r o x id e1 and p la t in is e d
a sb e sto s d esc rib ed by H ied erl and H ie d e rl (32) w ith th e in c lu s io n o f
s i l v e r w ire im m ediately b e fo re th e packing as w e ll as a t th e e n d .

When

burning th e io d id e , i t was found n ecessa ry to r e p la c e the s i l v e r w ire
a f t e r 3 or i* com bustions because a f t e r th a t tim e , im p u r itie s cam® through
w ith th e water which were d i f f i c u l t t o remove In th e p u r if ic a t io n pro*
ced u re.

The GQa o f combustion was p r e c ip ita te d as barium carbonate from

a barium hydroxide s o lu tio n i n a s p e c ia l trap in th e combustion t r a in .
The barium carbonate was c o ll e c t e d , washed w ith h ot water and d r ie d a t
110°C* f o r one hour.

To plat® th e barium carbonate an a lc o h o l slu r r y

o f th e compound was made on an aluminum d ish and th e a lc o h o l was evaporated
over a h e a t lamp * S in ce o n ly a comparison o f r a d i o a c t i v it i e s was d e sir e d
i n th e tra n sm eth ylation s t u d i e s , UO mg, o f barium carbonate were counted
u n t i l 1 0 ,0 0 0 cou n ts were obtained in each ca se and the count was not
c o rr ec ted fo r s e lf -a b s o r p t io n „
1 The le a d peroxid e was om itted in l a t e r combustions w ith e q u a lly
good r e s u l t s .

10

A tra p immersed i n a s o l i d carbon dioxide-© © thyl c e llo s o lv e bath
was p la ced i n th e combustion t r a in b efo re th e barium hydroxide s o lu tio n
t o f r e e z e ou t th e ou ter o f combustion*

The water was p u r ifie d mod i t s

deuterium c o n ten t determ ined by th e grad ien t tube method d e scrib ed by
Lindaretrora^lang e t al* (3 3 )*

The water from th e combustion was tra n s­

fe r r e d from th e trap to a pyrex tube ( i n t e r i o r diam eter about $ ram. and
le n g th 12 ora,) se a le d a t one end and co n ta in in g about 3 mg* o f sodium
p ero x id e and 3 mg* o f potassium permanganate c a r e fu lly introduced
p r e v io u sly w ith a lo n g th in paper fu n n el so th a t none o f th e rea g en ts
cam© i n c o n ta c t w ith th e tube w a lls* Then th e tube was q u ick ly b e n t in
o
th e m iddle a t a b$ a n g le and s e a le d . The w ater was d i s t i l l e d from th e
o x id is in g m ixture in to the r e c e iv in g am and then poured back again*
T h is d i s t i l l a t i o n procedure was rep eated s e v e r a l tim es t o in su re proper
p u r ific a tio n *

At th e end o f th e l a s t d i s t i l l a t i o n th e r e c e iv in g arm was

c u t o f f and th e water analysed Im m ediately a s d escrib ed below*
An H^O-BgO m ixture was stan d ard ised by determ ining th e d e n s ity o f
th e s o lu tio n by means o f a pycnometer and u sin g a m icro-balance fo r
w eighings and then reading th e per c e n t Da0 in th e s o lu tio n from th e
s t r a ig h t l i n e p l o t o f th e d e n s it ie s o f pure HaQ and pure DgO a g a in st per
c e n t o f D*0*

The atom per cen t e x c e ss D in th e water o f th e standard

was c a lc u la te d from th e p er cen t o f

0 in th e standard*

V arious second*

ary standards o f from 0 *07h6 to l.Obf* atom per e e n t e x ce ss V were
prepared by d ilu t in g th e primary standard w ith appropriate amounts o f
r e d i s t i l l e d H&0*

n

th e g r a d ien t tube In which th e deuterium analysed were made was
h a l f - f i l l e d w ith a m ixture o f r e d i s t i l l e d bromobenzene and o d o r le ss
k erosen e having a d e n s ity s l i g h t l y la r g e r than th e h e a v ie s t secondary
stan d ard .

The remainder o f th e tube was c a r e fu lly f i l l e d w ith another

m ixture o f th e se two liq u id s having a d e n s ity s l i g h t l y l e s s than th e
l i g h t e s t secondary sta n d a rd .

Both th e kerosene and broraobenaene were

d i s t i l l e d b efo re u se i n an a l l g la s s apparatus and th en d r ied over calcium
c h lo r id e .

The kerosene f r a c t io n b o ilin g from 210 t o 2i40°C# and th e

bromobenzen© f r a c t io n b o i l i n g a t 152 to l$ k °C , were u sed ,

The two so lu ­

t io n s in th e tube were mixed s l i g h t l y a t th e ir in terp h ase w ith a lo n g
g la s s rod term in atin g in a lo o p , and th e tube was allow ed t o stan d
s e v e r a l days i n a co n sta n t temperature bath t o e s t a b lis h a lin e a r d e n s ity
g r a d ie n t.

To determ ine th e atom per c e n t e x c e ss D in an unknown sample

v a rio u s secondary standard s o lu tio n s co v erin g th e in te r v a l o f d e n s ity
expected In th e unknown were introduced In to one s id e o f th e gradien t
tube w ith a m ic r o -p ip e tte and the unknown was introdu ced in to th e oth er
s id e .

At th e end o f a g iv en tim e , u s u a lly 0 ,5 hour* the d is ta n c e the

unknown had f a l l e n and th e d ista n c e s th e standards had f a l l e n were read
w ith th e a id o f a cath etom eter.

The atom par c e n t e x c e ss B o f th e unknown

was then o b ta in ed by sim ple in te r p o la tio n assuming a lin e a r r e la tio n s h ip
between th e d e n s ity o f th e water d r o p le ts and th e d ista n ce th e y had f a l l e n
i n th e medium*

A n a ly sis o f m eth io n in e, sy n th e siz e d from m ethyl io d id e

o f known deuterium c o n te n t, by th e g rad ien t tube method ju s t o u tlin e d
in d ic a te d th a t th e method was accurate w ith in a range o f about - 0 ,0 3

12

atom par c e n t deuterium w ith the average o f se v e r a l an alyses f a l l i n g
c lo s e t o th e known deuterium c o n te n t.
P rep aration o f th e P la n ts
The treatm ent o f th e p la n ts employed in th e se v e r a l s tu d ie s
d escrib ed i n t h i s in v e s t ig a t io n v a ried somewhat w ith the sep arate stu d y .
For th e s tu d ie s in v o lv in g n ic o tin e and the l ig n i n o f tobacco p la n t s .
N ic o tia n s r u s t i c s . v a r . humi l l s ». a high n ic o tin e s t r a i n , was u sed .
A v a r ie ty o f Hordeum vulgar® known as Bay b a r le y was used i n th e tr a n s­
m eth y la tio n study in v o lv in g b a r le y lig n in *

B arley and tobacco seed s

were p la n ted in f l a t s co n ta in in g verraiculit© ,* which provided a base
fo r th e growing p la n ts but gave no n u t r ie n t s .

The p la n ts were grown

u n t i l th e y had a tta in e d a h e ig h t o f 13-18 cm. (about one month fo r th e
b a r le y and from two t o th ree months f o r to b a c c o ).

The p la n ts were

watered tw ice a week w ith a n u tr ie n t s o lu tio n composed o f 1 g* MgS04 •
7BtfG, 1 g* K*$PQ4 s 5*8 g* CailSOg^'iiKflp and fou r l i t e r s o f tep w a ter.
T© prepare th e p la n ts fo r th e hydroponic a d m in istra tio n o f the
r a d io a c tiv e m a te r ia ls th e p la n ts were removed from th e f l a t s and th e
r o o ts were c a r e fu lly fr e e d o f v e n a ie u llte by soak in g and washing in tap
w a ter.

The r o o ts o f th e p la n ts were th en immersed i n a 0 .1 per cen t

s o lu t io n o f d eterg en t germ icide3 fo r 0*5 hour, w ith o c c a sio n a l a g it a t io n ,

3 V erm icu lite i s a com m ercially a v a ila b le h ea t expanded m ica .
3 Wyandotte d eterg en t germ icide No* 1528 was obtained from the
Wyandotte Chem icals C orp oration , Wyandotte, Michigan*

13

bo reduce th e b a c t e r ia l population*

A fter r in s in g under tap w ater, the

p la n ts were p laced in 12£ m l, erlenm eyer f la s k s co n ta in in g $0 m l. (25
ml* fo r th e b a r le y p la n ts ) o f an in organ ic n u tr ie n t medium prepared by
d i l u t i n g , w ith two p a r ts o f w a ter, one p art o f a sto c k s o lu tio n which
had th e fo llo w in g com p ositioni

w ater 1 l . j calcium n i t r a t e , 1 g . |

potassium c h lo r id e , 250 rog.j potassium dihydrogen phosphate, 250 mg*j
magnesium s u l f a t e , 250 ®g*j ammonium s u l f a t e , 250 mg.j f e r r i c c h lo r id e ,
2 mg.

The w eigh ts are o f the anhydrous s a l t s .

S ix drops o f one per

c e n t germ icide s o lu tio n were added t o each f l a s k .

Twice a day fo r two

m inutes a stream o f oxygen was p assed tlirough th e n u tr ie n t s o lu tio n to
p rovide a e r a tio n fo r th e r o o t system s and to preven t w ilt in g o f th e
p la n ts*

In th e s tu d ie s w ith calcium g ly c o la t e , o r n ith in e and ly s in e

th e treatment® w ith germ icide and oxygen were o m itte d .

In ste a d o f th e

s i x drops o f germ icide s o lu t io n , 0 .5 ml* o f an aureomycin a b lu tio n
(5 0 mg. per $0 ml* o f s o lu tio n ) was added to each f l a s k as a means o f
red u cin g the number o f r o o t m icroorganism s.
In th e f i r s t experim en ts w ith o r n ith in e and ly s in e the r a d io a c t iv it y
recovered i n th e is o l a t e d n ic o tin e was too lo w , when th e treatm ent o f
th e p la n ts o u tlin e d above was fo llo w e d , to perm it d egrad ative s tu d ie s on
the n i c o t i n e .

I t had been noted in th e e a r l ie r s tu d ie s th a t th e tobacco

p la n ts developed many new root© when they had been in th e n u tr ie n t
medium fo r a week or more.

S in ce n ic o tin e s y n th e s is has been shown to

occur i n th e r o o ts ( 1 6 ) , i t was decided t o fo r c e th e tobacco p la n ts to
develop new r o o t system s a f t e r t h e ir tr a n sfe r from th e v e r m ic u lite in

14

an attem pt to o b ta in an in crea sed s y n th e s is o f n ic o tin e and th e r e fo r e
an augm entation i n th e amount o f carbon*!!* i n the is o l a t e d n ic o t in e ,
t o in d u ce th e development o f new r o o t system s th e r o o ts o f th e tobacco
p la n ts were cu t o f f a fte r th e p la n ts had been removed from th e v e r m ic u lite .
T h ep la n ts were th en p laced i n th e erlerraeyer f la s k s c o n ta in in g th e
hydroponic medium d escrib ed e a r l i e r .

At th e end o f two weeks the p la n ts

had produced co m p letely new r o o t system s and a t t h i s tim e th e radio-*
a c tiv e m a te r ia ls were added as w i l l be d escrib ed l a t e r .

Bo improvement

in t h i s procedure occurred when th e cu t p la n ts were tr e a te d w ith a com­
m ercia l p la n t hormone powder4 b efo re b ein g p laced in th e in organ ic
n u tr ie n t s o lu tio n ; so the hormone treatm ent was not u t i l i z e d .
During th e ad m in istra tio n o f th e r a d io a c tiv e compounds th e p la n ts
were grown in a hood.

Two 36-ineh* 30-w att flu o r e s c e n t tub es and a 100-

w a tt in ca n d escen t bulb were p laced about 11* in ch es above th e top s o f
th e p la n ts as a source o f illu m in a tio n .

A l i g h t in t e n s it y a t th e l e v e l

o f th e upper le a v e s o f about 200 fo e t-c a n d le s was thus o b ta in ed .

The

l i g h t s were l e f t on 12 hours o u t o f 24 during th e e n tir e a d m in istra tio n
p e r io d .

A d d itio n a l n u tr ie n t s o lu tio n was added to th e f la s k s t o keep

th e volume c o n sta n t.
I s o l a t i o n and P u r i f i c a t i o n o f N ic o tin e
A fte r th e a d m in istra tio n period th e tobacco p la n ts were removed from
th e n u tr ie n t medium and th e r o o ts ware r in se d w ith d i s t i l l e d w a te r, the

4 The hormone powder employed was E ootone, which i s manufactured
by th e American Chemical P a in t C o ., Ambler, Pa.

15

9X8039 b ein g b lo tt e d o f f w ith c h e e s e c lo th .

The p la n ts then were cu t

In to sm all p ie c e s and im m ediately d r ied as r a p id ly as p o s s ib le under
In fra red h ea t lam ps.

The tem perature o f th e drying p la n ts reached about

8G°C, l a 15 m in u te s.

The d r ie d m a te r ia l was ground in a m ortar, mixed

w ith 20 per c e n t o f i t s w eigh t o f calcium h yd roxid e, and steam d i s t i l l e d
In a K jeld ah l f l a s k .

The d i s t i l l a t i o n was continued u n t i l no p r e c ip it a t e

was obtain ed when th e d i s t i l l a t e was t e s t e d w ith s i l ic o t u n g s t i c a cid
In d ic a tin g th a t no more n ic o tin e was coming o v e r.

The d i s t i l l a t e which

was c o lle c t e d in 5 m l. o f 6 N hyd roch loric acid was concen trated to a
sm a ll volume under reduced p r e ssu r e , and p u r ific a tio n o f the a lk a lo id
was accom plished by two s u c c e s s iv e a se o tr o p lc d i s t i l l a t i o n s w ith water
from a lk a lin e medium as d escrib ed by Smith (3 b ).

The a cid d i s t i l l a t e

was con cen trated t o dryness under reduced p ressu re and the n ic o tin e
h yd rochlorid e r e sid u e r e s u lt in g was d is s o lv e d in m ethanol p lu s a l i t t l e
w a te r.

Then a sa tu ra ted m ethanolic s o lu t io n o f p i c r i c acid was added in

ex cess.

A fte r stan d in g a sh o rt tim e th e p r e c ip it a t e o f n ic o tin e diplcr& te

was f i l t e r e d o f f , washed w ith methanol and r e c r y s t a lliz e d from h o t w ater.
<m.p. 22li-226°C .{ recorded 22li°C. ( 3 5 ) .)
T ra n sm e th y la tio n E xperim ents w ith Doubly l a b e le d M ethionine
A, N ic o tin e Study
A d m in is tra tio n o f m e th io n in e .

P re v io u s ly i t h as b e en shown t h a t

to b ac co p l a n t s ab so rb m eth io n in e from a n u t r i e n t s o l u t io n th ro u g h th e
r o o t sy stem s ( 3 ) ,

S ix ty to b ac co p l a n t s were p re p a re d f o r th e hydroponic

16

a d m in istra tio n o f th e la b e le d m ethionine as d escrib ed e a r lie r ,

They

were allow ed t o grow i n the n u tr ie n t s o lu tio n fo r nin e days to e s t a b lis h
th e r o o t system s .

On the ten th day th roe p la n ts were discard ed because

o f w i l t i n g , and then one m l. o f a s o lu tio n c o n ta in in g two mg. o f doubly
la b e le d m ethionine per m l. was added to th e n u tr ie n t s o lu tio n s in th e
f l a s k s c o n ta in in g th e remaining p la n t s .

During th e r e s t o f th e growing

p eriod th e p la n ts grew as was evidenced by in c r e a se in stem le n g th ,
p rodu ction o f le a v e s and growth o f r o o t s .

Four and nine days la t e r

2 mg. more o f la b e le d m ethionine were added t o th e f l a s k s making a t o t a l
o f 6 mg. o f la b e le d m ethionine fe d to each p la n t .

T his procedure was

n ecessa ry t o o b ta in a s u f f i c i e n t in co rp o ra tio n o f deuterium to be de­
t e c t e d a c c u r a te ly in th e is o la t e d n ic o t in e .

The la b e le d m ethionine had

an atom p e r cen t e x c e ss 0 o f 2 4 .5 and the t o t a l count r e c e iv e d by each
p la n t was 8 x 10* counts per m in u te.

T h irteen days a f t e r the adm inis­

t r a t io n o f th e f i r s t m ethionine th e p la n ts were removed from the f la s k s
and tr e a te d as d escrib ed e a r lie r f o r th e i s o l a t i o n o f th e n ic o t in e .
Exchange stu d y . — Although th e p o s s i b i l i t y o f an u n ca ta ly sed
exchange o f m ethyl groups between m ethionine and n ic o tin e during th e
fe e d in g p eriod and I s o la t io n o f the n ic o tin e from th e p la n ts seemed
rem ote, an experim ent was s e t up to check t h i s p o s s i b i l i t y .

Seventy mg.

o f n ic o t in e and 50 mg- o f C 14-m ethyiraethionine were d is s o lv e d i n 250
m l. o f a 0 ,0 5 H phosphate b u ffe r , pH 6 ,6 ,

A fter t h i s m ixture had sto o d

fo r e ig h t days a t room tem perature, th e n ic o tin e was sep arated by an
a zeo tro p ic d i s t i l l a t i o n w ith water and is o la t e d from th e d i s t i l l a t e as
th e d lp ic r a t e .

The d ip ic r a te was counted in an aluminum d is h in th e

17

flo w counter and no s ig n if ic a n t count o v er background was o b ta in ed ,
t h i s experim ent in d ic a te s th a t under c o n d itio n s comparable to th e con­
d it io n s o f th e fe e d in g experim ents th ere was no d e te c ta b le , u n ca ta ly sed
in terch an ge o f th e m ethyl groups between n ic o tin e and m eth io n in e.
R e s u lt s . — The atom per cen t e x c e ss deuterium and th e r a d io a c t iv it y
o f th e fe d m ethionine and th e is o la t e d n ic o tin e are p resen ted in columns
U ) and ( 0 ) o f f a b le X.

In column (@) i s shown th e per c e n t e x c e ss D

in th e m eth yl groups o f th e la b e le d m ethionine and d ip ie r a t e ,

The d a ta

in column (B) are v a lu e s c a lc u la te d on th e assumption th a t a l l o f the
e x c e ss deuterium i s lo c a te d in th e m ethyl groups o f th e r e s p e c tiv e com­
pounds.

In th e ca se o f th e m ethionine the compound was sy n th e sise d

from d eu terated m ethyl io d id e and hom ocystine, and the m ethyl group i s
th e o n ly p o s it io n where th e e x c e ss deuterium may be lo c a t e d .

In th e

ca se o f th e n ic o tin e d ip lc r a te i t has been shown th a t w ith in experim ental
erro r a l l o f th e earbon-li*, in n ic o t in e is o la t e d from p la n ts fed c * 4m ethyl m ethionine was lo c a te d in th e m ethyl group o f th e n ic o tin e ( 3) .
The above assum ption, th e r e fo r e , seems j u s t i f i e d in t h i s stu d y .

By

s im ila r argument th e r a d io a c t iv it y o f th e m ethyl groups o f th e two
compounds I s assumed t o be th e same as th e r a d io a c t iv it y o f th e r e ­
s p e c tiv e compounds.

The r a d io a c t iv it y o f th e compounds and m ethyl

groups are given in columns (C) and (D) r e s p e c t iv e ly .
The r a t i o s o f th e deuterium t o th e r a d io a c t iv it y in the m ethyl
group o f th e n ic o tin e d ip lc r a te and th e m ethionine are shown in th e
column s u b t it le d (B/B) in Table X.

In th e l a s t column i t I s shown th a t

th e r a t io o f deuterium t o C** in th e m ethyl group o f th e n ic o t in e

18

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19

dip icrat® I s 9k per e e n t o f th e r a t i o o f deuterium to G14 in th e m ethyl
group o f th e m eth io n in e.

T his percentage rep resen ts th e per cen t o f the

deuterium , o r ig in a lly p resen t in th e m ethionine m ethyl group, which was
tr a n sfe r r e d w ith th e m ethyl carbon.

I f tran sm eth ylation occurs th e n ,

o f c o u r se , 100 per c e n t o f th e deuterium would remain in the m ethyl
group.

However, i f th e m ethyl group o f m ethionine i s o x id ise d to th e

s t a t e o f formaldehyde and l a t e r reduced o n ly 67 per cen t o f th e o r ig in a l
deuterium can remain w ith th e m ethyl carbon.

Since 9U per cen t o f the

deuterium remained w ith the m ethyl carbon during i t s tr a n s fe r from
m ethionine In th e p r e se n t stu d y , i t may be concluded th a t a la rg e pro­
p o r tio n o f the m ethyl groups, which are tr a n sfe r r e d from m eth io n in e, are
tra n sfe r r e d through a tran sm eth ylation p r o c e s s ,
B . l ig n i n Study
A d m in istration o f m eth ion in e. — F lo k str a (8 ) has demonstrated
t h a t b a r ley p la n ts absorb m ethionine from a n u tr ie n t s o lu tio n through
th e ro o t system s and so th e hydroponic technique o f ad m in isterin g
m ethionine was used In th e p resen t stu d y .

S ix ty b a r le y p la n ts were

prepared f o r th e fe e d in g experiment as was o u tlin e d e a r l i e r .

One m l. o f

a s o lu tio n co n ta in in g two mg. o f doubly la b e le d m ethionine p er m l. was
added to each fla sk ; At th e end o f f i v e days two more mg. o f la b e le d
m ethionine were added t o each f la s k making a t o t a l o f four mg. o f
m ethionine r e c e iv e d by each p la n t .
per cen t e x c e s s D o f
was S . 6 x 10s c .p j a .

The la b e le d m ethionine had an atom

and the t o t a l count r e c e iv e d by each p la n t
Twelve days a f t e r th e ad m in istra tio n o f th e f i r s t

20

m ethionine the p la n ts were removed from th e n u tr ie n t s o l u t i o n , the r o o ts
were d iscard ed and th e rem aining p a r t o f th e p la n ts was dried *

The

d ried m a te r ia l was th en su b jected t o th e treatm ent d escrib ed below fo r
th e i s o l a t i o n o f th e l i g n i n .
In th e e a se o f the l i g n i n stu d ie s on tobacco p la n ts th e p la n t
m a t e r ia l, which had been steam d i s t i l l e d t o recover the n ic o tin e as out­
lin e d e a r l i e r , was d ried again and su b jected t o th e lig n in i s o l a t io n
proced u re,
I s o la t io n o f l i g n i n , — F lo k str a (8 ) has d escrib ed a method f o r the
i s o l a t i o n o f l i g n i n from f r e s h ly d ried p la n t m a te r ia l, and h is procedure
was u sed i n th e p resen t stu d y .

The procedure c o n s is t s o f a s e r i e s o f

s o lv e n t e x tr a c t io n s , which were performed on th e p la n t t is s u e in a Waring
B len d e r, fo llo w ed by treatm ent w ith s u lf u r ic a c id .

The s e r i e s o f so lv e n t

e x tr a c tio n s as su g g ested by M&oDougall and Delong ( 36) inclu ded two 1 5 m inute e x tr a c tio n s w ith eth er ^ saturated water to remove some n itr o g en
c o n ta in in g m a t e r ia ls , one 20-m inute e x tr a c tio n with f i v e per cen t a c e t ic
a c id In water fo r th e removal o f carb oh yd rates, and two 15-m inute
e x tr a c tio n s w ith ethanol-bencene (1 + 2) to remove l i p i d s .

These s o lv e n t

e x tr a c tio n s were fo llo w ed by treatm ent o f th e rem aining whit©, fib r o u s
o
m a te r ia l w ith ?0 per c e n t s u lfu r ic a cid fo r 18 hours a t $ C. The s u lf u r ic
a c id

was then d ilu te d to 3 per c e n t anfi the m ixture b o ile d g e n tly f o r two

h o u rs, keeping th e volume c o n sta n t by adding w ater.

A fter c o o lin g , th e

l i g n i n , tdaich s e t t l e d o u t , was f i l t e r e d , washed w ith d i s t i l l e d water
and d r ied in a vacuum d e s ic c a to r fo r fu tu re u s e .

21

D em ethylation o f l i g n i n . — For & comparison o f th e C14 and D in
th e m ethyl group o f th e fe d m ethionine t o th e

and 0 In th e m ethoxy!

group o f th e is o la t e d l i g n i n , which has an unknown chem ical s tr u c tu r e ,
i t was n ecessa ry to o b ta in a compound w ith a w e ll c h a ra cterized
str u c tu r e by degrading th e lig n in *

Sin ce o n ly th e m ethyl groups i n th e

l ig n i n were o f i n t e r e s t i n the p r e se n t stu d y , th e lig n in was dem eihylated
u t i l i z i n g F lokstra* s m o d ifica tio n (8 ) o f th e P h i l l i p s 1 method (3?)*

The

dem eth ylation apparatus employed here was the m odified form o f the P re g l
apparatus (3 8 ) d escrib ed by Brown ( 2 8 ) .

About 60 mg. o f the lig n in to

be deraeihylated was weighed on c ig a r e t t e pap er, which p r e v io u sly had
been shown t o y ie ld no m ethyl io d id e in the dem ethylation procedure,
and was introdu ced in t o the r e a c tio n fla sk *

Then two ml* o f p h en ol,

which acted as a s o lv e n t fo r th e l i g n i n , and fo u r m l. o f U7.3 Per oent
p e r io d ic a c id were p la c ed i n the r e a c tio n v e s s e l .

A gas-w ashing bubbler

c o n ta in in g 1 .5 m l. o f th e 5 par cen t GdS04-B&3SaOs so lu tio n , recommended
by P reg l to remove any h yd riod ic a c id and io d in e ca rried ovei; was
attach ed to th e r e a c tio n v e s s e l .

A tube from th e bubbler le d to th e

r e c e iv in g v e s s e l , which contained a f i v e per cen t s o lu tio n o f t r i e t h y l amine in e th a n o l, co o led in a m ethyl c e l lo s o lv e - s o l i d carbon d io x id e
o
bath t o about -7 5 C.
During th e dem ethylation procedure th e r e a c tio n f la s k was heated
i n a copper oxid e b a th .

A stream o f n itr o g e n was slo w ly run in through

th e s id e arm o f th e r e a c tio n f l a s k . The temperature o f th e bath was
o
o
k ep t a t about 150 C . fo r 1*5 m in u tes, then r a ise d t o 200 and h e ld fo r
30 m in u te s.

The bath was allow ed to c o o l fo r 15 m inutes during which

22

tim e a more rap id str e a a o f n itr o g en was passed through th e ap p aratu s.
Hescfe th e t i p o f th e d e liv e r y tube was r in se d w ith eth a n o l in to th e
r e c e iv in g v e s s e l , which was then stoppered and allow ed to stand over­
n ig h t a t room tem perature.

The naaet day th e e th a n o lic s o lu tio n was

taken n e a r ly t o dryness by h eatin g and the l a s t tr a c e s o f eth a n o l and
trie th y la m ln e were removed in a vacuum d e sic c a to r le a v in g th e w h ite ,
s o l i d methyltrietbylammonium io d id e .

The is o to p e con ten t o f th e io d id e

was measured by th e method already d escrib ed in th e s e c t io n on the de­
term in a tion o f is o t o p e s .
R e s u lt s . ** The data from two t r i a l s in t h i s p a r tic u la r stu dy are
p resen ted in Table 1 1 .

S in ce a l l o f th e deuterium in th e compounds

analyzed i s p r e se n t in th e m ethyl group o f the r e s p e c tiv e compounds,
th e v a lu es i n column (3 ) are c a lc u la te d from the v a lu e s in column (A)
by em ploying th e appropriate con version f a c t o r .

The r a d io a c t iv it y o f

th e m ethyl group® i s th e same as the r a d io a c t iv it y o f the r e s p e c tiv e
compounds so th e v a lu es i n column (I>) are obtained d ir e c t ly from th e
v a lu e s in column (C ),

I t w i l l be noted th a t th e 3 t o C14 r a t io obtained

i n th e m etly 1 t r iethylammonium io d id e fra® th© b a r le y l ig n i n m ethoxyl
groups was about 9k p e r cen t o f th e 3 to C14 r a t io in th e m ethyl group
o f th e fe d m eth io n in e .

T h is percentage again r e p r e se n ts th® per cen t

o f th e deuterium , o r ig in a lly p resen t in th e m ethionine m ethyl group,
which was tr a n sfe r r e d w ith th e m ethyl carbon*

The D to Cu r a t io in

th e m etlyltriethylsm m oBium io d id e obtain ed from l ig n i n is o la t e d from
tob acco p la n ts was about 9$ per cen t o f th e D t o G14 r a t io in th e fe d
m ethionine as a sc e r ta in e d from two independent deuterium a n a ly s e s .

23

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As was In d ica ted e a r lie r i f a l l o f th e m ethyl groups o f m ethionine had
been o x id iz e d to th© s t a t e o f form aldehyde, th e l e a s t p o s s ib le o x id a tio n ,
and th en reduced to g iv e th© m ethoxyl groups o f l i g n i n , the r a t io o f D
t o C*4 in th e m ethyl o f th© I s o la te d quaternary s a l t would be 67 per cen t
o f th e D to Cw r a t i o o f th© fe d m eth ion in e.

I t w i l l be noted th a t the

r a t io o f D to C1* in th e m ethyl group o f th e quaternary io d id e in a l l
c a se s i s w e ll over 67 per cen t o f the 0 to Cw r a tio in th e fed m eth ion in e.
Sin ce t h i s i s th e ca se i t may be concluded th a t some d ir e c t tr a n sfe r o f
th e m ethyl group, i . e . tra n sm eth y la tio n , occurred from th e su lfu r o f
m ethionine t o th e oxygen o f lig n in in b a rley and tobacco p la n t m etabolism .
In t h i s con n ection i t should be p oin ted out th a t F lo k str a (8 ) has shown
th a t th e r a d io a c t iv it y recovered frem th e l ig n i n by dem ethylation was
p r e se n t i n th e m ethoxyl groups o f th e lig n in and not in H-methyl or
Snmethyl groups, which might be p resen t in im p u ritie s i n th e l i g n i n .
M ethylation Experiments With Calcium G ly eo la te
S ev eral s tu d ie s in t h i s lab oratory ( 3 , li,13) have shown th a t tobacco
p la n ts can absorb variou s organic compounds through t h e ir r o o t system s
from a n u tr ie n t s o lu tio n ,

th e hydroponic procedure o f ad m in isterin g

la b e le d compounds th e r e fo r e was adopted i n th e p resen t stu dy s in c e i t was
d e s ir e d to d u p lic a te the p reviou s s tu d ie s as c l o s e l y as p o s s ib le in order
t o make v a lid comparisons between th e g ly e o la te experim ent and th e former
ex p erim en ts.

th e calcium s a l t o f g ly c o lic a cid was used in th e p resen t

stu d y not o n ly because I t occurred as th e f i n a l product o f th e g ly e o la te

25

s y n th e s is m entioned e a r lie r b u t Also because I t 1® A more con ven ien t
compound to handle than g ly c o lic a c id I t s e l f *

B efore th e m eta b o lic

s t u d ie s w ith g ly e o la te cou ld be u n d ertak e i t was n ecessa ry t o a s c e r ta in
whether or n ot th e g ly e o la te would be absorbed by th e p la n ts and a ls o
whether o r n ot mi cr©organisms would d e str o y th e g ly e o la te in th e
n u tr ie n t so lu tio n *
Uptake o f calcium g ly e o la t e .

t o determ ine th e r a te o f g ly e o la te

uptake by th e r o o t system s a s e n s it iv e method fo r th e a n a ly s is o f
g ly e o la t e s o lu tio n s was needed*

The method o f a n a ly s is chosen was th a t

o f C a lk in s ( 3 9 ) , a c o lo r im e tr ic method based on the form ation o f a
v io le t - r e d c o lo r when g ly c o lic a cid and 2 ,7 dihydroixynaphthalen© are
h eated to g eth er i n concen trated s u lfu r ic acid*

The s o lu tio n to be

analyzed was made 2 H w ith r e s p e c t t o s u lf u r ic acid and 0*2 ml* was
in trod u ced in t o a t e s t tube*

The tube was oooled i n an i c e bath and

then 2 ml* o f a 0*01 per cen t s o lu tio n o f 2 ,7 dihydroxynaphthalene were
added from a m icroburet*

The c o n ten ts o f th e tube were made homogeneous

by shaking and th en heated in a b o ilin g water bath fo r 20 m inutes * A fter
c o o lin g again in an l e e b a th , th e s o lu tio n was d ilu te d w ith four ml* o f
2 H s u lf u r ic s o l d , shaken v ig o r o u sly a f t e r th e h eat o f r e a c tio n had
su b sid ed and th e c o lo r read a t 530 mp. w ith a Beckman sp ectrop hotom eter.
Various standard s o lu tio n s c o n ta in in g from 10 to 70 microgrsnis o f calcium
g ly e o la t e per ml* were used t o prepare a standard curve*
To t e s t th e uptake o f g ly e o la te fou r p la n ts were prepared as
d escrib ed p r e v io u s ly , u sin g kO ml* o f n u tr ie n t s o lu t io n , 2 .5 m l. o f
calcium g ly e o la te standard (1 m g ./m l.) and 0*5 m l. of aureomycin s o lu tio n

26

(X m g./asl.) «

The aureomycin e f f e c t i v e l y in h ib ite d th e growth o f

b a c te r ia ( k ,1 3 ) .

Four oth er f la s k s prepared in the same manner were

in o c u la te d fey the a d d itio n o f afoot fragnent® , and four f la c k s c o n ta in ­
in g o n ly th e th ree so lu tio n * were need m co n tro l* .

At th e end o f 1|8

hour* th e p la n t* were removed, th e root* were r in se d and a l l s o lu tio n *
were f i l t e r e d through Whatman number 1*2 f i l t e r paper,
were an alysed fo r g ly e o la te as o u tlin e d e a r l i e r .

The f i l t r a t e s

I t was found th a t

a t th e end o f i*8 hours over 95 per cen t o f th e g ly e o la te had disappeared
from th e n u tr ie n t s o lu tio n in which p lan t* had been p la c e d .

There was

no l o s s due t o th e growth o f m icroorganisms s in c e over 90 per c e n t o f
th e g ly e o la te was recovered in both th e c o n tr o ls and th e in o c u la te d
s o lu t io n * .

In a d d itio n sample* o f n u tr ie n t s o lu t io n , which had served

a s m edia fo r th e a d m in istration o f r a d io a c tiv e g ly e o la t e , were evaporated
t o dryness a fte r removal o f the p la n ts and th e resid u e was t e s t e d fo r
r a d io a c t iv it y .
r e s id u e .

There was no s ig n if ic a n t count over background, in th e

From th e s e r e s u lt s i t was concluded th a t g ly e o la te was r e a d ily

absorbed through th e r o o ts and th e re fo re th a t th e hydroponic technique
was e n t i r e ly s a t is f a c t o r y f o r i t s a d m in istra tio n .
A d m in istration o f th e r a d io a c tiv e g ly e o la t e ,

The tobacco p la n ts

were prepared fo r th e hydroponic a d m in istration o f th e g ly e o la te as was
o u tlin e d e a r l i e r .

In th e f i r s t t r i a l each o f 30 tobacco p la n ts was g iv en

1 ,2 7 mg. o f calcium g ly e o la te (corresponding t o the 2 mg. o f m ethionine
adm in istered by Brown ( 3 ) ) having a r a d io a c t iv it y o f 1 .8 7 x 10^ e .p ja .®
® T h is was determined by countin g an ” i n f i n i t e l y thin" la y e r
(0 .0 0 9 mg ./sq u are cm J in th e flo w co u n ter.

27

S in ce an a c t i v i t y o f 1 x 106 c .p«m» was adm inistered in th e p reviou s
m stfaylation s t u d ie s , th e Tirial I d ata p resen ted in Table I I I have been
co rr ec ted t o correspond t o a dosage o f 1 x 10® c .p jn .

In T r ia l I I 25

p la n ts each r e c e iv e d 1*27 mg. o f calcium g ly e o la te having a count o f
1*06 oe 10* e*p*m* T r ia l XXX was conducted in th e same manner as T r ia l II*
At th e end o f one week th e p la n ts were removed from the f la s k s and the
n ic o tin e was is o la t e d as th e d ip ic r a te , according to the procedure d i s ­
cu ssed e a r lie r ,a n d found to be r a d io a c tiv e .
D em ethylation o f th e n ic o t in e * — As In th e p a s t m etb ylation s tu d ie s
i t was n ecessa ry i n the p re sen t case to determine whether or n o t th e
r a d io a c t iv it y o f th e n ic o tin e was lo c a l i s e d in th e m ethyl group.

The

derae t h y la tio n procedure used to e s t a b lis h t h i s p o in t was the Brown m o d ifi­
c a tio n (2 8 ) o f th e P reg l { 38) m ethyl- and eth yl-irain o d eterm in ation .
U sin g t h i s procedure th e m ethyl group i s is o la t e d as m e th y ltr ie th y lammonlwm i o d id e , a s o l i d compound s u ita b le fo r c o u n tin g .
Two hundred mg. o f n ic o tin e d ip lc r a te were decomposed w ith sodium
hydroxide and th e n ic o tin e was recovered by a zeotrop ic d i s t i l l a t i o n w ith
water through a Widmer column*

The a c id if ie d d i s t i l l a t e was concen trated

under reduced p r e s s u r e , the con cen tration b ein g com pleted in th e r e a c tio n
f l a s k o f th e d em eth ylation apparatus.

To th e n ic o t in e hydrochloride in

th e r e a c tio n f l a s k were added 1*5 mg. o f ammonium io d id e , two drops o f
f i v e p er cen t gold c h lo r id e s o lu tio n and th ree x l . o f h yd riod ic a cid
( s p e c i f i c g r a v ity 1*5)*

The gas washing v e s s e l in th e apparatus con tain ed

1*5 m l. o f th e f i v e per cen t Cd$04-WaaSapa s o lu t io n d escrib ed by P regl*

28

The r e c e iv e r contained a f i v e per cen t s o lu tio n o f t r i e th y lam in e in
eth a n o l and was co o led i n a s o lid carbon d ioxid e-m eth yl c e llo s o lv e b a th ,
Itfhen th a t apparatus was com p letely assembled a alow stream o f
n itr o g e n was p assed through, and th e r e a c tio n f la s k was heated in a
copper 03d.de bath to 200°C, in 20 to 2$ m in u tes.

The temperature was

th en r a is e d s lo w ly t o 350**60°C. and h e ld th ere fo r kS m in u te s.

The h eat

was removed and as th e apparatus co o led i t was flu sh ed w ith a continuous
stream o f n itr o g e n .

A fter the apparatus had cooled to room tem perature

th e d e liv e r y tube was r in se d w ith eth an ol in to th e r e c e iv in g v e s s e l ,
which was th en sto p p ered , shaken, and allow ed to stand ov ern ig h t a t room
tem perature.

The next day most o f th e e th a n o lic s o lu tio n was evaporated

by h ea tin g and the l a s t tr a c e s o f e th a n o l and trie th y la m in e were removed
i n a vacuum d e s ic c a to r .

A w h ite , c r y s t a llin e r e sid u e o f m e th y ltr ie th y l-

aramonium io d id e was reco v ered .
The io d id e was d is so lv e d in a sm all amount o f eth a n o l and tr a n sfe r r e d
to a ta re d aluminum counting d is h .

The eth a n o l was evaporated and the

d is h weighed to acquire the w eight o f th e p la te d compound.

The countin g

o f th e sample fo llo w ed the procedure already o u tlin e d b e fo r e .
R e s u lt s .

The counts per minute per roM fo r both the n ic o tin e

d ip ic r a te and th e m ethyltriethylaaasonium io d id e are p resen ted in Table I U
f o r th ree experim ental ru n s.
From t h i s d ata i t can b© seen th a t m o st, i f n ot a l l , o f th e radio-*
a c t i v i t y o f th e n ic o tin e i s lo c a liz e d in the m etbyl group o f th a t compound.
The r e l a t i v e l y h ig h valu es o f T r ia l I may be due to sea so n a l in flu e n c e s
s in c e th e p la n ts were grown a t d if f e r e n t tim es o f th e y e a r .

29

TABLE I I I
LOCATION OF RADIOACTIVITY IN THE NICOTINE MOLECULE AFTER THE
ADMINISTRATION OF CALCIUM GLICOLATE-2-C **

T r ia l
No.

No. o f
P la n ts

Maximum S p e c ific A c tiv ity
(c .p.m./nM)
N ic o tin e D ip icra te M e th y ltr ie tliy lammonium io d id e

Per Cent
Recovery in
M ethyl Group

30

7 .6 5 x 1 0 *

7 .8 1 x 1 0 *

106

n

25

2.20

10*

2.08 x 10*

95

m

25

3-7-U x 10*

3 .5 3 x 10*

9k

I

x

B io sy n th e tic S tu d ies w ith O rnithine- 2 -C*4
S e v e r a l in v e s tig a to r s have f e l t th a t o r n ith in e or compounds b io ­
l o g i c a l l y r e la te d to o r n ith in e may p la y an im portant r o le in the b io sy n ­
t h e s i s o f n ic o tin e as was p oin ted out e a r l i e r .

However, up to the

p r e se n t tim e no one has been ab le to demonstrate c o n c lu s iv e ly th a t
o r n ith in e can serve as a precursor o f n ic o tin e in th e tobacco plan t*
I t th e r e fo r e seemed o f in t e r e s t t o e s t a b lis h whether or not o r n ith in e
could be Incorporated in t o the n ic o tin e m olecule in tobacco p la n ts by
a d m in isterin g o r n ith in e la b e le d w ith ca rb o n -lli.
Uptake o f o r n ith in e h yd roch lorid e. — As in th e other fe e d in g e x p e r i­
ments i t was n ecessa ry to fin d a con ven ien t method o f ad m in isterin g th e
la b e le d o r n ith in e to th e p la n ts .

S in ce th e hydroponic ad m in istration

o f compounds had been s u c c e s s fu l in th e p a st w ith m ethyl group p recu rsors ,
i t was attem pted in t h i s stu dy a ls o .

30

In t e s t in g th e absorp tion o f o r n ith in e by tobacco r o o ts the
n in hyd rin method o f B ergd oll and Doty (Uo) , o r ig in a lly used fo r the
a n a ly s is o f ly s in e s o lu t io n s , was employed in th e p r e se n t study f o r the
a n a ly s is o f o r n ith in e in th e n u tr ie n t medium,

A standard curve was

prepared by m ixing to g eth er th r e e m l, o f variou s standard o r n ith in e
h yd roch lorid e s o lu tio n s (10 t o 7 0 <y per m l* ), one m l, o f ninhydrin s o lu «
t lo n and f i v e m l, o f g ly c e r o l in la rg e t e s t tubes graduated a t 35 ml*
The standard s o lu tio n s contained 15 ml* o f phosphate b u f f e r ,7 pH 7 .U ,
per 100 ml* o f s o lu t io n .

A fte r m ixing thoroughly th e tubes se r e p laced

i n a b o ilin g water bath fo r 30 m in u tes.

At the end o f t h i s time the

tu b es were removed, d ilu te d to 35 ml* w ith 95 per cen t eth an ol and the
p er c e n t tra n sm issio n read a t £1*0 raja w ith a Beckman spectrophotom eter,
A s t r a ig h t l i n e was obtained when th e logarithm o f th e per cen t tr a n s­
m issio n was p lo tt e d a g a in st th e concen tration*
S ix 125 b*1* erleia&eyer f la s k s were s e t up w ith 1*0 m l, o f th e
n u tr ie n t s o lu t io n , 0 ,5 ®1. o f aureemycin s o lu tio n (1 mg ,/m l,) and th r ee
m l, o f standard o r n ith in e hydrochloride s o lu tio n (1 ,2 m g . / a l , ) .

P la n t s ,

whose r o o t system s were fr e e d o f v e rm icu lite and r in s e d , were p laced in
two o f th e fla s k s *

Two other f la s k s were in o c u la te d with ro o t fragm ents

and th e rem aining two fla s k s were used as c o n tr o ls in th e experim ent,
The f la s k s were p la c ed i n th e hood fo r 1*8 hours and the l i g h t s were kept
6 T his s o lu tio n was prepared fr e sh each day by d ilu tin g 125 mg.
o f ninhydrin (trik etoh yd rin d en e) and 6,57 g . o f sodium c h lo r id e
to 25 rol* w ith d i s t i l l e d w ater,
7 Th© phosphate b u ffer was prepared by a d ju stin g 250 m l. o f 0.1*
M KHfcPO* to pH 7*1* w ith 80 m l, o f 1 H HaOH and d ilu t in g to £00
m l, w ith d i s t i l l e d w ater.

31

on 12 hours e&oh day.

At the end o f t h is p er io d th e p la n ts were removed

from th e f la s k s and th® r o o ts were washed w ith a f in e stream o f water
in t o th e f l a s k s from which they had been removed.

The n u tr ie n t s o lu tio n s

were f i l t e r e d through Whatman number 1*2 f i l t e r paper.

Seven and o n e -h a lf

m l. o f phosphate b u f f e r , pH 7.1*, were added to th e f i l t r a t e s and the
s o lu tio n s were d ilu te d to £0 m l. w ith d i s t i l l e d water in volum etric f l a s k s .
The a n a ly s is fo r o r n ith in e in th e se s o lu tio n s was conducted as d escrib ed
above fo r th e p rep aration o f the standard c u r v e .

In both th e c o n tr o ls

and the s o lu tio n s in o c u la te d w ith r o o ts alm ost 100 per cen t o f th e
o r n ith in e was recovered w h ile in the so lu tio n s which had been exposed to
th e p la n ts o n ly f i v e per cen t o f th e o r n ith in e rem ained.

In a 1*8 hour®

p eriod , then, p r a c t ic a lly a l l o f th e o r n ith in e was assumed t o be absorbed
by the tobacco p la n t s , and th ere was no evidence o f o r n ith in e d e str u c tio n
by m icroorganism s.

As an added check th e n u tr ie n t s o lu tio n in one o f

th e erlenm eyer f la s k s from a C14-la b e le d o r n ith in e feed in g experiment was
evaporated to dryness and the resid u e was t e s t e d fo r r a d io a c t iv it y .
There was on ly a s l i g h t count above background in d ic a tin g e s s e n t i a l ly
com plete ab sorp tion o f th e amino a c i d from the s o lu t io n .
A d m in istration o f th e r a d io a c tiv e o r n ith in e . — I n i t i a l l y a procedure
s im ila r t o th a t employed in th e a d m in istration o f th e doubly la b e le d
m ethionine m entioned e a r lie r was follow ed in th e fe e d in g o f the la b e le d
o r n it h in e ,

The p la n ts were prepared as o u tlin e d b e fo r e .

They were

allow ed t o grow in th e n u tr ie n t s o lu tio n four days and then each p la n t
was g iv e n G.25 mg, o f D L -ornithine-2-C 14 hydrochloride having an a c t i v i t y

32

o f it x 10* e ,p .m .

A fter e le v e n days the p la n ts were removed, and th e

n ic o t in e was is o la t e d as th e d ip ic r a t e .

The n ic o tin e d ip ic r a te

p o sse sse d an a c t i v i t y o f 8 ,0 x 103 e.p.m/raM.

However, on ly about 200

t o 300 mg, o f n ic o tin e d ip ic r a te are u s u a lly obtained froan th e number
o f p la n ts employed in th ese s t u d i e s , and i t was found th a t a t l e a s t
two g . o f the p ic r a te were req u ired fo r th e degradation s t u d ie s .

I t was

n ec essa ry therefor© e it h e r to use many more p la n ts or to in c re a se th e
r a d io a c t iv it y o f th e n ic o tin e is o l a t e d .

The former i s not too f e a s ib le

because o f the expense o f the la b e le d compound so th e l a t t e r a lte r n a t iv e
was e x p lo r e d .
In order to in c re a se the amount o f la b e le d o r n ith in e incorporated
in t o th e n ic o tin e m olecule i t was n ecessary to in crea se th e amount o f
n ic o tin e sy n th e siz e d by the p la n ts during th e fe e d in g p e r io d .

T his was

accom plished by removing th e o ld r o o ts froan th© p la n ts b efo re p la c in g
them i n th e n u tr ie n t s o lu tio n and thereby fo r c in g th e p la n ts to produce
e n t ir e ly new r o o t system s p rio r t o th e a d m in istration o f the r a d io a c tiv e
o r n it h in e .
In experim ent number two the r o o ts o f It6 p la n ts were removed, and
th e p la n ts were placed in e r le m e y e r f la s k s co n ta in in g 5>Q m l. o f n u tr ie n t
s o lu t io n and 0 .5 m l. o f th e aureomycin s o lu t io n .

As was m entioned

e a r l ie r some p la n ts were tr e a te d w ith a p la n t hormone, but s in c e no
b e t t e r r e s u lt s were obtain ed than w ith the u n trea ted p la n t s , th e hormone
treatm ent was d isc o n tin u e d .

At the end o f two weeks the p la n ts had

developed r a th er e x te n siv e r o o t system s so 37 o f th e b e s t appearing
p la n ts were chosen fo r th e a d m in istration o f the la b e le d o r n ith in e .

33

Each p la n t r e c e iv e d 0*25 mg. o f o r n ith in e - 2 -C1,4 hydrochloride which had
an a c t i v i t y o f U x 10® c,p*m*

At th e end o f f i v e days each p la n t was

su p p lied w ith another 0*25 mg. o f la b e le d o r n ith in e .

Hine days a f t e r

t h i s l a s t a d m in istra tio n th e p la n ts ware removed from the f la s k s and the
n ic o t in e was is o la t e d as has been d escrib ed p reviou sly*

The n ic o tin e

d ip ic r a te was counted and p o sse ssed an a c t i v i t y o f 1 .2 1 x 10® c«p*m./foM*
T his count r e p re sen ts over a t e n - f o ld in c re a se i n th e r a d io a c t iv it y o f
th e n ic o t in e when compared w ith th e count ob tain ed in the f i r s t e x p e r iraent.

I t was th e r e fo r e p o s s ib le t o d ilu t e th e r a d io a c t iv it y o f the

n ic o t in e d ip lc r a te te n tim es by m ixing one

p a r t o f C14-n ic o tin e d ip ic r a te

w ith 9 p a r ts o f n o n -ra d io a ctiv e n ic o tin e d ip lc r a te t o o b ta in s u f f i c i e n t
m a te r ia l fo r th e degradations*

The data presen ted in Table XV are th e

counts d erived from th e d ilu te d sam ple.

The th ir d experim ent was con­

ducted i n th e same manner as th e second experim ent e x ce p t th a t h8 p la n ts
were g iv e n th e la b e le d o r n ith in e .

The r a d io a c t iv it y o f th e d ip lc r a te

is o la t e d was a lso d ilu te d te n tim e s .
D egradation o f th e r a d io a c tiv e n ic o t in e . —* 1) O xidation w ith
potassium permanganate*

Of th e se v e r a l methods mentioned in th e l i t e r a ­

tu r e fo r th e o x id a tio n o f n ic o tin e to n ic o t in ic a cid the potassium
permanganate method d escrib ed by L a ib lin (lil) appeared to be more
a p p lic a b le i n th e p resen t stu dy than th e o th e r s .

In t h i s method C0a

from th e o x id a tio n remains in th e o x id a tio n m ixture as p ot as a im bicarbon­
a te and can be recovered l a t e r fo r countin g by a c id ify in g th e medium
and c o ll e c t i n g th e C03 as barium carbonate by bubbling through barium
h y d ro x id e.

3U

t o ca rry out th e o x id a tio n th e n ic o tin e (O jtf g .) from 1 .8 g . o f
the d ilu t e d , r a d io a c tiv e n ic o tin e d ip ic r a te was recovered by the
a se o tr o p ic d i s t i l l a t i o n w ith water p r e v io u sly d escrib ed excep t th a t no
h y d ro ch loric a c id was added t o th e r e c e iv in g v e s s e l ,

th e 250 m l. o f

d i s t i l l a t e o b ta in ed were tr e a te d w ith 100 m l. o f KMn04 s o lu tio n ( 3 g . A 00
ml* d i s t i l l e d w a te r ), which were added in 5 m l. amounts a t in t e r v a ls o f
3*5 m in u te s, a t r o m tem perature.

A fte r each a d d itio n th e m ixture was

th orou gh ly mixed u n t i l th e permanganate c o lo r had disappeared.

Toward

th e end o f th e a d d itio n th e permanganate c o lo r p e r s is te d fo r lo n g er
p e r io d s , and th e m ixture was heated on th e steam bath fo r 3 to h hours
t o com plete th e o x id a tio n .

At th e end o f t h i s time th e s o lu tio n was

removed from th e steam bath and allow ed t o c o o l.

The s o lu tio n was then

f i l t e r e d and th e MnOa was washed thoroughly w ith hot w ater.

The wash­

in g s and f i l t r a t e were combined and concen trated t o dryness uhder reduced
p ressu re.
The r e sid u e rem aining a f t e r th e co n cen tra tio n c o n s is te d m ainly o f
the potassium s a l t o f n ic o t in ic so ld and potassium b ica rb o n a te.
Although th e b icarb on ate was not I d e n tif ie d as such i t seems probable
th a t under th e c o n d itio n s o f th e o x id a tio n th e C0a i s p resen t m ainly
a s potassium b icarb on ate and not potassium ca rb o n a te.

For purposes o f

i d e n t i f i c a t i o n , cou n tin g and fu r th e r degradation i t was d e sir e d to i s o ­
l a t e th e n ic o t in ic a c id as th e f r e e a c id .

I n i t i a l l y th e r e sid u e was

d is s o lv e d in 1*0 m l, o f water and tr e a te d w ith Dowex-5 0 io n exchange
r e s i n ( a r e s in c o n ta in in g s u lfo n ic acid groups) in th e a c id form to
remove th e potassium io n s and fr e e th e n ic o t in ic a c id .

However, the

35

io n exchange r e s in Adsorbed moat o f th e n ic o t in ic a cid as w e ll as th e
potassium io n s so t h i s procedure was abandoned*
Another proced ure, which was used t o I s o la t e th e n ic o t in ic acid
i n easperimont number two rep orted i n f a b le I ? , was based on the r e la ­
t i v e i n s o l u b i l i t y o f th e copper s a l t o f n ic o t in ic acid*

The r esid u e

o f potassium n ic o tin a t e and potassium bicarbonate was again d is s o lv e d
i n hO m l. o f w a te r, tr e a te d w ith a cid to remove th e bicarbonate and
th en n e u tr a lis e d w ith ammonium hydroxide.

A sa tu ra te d s o lu tio n o f

copper s u lf a t e was then added slo w ly and a b lu e p r e c ip it a t e was ob ta in ed .
T h is p r e c ip it a t e o f copper n ic o tin a te was c o lle c t e d and washed th orou gh ly.
To f r e e th e n ic o t in ic acid th e copper s a l t was suspended in water and
tr e a t e d w ith hydrogen s u l f i d e .

The copper s u lf id e p r e c ip ita te was

f i l t e r e d o f f and the f i l t r a t e was d e c o lo r iz ed w ith charcoal and th en
evaporated to a sm a ll volume on th e steam b a th .
n i c o t in ic a c id p r e c ip ita t e d .

Upon c o o lin g th e

However, o n ly 16 mg. o f th e n ic o t in ic a cid

were recovered so fa r th e r p u r if ic a t io n was not p o s s ib le .

The apparent

low reco v ery o f counts in t h i s sample o f n ic o t in ic a c id may be due not
o n ly t o erro rs i n counting t h i s sm all a sample but a ls o to some impuri­
t i e s in th e n i c o t in ic acid i t s e l f .
Sin ce th e y i e l d o f n ic o t in ic acid was very low when th e copper
s u l f a t e procedure was fo llo w e d , a fu rth er search was mad© f o r a b a tte r
p r e c ip it a t in g a g e n t.

F in a lly th e p r e c ip it a tio n o f th e s i l v e r s a l t was

adopted as th e b e s t procedure.

The r esid u e o f potassium bicarb onate

and potassium n ic o tin a te d is s o lv e d i n itO m l. o f water was f i r s t a c id if ie d
w ith d ilu t e n i t r i c a cid to lib e r a t e the C03 .

Next th e s o lu tio n was

36

n e u tr a lis e d w ith d ilu t e ammonium hydroxide and th e s i l v e r s a l t o f
n ic o t in ic a c id p r e c ip ita te d by adding 0 ,1 M AgN03 u n t i l no more pre­
c i p i t a t e form ed.

The s i l v e r s a l t was c o ll e c t e d , washed w ith d i s t i l l e d

water and suspended in 50 m l, o f w a te r.

The su sp en sion was tr e a te d

w ith hydrogen s u lf id e and th e p r e c ip ita t e o f s i l v e r s u lfid e removed by
filtr a tio n .

The f i l t r a t e was d e c o lo r ise d w ith charcoal and then evapo­

r a te d t o a sm a ll volume on th e steam b a th .

Upon c o o lin g th e n ic o t in ic

a c id p r e c ip ita te d as f i n e , white n e e d le s .

I t was c o ll e c t e d , r e c r y s t a l­

l i s e d from h ot water and d ried in a vacuum d e s ic c a to r .
C0H*NOat

C, 58.53#! H, b .09#j H, 11.38#! foundt

A n al.

O aled. fo r

C, 5 8 .7 2 # | H, 3-98%\

S , 11.86#.*
The n ic o t in ic a c id was p la te d and counted as has been d escrib ed
e a r lie r .

Since th e re was not enough n ic o t in ic acid recovered to prepare

a s e lf -a b s o r p t io n cu rv e, th e count from th e n ic o t in ic a c id was c o rrected
f o r s e lf -a b s o r p t io n u sin g th© s e lf-a b s o r p tio n curve prepared fo r th e
n ic o t in e d i p lc r a t e .

T h is procedure may introduce some error b a t th e

magnitude o f th© error i s probably low ( k 2 ) .
The CO2 lib e r a te d by the a c id if ic a t io n o f th e potassium n ic o tin a te
and potassium bicarbonate s o lu tio n s was always swept in to a sa tu ra ted
s o lu tio n o f barium hydroxide by a stream o f n itr o g e n .

Th© barium carbon­

a te p r e c ip it a t e was c o ll e c t e d , washed w ith h o t d i s t i l l e d water and
d r ied a t 110°C . fo r one hou r.

In determ ining th e r a d io a c t iv it y o f th e

barium carbonate th e procedure o u tlin e d p r e v io u sly was u sed .
a The m ieroan alyses rep orted i n t h i s t h e s is were performed by
th© Clark M icro a n a ly tic a l L aboratory, Urbmna, I l l i n o i s .

37

2)

D ecarboaylation o f th© r a d io a c tiv e n ic o t in ic a c id .

S in ce the

n i c o t in ic acid obtain ed from th e o x id a tio n o f th e r a d io a c tiv e n ic o tin e
p o s se ss e d co n sid er a b le r a d io a c t iv it y , i t was n ecessary t o determ ine the
lo c a t io n o f t h i s source o f r a d io a c t iv it y i n th e n ie o t in ic a cid m olecule
i f p o s s ib le .

To a sc e r ta in 'whether or n ot any o f th e carbon**lU was

lo c a t e d i n th e carb osyl group th e n ie o t in ic a c id was decarb oxylated.
I n th e e a r ly work on th e determ ination o f the str u c tu r e o f n ic o tin e i t
was found th a t th e dry d i s t i l l a t i o n o f th e calcium s a l t o f n ic o t in ic
a c id mixed w ith calcium hydroxide y ie ld e d pyridine and calcium carbon­
a te (1+3) ,

I n th e p resen t stu dy the n ic o t in ic a c id (u s u a lly about $0 mg.)

was mixed w ith an e x ce ss o f calcium oxide and p laced in the s p e c ia lly
co n stru cted f l a s k used in th e dem ethylation apparatus,

A d e liv e r y tube

le d from th e r e a c tio n f l a s k in to a c o n ic a l c e n tr ifu g e tube c o n ta in in g
$ m l, o f a sa tu ra ted s o lu tio n o f p ic r ic acid in m ethanol.

To sweep th e

p y r id in e in to th e r e c e iv e r a slow stream o f n itr o g e n was passed through
th e d i s t i l l a t i o n apparatus.

The f l a s k was heated slo w ly a t f i r s t w ith

a m icro-burner and then more ra p id ly as th e d i s t i l l a t i o n p roceeded.
Toward th e end o f th e d i s t i l l a t i o n th e f l a s k was heated v ig o r o u sly w ith
a bunsen burn er.

A fte r about twenty m inutes the h ea tin g was stopped and

th e f l a s k was allow ed to c o o l .

The d e liv e r y tube was removed and r in se d

s e v e r a l tim es w ith methanol in to th e r e c e iv in g tu b e .
p r e c ip it a te d as th e p lc r a te alm ost im m ediately.

The p y r id in e

A fte r stan d in g about

t h i r t y m inutes th e p y rid in e p ic r a te was c o lle c t e d on a s in te r e d g la s s
f u n n e l, washed s e v e r a l tim es w ith methanol and d r ied in a vacuum d e s ic ­
c a to r ,

For fu r th e r p u r if ic a t io n i t was r e c r y s t a lliz e d from h o t w ater.

38

£ i *k*

M ta d . t o r Cu H^I40 7 j

C, k t M $ i Hf 2.60#* M, 1 8 .l8 * j foundt

Cf it 2 ,9 l$ | Bf 2 .5 1 $ ; S , 18*03$.

The p y rid in e p ic r a te was counted and

found t o c o n ta in no r a d io a c t iv it y , and th e r e fo r e fu rth er degradation
o f th e p y r id in e r in g was unnecessary in th e p r e sen t stu d y .
The r a d io a c t iv it y o f th e n ic o t in ic a c id th en was lo c a te d s o l e l y
i n th e carb oxyl group.

As an added check th e carb oxyl carbon was

recovered from th e calcium carbonate r e sid u e in th© r e a c tio n f l a s k .
The r e a c tio n f l a s k was flu sh e d out w ith n itr o g en and then a d e liv e r y
tube le a d in g in to a sa tu ra ted aqueous s o lu tio n o f barium hydroxide was
connected t o th e f l a s k .

B ilu te n i t r i c a c id was added in th e s id e arm

o f th e f la s k a t in t e r v a ls and was fo r c e d in to th e f l a s k by recon n ectin g
th e tube t o th e n itro g en tank and p a ssin g n itro g en gas through th e
app aratu s.

The lib e r a te d C03 was swept in to the barium hydroxide so lu ­

t i o n by th e n itr o g e n g a s.

The r e s u lt in g barium carbonate p r e c ip ita te

was recovered and counted as has been d escrib ed p r e v io u s ly .
3)

D em ethylation o f th e r a d io a c tiv e n ic o t in e .

Although as w i l l be

shown l a t e r e s s e n t i a l l y a l l o f th e r a d io a c t iv it y from th e n ic o tin e was
recovered i n th e n ic o t in ic a c id and barium carbonate from th e n ic o t in e permanganate s e d a t i o n , th© p o s s i b i l i t y th a t seme r a d io a c t iv it y m ight
r e s id e i n th e H-methyl group was in v e s tig a te d n e v e r th e le s s .

In a d d itio n

i t was o f in t e r e s t to compare th e r a te o f in co rp o ra tio n o f th e alpha carbon
o f o r n ith in e in to th e m ethyl group o f n ic o tin e w ith th e r a te o f incorpora­
t io n o f p r e v io u sly stu d ie d compounds.

To a s c e r ta in whfether or n ot the

m ethyl group con tain ed any carbon-lit th e n ic o tin e from a 200 r'g. sample

39

o f th e d ilu t e d , r a d io a c tiv e n ic o tin e d ip ic r a te was dem ethylated in the
same manner aa waa o u tlin e d in th e calcium g ly e o la te stu d y .
R e su lts > — The d ata ob tain ed from two experim ents in which an
attem pt t o lo c a t e th e r a d io a c t iv it y in n ic o tin e a fte r feed in g o r n ith in e a-C14 t o tobacco p la n ts waa made are p resen ted in Table I ? ,

The o x id a -

t io n o f n ic o tin e w ith potassium permanganate in n e u tr a l s o lu tio n y ie ld s
potassium b ic a rb o n a te , n ic o t in ic acid and methyl amine,

The potassium

bicarb on ate presumably a r is e s from th e 3 ,k and 5 carbons o f the
p y r r o lid in e r in g although i t i s not d e f i n i t e l y known whether or not
any o f th e m ethyl carbon I s o x id is e d to carb on ate.

In t h i s connection

i t should be noted th a t L a ib lin (U l) waa ab le to I s o la t e m ethyl amine
from th e o x id a tio n m ixture , and th ere la always a atrong odor o f m ethyl
amine during th e o x id a tio n procedure.

In th e p resen t stu dy i t has been

assumed th a t e s s e n t i a l l y o n ly th e 3 , h and 5 carbons o f th e p y r r o lid in e
r in g o f n ic o tin e are recovered in the potassium b ica rb o n a te.

T h erefo re,

s in c e th ree m oles o f potassium bicarbonate are obtain ed in the o x id a tio n ,
th e o r ig in a l count o f th e barium carbonate recovered from the potassium
bicarb on ate i s m u ltip lie d by th ree to make i t comparable to the count o f
th e n ic o t in e .

The counts o f th e barium carbonate c a lc u la te d in t h i s

manner are g iv en i n column (B) fo r the two exp erim en ts.

I t w i l l be noted

th a t around 50 per cen t o f th e t o t a l count o f th e n ic o tin e d ip ic r a te i s
reco v ered in th e barium carbonate In each e a s e .

T h erefo re, about h a lf o f

th e r a d io a c t iv it y o f th e o r ig in a l n ic o tin e r e s id e s somewhere in th e 3 ,
h and 5 p o s itio n s o f the p y r r o lid in e r in g .

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In column (C) th e p a d to e fitiv ity o f “the n ic o t in ic a c id obtained
from th e permanganate o x id a tio n o f th e n ic o tin e i s p re sen ted .

In e x p e ri­

ment number two 36 per cen t o f th e r a d io a c t iv it y o f th e o r ig in a l
n ic o t in e i s recovered i n the n ic o t in ic a cid w h ile in experim ent number
th r ee th e r e i s a reco v ery o f

p er cen t o f th e t o t a l co u n t.

The r e ­

covery o f th e r a d io a c t iv it y in th e n ic o t in ic acid o f experiment number
two app arently i s low sin c e th e t o t a l recovery o f counts in the barium
carbonate and n ic o t in ic acid in experiment number two i s about 88 per
c e n t o f th e count p r esen t in th e o r ig in a l n ic o tin e w h ile in experiment
number th r e e 100 per cen t o f the r a d io a c t iv it y o f th e n ic o tin e i s
recovered in th e barium carbonate and n ic o t in ic a c id .

T h erefore, I t

may be concluded th a t w ith in experim ental erro r h a lf o f th e r a d io a c t iv it y
o f th e n ic o tin e recovered from p la n ts fe d o r n ith in e -2 -6 14 i s lo c a te d
i n th e n ic o t in ic a cid obtained from th a t n ic o tin e and h a l f i s lo c a te d
somewhere in th e 3 , h and 5 p o s itio n s o f th e p y r r o lid in e r in g .

I t w ill

be n ec essa ry to perform fu r th e r degradations on th e r a d io a c tiv e n ic o tin e
t o a s c e r ta in where th e cerbon-lU i s lo c a te d i n th e 3 , U and 5 p o s itio n s
o f th e p y r r o lid in e r in g .

A procedure i s b eing developed a t th e p resen t

tim e in t h i s la b o ra to ry whereby th e 3 , U and $ carbons o f th e p y r r o lid in e
r in g may be recovered in d iv id u a lly fo r co u n tin g .

As w i l l be d isc u sse d

i n more d e t a i l l a t e r I t seems very l i k e l y th a t the lo c a t io n o f th e ra d io ­
a c t i v i t y w i l l e v e n tu a lly prove t o be in th e 5 p o sitio n *
Sin ce th e n ic o t in ic a cid was found to be r a d io a c tiv e , i t was
n ec essa ry to degrade I t in an attem pt to lo c a te th e p o s itio n o f th e
r a d io a c tiv e carbon.

As has already been mentioned th e f i r s t degradation

U2

attem pted on th e n ic o t in ic acid was a decarb oxylation to y ie ld
p y r id in e and carbon d io x id e .

The r e s u lt s o f t h i s degradation are

p r e sen ted i n columns (D) and (E) * I t w i l l be noted th a t no r a d io ­
a c t i v i t y was recovered in th e p yrid in e p ie r ate in d ic a tin g th a t a l l o f
th e earbon-li* p resen t in th e n ic o t in ic acid was lo c a te d i n th e carboxyl
group.

The data presented in column (E) confirm t h i s fin d in g s in c e

e s s e n t i a l l y a l l o f th e r a d io a c tiv ity o f th e n ic o t in ic acid i s recovered
i n th e barium carbonate derived from the carboxyl group.

Prom th e se

r e s u l t s i t i s now known th a t the 2 p o s itio n in th e p y r r o lid in e r in g
c o n ta in s approxim ately h a lf o f the r a d io a c tiv ity p resen t in th e o r ig i­
n a l n ic o tin e w h ile th e oth er h a lf i s lo c a te d o u tsid e o f the 2 p o s itio n
but s t i l l in the p y r r o lid in e r in g .
In column (F ) th e counts o f the meth y I t r i e t h y l ammonium io d id e
ob tain ed from the dem ethylation o f th e r a d io a c tiv e n ic o tin e are g iv e n .
I t can be seen th a t l e s s than two per cen t o f the t o t a l r a d io a c t iv it y
o f th e n ic o tin e i s lo c a te d in the methyl group.

The r a te o f incorpora­

t i o n o f th e alpha carbon o f o r n ith in e in to th e methyl group o f n ic o tin e
i s markedly low er than the r a te o f in corp oration o f th e m ethyl groups
o f m ethionine (3 ) and c h o lin e (U ), th e alpha carbon o f g ly c in e (13)
and g ly c o l ate and th e carbon o f formate ( 3) * In t h i s connection i t
should be p oin ted out th a t th e p la n ts In th e presen t stu dy r e c e iv e d a t
l e a s t e ig h t tim es th e amount o f r a d io a c t iv ity adm inistered to the
p la n ts i n p a st m eth ylation s tu d ie s and were grown fo r a lon ger p erio d
o f tim e in co n ta ct w ith the ca rb o n -lk .

h3

B io sy n th e tic S tu d ies With L ysin e-2-C 14
I t has been shown th a t ly s in e can be converted to p lp e c o lic a c id
( p ip e r id in e -2 -c arb o x y lic a c id ) In both p la n ts (hh) and arritnaiat (ij.5)»
I t seemed co n ceiv a b le th a t p lp e c o lic acid m ight g ive r i s e to th e p y r i­
d in e r in g o f th e n ic o tin e m olecule i f th e p lp e c o lic a cid were f i r s t
dehydrogenated and d ecarb oxylated .

Thus ly s in e through con version to

p lp e c o lic acid cou ld be a precursor fo r th e p yrid in e r in g p o rtio n o f
th e n ic o t in e m o le c u le .

To determine whether or not ly s in e could se rv e

as a p recu rsor o f th e n ic o tin e m o lec u le , ly s in e -2 -C X4 was adm inistered
to I n t a c t tobacco p la n ts u sin g the same procedures as were fo llo w ed in
th e o r n ith in e exp erim en ts.
I t was n ecessary f i r s t to show th a t ly s in e could be absorbed from
th e n u tr ie n t medium by th e tobacco p la n ts .

U sing th e same a n a ly tic a l

procedure employed f o r th e o r n ith in e a n a ly s e s, i t was found th a t a t the
end o f kB hours about 100 per cen t o f th e ly s in e was recovered both in
th e f la s k s in o c u la ted w ith the r o o ts and in th e c o n tr o l f la s k s w h ile
o n ly 10 per cen t o f th e ly s in e was recovered i n th e f la s k s con tain in g
th e p l a n t s .

I t was concluded th a t w ith in th e fe e d in g p eriod p r a c t ic a lly

a l l o f th e ly s in e would be absorbed by the tobacco p la n t s .

In a d d itio n

th e co n ten ts o f one o f th e f la s k s used i n ad m in isterin g th e r a d io a c tiv e
ly s in e was evaporated to dryness a fte r the growing p e r io d , and when
th e r e sid u e was te s t e d fo r r a d io a c t iv it y th ere was on ly a s l i g h t count
above background in d ic a tin g p r a c t ic a lly com plete disappearance o f the
amino a c id from the n u tr ie n t medium.

There was no evid en ce o f the growth

o f m icroorganism s i n th e n u tr ie n t s o lu tio n s .

Uh

When 0 , 2 5 Kg. o f D L -lysin e-2-C 14 hydrochloride co n ta in in g it x 10®
c .p .m . was adm inistered to 20 tobacco p la n ts under the same c o n d itio n s
as were d escrib ed in experiment number one w ith th e o r n ith in e - 2 -C14
h y d ro ch lo rid e, th e n ic o tin e I s o la te d from th e p la n ts p o ssessed an in ­
s i g n if i c a n t count over background.

Under th e se co n d itio n s no d e te c t­

a b le amount o f ly s in e i s incorp orated in to th e n ic o tin e m o lecu le.
When th e ly s in e la b e le d w ith carbon-lit was adm inistered employing
th e technique used fo r experim ents number two and number th ree in the
o r n ith in e s t u d i e s , th e n ic o tin e which was I s o la te d from the p la n ts as
th e d lp ic r a te p o ssessed a count o f 2 .0 0 x 10a c .p jb ./k M .

The n ic o tin e

i s o l a t e d from p la n t s , which were fe d o r n ith in e -2 -C14 under comparable
c o n d it io n s , p o sse sse d a count o f 1 .2 1 x 106 e.p.m./mM.

The r a te o f

in co rp o ra tio n o f o r n ith in e in to the n ic o tin e m olecule then i s about
60 tim es th e r a te o f in co rp o ra tio n o f ly s in e when th e two were admin­
is t e r e d under e s s e n t i a l l y id e n tic a l c o n d itio n s .

Because o f t h i s low

r a te o f l y s in e in co rp o ra tio n i t was concluded th a t ly s in e does not give
r i s e to n ic o t in e through a d ir e c t pathway but on ly in d ir e c t ly .

Since

th e r a d io a c t iv it y o f the n ic o tin e d lp ic r a te is o la t e d from th e p la n ts
fe d ly s in e was so lo w , i t was not p o s s ib le to o b ta in s u f f ic i e n t m a teria l
by m ixing u n la b eled n ic o tin e d ip ic r a te w ith the la b e le d sample fo r
d egrad ation s tu d ie s so th e degradation procedure used in the o r n ith in e
esqperlments was n ot undertaken in th e p re sen t stu d y .
One way in which th e alpha carbon o f ly s in e could en ter the n ic o ­
t in e m olecule i s through th e conversion o f ly s in e to glutamate or some
o th e r compound c lo s e l y r e la te d to o r n ith in e .

Borsook and co-workers (1*6)

ks

have dem onstrated th a t guinea p ig l i v e r hamogen&tes are ab le to con v e r t l y s i n e t o g lu ta r lc acid*

GELutaric acid through o x id a tio n to

GC~ketoglutaric a cid fo llo w ed by a transam ination r e a c tio n could in
tu r n be con verted to glutam ic a c id .

More r e c e n tly M ille r and B ale (U7)

have shown t h a t D L *lyelne-6*C*4 i s converted to glutam ic a c id , argin in e
and o r n ith in e in in t a c t d o g s, and th ey su g g est th a t g lu ta r a te or o(~ketog lu ta r a te i s th e l i k e l y common Interm ediate fo r th e form ation o f th ese
th r ee amino a c id s from l y s i n e .

Glutamic acid has been shown to a c t as

a precursor o f p r o lin e and argin in e in r a ts ( 1*8) and o f p r o lin e in
B* c o l l ( ! i9 ) .

In a d d itio n Flncham ($0) has demonstrated th a t enzyme

p rep a ra tion s from Neuroapora c ra ssa m yeelia can c a ta ly s e th e sy n th e sis
o f o r n ith in e from glutam ic -T- semialdehyde and a ls o from glutamate*
Although th e re la no evidence a t p resen t th a t ly s in e can be converted
t o glutam ic acid and th en to p r o lin e or o r n ith in e in h igh er p la n ts i t
seems q u ite p o s s ib le th a t t h i s conversion could occu r.
th e r e fo r e one mechanism, which would be c o n s is te n t w ith th e above
f in d in g s end which would e x p la in the in corp oration o f th e alpha carbon
o f ly s in e in t o th e n ic o tin e m o lecu le, could be based on th e su p p o sitio n
th a t ly s in e i s f i r s t converted to o r n ith in e or some c lo s e l y r e la t e d com­
pound b efo re b ein g incorporated in to th e n ic o tin e m olecule in th e
i n t a c t tobacco p la n t.
In view o f th e r e s u lt s o f th e o r n ith in e experim ent and the sim i­
l a r i t y between ly s in e and o r n it h in e , i t i s in t e r e s t in g to sp e c u la te
on th e o r ig in o f th e a lk a lo id anabasine ( o t-(/3 -p y r id y l) - p ip e r id in e )

U6

i n tobacco p la n ts*

H * in ( £ l) has su ggested th a t anabasine i s formed

from n ic o tin e by the expansion o f th e 5«member@d p y r r o lid in e r in g a t
th e expense o f th e m ethyl group.

However, i t seesns more l i k e l y th a t

th e p ip e r id in e r in g o f anabasine i s formed from ly s in e in a manner
analogous t o th e form ation o f th e p y r r o lid in e r in g o f n ic o t in e .

U7

DISCUSSION
Tr angme th y la tio n stud l e a . — The r e s u lt s o f th e experim ents in
which m ethionine doubly la b e le d in th e m ethyl group w ith carbon-lit and
deuterium was adm inistered to b a r ley and tobacco p la n ts provide e v i­
dence th a t th e in term o lecu lsr tr a n sfe r o f methyl groups i s a r e a c tio n
i n th e m etabolism o f high er p la n t s .

The widespread occurrence o f

m ethylated compounds in th e p la n t kingdom in d ic a te s the importance o f
tra n sm e th y la tio n r e a c tio n s In th e m etab olic p r o c esses o f h igh er p la n t s .
I t should be p oin ted ou t t h a t tran sm eth ylation a t l e a s t where m ethionine
and c h o lin e are in v o lv ed i s probably n o t the on ly r e a c tio n which can
g iv e r i s e to m ethyl groups in high er p la n ts s in c e there i s evidence th a t
th e alpha carbon o f g ly c in e may be incorp orated in to th e m ethyl group
o f n ic o t in e a t a f a s t e r r a te than th e m ethyl carbons o f e ith e r m ethionine
or c h o lin e ( 13)»
The q u estio n as to whether or not the o r ig in o f th e m ethyl group
o f n ic o t in e through tran sm eth ylation from m ethionine a c tu a lly r ep re sen ts
a n e t b io s y n th e s is o f n ic o tin e s t i l l remains improved.

In t h i s con­

n e c tio n I t i s o f i n t e r e s t to con sid er Dawson* s work on th e form ation o f
n o rn ico tin e in H iootian a g lu tin o s a ( l l ) ,

I t was shown th a t n ic o tin e i s

s y n th e siz e d in th e r o o ts o f S , g lu tin o sa and then tr a n slo c a te d to th e
a e r ia l p o r tio n s o f th e p la n t where i t I s dem ethylated to form n o r n ic o tin e .
In an attem pt t o r ev e rse th e dem ethylation r e a c tio n and form n ic o tin e
from n o r n ic o tin e and some m ethyl precursor in H. g lu t in o s a , Dawson

1*8

r e p o r te d in a l a t e r p u b lic a tio n ( 52) th a t there was no d e te c ta b le
s y n t h e s is o f n ic o tin e when n o r n ic o tin e alone or when n o rn ico tin e and
e it h e r o h o lin e o r m ethionine were adm inistered t o e x c ise d le a v e s , which
were made e s s e n t i a l l y f r e e o f a lk a lo id by g r a ftin g g lu tin o s a sh o o ts on
tomato r o o t s t o c k s ,

From th e se r e s u lt s th e co n clu sio n was drawn th a t

th e pathway Of n ic o tin e dem ethylation c o n ta in s a t l e a s t one ir r e v e r s ib le
ste p *

I t I s d ou b tfu l th a t t h i s s it u a t io n ob tain s i n H. r u s t ic a . th e

p la n t u sed i n th e p resen t stu d y , s in c e th ere appears t o be e s s e n t i a l ly
no n o r n ic o tin e i n H* r u s t ic a ( 5 3 ) .

H * in (5 l,5 U ) has confirmed Dawson's

f in d in g s concerning th e form ation o f n o rn ico tin e i n K. g lu tin o s a . and
although th e accep tor o f th e methyl group from n ic o tin e was not i d e n t i f i e d ,
i t was indie& ted th a t n ic o tin e may p a r tic ip a te in th e general metabolism
o f th e p la n t .

More r e c e n tly Kusln and Merenova (5 5 ) have shown th a t

when e x c ise d tobacco le a v e s are exposed to CO* la b e le d w ith carbon-lU
th e n ic o tin e is o la t e d from th e se le a v e s i s r a d io a c tiv e .

Moreover a l l

o f th e radiocarbon i s lo c a te d i n th e m ethyl group o f th e n ic o tin e *
The authors conclude th a t th e le a v e s o f H icotian a thus carry on tr a n s m ethyl a tio n s w ith the p a r tic ip a tio n o f n ic o t in e .

In a d d itio n Culp (56)

i n t h i s la b o r a to ry has found th a t in t a c t H* r u s t i c s p la n ts in j e c t e d
w ith a s o lu tio n o f sodium bicarbonate la b e le d w ith carbon-lit produce
n ic o t in e w ith a s l i g h t l y la r g e r amount o f radiocarbon in the m ethyl
group than in th e r e s t o f th e n ic o tin e m o lec u le.

The se v e r a l s tu d ie s

m entioned above in d ic a te th a t th e m e tly l group o f n ic o tin e may be l a b i l e
a t l e a s t to a c e r ta in e x te n t .

In view o f th e se fin d in g s i t i s d i f f i c u l t

U9

a t p r e se n t to sa y whether th e tran sm ethylation r e a c tio n observed in
th e p r e se n t stu d y le a d s to a n et sy n th e sis o f n ic o tin e or whether i t
I s m erely in v o lv ed in th e general tran sm eth ylation r e a c tio n s which are
o p e r a tiv e in th e le a v e s o f th e p la n t.

Since i t has been shown th a t

n ic o tin e i s sy n th e sise d m ainly in th e r o o ts ( 16) , one approach to t h i s
q u e stio n would be to a sc e r ta in whether or not a n e t s y n th e s is o f n ic o ­
t i n e co u ld be ob tain ed by in cu b atin g ro o t enzyme preparations w ith
m ethionine and n o r n ic o tin e .

Such a study i s in progress a t the p resen t

tim e in t h i s la b o r a to ry .
The s tu d ie s on the lig n in o f b a rley and tobacco p la n ts provid e the
f i r s t c o n c lu siv e evidence th a t m ethyl groups can be tra n sfe rr ed in t a c t
from s u lfu r to oxygen i n m etab olic p r o c e s s e s .

The ob servation s in the

p r e se n t stu d y along w ith th e fin d in g o f Kirkwood, e t a l . (£ 7) th a t the
m ethyl carbon o f m ethionine may be a precursor o f th e methoxyl carbon
o f r ic ln in e in c a sto r beans and th e raetbylenedioxy groups o f protopin e
In D icen tra h y b r id s, would len d support to the h yp oth esis th a t the
acceptance o f m ethyls by oxygen, a r e a c tio n not observed in animal me­
ta b o lism , i s a gen eral r e a c tio n in p la n t m etabolism .
Xt i s o f i n t e r e s t to examine whether th e tr a n sfe r o f m ethyl groups
dem onstrated in t h i s study rep resen ted sy n th e s is o f p a rt o f th e l i g n i n
m olecule or m erely some type o f exchange r e a c tio n s im ila r to th a t which
may have been encountered in th e n ic o tin e stu d y .

Stone (5 8 ) has shown

th a t in wheat p la n ts fe d C^Oa fo r a sh o rt tim e th e t o t a l a c t i v i t y
acquired by th e syringaldehyde p ortion o f l ig n in during th a t time r e ­
mained c o n sta n t throughout th e growth o f th e p lan t 9 th a t i s , th e l ig n i n

50

( a s p eja w w a ted by syringaldehyde) was an end product i«<i was nob &
p a r t ic ip a n t i n fa r th e r m etab olic r e a c tio n s o f th e p la n t.

M oreover,

S to n e dem onstrated th a t p a r t o f th e r a d io a c tiv ity i n th e l ig n i n de­
g ra d a tio n p rodu cts was lo c a te d i n th e methoayl groups.

Stone *e vork

would seem to show th a t th e d ir e c t tr a n sfe r o f m ethyl groups demon­
s t r a t e d in th e p resen t stu d y was a r e a c tio n in th e sy n th e sis o f l i g n i n .
M ethylation stu d y . —

The r e s u lt s o f th e study w ith calcium

g ly c o la t e - 2-G14 in d ic a te th a t th e alpha carbon o f g ly c o lic so ld I s
capable o f se rv in g as a source o f m ethyl groups in th e metabolism o f th e
tob acco p la n t .

T h is I s in c o n tr a st to th e s tu d ie s mentioned in th e

In tr o d u c tio n where i t was p o in ted out th a t se v e r a l is o la t e d system s
from b oth p la n ts ( i h ) and anim als (1 5) were unable to use g ly c o lic a c id
i n m eth y la tin g gu& nldineacetic a d d .

In th e p resen t stu dy a l l o f th e

r a d io a c t iv it y o f th e n ic o tin e w ith in experim ental error was recovered
In th e m ethyl group in d ic a tin g th a t under the c o n d itio n s o f th e e x p e r i­
ment none o f th e alpha carbon o f g iy e o la te e n ter s th e r in g p o r tio n s o f
th e n ic o tin e m o le c u le .
The g iy e o la te alpha carbon i s more r a p id ly incorp orated in to th e
n ic o t in e m eth yl group than e ith e r formate ( 3) or biearb onate ( 56) but
th e d iffe r e n c e between th e r a te s o f in corp oration o f the m ethyl group
o f m eth ionine ( 3) and o f th e alpha carbon o f g iy e o la te i s so sm all a
d e f in it e c o n c lu sio n cannot be drawn about which o f th e two e n te r s more
r a p id ly .

On th e oth er hand the r a te o f en try o f th e alpha carbon o f

g ly c in e ( 1 3) i s somewhat high er than th e r a te o f en try o f th e g iy e o la te

51

a lp h a carbon*

I t therefor© seems probable th a t th e g ly cin e i s not

con verted t o g ly c o lic a c id before th e alpha carbon i a incorporated
in t o th e m ethyl group o f n ic o tin e *

H sm ill (13) has su ggested two

o th e r p o s s i b i l i t i e s f o r th e mechanism o f m eth ylation by th e alpha
/
carbon o f g ly c in e which seem more probable in view o f th e p resen t fin d ­
in g s*

th e s e are*

l ) g ly c in e may be o x id a tiv e ly deaminated t o g iv e

g ly o x y lie a c id th e alpha carbon o f which i s th en reduced w ithout going
t o format©, or 2) the n itr o g en o f g ly c in e may be incorporated d ir e c t ly
in t o th e p y r r o lid in e r in g o f n ic o tin e and th e alpha carbon remains to
form th e m ethyl group.

The l a t t e r proposal i s not to o a t t r a c t iv e i n

view o f th e experim ents w ith o r n ith in e -2 -C i4 to be d isc u sse d l a t e r ,
b u t th e form er proposal i s a d e f i n i t e p o s s i b i l i t y .
I t seems h ig h ly improbable th a t th e alpha carbon o f g iy e o la te i s
co n v erted t o form ate b efo re i t s in corp oration in to th e n ic o tin e m ethyl
group , but as was su ggested above the g iy e o la te may be converted f i r s t
to g ly o x y lic a c id , th e carbonyl group o f which could g iv e r i s e to
m eth yl groups through r e d u c tio n .

There i s ample evid en ce th a t g ly c o lic

a c id can be o x id is e d by h igh er p la n ts t o y ie ld g ly o a y lic acid ( 5 9 ) , and
although t h i s o x id a tio n o f g iy e o la te to g ly o a y la te seems to be a round­
about pathway fu r th e r s t u d ie s are n ecessary t o determ ine i t s v a l i d i t y .
The rat© o f in co rp o ra tio n o f the carbonyl carbon o f g ly o x y lic a c id in to
th e n ic o tin e m ethyl group i s being stu d ied i n t h i s lab oratory a t the
p r e se n t tim e , and the r e s u l t s should a id i n c la r if y in g t h i s s it u a t io n .
A fu r th e r p o s s i b i l i t y concerning th e m eth ylatin g c a p a c ity o f g ly ­
c o l i c a c id should be mentioned in view o f the fin d in g s o f T olb ert and

52

Cohan t h a t th e alpha carbon o f g ly c o lic a cid can give r i s e to the
b e ta carbon o f s e r in e i n b a r ley and wheat le a v e s ( 60) .

I t i s known

th a t th e b e ta carbon o f se r in e can serv e as a m ethyl group precursor
fo r m eth io n in e, c h o lin e and c r e a tin in e in h igh er animals ( 6 1 ) , and i t
i s c o n ceiv a b le th a t a t l e a s t p a rt o f the g ly c o lic acid may en ter th e
n ic o t in e m olecu le by way o f s e r in e In higher p la n t s .

The a d m in istra tio n

o f serin e*3*C *4 to tobacco p la n ts I s being conducted now in t h i s
la b o r a to ry and th e r e s u l t s should g iv e some in d ic a tio n as to th e p la u s­
i b i l i t y o f such a pathway.
F in a ll y , th e p o s s i b i l i t y o f th e alpha carbon o f g ly c o lic a cid
b e in g converted to formaldehyde b e fo re i t s conversion to m ethyl groups
should be p oin ted o u t.

Prelim inary data i n t h i s lab oratory in d ic a te s

th a t formaldehyde e n te r s the n ic o tin e m olecule more r a p id ly than any
p r e v io u sly t e s t e d compound, but i t s t i l l i s n ot known whether a l l o f
th e radiocarbon o f th e n ic o tin e r e s id e s i n the m ethyl group ( 62) .
G ly c o lic a c id i s found i n sm all amounts in h igh er p la n ts ( 63) , and
as y e t i s th e o n ly two carbon organic a c id , which has been found to
accumulate carbon~li* during sh ort t in e p h o to sy n th esis w ith C140 a ( 6h ) .
The alpha carbon o f g ly c o lic acid may th e re fo re be a ra th er im portant
sou rce o f m ethyl groups in higher p la n t s .
S tu d ie s w ith o m ith in e-2 -G **. — As has already been poin ted out
h a l f o f th e radiocarbon p resen t in n ic o tin e from p la n ts fe d o r n ith in e *
2-C14 i s lo c a te d in th e 2 p o s itio n o f th e p y r r o lid in e r in g .

The o th er

h a lf o f th e radiocarbon i s lo c a te d somewhere in th e 3 , I4 and $ p o s i t i o n s ,
and although I t i s n o t known d e f i n i t e l y th a t th e 5 p o s itio n c o n ta in s

53

th e f f i i i l i i i n g h a l f o f "the p sd io a o tiv itjr t h i s appears t o be the l o gi c al
p o s itio n ,,

T h is would c a l l fo r a sym m etrical in term ed iate in 'Use f o r ­

m ation o f th e p y r r o lid in e r in g o f n ic o tin e from th e o r n ith in e m olecule
as i s shown In Scheme I ,
o r n ith in e

sym m etrical interm ed iate

p y r id in e r in g
precu rsor
"T
n ic o tin e

Scheme 1

There are se v e r a l p o s s i b i l i t i e s fo r th e form ation o f a symmetri­
c a l m olecu le from o r n it h in e .

The f i r s t p o s s i b i l i t y would be th e form­

a t io n o f th e sym m etrical diam ine, p u tr e s c in e , d ir e c t ly by th e decarboxy­
l a t i o n o f o r n ith in e ,

A second pathway, which I s more in d ir e c t , would

in v o lv e th e o x id a tiv e deamination o f o r n ith in e to g ive p o s s ib ly glutam ic
sem iald eh yde, which on e y c liz & tio n could y ie ld
c a r b o x y lie a c i d .

a } -p y r r o lin e -5

The l a t t e r may be decarboxylated to g ive p y r r o lin e ,

which would become sym m etrical by s h i f t i n g th e double bond, or i t may
f i r s t be reduced to g iv e p r o lin e , which would y ie ld p y r r o lid in e , a
sym m etrical m o le c u le , when d ecarb oxylated .

F in a ll y , the p o s s i b i l i t y

o f o r n ith in e b ein g converted by d ecarb oxylation fo llo w ed by o x id a tiv e

5U

dsSBtinstlofi t o o itb o r su ecliyiloidolydQ or su c c in ic so ld may be non*
tio n e d although i t Seem8 l e s s l i k e l y than th e two p r e v io u sly mentioned
p o s s ib ilitie s .

The se v e r a l p o s s i b i l i t i e s are i llu s t r a t e d in Scheme IX .

Although p u tr e sc in e i s g e n e ra lly considered a product o f the
a c t i v i t y o f m icroorganism s, th e r e i s some evidence th a t diamines may
p la y some r o le i n th e metabolism o f h igh er organism s.
B e st and

McHenry

Z e lle r (6 $ ) and

(6 6 ) have rep orted th e occurrence o f a diem ine-

o x id a se system in animal t i s s u e s .

More r e c e n tly Cromwell ( 67) has

found t h a t p u tr e sc in e stim u la te s th e sy n th e s is o f th e a lk a lo id ,
hyoseyamin©, when adm inistered to both Atrppa belladonna L. and Datura
stramonium 1*

In a l a t e r p u b lic a tio n (6 8 ) Cromwell rep orted the

p rep a ra tio n o f an enzyme system , which was capable o f o x id iz in g
p u tr e sc in e w ith th e form ation o f ammonia and an aldehyd e, from th e
t i s s u e s o f A. b ella d o n n a .

Furthermore he was able to i s o l a t e sm all

amounts o f p u tr e sc in e from th e le a v e s and upper stems o f A. belladonna
and D, stramonium.

On th e b a s is o f h is r e s u l t s Cromwell p o stu la ted

th a t p u tre sc in e could be converted t o e ith e r suecindialdehyde or
T'-amlno b u tyrald eh yde, which could then be incorp orated in to th e
byoscyamine m o le c u le .

I f p u trescin e i s consid ered as an in term ed iate

i n th e form ation o f th e p y r r o lid in e r in g o f th e n ic o tin e m o lec u le,
th en i t seems n ec essa ry t o p o s tu la te an o x id a tiv e or p o s s ib ly a hydro­
l y t i c deam ination o f th e p u tr e sc in e b efore e y c llz a t lo n can take p la c e
as Cromwell has done in th e hyoscyarain© stu d y .
I f on© amino group o f p u tre sc in e i s l o s t by o x id a tiv e deam ination,
7^amino butyraldehyde r e s u l t s .

The l a t t e r could c y c liz e to give

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55

p y r r o lin e o r i t may condense d ir e c t ly w ith a p y rid in e r in g p recu rsor
and toe c y c lis e d la te r *

On th e oth er hand i f o x id a tiv e deam ination

occu rs a t tooth ends o f the p u trescin e m olecule then succlndialdehyde
r e s u lt s *

T h is dialdebyde m ight then condense w ith e it h e r ammonia or

m eth yl amine to g iv e a 2 # S dlhydroaqr p y r r o lid in e d e r iv a tiv e .

The

l a t t e r compound must undergo a dehydration and red u ctio n toefore the
n ic o t in e m olecu le i s f i n a l l y formed*

P u trescin e i s th e re fo re a con­

c e iv a b le in term ed ia te i n th e b io s y n th e sis o f n ic o tin e s in c e i t i s
sym m etrical and could undergo fu r th e r r e a c tio n s which would le a d t o
th e p y r r o lid in e r in g .
Perhaps a more l i k e l y pathway fo r th e in corp oration o f o r n ith in e
in t o th e n ic o t in e m olecule i s through th e form ation o f p r o lin e or some
compound c lo s e l y r e la te d to p rolin e*

The mechanism fo r th e con version

o f o r n ith in e t o p r o lin e in animals has been rath er w e ll e s ta b lis h e d .
Schoenheimer and co-workers (69) have found th a t d euterated o r n ith in e
g iv e s r i s e t o la b e le d p r o lin e in m ice * Schemes, which In volve the
co n v ersio n o f o r n ith in e t o e it h e r

of-k eto- S -amino v a le r ic a cid or

glutam ic sem ialdehyde fo llo w ed by c y c liz a t io n t o g iv e

a

1 p y rro lin e*

5 -c a r b o x y lic a c id and r ed u ctio n o f th e l a t t e r t o g iv e p r o lin e , have
b een proposed toy se v e r a l in v e s tig a to r s to e x p la in th e con version o f
o r n ith in e t o p r o lin e in animals ( 7 0 ) .

More r e c e n t ly , S te tte n (71)

u s in g o r n ith in e la b e le d i n e ith e r th e alpha p o s it io n or th e d e lt a
p o s it io n w ith II16 has shown th a t th e d e lt a amino group o f o r n ith in e i s
l o s t in s te a d o f the alpha amino group so th a t glutam ic sem ialdehyde i s
th e more l i k e l y in term ed iate i n th e conversion o f o r n ith in e to p r o lin e

56

i n mice*

I t seems very l i k e l y th a t high er p la n ts could a ls o convert

o r n ith in e t o p r o lin e although th ere i s no co n c lu siv e evidence to sup—
p o r t t h i s su p p o sitio n a t th e presen t tim e .

I f tobacco p la n ts are able

t o perform t h i s c o n v ersio n , there are two sym m etrical in term ed iates
which cou ld r e s u l t , nam ely, p y r r o lin e , which arise® from th e decarboatyla t io n o f

A f pyrro lin e - 5 - e arb oxylic a c id , or p y r r o lid in e from the

d eca rb o x y la tio n o f p r o lin e .

A condensation between the p yrid in e r in g

p recu rsor o f n ic o tin e and p y rro lin e or p y r r o lid in e or p o s s ib ly one o f
t h e i r m e ta b o lite s would then be n ecessary fo r the form ation o f th e
n ic o tin e m o le c u le .
The f i n a l p o s s i b i l i t y su ggested fo r th e form ation o f a sym m etrical
in term ed ia te from o r n ith in e i s the conversion o f o r n ith in e to s u c c in d iaLdehyde or s u c c in ic a c id .

Both o f th ese conversion s in v o lv e th e l o s s

o f a l l th e n itr o g e n o f o rn ith in e and might provide a means fo r the
entrance o f th e amino group along w ith the alpha carbon o f g ly cin e
in t o th e N-methyl group o f n ic o tin e as was su ggested by HamUl (13) •
The su ccln d iald eh yd e might be formed by th e o x id a tiv e deamination o f
p u tr e sc in e as has already been mentioned whereas the s u c c in ic acid
m ight a r is e from
The

of -k o to g lu ia r ic acid by o x id a tiv e dec a rb o x y la tio n .

d - k e t o g lu t a r ic a cid i s d erived from glutam ic a c id , which can

S&ise from o r n ith in e (69)*
There i s no b a s is fo r choosing among th e se variou s p rop osals a t
p r e se n t so th ey oan o n ly be su ggested as h y p o th e tic a l schem es.
In view o f th e p resen t fin d in g s T r ie r f a (22) proposal th a t n ic o tin e
I s d eriv ed from a s in g le amino a c id , p r o lin e , i s on ly p a r t i a ll y c o r r e c t.

57

I t i s tr u e th a t th e p y r r o lid in e r in g may a r is e from p r o lin e but th e
p y r id in e r in g apparently does n o t.

Robinson had su ggested th a t

o r n ith in e was a l i k e l y precursor o f th e p y r r o lid in e r in g in n ic o tin e
i n 1917 ( 2 l ) * I t la in t e r e s t in g th a t he su ggested an Nmhethyl compound
(N -m etlyl-2-hydroxypyTr o lid in e ) as th e compound, which would condense
w ith th e p y rid in e r in g precursor (aceton e d ic a rb o x y lie a cid i n h is
schem e)« As y e t i t i s not known e x a c tly when th e m eth ylatlon r e a c tio n
ta k e s p l a c e , b efore th e p y r r o lid in e and p yrid in e r in g s are join ed or
a f t e r th e y are jo in e d .

In any even t i t i s p o s s ib le as Robinson su g g e sts

t h a t th e p y r r o lid in e r in g precursor may be m ethylated previous to th e
co n d en sation w ith th e p yrid in e r in g p recu rsor.
Mortimer ( 23) has su ggested th a t p art or a l l o f the s id e ch ain o f
kyuurentne, which can be d erived from tryptophan in l iv i n g organisms
( 7 2 ) , may be in corp orated in to th e p y r r o lid in e r in g .

I t i s c le a r th a t

t h i s i s n ot to o l i k e l y in view o f th e p resen t f in d in g s .

Indeed Bowden

has already shown th a t th e b e ta carbon i n the s id e ch ain o f tryptophan
I s n o t in corp orated in to th e n ic o tin e m olecule ( 2 6 ) .

The p resen t study

a ls o sag&ains th e fin d in g o f Dawson and co-workers th a t the carboxyl
carbon o f n ic o t in ic a c id I s not a v a ila b le fo r n ic o tin e sy n th e s is (25 ) •
n e v e r t h e le s s , th e p o s s i b i l i t y th a t tryptophan or n ic o t in ic a cid may
g iv e r i s e t o th e p yrid in e r in g o f n ic o tin e i s not r u led o u t.

I t has

r e c e n tly been su ggested (7 3 ) th a t some s o r t o f a condensation between
n ic o t in ic a c id and N ~raethylpyrrolidine, which has been found in the
a lk a lo id f r a c t io n from tobacco p la n ts (7h ) , would seem to be a l o g ic a l

58

s t e p In th e s y n th e s is o f n ic o t in e .

Trl®* (22) had proposed a sim ila r

con d en sation between n ic o t in ic a cid and p r o lin e many yeans b efore ,
b u t th e prolin© m olecule i t s e l f p o s se ss e s asymmetry, and t h i s con*
d e n sa tio n would be in c o n s is te n t w ith th e fin d in g s o f th e p re sen t stu d y .
A co n d en sation between n ic o t in ic a c id and some p y r r o lid in e r in g pro-*
cu rso r would be e n t ir e ly c o n s is te n t w ith th e fin d in g s o f the p resen t
s tu d y , b u t ouch a condensation would n e c e s s a r ily in v o lv e th e l o s s o f
th e ea rb o sy l group o f n ic o t in ic a cid and th ere seems to be no precedent
f o r such a r e a c tio n .
One fu r th e r p o in t should be mentioned in con n eetion w ith th e union
betw een th e p y r r o lid in e and p yrid in e r in g s , and th a t i s th a t o n ly
1 - n t c o t in e i s formed i n nature*

Since th e asymmetric carbon atom In

n ic o t in e I s lo c a te d i n th e p y r r o lid in e r in g , e ith e r th e r in g precursor
p o s s e s s e s an asymmetric carbon or th e condensation o f the p yrid in e and
p y r r o lid in e r in g s r e s u lt s in an asymmetric s y n t h e s is .

I f an asymmetric

p y r r o lid in e m olecu le i s formed before th e condensation i t must be formed
from a sym m etrical compound to be c o n s is te n t w ith th e r e s u lt s o f th e
p r e se n t stu d y .

Such a compound could be 2-hyd rosypyrrolidine which

m ight a r is e e it h e r from th e hydration o f th e double bond o f p y r r o lin e
o r th e oxLdatinn o f p y r r o lid in e .

I f 2-hydroxypyrrolidine i s formed

th e n i t i s c o n ceiv a b le th a t th e n ic o tin e r in g str u c tu r e could be formed
by s p l i t t i n g ou t a m olecule o f water between p yrid in e and 2-hydro:xyp y r r o lid in e .
At p r e sen t i t i s im p ossib le to propose a d e f in it e pathway fo r the
b io s y n th e s is o f n ic o tin e sin c e th e nature o f th e precursor o f the

59

p y r id in e r in g la unknown.

In a d d itio n th e nature o f th e immediate

precu rsor o f th e p y r r o lid in e r in g i s n ot c le a r so th a t con sid erab ly
more

study

i s req u ired b e fo re a d e f i n i t iv e mechanism can be proposed.

F i n a l l y , th e q u estion a r is e s as to -whether o rn ith in e i s a n atu ra l
m e ta b o lite in tobacco p la n t s .

James (75) has found th a t both o r n ith in e

and a r g in in e when adm inistered t o detached le a v e s o f belladonna p la n ts
g iv e r i s e t o an in c re a se d s y n th e s is o f th e tropane a lk a lo id s .

Moreover,

th e in c r e a se in a lk a lo id when o r n ith in e was fe d was apparently in
e x c e ss o f th e in c r e a se when argin in e was f e d .

Janes was a ls o able t o

show th e presence o f 1 -o r n ith in e i n th e f r e e s t a t e in young sh oots o f
b ellad on n a p l a n t s .

A rginase has a lso been found in the e x tr a c ts o f

r o o ts and sh o o ts o f b ellad on n a, tobacco and tomato ( 7 6 ) .

Since argin in e

i s known t o occur i n the p r o te in s o f tobacco p la n ts ( 7 7 ) , i t seems
e n t i r e l y p o s s ib le th a t o r n ith in e could be produced from argin in e through
th e a c tio n o f argin ase and th e re fo re could be a v a ila b le fo r th e synthe­
s i s o f n ic o t in e .

m m m

60

SUMMARY
X. A stu d y in which m eth ion in e, doubly la b e le d in the m ethyl
group w ith G*4 and deuterium , was adm inistered to in t a c t tobacco p la n ts
has e s ta b lis h e d th a t th e m ethyl group o f m ethionine can g iv e r i s e to
th e m ethyl group o f n ic o tin e throu^ i tran sm eth ylation .
2 . The m ethyl group o f m ethionine was shown t o be tra n sferred as
a u n it to form th e m ethoayl groups o f l ig n in in b a r ley and to b a c co , a
r e a c t io n , which in v o lv e s th e d ir e c t tr a n s fe r o f m ethyl groups from
s u lf u r t o osygen .

Xt was in d ic a ted t h a t th e in corp oration o f th e methyl

group o f m ethionine In to l ig n i n rep resen ted a sy n th e sis o f p art o f the
l i g n i n m o leo u le .
3 . A fte r th e ad m in istra tio n o f calcium g ly c o la te -2 -C 14 to tobacco
p la n t s , r a d io a c tiv e n ic o tin e has been i s o l a t e d .

Most i f n ot a l l o f th e

r a d io a c t iv it y has been shown to r e s id e i n th e m ethyl group.
li.

Tobacco p la n ts fe d orn ith in e-2*C 14 hydrochloride produce n ic o t in e ,

which has been shown t o be r a d io a c tiv e .

Xt has been found th a t h a lf o f

th e carbon-lU in th e i s o l a t e d n ic o tin e i s lo c a lis e d in th e 2 p o s itio n o f
th e p y r r o lid in e r in g o f the n ic o t in e .

The other h a lf o f th e radio*

a c t i v i t y has b een p o stu la te d t o r e s id e i n th e $ p o s itio n o f th e p y r r o li­
d in e r in g , and th e r e fo r e i t was concluded th a t a sym m etrical in term ediate
i s formed from o r n ith in e b efo re i t s in co rp o ra tio n in to the p y r r o lid in e
r in g o f n i c o t in e .

None o f th e r a d io a c t iv ity has been found In the

61

p y r id in e r in g o f n ic o t in e , and l e a s than two p ercen t o f the rad io­
a c t i v i t y o f th e n ic o tin e has been found in the m ethyl group,
5.

h y sin e-2 -C 3,4 hydrochloride when adm inistered to tobacco p la n ts

under th e same c o n d itio n s as were employed in th e o r n ith in e study g iv es
r i s e t o n ic o tin e p o s se ss in g con sid erab ly l e s s r a d io a c t iv it y .

I t has

been p o s tu la te d th a t ly s in e i s not incorporated d ir e c t ly in to th e
n ic o t in e m olecule b u t i s f i r s t converted t o some precursor o f p r o lin e
o r o r n ith in e sueh as glutam ic a c id .

M PM M SES

62

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67

APPMDIX
The form ula u sed in c o r r e c tin g the observed count to zero scrapie
th ic k n e ss west
0© » M
W «b
where

* maximum s p e c i f ic a c t i v i t y ( cou n ts/m in u te/feillim o le)
G© * observed count ( counts/m inute)
M * m olecular w eight o f compound
W » w eight o f sample counted
b

* f r a c t io n o f maximum a c t i v i t y a t th e sample th ic k n ess used
(7 ) —* obtain ed from s e lf-a b s o r p tio n cu rv e.

Sample c a lc u la tio n *
B ic o tin e d ip ic r a te — 0© * 23£#2 c #p jm .,

W » 67.3 rag.,

M « 620, T * 2 3 .8 m g./cm *, b * 0 .2 7 .
*

8 .0 3 x 10* c.p.m ./aJI