NMR and Macromolecules - American Chemical Society

15

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 22, 2015 | http://pubs.acs.org Publication Date: March 28, 1984 | doi: 10.1021/bk-1984-0247.ch015

Structural Characterization of Naturally Occurring cis-Polyisoprenes YASUYUKI TANAKA Department of Material Systems Engineering, Faculty of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184, Japan The chemical structure of naturally occurring cis polyisoprenes was determined by 13C NMR spectroscopy using acyclic terpenes and polyprenols as model compounds. The arrangement of the isoprene units along the polymer chain was estimated to be i n the order: dimethylallyl terminal unit, three trans units, a long block of cis units, and cis isoprenyl terminal unit. This result demonstrates that the biosynthesis of cis-polyisoprenes i n higher plants starts from trans,trans,trans-geranylgeranyl pyro phosphate. Polyisoprenes are synthesized by thousands of plant species c o v e r i n g most g e n e r i c f a m i l i e s . Usually, the polyisoprenes are rubbery and have p r e d o m i n a n t l y a cis-1,4 structure. Only a r e l a t i v e l y f e w p l a n t s p e c i e s p r o d u c e trans - 1 , 4 p o l y i s o p r e n e s . In e a r l y studies the presence o f the 3,4 structure i n natural rubber was e s t i m a t e d a t 1 - 2 % b y i n f r a r e d a n a l y s i s . T h i s c o n c l u s i o n was challenged after studies utilizing 1 H NMR s p e c t r o s c o p y , which r e p o r t e d t h a t n a t u r a l r u b b e r ( f r o m Hevea brasiliensis) and g u t t a percha a r e a t l e a s t 9 9 . 0 - 9 9 . 5 % cis-1,4 a n d trans-1,4 polyiso prenes . The 3 , 4 i s o m e r i c s t r u c t u r e was p r e s e n t i n l e s s t h a n t h e minimum d e t e c t a b l e amount ( 1 , 2 ) . Guayule rubber from Parthenium argentatum w a s f o u n d t o h a v e a s t r u c t u r e n e a r l y 1 0 0 % cis-1,4 and i d e n t i c a l t o t h a t o f n a t u r a l r u b b e r a s d e t e r m i n e d u s i n g 3 0 0 MHz 1 H NMR spectroscopy (3). The d i f f e r e n c e s i n physical properties between n a t u r a l r u b b e r a n d s y n t h e t i c cis-1,4 p o l y i s o p r e n e s have been a s c r i b e d i n p a r t to the s t r u c t u r a l p u r i t y o f the repeating units. S y n t h e t i c p o l y i s o p r e n e s prepared w i t h A l - T i c a t a l y s t s were f o u n d t o b e 99% cis-1,4, t h e r e m a i n d e r b e i n g 0 - 0 . 7 % trans-1,Λ and 0.3-1.0% 3,4. The p r e s e n c e o f b r a n c h e s and c r o s s - l i n k i n g h a s been considered to be another characteristic of natural rubber. H o w e v e r , l i t t l e s t r u c t u r a l i n f o r m a t i o n i s known a b o u t t h e t e r m i n a l u n i t s , b r a n c h i n g , and s o - c a l l e d abnormal groups i n n a t u r a l r u b b e r and how t h e y r e l a t e t o s p e c i f i c p h y s i c a l p r o p e r t i e s .

0097 6156/84/0247 0233S06.00/0 © 1984 A m e r i c a n C h e m i c a l S n e i e t v

In NMR and Macromolecules; Randall, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

NMR


234

AND MACROMOLECULES

The b i o s y n t h e s i s o f n a t u r a l r u b b e r h a s been s t u d i e d f r o m t h e viewpoint o f an e l u c i d a t i o n o f i n i t i a t i o n and propagation mechanisms m a i n l y by t r a c e r t e c h n i q u e s . The s t e p s i n the formation of isopentenyl pyrophosphate from acetyl-coA v i a m e v a l o n a t e a r e now w e l l e s t a b l i s h e d i n t h e in v i t r o s y n t h e s i s o f rubber. I t has a l s o been c o n f i r m e d t h a t c h a i n e x t e n s i o n o c c u r s on t h e s u r f a c e o f e x i s t i n g r u b b e r p a r t i c l e s by s u c c e s s i v e a d d i t i o n s o f i s o p e n t e n y l p y r o p h o s p h a t e t o b u i l d up c h a i n s o f 5000-7000 isoprene u n i t s (4,5). The i n i t i a t i o n s t e p o f r u b b e r f o r m a t i o n , however, r e m a i n s unknown due t o t h e l a c k o f d e t a i l e d i n f o r m a t i o n concerning the d i r e c t precursor of the chain extension. This paper d e s c r i b e s t h e s t r u c t u r a l a n a l y s i s o f n a t u r a l l y o c c u r r i n g c i s -1,4 polyisoprenes using 13C NMR spectroscopy. F i r s t , t h e s t r u c t u r a l c h a r a c t e r i z a t i o n o f p o l y p r e n o l s , which a r e l i n e a r i s o p r e n o i d compounds c o n t a i n i n g 30 t o 100 c a r b o n s , was c a r r i e d o u t on t h e b a s i s o f i n f o r m a t i o n o b t a i n e d f r o m a c y c l i c terpenes h a v i n g v a r i o u s c i s and trans i s o p r e n e u n i t s as model compounds. This method was a l s o a p p l i e d t o t h e s t r u c t u r a l analysis of polyisoprenes. The e l u c i d a t i o n o f t h e s t r u c t u r e o f t h e end g r o u p s and t h e a r r a n g e m e n t o f i s o p r e n e u n i t s p r o v i d e s i n f o r m a t i o n on t h e mechanism o f t h e b i o s y n t h e s i s o f p o l y p r e n y l compounds i n n a t u r e . 13C

NMR A n a l y s i s o f Model Compounds

By a n a l o g y w i t h t h e mechanism o f t h e b i o s y n t h e s i s o f f a r n e s y l p y r o p h o s p h a t e and all-trans t e r p e n o i d s , i t was deduced t h a t r u b b e r formation p r o c e e d s by t h e s u c c e s s i v e a d d i t i o n o f i s o p e n t e n y l pyrophosphate t o d i m e t h y l a l l y l pyrophosphate ( 4 ) . f ^C=CHCH 0PP + η CH =CCH CH 0ΡΡ Un ^ d d d d CH

3

3 N

»

o

CH CH

' 3

C=CHCH -(CH C=CHCH )-0ΡΡ d d d η

( I ) (0ΡΡ: p y r o p h o s p h a t e )

According to this mechanism, n a t u r a l r u b b e r c h a i n s a r e e x p e c t e d t o have one d i m e t h y l a l l y l t e r m i n a l u n i t and one i s o p r e n y l pyrophosphate t e r m i n a l u n i t ; t h e l a t t e r may g i v e r i s e t o a h y d r o x y l group by h y d r o l y s i s . From t h i s p o i n t o f v i e w , a c y c l i c terpenes i n t h e g e n e r a l i z e d s t r u c t u r e ( I I ) may be a p p r o p r i a t e models f o r t h e s t r u c t u r a l c h a r a c t e r i z a t i o n o f n a t u r a l p o l y i s o p r e n e s by 13C NMR s p e c t r o s c o p y . ψ3 f3 ^C=CHCH -(CH C=CHCH ) CH C=CHCH 0H Un d d d d d 3 n-2 ω-terminal internal a-terminal CH

n

n

n

o

(II)


15.

TANAKA

Naturally Occurring

Table I . Model compounds f o r 13C NMR assignment compounds ( 8 ) .


n=2 ω-Τ-ΟΗ (geraniol) ω-C-OH (nerol)

235

cis-Polyisoprenes

of p o l y i s o p r e n o i d

n=3

n=4

ω-Τ-Τ-ΟΗ (farnesol) ω-1-C-OH ω-C-T-OH ω-C-C-OH

ω-Τ-Τ-Τ-ΟΗ (geranylgeraniol) ω-C-T-T-OH ω-Τ-e-T-OH ω-C-C-T-OH ω-Τ-Τ-C-OH ω-C-T-C-OH ω-Τ-C-C-OH ω-C-C-C-OH

The a b b r e v i a t i o n ω, τ, and C correspond to ω-terminal» trans, cis u n i t s , r e s p e c t i v e l y .

and

Geraniol and n e r o l (n=2), farnesol isomers (n=3), and g e r a n y l g e r a n i o l isomers (n=4) having various combinations of trans and cis isoprene u n i t s were used as model compounds, as l i s t e d i n Table I . Here, the geometric isomers of f a r n e s o l were i s o l a t e d from s y n t h e t i c f a r n e s o l , which i s a mixture of four isomers, by l i q u i d chromatography (6) . In a s i m i l a r way, the g e r a n y l g e r a n i o l isomers were separated from a mixture prepared by the isomeriza tion of n a t u r a l l y o c c u r r i n g trans,trans,trans-geranylgeraniol under UV i r r a d i a t i o n ( 7). The a l i p h a t i c carbon s i g n a l s , observed at 50.1 MHz i n these compounds, were assigned through a c o n s i d e r a t i o n of chemical s h i f t c o r r e l a t i o n s among these compounds as well as by the usual 13C NMR techniques (8) . In these compounds, the methyl carbon atoms i n the i n t e r n a l trans and cis u n i t s resonated at 16.0 and 23.4 ppm, r e s p e c t i v e l y , while those i n the ω-terminal u n i t resonated at 17.6 and 25.6 ppm. The C - l methylene carbon atoms i n the cis and trans units showed s i g n a l s p l i t t i n g s r e f l e c t i n g the geometric isomerism of the u n i t l i n k e d to the C - l methylene carbon atom, that i s , r e f l e c t i n g the dyad sequences of cis and trans u n i t s . Here, the carbon atoms are designated as f o l l o w s : 5 C -C-C=C-C12 3 4 The C - l methylene carbon atoms i n trans u n i t s resonated at 39 .6 ppm (trans-trans( α ) ) , 39.7-39.8 ppm ( ω - t r a n s and trans trans), 39.9 ppm (cis-trans(a)) and 40.0 ppm (cis-trans), while those i n cis u n i t s resonated at 32.0-32.1 ppm (trans-cis , ω-cis, and trans-cis( a ) ) and 32.3-32.4 ppm (cis-cis and cis-cis(a)). In f


NMR AND MACROMOLECULES


236

these c o r r e l a t i o n s i t was observed that the ω-terminal u n i t has the same s h i e l d i n g e f f e c t on the subsequent C-l methylene carbon atom as the i n t e r n a l trans unit. The C-4 methylene carbons i n the trans and cis α-terminal u n i t s gave s i g n a l s a t 59.4 and 59.1 ppm, r e s p e c t i v e l y . The geometric isomerism of the u n i t l i n k e d to the ω-terminal u n i t could be determined by the observed chemical s h i f t o f the ω C-2 carbon atom; the ω - t r a n s linkage showed about 0.2-0.4 ppm u p f i e l d s h i f t compared with that i n the ω-cis linkage. The assignment o f the s i g n a l s c h a r a c t e r i s t i c o f the terminal u n i t s and the alignment of isoprene u n i t s i n the geranylgeraniol isomers are l i s t e d i n Table I I . S t ru c t u r a l C har ac t e r i ζ a t i on o f Ρolypggnols Polyisoprenoid alcohols c o n s i s t i n g o f 9 to 20 isoprene u n i t s have a widespread occurrence as i n d i c a t e d by t h e i r presence i n the leaves of higher p l a n t s , mammalian t i s s u e s , and microorganisms. Most of the polyprenols i s o l a t e d from higher plants c o n s i s t o f trans and cis isoprene u n i t s with the exception o f s o l a n e s o l , which i s composed o f all-trans isoprene u n i t s . The arrangement o f trans and cis u n i t s i n these polyprenols has been determined from a consideration o f the mechanism o f the formation o f b e t u l a prenols, C(30)-C(45), isolated from wood tissue of Betula verrucosa (9) . However, up to the present there has been no d i r e c t evidence to prove the l o c a t i o n o f the i n t e r n a l trans and cis units. The arrangement o f the trans and cis units i n typical polyprenols was determined according to information obtained from the 13C NMR study o f a c y c l i c terpenes mentioned above. Polyprenol-11 C(55), i s o l a t e d from the leaves of Ficus elastica, was found to contain the ω-terminal u n i t , three i n t e r n a l trans units, six i n t e r n a l cis u n i t s , and a cis α-terminal u n i t from 1H NMR observations. Polyprenol-12 C(60), i s o l a t e d from Ficus elastica and silkworm feces, showed a s i m i l a r composition except that there were seven i n t e r n a l cis u n i t s . As shown i n F i g . 1, the s i g n a l due to the C-l methylene carbon atom i n cis u n i t s i s s p l i t i n t o two peaks a t 32.37 and 32.12 ppm, which were assigned to the cis u n i t s i n the cis-cis (cis-cis{a))

and trans-cis

(ω-cis)

linkages, respectively.

On the

other hand, the C-l methylene carbon atom i n trans u n i t s showed a s i n g l e peak a t 39.86 ppm, corresponding to the trans u n i t i n the trans-trans

( ω -trans

)

linkage.

The

presence

o f the

^-trans

linkage was confirmed by the c h a r a c t e r i s t i c C-2 o l e f i n i c carbon s i g n a l o f the ω-terminal u n i t a t 131.20 ppm. The CH OH s i g n a l at 59.00 ppm i n d i c a t e d that the α-terminal u n i t has the cis configuration. The observed i n t e n s i t y r a t i o of the trans-trans (ω-trans)

0.9

j

trans-cis,

and cis -cis

(cis-eis(ct))

signals

was 2.8 :

: 6.3 f o r polyprenol-11 and 2.7 : 1.2 : 7.1 f o r polyprenol-12.



131.47

131.51

131.49

131.55

131 .22

131 .30

131,.24

131,.29

ωΤ

59.47 59.43 59.39 59.43

T

59.08 59.06 59.06 59.08

C

2

α C-4 (-CH 0H)

40,.01

40,,01

CT a

39,.72 39,.76

39,.78

TT ωΤ

TT a

39,.58 39 .76 39,.60

2

C - l (-CH ~)

39,.89 39 .88 39,.78

CT

trans

2

CC CC a

TC TC

a

C - l (~CH ~)

units,

32,.34 32.03 32.01 32.03

32..30 32.04 32..29 31.97 32.03 32.07

cis

isomers ( 8 ) .

*The a b b r e v i a t i o n s ω, Τ, and C correspond to ω-terminal, trans, and cis respectively. The s u b s c r i p t α means the u n i t with hydroxyl group.

CCT-OH TCT-OH CTT-OH TTT-OH

CCC-OH TCC-OH CTC-OH TTC-OH

coC

ω C-2 (=CH-)

T a b l e I I . C h e m i c a l s h i f t s o f 13C NMR s i g n a l s i n g e r a n y l g e r a n i o l


NMR A N DMACROMOLECULES

238 These s i g n a l s values. On t h e polyprenols-11 i n t e r n a l trans units aligned

showed T^ v a l u e o f l e s s t h a n 1 s and n e a r l y f u l l NOE b a s i s o f these findings, i t i s concluded that and -12 a r e composed o f t h e ω-terminal, t h r e e , s i x o r s e v e n i n t e r n a l c i s , and c i s α-terminal i n t h a t o r d e r a s shown b e l o w :

H H C H C H H C H ι 3ι 3j ι 3ι ι /C=CCH -(CH C=ÇCH_)-(CH C=CCH )-CH C=CCH OH (III) CH 2 2 ^ 2 2 ,7 2 ω-terminal trans cis c i s α-terminal


r H

CH

n u

0

0

0

2

3

3

2

6

T h i s a l i g n m e n t o f t h e trans and c i s u n i t s c l e a r l y i n d i c a t e s t h a t t h e s e p o l y p r e n o l s a r e s y n t h e s i z e d in vivo by t h e c i s a d d i t i o n o f isopentenyl pyrophosphate to trans,trans,tpans-geranylgeranyl pyrophosphate ( 1 0 ) . S i m i l a r l y , p o l y p r e n o l s - 1 5 t o -20, i s o l a t e d from t h e l e a v e s o f Ginkgo v i l o b a , showed t h e same s p l i t t i n g o f C - l m e t h y l e n e c a r b o n s i g n a l s , as shown i n F i g . 2. From t h e i n t e n s i t y r a t i o s o f t h e s e s i g n a l s i t was f o u n d t h a t t h e s e p o l y p r e n o l s c o n t a i n e d two i n t e r n a l trans and 11 t o 16 i n t e r n a l c i s u n i t s a l i g n e d as g i v e n i n ( I V ) (11). H H C H C H CH I Sj 3,| C=CCH_-(CH C=CCH_)-(CH_C=CCH )CH 2 2 2 2 ^ 2 _ n u

y

Q

3

31

Q

à

ω-terminal

H C H

2

n

trans

cis

1

-CH_C=CCH 0H ^ 2 2 Q

1

6

(IV)

cis a-terminal

The p o l y p r e n o l s composed o f trans and c i s i s o p r e n e u n i t s isolated so f a r have been classified into two t y p e s : t h e f i c a p r e n o l t y p e and t h e b e t u l a p r e n o l - t y p e , c o n t a i n i n g t h r e e and two i n t e r n a l trans u n i t s , r e s p e c t i v e l y ( 1 2 ) . The f i n d i n g s d e s c r i b e d above s u g g e s t t h a t trans trans trans-geranylgerany1 pyrophosphate o r t r a n s , t r a n s - f a r n e s y l p y r o p h o s p h a t e a c t s as an i n i t i a t o r o f t h e biosynthesis of polyprenols. f

t

Structure of Naturally Occurring

Cig-Polyisoprenes

The c h e m i c a l shifts o f the c h a r a c t e r i s t i c carbon s i g n a l s i n acyclic t e r p e n e s , p o l y p r e n o l s , and c i s - t r a n s i s o m e r i z e d poly i s o p r e n e s a r e p l o t t e d i n F i g . 3. H e r e , t h e c h e m i c a l s h i f t s a r e c o r r e l a t e d u s i n g t h e ω C-5 m e t h y l c a r b o n s i g n a l a t 17.66 ppm as an i n t e r n a l standard (except f o r isomerized polyisoprenes) i n order t o compensate f o r t h e e f f e c t o f s o l u t i o n c o n c e n t r a t i o n . It is c l e a r that these chemical s h i f t s a r e independent o f the c h a i n l e n g t h o f t h e compounds and c a n be u s e d f o r t h e d e t e r m i n a t i o n o f t h e a r r a n g e m e n t o f i s o p r e n e u n i t s as w e l l as t h e t e r m i n a l u n i t s i n v a r i o u s i s o p r e n o i d compounds ( 8 ) . Cis-polyisoprene isolated from the leaves o f goldenrod (Solidago altissima) was s e p a r a t e d i n t o two f r a c t i o n s by GPC, S - l (Mn=76,000) and S-2 (Mn=120,000). The 13C NMR s p e c t r a o f b o t h


TANAKA

Naturally Occurring cis- Polyisoprenes

cis-cis ω -trans


trans-trans

trans-cis

40.0

32

39.5

ppm(TMS)

F i g u r e 1. C - l m e t h y l e n e c a r b o n s i g n a l s i n p o l y p r e n o l - 1 1 f r o m Ficus e l a s t i c a . Reproduced w i t h p e r m i s s i o n from R e f . 1 0 . C o p y r i g h t 1979, The B i o c h e m i c a l S o c i e t y .

cis-cis

trans-trans ω -trans

40

39

trans-cls

33

32

ppm(TMS)

F i g u r e 2. C - l metylene carbon s i g n a l s i n p o l y p r e n o l - 1 8 f r o m Ginkgo viloba. Reproduced w i t h p e r m i s s i o n from R e f . 1 1 . C o p y r i g h t 1 9 8 3 , The B i o c h e m i c a l S o c i e t y .



240


f r a c t i o n s showed f i v e s i g n a l s corresponding to the carbon atoms i n the cis isoprene u n i t . In the expanded spectrum of S - l , obtained by 34,730 accumulations, small s i g n a l s were observed, as shown i n F i g . 4. The s i g n a l at 59.00 ppm was assigned to the CH^OH carbon atom i n the cis α-terminal u n i t from the r e l a t i o n s h i p shown i n F i g . 3. S i m i l a r l y , the s i g n a l s at 17.67 and 16.00 ppm were ascribed to the C-5 methyl carbon atoms i n the ω -terminal and i n t e r n a l trans u n i t s , r e s p e c t i v e l y . The C - l methyl carbon s i g n a l due to the i n t e r n a l trans u n i t s was observed at 39.74 ppm, which i s c h a r a c t e r i s t i c of the trans u n i t s i n the trans-trans and ω-trans linkage. The C-2 o l e f i n i c carbon atom i n the ω-terminal u n i t resonated at 131.14 ppm, showing the presence of the ω-trans linkage. The absence of the s i g n a l c h a r a c t e r i s t i c of the cis-trans linkage, which i s presumed to resonate around 39.9 ppm, i n d i c a t e s that a l l the trans u n i t s are s i t u a t e d f o l l o w i n g the ω-terminal u n i t . Although the C - l methylene carbon s i g n a l due to the trans-cis linkage was not observed because of overlap with the strong s i g n a l from the cis-cis linkage, the f i n d i n g s shown above s t r o n g l y support the idea that a block of cis u n i t s i s l i n k e d to the ω-(trans) - sequence. S i m i l a r l y , small s i g n a l s due to the ω-terminal, czs α-terminal, and i n t e r n a l trans u n i t s were observed i n the spectrum of the sample S-2. The r e l a t i v e i n t e n s i t i e s of these s i g n a l s , together with the NOE and T^ values, are l i s t e d i n Table I I I . The i n t e n s i t y r a t i o from the s i g n a l s of the ω-terminal u n i t ( ω C-5) and α -terminal u n i t (α C-4) was 0.8-0.9 f o r both samples. The i n t e n s i t y r a t i o of the corresponding s i g n a l s i n polyprenol-11 was 0.84, i n which case the d i f f e r e n c e i s ascribed to a T^ of 7.5 versus 1.9 s f o r the ω C-5 and α C-4 carbon atom, r e s p e c t i v e l y , as well as to the NOE values. Therefore, i t seems resonable to assume that polymers S - l and S-2 contain the same amount of ω - and α-terminal u n i t s . The number of i n t e r n a l trans u n i t s determined from the i n t e n s i t y r a t i o of the trans C - l and α C-4 methylene carbon s i g n a l s was 3.4 and 3.6 f o r S - l and S-2, r e s p e c t i v e l y . On the other hand, 2.5 trans u n i t s were estimated f o r both samples from the i n t e n s i t y r a t i o of the trans C-5 and α C-4 carbon s i g n a l s . The former i s considered to be a more r e l i a b l e value i n view of the f a c t that the C-5 carbon atom shows smaller NOE and longer Τ values than the trans C-l methylene carbon atom i n polyprenols-11 and -12. This i n d i c a t e s that the polymers contain approximately three to four i n t e r n a l trans units. The number of the i n t e r n a l cis u n i t s determined from the i n t e n s i t y r a t i o of e i t h e r the cis C - l or C-4 methylene s i g n a l to the α C-4 methylene carbon s i g n a l , was approximately 1000 and 2200 f o r the polymers S - l and S-2, respectively. S i m i l a r NOE and shorter T^ values f o r these carbon atoms i n d i c a t e that an almost quantitative evaluation i s p o s s i b l e f o r the i n t e n s i t y r a t i o s between these methylene carbon s i g n a l s . The degree of polymeri z a t i o n determined from the α-terminal u n i t agrees with that from GPC f o r these samples, i . e . , 1100 f o r S - l and 1800 f o r S-2. This



TANAKA

Isoprene

ω C-2

Units

α C-4

C-5

C-1

u>C u?T

2


3

4

1

9

1

11 12 17 18 Polymer 132

1 3 1 60

59 40

39

32

26

25 24

2 3 18

ppm(TMS)

Figure 3. Chemical s h i f t s of the s i g n a l s c h a r a c t e r i s t i c of the arrangement of the isoprene u n i t s (8, 13).

cis

CIS -CH

-CH

2

cis 2

-CH

3

-CH

3

cis α -CH

-CH OH 2

3

Χ: X 8 , Y : X 2 5 6

LJJLX 60

50

40

30

X 2 , Y: X 1 2 8

20

ppm(TMS)

Figure 4. A l i p h a t i c carbon s i g n a l s i n c i s - p o l y i s o p r e n e from Solidago altissima observed with a pulse r e p e t i t i o n time of 7 s f o r 45° pulse (^denotes s i g n a l s due to i m p u r i t i e s ) . Reproduced with permission from Ref. 13. Copyright 1983, The American Chemical S o c i e t y .


242


Table I I I . R e l a t i v e i n t e n s i t i e s o f the a l i p h a t i c carbon s i g n a l s (13) . ( * V a l u e s d e t e r m i n e d f o r p o l y p r e n o l - 1 1 as a model compound) Chemical

shift

A s s i |inment


S-2

S-l α trans cis cis cis ω trans

59.00 39.74 32.23 26.43 23.41 17.67 16.00

C-4 C-l C-l C-4 C-5 C-5 C-5

NOE

Relative intensity

(ppm)

2.62* 2.62* 2.71 2.78 2.49 2.22* 2.22*

1 3.6 2200 2200 2100 0.9 2.5

1 3.4 1000 1000 980 0.8 2.5

T

i (s) 1.9* 0.9* 0.7 0.8 1.7 7.5* 3.9*

i s important evidence t h a t these polymers a r e l i n e a r p o l y i s o p r e n e s h a v i n g ω- and α-terminal u n i t s . These facts clearly indicate that the cis-polyisoprene i s o l a t e d f r o m g o l d e n r o d h a s t h e a l i g n m e n t o f i s o p r e n e u n i t s as shown i n (V) ( 1 3 ) . H H C H C H H C H j 3ι 3ι ι 31 ι /C=CCH -(CH C=ÇCH )-(CH C=CCH )-CH C=CCH 0H 3 H m η ω-terminal trans cis cis a - t e r m i n a l CH C

H

2

2

2

2

2

2

2

(V)

(m=3-4 and n=1000-2200) A similar alignment o f i s o p r e n e u n i t s was o b s e r v e d f o r c i s - p o l y i s o p r e n e i s o l a t e d f r o m t h e l a t e x o f Ficus elastica. This is significant evidence clarifying t h e d e t a i l e d mechanism o f rubber formation i n higher p l a n t s . The p r e s e n c e o f t h e s e q u e n c e c o n s i s t i n g o f t h r e e t o f o u r trans u n i t s l i n k e d t o t h e ω-terminal u n i t demonstrates t h a t the primer o f c i s - p o l y i s o p r e n e i s a p r e n y l p y r o p h o s p h a t e p o s s e s s i n g an a l l - t r a n s c o n f i g u r a t i o n . Geranyl g e r a n i o l i s one o f t h e most common p r e n y l compounds i n n a t u r e , whereas g e r a n y l f a r n e s o l ( C ( 2 5 ) trans,trans,trans,trans) occurs only r a r e l y . Therefore, i t c a n be presumed t h a t t h e c h a i n e x t e n s i o n t o t h e cis p o l y m e r o c c u r s by t h e a d d i t i o n o f i s o p e n t e n y l p y r o - p h o s p h a t e t o all-trans g e r a n y l g e r a n y l p h r o p h o s p h a t e , a s shown i n F i g . 5. I t i s w o r t h n o t i n g t h a t t h i s s t r u c t u r e i s i d e n t i c a l t o t h a t o f f i c a p r e n o l - t y p e p o l y p r e n o l s i s o l a t e d mainly from t h e l e a f t i s s u e s o f a n g i o s p e r m s , e x c e p t f o r t h e number o f cis u n i t s . On the other hand, the low molecular weight fraction (Mn=10,000) o f n a t u r a l r u b b e r , w h i c h was o b t a i n e d by f r a c t i o n a t i o n o f Hevea r u b b e r purified by d e p r o t e i n i z a t i o n o f a c o m m e r c i a l l a t e x , showed s m a l l s i g n a l s c h a r a c t e r i s t i c o f t h e C - l m e t h y l e n e and C-5 m e t h y l c a r b o n atoms i n i n t e r n a l trans u n i t s a t 39.80 ppm and 16.02 ppm, r e s p e c t i v e l y , as shown i n F i g . 6. The s i g n a l s due t o t h e t e r m i n a l u n i t s were n o t d e t e c t e d a t 59.0 ppm and 17.7 ppm,


TAN A Κ A

243


IPP


O-PP

c i s a d d . xn

trans add. x 3

DMAPP ι

ψ

/n^/^o-PP

*'

V

\v V

OPP

40-PP

3

ri-1|

trans

ω-end

cis

cis-a-end

n=1000 or 2200 F i g u r e 5. (13, 1 4 ) .

B i o s y n t h e s i s mechanism o f c i s - p o l y i s o p r e n e s

cis

cis

-CH

CH ι "k

C

2

cis

-CH -CH 2

3

3 3

W

C

H

bΟa

60

"

*

trans

trans-tran»

CH2

I " i 50

40

-CH

y 30

20

3

10

ppm(TMS)

F i g u r e 6. A l i p h a t i c c a r b o n s i g n a l s i n c i s - p o l y i s o p r e n e f r o m Hevea brasiliensis ( n a t u r a l r u b b e r ) (^denotes s i g n a l s due t o i m p u r i t i e s ) ( 1 4 ) .


N M R A N D MACROMOLECULES


244

whereas the s i g n a l s from epoxide group were observed a t 64.51 and 60.76 ppm. The trans C - l methylene carbon s i g n a l showed the chemical s h i f t c h a r a c t e r i s t i c of the trans -trans and ω-trans linkages and had a r e l a t i v e i n t e n s i t y of approximately 1/500 compared to the c i s C - l methylene carbon s i g n a l . Taking into account the degree o f p o l y m e r i z a t i o n of the sample, one expects about three trans u n i t s per molecule to occur as an i s o l a t e d sequence (14). These f a c t s suggest that natural rubber i s synthesized in vivo i n a s i m i l a r manner to goldenrod rubber and that the terminal units subsequently undergo some p a r t i c u l a r r e a c t i o n to form f u n c t i o n a l groups. One may speculate that the r e a c t i v e f u n c t i o n a l groups are r e s p o n s i b l e f o r the formation of branches and c r o s s - l i n k s , which are believed to occur i n s i g n i f i c a n t amounts i n n a t u r a l rubber. Very r e c e n t l y , Archer et al. found that a l l - t r a n s geranylgeranyl pyrophosphate can a c t as an i n i t i a t o r to synthesize new rubber molecules (15). This r e s u l t s t r o n g l y supports the suggested mechanism f o r the formation of n a t u r a l rubber as described above.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Golub, Μ. Α.; Fuqua, F. Α.; Bhacca, Ν. S. J . Am. Chem. Soc. 1962, 84, 498 (1962). Chen, H. Y. J . Polym. Sci. 1966, B4, 891. Campos-Lopez, E . ; Palacios, J . J . Polym. Sci. Polym. Lett. Ed. 1976, 14, 1561. Lynen, F . ; Henning, U. Angew Chem. 1960, 72, 820. Archer, B. L . ; Ayrey, G.; Cockbain, Ε. G.; McSweeney, G. P. Nature (London) 1961, 189, 663. Sato, H; Kageyu, A.; Miyashita, Κ.; Tanaka, Y. J. Chromatogr. 1982, 237, 178. Tanaka, Y. ; Sato, H.; Kageyu, A. Polym. Prep. Japan 1981, 30, 1834. Tanaka, Y.; Sato, H.; Kageyu, A. Polymer 1982, 23, 1087. Wellburn, A. R.; Hemming, F. W. Nature (London) 1966, 212, 1634. Tanaka, Y.; and Takagi, M. Biochem. J. 1979, 183, 163. Ibata, Κ.; Mizuno, M.; Takigawa, T.; Tanaka, Y. Biochem. J. 1983, 213, 305. Hemming, F. W. "Biochemistry of Lipids (Biochemistry Series 1)"; Goodwin, T. W., Ed; Butterworth: London, 1974; Vol. 4, p. 39-98. Tanaka, Y.; Sato, H.; Kageyu, A. Rubber Chem. Technol. 1983, 56, 299. Tanaka, Y. Polym. Prep. Japan 1983, 32, 75. Archer, Β. L., personal communication.

RECEIVED

November 3, 1983


NMR and Macromolecules - American Chemical Society

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