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P e r g a m o n 0 1 4 6 - 6 3 8 0 ( 9 4 ) 0 0 0 7 8 - 6Org. Geochera. Vo l. 22, No. 6, pp. 991-1004, 1994Cop yright 1994 ElsevierScience LtdPrinted in Great Britain. All rights reserved0146-6380/94 $7.00 + 0.00

Extended sa turated and m onoarom at ic t r i c yc l i c t erpeno id c arboxy l i ca c i d s f o u n d i n T a s m a n i a n t a s m a n i t e

D. A . AZE VEDO, F . R . AQUINO NE T O and B . R . T . S I M O N E I T 2 .Institu to de Quimica, Centro de Tccnologia, Bloco A, Sala 607, Universidade Fed eral do R io de Janeiro,Ilba do Fund~.o, Rio de Janeiro, 21949-900, Brazil and 2petroleum and Environmental Geochem istryGr ou p, College of Oceanic and A tmospheric Sciences, Oregon S tate University, Corvallis , OR 97331,U.S.A.

( R e c e i v e d 2 3 N o v e m b e r 1 9 9 3; r e t u r n e d f o r r e vi si on 2 5 J a n u a r y 1 9 94 ; a c c e p t e d 1 J u n e 1 9 9 4)A bs tra ct- -A series of tricyclic terpenoid carboxylic acids (C20--C40) was fo und in the acidic fraction ofTasmanian tasmanite bitumen, occurring as a mixture of stereoisomers with mainly the 13~(H),14a(H)-and 13~(H),14a(H)-configurations. These dominant acidic tricyclic consituents have the same carbonskeleton as the ubiquitous tricyclic terpan e biomarkers. A no vel series of ring-C m ono arom atic tricyclicterpenoid carboxylic acids was also characterized. The series ranges from Ct9 to C39 and is the acidiccounterpart of the recently described series of monoaromatic tricyclic terpanes.K e y w o r d s - - t r i c y c l i c terpenoid carboxylic acids, aro ma tic tricyclic terpenoid carboxylic acids, tasmanite,b iomarke rs

INTRODUCTION

T a s m a n i a n t a s m a n i t e ( P e r m i a n , 2 2 0 - 2 7 5 x 1 0 6 yr ) i sc o m p o s e d o f c o m p r e s s e d d i s k s o f t a s m a n i t e s o r t a s -m a n i t i d s ( N e w t o n , 1 8 75 ). T h e s e u n i c e l l u l a r o r g a n -i s m s a r e t h o u g h t t o b e g r e e n a lg a e ( z o o s p o r a n g i a o fP r a s i n o p h y c e a n a l g a e , T a p p a n , 1 9 80 ; S a r j e a n t , 1 9 89 ).T h e t a s m a n i t i d s a r e u n i q u e i n t h a t t h e y a r e d e r i v e df r o m a s i n g l e s p e c i e s a n d h e n c e y i e l d s i m p l e b i o -m a r k e r m i x t u r e s th a t c a n b e r e l a t e d t o t h e p r e c u r s o ro r g a n i s m s ( B u r l i n g a m e e t a l . , 1 9 6 9 ; S i m o n e i t a n dB u r l i n g a m e , 1 9 7 3 ; S i m o n e i t e t a l . , 1990, 1993).

T h e T a s m a n i a n t a s m a n i t e i s o f p a r t i c u l a r i n t e r e s tb e c a u s e i t s b i t u m e n i s c o m p o s e d m a i n l y o f c o m -p o u n d s w i t h t h e s a m e t r ic y c l ic t e r p e n o i d s k e l e t o n i na l l f r a c t io n s s t u d i e d ( s a tu r a t e d , u n s a t u r a t e d a n d a r o -m a t i c h y d r o c a r b o n s ) . T h e s a t u r a t e d p lu s m o n o u n s a t -u r a t e d h y d r o c a r b o n f r a c t io n c o m p r i s e s p r i m a r i l y t h eu b i q u i t o u s e x t e n d e d t r ic y c l ic te r p a n e s e ri e s (A q u i n oN e t o e t a l . , 1 9 9 2 ; S i m o n e i t e t a l . , 1990) and two l e s sab un da nt series o f A 9{11) an d A ~3t~4}-unsaturated t r i -c y c l i c t e r p e n e s ( A z e v e d o e t a l . , 1991, 1994). Thea r o m a t i c h y d r o c a r b o n f r a c t i o n i s c h a r a c t e r i z e d b y as e ri e s o f e x t e n d e d r i n g - C m o n o a r o m a t i c t r ic y c l ict e r p a n e s ( A z e v e d o e t a l . , 1990, 1992).

T r i c y c li c t e r p a n e h y d r o c a r b o n s h a v e b e e n s t u d i e de x t e n si v e l y a n d a r e f o u n d i n m a n y p e t r o l e u m s a n ds e d im e n t s . O n e o f t h e i r p o t e n t i a l g e o c h e m i c a l p r e c u r -s o r s c o u l d b e t h e c o r r e s p o n d i n g t r i c y c li c c a r b o x y l i ca c i d s . T h e r e a r e f e w c i t a t i o n s o f s a t u r a t e d t r i c y c l i cc a r b o x y l i c a c id s i n g e o l o g i c al s a m p l e s a n d n o n e o f*Author for correspondence

t h e i r a r o m a t i c o r u n s a t u r a t e d a n a l o g s . T h e y w e r e f ir s tr e p o r t e d b y S i m o n e i t a n d B u r l i n g a m e ( 1 9 7 3 ) i n t h ec h e m i c a l o x i d a t i o n p r o d u c t s o f t a s m a n i t e k e r o g e n .T h i s w a s f o l l o w e d b y S e i f e r t ( 19 7 5 ) w i t h t h e i d e n t i f i -c a t i o n o f C 2 ~ - a n d C 2 4 - tr ic y c li c t e r p e n o i d c a r b o x y l i ca c i d s i n a C a l i f o r n i a p e t r o l e u m , b a s e d o n t h e i r r e -d u c t i o n t o t h e c o r r e s p o n d i n g h y d r o c a r b o n s a n da n a l y s i s o f t h e m a s s s p e c t r a . H o w e v e r , t h e s e s t r u c -t u r e s w e r e n o t f u l ly c h a r a c te r i z e d . T h i s w a s d o n e b yC y r a n d S t r a u s z (1 9 83 , 1 9 84 ). T h e y d e t e r m i n e d t h es t r u c t u r e s o f t h e c a r b o x y l i c a c i d s f r o m C2~ t o C 26 b yt h e i r t r a n s f o r m a t i o n i n t o t h e c o r r e s p o n d i n g h y d r o -c a r b o n s a n d c o m p a r i s o n w i t h t h e n a t u r a l t e r p a n e s ,i s o l a t e d f r o m A l b e r t a o i l s a n d d e p o s i t s . T h e h y d r o -c a r b o n o b t a i n e d f r o m t h e m e t h y l e s t e r o f t h e C 2 0c a r b o x y l i c ac i d , h o w e v e r , d i d n o t c o r r e s p o n d t o t h et r ic y c l ic t e r p a n e , f u r t h e r m o r e , t h e n a t u r a l e s t e r d i dn o t m a t c h a n y o f t h e f o u r p o s s i b l e i s o m e r s a t C - 1 3a n d C - 1 4 . O n t h e b a s i s o f t h i s e v i d e n c e a n d t h ei n f o r m a t i o n o b t a i n e d f r o m t h e m a s s s p e c t r a l f r a g -m e n t a t i o n p a t t e r n o f t h e C z0 e s t e r a n d t h e c o r r e -s p o n d i n g a l k a n e , a n e w s t r u c t u r e w a s p r o p o s e d ,w h i c h w a s c o n f i r m e d r e c e n t l y b y s y n t h e s i s o f t h es t a n d a r d s ( K a u f m a n e t a l . , 1988).

W e n o w r e p o r t t h e i d e n t i f i c a t io n o f a f a m i l y o fe x t e n d e d t r ic y c l ic t e r p e n o i d c a r b o x y l i c a c i d s f o u n d i nt h e a c id i c f r a c t io n o f t h e b i t u m e n f r o m T a s m a n i a nt a s m a n i t e . A l es s a b u n d a n t s e ri e s o f e x t e n d e d r i n g - Cm o n o a r o m a t i c t r ic y c li c t e r p e n o i d c a r b o x y l i c ac i d s i sa l s o r e p o r t e d . T h e y a r e r e l a t e d t o t h e a l r e a d y k n o w nt r i c y c l ic t e r p a n e h y d r o c a r b o n s e r ie s , a s w e l l a s t o t h er e c e n t l y d i s c o v e r e d r i n g - C m o n o a r o m a t i c t r i c y c l i ct e r p a n e s .

991

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992 D.A. AZEVEDO / al.

I ~ t ( 8 ) I6 0 gi ( 0 H 2 C l2 C H o O H )

a n a m , , n ( a ) II 7 4 & 3 ,m g ( S : 1 . 2 4 % ) I

,I 7 0 . 1 m g II ( s : o . 8 6 % ) 1I ~ B : , . , % I . I

A l i q u o t I101 , 4 r ag ,I ( M c C a r t h y D u t h ie C h r o m . )

IA c i d s ( / I t )2 1 . 5 r n g( S : 0 . 26%)( B : 2 1 . 2 % )

IH o n o l r b o x y l l c A c i d s( a s m e t h y l e s t e r s ) i3 , 4 m g( S 0 . 0 4 2 % )( B 3 . 3 % )( A t 5 . 8 % )

Io t n ~ A a =( a s m e t h y l e s t e r s )1 7 . 0 m g( S 0 . 2 1 % )( B t 6 . 8 % )( A 7 9 . 1 % )Fig. 1. Yields of various fractions from sediment andbitumen of Tasmanian tasmanite.

E X P E R I M E N T A L

The Tasmanian tasmanite (Permian) was collectedby B.R.T.S. and T. C. Hoering in an aban doned shalemine on the Mersey River, 3 miles south of Latrobe,Tasmania (latitude 4117'S, longitude 14627'E andelevation 35 m). A sample (60 g, Fig. 1), was washedwith dichloromethane, powdered in an agate mortar,and extracted four times with dichloromethane/methano l (9:1) in an ultrasonic bath. The solvent wasremoved under vacuum on a rotary evaporator andan aliquot of the bitumen (101.4mg, Fig. 1) wassubjected to McCarthy and Duthie chromatographyon a SiO2/KOH column with elution of the acids bya solution of 10% formic acid in diethyl ether, afterremoval of the neutrals with diethyl ether (Loureiroe t a l . , 1986; McCarthy and Duthie, 1962). The acidfraction was concentrated and treated with dia-zomethane in diethyl ether to esterify the acids. Themonocarboxylic acid methyl esters were purified bysilica gel (Merck) thin-layer chromatography (TLC)using hexane/dichloromethane (7:3) as eluent andmethyl abietate as elution standard. The band corre-sponding to the methyl esters was scraped off theTLC plate after visualization under UV-light(254 nm) and eluted with diethyl ether. The yields ofthe various fractions are shown in Fig. 1.

The monocarboxyl ic acid methyl esters were ana-lyzed by high resolution gas chromatography(HRGC) on a glass capillary column (SE-54,25 m 0.30 mm, dr = 0.25/~m) using hydrogen ascarrier gas and temperature programming from80-30@C at 10/min. The same chromatographic

conditi ons were used for the HRGC- MS- C analysison a Hewlett-Packard Model 5987A instrument, withlinear s canning (50-500 Da, 1.87 s/decade), electronimpact ionization (70eV) and also SIM analysis(single ion monitoring) for detection of the highermembers of the series (C30-C~6) (Condi tion A). TheSIM analysis was also done by high-temperature-HRGC on an Ultra 2 fused silica column (Hewlett-Packard- -5.5 m x 0.20 mm, df = 0.33/~m) and tem-perature programming from 90-370C at 20C/min(Condition B) for better visualization of the highermembers of the series (C36-C40).

L UZC O==

n C [ 2 n C i 6i I I ~ i nC e ZT T I Cl l F E l ; l i c . ,

n 2a . c . n C , . io c ~ m / z 7 4, J j ~ c 8l l t i } d J~ . . , ( J , i ~ . .

C i C ~ 4 Ci~ m / z 8 8, c , 5

LD [ c J 5 iC z m / z I 0 1

h i , - - i , i i , t1

C I~ T i , j .~ 6 rE ~ ~ m /z 191C ~ 4 T

, r I , i , r , i , i , r , [ i , r ' i , J, I

C,gA m / z 1 4 5 + 2 0 5 +F , IC2~I , C ~ 6

4 6 8 I 0 ~ e 1 4 ~ 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0l (min)

Fig. 2. (A) Total ion current (TIC) trace of the acid fraction(as methyl esters) of Tasmanian tasmanite bitumen; (B) m / _74 fragmentogram, key ion characteristic for normal car-boxylic acid methyl esters; (C) r n / z 88 fragmentogram, keyion characteristic for 2-methyl-isoprenoid carboxytic acidmethyl esters; (D) m / z 01 fragmentogram, key ion charac-teristic for 3-methyl-is0prenoid carboxylic acid methyl es-ters; (E) m / z 191 fragmentogram, key ion characteristic fortricyclic terpenoid earboxylic acid methyl esters; (F) com-posite fragmentogram m / z 145,203,217,231,245,273, 287,329, 343, plus 357) for the aromatic tricyclic terpenoidcarboxylic acid methyl esters.

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Tricyclic terpenoidS t a n d a r d m e t h y l 1 3 ~ ( H ) , I 4 f l ( H ) - a n d 1 3 f l( H ) ,

1 4 f l ( H ) - d i h y d r o i s o c o p a l a t e ( C ' a n d D ' i n F i g . 6 ) w e r eo b t a i n e d b y c y c l i z a t io n o f m e t h y l c o p a l a t e i n f o r m i ca c i d ( H e i s s l e r a n d L a d e n b u r g e r , 1 9 8 8 ). T h e y a r e t h ee n a n t i o m e r s o f t h e C 20 t r i c y c l i c t e r p a n e a c i d m e t h y le s te r s . ( E n a n t i o m e r s h a v e t h e s a m e r e t e n t i o n t i m e i nG C a n a l y s i s , i . e . t h e y c o e l u t e . N o t e : U s u a l l y g e o -c h e m i c a l l i t e r a t u r e r e fe r s t o t h e s t e r e o c h e m i s t r y o f t h eena n t io m ers , in th i s ca s e f l~ and ~ t~ t, an d th i s wi l l beu s e d i n t h e d i s c u s s i o n o f o u r r e s u l t s ) . C o p a l i c a c i dw a s i s o l a t e d f r o m c o p a i b a o i l , c y c l iz e d a n d t h e n

carboxy lic acids 993r e d u c e d o n P d / C 5 % i n h e x a n e ( C y r a n d S t r a u s z ,1 9 8 3 ) o r o n P t O 2 i n m e t h a n o l / e t h y l a c e t a t e ( 5 : 1 )( I m a m u r a a n d R f i v e d a , 19 80 ) t o o b t a i n o n l y t h e / / pi s o m e r . T h e f l / / s t a n d a r d w a s c h a r a c t e r iz e d b y p r o t o nn u c l e a r m a g n e t i c r e s o n a n c e a n d m a s s s p e c t r o m e t r y .V e a d e i r o i c a c i d m e t h y l e s t e r ( 1 4 - e t h y l p o d o c a r p a -8 , 1 1 ,1 3 - t r ie n e - 1 3 - ca r b o x y m e t h y l e s t er ) s t a n d a r d w a si s o l a te d f r o m Vellozia f lavicans a n d c h a r a c t e r i z e d b yP i n c h i n a n d c o w o r k e r s ( 19 7 8) . T h e s e s t a n d a r d s w e r ec o i n j e c te d w i t h t h e m o n o c a r b o x y l i c a c i d m e t h y l e s t e rf r a c t i o n o n a S E - 5 4 c o l u m n ( s ee a b o v e ) a n d a n O V - 1 7

J A ~ T 1 1 1 1 4 2 2 2 7 3 2 C 0 2 ~ [ 3

, ,~ '; . . . . . . . . . . . ',--, 3 , , - 474C C32T

~ m/z4 8 8c)./}OC I r T I t i I I I I K I I I I i I I I~ ~~ Z

I I l l l l I I I I I I I I I I I I

C~Tm / z516

m / z5 3 0I I I i i i i I i ~ i ; i i i i i i i w

C 3 S T G m / z

16 20 24 2 8 3 2 3 6 4 0t~ (m in)

Fig. 3. Single ion m onito ring (SIM ) of the methyl ester fraction of Tasmanian tasmanite bitumen(Condition A): (A) m / z 191 fragmentogram, characteristic fo r the satu rated tricyclic terpenoid carboxylicacid methyl esters; (B-G ) mass fragmentograms of the molecular ions of the higher members o f the series(C31-C36).

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60OOO-4 0 0 0 0 - C z 0

zoo(X)o~ ~ ~ I ~

n J l li i I I

- tO0806o40 m / z 1 9 1

- I I a = c = AJ~

4 O 0 0 1O , I iZ 11 0 0 0 1O ,

{,- lOOO00 . s 1/ ) o ,

r r

~ n--

4 0 0O ,

2 o 0O i

o : t

I I 1

i I I I

B~' , , I. , , a~ Z2~ T - I ~ I I I

. . o . ~ z s

e , - .. . .( . . . o . o C z 4R / S R / S~ . I c ~ C = 5

R / S '

R I S 6 ~ . , ~ C z sI I l ' l ~ l ~ I I I I I i

[ . " ~ c ," 1 c . .

I I I I I | | l | i I |

; i i I I i I I : I

1 6 1 9 2 2 2 5tR ( m i n

20

0

8040, 0400

I 0I I 0I :b O

I :I 0, 0

40, 0

40i ' 0} o o

40~ 0i R /S 8 o

. i ~ , , 40028

m/z 320

m/z 334

m / z 3 4 0

m / z 3 6 2

m / z 3 7 6

m h 3 9 0

m / z 4 0 4

m/z 4 1 8

m h 4 3 2

m/z 4 4 6

F ig . 5 . I o n m a p o f t r i c y c l i c t e r p e n o id c a r b o x y l i c a c id m e th y l e s t e r s f o r C 2 04 2 29 . K e y t o s t e r e o i s o m e r s (s e ea l s o F ig . 7 ) : fl~t -C 20 = 1 3 f l (H ) , 14~ t ( H ) - t r i c y c l i c t e r p e n o id c a r b o x y l i c a c id m e th y l e s t e r w i th 2 0 c a r b o n a to m s(see Fig. 6).

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996 D.A. AZEVEDO t al.

. I C O 2 H/ \ I ~ ( A ) O2' "o q ( B )

CO2H

/ cto. (A')

CO2H

13o. (B') . x~ CO2HI~ (c)

C O 2 Hcte{ D)

CO2H

a 1 3 ( C ' )

CO2H

I~ (O )Fig. 6. Structures of the four stereolsomerlc C20saturated tricyclic terpenoid carboxylic acids: A13fl(H),I4/~(H); B 13ct(H),t4f(H); C 13#(H),I4~t(H);D- -t 3~t(H),14a (H), and their enantiomers: A"13c~(H),14ct(H); B' 13fl(H),140t(H); C' 13~(H),I4~(H);D' 13fl(H),14fl(H).The pair ofenantiomers are not separ-ated by the achiral GC conditions employed.

presence of 13fl(H),14et(H)-C20 and 13ct(H),14ct[H)-C20 was confirmed by coinjection of the syntheticstandards (see Experimental) with the acid fractionon SE-54 and OV-17 glass capillary columns. All fourisomers of the C2~ homolog are observed: Bfl, ~fl, fla,act (Fig. 6). The stereoisomeric distribution wassuggested by comparison with that observed for thetricyclic terpane series by Chicarelli e t a L (1988) andthe observed e lut ion order of the t iff and ~tfl-C20standards . For C22 and C2s only the flct and ct~t somerswere observed (see Experimental for clarification ofthe conflicting stereoisomer nomenclature for thestandards and the geological series).

Resolut ion of the R/S fl~t isomers for the C24 to C2epseudohomologs increases with higher carbon num-bers (Fig. 7). It is interesting to note that in thetricyclic terpane hydrocarbon series the C24 homologdoes not show resolved R/S stereoisomers becausethe carbon at position 22 is achiral by being linked totwo methyl groups. In the C24 tricyclic terpenoidcarboxylic acid the carbon at position 22 is chiralbecause it is linked to a methyl and a carboxy group.In the GC trace it shows a broadened peak for thetwo resolved isomers. This is the same behaviour as

observed for the tic(-C:5 tricyclic terpane, which is thefirst pseudohomolog of the saturate hydrocarbonseries with a chiral carbon in the side chain.These results differ from those of Cyr and Strausz(1983, 1984). Their tricyclic carboxylic acid concen-trate of the Alberta oil sands consisted primarily ofthe C20 and Cz3 and the diastereomeric C24 to C2~homologs . The C2~ and the pair of C24 diastereomericacids comprised about 75% of the series. They did notobserve coelution of the [3c~ and ~tct-C:0 standards withtheir sample and thus proposed another structure lbrthe C20 compound, which was confirmed recently byKaufman e t a l . (1988). They retained the tricyclicterpenoid structure for the C2rC26 homologs.The mass spectra o f the C20~$29 members of thetricyclic terpenoid carboxylic acids (as methyl esters.no C2v) are shown in Fig. 8. The mass spectrometricfragmentation pattern (Fig. 9) consists mainly of M +.[M 15] +, m / z 191 (base peak), m / z 261 (from theloss of the side chain at C-14), m / z 313 (from cleavageat C-22 of the isoprenoidal side chain, for homologs>C24), and smaller fragment ions ( m / z 109, m / z 123)probab ly derived from fragmentation of rings A andB, as was also observed by Cyr and Strausz (1983,1984).M o n o a r o m a t i c t r i c v c l i c t e r p e n o i d c a r b o x y l i c a cidL~

A novel series ol ring-C monoaromatic tricyclicterpenoid carboxylic acids was found in the acidfraction of this bitumen. The SIM analysis shows thatthe series ranges from C~9 to at least C39 (Fig. 10),with the C~9 homolog being the most abundant.

The mass spectrum of the C t9 ring-C monoaro -matic tricyclic terpenoid carboxylic acid (as methylester) is shown in Fig. 11 Inspection of the massspectra of the homologous senes shows the samefragmentation pattern as the recently described rmg-C monoaromatic tricyclic terpane series, differingonly by 58 mass units (CO2Me group attached at theC-14 side chain). The principal fragments include thearomatic C-ring plus the side chain with the CO 2 Megroup, which shift by 14 mass units up to C24 fromone homolog to the next. For compounds greaterthan C24, the fragmentat ion is dominated by cleavageof the side chain resulting in a base peak at m z 145.analogous to the monoaromatic tricyclic terpanes(Azevedo e t a l . . 1990).

A suitable representation of the series up to C~0 isobtained by plotting the composite fragmentogram ofions m / z 145. 203. 217. 231. 245, 273. 287. 329. 343.plus 357 (key ions of the series. Fig. 12). It should benoted that the surprisingly intense C26 homolog(suggested by its relative retention time vs C25 andC28, molecular ion and key fragments) may be due tocoelution as the composite fragmentogram involves10 different ions. Also the C~9 homolog a t 16.8 min(Fig. 12) has an identical mass spectrum as the otherisomer, which points to a probable C-13 carboxymethyl structure. By analogy with the interpretationof the mass spectra of the ring-C monoaromatic

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Tricyclic terpenoid carboxy lic acids 997

~

* ~ I c 2 , R / s' '1 1' " ' 1 I I1 ~ I i l

1 9 . 0 1 9 . 5 2 0 . 0 2 @ . 5 2 1 . 0 2 1 . 5 : : = . g 2 2 . 5 2 3 . 0 2 3 . 5 2 4 . e

. o , t l ~ / ~ l % s R /s r rV z 3 ~8 e ~ ]

1 9 . 0 1 9 . 5 2 8 .e l 2 @ . 5 2 1 . @ 2 1 . 5 2 2 . 0 2 2 . 5 2 3 . 0 2 3 . 5 2 4 . 0|m

c ~ a H I S I m l z3 0 0 -. . ~ i \ ~ 1 4 0 41 5 0

5~ I

1 9 . 0 1 9 . 5 2 g . e : : '@ . 5 2 1 . g 2 1 . 5 2 2 , e 2 2 . 5 2 3 . e ~ 3 . 5 2 4 . e

t R ( m i n )Fig. 7. Expanded mass fragmentogram s displaying the isomers of C24-C26. No te the gre ater resolutionof the #~ R/S isomers when going from C24 to C26.

t r i c y c l ic t e r p a n e s e r i e s ( A z e v e d o et aL, 1990) , we cana l s o c l a s s i f y t h e s e f r a g m e n t s i n t o 7 g r o u p s :

( i) G r o u p h m o l e c u l a r i o n M + ' ;( i i ) Gr ou p 2 : [M - 15] + , Schem e 1 ;

( i i i ) G r o u p 3 : m / z 69 , Scheme 1 ;( iv ) G r o u p 4 : [ M - 1 1 1 ] a n d [ M - 9 7 ] + p a ir ,r a n g i n g f r o m m / z 1 8 9 a n d m / z 203, + 14n(n = 0 , 1 , 2 , 3 . . . ) r e s pec t ive ly , Sche me 2 ;

( v ) G r o u p 5 : f o u r h o m o l o g o u s io n s o f d e c r e a s i n gi n t e n s it y , r a n g i n g o v e r m / z 215, 229, 243, 257p l u s 1 4 n; [ M - 8 5 ] + t o [ M - 4 3 ] +, re s pe c t-i v e l y , S c h e m e 3 ;

( v i ) G r o u p 6 : f r a g m e n t s o f m / z 145 and 159 duet o l o s s o f t h e s i d e - c h a in f ro m G r o u p 4 f r a g -m e n t s , S c h e m e 4 ;

( v i i ) G r o u p 7 : f r a g m e n t s o f m / z 2 5 5 a n d 2 4 1 d u et o t h e l o s s o f t h e s i d e c h a i n f r o m M . a n d[M - 15] + ions r e s pec t iv e ly , Schem e 5 .

T h e C O : M e g r o u p i s i n t e r p r e t e d t o b e a t t a c h e d t ot h e s i d e c h a i n a t t h e C - 1 4 p o s i t i o n . T h e r e i s c o n s i d e r -a b l e e v i d e n c e f o r t h i s c o n c l u s i o n :

( a ) A s t a n d a r d c o m p o u n d , v e a d e i r o i c a c i d (t h eC O 2 M e g r o u p i s a t t a c h e d a t C - 1 3 ) w a s c o in -j e c t e d w i t h t h e a c i d f r a c t io n . I t s m a s s s p e c t r u m

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. . a l . d J , _ L J l 1 , + + . . . ' + I t + . + +. . . . . . + . . . . . . + + : i + i + '+ . . . ~ ' ~ . . . . . . . . . . ~ + + i . . . . . . + + + . . . . . + k + . . . . . .

: /k7711Z~ e~ a

m/zFig. 11. Mass spectrum of the C~9 ring-C m ono arom atic tricyclic terpenoid carboxylic acid methyl ester.

( b )

m a t c h e s w i t h t h a t o f o n e m e m b e r o f t h e se r ie st h e C20 c o m p o u n d . H o w e v e r , t h e y d o n o tc o e l u t e ( m e t h y l v e a d e i r o a t e h a s a s h o r t e r r e t e n -t i o n t i m e ) , e x c l u d i n g C - 1 3 a s a s i t e f o r t h eC O 2 M e .T h e k e y i o n s o f t h e s e r i e s a r e e x p l a i n e d b y t h ew e l l p r o v e n r e a r r a n g e m e n t s o f r i n g s A a n d Bt o p r o d u c e i n d a n y l c a t i o n s . T h e y d i f f e r b y 5 8m a s s u n i t s ( C O 2M e g r o u p ) f r o m t h e k e y i o n so f t h e r i n g - C m o n o a r o m a t i c t r i c y c li c t e r p a n es e r ie s ( A z e v e d o e t a l . , 1990) . T h i s in d ica te s tha tt h e C O 2 M e g r o u p m u s t b e p r e s e n t i n t h ei n d a n y l c a t i o n s , a t t a c h e d e i t h e r d i r e c t l y t o t h e

a r o m a t i c C - r i n g o r i n t h e s i de c h a in a t t a c h e d a tC-14 .( c ) F o r h i g h e r m e m b e r s o f t h e s e r i e s , > C 2 4 , t h e

f r a g m e n t a t i o n i s d o m i n a t e d b y c l e a v a g e o f t h es i d e c h a i n r e s u l t i n g i n a p e a k a t m / z 145 ( s amef r a g m e n t t h a t o c c u r s i n t h e m o n o a r o m a t i ct r i c y c l ic t e r p a n e s e r ie s ) .

( d ) T h e p r e s en c e o f a f r a g m e n t a t m / z 255 in them a s s s p e c t r a o f t h e s e r i e s ( l o s s o f t h e s i d ec h a i n ) e x c lu d e s t h e p o s s i b i l it y o f a C O z M eg r o u p a t t a c h e d t o t h e t i n g s .

( e ) C o m p a r i s o n w i t h t h e s a t u r a t e d t r i c y c l i c t e r -p e n o i d c a r b o x y l i c a c i d s, w h i c h a r e t h e m a i n

iz203145 I c 9 CO2CH32172 3 124s ( I ) C 3 o

t 'w 273 ~ ; t9 C26I c c28287

J + 7 1 .l.j, , J iI i~ e = 1 . e i4 .e i~' e mac e ++e".'e" ~ e a4".~e Id.i" e+ e : ; ,

tR ( m i n )Fig. 12. Composite fragmentogram ( m / z 145, 203 , 217 , 231, 245, 273, 287, 329, 343, plus 357) fo r thearom atic tricyclic terpenoid carboxylic acid methyl esters of Tasmanian tasmanite bitumen.

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Tricyclic terpenoid carboxy lic acids 1001G R O U P 2

G R O U P 3

C H 3_1

n C O 2 M e ~ ~ ~ ~ C 0 2 M e( M - 1 5 ] + m / z S 9

Scheme 1. Fragm ents [M - 15]+ (Group 2) and m / z 69 (Group 3).

cyc l i c cons t i t u t en t s o f t he ac id f rac t i on andw h o s e C O 2 M e g r o u p w a s d e t e r m i n e d t o b ea t t ached to t he s ide cha in a t t he C-14 pos i t i on(see above) .( f ) O t h e r p o s i t io n s u s u a l l y f o u n d t o b e f u n c t i o n a l -i z e d i n n a t u r a l t e r p e n o i d c o m p o u n d s a r e a tC - 1 8 , C - 1 9 a n d C - 2 0 . N e v e r th e l es s , t h o s e c o m -p o u n d s h a v e c o m p l e t e ly d i f fe r e n t m a s s s p e c t r a( P i n t o e t a l . 1987; Simonei t , 1977; Pat i tucci ,1983).

The re fo re , s t ruc tu re I (F ig . 12) was t en t a t i ve ly a ss ign e d f o r t h i s m o n o a r o m a t i c t r i c y c l i c t e r p e n o i d c a r -boxy l i c ac id se r i e s based on the p rev ious i n t e rp re t -a t i on .

CONCLUSIONA no ve l ser ie s o f m on oa rom a t i c t r i cyc l ic ca r -b o x y l i c a c i d s w a s f o u n d i n i m m a t u r e T a s m a n i a n

t a smani t e b i t umen and was cha rac t e r i zed a s a se r i e so f 1 3 - m e t h y l - 1 4 - c a r b o x y a l k y l p o d o c a r p a - 8 , 1 1 , 1 3 -t rienes. Th e ser ies range s fr om C~9 to C 39, wi th am a x i m u m a t C ~ 9 , a n d i s t h e a c id i c c o u n t e r p a r t o fthe recen t ly desc r ibed se ri es o f mo no a ro m a t i c t r i -cyc l i c t e rpanes . The ex t ended t r i cyc l i c t e rpeno idca rboxy l i c ac ids o f th i s b i t ume n a re r e l a t ed t o t heub iqu i to us t r icyc l i c t e rpane se ri es . The ma in cyc l icc o n s t i tu e n t s o f t h e m o n o c a r b o x y l i c a c i d f r a c ti o n a r ethe C20-C4o ho mo logs o f t he se ri es o f t r icyc l ic t e r -peno id ca rboxy l i c ac ids . A pa i r o f s t e reo i somers( fl ct and a~) w as obse rved fo r a ll pseud oho mo logs ,C20-C26 , excep t fo r C2I , which cons i s t ed o f a lli somers, i .e . t i f f o t f l f l ~ t and ~tc t. The d i a s t e reom erswere a l so re so lved fo r t he R/S f lc t ho mo logs t> C24 .N o r m a l a l k a n o i c a c i d s r a n g i n g f r o m C 9 t oC 3 0, w i t h a c a r b o n n u m b e r m a x i m u m a t C ~ 6 , a n dCj4, C ~5 , C]9 and C20 i sopreno id ac ids a re

G R O U P 4

. ~ n C O a M e.

~ ~ C ~+ ~ r" l n m / z 1 8 9 ( n = O )[ M - 1 1 1 ] +

m / z 2 0 3 ( n . o )[ M - 9 7 ] +

Scheme 2. Ring A/B rearrangements give rise to a p air o f fragments derived from m / z 189 and 203, plus14n (Group 4).

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1002G R O U P 5

D. A. AZEVEDO et al.

/ n

O=Me , L O2Me m/z21S n=0) [ M - 8 5 ] +

[M - 71]

n C O = M e

- 57]+

[ M - 4 3 ] *

S c h e m e 3 . R i n g A / B r e a r r a n g e m e n t s g i v e r i se t o f o u r f r a g m e n t s d e ri v e d f r o m m z 215 , 229 , 243 , 257 , p lus1 4 n ( G r o u p 5 ) .G R O U P 6

+ ~ ~ ~ n C O ~ 4en CO =Me

+ ~ m / z 1 4 5

n CO=Me

m / z 1 5 9S c h e m e 4 . F r a g m e n t s d u e t o l o s s o f th e s i d e c h a i n g i v e ri se t o i o n s m/z 1 4 5 a n d 1 5 9 ( G r o u p 6 ).

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Tricyc l ic t e rpen oid carboxy l ic ac ids 1003G R O U P 7

~ C O 2 M e . . ~ n C O 2 M e ~ ~ ~ 55J , - .C H 3~ C 0 2 M e . . . ~ n C O 2 M e J ,~ . C HS c h e m e 5 . F r a g m e n t s d u e t o l o s s o f t h e s id e c h a i n f r o m M + ' o r [ M - 1 5]+ g ive r i se to ion s m / z 2 5 5 a n d241 (Group 7) .

a l s o s ig n i f ic a n t c o m p o n e n t s o f t h e f r a c t io n a n d c o u l db e i n d i c a t i v e o f t h e m a r i n e i n p u t .

T h e e x t e n d e d t r i c y c l i c t e r p e n o i d c a r b o x y l i c a c i d sa r e i n t e r p r e t e d t o b e b i o m a r k e r s o f t a s m a n i t i d s ,w h i c h a r e u b i q u i t o u s k e r o g e n m a c e r a l s i n p e t r o l e u ms o u r c e r o c k s ( T a p p a n , 1 9 8 0; T e i c h m i i l l e r , 1 98 6 ) .T h u s , s u c h b i o m a r k e r s c a n c o n t r i b u t e t o t h e o v e r a l lb i t u m e n m i x t u r e a n d m a y p r o v e t o b e u s e f u l a ss o u r c e i n d i c a t o r s f o r c e r t a i n m a r i n e o r g a n i c m a t t e r( D e G r a n d e e t a l . , 1 9 9 1 , 1 9 9 3 ) .A s s o c i a t e E d i t o r - - K . P E TE R SA c k n o w l e d g e m e n t s - - W e t h a n k F I N E P a n d C N P q f o r f i na n -c i a l s u p p o r t t o c o n d u c t t h i s r e s e a r c h a n d D r P . A l b r e c h t a n dDr K. E . Pe te rs for the i r rev iews .

R E F E R E N C E S

A q u i n o N e t o F . R . , T r i g i i i s J . , A z e v e d o D . A . , R o d r i g u e sR . a n d S i m o n e i t B . R . T . ( 1 9 9 2 ) O r g a n i c g e o c h e m i s t r y o fg e o g r a p h i c a l l y u n r e l a t e d T a s m a n i t e s . O r g . G e o c h e m . 18,791--808.A z e v e d o D . A . , A q u i n o N e t o F . R . a n d S i m o n e i t B . R . T .( 1 9 9 0 ) M a s s s p e c t r o m e t r i c c h a r a c t e r i s t ic s o f a n o v e l s e r ie so f r i n g - C m o n o a r o m a t i c t r i c y c l i c t e r p a n e s f o u n d i n T a s -m a r t i a n t a s m a n i t e . Org . Ma ss Sp ec tro m. 2 5 , 475-480 .A z e v e d o D . A . , A q u i n o N e t o F . R . a n d S i m o n e i t B . R . T .( 1 9 9 1 ) D o u b l e b o n d l o c a t i o n i n a s e r i e s o f m o n o u n s a t u -ra ted t r i cyc l ic t e rpanes . 1 2 th In t . Ma s s Sp ec tro m. Co n f . ,A m s t e r d a m .A z e v e d o D . A . , A q u i n o N e t o F . R . , S i m o n e i t B. R . T . a n dPin to A. C. (1992) Novel se r ies of t r i cyc l ic a romat ict e r p a n e s c h a r a c t e r i z e d i n T a s m a n i a n t a s m a n i t e . O r g .G e o c h e m . 18, 9-16.A z e v e d o D . A . , A q u i n o N e t o F . R . a n d S i m o n e i t B . R . T .( 1 9 9 4 ) M a s s s p e c t r o m e t r i c c h a r a c t e r i s t i c s o f t w o n o v e ls e r ie s o f r i n g - C m o n o u n s a t u r a t e d t r ic y c l ic t e rp e n e s f o u n di n T a s m a n i a n t a s m a n it e . O r g . M a s s S p e c t r o m In press .Bur l ingame A. L . , Wszolek P . C . and S imonei t B . R . T .( 1 9 6 9 ) T h e f a t t y a c i d c o n t e n t o f t a s m a n i t e s . I n Ad va n cesin Org a n ic Geo ch emis t ry 1 9 6 8 ( E d i t e d b y S c h e n c k P . A .and Havenaar I . ) , pp . 131-155 . Vieweg, Braunschweig .

C h i c a r e l li M . I . , A q u i n o N e t o F . R . a n d A l b r e c h t P . (1 9 88 )Ocu rrence o f four s te reo isome r ic t r i cyc l ic t e rpa ne se r ies ini m m a t u r e B r a z i l i a n s ha l e s. G e o c h i m . C o s m o c h i m . A c t a 52,1955-1959.Cyr T . D . and S t rausz O. P . (1983) The s t ruc ture s of t r i cyc lict e r p e n o i d c a r b o x y l i c a c id s a n d t h e i r p a r e n t a l k a n e s i n t h eA l b e r t a o i l s a n d s . J . Ch em. So c . , Ch em. Co mmu n .1028-1030.Cyr T . D. and S t rausz O. P . (1984) Bound carboxyl ic ac idsin the Alber ta o i l sands . O r g . G e o c h e m . 7, 127-140.D e G r a n d e S . M . B . , A q u i n o N e t o F . R . a n d M e l l o M . R .( 1 9 9 1 ) T a n d e m m a s s s p e c t r o m e t r y a n a l y s i s o f t r i c y c l i cte rpanes . Pro c . 1 2 th In t . Ma ss Sp ec tro m. Co n f . , p . 4 05 .A m s t e r d a m .D e G r a n d e S . M . B . , A q u i n o N e t o F . R . a n d M e U o M . R .(1993) Extended t r i cyc l ic t e rpanes in sed iments and pe t -r o l e u m s . O r g . G e o c h e m . 20, 1039-1047.H e i s s l e r D . a n d L a d e n b u r g e r C . ( 1 9 8 8 ) S y n t h e s i s o f ( + ) -t r i c y c l o h e x a p r e n o l , a p o s s i b l e p r e c u r s o r o f a f a m i l y o ft r icyc l ic geo te rpanes , and synthes i s o f an i somer . T e t r a -h ed ro n 44, 2513-2521.l m a m u r a P . M . a n d R f i v e d a E . A . ( 19 8 0) T h e C - 1 3 c o n f i g u r -a t i o n o f t h e b r o m i n e - c o n t a i n i n g d it e r p e n e i s o a p ly s i n - 2 0.S y n t h e s is o f d e b r o m o i s o a p l y s i n - 2 0 a n d i t s C - 13 e p i m e r .J . Org . Ch em. 45, 510-515 .K a u f m a n T . S . , G o n z a l e z - S i e r r a M . a n d R r v e d a E . A .(1988) Synthes i s o f an Alber ta o i l sand b i tumen C20t r icyc lic carbox yl ic ac id bea t in g a nove l d i te rp enoid ske l -e ton . J. Chem. Soc. Perkin Trans. I , 2323-2327.L o u r e i r o M . R . B . , A q u i n o N e t o F . R . a n d C a r d o s o J . N .(1986) Anfi li se da m at r r ia o rgf in ica de or igem se d ime ntar .I . I s o l a m e n t o d e ~ i c id o s c a r b o x i l ic o s e h i d r o c a r b o n e t o salif~iticos e aro mfit ico s. Q u i m . N o v a 9, 15-19.M c C a r t h y R . D . a n d D u t h i e A . H . ( 1 9 6 2 ) A r a p i d q u a n t i -t a t i v e m e t h o d f o r t h e s e p a r a t i o n o f f r e e fa t t y a c i d s f r o mother l ip ids . J . L ip id Res . 3, 117-119.N e w t o n E . T . (1 8 75 ) O n " ' T a s m a n i t e " a n d A u s t r a l i a n" W h i t e C o a l " . G e o l . M a g . 12, 337-342.Pa t i tucc i M. L . (1983) Cons t i tu in tes qu imicos de Vello:ialeptopetala . M . S c . T h e si s , U n i v e r s i d a d e F e d e r a l d o R i o d e

J a n e i r o .P i n c h i n R . , M a y e r L . M . U , a n d P i n t o A . C . ( 1 9 7 8 )V e a d e i r o l a n d v e a d e i r o i c ac i d , t w o n o v e l d i t e r p e n e s f r o mVe l lo z ia f la v ica n s . Ph y to ch emis t ry 17, 1671-1672.Pin to A. C. , Zocker D. H. T . , Quei roz P . P . S . and KelecomA. (1987) Di te rpenoids f rom Ve l lo z ia f la v ica n s . Ph y to -ch emis t ry 26, 2409-2411.

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