Chem. Phys. Lipids I (1967)407-423 t ~ North-Holland Publ. Co., Amsterdam

S Y N T H E S I S A N D C H A R A C T E R I Z A T I O N OF

T H E C O M P L E T E SERIES OF M E T H Y L E N E - I N T E R R U P T E D

c i s , c i s - O C T A D E C A D I E N O I C A C I D S *

WILLIAM W. CHRISTIE** and RALPH T. HOLMAN

University ~[" Minnesota, The Hormel Institute,

Austin, Minnesota

Received 14 June 1967

The complete series of methylene-interrupted cis,cis-octadecadienoic acids (analogues of linoleic acid), from the 2,5- to 14,17-isomer, has been synthesized. The 4,7- to 10,13- isomers were prepared by coupling a I-bromoalk-2-yne with the di-Grignard complex of an ¢,)-acetylenic acid and semi-hydrogenation of the resulting diynoic acid to a cis,eis- dienewith Lindlar's catalyst. The 2,5- and 3,6-isomers and the 11,14- to 14,17-isomers were synthesized by first preparing the corresponding diacetylenic alcohols which were then oxidized to the acids and semi-hydrogenated. Purity of the intermediates and final compounds was established by gas-liquid and thin-layer chromatography. The compounds were characterized by melting points, critical solution temperatures, infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy.

Hydrogenation followed by gas chromatography confirmed the chain length and indi- cated absence of branched chain material. The positions of the double bonds were con- firmed by ozonolysis. The 2,5-isomer has distinguishing features in its mass spectrum but only subtle distinctions can be detected in the spectra of the rest. The NMR spectra of the 2,5- to 5,8- and 1 I, 14- to 14,17-isomers, however, all have features which uniquely identify them, though the 6,9- to 10,13-isomers have spectra which are virtually identical.

Introduction

Linole ic acid or cis,cis-9,12-octadecadienoic acid was shown in 1929 to be

essential to an ima l nu t r i t ion l) and is f o u n d in all n o r m a l an ima l tissues. The

5,8-, 6,9-, 7,10- and 8 ,11- isomers , i n c o r p o r a t i n g a s imi lar me thy lene -

in t e r rup ted d iene system, have also been found in na tu re e ~i). M a n y synthe-

ses o f l inoleic acid have been r epo r t ed and are listed in O s b o n d ' s recent

reviewT); and the 8,11-, 10,13- and l l , 1 4 - o c t a d e c a d i e n o i c ac ids have also

been synthes ized 8,:t). The 12,15-isomer has been p r o d u c e d by part ial r educ t ion

o f l inolenic acid but does not a p p e a r to have been isola ted in the pure state 10).

* This investigation was supported in part by a PHS research grant No. HE03559 from the National Institutes of Health, Public Health Service.

** Present address: Hannah Dairy Research Institute, Ayr, Scotland.

408 ~A,'ILLIAM W . C H R I S T I E A N D R A L P H T. H O L M A N

The present communication describes the syntheses of all possible methylene-interrupted cis,cis-octadecadienoic acids by new or improved procedures. These substances were prepared for metabolic studies to eluci- date the structure essential for the biological activity of linoleic acid. Their physical and spectroscopic properties are reported.

A synthesis of linoleic acid readily extendable to other isomers has been describedU). A l-bromo-2-alkyne was condensed with the di-Grignard complex of an c0-acetylenic acid using cuprous cyanide as catalyst in tetra- hydrofuran solution and the resulting diynoic acid reduced to the cis,cis- diene by semi-hydrogenation over kindlar's catalyst l,_,). Using this method, the 4,7-, 5,8-, 6,9-, 7,10-, 8,11- and 10,13-isomers were obtained. The attempted preparation of the 2,5- and 3,6-isomers by this procedure gave a mixture of products, possibly, in the latter case because of the formation of branched-chain compounds 13). However, the required diacetylenic acids were obtained by reaction of the 1-bromoalk-2-yne with the Grignard complex of the tetrahydropyran derivative of the appropriate eJ-acetylenic alcohol. After hydrolytic removal of the tetrahydropyranyl group, a crystalline CI s diynoic alcohol was obtained which was oxidized in good yield to the diacetylenic acid with chromic acid in aqueous acetone14). The 11,14-, 12,15-, 13,16- and 14,17-isomers were also prepared by this method when the di-Grignard complexes of the appropriate ~o-acetylenic acids failed to react with the bromoalkynes.

Lindlar's catalyst 1~) was used for the semi-reduction of acetylenic bonds. Hydroboration with bis-(2-methyl-3-butyl)-borane 1'~) gave good yields of stereochemically pure products with monoynes but failed with diynes. The purity of the compounds was checked by gas-liquid chromatography (GLC) and thin-layer chromatography (TLC). The positions of the double bonds were established by ozonolysis. GLC examination of the fully hydrogenated products confirmed the chain length and indicated absence of any homolo- gues or branched-chain isomers.

Experimental Melting points and critical solution temperatures x6) were determined

under a microscope equipped with Kofler hot or cold stages and are cor- rected. Infrared spectra were recorded with a Beckman IR 8 spectrophotometer in carbon tetrachloride solution. UV spectra were measured using a Beckman DK 2 spectrophotometer. All reactions involving unsaturated compounds were carried out under nitrogen. Elemental analyses were made on all hitherto unreported compounds by Clark Microanalytical Laboratories, Urbana, Illinois.

Mass spectra were obtained on a Hitachi Perkin-Elmer RMU-6D single

METHYLENE-INTERRUPTED Cis,cis-OCTADECADIENOIC ACIDS 409

f o c u s i n g i n s t r u m e n t . A l i qu id i n j e c t i o n sys t em was used, a s u b o v e n h e a t i n g

t h e s a m p l e to 160°C to p r o d u c e t h e v a p o r a t a p p r o x i m a t e l y 5 x 10 - 7 T o r t

w h i c h was i o n i z e d a t a s t a n d a r d p o t e n t i a l o f 70 eV. N M R s p e c t r a were

o b t a i n e d wi th 15 ~o s o l u t i o n s o f the m e t h y l es te r s in c a r b o n t e t r a c h l o r i d e o n

a P e r k i n - E l m e r R-10 s p e c t r o m e t e r o p e r a t i n g a t 60 Mc/s . C h e m i c a l sh i f t s

were m e a s u r e d in p .p .m, d o w n f i e l d f r o m i n t e r n a l t e t r a m e t h y l s i l a n e ( r = 10).

l- t t ydro.xo,-2-alk ynes

T h e phys i ca l c o n s t a n t s a n d yie lds o f t he se c o m p o u n d s a re de t a i l ed in

t a b l e 1. T h e d i h y d r o p y r a n d e r i v a t i v e s o f t he a l c o h o l s were s y n t h e s i z e d in

TABLE 1

l-Hydroxy-2-alkynes CHa(CH2),,.C~C.CH20H

nl Ref. b.p. ( C ) m.p. ( 'C) hi) e~

0 ~ 143 145 (760 mm) - 1.4583 I l~ 145-150 (760 ram) - 1.4486 2 " 84-87 (20 ram) - 1.4500 3 ~' 52-55 (0.6 mm) - 1.4510 5 b 64-68 (0.6 mm) - 1.4556 6 ,i 76-80 (0.4 ram) - 1,4564 7 " 97 103 (0.3 ram) - 1,4546 8 i, 102 107 (0.3 ram) 30-31 9 f,~ - 37-38 -

10 ,t.~,l~ _ 41-42 - I I g.i _ 42-43 -

Yield ('!i;)

72 84 60 70 63 62 68 52 50 72 91

a G. Smets, Acad. roy. Beige., Classe sci., Mem. 21 (1947) 3. b Tchao Yin Lai, Bull. Soc. chim., France 53 (1933) 682. e M. S. Newman and J. H. Wotiz, J. Am. Chem. Soc. 71 (1949) 1292. ,l Calcd. for C10Ht80: C 77.87 H 11.76

Found: C 77.74 H 11.90. e Calcd. for CllH200: C 78.51 H I 1.96

Found: C 77.36 H 11.83. r Calcd. for CaaH240: C 79.53 H 12.32

Found: C 79.42 H 12.07. g Recrystallized from light petroleum. h D. E. Ames, A. N. Covell and T. G. Goodburn, J. Chem. Soc. (1963)5889. J W.G. Knipprath and R. A. Stein, Lipids I (1966) 82.

e s sence by C o n i a ' s m e t h o d 17) a n d c o n v e r i e d to t he a l c o h o l s by ac id h y d r o -

lysis i s ) w i t h o u t f u r t h e r pu r i f i c a t i on . S o d i u m in l iqu id a m m o n i a was used

for t h e s h o r t e r c h a i n c o m p o u n d s ( m = 0 - 5 ) a n d l i t h i u m in l iqu id a m m o n i a :

t e t r a h y d r o f u r a n (3 :1) fo r t he l o n g e r ones ( m = 6 - 1 1 ) . T h e p r e p a r a t i o n o f

u n d e c - 2 - y n - l - o l is d e s c r i b e d as a n e x a m p l e .

2 - ( P r o p - 2 ' - y n - l ' - y l o x y ) - t e t r a h y d r o p y r a n 19) (70 g, 0.5 M ) was a d d e d wi th

s t i r r i n g to l i t h a m i d e ( f r o m l i t h i u m , 3.5 g, 0 .52 M ; fe r r i c n i t r a t e , 0.05 g)20)

410 WILLIAM W. CHRISTIE AND RALPH T. HOLMAN

in l iqu id a m m o n i a " t e t r a h y d r o f u r a n (3:1; 1000 ml). Af t e r 1 hr, octyl b r o m i d e

(96 g, 0.5 M) was a d d e d and the so lu t ion s t i r red for 8 hr be fo re the a m m o n i a

was a l lowed to evapora t e . W a t e r (100 ml) was added and the p r o d u c t

ex t rac ted with ether. The e therea l layer was washed with water , dr ied over

s o d i u m sulfa te and so lvent e v a p o r a t e d . The c rude p r o d u c t was d i sso lved

in m e t h a n o l (500 ml), p - to luenesu l fon ic ac id (3.0 g) added and the so lu t ion

ref luxed for 2 hr. Af t e r coo l ing , the reac t ion mix tu re was p o u r e d into a

large v o l u m e of wa te r and ex t rac ted with ether . T h e e therea l layer was washed

with water , s o d i u m b i c a r b o n a t e so lu t ion (2'/,,) and water . Af t e r d ry ing and

r emova l o f solvent , u n d e c - 2 - y n - l - o l (57 g) was ob t a ined by d is t i l la t ion (see

table 1). T h e inf ra red s p e c t r u m had p r o m i n e n t bands at 1005 cm

( C ~ C ' C H 2 O H ) and 3650 and 3520 c m -1 ( - - O H ) .

l-Bromo-2-alkynes

The physical cons t an t s and yields o f these c o m p o u n d s are de ta i led in

table 2. T h e p r e p a r a t i o n o f l - b r o m o - u n d e c - 2 - y n e is desc r ibed as an ex-

a m p l e e l ) .

P h o s p h o r u s t r i b r o m i d e (34 g) was a d d e d ove r ½ h o u r to u n d e c - 2 - y n - l - o l

TABLE 2

I-Bromo-2-alkynes CHa(CH2)m .C=_C-CH2Br

m Ref. b.p. (C) hi) e:' Yield ('~,~)

0 ~ 123-125 (760 ram) 1.5034 40 I ~' 146-149 (760 mrn) 1.4921 70 2 " 3~31 (0.75mm) 1.4877 47 3 " 79 81 (16 ram) 1.4840 57 5 t, 48-50 (0.2 rnm) 1.4842 79 6 ,L 82 84 (1.4 ram) 1.4828 60 7 " 90-92 (l.25mm) 1.4803 69 8 b 92 97 (0.3 ram) 1.4774 57 9 r 99 101 (0.3 ram) 1.4794 59

10 ~ 123 126 (I ram) 1.4770 75 11 l~ 123 127 (0.5 ram) 1.4749 56

" A. D. Petrov and G. I. N ikishin, Dokl. Akad. Nauk. SSSR 93 (1953) 1049. See Ref. el).

~' Footnote c, table 1. d Calcd. for C10H17Br: C 55.31 H 7,89 Br 36.80

Found: C 55.12 H 7,65 Br 37.40 e Calcd. for CllH19Br: C 57.15 H 8.28 Br 34.57

Found: C 53.67 H 8.01 Br 38.32 Calcd. for ClzHz3Br: C 60.23 H 8.94 Br 30.83 Found: C 59.88 H 8.71 Br 31.39

g Ref. h, table 1 h Calcd. for C15HzTBr: C 62.71 H 9.47 Br 27.82

Found: C 62.33 H 9.38 Br 27.29

M E T H Y L E N E - I N T E R R U P T E D Cis,cis-OCTADECAD1ENOIC A C I D S 411

(56.5 g) in ether (750 ml) containing pyridine (5 ml). The solution was refluxed for 3 hr, then poured into ice water. The aqueous layer was extracted with ether and the combined ethereal layers washed with sodium bicarbonate

O / solution (5 ,o) and water. After drying and removal of solvent, 1-bromo- undec-2-yne (54.3 g) was obtained by distillation (table 2). The infrared spectrum had characteristic bands at 2230 cm -~ ( C-=CCH2Br) and 1210cm -I ( C Br).

o)-Acetylenic acids and alcohols

Short-chain co-acetylenic acids (C5-Co) were prepared by the methods described in the literature. Longer acids (C~ 2-C~ 5) were synthesized by chain extension of undec-10-ynoic acid. Undec-10-ynoic acid was prepared by bromination-dehydrobromination of undec-10-enoic acid')')), though by using liquid ammonia: tetrahydrofuran (5:1) as solvent, much better yields

D were obtained. Undec-10-yn-l-ol (b.p. 86-89'C, 0.3 mm; n25, 1.4898) ~3) was obtained by lithium aluminium hydride reduction of this, and was converted to the methanesulfonate for chain lengthening by I, 2, 3 or 4 carbon atoms.

Dodec- 1 l-yn- I-ol

Methanesulfonyl chloride (42 g, 0.37 M) was added to undec-10-yn-l-ol (55 g, 0.33 M) in pyridine (120 g) in an ice bath at such a rate that the temperature did not exceed 30~'C at any time. The mixture was stirred at atemperature below 20°C for 3 hr after which it was diluted with sufficient hydrochloric acid in ice water to form the pyridine salt. The solution was extracted with ether and washed thoroughly with water, sodium bicarbonate solution (5'~(,) and water. After drying, the solvent was removed and undec- 10-ynyl-l-methanesulfonate (74 g,91 "/)/o was obtained by crystallization from methanol at - 50~C.

To a solution of sodium cyanide (40 g) in water (65 ml) was added ethanol (260 ml) and the methanesulfonate. The solution was refluxed over- night after which sodium hydroxide (50 g) was added and the refluxing continued for a further 24 hr. Water was added to the cooled mixture and the solution extracted with ether to remove neutral material. The aqueous layer was acidified with hydrochloric acid then extracted with ether. The ether layer was dried, solvent removed and dodec-I l-ynoic acid (51.9 g, 88 i~;, m.p. 47-49 C) '-'4) obtained by crystallization from light petroleum at

o / - 2 0 ' C . Dodec-II-yn-l-ol (40.9 g, 89/0), prepared by lithium aluminum hydride reduction of the acid, had b.p. 83-86 °C (0.05 mm) and m.p. 26-28 "C.

Calcd. for CI2H220: C 79.06 H 12.16 Found: C 79.20 H 12.12

412 W I L L I A M W . CHRISTIE A N D R A L P H T. H O L M A N

Tridec- 12-yn-l-ol

Sodium (6 g) was dissolved in dry ethanol (250 ml), diethyl malonate (35 g) was added and the mixture heated under reflux for 1 hr. Undec-10- ynyl-l-methanesulfonate (71.4 g) was added, the solution warmed until reaction started and left at room temperature overnight. The solid was filtered off and most of the solvent removed on a rotary evaporator. Water was added and the solution extracted. The oil so obtained was hydrolyzed by refluxing with potassium hydroxide (60 g) in ethanol (250 ml) for 1 hr. The solution was acidified with dilute hydrochloric acid and extracted with ether. The crude di-acid was decarboxylated by heating to 150~'C for 2 hr and tridec-12-ynoic acid (45.6 g, 65 ~o; m.p. 54-55 '~C)z4) obtained by crystalliza- tion from light petroleum at - 20~'C.

Tridec-12-yn-l-ol (32.4 g, 89')~), prepared by lithium aluminum hydride reduction of the acid, had b.p. 95-98 °C (0.3 ram) and m.p. 30-32°C.

Calcd. for C13H240: C 79.53 H 12.32 Found: C 79.72 H 12.58

Tetradec- 13-yn- l-ol

Tetradec-13-ynoic acid, prepared by chain extension of tridec-12-yn- 1-ol, had m.p. 53-55:'C.

Calcd. for C14H2402: C 74.95 H 10.78 Found: C 74.50 H 10.85

Tetradec-13-yn-l-ol, prepared by lithium aluminium hydride reduction of the acid, had b.p. 112-115 °C (0.5 mm) and m.p. 34-36'~'C.

Calcd. for C14H260: C 79.94 H 12.46 Found: C 79.14 H 12.58

Pen tadee- 14-yn- l-ol

Pentadec- 14-ynoic acid, prepared by chain extension of tridec- 12-yn- l-ol, had m.p. 68-70°C.

Calcd. for C15H2602 : C 75.58 H 10.99 Found: C 75.59 H 11.25

Pentadec-14-yn-l-ol, prepared by lithium aluminium hydride reduction of the acid, had m.p. 35-36°C.

Calcd. for C15H280: C 80.29 H 12.58 Found: C 80.31 H 12.72

Dihydropyran derivatives

The preparation of the dihydropyran derivative of dodec-l l-yn-l-ol is described as an example. Dihydropyran (25 g) was added to dodec-I 1-yn-l-ol

METHYLENE-INTERRUPTED C[s~cis-OCTADECADIENOIC ACIDS 413

(40.5 g) and phosphorus oxychlor ide (0.25 g) in an ice bath. The solut ion

was st irred at r oom tempera tu re for 3 hr, then po tass ium hydroxide solut ion

(5~0) added and the produc ts extracted with ether and the ethereal layer

washed with water. The ether solut ion was dr ied and solvent and excess

d i h y d r o p y r a n removed on a ro ta ry evapora tor . The p roduc t could not be

purified by low tempera tu re crysta l l izat ion or by dis t i l la t ion but could be

ob ta ined via column c h r o m a t o g r a p h y on Florisi l with benzene as elut ing

solvent in a sufficiently pure state for fur ther use.

Synthesis of diacetylenic acids' .from acidic intermediates

4,7-, 5,8-, 6,9-, 7,10-, 8,11- and 10,13-octadecadiynoic acids were p repared

f rom the app rop r i a t e o)-acetylenic acids. Where the d iynoic acid could not

be purified by crysta l l izat ion it was conver ted to the methyl ester (5~o

methanol ic HCI, 24 hr at r o o m tempera ture) , compara t ive ly volat i le inter-

mediates were disti l led off and the p roduc t purified by column c h r o m a t o -

graphy on Florisi l with benzene as elut ing solvent. Table 3 gives the physical

constants of the diacetylenic acids or methyl esters. The comple te synthesis

of 6 ,9-octadecadiynoic acid is given as an example.

TABLE 3

Octadecadiynoic acids and esters

Found a,h,e Acid or ester m.p. CC) Yield (°(,)

C H

2,5-Octadecadiynoic acid 53-54 56 78.16 9.99 3,6-Octadecadiynoic acid 84 85 56 78.30 10.11 6,9-Octadecadiynoic acid 33-34 59 76.45 9.61

11,14-Octadecadiynoic acid 63-64 58 76.96 9.95 12,15-Octadecadiynoic acid 55 57 75 77.54 10.20 13,16-Octadecadiynoic acid 83-85 66 77.42 10.32 Methyl 4.7-octadecadiynoate - 52 77.61 10.10 Methyl 5,8-octadecadiynoate - 23 78.14 I 0.14 Methyl 7,10-octadecadiyonate - 34 78.70 10.25 Methyl 8,11-octadecadiynoate 32 78.50 10.26 Methyl 10,13-octadecadiynoate - 27 77.05 9.59 Octadeca-14-yn-17-enoic acid '~ 55 56 77 77.92 10.69

~ Octadecadiynoic acids. Calcd. for C18H2802 : C 78.2 l, H 10.21. b Methyl octadecadiynoates. Calcd. for CloHz002: C 78.57, H 10.41. " Octadeca- 14-yn-17-enoic acid. Calcd. for C18H3002: C 77.65, H 10.86. ,l Methyl octadeca- 14-yn- 17-enoate had m.p. 12.5 13.5 °C.

6,9-Octadecadiynoic acid

Hept-6-ynoic acid 2a) (30 g) was a d d e d slowly to ethyl magnes ium b romide

(from Mg, 11.9 g; Et Br, 57 g) in t e t r ahydrofuran (500 ml) at 5 °C and the solut ion s t i r red vigorously at r o o m tempera tu re for 2 hr. Cuprous cyanide

4 1 4 WILLIAM W. CHRISTIE AND RALPH T. HOLMAN

(I .25 g) was added, followed 30 rain later by l -bromo-undec-2-yne (31.3 g),

and the mixture stirred at room temperature for 36 hr. Sulfuric acid (12 N,

200 ml) was added slowly and the solution extracted with ether and washed with sodium chloride solution (10'~/~,) to remove mineral acid. The ether layer

was extracted with ammonia (2 N containing 3 ...... ,,, sodium chloride, 3 x 200 ml) and the ammonia extract shaken with ether to eliminate neutral material.

The aqueous layer was acidified and extracted with ether. Alter drying, the

solvent was removed and 6,9-octadecadiynoic acid (22.2 g, 59~',~i) obtained

as white needles by crystallization from light petroleum (2 X).

Srnthesis q[" diacetylenic acids via the corresponding alcohols

The 2,5-, 3,6-, 11,14-, 12,15- and 13,16-octadecadiynoic acids and octadeca-

14-yn-17-enoic acid were synthesized by first preparing the corresponding

acetylenic alcohols and then oxidizing these with chromic acid 14) to the

acids. The physical constants of the C~8 alcohol intermediates are listed in table 4. The synthesis of 11,14-octadecadiynoic acid is described in detail as

an example.

TABLI- 4

Octadecadiynols

Alcohol m.p. ~ C) Yield ('!;i) Found a. b

C H

Octadeca-2,5-diyn-1-ol 46 47 67 82.16 I 1.33 Octadeca-3,6-diyn-l-ol 42 43 64 82.63 II .27 Octadeca-1 I, 14-diyn-l-ol 38 40 30 82.26 I 1.38 Octadeca- 12,15-diyn- I -ol 40 41 38 82.47 I 1.68 Octadeca- 13,16-diyn- 1 -ol 56 58 37 82.19 I 1.82 Octadeca- 14-yn- 17-en- I-ol 38 39 51 81.74 12.05

" Octadecadiynols. Calcd. for CasH:mO:C 82.38, H I 1.52. ~' Octadeca- 14-yn- 17-en-1 -ol. Calcd. for CtsHa20 : C 81.25, H 12.20.

Octadeca- 11,14-diyn- l-ol

2-(1 l ' -Dodecynyloxy)- te t rahydropyran (158.8 g) was added slowly to ethyl

magnesium bromide (from Mg, 5.25 g, Et Br, 25.7 g) in te t rahydrofuran (200 ml) at 0"C and the whole stirred at room temperature for 2 hr. Anhydrous cuprous cyanide (`1.0 g) was added followed, 30 min later, by l -bromohex-2- yne (38 g) and the solution refluxed overnight. Saturated ammonium chloride solution (250 ml) was added to the cooled solution and the mixture extracted with ether. The ethereal layer was washed with more ammonium chloride solution and dried. After removal of the solvent, the crude product was hydrolyzed with p-toluene-sulfonic acid in methano118). Octadec- l l ,14- diyn-l-ol (17.2 g, 30~o) was obtained by crystallization (3 X) f rom light

METHYLENE-INTERRUPTED cis,¢is-OCTADECADIENOIC ACIDS 415

petroleum. Its IR spectrum did not have bands at 3320 c m - ~ and 21 l0 c m - l

( - - C = CH) indicating absence of starting material.

11,14-Octadecadiynoic acid

Chromium trioxide (10 g) in water (35 ml) and conc. sulfuric acid (8 ml) was added over 1 hr to a vigorously stirred solution of octadeca- 1 l, 14-diyn- 1-ol (17 g) in acetone (350 ml) at Oc'C. The mixture was allowed to warm up to room temperature and was stirred for a further 4 hr. It was then poured into water and extracted with ether. The ether was washed free of inorganic acid and extracted with aqueous ammonia (2 N containing 37/~, sodium chloride, 3 x 150 ml portions). The combined ammonia layers were washed with ether, acidified with sulfuric acid (2 N) and the acidic product extracted with ether. After drying, the solvent was removed and octadeca-ll ,14-

diynoic acid (10.4 g, 58 ~o) was obtained as white needles by crystallization from light petroleum.

Methyl cis, cis-octadecadienoates

The diynoic acids were converted to methyl esters (methanolic HC1, 5 "/" / o ,

room temperature overnight) and purified by repeated chromatography on Florisil, with benzene as eluting solvent, to remove oxidized material which acts as a catalyst poison and which must be removed before hydrogenation.

TABLE 5

Critical solut ion temperatures , elemental analyses and melt ing points of isomeric methyl cis, c&-octadecadienoates

C.S.T. Found" m.p. ( C ) Ester

CC) C H of acid

N a A b Methyl 2 ,5-octadecadienoate 52.5 40 77.49 I 1.53 34 36 Methyl 3 ,6-octadecadienoate 21.5 2.5 77.55 11.48 22 23 Methyl 4 ,7-octadecadienoate 24 5.5 77.20 I 1.19 I 1-12 Methyl 5 ,8-octadecadienoate 22 4 77.32 11.44 8 - - 9 Methyl 6,9-octadecadienoate 19 --1.5 76.89 10.99 -- 10.5--- I 1.5 Methyl 7, I 0-octadecadienoate 18 --2.5 77.14 11.34 15 16 Methyl 8, I 1 -octadecadienoate 18 --2.5 77.18 11.39 - - 1 5 . 5 - - - 1 6 Methyl 9,12-octadecadienoate 'q8 2.5 - 8 - - 9 Methyl 10,13-octadecadienoate 18 --2.5 77.28 I 1.22 10.5 -I I Methyl I 1,14-octadecadienoate 19 --2.5 77.34 I 1.48 4.5 5.5 Methyl 12,15-octadecadienoate 19 --2.5 77.94 I 1.80 18 18.5 Methyl 13,16-octadecadienoate 19.5 -- 1 77.76 I 1.73 20.5-21.5 Methyl 14,17-octadecadienoate 18 0 77.61 11.56 37 37.5

Critical solut ion tempera ture (ni t romethane) . b Critical solut ion tempera ture (acetonitrile). c A n a l . Calcd. for C19Ha402: C 77.50, H 11.64. d Purchased f rom The Hormel Insti tute, Aus t in , Minn.

416 W I L L I A M W. CHRISTIE A N D R A L P H To H O L M A N

The esters in methanol were hydrogenated over Lindlar's catalyst x2) until close to the theoretical uptake of hydrogen was indicated, then quinoline was added and the remainder of the reaction monitored by GLC analysis. After removal of catalyst and solvent, the product was purified by Florisil chromatography. Yields of 90-95 % of products were obtained containing 2-5 % monoenoic esters as impurities as estimated by GLC (20}0 ethylene glycol succinate, 2 % phosphoric acid columns) and argentation thin-layer chromatography26). Infrared spectroscopy indicated less than 1 i'~, trans unsaturation and UV spectroscopy less than 1% conjugated double bonds in all cases. Small samples (50 rag) for physical measurements were purified by argentation thin-layer chromatography and larger amounts (2 g) by chro- matography on acid-washed Florisil impregnated with silver nitrate='V,2s). Acids were prepared from the methyl esters by refluxing with 5 ~.,; ethanolic potassium hydroxide for 45 rain followed by recrystallization of the extracted products from light petroleum at - 6 0 :C. The physical constants of the octadecadienoic esters and acids are detailed in table 5.

Complete Io'drogenation oJ" methyl octadecadienoates

The dienoic esters were hydrogenated in methanol solution with palladium oxide as catalyst, and a single product indistinguishable in chromatographic behavior (GLC) from methyl stearate was obtained in all cases, indicating absence of branched chain material or homologues.

Ozonolysis

The methyl octadecadienoates were reacted with a solution of ozone in pentane at - 6 5 ° C and the ozonides reduced with triphenylphosphinee9). The resulting aldehydes and aldesters were examined by GLC and identified by comparison of retention times with those of known standards. In each case, only 1-30j/o of fragments other than those expected were found.

Discussion

Physical measurements indicated little unusual in the properties of the natural 9,12-isomer in comparison to adjacent members of the series. The melting points of the acids (table 5) are lowest in those isomers in which the double bonds are centrally located in the chain, and rise as the double bonds move towards either end of the chain. There is a slight discontinuity in this trend at the 9,12-isomer. Critical solution temperatures (CST) for the isomeric series reveal that as the unsaturation is moved from the methyl end of the molecule, the isomeric methyl octadecadienoates have similar critical solution temperatures until the 6,9-isomer is reached. The CST value reaches a maximum at the 4,7-isomer, drops slightly for the 3,6-compound

MEFHYLENE-INTERRUPTED cis,cis-OCTADECADIENOIC ACIDS 417

and rises sharply with the 2,5-isomer, probably because the conjugation between the double bond and carboxyl group of this compound has a considerable effect on its solution properties.

InJmred spectra

The infrared spectra of the methyl octadecadienoates are quite similar, and show little difference from that of natural methyl linoleate. In the spectrum of the 2,5-isomer, however, there is a distinctive band at 1630 cm- J ( - - C H = C H . C O z C H 3 )and in that of the 14,17-isomer, prominent bands occur at 3110, 1630, 990 and 910 cm -I ( - - C H = C H 2 ) . In the series of methyl octadecadiynoates also, the spectra differ little from the norm though the 2,5-isomer has characteristic bands at 2230 cm J, 1300 cm-~ and 953 c m - ' fi'om interactions between the triple bond and carboxyl group.

Mass spectrometry

Mass spectra of a number of monoenoic fatty acid methyl esters have been recorded including many of the octadecenoates 30), all the hexenoates 31) and nonenoates32). Though minor differences are found in the spectra of the shorter monoenoic esters, 2-isomers alone have features in their spectra which differentiate them completely from the rest 30 3z). Mass spectra of few polyenoic esters are reported, but there are distinct differences in the spectra of methyl 6,9,12-octadecatrienoate and methyl 9,12,15-octadeca- trienoate34), which are best explained if the double bonds are less mobile than is apparently the case for monoenoic esters.

In the spectrum of methyl linoleate30), hydrocarbon peaks predominate. The base peak is at m/e=67 with other major low molecular weight frag- ments at m/e=81, 95, 55 and 41. The peak at m/e=263 (loss of CH3 O - ) is higher than that at m/e = 262 (M--CH3OH), the base peak in the spectrum of methyl oleate. Fragments of m/e = 74 and 220 (M-74) from the McLafferty rearrangement a~) are much smaller than the corresponding ions in the spectrum of methyl oleate. The peak at m/e = 91 (12}i; of base peak) may be due to tropylium ions 36) formed by a complex rearrangement involving cyclization. This peak becomes much larger in the spectra of polyunsaturated esters 30).

The spectra of the synthetic dienoic esters are qualitatively very similar to the published spectrum of methyl linoleate, with the exception of the 2,5-isomer. Fig. I contains the spectra of four typical isomers and table 6, the relative abundances of some of the more significant ions.

The base peak in most of the isomers is at m/e= 67 and low molecular weight hydrocarbon ions predominate. Ions more typical of saturated esters at m/e=43, 57, 69 and 83 are more prominent in the isomers with double

M

52;

,)

Fig.

I.

ME

TH

YL

i 1 !

2,5-

OC

TA

DE

CA

DIE

NO

AT

E

70

eV

=

ME

TH

YL

5,8

-OC

TA

DE

CA

DIE

NO

AT

E

I i !

ME

TH

YL

10,1

3- O

CT

AD

EC

AD

I EN

OA

T E

L I

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~ :

ii I

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:- :

2:",

ME

TH

YL

13,1

6- O

CT

AD

EC

AD

IEN

OA

TE

~5

i1: L p.

Mas

s sp

ectr

a at

70

eV o

f fo

ur

rep

rese

nta

tiv

e m

eth

yl

oct

adec

adie

no

ates

in

wh

ich

th

e u

nsa

tura

tio

n i

s ca

rbo

ny

l-co

nju

gat

ed,

mid

-ch

ain

or

nea

r th

e en

d o

f th

e ch

ain

.

METHYLENE-INTERRUPTED cis,cis-OCTADECAD1ENOIC ACIDS

TABLE 6 Major fragments in the mass spectra of isomeric methyl cis, cis-octadecadienoates

Relative abundances

M M-31 M-32 M-74 Isomer (294) (263) (262) (220) 74 91

2,5- 31 3 1 19 12 3,6- I 1 2 18 25 4,7- 27 I1 8 49 26 25 5,8- 41 5 14 26 28 26 6,9- 33 8 10 10 16 18 7,10- 31 11 10 7 17 20 8,11- 27 12 8 5 14 I1 9,12- 12 5 3 2 21 12

10,13- 25 4 9 3 40 8 11,14- 26 12 7 3 13 7 12,15- 7 6 4 2 14 9 13,16- 5 3 1 1 74 5 14,17- 19 11 8 4 37 3

419

bonds close to the carboxyl group. The molecular ion is at a max imum with

the 5,8-isomer and drops in a fairly regular progress ion for isomers with

double bonds more remote f rom the carboxyl group. A similar progress ion

occurs with the peaks at m/e = 91 and m/e = M-74, though the 4,7-isomer has

the max imum in this case. The peak at m/e = 74 is variable but becomes quite

large in the spectra o f some of the isomers in which the double bonds are

more remote f rom the carboxyl group. In some isomers, the ion m / e = M-31

is greater than that at m / e = M-32, though the total of these is approx i -

mate ly cons tan t and no pat tern is appa ren t in the variat ions. In general ,

however, only minor differences are found in the mass spectra with change in

double bond posi t ion and none which posit ively identify any par t icu lar

isomer.

The spectrum of methyl 2 ,5-octadecadienoate , however, is quite dist inct

and contains many features a l ready descr ibed for cis-2-unsaturated

esters3°-34). Cyclic s tructures have been p roposed for ions at m / e = l l l

and 113 31) and studies with deuter ium labelled compounds reinforce these

rationalizations3X,32). A p rominen t peak at m / e = 100 presents some diffi-

culties, however, and an open-chain ion of the fo rm:

CH CH /

CHaO C CH~ \

O + \

H

TA

BL

E

7

Prin

cipa

l fe

atur

es i

n th

e nu

clea

r m

agne

tic

reso

nanc

e sp

ectr

a of

iso

mer

ic m

ethy

l ci

s,ci

s-oc

tade

cadi

enoa

tes

Isom

er

--C

H

:: C

H--

--

OC

H3

--C

He-

- x

CH

2 --

(CH

2)~,

--

--C

H3

> 2,

5-

~ 4.

2 (4

)~

6.37

(3)

a 6.

60 (

2) ~

8.00

(2)

~ 8.

75 (

20p

9.12

(3)

~ 3,

6-

4.58

(4)

6.

37 (

3)

7.22

(2)

6.

97 (

2)

8.00

(2)

8.

73 (

18)

9.11

(3)

.~

4,

7-

4.70

(4)

6.

40 (

3)

7.23

(2)

7.

70 (

4)

8.00

(2)

8.

72 (

16)

9.10

(3)

=

5,8-

4.

72 (

4)

6.41

(3)

7.

26 (

2)

7.95

(6)

8.

30 (

2)

8.72

(14

) 9.

11 (

3)

6,9-

4.

69 (

4)

6.40

(3)

7.

25 (

2)

7.97

(6)

8.

71 (

16)

9.11

(3)

7,

10-

4.71

(4)

6.

40 (

3)

7.26

(2)

7.

92 (

6)

8.70

(16

) 9.

11 (

3)

> 8,

11-

4.72

(4)

6.

40 (

3)

7.26

(2)

7.

88 (

6)

8.67

(16

) 9.

10 (

3)

z 9,

12-

4.72

(4)

6.

40 (

3)

7.25

(2)

7.

88 (

6)

8.67

(16

) 9.

10 (

3)

>~

10,1

3-

4.73

(4)

6.

40 (

3)

7.27

(2)

7.

88 (

6)

8.69

(16

) 9.

09 (

3)

.~

11,1

4-

4.71

(4)

6.

41 (

3)

7.27

(2)

7.

90 (

6)

8.69

(16

) 9.

09 (

3)

~:

12,1

5-

4.71

(4)

6.

40 (

3)

7.27

(2)

7.

90 (

6)

8.69

(16

) 9.

02 (

3)

13,1

6-

4.70

(4)

6.

40 (

3)

7.25

(2)

7.

90 (

4)

8.72

(18

) 8.

38 (

3)

o 14

,17-

~

4.65

(5)

6.

41 (

3)

7.25

(2)

7.

90 (

4)

8.72

(20

) -

Z

Figu

re i

n br

acke

ts d

enot

es n

o. o

f pr

oton

s.

Che

mic

al s

hift

s ar

e ex

pres

sed

in p

.p.m

, do

wnf

ield

fro

m i

nter

nal

tetr

amet

hyls

ilan

e (r

= 1

0).

METHYLENE-INTERRUPTED cis,cis-OCTADECADIENOIC ACIDS 421

seems the most likely possibility. In the absence of isotope studies, the nature of a distinctive peak at m/e = 139 is uncertain but it is probably related to the prominent oxygenated peaks.

Nuclear magnetic resonance spectra (NMR)

The NMR spectra (proton) of a large number of fatty acids or their methyl esters have been obtained and are comprehensively described by HopkinsaT). NMR spectra of other polyunsaturated fatty acids have since been reported as,ag) as have the spectra of the complete series of methyl cis-octadecenoates (A 2--3 17) 40).

The NMR spectrum of methyl linoleate has six principal featuresag), a multiplet at 4.723 for the olefinic protons, a sharp singIet at 6.40~ for the protons on the methoxyl group, a triplet at 7.253 for the methylene group between the double bonds, a complex multiplet at 7.883 for the protons to the double bonds and carboxyl group, a broad band at 8.67 for the chain methylene protons and a partially resolved triplet at 9.10~ for the terminal methyl protons. Table 7 details the chemical shifts of these protons in all the methylene-interrupted cis,cis-octadecadienoates.

The 6,9- to 10,13-isomers have NMR spectra which are virtually identical to that reported for methyl linoleate and cannot be distinguished by this means. The spectra of the remaining isomers, however, have distinctive features which permit their identification.

in the spectrum of methyl 2,5-octadecadienoate, the C-2 olefinic proton gives a doublet at 4.25~, and the C-3, two overlapping triplets centered at 3.81 3 and 4.0as, a°). The olefinic protons on C-5 and C-6 give a multiplet centered at 4.65 ~. The signal for the diallylic methylene protons is a triplet shifted downfield to 6.60z (J =6.0 as opposed to 4.8 in methyl linoleate).

In the spectrum of methyl 3.6-octadecadienoate, the A 3 olefinic protons give a triplet at 4.48~4°), whereas the A 6 olefinic protons give a multiplet at 4.67~. The protons on C-2 produce a doublet at 6.973. The diallyl methylene protons give a triplet at 6.23 T.

With the 4,7-isomer, the protons on C-2 and C-3 give a distinctive signal at 7.703a9,4°). In the spectrum of methyl 5,8-octadecadienoate, there is a signal at 8.303 for the protons fl to the double bond and carboxyl group on C-3a9,4°). It also has only 14 polymethylene protons whereas most of the others have 16 (the 2,5- and 14,17-isomers have 20 and the 3,6- and 13,16- isomers have 18).

The spectra of the 11,14-, 12,15- and 13,16-isomers can be distinguished by the signal of the terminal methyl protons. With the 11,14-isomer, it is a distinct symmetrical triplet (J=6.0) at 9.09T, whereas with the 2,5- to 10,13-isomers, it is a poorly resolved distorted triplet a7, 40). [n the spectrum

422 W I L L I A M W . CHRISTIE A N D R A L P H T. H O L M A N

of methyl 12 ,15-oc tadecad ienoa te , the t r ip le t is at 9 .02z ( J : 7 . 8 ) 37,4°,41)

and in tha t o f me thy l 13 ,16-oc tadecad ienoa te , a d o u b l e t at 8.38~ is f ound 40,42).

In the spec t rum of methy l 14 ,17-oc tadecad ienoa te , a d o u b l e t at 5 .05z is

obse rved for the olefinic p ro tons on C-18. The mul t ip le t usual ly found for

the C-17 p r o t o n at 4 .3-4.4z 37,4°) is par t ia l ly s u b m e r g e d unde r the mul t ip le t

at 4.65~ for the C-14 and C-15 olef inic p ro tons , The re are, o f course , no

t e rmina l methyl p ro tons .

Acknowledgments

The a u t h o r s are i ndeb t ed to W. E. Brugger for the synthes is o f s o m e o f the

in te rmedia tes , H. Hayes for o p e r a t i o n o f the mass s p e c t r o m e t e r and M i n e r v a

V. G a n for p e r f o r m i n g the h y d r o g e n a t i o n s and ozonolyses .

References

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(1965) 372 17) J. M. Conia, Bull. Soc. Chim. France (1955) 1449 18) S. S. Nigam and B. C. L. Weedon, J. Chem. Soc. (I 956) 1049 19) H. B. Henbest, E. R. H. Jones and 1. M. S. Walls, J. Chem. Soc. (1950) 3646 20) T. H. Vaughn, R. R. Vogt and J. A. Nieuwland, J. Am. Chem. Soc. 56 (1934) 2120 21) Tchao Yin Lai, Bull. Soc. Chim. France 53 (1933) 1553 22) N. A. Khan, Org. Syn. Coll. Vol. IV. (1963) 969 23) A. Oskerko, Chem. Ber. 70B (1937) 55 24) M. Y. Kraft and E. G. Popova, Zh. Obshch. Khim. 27 (1957) 907 25) W. R. Taylor and F. M. Strong, J. Am. Chem. Soc. 72 (1950) 4263 26) L.J. Morris, Chem. Ind. London (1962) 1238 27) R. L. Anderson and E. J. Hollenbach, J. Lipid Res. 6 (1965) 577 28) D. Willner, Chem. Ind. London (1965) 1839 29) R. A. Stein and N. Nicolaides, J. Lipid Res. 3 (1962) 476

METHYLENE-INTERRUPTED C/s,£is-OCTADECADIENOIC ACIDS 423

30) B. Hallgren, R. Ryhage and E. Stenhagen, Acta Chem. Scand. 13 (1959) 845 31) W. K. Rohwedder, A. F. Mabrouk and E. Selke, J. Phys. Chem. 69 (1965) 1711 32) T. M. Groff, H. Rakoffand R. T. Holman, unpublished data 33) R. Ryhage, S. Stfillberg-Stenhagen and E. Stenhagen, Arkiv Kemi 18 (1961) 179 34) R. T. Holman and J. J. Rahm, In: Progress in the chemistry of fats and other lipids,

Vol. IX, R. T. Holman ed., Pergamon Press, Oxford-New York (1966) p. 15 35) F. W. McLafferty, Anal. Chem. 31 (1959) 82 36) P. N. Rylander, S. Meyerson and H. M. Grubb, J. Am. Chem. Soc. 79 (1957) 842 37) C. Y. Hopkins, In: Progress in the chemistry of fats and other lipids, Vol. VIII,

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