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METHODS

Preparation of cis, cis, cis-5,8,11-Eicosatrienoic Acid from Arachidonic Acid AMITABHA GHOSH, MALATI KOLEY and J. DUTTA, Department of Chemistry, Bose Institute, 93/I A.P.C. Road, Calcutta 700 009, India

ABSTRACT

Arachidonic acid was reduced by hydrazine to yield isomeric eicosatrienoic acids with other product~ Methyl cis,cis,cis-5,8,11-eicosatrienoate was isolated from the products by silver ion chro- matography and preparative gas liquid chromatography in 8% yield. The structure was confirmed by spectral studies and oxidative degradation. Lipids 17:314-316, 1982.

INTRODUCTION

It was first shown by Mead and Salton and Fulco and Mead (1,2) that eicosa-5,8,11- trienoic acid (20:3609) was derived from oleic acid (18:1 609), sometimes called Mead's acid. The structure of this eicosatrienoic acid (20:36o9) was also established by Mead and Salton (1). It is well established that, under essential fatty acid (EFA)-deficient conditions, the arachidonic acid (20:4606) level decreases and there is a considerable increase of 20:3609 in different tissues of rat (3-5), which normally contains a negligible amount of the lat ter acid. Although it is quite clear that EFA deficiency is responsible for accumulation of 20:3609 acid, very little is known about the effect of this acid on various metabolic functions, because of the very limited availability of this acid. The significance of 20:3609 acid in EFA deficiency has been currently reviewed (6).

A novel, general and flexible method for the synthesis of some EFA was described by Osbond et al. (7). Their method consisted of stereospecific partial reduction of the corres- ponding polyacetylenic acids. These were prepared by the condensation of various propargyl bromides with the di-Grignard derivatives of r acids. Subsequently, various polyethenoid fatty acids were synthe- sized for metabolic studies, notably by Sprech- er (8-10), with some modification of the original method of Osbond et al. (7). Synthesis of 20:3606 has also been reported (9). For the purpose of some metabolic studies with 20: 36o9, we have developed a simple and very short route to its preparation, by the partial reduction of 20:4606, which is easily available.

MATERIALS AND METHODS

Arachidonic acid (20:4606) and methyl eicosa-8,11,14-trienoate (20:3606) were pur- chased from Nu-Chek-Prep. Inc., Elysian, MN. All the reagents and solvents were of analytical grade. Solvents were dried and distilled before use .

Hydrazine Hydrate Reduction

To 100 mg of arachidonic acid in 50 ml of aq (50%) ethanol, 1 ml of hydrazine hydrate (99%, BDH, England) was added and stirred vigorously at 50 C (11). Five-ml aliquots were withdrawn at intervals of 1 hr up to 7 hr. From each of the aliquots, fat ty acids were extracted with diethyl ether, methyla ted with diazometh- ane (12) and analyzed by gas liquid chromatog- raphy (GLC). For a preparative scale, 500 mg of arachidonic acid was dissolved in 250 ml of aq ethanol, 3 ml of hydrazine hydrate was added and the mixture was stirred vigorously for 5 hr at 50 C. The fat ty acids were extracted and methylated by diazomethane.

Argentation Column Chromatography

The mixed methyl esters obt'ained by the partial reduction of 20:4606 acid was charged in a glass column (50 cm x 3 cm) containing silver nitrate (20%) coated silicic acid (150 g) and eluted with a solvent system of light petroleum (40-60 C) containing increasing amounts of diethyl ether (13). The column chromatography was monitored by GLC, using a 10% DEGS column (6 ft x 0.25 in.).

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METHODS

Isolation of 20:3~9 by Preparative GLC

The pooled triene fraction obtained from argentation column chromatography was sub- jected to preparative GLC using glass columns (2.1 m x 8 mm) packed with 15% DEGS on Diatomite C (AW, DMCS-treated, 60-70 mesh) obtained from Pye Unicam Ltd., England. The purity of 20:36o9 acid isolated was checked by analytical GLC. The overall yield was 8% of arachidonic acid.

Determination of Double Bond Position of 20:3~9

Thirty mg of 20:3609 was reduced by hydrazine hydrate (11)for 3.5 hr. After extrac- tion and methylation of the reduced products, the monoenes were isolated by silver ion TLC (14) and were subjected to permanganate- periodate oxidation (15). The fatty acids were recovered and methylated. The mono- and dicarboxylic acids were separated on thin layer chromatographic (TLC) plate, recovered, and analyzed separately for their chain lengths (16).

Nuclear Magnetic Resonance (NMR), Mass Spectrometry (MS) and Elemental Analysis

The tH NMR (CHC13, d) 0.9 ppm (t, 3H, CHa CH2), 1.3 (broad, 10H, C H2), 1.7 (m, 4H, CH2 CH2 CH = CH) 2.0 (d, 4H, CH 2 CH = CH) 2.25 (t, 21t, CH2COO), 2.8 (t, 4H, = CHCH2CH =) 3.7 (s, 3H, C HaOCO) , 5.3 (t, 6H, C_H = CH). The MS had a parent ion peak at m/e 320. Elemental analysis of methyl eicosa- trienoate, C21H~O2, requires C, 78.69; H, 11.32 and 0, 9.98%; found C, 78.42; H, 11.20 and 0, 9.94%.

RESULTS AND DISCUSSION

The GLC analyses of the reduction product of 20:4606 at 5 hr, which showed maximal production of 20:3609 along with other prod- ucts, are presented in Table 1.

It is known that hydrazine randomly reduces polyethenoid fatty acids without double bond movement or stereomutation (17). Theoret- ically, 15 products are possible altogether, of

315

which there should be 4 trienes, 6 dienes, 4 monoenes and 20:0. In the present study, traces of 20:0 were found along with some unreduced 20:4606. The main peaks in the GLC tracing had shoulders, indicating the presence of other compounds. With the help of pub- lished equivalent chain length (ECL) values (18,19) on the DEGS column and using authen- tic 20:3606, it was possible to identify 20:3609 and 20:3606. The triene band isolated by silver ion column chromatography contained pri- marily 20:3609 and 20:3606 with some con- taminants, probably the other triene isomers. Isolation of 20:3609 by preparative GLC produced a perfect Gaussian peak with an ECL of 21.2 by analytical GLC on a 10% DEGS column.

The NMR data support the presumption that the FAME in question contains 3 double bonds, interrupted by 2 methylene groups. The MS of methyl arachidonate, its fully reduced product, and of the purified 20:3609 gave molecular ion peaks at 318, 326 and 320, respectively, and confirmed the presence of 3 double bonds in the purified 20:3609.

The GLC of the mono- and dicarboxylic acid methyl esters obtained by the permanga- nate-periodate oxidation of 20:3609 produced peaks due to 9-, 12- and 15-carbon chain monocarboxylic acids and 5-, 8- and 11-carbon chain dicarboxylic acids. These facts unam- biguously indicate that 20:3609 contained double bonds at the 5-, 8- and ll-positions.

Table 1 shows that, under optimal condi- tions, the percentages of 20:3609 and 20:3606 are 31 and 37, respectively. Although low, the overall 8% yield of 20:3609 is encouraging in the sense that the production of 20:3609 through animal experiments or by total syn- thesis are long and expensive routes.

ACKNOWLEDGMENT

Authors are t h a n k f u l to Prof. S.C. Bhattacharyya, Director, and Prof. A. Sen, Chairman, Department of Chemistry, for providing research faci l i t ies and to Indian Council of Medical Research for providing a fellowship to M. Koley.

TABLE 1

Peak 1 2 3 4 5

Components 2 0 : 0 2 0 : 1 20:3to9 20:3to6 20:4~6 20:2

% (w/w) 1 l 1 31 37 20 ECL 20.0 20.5 21.2 21.8 22.5

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316 METHODS

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[Received August 11, 1981 ]

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