Synthesis of conjugated polyunsaturated fatty acids

Details Synthesis of conjugated polyunsaturated fatty acids Synthesis of conjugated polyunsaturated fatty acids

1998-10-20

2001-11-13

Carr, Deborah D. (Department: 1621)

Organic compounds -- part of the class 532-570 series

Organic compounds

Fatty compounds having an acid moiety which contains the...

C554S223000, C554S224000

Reexamination Certificate

active

06316645

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
Not Applicable
BACKGROUND OF THE INVENTION
This invention relates generally to conjugated fatty acids and, in particular, to a method of making such conjugated fatty acids and isolating specific isomers thereof.
In recent years, substantial interest has arisen in conjugated polyunsaturated fatty acids, especially conjugated linoleic acid. Polyunsaturated fatty acids are those fatty acids that have two or more double bonds between carbon atoms in their hydrocarbon chain. These acids are usually vegetable-derived and consist of hydrocarbon chains of 18 or more carbon atoms. Of these acids, linoleic, linolenic and arachidonic acid are the so-called essential fatty acids.
The positioning of the double bonds in the hydrocarbon chain is typically not in a conjugated, i.e., alternating double bond-single bond-double bond, manner. For example, linoleic acid is an octadecadienoic fatty acid having an eighteen carbon chain with two double bonds (18:2), one between carbons 9 and 10 and one between carbons 12 and 13, in which the configuration about each double bond is the cis configuration, i.e., cis-9,cis-12-octadecadienoic acid (or c9,c12-octadecadienoic acid). Linolenic acid is also an eighteen carbon acid but with three double bonds (18:3) at carbons 9, 12 and 15 in which all three double bonds have in the cis configuration, i.e., c9,c12,c15-octadecadienoic acid. Changing the position of the double bonds, e.g., conjugation, gives rise to many positional and geometric (i.e., cis-trans) isomers.
Conjugated linoleic acid (CLA) is a collective term for positional and geometric isomers of linoleic acid having a conjugated double-bond system starting at carbon 9, 10 or 11. For example, one CLA positional isomer has double bonds between carbons 9 and 10 and carbons 11 and 12 (i.e, 9,11-octadecadienoic acid); another has double bonds between carbons 10 and 11 and carbons 12 and 13 (i.e., 10,12-octadecadienoic acid), each with several possible cis and trans isomers. Because of cis/trans isomerism, the 9,11 and 10,12 CLA's can have eight geometric different isomers, i.e., cis-9, cis-11; cis-9,trans-11; trans-9,cis-11; trans-9,trans-11; cis-10,cis-12; cis-10,trans-12; trans-10,cis-12 and trans-10, trans-12.
Although a conjugated structure is not usual in fatty acids, the existence of CLA has been known for many years, and CLA is found naturally in milk, dairy products and meat for ruminants because of its formation as an intermediate of biohydrogenation by anaerobic bacteria in the rumen. The cis-9,trans-11 and the trans-10,cis-12 isomers appear to be the most abundant isomers.
The cis-9,trans-11 isomer has been shown to be the first intermediate in the biohydrogenation of linoleic acid by the anaerobic rumen bacterium
Butyrvibrio fibrisolvens.
This reaction is catalyzed by the enzyme linoleate isomerase which converts the cis-
12
double bond of linoleic acid to a trans-11 double bond. (C. R. Kepler et al., 241
J. Biol. Chem.
(1966) 1350.) It has also been found that the normal intestinal flora of rats can convert linoleic acid to the cis-9,trans-11 isomer. The reaction does not, however, take place in animals lacking the required bacteria. Therefore, CLA is largely a product of microbial metabolism in the digestive tract of primarily ruminants, but to a lesser extent in other mammals and birds.
Interest in CLA has increased because of reports that dietary CLA reduces carcinogenesis, atherosclerosis and body fat in laboratory animals (see, e.g., B. F. Haumann, 1
Inform
(1996) 152; C. Steinhart, 73
J. Chem. Ed.
(1996) A302). To date, there is no conclusive evidence as to which isomer or isomers of the many CLA isomers is the active component(s). However, it is generally assumed that the active isomer is the major isomer, i.e., the cis-9,trans-11 isomer, found in dairy products. Whether eaten in the diet or synthesized in the digestive tract, CLA is absorbed from the gut and distributed throughout the body wherein the cis-9,trans-11 isomer is incorporated into blood lipids, cell membranes and fat tissue.
CLA has been found to be an in vitro antioxidant, and in cells, it protects membranes from oxidative attack. In relation to other important dietary antioxidants, it quenches singlet oxygen less effectively than &bgr;-carotene but more effectively than &agr;-tocopherol. It appears to act as a chain terminating antioxidant by chain-propagating free radicals.
Currently, commercial sources of CLA are produced by alkaline isomerization of linoleic acid, i.e., by heating linoleic acid with sodium hydroxide in ethylene glycol at high temperatures. Commercial samples so produced yield complex mixtures of many different isomers. (P. I. Nichols, Jr. et al., 73
J. Am. Chem.
(1951) 247.) In a recent report (N. Sehat et al., 33
Lipids
(1998) 217), using silver-ion impregnated high performance liquid chromatography, a commercial sample of CLA was separated into twelve separate peaks. Other methods of preparation have been reported; see, e.g., U.S. Pat. No. 4,381,264 issued to Struve which utilizes sulfur dioxide in the presence of soap-forming bases but appears to produce primarily trans-trans conjugated fatty acids; U.S. Pat. No. 4,164,505 issued to Krajca which discloses a flow process for conjugating unsaturation of fatty acids using alkali metal hydroxides; WO 97/18320, a PCT published application disclosing a process for the preparation of materials with a high content of long chain polyunsaturated fatty acids; Mounts et al.; 5
Lipids
997 (1970). As some of the isomers may give rise to undesirable side effects (namely, 11,13 isomers), an alternative method that affords only the desirable isomers is clearly warranted.
Conjugation of linolenic acid with its three double bonds affords the possibility of two conjugated double bond systems; namely, conjugated diene isomers and conjugated triene isomers. The conjugated diene isomers are essentially CLA equivalents. A conjugated triene isomer, pseudo-eleostearic acid, 10,12,14-octadecadienoic acid, has long been reported, formed by partially converting linolenic acid with alcoholic alkalies (Kass and Burr, 61
J. Am. Chem. Soc.
(1939) 3292).
Despite recognition of the need for isomeric purity of CLA and other conjugated fatty acids, the prior art has produced very little in the way of a practical technique for such synthesis and isomeric separation.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a method of preparation of conjugated polyunsaturated fatty acids. The conjugated fatty acids are suitably conjugated diene and conjugated triene fatty acids. Conjugated diene fatty acids include conjugated linoleic acid (CLA) and conjugated diene linolenic acid. Conjugated triene fatty acids include fully conjugated linolenic acid. The method of the present invention is carried out under relatively mild conditions compared to the refluxing conditions of the currently used alkaline isomerization.
The foregoing, and other advantages of the present invention, are realized in its most general aspect thereof in method comprising the steps of deprotonating a polyunsaturated fatty acid with a superstrong base, e.g., pK>30, and then reprotonating the fatty acid to yield the desired conjugated isomers. The polyunsaturated fatty acid substrate has two or more double bonds in its hydrocarbon chain. The deprotonating step selectively removes diallylic hydrogens from the fatty acid, and is carried out at low temperature. The reprotonating step is accomplished with reaction with a strong acid. Isomeric purity can then be achieved by reaction of the conjugated isomers with a regioselective lipase that can discriminate between isomers to yield the substantially pure desired conjugated isomer.
In an illustrated embodiment, the invention provides a method of preparing stereospecific isomers of linoleic acid, including deprotonating linoleic acid with the superstrong base, and then reprotonating it to substantially yield only two major isomers, cis-9,trans-11 and cis-12,trans-10. These isomers are then treated

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