Enantiomeric Separation of Triacylglycerols Consisting of Three Different Fatty Acyls and Their Chiral Chromatographic Elution Behavior.

Chromatographic separation of triacylglycerol (TG) enantiomers is a highly challenging task of analytical chemistry because of the similar physicochemical properties. The analysis of chiral TGs is crucial for improving the knowledge of lipid biochemistry and for understanding the nutritional properties of fats and oils. Thus, this study aimed to systematically investigate the chiral resolution of TGs consisting of three different fatty acyls (FAs). Thirty-three asymmetric TG enantiopairs, including 49 synthesized enantiopure TGs and racemic TGs, were analyzed with a recycling chiral HPLC system. Twenty-six enantiopairs were successfully separated. Overall, having both unsaturated and saturated FAs in the outer positions or a difference in carbon chain length between two saturated FAs at the outer positions favored the separation of enantiomers. The retention time at separation correlated negatively with the sn-3 carbon number of the early eluting enantiomer and positively with the carbon number difference between sn-1 and sn-3. When the samples were studied in separate groups based on unsaturation and regioisomers, both the acyl carbon number and the degree of unsaturation of FAs in all three positions influenced the separation and elution behavior of chiral TGs, indicating an active role of both intermolecular interactions and steric hindrances. This is the first systematic study of the chiral separation of TGs consisting of three different FAs using a large number of enantiopairs. The novel findings on the behavior of TG enantiomers in a chiral environment provide important guidance and reference for a stereospecific study of the chemistry and biochemistry of natural lipids.

A New Approach in Lipase-Octyl-Agarose Biocatalysis of 2-Arylpropionic Acid Derivatives.

The use of lipase immobilized on an octyl-agarose support to obtain the optically pure enantiomers of chiral drugs in reactions carried out in organic solvents is a great challenge for chemical and pharmaceutical sciences. Therefore, it is extremely important to develop optimal procedures to achieve a high enantioselectivity of the biocatalysts in the organic medium. Our paper describes a new approach to biocatalysis performed in an organic solvent with the use of CALB-octyl-agarose support including the application of a polypropylene reactor, an appropriate buffer for immobilization (Tris base-pH 9, 100 mM), a drying step, and then the storage of immobilized lipases in a climatic chamber or a refrigerator. An immobilized lipase B from Candida antarctica (CALB) was used in the kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification with methanol, reaching a high enantiomeric excess (eep = 89.6 ± 2.0%). As part of the immobilization optimization, the influence of different buffers was investigated. The effect of the reactor material and the reaction medium on the lipase activity was also studied. Moreover, the stability of the immobilized lipases: lipase from Candida rugosa (CRL) and CALB during storage in various temperature and humidity conditions (climatic chamber and refrigerator) was tested. The application of the immobilized CALB in a polypropylene reactor allowed for receiving over 9-fold higher conversion values compared to the results achieved when conducting the reaction in a glass reactor, as well as approximately 30-fold higher conversion values in comparison with free lipase. The good stability of the CALB-octyl-agarose support was demonstrated. After 7 days of storage in a climatic chamber or refrigerator (with protection from humidity) approximately 60% higher conversion values were obtained compared to the results observed for the immobilized form that had not been stored. The new approach involving the application of the CALB-octyl-agarose support for reactions performed in organic solvents indicates a significant role of the polymer reactor material being used in achieving high catalytic activity.

Chemoenzymatic Synthesis of ABC-Type Enantiostructured Triacylglycerols by the Use of the p-Methoxybenzyl Protective Group.

This report demonstrates the first asymmetric synthesis of enantiopure structured triacylglycerols (TAGs) of the ABC type presenting three non-identical fatty acids, two of which are unsaturated. The unsaturated fatty acids included monounsaturated oleic acid (C18:1 n-9) and polyunsaturated linoleic acid (C18:2 n-6). This was accomplished by a six-step chemoenzymatic approach starting from (R)- and (S)-solketals. The highly regioselective immobilized Candida antarctica lipase (CAL-B) played a crucial role in the regiocontrol of the synthesis. The synthesis also benefited from the use of the p-methoxybenzyl (PMB) ether protective group, which enabled the incorporation of two different unsaturated fatty acids into the glycerol skeleton. The total of six such TAGs were prepared, four constituting the unsaturated fatty acids in the sn-1 and sn-2 positions, with a saturated fatty acid in the remaining sn-3 position of the glycerol backbone. In the two remaining TAGs, the different unsaturated fatty acids accommodated the sn-1 and sn-3 end positions, with the saturated fatty acid present in the sn-2 position. Enantiopure TAGs are urgently demanded as standards for the enantiospecific analysis of intact TAGs in fats and oils.

Influence of Dietary Triacylglycerol Structure on the Accumulation of Docosahexaenoic Acid [22:6(n-3)] in Organs in a Short-Term Feeding Trial with Mildly Omega-3 Deficient Rats.

SCOPE: To study the effect of positional distribution of docosahexaenoic acid (DHA) in dietary triacylglycerols (TAG) on the tissue fatty acid content and composition of mildly (n-3) deficient rats. METHODS AND RESULTS: In a 5-day feeding trial, mildly (n-3) deficient rats received 360 mg daily structured TAGs: sn-22:6(n-3)-18:0-18:0, sn-18:0-18:0-22:6(n-3), sn-18:0-22:6(n-3)-18:0, or tristearin. A fifth group receives standard (n-3) adequate feed AIN-93G from birth till the end of the trial. The DHA-fed groups show significantly higher DHA levels in the liver and visceral fat compared to the tristearin or normal feed groups showing that the dose and the short feeding period of DHA were sufficient to restore the DHA content in the organs of (n-3) deficient rats. Feeding sn-1 DHA resulted in higher levels of DHA in the liver TAG compared to sn-3 DHA feeding, although the difference did not reach statistical significance. CONCLUSION: These findings indicated a possible difference in the tissue accumulation and/or metabolic fate of DHA from the sn-1 and sn-3 positions of TAG.

Metabolomic Investigation of Brain and Liver in Rats Fed Docosahexaenoic Acid in Regio- and Enantiopure Triacylglycerols.

SCOPE: N-3 polyunsaturated fatty acids (n-3 PUFAs) play important roles in cognitive functions. However, there is a lack of knowledge on the metabolic impact of regio- and stereo-specific positioning of n-3 PUFAs in dietary triacylglycerols. METHODS AND RESULTS: Rats in a state of mild n-3 PUFA deficiency are fed daily with 360 mg triacylglycerols containing DHA (docosahexaenoic acid) at sn (stereospecific numbering)-1, 2, or 3 positions and 18:0 at remaining positions, or an equal amount of tristearin for 5 days. Groups fed with n-3 deficient diet and normal n-3 adequate diet are included as controls. The metabolic profiles of the brain and liver are studied using NMR (nuclear magnetic resonance)-based metabolomics. Several metabolites of significance in membrane integrity and neurotransmission, and glutamate, in particular, are significantly lower in the brain of the groups fed with sn-1 and sn-3 DHA compared to the sn-2 DHA group. Further, the tristearin and DHA groups show a lower lactate level compared to the groups fed on normal or n-3 deficient diet, suggesting a prominent role of C18:0 in regulating energy metabolism. CONCLUSION: This study sheds light on the impact of stereospecific positioning of DHA in triacylglycerols and the role of dietary stearic acid on metabolism in the brain and liver.

Asymmetric Synthesis of Methoxylated Ether Lipids: Total Synthesis of Polyunsaturated C18:3 Omega-3 and Omega-6 MEL Triene Derivatives.

The asymmetric synthesis of polyunsaturated triene C18:3 n-3 and C18:3 n-6 methoxylated ether lipids (MEL) of the 1-O-alkyl-sn-glycerol type is described as possible structural candidates for a triene C18:3 MEL of an unknown identity found in a mixture of shark and dogfish liver oil. Their C18:3 hydrocarbon chains constitute an all-cis methylene skipped n-3 or n-6 triene framework, along with a methoxyl group at the 2'-position and R-configuration of the resulting stereogenic center. The methoxylated polyenes are attached by an ether linkage to the pro-S hydroxymethyl group of the glycerol backbone. The syntheses were based on the polyacetylene approach that involves a semi-hydrogenation of the resulting triynes. Both syntheses were started from our previously described enantio- and diastereomerically pure isopropylidene-protected glyceryl glycidyl ether, a double-C3 building block that was designed as a head group synthon for the synthesis of various types of MELs.