Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The asymmetric synthesis of a triene C18 : 3 n‐8 methoxylated ether lipid (MEL) of the 1‐O‐alkyl‐sn‐glycerol type is described by two different routes. The C18 : 3 hydrocarbon chain is attached by an ether linkage to the pro‐S hydroxymethyl group of the glycerol backbone, and constitutes an all‐cis methylene skipped triene framework, along with a methoxyl group in the 2'‐position and R‐configuration at the resulting chiral centre. The initial synthesis was based on the polyacetylene approach involving a semi‐hydrogenation of the resulting triyne. A modified approach based on a combined polyacetylene‐Wittig strategy was also successfully executed and an attempt made to compare and evaluate these strategies. Both syntheses were started from our previously described enantio‐ and diastereomerically pure isopropylidene‐protected glyceryl glycidyl ether, a double‐C3 building block designed as a head group synthon for synthesis of various types of MELs which was shown to suit the Wittig‐based approach very well. [ABSTRACT FROM AUTHOR]

Thermal stability is one of the essential parameters characterizing biocatalysts with potential applications in the chemical and pharmaceutical industries. Therefore, it is extremely important to develop standardized procedures for enzyme stability studies. The paper attempts to assess the thermal stability of immobilized lipases in aqueous buffers: lipase B from Candida antarctica (CALB) and lipase from Candida rugosa (CRL-OF) immobilized on the Octyl-Sepharose CL-4B carrier. As part of the optimization conditions of the immobilization, the influence of time on the catalytic activity and lipase loading, as well as the effect of temperature on lipase activity (optimal incubation—14 h at 4 °C), was determined. The thermal stability test procedure was carried out for 7 days using a climatic chamber (65 °C) and a refrigerator (4 °C). The studies of immobilized lipases included the assessment of the impact of various solvents (water, citrate buffer, 1,2-dichloropropane—DCP), temperature, light in the visible spectral range (400–800 nm), and additions of calcium ions. The highest value of residual activity (564.5 ± 21.6%) was received by storing the immobilized CALB in citrate buffer (pH 4.0, 500 mM) with the addition of calcium ions (Ca2+). On the other hand, residual activity values for immobilized CRL-OF after storage in the climatic chamber were lower than 5%. A combining of techniques: immobilization onto the support in high ionic strength and low pH, with a technique of extremally high-temperature applied in a climatic chamber, with the addition of Ca2+ allowed to achieve of excellent thermal stability of the immobilized CALB, with increasing of catalytic activity more than five-fold. Additionally, performing studies on the thermal stability of the tested lipases using a climatic chamber seems to be particularly promising in the context of unifying and standardizing storage guidelines, enabling the comparison of results between different laboratories, as well as enhancing catalytic activity. [ABSTRACT FROM AUTHOR]

The application of the climatic chamber presented in this paper to assess the storage stability of immobilized lipases is a new approach characterized by the potential of unifying the study conditions of biocatalysts created in various laboratories. The data achieved from storing lipases in the climatic chambers may be crucial for the chemical and pharmaceutical industry. Our paper describes the developed protocols for immobilization via interfacial activation of lipase B from Candida antarctica (CALB) and lipase OF from Candida rugosa (CRL-OF) on the Octyl-Sepharose CL-4B support. Optimization included buffers with different pH values of 4–9 and a wide range of ionic strength from 5 mM to 700 mM. It has been shown that the optimal medium for the CALB immobilization process on the tested support is a citrate buffer at pH 4 and high ionic strength of 500 mM. Implementing new optimal procedures enabled the hyperactivation of immobilized CALB (recovery activity 116.10 ± 1.70%) under the applicable reaction conditions using olive oil as a substrate. Importantly, CALB storage stability tests performed in a climatic chamber under drastic temperature and humidity conditions proved good stability of the developed biocatalyst (residual activity 218 ± 7.3% of dry form, after 7 days). At the same time, the low storage stability of CRL OF in a climatic chamber was demonstrated. It should be emphasized that the use of a climatic chamber to test the storage stability of a dry form of the studied lipases immobilized on Octyl-Sepharose CL-4B is, to our knowledge, described for the first time in the literature. [ABSTRACT FROM AUTHOR]

This report describes synthesis of novel structured triacylglycerols (TAGs) and diacylglyceryl ethers (DAGEs) of the 1- O-alkyl- sn-glycerol type constituting EPA and DHA as the sole fatty acids. One of two TAGs was comprised of a pure EPA located at the 1,3-positions with pure DHA at the 2-position and the other had it reversed with a pure DHA at the 1,3-positions and pure EPA at the 2-positions. The similarly structured DAGEs were derived from chimyl-, batyl-, and selachyl alcohols constituting pure EPA at their sn-3 position with pure DHA at the sn-2 position as well as the opposite composition with pure DHA at the sn-3 position and EPA at the sn-2 position, the total of six such DAGE products. The syntheses of these compounds were brought about by a two-step chemoenzymatic process involving a highly regioselective immobilized Candida antarctica lipase to incorporate EPA or DHA activated as acetoxime esters exclusively into the 1,3-positions of glycerol and the sn-3 position of the 1- O-alkyl- sn-glycerols. The second PUFA acyl groups were subsequently introduced to the remaining 2-positions by EDCI coupling agent to accomplish the title compounds highly efficiently in very high to excellent yields (86-92%). Practical applications: The work described is based on a previously reported methodology to introduce n-3 PUFAs activated as oxime esters exclusively into the terminal 1,3-positions of glycerol and 1- O-alkyl- sn-glycerols to prepare reversed structured TAGs and DAGEs by use of immobilized Candida antarctica lipase. The methodology is ideal for introducing isotopically labeled fatty acids into predetermined positions of TAGs and DAGEs. Novel structured triacylglycerols (TAGs) and diacylglyceryl ethers (DAGEs), constituting EPA and DHA as the sole fatty acids, were obtained by a two-step chemoenzymatic synthesis comprised of an exclusive incorporation of EPA or DHA, activated as acetoxime esters, into the terminal positions of glycerol and 1- O-alkyl- sn-glycerols by use of immobilized Candida antarctica lipase, and a subsequent introduction of a second PUFA acyl group to the remaining 2-positions by EDCI coupling agent. [ABSTRACT FROM AUTHOR]

This report describes the synthesis of reversed structured 1-O-alkyl-2,3-diacylsn-glycerols (DAGEs) possessing a pure saturated even number fatty acid (C6:0-C16:0) at the sn-2 position along with a pure EPA or DHA located at the terminal sn-3 position of the glycerol backbone of chimyl, batyl and selachyl alcohols. These adducts were synthesized by a highly efficient two-step chemoenzymatic process involving an immobilized Candida antarctica lipase to introduce pure EPA and DHA activated as oxime esters exclusively to the sn-3 terminal position of enantiopure chimyl, batyl and selachyl alcohols in excellent yields. The saturated fatty acids were subsequently incorporated to the remaining sn-2 position of the resulting 3-monoacylglyceryl ethers (3-MAGEs) using EDAC coupling agent in the presence of DMAP in very high to excellent yields (85%-98%). No losses of enantiomeric composition were observed during these processes. The multiple utilities of the resulting focused library of reversed structured DAGEs are discussed including how such compounds may possibly be utilized within the pharmaceutical area. [ABSTRACT FROM AUTHOR]

Abstract: A novel approach has been developed for activating the highly bioactive long-chain n-3 polyunsaturated fatty acids EPA and DHA as oxime esters and incorporating them exclusively to the end-positions of glycerol and enantiopure 1-O-alkylglycerols. The Candida antarctica lipase B was observed to display a superb regioselectivity when using the acetoxime esters of EPA and DHA as acyldonors under mild condition to keep acyl-migration side-reaction under complete control. Regiopure 1,3-diacylglycerols, 1-O-alkyl-3-acyl-sn-glycerols and their antipodes possessing EPA and DHA were afforded in very high purity and yields. [Copyright &y& Elsevier]

Background: n-3 polyunsaturated fatty acids, namely docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), reduce the risk of cardiovascular disease and can ameliorate many of obesity-associated disorders. We hypothesised that the latter effect will be more pronounced when DHA/EPA is supplemented as phospholipids rather than as triglycerides. Methodology/Principal Findings: In a 'prevention study', C57BL/6J mice were fed for 9 weeks on either a corn oil-based high-fat obesogenic diet (cHF; lipids ∼35% wt/wt), or cHF-based diets in which corn oil was partially replaced by DHA/EPA, admixed either as phospholipids or triglycerides from marine fish. The reversal of obesity was studied in mice subjected to the preceding cHF-feeding for 4 months. DHA/EPA administered as phospholipids prevented glucose intolerance and tended to reduce obesity better than triglycerides. Lipemia and hepatosteatosis were suppressed more in response to dietary phospholipids, in correlation with better bioavailability of DHA and EPA, and a higher DHA accumulation in the liver, white adipose tissue (WAT), and muscle phospholipids. In dietary obese mice, both DHA/EPA concentrates prevented a further weight gain, reduced plasma lipid levels to a similar extent, and tended to improve glucose tolerance. Importantly, only the phospholipid form reduced plasma insulin and adipocyte hypertrophy, while being more effective in reducing hepatic steatosis and low-grade inflammation of WAT. These beneficial effects were correlated with changes of endocannabinoid metabolome in WAT, where phospholipids reduced 2-arachidonoylglycerol, and were more effective in increasing anti-inflammatory lipids such as N-docosahexaenoylethanolamine. Conclusions/Significance: Compared with triglycerides, dietary DHA/EPA administered as phospholipids are superior in preserving a healthy metabolic profile under obesogenic conditions, possibly reflecting better bioavalability and improved modulation of the endocannabinoid system activity in WAT. [ABSTRACT FROM AUTHOR]

Abstract: The naturally occurring 1-O-alkyl-sn-glycerols and their methoxylated congeners, 1-O-(2′-methoxyalkyl)-sn-glycerols, are biologically active compounds, ubiquitously found in nature as diacyl glyceryl ether lipids and phosphoether lipids. The chief objective of this article is to provide a comprehensive and up to date review on such ether lipids. The occurrence and distribution of these compounds in nature are extensively reviewed, their chemical structure and molecular variety, their biosynthesis and chemical synthesis and, finally, their various biological effects are described and discussed. An unprecedented biosynthesis of the 2′-methoxylated alkylglycerols is proposed. The first synthesis of enantiopure (Z)-(2′R)-1-O-(2′-methoxyhexadec-4′-enyl)-sn-glycerol, the most prevalent 2′-methoxylated type alkylglycerol present in cartilaginous fish, is described. It was accomplished by a highly convergent five step process. [Copyright &y& Elsevier]

Abstract: Synthesis of symmetrically structured triacylglycerols possessing bioactive n−3 polyunsaturated fatty acids (eicosapentaenoic acid or docosahexaenoic acid) at the 2-position and a short-chain fatty acid (C2, C4, C6) located at the end-positions by a highly efficient two-step chemoenzymatic process is described. Full regiocontrol devoid of any acyl-migration side reactions was obtained in both a lipase promoted step to introduce the short-chain fatty acids exclusively into the primary alcohol positions of glycerol using activated vinyl esters at low temperature and a subsequent coupling reaction involving free EPA and DHA using EDAC as a coupling agent. [Copyright &y& Elsevier]

A highly efficient chemoenzymatic method for the synthesis of enantiopure ABC-type asymmetrically structured triacyl�glycerols has been developed starting from enantiopure (S)-solketal and involving two lipase steps. [ABSTRACT FROM AUTHOR]

This paper focuses on a detailed evaluation of commercially available immobilized lipases and simple monohydric alcohols for the production of alkyl esters from sunflower oil by enzymatic alcoholysis. Six lipases were tested with seven alcohols, including straight and branched-chain primary and secondary alcohols. The reactions were conducted in a batch stirred reaction vessel using stoichiometric amounts of substrafes under solvent-free conditions. Dramatic differences in alcoholysis performance were observed among the different lipases. For most of the alcohols, Novozym 435 produced the highest yield of FA alkyl esters, with yields well over 90% for methanol, absolute ethanol, and 1-propanol. Overall, 96% ethanol was the preferred alcohol for all lipases except Novozym 435, and ethanolysis reactions reached the maximal conversion efficiency. Increasing the water content in the system resulted in an increased degree of conversion for all lipases except Novozym 435. The secondary alcohol 2-propanol significantly reduced the alcoholysis reaction with all lipases; however, the branch-chain isobutanol was more advantageous than linear 1-butanol for Novozym 435, Lipozyme RM IM, and Lipase PS-C. Many commercial immobilized lipases are highly efficient and promising for the production of alkyl esters, offering high reaction yields and a simple operation process. [ABSTRACT FROM AUTHOR]

Abstract: A highly efficient two-step chemoenzymatic synthesis of enantiopure structured ether lipids of the 1-O-alkyl-2,3-diacyl-sn-glycerol type has been developed. Chimyl, batyl and selachyl alcohols possessing pure saturated fatty acid (SFA) attached to the sn-3 position and pure EPA and DHA attached to the sn-2 position were obtained under full regiocontrol. This was offered by mild conditions and a highly efficient lipase that operated at room temperature. High-resolution 1H NMR spectroscopy was used to monitor the progress of the reactions and to evaluate the full regiocontrol of the reactions involved by keeping track of all prospective adducts involved in these reactions. This was extended to preparation of a focused library of eight monoacyl intermediate adducts for all even-numbered SFA ranging from C2–C16 and the corresponding EPA and DHA structured diacyl glycerol ethers (DAGE) products for chimyl, batyl and selachyl alcohols, the total of 72 compounds. [Copyright &y& Elsevier]

Highlights • DHA was efficiently absorbed in Sprague-Dawley rats with mild n-3 deficiency. • No significant differences in the absorption between DHA in sn -1 and sn -3 positions. • Less fecal DHA from the sn -2 position compared with the sn -1 and sn -3 positions. • 5-Day intervention enough to modify the fatty acid profile of plasma phospholipids. Abstract Lack of synthetic enantiospecific triacylglycerols (TAGs) has hindered our understanding of the impact of TAG structure on the absorption and metabolic fate of fatty acids (FAs). In a five-day feeding trial with mildly (n-3) deficient rats, the bioavailability of docosahexaenoic acid [22:6(n-3), DHA] and stearic acid (18:0) from the two different enantiomers of TAG: sn -22:6(n-3)-18:0-18:0 and sn -18:0-18:0-22:6(n-3), and their regioisomeric TAG: sn -18:0-22:6(n-3)-18:0 was compared. Less secretion of fecal DHA was detected from the sn -2 position compared with the sn -1 and sn -3 positions, but no difference was found in DHA content of the fasting plasma or in the weight of the body or organs. 18:0 was lost to feces mainly as cleaved from the primary positions but also as glycerol-bound. The 5-day intervention in rats was long enough to modify the fatty acid profile of plasma phospholipids. [ABSTRACT FROM AUTHOR]