# Two-Step Enzymatic Synthesis of Structured Triacylglycerglycerols from ARASCO+
## 1 Introduction  
Arachidonic acid (ARA, 20:4n-6) is a polyunsaturated fatty acid (PUFA) of the \(n\)-6 family that is biosynthesized from its metabolic precursor, the essential fatty acid linoleic acid (LA, 18:2n-6), by the enzymes \(\Delta\)5 and \(\Delta\)6 desaturase [1]. ARA is considered conditionally essential because the limited rate of synthesis by these desaturases may not be sufficient to meet the physiological requirements in humans [2]. This FA is predominantly found as part of the structural lipids of the cell and intracellular membranes and plays an important role as a direct precursor of bioactive lipid mediators called eicosanoids, which are produced by enzymatic metabolic pathways; Prostaglandins and thromoboxanes are produced by the cyclooxygenase (COX) pathway; leukotrienes, lipoxins, and hydroperoxyeicosatetraenoic acid are produced by the lipoxygenase (LOX) pathway, whereas epoxyeicosatetraenoic acids and 19,20-hydroxyeicosatetraenoic acid are produced by the cytochrome P450 pathway [3]. Eicosanoids play several roles in many physiological processes in the human organism such as regulating immune responses, maintaining vascular stability, and promoting platelet aggregation [3, 4]. ARA metabolism is also involved in the modulation of inflammation, blood pressure, hypertension, and diabetes mellitus, among others [3, 5, 6]. ARA is known to be essential for brain and retinal development during gestation and infancy [7]. This PUFA, along with docosahexaenoic acid (DHA, 22:6n-3), is preferentially delivered to the brain during the first year of life through breast milk, which contains approximately 0.5% ARA by weight [7]. The EFSA defines 140 mg as the adequate daily intake of ARA for infants up to 6 months [8]. However, it has been estimated that the average dietary intake of ARA in infants is 65 mg/d, mainly from breast milk, with some countries having lower intakes, such as the United States (4 and 20 mg/d for infants at 9 and 12 months, respectively) and Belgium (17 mg/day for preschool children) [8]. Infant formulas are supplemented with ARA to provide this PUFA to infants who do not receive sufficient ARA from breast milk. To mimic the lipid composition of human milk, PUFA are supplemented as triacylglycerlycerols (TAG) in infant formulas, since TAGs make up more than 98% of the total lipids in human milk. The bioaccessibility of ARA is enhanced when it is located at the _sn_-2 position of the glycerol backbone and is preferentially taken up by the intestinal entrocytes after the TAG hydrolysis by the pancreatic lipase to _sn_-2 monoacylglycerolycerols (_sn_-2 MAG) and free fatty acids (FFA) [1]. Furthermore, medium-chain FA (MCFA, 8-12 carbon length) located at the _sn_-1,3 positions of the glycerol backbone are readily released by gastric and pancreatic lipases, directly absorbed by the entrocytes, and transported through the portal vein to the liver for oxidation [9]. Therefore, ARA-containing structured TAG (STAG) at the central position and MCFA at the outer positions (MLM-type STAG) seems to be a suitable alternative for ARA supply via infant formula. STAG are synthesized by either chemical or enzymatic processes. However, enzymatic methods have advantages over their chemical counterparts, such as milder reaction conditions, lower residual solvents, higher selectivity, and simpler product purification methods [10]. Several attempts have been made to synthesize STAG with ARA and MCFA enzymatically. For example, one study described a two-step method to incorporate caprylic acid (CA, 8:0) into triarchidonoyl glycerol (Tri-ARA) previously prepared by reacting glycerol and arachidonic acid at a 1:3 molar ratio with 5% of Novozym 435 lipase at 40 \({}^{\circ}\)C for 24 h, achieving 89% yield of Tri-ARA [11]. These authors were able to incorporate 63.8 mol% of ARA into the STAG after three synthesis cycles using Tri-ARA and CA as substrates at a 1:15 molar ratio, a 7% _Rhizopus delmar_ lipase load (based on the weight of substrates), 30 \({}^{\circ}\)C and a 48 h reaction time.