Bioorganic Chemistry

Cyclooxygenase-2 and several lipoxygenases convert polyunsaturated fatty acids into a large variety of products. During inflammatory processes, these enzymes form several distinct families of specialized pro-resolving lipid mediators possessing potent anti-inflammatory and pro-resolving effects. These mediators have attracted a great interest as leads in drug discovery and have recently been the subject of biosynthetic pathway studies using docosahexaenoic and n-3 docosapentaenoic acid as substrates. Herein we present enzymatic studies with cyclooxygenase-2 and 5, 12-and 15-lipoxygenase enzymes using 3-oxa n-3 DPA as a synthetic mimic of n-3 docosapentaenoic acid. Structural elucidation based on data from RP-HPLC UV and LC/MS-MS experiments enabled the identification of novel enzymatically formed products. These findings constitute the basis for further biosynthetic studies towards understanding the mechanisms regulating substrate utilization in the biosynthesis of specialized pro-resolving lipid mediators.

SPMs are biosynthesized during the resolution phase of the acuteinflammatory response [5].The biochemical pathways for some SPMs have been established [2c,2d,6] opening up new research areas related to the many disease states associated with inflammation and drug discovery efforts [7].The biosynthesis [8,9] of protectin D1 has been established and is presented in Scheme 1.
The biosynthetic formation of and metabolism of PD1 n-3 DPA , a congener of protectin D1 formed from n-3 DPA (4), was recently presented [10].Of interest, an increased number of n-3 DPA derived SPMs have been reported [11,12] and submitted to biological testing [13] over the last five years.These and other SPMs have attracted attention in drug discovery towards developing small molecular resolution agents and immunoresolvents [7,14].Moreover, the increased understanding that SPMs are actively biosynthesized products acting as resolution agonists is considered a biomedical paradigm shift [15].However, the secondary metabolism of these potent pro-resolution agents has been less studied [2c,4a,16].Hence, new knowledge on such metabolic enzymatic processes is central for the development of SPM based pro-resolution agonists as remedies in inflammation driven human diseases.As of today, it has been reported that protectin D1 undergoes enzymatic oxidation at C-22 to produce 22-OH-PD1 that carries potent anti-inflammatory and pro-resolving bioactions [17].The eicosaoxido reductase C-10 keto product of PD1 has also been reported [2c,4a].Moreover, SPMs, as all PUFAs, undergo β-oxidation metabolism [4a,18].The double bond geometry of the specific classes of the SPMs reflects their biochemical origin, but also their stereospecific bioactions as agonists towards different G protein-coupled receptors (GPCRs) [19].Hence, access to novel SPMs mimetic possesing pro-resolution and antiinflammatory bioactions resistant to enzymatic oxidative metabolism or catabolic degradation is of great interest to the biomedical research M. K. Pangopoulos, et al. Bioorganic Chemistry 96 (2020) 103653 community [13,14].Towards such aims we prepared the n-3 DPA (4) mimetic 5, named 3-oxa n-3 DPA, see Fig. 2. Earlier Hamberg and coworkers have shown that 3-oxalinolenic acid is efficiently oxygenated by soybean lipoxygenase-1 into 3-oxa-oxylipins [20].These studies provided inspiration for the design as well as the synthetic and enzymatic results presented herein.
UV and 13 C NMR spectral data analyses confirmed that the all-Z double bond configuration was present in 5 [23].

Biological evaluations 2.2.1. Enzymatic studies and LC/MS-MS identification of products
The enzymatic studies with different oxygenase enzymes were then investigated, Scheme 3.
The 15-LOX enzyme is involved in the biosynthesis of the protectins [9,10] and some resolvins [2c,2d,4a].Hence, we investigated the formation of mono-hydroxylated products in the presence of soybean 15-LOX.LC/MS-MS metabololipidomics paved the way for the identification of a mono-hydroxylated PUFA product, based on the m/z fragmentation pattern.In the MS/MS-spectrum fragments associated with m/z = 347 (M-H), 329 (M-H-H 2 O) and 303 (M-H-CO 2 ) were observed, along with peaks for m/z = 277 and 249 (Fig. 3D).The UV chromophores (λ max = 237 nm) observed for 12-15 were all characteristic for a E,Z-diene conjugated double bond system, see right panels in Fig. 3.
Human lipoxygenases catalyze the incorporation of molecular oxygen at a 1(Z),4(Z)-pentadienyl moiety in PUFAs resulting in the formation of an S-configured hydroperoxy E,Z-conjugated fatty acid that is reduced to the corresponding alcohol [26].Based on this, the configuration of the secondary alcohols in 12, 14 and 15 should all be S. On the other hand, COX-2 enables oxygenation of DHA and n-3 docosapentaenoic to produce R-configured PUFA-alcohols [4a,12].Future studies will establish the absolute configuration of the four novel PUFA-products presented herein.
Recently it was reported that protectin D1 undergoes rapid β-oxidation metabolism in vitro by human hepatoma cells [27].Sala and coworkers identified two products formed 30 min after incubation of protectin D1, as the result of one cycle or two cycles of β-oxidation metabolism, respectively.These authors confirmed the presence of minor amounts of the known 22-OH-PD1 metabolite [9], but no further ω-metabolism of 22-OH-PD1 was observed [27].Interestingly, the tetranor C18 metabolite of protectin D1 was the only metabolite that maintained the bioactivity of the protectin D1 [4], inhibiting neutrophil chemotaxis in vitro and neutrophil LTB 4 -induced chemotaxis in vivo.Overall, these studies support the design, synthesis and enzymatic studies on 3-oxa n-3 DPA (5) reported herein, but also provide a rational for protectin D1 analogs resistant to β-oxidation metabolism.Towards such aims, the biosynthesis of 17-OH-3-oxa n-3 DPA (15) presented herein, is of particular interest.

Conclusions
To summarize, 3-oxa n-3 DPA (5) was stereoselectively synthesized in multi-milligram amounts and in chemical purity of > 97%.We have shown that 3-oxa n-3 DPA ( 5) is a substrate for 5-LOX, 12-LOX, 15-LOX and COX-2, yielding mono-hydroxylated products as identified using LC/MS-MS analysis.These enzymes are essential in the biosynthesis of all SPMs reported to date.SPMs [7,13], as well as other oxygenated PUFA-products [28], exhibit potent and interesting pharmacological actions.These lipids are of interest as lead compounds in resolution pharmacology and drug development.Scheme 3. Enzymatic investigations using 3-oxa n-3 DPA (5) as substrate.

LC-MS/MS-based lipidomics
Collected fractions were analyzed using a Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC autoinjector, paired with a QTrap 5500 (ABSciex) using the same column and gradient as above.QTrap 5500 was operated in negative ionization mode using a multiple reaction monitoring (MRM) method coupled with information-dependent acquisition and enhanced product ion scan as previously described [30] using specific MRM transitions

Fig. 1 .
Fig. 1.Chemical structures of PUFAs, outline of families of SPMs and classical eicosanoids.