Total Synthesis and Bioactivity Investigation of a Chiral Diacylglycerol

Total Synthesis and Bioactivity Investigation of a Chiral Diacylglycerol | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Total Synthesis and Bioactivity Investigation of a Chiral Diacylglycerol Xuemei Liao, Xin Cheng, Ruirong Zhuang, Beidou Zhou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6896088/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Compound 1 , a chiral diglyceride metabolite found in both humans and fungi, was targeted for total synthesis. A seven-step synthetic route was developed, affording compound 1 in 2.33% overall yield. The key steps involved: (1) selective protection of the terminal hydroxyl group of chiral ketal 2 with a sterically hindered benzyl group, followed by removal of the ketal moiety to generate benzyl ether 4 ; (2) protection of the terminal hydroxyl group of benzyl ether 4 with a bulky silyl protecting group, and subsequent esterification of the remaining free hydroxyl with erucic acid to yield ester 6 ; (3) removal of the silyl protecting group from ester 6 , followed by esterification of the liberated hydroxyl group with pentadecanoic acid, affording ester 8 ; and (4) selective deprotection of the benzyl group of ester 8 to furnish compound 1 . In silico screening using three databases identified 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) as a potential biological target of compound 1 . However, in vitro HMGCR inhibition assays demonstrated that compound 1 did not significantly reduce cholesterol levels in human blood. These results contribute to the chemical synthesis and biological evaluation of chiral diglycerides and provide a foundation for future investigations in this area. Biological sciences/Biochemistry Biological sciences/Molecular biology Physical sciences/Chemistry/Organic chemistry Physical sciences/Chemistry/Synthesis total synthesis chiral diglyceride HMG-CoA reductase Figures Figure 1 Figure 2 Figure 3 1. Introduction Diglycerides are formed by combining two fatty acid chains with a glycerol molecule via ester bonds. They exist primarily as 1,2-diglycerides and 1,3-diglycerides. Diglycerides are generated either enzymatically from phosphatidic acids by phosphatidic acid phosphatase in the endoplasmic reticulum, or by triacylglycerol lipase during lipolysis. They serve as crucial substrates in the biosynthesis of triglycerides (for energy storage) and phospholipids like phosphatidylcholine (lecithin) and phosphatidylethanolamine [ 1 , 2 ]. Diglycerides function as key second messengers in activating numerous signaling cascades, with the stereochemistry of their isomers dictating distinct metabolic roles. Diglyceride kinases tightly regulate diglyceride signaling by phosphorylation, ensuring controlled availability. Critically, diglycerides activate protein kinase C, enhancing downstream serine and threonine phosphorylation [ 3 ]. Moreover, diglyceride dysregulation is implicated in a broad range of pathologies. Ectopic diglyceride accumulation disrupts insulin signaling, and elevated levels in the liver and muscles correlate with insulin resistance in obese animal models [ 4 ]. Furthermore, diglycerides are implicated in cancer, nervous system signaling, and immune dysregulation. Thus, understanding diglyceride biology is essential for developing effective therapeutic strategies. A chiral diglyceride (designated as compound 1 ) can be described as diglyceride (15:0/22:1(13Z)/0:0), diglyceride (15:0/22:1), or [(2S)-1-hydroxy-3-pentadecanoyloxypropan-2-yl] (Z)-docos-13-enoate (among other nomenclature). (See Fig. 1 ). The numerical designations indicate the fatty acyl composition. For example, "15:0" signifies a fatty acyl group with 15 carbon atoms and no unsaturation, while "22:1(13Z)" indicates a fatty acyl group with 22 carbon atoms containing a Z -configured double bond between the 13th and 14th carbons. "0:0" indicates the absence of a third fatty acyl group on the diglyceride. This compound is found as a mammalian metabolite in humans (Homo sapiens) – specifically in blood plasma – and as a fungal metabolite in Baker's yeast (Saccharomyces cerevisiae ). To facilitate studies of compound 1 's biological activity, a total synthesis route was investigated. 2. Results and Discussion 2.1 Synthesis The synthesis commenced with the chiral raw material ( S )-(+)-1,2-isopropylidene glycerol ( 2 ), procured from Bide Medicine. Reaction with benzyl bromide in the presence of potassium hydroxide and 18-crown-6 (as a phase-transfer catalyst) in anhydrous tetrahydrofuran afforded benzyl ether 3 [5.6]. The 1,2-isopropylidene protecting group of 3 was then removed by treatment with a 60% aqueous solution of acetic acid at 60–65°C, yielding benzyl ether 4 [5.6]. The syntheses of compounds 3 and 4 have been described in the literature [ 5 ]. Selective protection of the primary hydroxyl group of 4 was achieved using tert-butyldimethylchlorosilane (TBSCl) and imidazole as a base in anhydrous dichloromethane, affording silyl ether 5 . The remaining secondary hydroxyl group of 5 was then esterified with erucic acid, using dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) as coupling reagents in anhydrous dichloromethane, to furnish ester 6 [5.6]. Deprotection of the tert-butyldimethylsilyl (TBS) group of 6 was accomplished with tetra-n-butylammonium fluoride (TBAF) in tetrahydrofuran, generating alcohol 7 . Subsequent esterification of the free hydroxyl group of 7 with pentadecanoic acid, again employing DCC/DMAP in anhydrous dichloromethane, provided ester 8 . Finally, cleavage of the benzyl ether moiety of 8 was effected with boron trichloride (BCl 3 ) in anhydrous dichloromethane at low temperature (-78°C to -40°C), yielding the target compound 1 . Importantly, under these conditions, the ester linkages and the double bond remained intact [5.6]. The overall yield of compound 1 for the seven-step sequence was 2.33%. See Fig. 2 for a schematic representation of the synthetic route. 2.2 Biological activity 2.2.1 Target prediction of compound 1 To identify potential targets of compound 1 , in silico predictions were performed using the SwissTargetPrediction, PharmMapper, and TargetNet databases. After removing duplicate entries, a total of 377 unique targets were identified. Rigorous probability thresholds were applied: targets from SwissTargetPrediction were included if their probability score was ≥ 0.05, TargetNet targets required a score ≥ 0.002, and PharmMapper targets needed a normalized fit score ≥ 0.5. Cross-validation of these three databases (SwissTargetPrediction, PharmMapper, and TargetNet) revealed 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) as a common potential target (Fig. 3 ). HMGCR is a key enzyme in the cholesterol biosynthesis pathway, catalyzing the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) to mevalonic acid (MVA). It is the primary target of statins and other cholesterol-lowering medications. Given the presence of compound 1 in human plasma, we investigated its potential interaction with HMGCR and its possible effects on cholesterol synthesis in vivo. 2.2.2 Determination of the activity of HMG-CoA reductase inhibition HMGCR is a transmembrane glycoprotein located in the endoplasmic reticulum that catalyzes the conversion of HMG-CoA to mevalonate and coenzyme A (CoA) using two reducing equivalents from NADPH. This reaction represents the rate-limiting step in cholesterol biosynthesis. During the reaction, NADPH is oxidized to NADP + . NADPH exhibits an absorbance peak at 340 nm, while NADP + does not absorb at this wavelength. This difference is exploited to screen for compounds that inhibit HMGCR activity. Pravastatin was included as a positive control. The results, shown in Table 1 , indicate that compound 1 , at a concentration of 50 µM, did not demonstrate significant HMGCR inhibitory activity. This suggests that compound 1 does not reduce cholesterol production through HMGCR inhibition. Table 1 Inhibitory activity of samples on HMGCR Samples concentartion (µM) percentage inhibition (%) Pravastatin 0.25 88.20 ± 1.71 1 50 0.20 ± 2.45 3. Conclusion Compound 1 was efficiently synthesized via a multi-step route from readily available fatty acids and a chiral ketal obtained from the chiral pool, employing a sequence of protection and deprotection steps. This synthetic route offers a potential template for the preparation of similar chiral glycerides. Importantly, our findings demonstrate that compound 1 does not reduce human blood cholesterol concentrations through HMGCR inhibition. This result provides a valuable theoretical framework for the design of alternative therapeutic approaches in the development of health supplements and lipid-lowering medications. 4. Experimental 4.1 Chemistry The chemical reagents were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) or Shanghai Bidd Pharmaceutical Technology Co., Ltd. (Shanghai, China) and used as received. IR spectra were recorded using KBr pellets (Bruker, Bremen, Germany). 1 H and 13 C NMR spectra were acquired on a Bruker spectrometer (500 MHz for 1 H and 125 MHz for 13 C; Bruker, Fällanden, Switzerland). The optical rotation was measured using a WZZ-2B automatic polarimeter at a wavelength of 589 nm (Sodium D line; Shanghai Yidian Physical Optical Instrument Co., Ltd, Shanghai, China). 4.2 Synthesis 4.2.1 Synthesis of compound 3 ( S )-(+)-1,2-Isopropylidene glycerol ( 2 ) (enantiomeric purity: 98%, 25 mL, 2.0 mmol), 18-crown-6 (0.05 g, 0.19 mmol), and anhydrous tetrahydrofuran (6 mL) were combined and stirred at room temperature for 40 min. The mixture was then heated to 60°C using an electric organic synthesizer, and benzyl bromide (0.25 mL, 2.0 mmol) was added dropwise. The reaction was stirred for several hours, with progress monitored by thin layer chromatography until equilibrium was reached. The reaction mixture was allowed to cool to room temperature and subsequently poured into water. The aqueous phase was extracted with ethyl acetate (3 × 10 mL), and the combined organic extracts were washed with saturated brine (3 × 8 mL) and dried over anhydrous Na 2 SO 4 . The solvent was removed under reduced pressure to afford the crude product, which was purified by medium-pressure preparative chromatography using ethyl acetate/petroleum ether as the eluent, yielding compound 3 . ( S )-4-((Benzyloxy)methyl)-2,2-dimethyl-1,3-dioxolane ( 3 ): colorless oil; yield 48%; [α]20 D = + 0.59° (c = 0.13 mg/mL, CHCl 3 ); IR (KBr) v max 711, 748, 1029, 1278, 1398, 1451, 1495, 1601, 2876, 2935 cm − 1 ; 1 H-NMR (500 MHz, acetone-d 6 ): δ 1.27 (d, 3H, CH 3 -), 1.32 (d, 3H, CH 3 -), 2.03–2.05 (acetone-d 6 ), 2.81 (s, H 2 O), 3.48 (dd, 1H, J = 5.2, 9.8 Hz, -CH 2 -OBn), 3.55 (dd, 1H, J = 5.3, 9.8 Hz, -CH 2 -OBn), 3.71 (dd, 1H, J = 6.3, 8.2 Hz, O-CH 2 -Ar), 4.03 (dd, 1H, J = 6.4, 8.2 Hz, O-CH 2 -Ar), 4.24 (m, 1H, H-4), 4.55 (s, 2H, -OCH 2 Ar), 7.26–7.35 (m, 5H, Ar-H); 13 C NMR (125 MHz, acetone-d 6 ): δ 25.7, 27.1, 67.4, 72.0, 73.7, 75.6, 109.5, 128.2, 128.3, 129.0 (C-3’’, C-5’’), 139.6; HRMS(ESI) m/z calcd for C 13 H 19 O 3 [M + H] + 223.1329, found 223.1335. The optical value of compound 3 reported by the literature [ 5 ] was [α]20 D = + 15.5° (c = 0.5, CHCl 3 ). This reference did not indicate the concentration unit of the optical value. 4.2.2 Synthesis of compound 4 A solution of compound 3 (241 mg, 1.09 mmol) in 60% aqueous acetic acid (6 mL) was heated to 65°C using an electric organic synthesizer. The reaction mixture was stirred at this temperature for several hours, with the progress monitored by thin layer chromatography until the reaction reached equilibrium. The solution was then allowed to cool naturally to room temperature. The organic components were extracted with ethyl acetate (3 × 12 mL). The combined ethyl acetate extracts were washed three times with 10% sodium carbonate solution (3 × 10 mL) followed by three washes with saturated saline (3 × 10 mL). The organic layer was then dried over anhydrous sodium sulfate (Na 2 SO 4 ). The solvent was removed by rotary evaporation under reduced pressure to afford the crude product, which was purified by medium-pressure preparative chromatography using a gradient of ethyl acetate in petroleum ether as the eluent, yielding compound 4 . ( R )-3-(Benzyloxy)propane-1,2-diol ( 4 ): yield 56%; [α]20 D = + 0.22° (c = 0.10 mg/mL, CHCl 3 ); IR (KBr) v max 706, 736, 1027, 1106, 1277, 1369, 1447, 1494, 1579, 1601, 2874, 2929, 3372 cm − 1 ; 1 H NMR (500 MHz, acetone-d 6 ) δ 2.03–2.05 (acetone-d 6 ), 2.85 (s, H 2 O), 3.46–3.60 (m, 4H, 2 × H-1, 2 × H-3), 3.76–3.81 (m, 1H, H-2), 4.53 (s, 2H, -OCH 2 Ar), 7.24–7.36 (m, 5H, Ar-H); 13 C NMR (125 MHz, acetone-d 6 ): δ 64.5, 71.8, 72.9, 73.6, 128.1 (C-2’’, C-6’’), 128.3, 129.0 (C-3’’, C-5’’), 139.8; HRMS(ESI) m/z calcd for C 10 H 15 O 3 [M + H] + 183.1016, found 183.1013. The optical value compound 4 reported by the literature [ 5 ] was [α]20 D = + 16.5° (c = 0.5, CHCl 3 ). This reference also did not indicate the concentration unit of the optical value. 4.2.3 Synthesis of compound 5 A mixture of compound 4 (186 mg, 1.02 mmol), imidazole (69 mg, 1.00 mmol), tert -butyldimethylchlorosilane (0.18 mL, 1.0 mmol), and anhydrous dichloromethane (6 mL) was stirred at room temperature for several hours. The reaction was monitored by thin layer chromatography until equilibrium was reached. The organic portion was extracted with dichloromethane (3 × 10 mL), washed with saturated brine (3 × 10 mL), and dried over anhydrous Na 2 SO 4 . The solvent was removed by evaporation under reduced pressure to obtain the crude product, which was purified by medium-pressure preparative chromatography using ethyl acetate/petroleum ether as eluent to afford compound 5 . ( S )-1-(Benzyloxy)-3-((tert-butyldimethylsilyl)oxy)propan-2-ol ( 5 ): colorless oil; yield 61%; [α]20 D = + 0.39° (c = 0.21 mg/mL, CHCl 3 ); IR (KBr) v max 697, 741, 1040, 1070, 1205, 1366, 1452, 1497, 1654, 2865, 2926, 3367 cm − 1 ; 1 H NMR (500 MHz, acetone-d 6 ) δ 0.07 (d, 6H, 2 CH 3 -), 0.89 (d, 9H, 3 CH 3 -), 2.03–2.05 (acetone-d 6 ), 2.82 (s, H 2 O), 3.47 (dd, 1H, J = 5.8, 9.7 Hz, H-1), 3.56 (dd, 1H, J = 4.8, 9.1 Hz, H-1), 3.64 (dd, 1H, J = 5.5, 8.2 Hz, H-3), 3.68 (dd, 1H, J = 5.3, 8.3 Hz, H-3), 3.75–3.79 (m, 1H, H-2), 4.53 (s, 2H,-OCH 2 Ar), 7.24–7.36 (m, 5H, Ar-H); 13 C NMR (125 MHz, acetone-d 6 ) δ 18.9 (2 CH 3 -Si), 26.2 (3 CH 3 -), 65.4, 71.7, 72.4, 73.6, 128.1, 128.3, 129.0 (C-3’, C-5’), 139.6; HRMS(ESI) m/z calcd for C 16 H 27 O 3 Si [M - H] − 295.1735, found 295.1741. 4.2.4 Synthesis of compound 6 A mixture of compound 5 (297 mg, 1.00 mmol), erucic acid (598 mg, 1.50 mmol), dicyclohexylcarbodiimide (DCC, 316 mg, 1.50 mmol), and 4-dimethylaminopyridine (DMAP, 15 mg, 0.12 mmol) in anhydrous dichloromethane (10 mL) was stirred at room temperature for several hours. The reaction was monitored by thin layer chromatography until equilibrium was reached. The resulting solids were filtered off, and the solvent was removed under reduced pressure. The crude product was then purified by medium-pressure preparative chromatography using ethyl acetate/petroleum ether as eluent to yield compound 6 . ( S )-1-(Benzyloxy)-3-((tert-butyldimethylsilyl)oxy)propan-2-yl ( Z )-docos-13-enoate ( 6 ): colorless oil; yield 53%; [α]20 D = + 1.02° (c = 0.084 mg/mL, CHCl 3 ); IR (KBr) v max 695, 734, 778, 833, 1096, 1248, 1366, 1459, 1740, 2852, 2925 cm − 1 ; 1 H NMR (500 MHz, acetone-d 6 ) δ 0.065 (s, 6H, 2 × CH 3 -Si), 0.88 (s, 12H, -C(CH 3 ) 3, CH 3 −22’), 1.28–1.36 (m, 30H, 2 × H-3’, 2 × H-4’, 2 × H-5’, 2 × H-6’, 2 × H-7’, 2 × H-8’, 2 × H-9’, 2 × H-10’, 2 × H-11’, 2 × H-16’, 2 × H-17’, 2 × H-18’, 2 × H-19’, 2 × H-20’, 2 × H-21’), 1.57–1.63 (m, 2H, 2 × H-15’), 2.01–2.05 (acetone-d 6 , 2 × H-12’), 2.28–2.81 (dt, 2H, J = 1.0, 7.7 Hz, 2 × H-2’), 2.81 (s, H 2 O), 3.60–3.66 (m, 2H, 2 × H-1), 3.75–3.82 (m, 2H, 2 × H-3), 4.51–4.56 (m, 2H, -OCH 2 Ar), 5.03–5.07 (m, 1H, H-2), 5.34–5.35 (m, 2H, H-13’, H-14’), 7.27–7.35 (m, 5H, Ar-H); 13 C NMR (125 MHz, acetone-d 6 ) δ -5.3 (-Si-(CH 3 ) 2 ), 14.4, 18.8 (3 CH 3 - in t-Bu), 23.3, 25.7, 26.2, 27.8, 29.3 (C-4’, C-5’, C-19’), 29.6 (C-6’, C-7’, C-8’), 29.7 (C-9’, C-10’, C-17’, C-18’), 30.0 (C-11’, C-16’), 30.4 (tertiary C in t-Bu), 29.3–30.4 (acetone-d 6) , 32.6, 34.8, 62.6, 69.3, 73.6 (C-2, -OCH 2 Ar), 128.2 (C-2’’, C-4’’, C-6’’), 129.0 (C-3’’, C-5’’), 130.5 (C-13’, C-14’), 139.5, 173.2; HRMS(ESI) m/z calcd for C 38 H 69 O 4 Si [M + H] + 617.4960, found 617.4962. 4.2.5 Synthesis of compound 7 A mixture of compound 6 (617 mg, 1.00 mmol), tetra-n-butylammonium fluoride (TBAF, 1.3 mL, 1.30 mmol), and tetrahydrofuran (THF, 6 mL) was stirred at room temperature for several hours. The reaction was monitored by thin layer chromatography until equilibrium was reached. The reaction mixture was then poured into water (25 mL) and extracted with ethyl acetate (3 × 10 mL). The combined organic extracts were washed with saturated brine (3 × 10 mL), dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure to afford the crude product. Purification by medium-pressure preparative chromatography using ethyl acetate/petroleum ether as eluent gave compound 7 . ( R )-1-(Benzyloxy)-3-hydroxypropan-2-yl ( Z )-docos-13-enoate ( 7 ): colorless oil; yield 87%; [α]20 D = + 1.11° (c = 0.076 mg/mL, CHCl 3 ); IR (KBr) v max 698, 731, 1017, 1094, 1162, 1252, 1371, 1447, 1733, 2852, 2921, 3453 cm − 1 ; 1 H NMR (500 MHz, acetone-d 6 ) δ 0.87 (t, 3H, J = 6.9 Hz, CH 3 -), 1.29–1.35 (m, 28H, 2 × H-4’, 2 × H-5’, 2 × H-6’, 2 × H-7’, 2 × H-8’, 2 × H-9’, 2 × H-10’, 2 × H-11’, 2 × H-16’, 2 × H-17’, 2 × H-18’, 2 × H-19’, 2 × H-20’, 2 × H-21’), 2.02–2.05 (m, acetone-d 6 , 4H, 2 × H-12’, 2 × H-15’), 2.28 (t, 2H, J = 7.5 Hz, 2 × H-2’), 2.82 (s, H 2 O), 3.53 (dd, 2H, J = 0.8, 5.5 Hz, 2 × H-1), 3.98 (m, 1H, H-2), 4.06–4.10 (m, 2H, H-3, HO-3), 4.15 (dd, 1H, J = 4.6, 11.2 Hz, H-3), 4.54 (s, 2H, -OCH 2 Ar), 5.33–5.35 (m, 2H, H-13’, H-14’), 7.27–7.36 (m, 5H, Ar-H); 13 C NMR (125 MHz, acetone-d 6 ) δ 14.3, 23.3, 25.6, 27.7 (C-12’, C-15’), 29.3 (C-4’, C-5’, C-19’), 29.6 (C-6’, C-7’, C-8’), 29.8 (C-9’, C-10’, C-17’, C-18’), 29.9 (C-11’, C-16’), 29.3–30.4 (acetone-d 6 ), 32.6, 34.5, 66.2, 69.1, 72.3, 73.7, 128.2 (C-2’’, C-6’’), 128.3, 129.0 (C-3’’, C-5’’), 130.5 (C-13’, C-14’), 139.6, 173.6; HRMS(ESI) m/z calcd for C 32 H 53 O 4 [M - H] − 501.3949, found 501.3946. 4.2.6 Synthesis of compound 8 A mixture of compound 7 (520 mg, 1.04 mmol), pentadecanoic acid (371 mg, 1.50 mmol), dicyclohexylcarbodiimide (DCC, 316 mg, 1.50 mmol), 4-dimethylaminopyridine (DMAP, 15 mg, 0.12 mmol), and anhydrous dichloromethane (10 mL) was stirred at room temperature for several hours. The reaction was monitored by thin layer chromatography until it reached completion. The resulting solids were filtered off, and the solvent was removed under reduced pressure to afford the crude product. Purification by medium-pressure preparative chromatography using ethyl acetate/petroleum ether as eluent yielded compound 8 . ( S )-1-(Benzyloxy)-3-(pentadecanoyloxy)propan-2-yl ( Z )-docos-13-enoate ( 8 ): colorless oil; yield 56%; [α]20 D = + 0.22° (c = 0.10 mg/mL, CHCl 3 ); IR (KBr) v max 698, 735, 1026, 1104, 1165, 1245, 1372, 1461, 1744, 2853, 2920 cm − 1 ; 1 H NMR (500 MHz, acetone-d 6 ) δ 0.88 (t, 6H, J = 6.8 Hz, H-22’, H-15’’), 1.29–1.34 (m, 50H, 2 × H-4’, 2 × H-5’, 2 × H-6’, 2 × H-7’, 2 × H-8’, 2 × H-9’, 2 × H-10’, 2 × H-11’, 2 × H-16’, 2 × H-17’, 2 × H-18’, 2 × H-19’, 2 × H-20’, 2 × H-21’, 2 × H-4’’, 2 × H-5’’, 2 × H-6’’, 2 × H-7’’, 2 × H-8’’, 2 × H-9’’, 2 × H-10’’, 2 × H-11’’, 2 × H-12’’, 2 × H-13’’, 2 × H-14’’), 1.56–1.61 (m, 4H, 2 × H-3’, 2 × H-3’’), 2.02–2.05 (m, acetone-d 6 , 4H, 2 × H-12’, 2 × H-15’), 2.26–2.32 (m, 4H, 2 × H-2’, 2 × H-2’’), 3.63–3.67 (m, 2H, 2 × H-1), 4.17 (dd, 1H, J = 6.6, 11.9 Hz, H-3), 4.36 (dd, 1H, J = 3.6, 11.9 Hz, H-3), 4.56 (m, 2H, -OCH 2 Ar), 5.22–5.26 (m, 1H, H-2), 5.31–5.38 (m, 2H, H-13’, H-14’), 7.26–7.36 (m, 5H, Ar-H); 13 C NMR (125 MHz, acetone-d 6 ) δ 14.4 (C-22’, C-15’’), 23.3 (C-21’, C-14’’), 25.6, 25.7, 27.8 (C-12’, C-15’), 29.3 (C-4’, C-5’, C-19’, C-4’’, C-5’’, C-12’’), 29.6 (C-6’, C-7’, C-8’, C-6’’, C-7’’, C-8’’, C-9’’, C-10’’, C-11’’), 29.8 (C-9’, C-10’, C-17’, C-18’), 29.9 (C-11’, C-16’), 29.3–30.4 (acetone-d 6 ), 32.6 (C-20’, C-13’’), 34.5, 34.7, 63.2, 69.2, 70.9, 73.6, 128.3, 129.1 (C-2’’’, C-6’’’), 130.5 (C-13’, C-14’, C-3’’’, C-5’’’), 139.2, 173.1, 173.3; HRMS(ESI) m/z calcd for C 47 H 83 O 5 [M + H] + 727.6235, found 727.6230. 4.2.7 Synthesis of compound 1 Compound 8 (120 mg, 0.16 mmol) was dissolved in anhydrous dichloromethane (4 mL) and cooled to -50°C. Boron trichloride (1.0 M in methylene chloride, 0.32 mL, 0.32 mmol) was added dropwise, and the solution was stirred at -50°C for 30–60 minutes, monitoring the reaction by thin layer chromatography until equilibrium was reached. The reaction was quenched with water, and the organic layer was extracted with dichloromethane, washed with saturated brine (3 × 10 mL), and dried over anhydrous Na 2 SO 4 . The solvent was removed under reduced pressure, and the crude product was purified by medium-pressure preparative chromatography (EtOAc/petroleum ether) to afford compound 1 . ( S )-1-Hydroxy-3-(pentadecanoyloxy)propan-2-yl ( Z )-docos-13-enoate ( 1 ): colorless oil; yield 55%; [α]20 D = + 0.16° (c = 0.10 mg/mL, CHCl 3 ); IR (KBr) v max 547, 718, 1179, 1381, 1427, 1467, 1733, 2850, 2919, 3496 cm − 1 ; 1 H NMR (500 MHz, acetone-d 6 ) δ 0.87 (t, 6H, J = 6.8 Hz, CH 3 -22’, CH 3 -15’’), 1.29 (m, 50H, 2 × H-4’, 2 × H-5’, 2 × H-6’, 2 × H-7’, 2 × H-8’, 2 × H-9’, 2 × H-10’, 2 × H-11’, 2 × H-16’, 2 × H-17’, 2 × H-18’, 2 × H-19’, 2 × H-20’, 2 × H-21’, 2 × H-4’’, 2 × H-5’’, 2 × H-6’’, 2 × H-7’’, 2 × H-8’’, 2 × H-9’’, 2 × H-10’’, 2 × H-11’’, 2 × H-12’’, 2 × H-13’’, 2 × H-14’’), 1.57–1.61 (m, 4H, 2 × H-3’, 2 × H-3’’), 2.03–2.05 (m, acetone-d 6 , 4H, 2 × H-12’, 2 × H-15’), 2.27–2.31 (m, 4H, 2 × H-2’, 2 × H-2’’), 3.67 (t, 2H, J = 5.8 Hz, 2 × H-1), 4.03 (t, 1H, J = 6.0 Hz, HO-1), 4.15 (dd, 1H, J = 6.0, 11.9 Hz, H-3), 4.35 (dd, 1H, J = 3.5, 14.9 Hz, H-3), 5.04–5.08 (m, 1H, H-2), 5.31–5.37 (m, 2H, H-13’, H-14’); 13 C NMR (125 MHz, acetone-d 6 ) δ 14.3 (C-22’, C-15’’), 23.3 (C-21’, C-14’’), 25.6 (C-3’, C-3’’), 27.7 (C-12’, C-15’), 29.3 (C-4’, C-5’, C-19’, C-4’’, C-5’’, C-12’’), 29.6 (C-6’, C-7’, C-8’, C-6’’, C-7’’, C-8’’, C-9’’, C-10’’, C-11’’), 29.8 (C-9’, C-10’, C-17’, C-18’), 29.9 (C-11’, C-16’), 29.3–30.4 (acetone-d 6 ), 32.6 (C-20’, C-13’’), 34.5, 34.7, 61.2, 63.1, 72.9, 130.5 (C-13’, C-14’), 173.2, 173.4; HRMS(ESI) m/z calcd for C 40 H 76 NaO 5 [M + Na] + 659.5591, found 659.5583. 4.3 Target prediction of compound 1 Compound 1 in sdf format were imported into the SwissTargetPrediction database ( http://swisstargetprediction.ch/ ) [7.8], PharmMapper ( http://www.lilab-ecust.cn/pharmmapper/ ) [ 9 – 11 ], and TargetNet ( http://targetnet.scbdd.com/ ) databases [ 12 ], respectively, then “homo sapiens” was select as the target species. Following the instructions, the targets of the compound 1 were obtained. 4.4 Experiment to inhibit the activity of HMG-CoA reductase The HMG-CoA reductase assay kit was purchased from Sigma-Aldrich (Shanghai, China). Samples were added to a 96-well microplate, followed by NADPH (final concentration 0.33 mg/mL), HMG-CoA substrate, and HMGCR (final concentration 5 µg/mL to 7 µg/mL), in that order. Each condition was tested in triplicate. A blank control (without drug) and a Pravastatin positive control were also included. The plate was incubated at 37°C for 10 minutes, and the optical density (OD) was measured at 340 nm using a microplate reader. The percentage inhibition of HMGCR was then calculated. Inhibition (%) = (1 - OD 340 nm of experimental well / OD 340 nm of blank well) × 100% Declarations Conflict of Interest The authors declare no conflict of interest. A data availability statement The data that support this study are available in the article and accompanying online supplementary material. Funding Declaration This research was funded by the Science and Technology Bureau of Putian City (Grant No. 2021S2001-9) and Fujian Shanhe Pharmaceutical Co., LTD (Grant No.2022AHX211(L)). Author Contribution XM Liao completed the total synthesis of chiral diacylglycerol and drafted the initial version of the article. X Chen and RR Zhuang jointly conducted the bioactivity assays of the target compound. BD Zhou provided the research methods and revised the paper. Data Availability The data that support this study are available in the article and accompanying online supplementary material. References Han, L. Y. et al. Diacylglycerol acyltransferase 3(DGAT3) is responsible for the biosynthesis of unsaturated fatty acids in vegetative organs of Paeonia rockii . Int. J. Mol. Sci. 23 , 14390 (2022). Foo, S., Cazenave-Gassiot, A., Wenk, M. R. & Oliferenko, S. Diacylglycerol at the inner nuclear membrane fuels nuclear envelope expansion in closed mitosis. J. Cell. Sci. 136 , 1–14 (2023). Shulga, Y. V., Topham, M. K. & Epand, R. M. Regulation and functions of diacylglycerol kinases. Chem. Rev. 111 , 6186–6208 (2011). Timmers, S., Schrauwen, P. & Vogel, J. D. Muscular diacylglycerol metabolism and insulin resistance. Physiol. Behav. 94 , 242–251 (2008). Xia, J. & Hui, Y. Z. Synthesis of a small library of mixed-acid phospholipids from D-mannitol as a homochiral starting material. Chem. Pharm. Bull. 47 , 1659–1663 (1999). Lu, B. L. et al. Investigating the individual importance of the pam 2 cys ester motifs on TLR2 activity. Eur. J. Org. Chem. 39 , 5415–5423 (2021). Daina, A., Michielin, O. & Zoete, V. SwissTargetPrediction: updated data and new features for efficient prediction of protein targets of small molecules. Nucleic Acids Res. 47 (W1), W357–W364 (2019). Gfeller, D., Michielin, O. & Zoete, V. Shaping the interaction landscape of bioactive molecules. Bioinformatics 29 (23), 3073–3079 (2013). Liu, X. F. et al. PharmMapper server: a web server for potential drug target identification via pharmacophore mapping approach. Nucleic Acids Res. 38 , W609–W614 (2010). Wang, X. et al. Enhancing the enrichment of pharmacophore-based target prediction for the polypharmacological profiles of drugs. J. Chem. Inf. Model. 56 , 1175–1183 (2016). Wang, X. et al. PharmMapper 2017 update: a web server for potential drug target identification with a comprehensive target pharmacophore database. Nucleic Acids Res. 45 , W356–W360 (2017). Yao, Z. J. et al. TargetNet: a web service for predicting potential drug-target interaction profiling via multi-target SAR models. J. Comput. Aided Mol. Des. 30 , 413–424 (2016). Additional Declarations No competing interests reported. Supplementary Files si2nd.docx Cite Share Download PDF Status: Published Journal Publication published 30 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 10 Jul, 2025 Reviews received at journal 09 Jul, 2025 Reviews received at journal 07 Jul, 2025 Reviewers agreed at journal 30 Jun, 2025 Reviewers agreed at journal 27 Jun, 2025 Reviewers agreed at journal 27 Jun, 2025 Reviewers agreed at journal 25 Jun, 2025 Reviewers invited by journal 25 Jun, 2025 Editor assigned by journal 25 Jun, 2025 Editor invited by journal 25 Jun, 2025 Submission checks completed at journal 17 Jun, 2025 First submitted to journal 17 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6896088","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":477595245,"identity":"dd246ad6-2550-4163-9298-4dabcc83272c","order_by":0,"name":"Xuemei Liao","email":"","orcid":"","institution":"Putian university","correspondingAuthor":false,"prefix":"","firstName":"Xuemei","middleName":"","lastName":"Liao","suffix":""},{"id":477595246,"identity":"6330e044-305c-4a00-9b37-5b16b3a83114","order_by":1,"name":"Xin Cheng","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Cheng","suffix":""},{"id":477595247,"identity":"53b4f04e-ada7-4d72-b366-500f4c1dc6e1","order_by":2,"name":"Ruirong Zhuang","email":"","orcid":"","institution":"Putian Lanhai Nuclear Medical Research Center","correspondingAuthor":false,"prefix":"","firstName":"Ruirong","middleName":"","lastName":"Zhuang","suffix":""},{"id":477595248,"identity":"1f97f464-031d-41d4-8444-7a4678392f16","order_by":3,"name":"Beidou Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBACxvbGxgcfKv7b2R9vIFILc8/hZsMZZ5iTGc4cIFIL+4z0NmneFmbGhhsJRGrhnZHYJjmzgY2ZcebjjTcYamyiCWqR7HnYbPFxBw8fs3RasQXDsbTcBkJaDNsTG2/OPCPBzCadYybB2HCYsBb7A4kN0rxtBow9kmeI1MLYkdgE1JLAOEOCh1gtPQdBgXwg2YAH6JcEYvzC2N7+EBiVB+wM2A9vvPGhxoawFmRgIJFAinKIFlJ1jIJRMApGwcgAAOIJRQOq10N6AAAAAElFTkSuQmCC","orcid":"","institution":"Putian university","correspondingAuthor":true,"prefix":"","firstName":"Beidou","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-06-15 02:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6896088/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6896088/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-14482-2","type":"published","date":"2025-08-30T15:57:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85645795,"identity":"418e7fd3-9f92-450d-be49-51c36cdc23c1","added_by":"auto","created_at":"2025-06-30 08:33:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11869,"visible":true,"origin":"","legend":"\u003cp\u003eThe chemical structure of the compound \u003cstrong\u003e1\u003c/strong\u003e and its number\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6896088/v1/11e3ab827c99c571346fdf69.png"},{"id":85645796,"identity":"c5db333e-ac4d-4eb6-8278-47a1897e2a28","added_by":"auto","created_at":"2025-06-30 08:33:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45847,"visible":true,"origin":"","legend":"\u003cp\u003eThe synthetic route of compound \u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6896088/v1/af86b4c5a18b1ef1562ed015.png"},{"id":85645646,"identity":"8ef6537f-db68-4596-a8a0-ebc1342da81b","added_by":"auto","created_at":"2025-06-30 08:25:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48798,"visible":true,"origin":"","legend":"\u003cp\u003eIntersection of targets predicted by three databases\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6896088/v1/31130f5a8d5ffb4fcf0661e9.png"},{"id":90344867,"identity":"9679a8f5-a5a8-407c-b339-d1e1472b400a","added_by":"auto","created_at":"2025-09-01 16:06:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":825306,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6896088/v1/53ae4992-01e4-45c3-b95b-0aa14b84e0ca.pdf"},{"id":85645653,"identity":"76a034fa-b83e-40d5-bec6-f20ebd4a6369","added_by":"auto","created_at":"2025-06-30 08:25:04","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1283528,"visible":true,"origin":"","legend":"","description":"","filename":"si2nd.docx","url":"https://assets-eu.researchsquare.com/files/rs-6896088/v1/0af617a2d549bf9097feaefc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Total Synthesis and Bioactivity Investigation of a Chiral Diacylglycerol","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDiglycerides are formed by combining two fatty acid chains with a glycerol molecule via ester bonds. They exist primarily as 1,2-diglycerides and 1,3-diglycerides. Diglycerides are generated either enzymatically from phosphatidic acids by phosphatidic acid phosphatase in the endoplasmic reticulum, or by triacylglycerol lipase during lipolysis. They serve as crucial substrates in the biosynthesis of triglycerides (for energy storage) and phospholipids like phosphatidylcholine (lecithin) and phosphatidylethanolamine [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDiglycerides function as key second messengers in activating numerous signaling cascades, with the stereochemistry of their isomers dictating distinct metabolic roles. Diglyceride kinases tightly regulate diglyceride signaling by phosphorylation, ensuring controlled availability. Critically, diglycerides activate protein kinase C, enhancing downstream serine and threonine phosphorylation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Moreover, diglyceride dysregulation is implicated in a broad range of pathologies. Ectopic diglyceride accumulation disrupts insulin signaling, and elevated levels in the liver and muscles correlate with insulin resistance in obese animal models [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, diglycerides are implicated in cancer, nervous system signaling, and immune dysregulation. Thus, understanding diglyceride biology is essential for developing effective therapeutic strategies.\u003c/p\u003e \u003cp\u003eA chiral diglyceride (designated as compound \u003cb\u003e1\u003c/b\u003e) can be described as diglyceride (15:0/22:1(13Z)/0:0), diglyceride (15:0/22:1), or [(2S)-1-hydroxy-3-pentadecanoyloxypropan-2-yl] (Z)-docos-13-enoate (among other nomenclature). (See Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The numerical designations indicate the fatty acyl composition. For example, \"15:0\" signifies a fatty acyl group with 15 carbon atoms and no unsaturation, while \"22:1(13Z)\" indicates a fatty acyl group with 22 carbon atoms containing a Z -configured double bond between the 13th and 14th carbons. \"0:0\" indicates the absence of a third fatty acyl group on the diglyceride. This compound is found as a mammalian metabolite in humans (Homo sapiens) \u0026ndash; specifically in blood plasma \u0026ndash; and as a fungal metabolite in Baker's yeast (Saccharomyces cerevisiae ).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo facilitate studies of compound \u003cb\u003e1\u003c/b\u003e's biological activity, a total synthesis route was investigated.\u003c/p\u003e"},{"header":"2. Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Synthesis\u003c/h2\u003e \u003cp\u003eThe synthesis commenced with the chiral raw material (\u003cem\u003eS\u003c/em\u003e)-(+)-1,2-isopropylidene glycerol (\u003cb\u003e2\u003c/b\u003e), procured from Bide Medicine. Reaction with benzyl bromide in the presence of potassium hydroxide and 18-crown-6 (as a phase-transfer catalyst) in anhydrous tetrahydrofuran afforded benzyl ether \u003cb\u003e3\u003c/b\u003e [5.6]. The 1,2-isopropylidene protecting group of \u003cb\u003e3\u003c/b\u003e was then removed by treatment with a 60% aqueous solution of acetic acid at 60\u0026ndash;65\u0026deg;C, yielding benzyl ether \u003cb\u003e4\u003c/b\u003e [5.6]. The syntheses of compounds \u003cb\u003e3\u003c/b\u003e and \u003cb\u003e4\u003c/b\u003e have been described in the literature [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Selective protection of the primary hydroxyl group of \u003cb\u003e4\u003c/b\u003e was achieved using tert-butyldimethylchlorosilane (TBSCl) and imidazole as a base in anhydrous dichloromethane, affording silyl ether \u003cb\u003e5\u003c/b\u003e. The remaining secondary hydroxyl group of \u003cb\u003e5\u003c/b\u003e was then esterified with erucic acid, using dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) as coupling reagents in anhydrous dichloromethane, to furnish ester \u003cb\u003e6\u003c/b\u003e [5.6]. Deprotection of the tert-butyldimethylsilyl (TBS) group of \u003cb\u003e6\u003c/b\u003e was accomplished with tetra-n-butylammonium fluoride (TBAF) in tetrahydrofuran, generating alcohol \u003cb\u003e7\u003c/b\u003e. Subsequent esterification of the free hydroxyl group of \u003cb\u003e7\u003c/b\u003e with pentadecanoic acid, again employing DCC/DMAP in anhydrous dichloromethane, provided ester \u003cb\u003e8\u003c/b\u003e. Finally, cleavage of the benzyl ether moiety of \u003cb\u003e8\u003c/b\u003e was effected with boron trichloride (BCl\u003csub\u003e3\u003c/sub\u003e) in anhydrous dichloromethane at low temperature (-78\u0026deg;C to -40\u0026deg;C), yielding the target compound \u003cb\u003e1\u003c/b\u003e. Importantly, under these conditions, the ester linkages and the double bond remained intact [5.6]. The overall yield of compound \u003cb\u003e1\u003c/b\u003e for the seven-step sequence was 2.33%. See Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e for a schematic representation of the synthetic route.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Biological activity\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Target prediction of compound \u003cb\u003e1\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo identify potential targets of compound \u003cb\u003e1\u003c/b\u003e, in silico predictions were performed using the SwissTargetPrediction, PharmMapper, and TargetNet databases. After removing duplicate entries, a total of 377 unique targets were identified. Rigorous probability thresholds were applied: targets from SwissTargetPrediction were included if their probability score was \u0026ge;\u0026thinsp;0.05, TargetNet targets required a score\u0026thinsp;\u0026ge;\u0026thinsp;0.002, and PharmMapper targets needed a normalized fit score\u0026thinsp;\u0026ge;\u0026thinsp;0.5.\u003c/p\u003e \u003cp\u003eCross-validation of these three databases (SwissTargetPrediction, PharmMapper, and TargetNet) revealed 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) as a common potential target (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). HMGCR is a key enzyme in the cholesterol biosynthesis pathway, catalyzing the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) to mevalonic acid (MVA). It is the primary target of statins and other cholesterol-lowering medications. Given the presence of compound \u003cb\u003e1\u003c/b\u003e in human plasma, we investigated its potential interaction with HMGCR and its possible effects on cholesterol synthesis in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Determination of the activity of HMG-CoA reductase inhibition\u003c/h2\u003e \u003cp\u003eHMGCR is a transmembrane glycoprotein located in the endoplasmic reticulum that catalyzes the conversion of HMG-CoA to mevalonate and coenzyme A (CoA) using two reducing equivalents from NADPH. This reaction represents the rate-limiting step in cholesterol biosynthesis. During the reaction, NADPH is oxidized to NADP\u003csup\u003e+\u003c/sup\u003e. NADPH exhibits an absorbance peak at 340 nm, while NADP\u003csup\u003e+\u003c/sup\u003e does not absorb at this wavelength. This difference is exploited to screen for compounds that inhibit HMGCR activity. Pravastatin was included as a positive control. The results, shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, indicate that compound \u003cb\u003e1\u003c/b\u003e, at a concentration of 50 \u0026micro;M, did not demonstrate significant HMGCR inhibitory activity. This suggests that compound \u003cb\u003e1\u003c/b\u003e does not reduce cholesterol production through HMGCR inhibition.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInhibitory activity of samples on HMGCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003econcentartion (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003epercentage inhibition (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePravastatin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e88.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eCompound \u003cb\u003e1\u003c/b\u003e was efficiently synthesized via a multi-step route from readily available fatty acids and a chiral ketal obtained from the chiral pool, employing a sequence of protection and deprotection steps. This synthetic route offers a potential template for the preparation of similar chiral glycerides. Importantly, our findings demonstrate that compound \u003cb\u003e1\u003c/b\u003e does not reduce human blood cholesterol concentrations through HMGCR inhibition. This result provides a valuable theoretical framework for the design of alternative therapeutic approaches in the development of health supplements and lipid-lowering medications.\u003c/p\u003e"},{"header":"4. Experimental","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Chemistry\u003c/h2\u003e \u003cp\u003eThe chemical reagents were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) or Shanghai Bidd Pharmaceutical Technology Co., Ltd. (Shanghai, China) and used as received. IR spectra were recorded using KBr pellets (Bruker, Bremen, Germany). \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were acquired on a Bruker spectrometer (500 MHz for \u003csup\u003e1\u003c/sup\u003eH and 125 MHz for \u003csup\u003e13\u003c/sup\u003eC; Bruker, F\u0026auml;llanden, Switzerland). The optical rotation was measured using a WZZ-2B automatic polarimeter at a wavelength of 589 nm (Sodium D line; Shanghai Yidian Physical Optical Instrument Co., Ltd, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Synthesis\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e4.2.1 Synthesis of compound \u003cb\u003e3\u003c/b\u003e\u003c/h2\u003e \u003cp\u003e(\u003cem\u003eS\u003c/em\u003e)-(+)-1,2-Isopropylidene glycerol (\u003cb\u003e2\u003c/b\u003e) (enantiomeric purity: 98%, 25 mL, 2.0 mmol), 18-crown-6 (0.05 g, 0.19 mmol), and anhydrous tetrahydrofuran (6 mL) were combined and stirred at room temperature for 40 min. The mixture was then heated to 60\u0026deg;C using an electric organic synthesizer, and benzyl bromide (0.25 mL, 2.0 mmol) was added dropwise. The reaction was stirred for several hours, with progress monitored by thin layer chromatography until equilibrium was reached. The reaction mixture was allowed to cool to room temperature and subsequently poured into water. The aqueous phase was extracted with ethyl acetate (3 \u0026times; 10 mL), and the combined organic extracts were washed with saturated brine (3 \u0026times; 8 mL) and dried over anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. The solvent was removed under reduced pressure to afford the crude product, which was purified by medium-pressure preparative chromatography using ethyl acetate/petroleum ether as the eluent, yielding compound \u003cb\u003e3\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e(\u003cem\u003eS\u003c/em\u003e)-4-((Benzyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (\u003cb\u003e3\u003c/b\u003e): colorless oil; yield 48%; [α]20 D\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.59\u0026deg; (c\u0026thinsp;=\u0026thinsp;0.13 mg/mL, CHCl\u003csub\u003e3\u003c/sub\u003e); IR (KBr) \u003cem\u003ev\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e 711, 748, 1029, 1278, 1398, 1451, 1495, 1601, 2876, 2935 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003csup\u003e1\u003c/sup\u003eH-NMR (500 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e): \u003cem\u003eδ\u003c/em\u003e 1.27 (d, 3H, CH\u003csub\u003e3\u003c/sub\u003e-), 1.32 (d, 3H, CH\u003csub\u003e3\u003c/sub\u003e-), 2.03\u0026ndash;2.05 (acetone-d\u003csub\u003e6\u003c/sub\u003e), 2.81 (s, H\u003csub\u003e2\u003c/sub\u003eO), 3.48 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.2, 9.8 Hz, -CH\u003csub\u003e2\u003c/sub\u003e-OBn), 3.55 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.3, 9.8 Hz, -CH\u003csub\u003e2\u003c/sub\u003e-OBn), 3.71 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.3, 8.2 Hz, O-CH\u003csub\u003e2\u003c/sub\u003e-Ar), 4.03 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.4, 8.2 Hz, O-CH\u003csub\u003e2\u003c/sub\u003e-Ar), 4.24 (m, 1H, H-4), 4.55 (s, 2H, -OCH\u003csub\u003e2\u003c/sub\u003eAr), 7.26\u0026ndash;7.35 (m, 5H, Ar-H); \u003csup\u003e13\u003c/sup\u003eC NMR (125 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e): \u003cem\u003eδ\u003c/em\u003e 25.7, 27.1, 67.4, 72.0, 73.7, 75.6, 109.5, 128.2, 128.3, 129.0 (C-3\u0026rsquo;\u0026rsquo;, C-5\u0026rsquo;\u0026rsquo;), 139.6; HRMS(ESI) m/z calcd for C\u003csub\u003e13\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e 223.1329, found 223.1335. The optical value of compound \u003cb\u003e3\u003c/b\u003e reported by the literature [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] was [α]20 D\u0026thinsp;=\u0026thinsp;+\u0026thinsp;15.5\u0026deg; (c\u0026thinsp;=\u0026thinsp;0.5, CHCl\u003csub\u003e3\u003c/sub\u003e). This reference did not indicate the concentration unit of the optical value.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e4.2.2 Synthesis of compound \u003cb\u003e4\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eA solution of compound \u003cb\u003e3\u003c/b\u003e (241 mg, 1.09 mmol) in 60% aqueous acetic acid (6 mL) was heated to 65\u0026deg;C using an electric organic synthesizer. The reaction mixture was stirred at this temperature for several hours, with the progress monitored by thin layer chromatography until the reaction reached equilibrium. The solution was then allowed to cool naturally to room temperature. The organic components were extracted with ethyl acetate (3 \u0026times; 12 mL). The combined ethyl acetate extracts were washed three times with 10% sodium carbonate solution (3 \u0026times; 10 mL) followed by three washes with saturated saline (3 \u0026times; 10 mL). The organic layer was then dried over anhydrous sodium sulfate (Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e). The solvent was removed by rotary evaporation under reduced pressure to afford the crude product, which was purified by medium-pressure preparative chromatography using a gradient of ethyl acetate in petroleum ether as the eluent, yielding compound \u003cb\u003e4\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e(\u003cem\u003eR\u003c/em\u003e)-3-(Benzyloxy)propane-1,2-diol (\u003cb\u003e4\u003c/b\u003e): yield 56%; [α]20 D\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.22\u0026deg; (c\u0026thinsp;=\u0026thinsp;0.10 mg/mL, CHCl\u003csub\u003e3\u003c/sub\u003e); IR (KBr) \u003cem\u003ev\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e 706, 736, 1027, 1106, 1277, 1369, 1447, 1494, 1579, 1601, 2874, 2929, 3372 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 2.03\u0026ndash;2.05 (acetone-d\u003csub\u003e6\u003c/sub\u003e), 2.85 (s, H\u003csub\u003e2\u003c/sub\u003eO), 3.46\u0026ndash;3.60 (m, 4H, 2 \u0026times; H-1, 2 \u0026times; H-3), 3.76\u0026ndash;3.81 (m, 1H, H-2), 4.53 (s, 2H, -OCH\u003csub\u003e2\u003c/sub\u003eAr), 7.24\u0026ndash;7.36 (m, 5H, Ar-H); \u003csup\u003e13\u003c/sup\u003eC NMR (125 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e): \u003cem\u003eδ\u003c/em\u003e 64.5, 71.8, 72.9, 73.6, 128.1 (C-2\u0026rsquo;\u0026rsquo;, C-6\u0026rsquo;\u0026rsquo;), 128.3, 129.0 (C-3\u0026rsquo;\u0026rsquo;, C-5\u0026rsquo;\u0026rsquo;), 139.8; HRMS(ESI) m/z calcd for C\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e 183.1016, found 183.1013. The optical value compound \u003cb\u003e4\u003c/b\u003e reported by the literature [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] was [α]20 D\u0026thinsp;=\u0026thinsp;+\u0026thinsp;16.5\u0026deg; (c\u0026thinsp;=\u0026thinsp;0.5, CHCl\u003csub\u003e3\u003c/sub\u003e). This reference also did not indicate the concentration unit of the optical value.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e4.2.3 Synthesis of compound \u003cb\u003e5\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eA mixture of compound \u003cb\u003e4\u003c/b\u003e (186 mg, 1.02 mmol), imidazole (69 mg, 1.00 mmol), tert -butyldimethylchlorosilane (0.18 mL, 1.0 mmol), and anhydrous dichloromethane (6 mL) was stirred at room temperature for several hours. The reaction was monitored by thin layer chromatography until equilibrium was reached. The organic portion was extracted with dichloromethane (3 \u0026times; 10 mL), washed with saturated brine (3 \u0026times; 10 mL), and dried over anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. The solvent was removed by evaporation under reduced pressure to obtain the crude product, which was purified by medium-pressure preparative chromatography using ethyl acetate/petroleum ether as eluent to afford compound \u003cb\u003e5\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e(\u003cem\u003eS\u003c/em\u003e)-1-(Benzyloxy)-3-((tert-butyldimethylsilyl)oxy)propan-2-ol (\u003cb\u003e5\u003c/b\u003e): colorless oil; yield 61%; [α]20 D\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.39\u0026deg; (c\u0026thinsp;=\u0026thinsp;0.21 mg/mL, CHCl\u003csub\u003e3\u003c/sub\u003e); IR (KBr) \u003cem\u003ev\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e 697, 741, 1040, 1070, 1205, 1366, 1452, 1497, 1654, 2865, 2926, 3367 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 0.07 (d, 6H, 2 CH\u003csub\u003e3\u003c/sub\u003e-), 0.89 (d, 9H, 3 CH\u003csub\u003e3\u003c/sub\u003e-), 2.03\u0026ndash;2.05 (acetone-d\u003csub\u003e6\u003c/sub\u003e), 2.82 (s, H\u003csub\u003e2\u003c/sub\u003eO), 3.47 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8, 9.7 Hz, H-1), 3.56 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8, 9.1 Hz, H-1), 3.64 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.5, 8.2 Hz, H-3), 3.68 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.3, 8.3 Hz, H-3), 3.75\u0026ndash;3.79 (m, 1H, H-2), 4.53 (s, 2H,-OCH\u003csub\u003e2\u003c/sub\u003eAr), 7.24\u0026ndash;7.36 (m, 5H, Ar-H); \u003csup\u003e13\u003c/sup\u003eC NMR (125 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 18.9 (2 CH\u003csub\u003e3\u003c/sub\u003e-Si), 26.2 (3 CH\u003csub\u003e3\u003c/sub\u003e-), 65.4, 71.7, 72.4, 73.6, 128.1, 128.3, 129.0 (C-3\u0026rsquo;, C-5\u0026rsquo;), 139.6; HRMS(ESI) m/z calcd for C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e27\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eSi [M - H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 295.1735, found 295.1741.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e4.2.4 Synthesis of compound \u003cb\u003e6\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eA mixture of compound \u003cb\u003e5\u003c/b\u003e (297 mg, 1.00 mmol), erucic acid (598 mg, 1.50 mmol), dicyclohexylcarbodiimide (DCC, 316 mg, 1.50 mmol), and 4-dimethylaminopyridine (DMAP, 15 mg, 0.12 mmol) in anhydrous dichloromethane (10 mL) was stirred at room temperature for several hours. The reaction was monitored by thin layer chromatography until equilibrium was reached. The resulting solids were filtered off, and the solvent was removed under reduced pressure. The crude product was then purified by medium-pressure preparative chromatography using ethyl acetate/petroleum ether as eluent to yield compound \u003cb\u003e6\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e(\u003cem\u003eS\u003c/em\u003e)-1-(Benzyloxy)-3-((tert-butyldimethylsilyl)oxy)propan-2-yl (\u003cem\u003eZ\u003c/em\u003e)-docos-13-enoate (\u003cb\u003e6\u003c/b\u003e): colorless oil; yield 53%; [α]20 D\u0026thinsp;=\u0026thinsp;+\u0026thinsp;1.02\u0026deg; (c\u0026thinsp;=\u0026thinsp;0.084 mg/mL, CHCl\u003csub\u003e3\u003c/sub\u003e); IR (KBr) \u003cem\u003ev\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e 695, 734, 778, 833, 1096, 1248, 1366, 1459, 1740, 2852, 2925 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 0.065 (s, 6H, 2 \u0026times; CH\u003csub\u003e3\u003c/sub\u003e-Si), 0.88 (s, 12H, -C(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3,\u003c/sub\u003e CH\u003csub\u003e3\u003c/sub\u003e\u0026minus;22\u0026rsquo;), 1.28\u0026ndash;1.36 (m, 30H, 2 \u0026times; H-3\u0026rsquo;, 2 \u0026times; H-4\u0026rsquo;, 2 \u0026times; H-5\u0026rsquo;, 2 \u0026times; H-6\u0026rsquo;, 2 \u0026times; H-7\u0026rsquo;, 2 \u0026times; H-8\u0026rsquo;, 2 \u0026times; H-9\u0026rsquo;, 2 \u0026times; H-10\u0026rsquo;, 2 \u0026times; H-11\u0026rsquo;, 2 \u0026times; H-16\u0026rsquo;, 2 \u0026times; H-17\u0026rsquo;, 2 \u0026times; H-18\u0026rsquo;, 2 \u0026times; H-19\u0026rsquo;, 2 \u0026times; H-20\u0026rsquo;, 2 \u0026times; H-21\u0026rsquo;), 1.57\u0026ndash;1.63 (m, 2H, 2 \u0026times; H-15\u0026rsquo;), 2.01\u0026ndash;2.05 (acetone-d\u003csub\u003e6\u003c/sub\u003e, 2 \u0026times; H-12\u0026rsquo;), 2.28\u0026ndash;2.81 (dt, 2H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.0, 7.7 Hz, 2 \u0026times; H-2\u0026rsquo;), 2.81 (s, H\u003csub\u003e2\u003c/sub\u003eO), 3.60\u0026ndash;3.66 (m, 2H, 2 \u0026times; H-1), 3.75\u0026ndash;3.82 (m, 2H, 2 \u0026times; H-3), 4.51\u0026ndash;4.56 (m, 2H, -OCH\u003csub\u003e2\u003c/sub\u003eAr), 5.03\u0026ndash;5.07 (m, 1H, H-2), 5.34\u0026ndash;5.35 (m, 2H, H-13\u0026rsquo;, H-14\u0026rsquo;), 7.27\u0026ndash;7.35 (m, 5H, Ar-H); \u003csup\u003e13\u003c/sup\u003eC NMR (125 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e -5.3 (-Si-(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e), 14.4, 18.8 (3 CH\u003csub\u003e3\u003c/sub\u003e- in t-Bu), 23.3, 25.7, 26.2, 27.8, 29.3 (C-4\u0026rsquo;, C-5\u0026rsquo;, C-19\u0026rsquo;), 29.6 (C-6\u0026rsquo;, C-7\u0026rsquo;, C-8\u0026rsquo;), 29.7 (C-9\u0026rsquo;, C-10\u0026rsquo;, C-17\u0026rsquo;, C-18\u0026rsquo;), 30.0 (C-11\u0026rsquo;, C-16\u0026rsquo;), 30.4 (tertiary C in t-Bu), 29.3\u0026ndash;30.4 (acetone-d\u003csub\u003e6)\u003c/sub\u003e, 32.6, 34.8, 62.6, 69.3, 73.6 (C-2, -OCH\u003csub\u003e2\u003c/sub\u003eAr), 128.2 (C-2\u0026rsquo;\u0026rsquo;, C-4\u0026rsquo;\u0026rsquo;, C-6\u0026rsquo;\u0026rsquo;), 129.0 (C-3\u0026rsquo;\u0026rsquo;, C-5\u0026rsquo;\u0026rsquo;), 130.5 (C-13\u0026rsquo;, C-14\u0026rsquo;), 139.5, 173.2; HRMS(ESI) m/z calcd for C\u003csub\u003e38\u003c/sub\u003eH\u003csub\u003e69\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eSi [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e 617.4960, found 617.4962.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e4.2.5 Synthesis of compound \u003cb\u003e7\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eA mixture of compound \u003cb\u003e6\u003c/b\u003e (617 mg, 1.00 mmol), tetra-n-butylammonium fluoride (TBAF, 1.3 mL, 1.30 mmol), and tetrahydrofuran (THF, 6 mL) was stirred at room temperature for several hours. The reaction was monitored by thin layer chromatography until equilibrium was reached. The reaction mixture was then poured into water (25 mL) and extracted with ethyl acetate (3 \u0026times; 10 mL). The combined organic extracts were washed with saturated brine (3 \u0026times; 10 mL), dried over anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and concentrated under reduced pressure to afford the crude product. Purification by medium-pressure preparative chromatography using ethyl acetate/petroleum ether as eluent gave compound \u003cb\u003e7\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e(\u003cem\u003eR\u003c/em\u003e)-1-(Benzyloxy)-3-hydroxypropan-2-yl (\u003cem\u003eZ\u003c/em\u003e)-docos-13-enoate (\u003cb\u003e7\u003c/b\u003e): colorless oil; yield 87%; [α]20 D\u0026thinsp;=\u0026thinsp;+\u0026thinsp;1.11\u0026deg; (c\u0026thinsp;=\u0026thinsp;0.076 mg/mL, CHCl\u003csub\u003e3\u003c/sub\u003e); IR (KBr) \u003cem\u003ev\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e 698, 731, 1017, 1094, 1162, 1252, 1371, 1447, 1733, 2852, 2921, 3453 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 0.87 (t, 3H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.9 Hz, CH\u003csub\u003e3\u003c/sub\u003e-), 1.29\u0026ndash;1.35 (m, 28H, 2 \u0026times; H-4\u0026rsquo;, 2 \u0026times; H-5\u0026rsquo;, 2 \u0026times; H-6\u0026rsquo;, 2 \u0026times; H-7\u0026rsquo;, 2 \u0026times; H-8\u0026rsquo;, 2 \u0026times; H-9\u0026rsquo;, 2 \u0026times; H-10\u0026rsquo;, 2 \u0026times; H-11\u0026rsquo;, 2 \u0026times; H-16\u0026rsquo;, 2 \u0026times; H-17\u0026rsquo;, 2 \u0026times; H-18\u0026rsquo;, 2 \u0026times; H-19\u0026rsquo;, 2 \u0026times; H-20\u0026rsquo;, 2 \u0026times; H-21\u0026rsquo;), 2.02\u0026ndash;2.05 (m, acetone-d\u003csub\u003e6\u003c/sub\u003e, 4H, 2 \u0026times; H-12\u0026rsquo;, 2 \u0026times; H-15\u0026rsquo;), 2.28 (t, 2H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 2 \u0026times; H-2\u0026rsquo;), 2.82 (s, H\u003csub\u003e2\u003c/sub\u003eO), 3.53 (dd, 2H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8, 5.5 Hz, 2 \u0026times; H-1), 3.98 (m, 1H, H-2), 4.06\u0026ndash;4.10 (m, 2H, H-3, HO-3), 4.15 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.6, 11.2 Hz, H-3), 4.54 (s, 2H, -OCH\u003csub\u003e2\u003c/sub\u003eAr), 5.33\u0026ndash;5.35 (m, 2H, H-13\u0026rsquo;, H-14\u0026rsquo;), 7.27\u0026ndash;7.36 (m, 5H, Ar-H); \u003csup\u003e13\u003c/sup\u003eC NMR (125 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 14.3, 23.3, 25.6, 27.7 (C-12\u0026rsquo;, C-15\u0026rsquo;), 29.3 (C-4\u0026rsquo;, C-5\u0026rsquo;, C-19\u0026rsquo;), 29.6 (C-6\u0026rsquo;, C-7\u0026rsquo;, C-8\u0026rsquo;), 29.8 (C-9\u0026rsquo;, C-10\u0026rsquo;, C-17\u0026rsquo;, C-18\u0026rsquo;), 29.9 (C-11\u0026rsquo;, C-16\u0026rsquo;), 29.3\u0026ndash;30.4 (acetone-d\u003csub\u003e6\u003c/sub\u003e), 32.6, 34.5, 66.2, 69.1, 72.3, 73.7, 128.2 (C-2\u0026rsquo;\u0026rsquo;, C-6\u0026rsquo;\u0026rsquo;), 128.3, 129.0 (C-3\u0026rsquo;\u0026rsquo;, C-5\u0026rsquo;\u0026rsquo;), 130.5 (C-13\u0026rsquo;, C-14\u0026rsquo;), 139.6, 173.6; HRMS(ESI) m/z calcd for C\u003csub\u003e32\u003c/sub\u003eH\u003csub\u003e53\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e [M - H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 501.3949, found 501.3946.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e4.2.6 Synthesis of compound \u003cb\u003e8\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eA mixture of compound \u003cb\u003e7\u003c/b\u003e (520 mg, 1.04 mmol), pentadecanoic acid (371 mg, 1.50 mmol), dicyclohexylcarbodiimide (DCC, 316 mg, 1.50 mmol), 4-dimethylaminopyridine (DMAP, 15 mg, 0.12 mmol), and anhydrous dichloromethane (10 mL) was stirred at room temperature for several hours. The reaction was monitored by thin layer chromatography until it reached completion. The resulting solids were filtered off, and the solvent was removed under reduced pressure to afford the crude product. Purification by medium-pressure preparative chromatography using ethyl acetate/petroleum ether as eluent yielded compound \u003cb\u003e8\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e(\u003cem\u003eS\u003c/em\u003e)-1-(Benzyloxy)-3-(pentadecanoyloxy)propan-2-yl (\u003cem\u003eZ\u003c/em\u003e)-docos-13-enoate (\u003cb\u003e8\u003c/b\u003e): colorless oil; yield 56%; [α]20 D\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.22\u0026deg; (c\u0026thinsp;=\u0026thinsp;0.10 mg/mL, CHCl\u003csub\u003e3\u003c/sub\u003e); IR (KBr) \u003cem\u003ev\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e 698, 735, 1026, 1104, 1165, 1245, 1372, 1461, 1744, 2853, 2920 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 0.88 (t, 6H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.8 Hz, H-22\u0026rsquo;, H-15\u0026rsquo;\u0026rsquo;), 1.29\u0026ndash;1.34 (m, 50H, 2 \u0026times; H-4\u0026rsquo;, 2 \u0026times; H-5\u0026rsquo;, 2 \u0026times; H-6\u0026rsquo;, 2 \u0026times; H-7\u0026rsquo;, 2 \u0026times; H-8\u0026rsquo;, 2 \u0026times; H-9\u0026rsquo;, 2 \u0026times; H-10\u0026rsquo;, 2 \u0026times; H-11\u0026rsquo;, 2 \u0026times; H-16\u0026rsquo;, 2 \u0026times; H-17\u0026rsquo;, 2 \u0026times; H-18\u0026rsquo;, 2 \u0026times; H-19\u0026rsquo;, 2 \u0026times; H-20\u0026rsquo;, 2 \u0026times; H-21\u0026rsquo;, 2 \u0026times; H-4\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-5\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-6\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-7\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-8\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-9\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-10\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-11\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-12\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-13\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-14\u0026rsquo;\u0026rsquo;), 1.56\u0026ndash;1.61 (m, 4H, 2 \u0026times; H-3\u0026rsquo;, 2 \u0026times; H-3\u0026rsquo;\u0026rsquo;), 2.02\u0026ndash;2.05 (m, acetone-d\u003csub\u003e6\u003c/sub\u003e, 4H, 2 \u0026times; H-12\u0026rsquo;, 2 \u0026times; H-15\u0026rsquo;), 2.26\u0026ndash;2.32 (m, 4H, 2 \u0026times; H-2\u0026rsquo;, 2 \u0026times; H-2\u0026rsquo;\u0026rsquo;), 3.63\u0026ndash;3.67 (m, 2H, 2 \u0026times; H-1), 4.17 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.6, 11.9 Hz, H-3), 4.36 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.6, 11.9 Hz, H-3), 4.56 (m, 2H, -OCH\u003csub\u003e2\u003c/sub\u003eAr), 5.22\u0026ndash;5.26 (m, 1H, H-2), 5.31\u0026ndash;5.38 (m, 2H, H-13\u0026rsquo;, H-14\u0026rsquo;), 7.26\u0026ndash;7.36 (m, 5H, Ar-H); \u003csup\u003e13\u003c/sup\u003eC NMR (125 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 14.4 (C-22\u0026rsquo;, C-15\u0026rsquo;\u0026rsquo;), 23.3 (C-21\u0026rsquo;, C-14\u0026rsquo;\u0026rsquo;), 25.6, 25.7, 27.8 (C-12\u0026rsquo;, C-15\u0026rsquo;), 29.3 (C-4\u0026rsquo;, C-5\u0026rsquo;, C-19\u0026rsquo;, C-4\u0026rsquo;\u0026rsquo;, C-5\u0026rsquo;\u0026rsquo;, C-12\u0026rsquo;\u0026rsquo;), 29.6 (C-6\u0026rsquo;, C-7\u0026rsquo;, C-8\u0026rsquo;, C-6\u0026rsquo;\u0026rsquo;, C-7\u0026rsquo;\u0026rsquo;, C-8\u0026rsquo;\u0026rsquo;, C-9\u0026rsquo;\u0026rsquo;, C-10\u0026rsquo;\u0026rsquo;, C-11\u0026rsquo;\u0026rsquo;), 29.8 (C-9\u0026rsquo;, C-10\u0026rsquo;, C-17\u0026rsquo;, C-18\u0026rsquo;), 29.9 (C-11\u0026rsquo;, C-16\u0026rsquo;), 29.3\u0026ndash;30.4 (acetone-d\u003csub\u003e6\u003c/sub\u003e), 32.6 (C-20\u0026rsquo;, C-13\u0026rsquo;\u0026rsquo;), 34.5, 34.7, 63.2, 69.2, 70.9, 73.6, 128.3, 129.1 (C-2\u0026rsquo;\u0026rsquo;\u0026rsquo;, C-6\u0026rsquo;\u0026rsquo;\u0026rsquo;), 130.5 (C-13\u0026rsquo;, C-14\u0026rsquo;, C-3\u0026rsquo;\u0026rsquo;\u0026rsquo;, C-5\u0026rsquo;\u0026rsquo;\u0026rsquo;), 139.2, 173.1, 173.3; HRMS(ESI) m/z calcd for C\u003csub\u003e47\u003c/sub\u003eH\u003csub\u003e83\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e 727.6235, found 727.6230.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e4.2.7 Synthesis of compound \u003cb\u003e1\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eCompound \u003cb\u003e8\u003c/b\u003e (120 mg, 0.16 mmol) was dissolved in anhydrous dichloromethane (4 mL) and cooled to -50\u0026deg;C. Boron trichloride (1.0 M in methylene chloride, 0.32 mL, 0.32 mmol) was added dropwise, and the solution was stirred at -50\u0026deg;C for 30\u0026ndash;60 minutes, monitoring the reaction by thin layer chromatography until equilibrium was reached. The reaction was quenched with water, and the organic layer was extracted with dichloromethane, washed with saturated brine (3 \u0026times; 10 mL), and dried over anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. The solvent was removed under reduced pressure, and the crude product was purified by medium-pressure preparative chromatography (EtOAc/petroleum ether) to afford compound \u003cb\u003e1\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e(\u003cem\u003eS\u003c/em\u003e)-1-Hydroxy-3-(pentadecanoyloxy)propan-2-yl (\u003cem\u003eZ\u003c/em\u003e)-docos-13-enoate (\u003cb\u003e1\u003c/b\u003e): colorless oil; yield 55%; [α]20 D\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.16\u0026deg; (c\u0026thinsp;=\u0026thinsp;0.10 mg/mL, CHCl\u003csub\u003e3\u003c/sub\u003e); IR (KBr) \u003cem\u003ev\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e 547, 718, 1179, 1381, 1427, 1467, 1733, 2850, 2919, 3496 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 0.87 (t, 6H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.8 Hz, CH\u003csub\u003e3\u003c/sub\u003e-22\u0026rsquo;, CH\u003csub\u003e3\u003c/sub\u003e-15\u0026rsquo;\u0026rsquo;), 1.29 (m, 50H, 2 \u0026times; H-4\u0026rsquo;, 2 \u0026times; H-5\u0026rsquo;, 2 \u0026times; H-6\u0026rsquo;, 2 \u0026times; H-7\u0026rsquo;, 2 \u0026times; H-8\u0026rsquo;, 2 \u0026times; H-9\u0026rsquo;, 2 \u0026times; H-10\u0026rsquo;, 2 \u0026times; H-11\u0026rsquo;, 2 \u0026times; H-16\u0026rsquo;, 2 \u0026times; H-17\u0026rsquo;, 2 \u0026times; H-18\u0026rsquo;, 2 \u0026times; H-19\u0026rsquo;, 2 \u0026times; H-20\u0026rsquo;, 2 \u0026times; H-21\u0026rsquo;, 2 \u0026times; H-4\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-5\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-6\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-7\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-8\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-9\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-10\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-11\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-12\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-13\u0026rsquo;\u0026rsquo;, 2 \u0026times; H-14\u0026rsquo;\u0026rsquo;), 1.57\u0026ndash;1.61 (m, 4H, 2 \u0026times; H-3\u0026rsquo;, 2 \u0026times; H-3\u0026rsquo;\u0026rsquo;), 2.03\u0026ndash;2.05 (m, acetone-d\u003csub\u003e6\u003c/sub\u003e, 4H, 2 \u0026times; H-12\u0026rsquo;, 2 \u0026times; H-15\u0026rsquo;), 2.27\u0026ndash;2.31 (m, 4H, 2 \u0026times; H-2\u0026rsquo;, 2 \u0026times; H-2\u0026rsquo;\u0026rsquo;), 3.67 (t, 2H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 2 \u0026times; H-1), 4.03 (t, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, HO-1), 4.15 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0, 11.9 Hz, H-3), 4.35 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.5, 14.9 Hz, H-3), 5.04\u0026ndash;5.08 (m, 1H, H-2), 5.31\u0026ndash;5.37 (m, 2H, H-13\u0026rsquo;, H-14\u0026rsquo;); \u003csup\u003e13\u003c/sup\u003eC NMR (125 MHz, acetone-d\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 14.3 (C-22\u0026rsquo;, C-15\u0026rsquo;\u0026rsquo;), 23.3 (C-21\u0026rsquo;, C-14\u0026rsquo;\u0026rsquo;), 25.6 (C-3\u0026rsquo;, C-3\u0026rsquo;\u0026rsquo;), 27.7 (C-12\u0026rsquo;, C-15\u0026rsquo;), 29.3 (C-4\u0026rsquo;, C-5\u0026rsquo;, C-19\u0026rsquo;, C-4\u0026rsquo;\u0026rsquo;, C-5\u0026rsquo;\u0026rsquo;, C-12\u0026rsquo;\u0026rsquo;), 29.6 (C-6\u0026rsquo;, C-7\u0026rsquo;, C-8\u0026rsquo;, C-6\u0026rsquo;\u0026rsquo;, C-7\u0026rsquo;\u0026rsquo;, C-8\u0026rsquo;\u0026rsquo;, C-9\u0026rsquo;\u0026rsquo;, C-10\u0026rsquo;\u0026rsquo;, C-11\u0026rsquo;\u0026rsquo;), 29.8 (C-9\u0026rsquo;, C-10\u0026rsquo;, C-17\u0026rsquo;, C-18\u0026rsquo;), 29.9 (C-11\u0026rsquo;, C-16\u0026rsquo;), 29.3\u0026ndash;30.4 (acetone-d\u003csub\u003e6\u003c/sub\u003e), 32.6 (C-20\u0026rsquo;, C-13\u0026rsquo;\u0026rsquo;), 34.5, 34.7, 61.2, 63.1, 72.9, 130.5 (C-13\u0026rsquo;, C-14\u0026rsquo;), 173.2, 173.4; HRMS(ESI) m/z calcd for C\u003csub\u003e40\u003c/sub\u003eH\u003csub\u003e76\u003c/sub\u003eNaO\u003csub\u003e5\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e 659.5591, found 659.5583.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Target prediction of compound \u003cb\u003e1\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eCompound \u003cb\u003e1\u003c/b\u003e in sdf format were imported into the SwissTargetPrediction database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [7.8], PharmMapper (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.lilab-ecust.cn/pharmmapper/\u003c/span\u003e\u003cspan address=\"http://www.lilab-ecust.cn/pharmmapper/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and TargetNet (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://targetnet.scbdd.com/\u003c/span\u003e\u003cspan address=\"http://targetnet.scbdd.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], respectively, then \u0026ldquo;homo sapiens\u0026rdquo; was select as the target species. Following the instructions, the targets of the compound \u003cb\u003e1\u003c/b\u003e were obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Experiment to inhibit the activity of HMG-CoA reductase\u003c/h2\u003e \u003cp\u003eThe HMG-CoA reductase assay kit was purchased from Sigma-Aldrich (Shanghai, China). Samples were added to a 96-well microplate, followed by NADPH (final concentration 0.33 mg/mL), HMG-CoA substrate, and HMGCR (final concentration 5 \u0026micro;g/mL to 7 \u0026micro;g/mL), in that order. Each condition was tested in triplicate. A blank control (without drug) and a Pravastatin positive control were also included. The plate was incubated at 37\u0026deg;C for 10 minutes, and the optical density (OD) was measured at 340 nm using a microplate reader. The percentage inhibition of HMGCR was then calculated.\u003c/p\u003e \u003cp\u003eInhibition (%) = (1 - OD\u003csub\u003e340 nm\u003c/sub\u003e of experimental well / OD\u003csub\u003e340 nm\u003c/sub\u003e of blank well) \u0026times; 100%\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003ch3\u003eA data availability statement\u003c/h3\u003e\n\u003cp\u003eThe data that support this study are available in the article and accompanying online supplementary material.\u003c/p\u003e\n\u003ch3\u003eFunding Declaration\u003c/h3\u003e\n\u003cp\u003eThis research was funded by the Science and Technology Bureau of Putian City (Grant No. 2021S2001-9) and Fujian Shanhe Pharmaceutical Co., LTD (Grant No.2022AHX211(L)).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXM Liao completed the total synthesis of chiral diacylglycerol and drafted the initial version of the article. X Chen and RR Zhuang jointly conducted the bioactivity assays of the target compound. BD Zhou provided the research methods and revised the paper.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support this study are available in the article and accompanying online supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHan, L. Y. et al. Diacylglycerol acyltransferase 3(DGAT3) is responsible for the biosynthesis of unsaturated fatty acids in vegetative organs of \u003cem\u003ePaeonia rockii\u003c/em\u003e. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 14390 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFoo, S., Cazenave-Gassiot, A., Wenk, M. R. \u0026amp; Oliferenko, S. Diacylglycerol at the inner nuclear membrane fuels nuclear envelope expansion in closed mitosis. \u003cem\u003eJ. Cell. Sci.\u003c/em\u003e \u003cb\u003e136\u003c/b\u003e, 1\u0026ndash;14 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShulga, Y. V., Topham, M. K. \u0026amp; Epand, R. M. Regulation and functions of diacylglycerol kinases. \u003cem\u003eChem. Rev.\u003c/em\u003e \u003cb\u003e111\u003c/b\u003e, 6186\u0026ndash;6208 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTimmers, S., Schrauwen, P. \u0026amp; Vogel, J. D. Muscular diacylglycerol metabolism and insulin resistance. \u003cem\u003ePhysiol. Behav.\u003c/em\u003e \u003cb\u003e94\u003c/b\u003e, 242\u0026ndash;251 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia, J. \u0026amp; Hui, Y. Z. Synthesis of a small library of mixed-acid phospholipids from D-mannitol as a homochiral starting material. \u003cem\u003eChem. Pharm. Bull.\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e, 1659\u0026ndash;1663 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu, B. L. et al. Investigating the individual importance of the pam\u003csub\u003e2\u003c/sub\u003ecys ester motifs on TLR2 activity. \u003cem\u003eEur. J. Org. Chem.\u003c/em\u003e \u003cb\u003e39\u003c/b\u003e, 5415\u0026ndash;5423 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaina, A., Michielin, O. \u0026amp; Zoete, V. SwissTargetPrediction: updated data and new features for efficient prediction of protein targets of small molecules. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e (W1), W357\u0026ndash;W364 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGfeller, D., Michielin, O. \u0026amp; Zoete, V. Shaping the interaction landscape of bioactive molecules. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e (23), 3073\u0026ndash;3079 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, X. F. et al. PharmMapper server: a web server for potential drug target identification via pharmacophore mapping approach. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e38\u003c/b\u003e, W609\u0026ndash;W614 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, X. et al. Enhancing the enrichment of pharmacophore-based target prediction for the polypharmacological profiles of drugs. \u003cem\u003eJ. Chem. Inf. Model.\u003c/em\u003e \u003cb\u003e56\u003c/b\u003e, 1175\u0026ndash;1183 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, X. et al. PharmMapper 2017 update: a web server for potential drug target identification with a comprehensive target pharmacophore database. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e45\u003c/b\u003e, W356\u0026ndash;W360 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao, Z. J. et al. TargetNet: a web service for predicting potential drug-target interaction profiling via multi-target SAR models. \u003cem\u003eJ. Comput. Aided Mol. Des.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 413\u0026ndash;424 (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"total synthesis, chiral diglyceride, HMG-CoA reductase","lastPublishedDoi":"10.21203/rs.3.rs-6896088/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6896088/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCompound \u003cb\u003e1\u003c/b\u003e, a chiral diglyceride metabolite found in both humans and fungi, was targeted for total synthesis. A seven-step synthetic route was developed, affording compound \u003cb\u003e1\u003c/b\u003e in 2.33% overall yield. The key steps involved: (1) selective protection of the terminal hydroxyl group of chiral ketal \u003cb\u003e2\u003c/b\u003e with a sterically hindered benzyl group, followed by removal of the ketal moiety to generate benzyl ether \u003cb\u003e4\u003c/b\u003e; (2) protection of the terminal hydroxyl group of benzyl ether \u003cb\u003e4\u003c/b\u003e with a bulky silyl protecting group, and subsequent esterification of the remaining free hydroxyl with erucic acid to yield ester \u003cb\u003e6\u003c/b\u003e; (3) removal of the silyl protecting group from ester \u003cb\u003e6\u003c/b\u003e, followed by esterification of the liberated hydroxyl group with pentadecanoic acid, affording ester \u003cb\u003e8\u003c/b\u003e; and (4) selective deprotection of the benzyl group of ester \u003cb\u003e8\u003c/b\u003e to furnish compound \u003cb\u003e1\u003c/b\u003e. \u003cem\u003eIn silico\u003c/em\u003e screening using three databases identified 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) as a potential biological target of compound \u003cb\u003e1\u003c/b\u003e. However, \u003cem\u003ein vitro\u003c/em\u003e HMGCR inhibition assays demonstrated that compound \u003cb\u003e1\u003c/b\u003e did not significantly reduce cholesterol levels in human blood. These results contribute to the chemical synthesis and biological evaluation of chiral diglycerides and provide a foundation for future investigations in this area.\u003c/p\u003e","manuscriptTitle":"Total Synthesis and Bioactivity Investigation of a Chiral Diacylglycerol","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-30 08:25:00","doi":"10.21203/rs.3.rs-6896088/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-10T05:57:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-09T06:07:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-07T08:59:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6364767239132892615021101730084801316","date":"2025-06-30T14:28:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262597836071387926778444038288250403400","date":"2025-06-28T00:45:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"327789315586982249281966308501644363826","date":"2025-06-27T11:01:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331216233000014269681629395500936652789","date":"2025-06-25T20:50:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-25T14:18:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-25T13:38:17+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-25T13:10:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-17T07:07:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-17T07:02:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b0bb06d0-16b3-4b17-a4e7-934c2e1262ae","owner":[],"postedDate":"June 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50718511,"name":"Biological sciences/Biochemistry"},{"id":50718512,"name":"Biological sciences/Molecular biology"},{"id":50718513,"name":"Physical sciences/Chemistry/Organic chemistry"},{"id":50718514,"name":"Physical sciences/Chemistry/Synthesis"}],"tags":[],"updatedAt":"2025-09-01T16:00:33+00:00","versionOfRecord":{"articleIdentity":"rs-6896088","link":"https://doi.org/10.1038/s41598-025-14482-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-30 15:57:26","publishedOnDateReadable":"August 30th, 2025"},"versionCreatedAt":"2025-06-30 08:25:00","video":"","vorDoi":"10.1038/s41598-025-14482-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-14482-2","workflowStages":[]},"version":"v1","identity":"rs-6896088","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6896088","identity":"rs-6896088","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}