Bioorganic Chemistry 65 (2016) 17–25

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Sonochemical enzyme-catalyzed regioselective acylation of flavonoid glycosides Ziaullah, H.P. Vasantha Rupasinghe ⇑ Department of Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, Nova Scotia B2N 5E3, Canada

a r t i c l e

i n f o

Article history: Received 29 November 2015 Revised 12 January 2016 Accepted 23 January 2016 Available online 23 January 2016 Keywords: Flavonoids Isoquercitrin Phloridzin Acylation Sonication-assisted irradiation Candida antarctica lipase B NMR spectroscopy

a b s t r a c t This work compares a highly efficient and alternative method of sonication-assisted lipase catalyzed acylation of quercetin-3-O-glucoside and phloretin-20 -glucoside, using Candida antarctica lipase B Ò (Novozyme 435 ), with a range of fatty acids. In this study, sonication-assisted irradiation coupled with stirring has been found to be more efficient and economical than conventional reaction conditions. Sonication-assisted acylation accelerated the reactions and reduced the time required by 4–5 folds. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Lipases, produced from microorganisms, play a pivotal role in industrial applications. They comprise an important group of enzymes that are used in biotechnology and have numerous industrial applications in dairy, food and pharmaceutical industries [1,2]. They are able to catalyze hydrolysis, alcoholysis, aminolysis, acidolysis, esterification and transesterification in different media [3–5]. The major advantages of using lipases over synthetic or semi synthetic methods are their wide range of substrates with higher degree of regio-, chemo- and stereo-selectivity under mild reaction conditions [6]. Lipases have gained considerable attention from food chemistry as well as biotechnology sectors for the synthesis of esters of natural flavors and food colors [7]. Recently, the term ‘‘natural” flavors have been defined as substances that can be extracted from natural sources such as plants, fruits or vegetables or prepared by enzymatic or microbial processes. Interestingly, it has been found that consumers are more convinced with the products if they are labeled as ‘‘natural” as compared to those labeled as ‘‘synthetic” [8]. Therefore, lipase catalyzed esterification has been investigated by many scientists to synthesize esters for industrial applications [9–13].

⇑ Corresponding author. E-mail address: [email protected] (H.P.V. Rupasinghe). http://dx.doi.org/10.1016/j.bioorg.2016.01.005 0045-2068/Ó 2016 Elsevier Inc. All rights reserved.

In principle, ultrasound is a mechanical rather than an electromagnetic wave. It is an oscillating sound wave with a frequency greater than the upper limit of the human hearing range (20 kHz). The ultrasound devices operate with frequencies from 20 kHz up to several GHz. Ultrasonication is considered as a ‘‘green” technology as well as it efficiently reduces the process time as compared to conventional mixing. Moreover, it is economical due to relatively low consumption of energy [14]. Ultrasonics has become an effective tool in green chemistry, synthetic organic chemistry, biological reactions and biotechnology during last few decades due to its capability of increasing the reaction rates and yields [15–17]. The reactions that require several days under conventional conditions can now be completed in few hours by this efficient and environmentally green technology. In general, ultrasound technology has the capability of increasing the interaction between phases by cavitations which results from the collapse of bubbles. The ultrasonic jet then disrupts the boundaries of the phases and causes exponential increase in emulsification [14,18]. In addition, the application of ultrasound to a suspension results in an increase in mixing and mass transfer [15,19]. Kwiatkowska et al. [17] has reported that the ultrasound frequency increases the geometric and electronic complementariness between substrate and enzyme interaction of the enzymesubstrate model. Nevertheless, it is notable that the influence of ultrasonics on the stability and activity of enzymes depends on the sonication parameters especially the power (frequency) of

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Ziaullah, H.P.V. Rupasinghe / Bioorganic Chemistry 65 (2016) 17–25

ultrasound. Some scientists have suggested that the ultrasound influence the reaction kinetics which is apparent from the reduction of esterification reaction time [20,21]. However, its application to enzyme catalysis is still hardly known and to the best of our knowledge the application of ultrasound-assisted technology for lipase-catalyzed synthesis of flavor esters is the latest of its kind [9–13,15–17,20–36]. The enzyme-catalyzed esterification and transesterification of flavonoid glycosides with various acyl donors under conventional conditions has been well documented in previous reports [22–36]. However, our present investigation provides an efficient, alternative and greener approach for lipase-catalyzed acylation of quercetin-3-O-glucoside (isoquercitrin) and phloretin-20 glucoside (phloridzin) with fatty acids of different chain lengths by using ultrasonic technology. Our present study has been supported by different analytical techniques including NMR spectroscopy. 2. Results and discussion 2.1. Chemistry Esterification of phloridzin and isoquercitrin with 20 different fatty acids of various chain lengths (C4–C18) were carried out at 40–45 °C in extra dry acetone along with ultrasonication or sonication coupled with stirring. The biocatalytic reactions were started Ò by the addition of Candida antarctica lipase B (Novozyme 435 ; purchased from Sigma–Aldrich (Mississauga, ON, Canada) into a solution of flavonoid glycosides and fatty acids in acetone, in the presence of flame dried molecular sieves (3 Å) (Scheme 1, Table 1). In the conventional method, the reactions were carried out according to our previously described procedure in Ziaullah et al. [37]. 2.2. Effect of temperature To study the effect of reaction temperature on the esterification, reactions were carried out under ultrasonic irradiation or ultrasonic irradiation coupled with stirring at different temperatures ranging from 40 to 70 °C. During optimization of this method we discovered that the activity of enzyme is at the optimum reaction

Ò

rate when reaction temperature is between 40 and 45 °C. When the temperature was further elevated to 60 °C, the activity of enzyme accelerated but on TLC it was noticed that further acylation occurs at some different site of the flavonoid glycosides. However, these trace amounts were not isolated due to their lower quantity. At elevated temperature, possibly the cavitation events increased which probably helped in increasing the mass transfer and thus consequently decreases the selectivity of enzyme. At above 60 °C we noticed that the activity of the enzyme decreases drastically. This decrease in the enzyme activity at elevated temperature could be due to thermal denaturation of enzyme. Therefore, considering the selectivity of the enzyme, conversion and feasibility, the optimum temperature found for the ultrasonic assisted synthesis was 40–45 °C. 2.3. Effect of power of ultrasound The different power grades of ultrasound are important factors for reactions under ultrasound irradiation. Herein, we selected two different powers of 20 kHz ultrasound, namely 100 and 120 W for our ultrasound-assisted reactions with intervals of 15–20 min each. The reactions accelerated with increase in the ultrasonic intensities which possibly affected the activity of enzyme. In case of lipase Amano PS-catalyzed acylations, Brenelli and Fernandes [38] have proved that rates of acyl transfer increased several folds by using ultrasonic technology as compared to conventional magnetic stirring [39,15,40]. In our case, at the experimental acoustic Ò power, the immobilized lipase (Novozyme 435 ) accelerated the reaction by 4–5 folds but did not show any deactivation at the optimum temperature. 2.4. Ultrasonic irradiation vs conventional stirring method These esterifications were conducted at optimized conditions to compare the conversion obtained for enzyme catalyzed transesterification with ultrasonic irradiation and ultrasonication coupled with conventional stirring. The results obtained are shown in Table 1. It was found that when the flavonoid glycoside esters are synthesized by ultrasonic irradiation coupled with stirring, the reactions complete in 2.5–4 h whereas with conventional

Scheme 1. (i) Immobilized lipase B, from Candida antarctica (Novozyme 435 ), 3 Å molecular sieves, ultrasonication, Acetone, 40–45 °C, 2–3.5 h or ultrasonication coupled with stirring (3–5 h); R = butyric, octanoic, dodecanoic, palmitic, oleic, stearic, linoleic, linolenic, eicosapentaenoic, docosahexaenoic acids or their corresponding esters.

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Ziaullah, H.P.V. Rupasinghe / Bioorganic Chemistry 65 (2016) 17–25 Table 1 Reaction time and yield for ultrasonication-based acylation of fatty acids with flavonoids. Flavonoids

Fatty acid

Structure of the product

Reaction time (h) Conventional [37]

Ultrasonication

Ultrasonication and Stirring

Yielda (%)

Phloridzin

Butyric acid (C4H8O2)

24

3.5

2

81b

Phloridzin

Octanoic acid (C8H16O2)

24

3.5

2

84b

Phloridzin

Dodecanoic acid (C12H24O2)

24

4

3

97

Phloridzin

Palmitic acid (C16H32O2)

24

4.5

3

97

Phloridzin

Stearic acid (C18H36O2)

24

4.5

3

96

(continued on next page)

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Ziaullah, H.P.V. Rupasinghe / Bioorganic Chemistry 65 (2016) 17–25

Table 1 (continued) Flavonoids

Fatty acid

Structure of the product

Reaction time (h) Conventional [37]

Ultrasonication

Ultrasonication and Stirring

Yielda (%)

Phloridzin

Oleic acid (C18H34O2)

24

4

2.5

93

Phloridzin

Linoleic acid (C18H32O2)

24

4

3

89

Isoquercitrin

Butyric acid (C4H8O2)

24

3

2

83b

Isoquercitrin

Octanoic acid (C8H16O2)

24

3

2.5

81b

Isoquercitrin

Dodecanoic acid (C12H24O2)

24

3.5

3

92

Isoquercitrin

Palmitic acid (C16H32O2)

24

4.5

3

93

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Ziaullah, H.P.V. Rupasinghe / Bioorganic Chemistry 65 (2016) 17–25 Table 1 (continued) Flavonoids

a b

Fatty acid

Structure of the product

Reaction time (h) Conventional [37]

Ultrasonication

Ultrasonication and Stirring

Yielda (%)

Isoquercitrin

Stearic acid (C18H36O2)

24

4.5

3

96

Isoquercitrin

Oleic acid (C18H34O2)

24

4

3

93

Isoquercitrin

Linoleic acid (C18H32O2)

24

4.2

3

91

There is no significant difference in yields of all the three methods. Compounds were volatile and some quantity lost during evaporation on rotavap.

stirring reactions completed in 24 h [40]. Thus, it can be postulated that ultrasonics induces an emulsion and mass transfer, which accelerates the reactions as compared to conventional stirring method. However, very little is known so far about the actual effect of ultrasound on enzyme behavior which requires a much deeper investigation to obtain more detailed and precise information.

purified by column chromatography, using silica gel 60 (0.040– 0.063 mm; 230–400 mesh, Merck, Darmstadt, Germany) and eluted with acetone toluene. The samples were analyzed by 1H NMR and 13 C NMR spectroscopic techniques in solvent DMSO-d6 or MeOD, on Bruker ADVANCE 300 MHz spectrometer. The IR data was collected on Nicolet 6700 FT-IR spectrometer.

2.5. Reusability of enzyme

2.7. Conclusion

Recycling of the enzyme is crucial factor to test the commercial viability of the enzyme catalyzed process. To assess the reusability of the enzyme, the immobilized enzyme was filtered from the reaction medium after completion of esterification (Table 2). The recovered biocatalyst was washed with acetone and dried for 18–20 h under phosphorous pentoxide (P2O5) in a desiccator and reused in a new fresh reaction to study the reusability extent. We reused the enzyme for 3-cycles but found that the conversion decreases gradually after each cycle. Nevertheless, it was noticed that the enzyme activity loss is due to physical breakage of enzyme supporting material during the sonication process.

The present work reports fourteen esters of two series of flavonoid glycosides with a range of fatty acids of various chain lengths under ultrasound irradiation as well as ultrasound irradiation couÒ pled with stirring while using Novozyme 435 in organic solvent. Our results proved the significance of ultrasonics for the selective esterification of flavonoid glycosides under greener and milder conditions. It has been found that if sonication is coupled with stirring it provides the most efficient enzyme catalyzed esterification method.

2.6. Analytical methods

3.1. General procedure for the biocatalytic esterification reaction of phloridzin and isoquercitrin (2–11 and 13–21)

The progress of esterification was monitored by thin-layer chromatography (TLC) on silica gel 60 F254 plates from Merck (Darmstadt, Germany), using a solvent system of acetone/toluene (40:60, few drops of acetic acid). The products were detected by using anisaldehyde as spray reagent. The acylated products were

3. Experimental

To flame dried 3 Å molecular sieves in an amber color vial, was added phloridzin (0.100 g; 0.23 mmol), butyric acid (0.06 ml, Ò 0.69 mmol), Novozyme 435 (0.300 g). It was followed by the addition of dry acetone (5 ml). The reaction was carried out in an

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Table 2 Reusability of enzyme and percentage yields.

a b c

Compound

Quantity of enzyme utilized (1st cycle) (g)a

Yield (1st cycle) (%)

Quantity of recovered enzyme (2nd cycle) (g)b

Yield (2nd cycle) (%)

Quantity of recovered enzyme (3rd cycle) (g)c

Yield (3rd cycle) (%)

2 3 4 5 6 7 8 9 10 11 12 13 14 15

0.300 0.302 0.303 0.302 0.304 0.302 0.301 0.302 0.303 0.304 0.303 0.303 0.302 0.303

81 84 97 97 96 93 89 83 81 92 93 96 93 91

0.293 0.295 0.297 0.296 0.297 0.296 0.294 0.292 0.294 0.295 0.293 0.296 0.295 0.294

76 77 89 90 91 87 83 76 75 84 86 88 84 83

0.284 0.286 0.290 0.289 0.287 0.288 0.284 0.283 0.283 0.284 0.283 0.288 0.284 0.283

68 69 80 83 82 82 77 68 67 77 80 79 75 74

1st cycle: enzyme utilized in the reaction mixture was fresh and dried. 2nd cycle: enzyme recovered and reutilized in the second cycle. 3rd cycle: enzyme recovered and re-reutilized in the third cycle.

ultrasonic bath of 20 kHz/1000 W (model 750D, VWR, West Chester, PA, USA) and exposed for 3.5–5 h under ultrasound irradiation alone (for 15–20 min each with 10 min interval in between) and for 2–3.5 h (for 15–20 min each with 10 min interval in between) under ultrasound irradiation coupled with stirring at 40–45 °C (Table 1). The progress of reaction was monitored by thin layer chromatography, followed by staining with anisaldehyde spray reagent and then heating at 110 °C. After completion of reaction, it was filtered, evaporated and chromatographed (acetone:toluene; 35:75–50:50) to get the pure butyric acid ester (2) of phloridzin. All other reactions followed the same procedure and produced esters 2–8 and 9–15, with yields in the range of 81–97%. The pure compounds were then analyzed by IR and NMR spectroscopy.

J = 10.8 Hz, H-6a00 ), 4.12 (dd, 1H, J = 10.8 Hz, 6.8 Hz, H-6b00 ), 3.62 (br t, 1H, J = 7.7 Hz, H-400 ), 3.49–3.17 (m, 6H, 2  Ha, H-200 , H-300 , H-500 , OH), 2.78 (br t, 2H, J = 7.3 Hz, 2  Hb), 2.27 (br t, 2H, J = 7.4 Hz, 2  H-200 ), 1.52–1.42 (m, 2H, 2  H-300 ), 1.24–1.16 (m, 8H, 4  CH2), 0.81 (br t, 3H, J = 6.3 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz): d 205.12 (CO), 173.30 (OCO), 165.83 (C-40 ), 164.92 (C60 ), 161.06 (C-20 ), 155.75 (C-4), 131.94 (C-1), 129.60 (C-2, C-6), 115.41 (C-3, C-5), 105.60 (C-10 ), 101.00 (C-100 ), 97.36 (C-30 ), 94.94 (C-50 ), 76.85 (C-300 ), 74.34 (C-500 ), 73.55 (C-200 ), 70.24 (C-400 ), 63.49 (C-600 ), 45.47 (Ca), 33.86 (C-2000 ), 31.58 (Cb), 29.52, 28.85, (C-4000 , C-5000 , C-6000 ), 24.85 (C-30 00 ), 22.50 (C-7000 ), 14.38 (C-8000 ).

3.1.1. (6-{3,5-Dihydroxy-2-[3-(4-hydroxyphenyl)propanoyl]phenoxy}3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl butyrate (2) Yield: 81%; light yellow liquid; Rf: 0.53 (acetone:toluene; 5:5: few drops of AcOH); IR (KBr) cm1: 3386, 2955, 2836, 2529, 2241, 2043, 1715, 1632, 1589, 1511, 1453, 1265, 1118, 1029, 916, 736, 653; 1H NMR (MeOD, 300 MHz): d 13.47 (s, 1H, ArOH), 10.56 (br s, 1H, ArOH), d 7.06 (d, 2H, J = 8.5 Hz, H-2, H-6), 6.71 (d, 2H, J = 8.5 Hz, H-3, H-5), 6.15 (d, 1H, J = 2.1 Hz, H-30 ), 6.00 (d, 1H, J = 2.1 Hz, 1H, H-50 ), 5.02 (br d, 1H, J = 7.5 Hz, H-100 ), 4.40 (dd, 1H, J = 11.8 Hz, J = 2.1 Hz, H-6a00 ), 4.27 (dd, 1H, J = 11.8 Hz, 6.2 Hz, H-6b00 ), 3.70–3.63 (br m, 1H, J = 7.7 Hz, H-400 ), 3.51–3.31 (m, 6H, 2  Ha, H-200 , H-300 , H-500 , OH), 2.88 (br t, 2H, J = 7.6 Hz, 2  Hb), 2.29 (br t, 2H, J = 7.4 Hz, 2  H-2000 ), 1.56 (m, 2H, 2  H-3000 ), 0.88 (br t, 3H, J = 7.4 Hz, CH3); 13C NMR (MeOD, 75 MHz): d 205.19 (CO), 173.92 (OCO), 166.14 (C-40 ), 164.48 (C-60 ), 160.77 (C-20 ), 154.98 (C-4), 132.46 (C-1), 129.02 (C-2, C-6), 114.73 (C-3, C-5), 105.59 (C-10 ), 100.60 (C-100 ), 97.07 (C-30 ), 94.48 (C-50 ), 76.89 (C-300 ), 74.24 (C-500 ), 73.28 (C-200 ), 70.11 (C-400 ), 62.95 (C-600 ), 45.57 (Ca), 35.46 (C-20 00 ), 29.53 (Cb), 18.03 (C-3000 ), 12.56 (C-4000 ).

3.1.3. (6-{3,5-Dihydroxy-2-[3-(4-hydroxyphenyl)propanoyl]phenoxy}3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl dodecanoate (4) Yield: 97%; light yellow solid; Rf: 0.53 (acetone:toluene; 5:5: few drops of AcOH); IR (KBr) cm1: 3382, 2943, 2837, 2525, 2246, 2048, 1714, 1635, 1596, 1517, 1458, 1264, 1119, 1027, 736, 658; 1H NMR (DMSO-d6, 300 MHz): d 13.49 (s, 1H, ArOH), 10.52 (br s, 1H, ArOH), 7.03 (d, 2H, J = 8.4 Hz, H-2, H-6), 6.65 (d, 2H, J = 8.4 Hz, H-3, H-5), 6.10 (d, 1H, J = 2.1 Hz, H-30 ), 5.95 (d, 1H, J = 2.1 Hz, 1H, H-50 ), 5.36 (br.s, 1H, OH), 5.29 (br s, 1H, OH), 5.26 (br s, 1H, OH), 4.98 (d, 1H, J = 7.2 Hz, H-100 ), 4.32 (br d, 1H, J = 10.2 Hz, H-6a00 ), 4.12 (dd, 1H, J = 10.2 Hz, 6.8 Hz, H-6b00 ), 3.61 (br t, 1H, J = 7.65 Hz, H-400 ), 3.48–3.19 (m, 6H, 2  Ha, H-200 , H-300 , H-500 , OH), 2.78 (br t, 2H, J = 7.3 Hz, 2  Hb), 2.26 (br t, 2H, J = 7.3 Hz, 2  H-200 ), 1.52–1.41 (m, 2H, 2  H-300 ), 1.25–1.13 (m, 16H, 8  CH2), 0.84 (br t, 3H, J = 6.4 Hz, CH3); 13C NMR (DMSOd6, 75 MHz): d 205.10 (CO), 173.29 (OCO), 165.89 (C-40 ), 164.94 (C-60 ), 161.09 (C-20 ), 155.75 (C-4), 131.93 (C-1), 129.59 (C-2, C-6), 115.41 (C-3, C-5), 105.57 (C-10 ), 101.04 (C-100 ), 97.38 (C-30 ), 94.95 (C-50 ), 76.86 (C-300 ), 74.34 (C-500 ), 73.55 (C-200 ), 70.25 (C-400 ), 63.52 (C-600 ), 45.47 (Ca), 33.86 (C-2000 ), 31.79 (Cb), 29.50, 29.39, 29.38, 29.21, 29,14, 28.90 (C-4000 , C-5000 , C-6000 , C-7000 , C-8000 , C-90 00 , C-10000 ), 24.85 (C-3000 ), 22.59 (C-11000 ), 14.42 (C-12000 ).

3.1.2. (6-{3,5-Dihydroxy-2-[3-(4-hydroxyphenyl)propanoyl]phenoxy}3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl octanoate (3) Yield: 84%; light yellow liquid; Rf: 0.53 (acetone:toluene; 5:5: few drops of AcOH); IR (KBr) cm1: 3387, 2951, 2835, 2519, 2241, 2044, 1715, 1637, 1593, 1516, 1451, 1266, 1112, 1025, 913, 739, 647; 1H NMR (DMSO-d6, 300 MHz): d 13.47 (s, 1H, ArOH), 10.56 (br s, 1H, ArOH), 7.03 (d, 2H, J = 8.3 Hz, H-2, H-6), 6.64 (d, 2H, J = 8.3 Hz, H-3, H-5), 6.10 (d, 1H, J = 1.6 Hz, H-30 ), 5.95 (d, 1H, J = 1.6 Hz, 1H, H-50 ), 5.36 (br.s, 1H, OH), 5.29 (br s, 1H, OH), 5.25 (br s, 1H, OH), 4.99 (d, 1H, J = 6.9 Hz, H-100 ), 4.32 (br d, 1H,

3.1.4. (6-{3,5-Dihydroxy-2-[3-(4-hydroxyphenyl)propanoyl]phenoxy}3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl palmitate (5) Yield: 97%; light yellow solid; Rf: 0.53 (acetone:toluene; 5:5: few drops of AcOH); IR (KBr) cm1: 3388, 2944, 2836, 2527, 2244, 2048, 1716, 1634, 1593, 1513, 1454, 1266, 1117, 1024, 918, 738, 659; 1H NMR (DMSO-d6, 300 MHz): d 13.49 (s, 1H, ArOH), 10.53 (br s, 1H, ArOH), 7.04 (d, 2H, J = 8.3 Hz, H-2, H-6), 6.65 (d, 2H, J = 8.3 Hz, H-3, H-5), 6.11 (br s, 1H, H-30 ), 5.95 (br s, 1H, H-50 ), 5.38 (br.s, 1H, OH), 5.37 (br s, 1H, OH), 5.26 (br s, 1H, OH), 4.99 (d, 1H, J = 6.81 Hz, H-100 ), 4.34 (br d, 1H, J = 11.5 Hz, H-6a00 ), 4.12 (dd, 1H,

Ziaullah, H.P.V. Rupasinghe / Bioorganic Chemistry 65 (2016) 17–25

J = 11.5 Hz, 6.7 Hz, H-6b00 ), 3.65 (br t, 1H, J = 8.3 Hz, H-400 ), 3.64– 3.18 (m, 6H, 2  Ha, H-200 , H-300 , H-500 , OH), 2.79 (br t, 2H, J = 7.2 Hz, 2  Hb), 2.27 (br t, 2H, J = 7.3 Hz, 2  H-200 ), 1.47 (m, 2H, 2  H-300 ), 1.27–1.16 (m, 24H, 12(CH2)), 0.85 (br t, 3H, J = 6.8 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz): d 205.09 (CO), 173.29 (OCO), 165.87 (C-40 ), 164.92 (C-60 ), 161.09 (C-20 ), 155.75 (C-4), 131.92 (C-1), 129.58 (C-2, C-6), 115.40 (C-3, C-5), 105.57 (C-10 ), 101.03 (C-100 ), 97.36 (C-30 ), 94.94 (C-50 ), 76.85 (C-300 ), 74.33 (C-500 ), 73.55 (C-200 ), 70.25 (C-400 ), 63.52 (C-600 ), 45.46 (Ca), 33.86 (C-2000 ), 31.79 (Cb), 29.54, 29.48, 29.38, 29.21, 29,14, 28.90 (C-4000 , C-5000 , C-6000 , C-7000 , C-8000 , C-9000 , C-10000 , C-11000 , C-12000 , C-13000 , C-14000 ), 24.85 (C-3000 ), 22.59 (C-15000 ), 14.42 (C-16000 ). 3.1.5. (6-{3,5-Dihydroxy-2-[3-(4-hydroxyphenyl)propanoyl]phenoxy}3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl stearate (6) Yield: 96%; light yellow solid; Rf: 0.53 (acetone:toluene; 4:6: few drops of AcOH); IR (KBr) cm1: 3383, 2944, 2832, 2523, 2247, 2043, 1719, 1631, 1591, 1512, 1450, 1269, 1113, 1028, 913, 737, 652; 1H NMR (DMSO-d6, 300 MHz): d 13.55 (s, 1H, ArOH), 9.18 (br s, 1H, ArOH), 7.06 (d, 2H, J = 8.4 Hz, H-2, H-6), 6.67 (d, 2H, J = 8.4 Hz, H-3, H-5), 6.13 (d, 1H, 1.8 Hz, H-30 ), 5.98 (d, 1H, J = 1.8 Hz, H-50 ), 5.46 (br.s, 1H, OH), 5.37 (br s, 2H, 2OH), 5.04 (d, 1H, H-100 ), 4.35 (br d, 1H, J = 11.6 Hz, H-6a00 ), 4.14 (dd, 1H, J = 11.6 Hz, 6.9 Hz, H-6b00 ), 3.65 (br t, J = 7.2 Hz, H-400 ), 3.46–3.20 (m, 6H, 2  Ha, H-200 , H-300 , H-500 , OH), 2.81 (br t, 2H, J = 7.5 Hz, 2  Hb), 2.30 (t, 2H, J = 7.2 Hz, 2  H-200 ), 1.49 (m, 2H, 2  H-300 ), 1.25–1.19 (m, 28H, 2(CH2)), 0.87 (br t, 3H, J = 6.9 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz): d 205.34 (CO), 173.44 (OCO), 166.05 (C-40 ), 165.13 (C-60 ), 161.30 (C-20 ), 156.03 (C-4), 132.25 (C-1), 129.72 (C-2, C-6), 115.74 (C-3, C-5), 106.18 (C-10 ), 101.55 (C-100 ), 97.79 (C-30 ), 95.44 (C-50 ), 77.30 (C-300 ), 74.77 (C-500 ), 73.94 (C-200 ), 70.68 (C-400 ), 63.81 (C-600 ), 45.61 (Ca), 34.17 (C-2000 ), 31.96 (Cb), 29.84, 29.34, 29.13 (C-4000 , C-50 00 , C-6000 , C-7000 , C-8000 , C-9000 , C-10000 , C-11000 , C-12000 , C-13000 , C-14000 , C-150 00 , C-16000 ), 25.08 (C-3000 ), 22.73 (C-17000 ), 14.53 (C-18000 ). 3.1.6. (6-{3,5-Dihydroxy-2-[3-(4-hydroxyphenyl)propanoyl]phenoxy}3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl (Z)-9octadecenoate (7) Yield: 93%; light brownish yellow spongy solid; Rf: 0.56 (Acetone:toluene; 4:6: few drops of AcOH); IR (KBr) cm1: 3383, 2927, 2855, 2253, 1714, 1627, 1599, 1514, 1455, 1264, 1203, 1078, 908, 734, 650; 1H NMR (DMSO-d6, 300 MHz): d 13.61 (s, 1H, OH), 10.66 (br s, 1H, OH), 9.25 (br s, 1H, OH), 7.09 (d, 2H, J = 8.7 Hz, H-2, H-6), 6.72 (d, 2H, J = 8.7 Hz, H-3, H-5), 6.17 (d, 1H, 1.8 Hz, H-30 ), 6.01 (s, 1H, H-50 ), 5.48–5.29 (m, 4H, H-9000 , H-10000 ), 5.04 (d, 1H, J = 6.9 Hz, H-100 ), 4.38 (br d, 1H, J = 11.4 Hz, H-6a00 ), 4.18 (dd, 1H, J = 11.4 Hz, 6.6 Hz, H-6b00 ), 3.67 (br t, 1H, J = 7.8 Hz, H-400 ), 3.55–3.23 (m, 6H, 2  Ha, H-200 , H-300 , H-500 , OH), 2.84 (br t, 2H, J = 6.9 Hz, 2  Hb), 2.32 (br t, 2H, J = 6.9 Hz, 2  H-200 ), 2.01– 1.98 (m, 4H, 2  H-800 , 2  H-1100 ), 1.54–1.50 (m, 2H, 2  H-300 ), 1.27–1.20 (m, 20H, 10(CH2)), 0.87 (br t, 3H, J = 6.9 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz): d 205.37 (CO), 173.41 (OCO), 166.13 (C-40 ), 165.15 (C-60 ), 161.34 (C-20 ), 156.06 (C-4), 132.30 (C-1), 130.30 (C-900 , C-1000 ), 129.73 (C-2, C-6), 115.78 (C-3, C-5), 106.25 (C-10 ), 101.63 (C-100 ), 97.86 (C-30 ), 95.50 (C-50 ), 77.37 (C-300 ), 74.83 (C-500 ), 73.98 (C-200 ), 70.74 (C-400 ), 63.85 (C-600 ), 45.63 (Ca), 34.19 (C-200 ), 31.97 (Cb), 29.83 (C-7000 , C-12000 ), 29.54 (C-15000 ), 29.33 (C4000 , C-5000 , C-14000 ), 29.20 (C-6000 , C-13000 , C-160 00 ), 27.33 (C-8000 , C11000 ), 25.10 (C-3000 ), 22.74 (C-17000 ), 14.50 (C-18000 ). 3.1.7. (6-{3,5-Dihydroxy-2-[3-(4-hydroxyphenyl)propanoyl]phenoxy}3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl (9Z,12Z)-9,12-octadecadienoate (8) Yield: 89%; light brownish yellow spongy solid; Rf: 0.57 (acetone:toluene; 4:6: few drops of AcOH); IR (KBr) cm1: 3396,

23

2928, 2856, 2253, 1711, 1628, 1599, 1515, 1454, 1378, 1263, 1204, 1173, 1080, 907, 733, 650; 1H NMR (DMSO-d6, 300 MHz): d 10.67 (br s, 1H, OH), 9.23 (br s, 1H, OH), 7.08 (d, 2H, J = 8.1 Hz, H-2, H-6), 6.70 (d, 2H, J = 8.1 Hz, H-3, H-5), 6.16 (d, 1H, J = 1.2 Hz, H-30 ), 6.01(d, 1H, J = 1.2 Hz, H-50 ), 5.47 (br s, 1H, OH), 5.40–5.27 (m, 5H, H-9000 , H-100 00 , H-12000 , H-13000 , OH), 5.04 (d, 1H, J = 6.9 Hz, H-100 ), 4.37 (br d, 1H, J = 11.4 Hz, H-6a00 ), 4.18 (dd, 1H, J = 11.4 Hz, 6.6 Hz, H-6b00 ), 3.66 (br t, 1H, J = 7.8 Hz, H-400 ), 3.57 (br s, 2  OH), 3.48–3.22 (m, 5H, 2  Ha, H-200 , H-300 , H-500 ), 2.83 (br t, 2H, J = 6.9 Hz, 2  Hb), 2.74 (br t, 2H, J = 5.4 Hz, 2  H-11000 ), 2.30 (br t, 2H, J = 7.5 Hz, 2  H-2000 ), 2.15–1.99 (m, 4H, 2  H-8000 , 2  H-14000 ), 1.53–1.48 (m, 2H, 2  H-3000 ), 1.37–1.18 (m, 14H, 7(CH2)), 0.86 (br t, 3H, J = 6.6 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz): d 205.42 (CO), 173.45 (OCO), 166.20 (C-40 ), 165.16 (C-60 ), 161.38 (C-20 ), 156.09 (C-4), 132.36 (C-1), 130.50 (C-9000 , C-130 00 ), 129.75 (C-2, C-6), 128.48 (C-10000 , C-12000 ), 115.84 (C-3, C-5), 106.33 (C-10 ), 101.68 (C-100 ), 97.94 (C-30 ), 95.56 (C-50 ), 77.43 (C-300 ), 74.89 (C500 ), 74.03 (C-200 ), 70.80 (C-400 ), 63.88 (C-600 ), 45.66 (Ca), 34.23 (C2000 ), 31.64 (Cb), 29.95 (C-7000 ), 29.73 (C-15000 ), 29.44 (C-40 00 ), 29.23 (C-5000 , C-60 00 , C-11000 ), 27.39 (C-8000 , C-14000 ), 25.99 (C-16000 ), 25.13 (C-3000 ), 22.65 (C-17000 ), 14.47 (C-18000 ).

3.1.8. (6-{[2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4Hchromen3-yl]oxy}-4,5-dihydroxytet-rahydro-2H-pyran-2-yl)methyl butyrate (9) Yield: 83%; yellowish solid; Rf: 0.42 (acetone:toluene; 1:1: few drops of AcOH); IR (KBr) cm1: 3453, 3276, 3072, 3003, 2927, 2859, 2543, 2247, 2131, 1989, 1834, 1763, 1659, 1450, 1363, 1307, 1203, 1169, 1053, 823, 737, 627; 1H NMR (DMSO-d6, 300 MHz): d 12.65 (br.s, 1H, ArOH), 7.54–7.50 (m, 2H, H-20 , H-60 ), 6.84 (br d, 1H, J = 9.0 Hz, H-50 ), 6.40 (d, 1H, J = 1.95 Hz, H-8), 6.22 (d, 1H, J = 1.95 Hz, H-6), 5.45 (d, 1H, J = 7.35 Hz, H-100 ), 5.29–4.99 (m, 3H, 3OH), 4.15 (br.d, 1H, J = 10.0 Hz, H-6a00 ), 3.97–3.91 (dd, 1H, J = 10.0 Hz, J = 6.87 Hz, H-6b00 ), 3.55–3.09 (m, 6H, H-200 , H-300 , H-400 , H-500 , 2OH), 1.95 (dd, 2H, J = 7.53 Hz, J = 3.48 Hz, 2  H-2000 ), 1.27–1.17 (m, 3H, 2  H-3000 , OH), 0.64 (br. t, 3H, J = 7.33 Hz, CH3); 13 C NMR (DMSO-d6, 75 MHz): d 177.80 (CO), 172.75 (OCO), 164.72 (C-7), 161.71 (C-5), 156.84, 156.78 (C-2, C-9), 148.98 (C-40 ), 145.31 (C-30 ), 133.39 (C-3), 121.94 (C-10 ), 121.49 (C-60 ), 116.53 (C-50 ), 115.58 (C-20 ), 104.27 (C-10), 101.01 (C-100 ), 99.14 (C-6), 93.95 (C-8), 76.71 (C-300 ), 74.57 (C-500 ), 74.36 (C-200 ), 70.51 (C-400 ), 63.34 (C-600 ), 35.66 (C-20 00 ), 18.25 (C-3000 ), 13.62 (C-4000 ).

3.1.9. (6-{[2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4Hchromen3-yl]oxy}-4,5-dihydroxytet-rahydro-2H-pyran-2-yl)methyl octanoate (10) Yield: 81%; yellowish solid; Rf: 0.42 (acetone:toluene; 1:1: few drops of AcOH); IR (KBr) cm1: 3458, 3277, 3078, 3009, 2925, 2856, 2549, 2246, 2135, 1987, 1833, 1768, 1653, 1459, 1368, 1309, 1206, 1165, 1058, 828, 736, 627; 1H NMR (DMSO-d6, 300 MHz): d 12.65 (br.s, 1H, ArOH), 7.54–7.50 (m, 2H, H-20 , H-60 ), 6.83 (br d, 1H, J = 9.0 Hz, H-50 ), 6.39 (d, 1H, J = 1.94 Hz, H-8), 6.19 (d, 1H, J = 1.94 Hz, H-6), 5.45 (br. dd, 1H, J = 7.35 Hz, H-100 ), 5.20 (br. s, 2H, 2OH), 4.17 (br.d, 1H, J = 10.0 Hz, H-6a00 ), 3.99–3.92 (dd, 1H, J = 10.0 Hz, J = 7.05 Hz, H-6b00 ), 3.35–3.08 (m, 6H, H-200 , H-300 , H-400 , H-500 , 2OH), 1.99–1.90 (m, 2H, 2  H-20 00 ), 1.27–1.01 (m, 12H, 5  CH2, 2OH), 0.82 (t, 3H, J = 6.84 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz): d 177.80 (CO), 172.88 (OCO), 164.62 (C-7), 161.70 (C-5), 156.84, 156.77 (C-2, C-9), 148.94 (C-40 ), 145.26 (C-30 ), 133.40 (C-3), 123.03 (C-10 ), 122.95 (C-60 ), 116.59 (C-50 ), 114.49 (C-20 ), 104.30 (C-10), 102.18 (C-100 ), 99.91 (C-6), 94.96 (C-8), 77.68 (C-300 ), 75.33 (C-500 ), 73.39 (C-200 ), 71.50 (C-400 ), 63.31 (C-600 ), 33.65 (C-2000 ), 31.96 (C-1400 ), 30.40, 28.74, 26.97 (C-4000 , C-5000 , C-60 00 ), 24.67 (C-3000 ), 22.51 (C-7000 ), 13.57 (C-8000 ).

24

Ziaullah, H.P.V. Rupasinghe / Bioorganic Chemistry 65 (2016) 17–25

3.1.10. (6-{[2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl]oxy}-4,5-dihy-droxy-tetrahydro-2H-pyran-2-yl)methyl dodecanoate (11) Yield: 92%; yellowish solid; Rf: 0.42 (acetone:toluene; 1:1: few drops of AcOH); IR (KBr) cm1: 3453, 3276, 3072, 3003, 2927, 2859, 2543, 2247, 2131, 1989, 1834, 1763, 1659, 1450, 1363, 1307, 1203, 1169, 1053, 823, 737, 627; 1H NMR (DMSO-d6, 300 MHz): d 12.62 (s, 1H, ArOH), 10.88 (br s, 1H, ArOH), 7.53–7.50 (m, 2H, H-20 , H-60 ), 6.82 (br d, 1H, J = 8.79 Hz, H-50 ), 6.39 (d, 1H, J = 2.04 Hz, H-8), 6.19 (d, 1H, J = 2.04 Hz, H-6), 5.46–5.41 (m, 2H, H-100 , OH), 5.22 (br. s, 2H, 2OH), 4.17 (br.d, 1H, J = 10.0 Hz, H-6a00 ), 3.99–3.93 (dd, 1H, J = 10.0 Hz, J = 7.01 Hz, H6b00 ), 3.35–3.08 (m, 5H, H-200 , H-300 , H-400 , H-500 , OH), 1.99–1.93 (m, 2H, 2  H-200 ), 1.27–0.95 (m, 19H, 9xCH2, OH), 0.83 (t, 3H, J = 6.45 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz): d 177.82 (CO), 172.90 (OCO), 164.64 (C-7), 161.74 (C-5), 156.84, 156.76 (C-2, C9), 148.95 (C-40 ), 145.28 (C-30 ), 133.44 (C-3), 121.89 (C-10 ), 121.51 (C-60 ), 116.57 (C-50 ), 115.54 (C-20 ), 104.29 (C-10), 101.11 (C-100 ), 99.11 (C-6), 93.89 (C-8), 76.74 (C-300 ), 74.60 (C-500 ), 74.37 (C-200 ), 70.54 (C-400 ), 63.36 (C-600 ), 33.66 (C-200 ), 31.80 (C-1000 ), 29.50, 29.35, 29.23, 29.01, 28.78 (C-400 , C-500 , C-600 , C-700 , C-800 , C-900 ), 24.68 (C-300 ), 22.59 (C-1100 ), 14.42 (C-1200 ). 3.1.11. (6-{[2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl]oxy}-4,5-dihydroxytet-rahydro-2H-pyran-2-yl)methyl palmitate (12) Yield: 93%; yellowish solid; Rf: 0.42 (acetone:toluene; 1:1: few drops of AcOH); IR (KBr) cm1: 3455, 3273, 3077, 3007, 2925, 2854, 2546, 2244, 2136, 1981, 1837, 1765, 1657, 1457, 1364, 1306, 1203, 1165, 1053, 828, 737, 625; 1H NMR (DMSO-d6, 300 MHz): d 12.62 (s, 1H, ArOH), 10.86 (br s, 1H, ArOH), 7.36–7.30 (m, 2H, H-20 , H-60 ), 6.83 (br d, 1H, J = 8.64 Hz, H-50 ), 6.39 (d, 1H, J = 2.01 Hz, H-8), 6.19 (d, 1H, J = 2.01 Hz, H-6), 5.46–5.40 (m, 2H, H-100 , OH), 5.22 (br. s, 2H, 2OH), 4.17 (br.d, 1H, J = 10.1 Hz, H-6a00 ), 3.99–3.93 (dd, 1H, J = 10.1 Hz, J = 7.02 Hz, H6b00 ), 3.33–3.06 (m, 5H, H-200 , H-300 , H-400 , H-500 , OH), 1.99–1.93 (m, 2H, 2  H-2000 ), 1.25–0.94 (m, 19H, 13  CH2, OH), 0.83 (t, 3H, J = 6.36 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz): d 177.83 (CO), 172.89 (OCO), 164.65 (C-7), 161.75 (C-5), 156.84, 156.77 (C-2, C9), 148.95 (C-40 ), 145.29 (C-30 ), 133.45 (C-3), 121.88 (C-10 ), 121.51 (C-60 ), 116.58 (C-50 ), 115.54 (C-20 ), 104.29 (C-10), 101.13 (C-100 ), 99.10 (C-6), 93.88 (C-8), 76.75 (C-300 ), 74.61 (C-500 ), 74.38 (C-200 ), 70.53 (C-400 ), 63.37 (C-600 ), 33.66 (C-2000 ), 31.79 (C-10000 ), 29.57, 29.49, 29.36, 29.23, 29.03, 28.80 (C-4000 , C-5000 , C-6000 , C-7000 , C-8000 , C-9000 , C-100 00 , C-11000 , C-12000 , C-13000 ), 24.69 (C-3000 ), 22.59 (C-15000 ), 14.40 (C-16000 ). 3.1.12. (6-{[2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4Hchromen-3-yl]oxy}-4,5-dihydroxytet-rahydro-2H-pyran-2-yl)methyl stearate (13) Yield: 96%; greenish solid; Rf: 0.42 (acetone:toluene; 1:1: few drops of AcOH); IR (KBr) cm1: 3449, 3271, 3070, 3007, 2926, 2854, 2559, 2250, 2124, 1997, 1830, 1767, 1655, 1454, 1366, 1308, 1203, 1172, 1057, 931, 822, 759, 731, 623; 1H NMR (DMSO-d6, 300 MHz): d 10.63–8.98 (br.s, 2H, ArOH), 7.56 (br.s, 2H, H-20 , H-60 ), 6.86 (br s, 1H, H-50 ), 6.41 (s, 1H, H-8), 6.22 (s, 1H, H-6), 5.47–5.26 (m, 3H, H-100 , 2OH), 4.20 (br.d, 1H, J = 10.5 Hz, H6a00 ), 4.00 (br s, 1H, H-6b00 ), 3.55–3.20 (m, 5H, H-200 , H-300 , H-400 , H-500 , OH), 2.00 (br s, 2H, H-200 , OH), 1.24–1.09 (m, 31H, 15  CH2, OH), 0.83 (br. s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz): d 178.14 (CO), 173.09 (OCO), 165.09 (C-7), 162.09 (C-5), 157.16 (C-2, C-9), 149.25 (C-40 ), 145.59 (C-30 ), 133.97 (C-3), 122.19 (C-10 ), 121.95 (C-60 ), 116.98 (C-50 ), 115.90 (C-20 ), 104.66 (C-10), 101.73 (C-100 ), 99.47 (C-6), 94.21 (C-8), 77.26 (C-300 ), 75.01 (C-500 ), 74.79 (C-200 ), 71.01 (C-400 ), 63.78 (C-600 ), 34.00 (C-2000 ), 31.99 (C-16000 ), 29.73 29.37, 29.23, 29.06 (C-4000 , C-50 00 , C-6000 , C-7000 , C-8000 , C-9000 , C-10000 ,

C-11000 , C-120 00 , C-13000 , C-14000 , C-15000 ), 24.95 (C-3000 ), 22.74 (C-17000 ), 14.51 (C-18000 ).

3.1.13. (6-{[2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4Hchromen-3-yl]oxy}-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-yl)methyl (Z)-9-octadecenoate (14) Yield: 93%; greenish yellow spongy solid; Rf: 0.58 (acetone:toluene; 1:1: few drops of AcOH); IR (KBr) cm1: 3373, 3065, 2947, 2836, 2497, 2251, 2121, 2042, 1898, 1763, 1640, 1596, 1560, 1459, 1448, 1229, 1165, 1026, 942, 739; 1H NMR (DMSO-d6, 300 MHz): d 11.45–9.01 (br.s, 2H, ArOH), 7.53 (d, 2H, J = 9.0 Hz, H-20 , H-60 ), 6.85 (d, 1H, J = 9.0 Hz, H-50 ), 6.41 (d, 1H, J = 1.8 Hz, H-8), 6.22 (d, 1H, J = 1.8 Hz, H-6), 5.48–5.25 (m, 5H, H-900 , H-1000 , H-100 , 2OH), 4.20 (br.d, 1H, J = 11.7 Hz, H-6a00 ), 3.99 (dd, 1H, J = 11.7 Hz, J = 7.2 Hz, H-6b00 ), 3.45–3.13 (m, 6H, H-200 , H300 , H-400 , H-500 , 2  OH), 2.06–1.97 (m, 6H, 2  H-20 00 , 2  H-8000 , 2  H-11000 ), 1.27–1.09 (m, 23H, 11  CH2), 0.85 (br t, 3H, J = 6.6 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz): d 178.12 (CO), 173.05 (OCO), 164.99 (C-7), 162.06 (C-5), 157.12 (C-2, C-9), 149.21 (C-40 ), 145.56 (C-30 ), 133.92 (C-3), 130.29 (C-900 , C-1000 ), 122.16 (C-10 ), 121.93 (C-60 ), 116.95 (C-50 ), 115.88 (C-20 ), 104.65 (C-10), 101.65 (C-100 ), 99.42 (C-6), 94.18 (C-8), 77.23 (C-300 ), 74.98 (C-500 ), 74.76 (C-200 ), 70.98 (C-400 ), 63.75 (C-600 ), 33.96 (C-2000 ), 31.92 (C-16000 ), 29.79 (2C), 29.49, 29.29 (3C), 29.13, 29.03 (C-4000 , C-5000 , C-6000 , C-7000 , C-12000 , C-13000 , C-14000 , C-150 00 ), 27.31 (C-8000 , C-11000 ), 27.90 (C-3000 ), 22.69 (C-17000 ), 14.49 (C-18000 ).

3.1.14. (6-{[2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo4Hchromen-3-yl]oxy}-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-yl) methyl (9Z,12Z)-9,12-octadecadienoate (15) Yield: 91%; greenish yellow spongy solid; Rf: 0.59 (acetone:toluene; 1:1: few drops of AcOH); IR (KBr) cm1: 3346, 2946, 2835, 2492, 2181, 2043, 1897, 1762, 1643, 1591, 1466, 1447, 1230, 1027, 941, 737, 700; 1H NMR (DMSO-d6, 300 MHz): d 11.46–8.97 (br.s, 2H, ArOH), 7.58 (br s, 2H, H-20 , H-60 ), 6.88 (br d, 1H, J = 6.9 Hz, H-50 ), 6.43 (s, 1H, H-8), 6.24 (s, 1H, H-6), 5.50–5.37 (m, 7H, H-90 00 , H-10000 , H-12000 , H-13000 , H-100 , 2OH), 4.22 (br.d, 1H, J = 10.5 Hz, H-6a00 ), 4.02 (dd, 1H, J = 10.5 Hz, J = 7.2 Hz, H-6b00 ), 3.55–3.20 (m, 6H, H-200 , H-300 , H-400 , H-500 , 2  OH), 2.78 (br s, 2H, 2  H-11000 ), 2.16–2.04 (m, 6H, 2  H-20 00 , 2  H-8000 , 2  H-14000 ), 1.29–1.11 (m, 17H, 8xCH2, OH), 0.88 (br s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz): d 178.12 (CO), 173.05 (OCO), 165.01 (C-7), 162.06 (C-5), 157.13 (C-2, C-9), 149.22 (C-40 ), 145.58 (C-30 ), 133.91 (C-3), 130.49 (C-9000 , C-10000 ), 128.45 (C-12000 , C-13000 ),122.17 (C-10 ), 121.93 (C-60 ), 116.96 (C-50 ), 115.89 (C-20 ), 104.65 (C-10), 101.64 (C-100 ), 99.43 (C-6), 94.19 (C-8), 77.22 (C-300 ), 74.99 (C-500 ), 74.77 (C-200 ), 70.98 (C-400 ), 63.75 (C-600 ), 33.96 (C-20 00 ), 31.57 (C-16000 ), 29.66 (C-15000 ), 29.38 (C-7000 ), 29.16 (C-50 00 ), 29.03 C-4000 , C-6000 ), 27.34 (C-8000 , C-14000 ), 25.94 (C-110 00 ), 24.91 (C-3000 ), 22.59 (C-17000 ), 14.48 (C-180 00 ).

Conflict of Interest None of the authors have any competing interests in the manuscript.

Acknowledgments We are thankful to the financial support of the Discovery Grant of Natural Sciences and Engineering Council (NSERC) of Canada and the Atlantic Innovation Funds (AIF) program of the Atlantic Canada Opportunities Agency (ACOA).

Ziaullah, H.P.V. Rupasinghe / Bioorganic Chemistry 65 (2016) 17–25

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