E exclusively helicid 6′-esters as characterized by 13C NMR and 1H NMR (Bruker DRX-400 NMR Spectrometer, Bruker Co., Germany) at one hundred MHz and 400 MHz, respectively, with DMSO-d6 being the solvent. Outcomes in the NMR spectroscopy are offered in Figure S1. Mass spectra have been recorded on LCQ Deca Xp (Thermo Finnigan) using ESI mode with ion spray voltage 3000 V. The sheath gas arbitrary flow was set at 15 arb. The capillary temperature and voltage were 250uC and 18 V, respectively. Final results from the mass spectra are given in Figure S3. Moreover, the HPLC chromatograms on the helicid ester derivatives are provided in Figure S2.Common Process for Enzymatic Acylation of HelicidIn a common experiment, helicid (0.02 mmol), Lipozyme TLL and fatty acid vinyl ester were added into two ml anhydrous THF and the mixture was incubated at a predetermined temperature in an orbital air-bath shaker (200 rpm). Aliquots were withdrawn at specified time intervals in the reaction mixture, and then diluted 50-fold with corresponding mobile phase prior to HPLC evaluation. Regioselectivity was defined because the molar ratio on the preferred solution for the total quantity of ester goods formed. All information are averages of experiments performed in triplicate. No chemical acylation of helicid was detectable in controls from which the lipase preparation was omitted.Operational StabilityAnhydrous THF (two ml), helicid (0.02 mmol), vinyl hexanoate (0.15 mmol) and enzyme (20 U) have been incubated at 200 rpm and 45uC for 1.five h. Then, the enzyme was separated by filtration, completely washed with reaction medium and added into fresh reaction mixture to catalyze the acylation of helicid with a new aliquot from the same volume of vinyl hexanoate. The procedure was repeated to receive the operational stability on the enzyme after up to 11 cycles of reaction.Helicid1 H NMR (400 MHz, DMSO-d6): d three.42?.50 (m, 3, H2’+ H3’+ H4′), three.67?.72 (m, 1, H5′), 3.74?.78 (apparent d, 1, J = 3.two Hz, H6′), three.96 (apparent d, 1, J = three.2 Hz, H6′), four.52 (t, 1, J = 5.7, six.6 Hz, OH6′), four.71 (d, 1, J = 7.four Hz, H1′), 5.01 (d, 1, J = 3.7 Hz, OH4′), 5.15 (d, 1, J = 6.8 Hz, OH3′), 5.27 (d, 1, J = 7.9 Hz, OH2′), 7.19 (d, 2, J = 8.7 Hz, H2+ H6), 7.87 (d, two, J = 8.7 Hz, H3+ H5), 9.89 (s, 1, OH7). 13C NMR (one hundred MHz, DMSO-d6): d 60.86 (C6′), 66.93 (C4′), 70.18 (C2′), 71.45 (C3′), 74.79 (C5′), 98.08 (C1′), 116.39 (C2+ C6), 130.45 (C4), 131.65 (C3+ C5), 162.38 (C1), 191.45 (C7).HPLC AnalysisThe reaction mixture was analyzed by RP-HPLC on a 4.six mm6250 mm (five mm) Zorbax SB-C18 column (Agilent Technologies Industries Co., Ltd., USA) employing an Agilent G1311A pump and also a UV detector at 270 nm. The mobile phase is really a mixture of water and methanol at 1.0 ml/min. The volumetric ratio of water to methanol plus the retention times for helicid and its 6′-O-monoester have been 60/40, three.8-Bromo-1,6-naphthyridine Order 210 and 6.3-Acetoxy-2-benzylpropanoic acid custom synthesis 808 min (acetylation), 60/40, 3.PMID:23667820 198 and ten.442 min (propionylation), 40/60, 2.657 and 4.578 min (butyrylation), 20/80, two.511 and three.921 min (hexanoylation), 20/ 80, two.509 and 4.797 min (caproylation), 20/80, two.512 and 7.704 min (decanoylation), 10/90, 2.409 and 5.189 min (lauroylation), 10/90, 2.413 and 7.498 min (myristoylation), respectively. A gradient elution with water/methanol of 40/60 (v/v) from 0 to three min, then water/methanol of 20/80 (v/v) at 5.0 min was utilized for crotonylation and methacryloylation. The retention times for helicid and its 6′-O-monoester have been 2.621, 4.029 (crotonylation) and 4.414 min (methacryloylation), respectively.Helicid 6′-a.
