Lipase from thermomyces lanuginosus

Lipase from Thermomyces lanuginosus (Lot# L0777, a liquid preparation with 72.75 ± 0.45 U/mg of protein determined by Ref. [16 (link)]), 4-morpholineethanesulfonic acid (MES), n-hexane, methanol, analytical or GC grade FAME (methyl undecanoate, methyl palmitate, methyl linoleate, methyl linolenate, methyl oleate, and methyl stearate) as analysis calibration standards were purchased from Sigma-Aldrich. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) were purchased from Thermo Fisher Scientific. Soybean oil (saponification value of 195.0 ± 0.1 mgKOH/g, acid value of 0.1794 ± 0.0009 mgKOH/g, average molecular weight of 864.1 ± 0.5 g/mol) was purchased from a local market (Taiyuan, China). All other chemicals were guaranteed or analytic grade and were used directly. Water used throughout this work was from a Milli-Q ultrapure water system.

All chemicals used were reagent grade or
better. Tyrosol (Ty), homovanillyl alcohol (Hva), bis(trichloromethyl)
carbonate (triphosgene), dichloromethane, 2-propanol, hexane, deuterated
dimethyl sulfoxide (d⁶-DMSO), tetrahydrofurane (THF), trifluoroacetic acid (TFA), phosphate
buffered saline (PBS) and lipase from Thermomyces lanuginosus (EC3.1.1.3, minimum 10⁵ units g^–1)
were obtained from Sigma-Aldrich (St. Louis, MO). Pyridine, hexane
and N,N-dimethylformamide (DMF)
were obtained from Fisher Scientific (Pittsburgh, PA).

(±)-3-Chloro-1-(chloroacetoxy)propylphosphonate
(±)-21 (5.463 g, 16.3 mmol) was dissolved in a mixture
of t-BuOMe and hexanes (36 mL, 1:1) and phosphate
buffer (25 mM, 120 mL).^{14 (link)} After the pH
had been adjusted to 7.0 using the autotitrator, lipase from Thermomyces lanuginosus (0.4 mL, ≥100 000
U/g, [3.1.1.3], Sigma) was added. The mixture was vigorously stirred
at room temperature, and pH of 7.0 was maintained by the addition
of NaOH (0.5 M). At a conversion of 40% by consumption of the base
(13.04 mL), the pH was brought to 4 by the addition of HCl (2 M).
The reaction mixture was extracted with EtOAc (3 × 100 mL). The
combined organic phases were washed with a saturated aq solution of
NaHCO₃ (50 mL), dried (MgSO₄), and concentrated
under reduced pressure. The residue was purified by flash chromatography
(hexanes/EtOAc, 1:2, chloroacetate: R_f 0.44; hydroxyphosphonate: R_f 0.15) to
give chloroacetate (R)-21 {2.918 g,
53%; [α]_D²¹ −21.4 (c 1.01, acetone)} and hydroxyphosphonate
(S)-20 {1.332 g, 5.14 mmol, 63%; [α]_D²¹ +31.7 (c 1.67, acetone)} as colorless liquids. The ee of 97% for
(+)-22 was determined by using(R)-(+)-(t-Bu)(Ph)P(O)(SH) as CSA and ³¹P NMR spectroscopy:^{29 (link),30 (link)} major singlet (1.00) at 22.9 ppm and minor one (0.015) at 22.7 ppm.