Supplementary materials for

Enantioselectiveammonolysis of phenylglycine methyl ester with lipase-Pluronicnanoconjugate in tertiary butanol

Xiaoling Wu, Rui Wang, Yifei Zhang, Jun Ge* and Zheng Liu*

Department of Chemical Engineering, Tsinghua University, Beijing 100084, China

Experimental

Materials

Candida antarctic lipase B(CALB),p-nitrophenyl butyrate,PlurpnicF-127,Dess-martin periodinane, and racemic phenylglycine methyl ester were purchased from Sigma-Aldrich.Ammonium carbamate and sodium cyanoborohydridem were purchased from Alfa Aesar.

Methods

Synthesis of CALB-Pluronicnanoconjugate

The CALB-Pluronic conjugate was synthesizedaccording to our previous study1. The hydroxyl group of Pluronic F-127 was first oxidized to aldehyde group by using Dess-Martin periodinane (the molar ratio of Dess-Martin to Pluronic F-127 was 5:1) as the oxidizing reagent. Then solvent was evaporated and the obtained viscous solution was filtered with diethyl ether and then dried undervacuum at room temperature.The obtained aldehyde-functionalized Pluronic was dissolved inphosphate buffer(10 mM,pH 7.0) and then mixed with an aqueous solution of CALB (5-10 mg/mL) (the molar ratio of the aldehyde group to the amine group of protein is 1.1:1). After 2 h reaction,sodium cyanoborohydride( NaCNBH3,10%wt of aldehyde-functionalized Pluronic) was added to the mixture and reacted for 15 hours at room temperature to reduce the Schiff base, followed by dialysis in phosphate buffer solution (10 mM, pH 7.0) to remove unreacted reagents. Finally, the powder of CALB-Pluronic conjugate was obtained by lyophilization.

Enzymatic activity assay

The hydrolytic activity of CALB and CALB-Pluronic conjugate was determined using 4-nitrophenyl butyrate(p-NPB) as the substrate.Briefly,p-NPB was first dissolved in acetone and then diluted with phosphate buffer(10 mM,pH 7.0) containing 1.25%(w/v) Triton X-100,giving a final concentration of 0.5 mM.The reaction was started by adding 50of enzyme solution (50in 10 mM phosphate buffer,pH 7.0) to 950of substrate solution and the increase of absorbance was detected at 348 nm using UV/Vis spectrophotometer.

The activity of CALB and CALB-Pluronic conjugate in organic solvents was determined by transesterification of hexanoic acid and n-butyl alcohol.During a run, dry power of native CALB or CALB-Pluronic conjugate with the same protein content of 0.5 mg was added into 5 mL of toluene solution containing 0.1 mol/L of fatty acid and 0.1 mol/L of alcohol.The reaction mixture was shaked at 40 oC and 200 rpm in a horizontal shaker. At specific times, the reaction was terminated by diluting with 10 mL of ethanol/acetone(1:1, v/v). The remaining free fatty acid in the reaction mixture was determined by titration with 0.05 mol/L of NaOH using phenolphthalein as the indicator. Conversion of1 μmol of fatty acid per minute in the assay conditions was defined as one enzyme unit activity (U·mg-1).

Enzymatic synthesis of R-phenylglycine amide

In a typical experiment,66 mg of racemic phenylglycine methyl ester and 125 mg of ammonium carbamatewas added in 2 mLoftertiary butanol (TBA) in a capped vial, followed by the addition of 20 mg of Novo 435 (or CALB-Pluronic with same protein content) to start the reaction.At specific times,sample was taken for HPLC analysis.

HPLC analysis

HPLC analysis was carried out on a SHIMADZU HPLC system by using a Chiralcel® OD column (2504.6mm,5). The mobile phase was the mixture of hexane and isopropanol(9:1,v/v). The analysis was performed at 1 mL/min and the absorbance was detected at215nm and 254nm.

Reference

[1] J. Zhu, Y. Zhang, D. Lu, R. N. Zare, J. Ge and Z. Liu, Chemical Communications, 2013, 49, 6090-6092.