One-Pot Deracemization of sec-Alcohols: Enantioconvergent Enzymatic Hydrolysis of Alkyl Sulfates Using Stereocomplementary Sulfatases

Given the fact that the theoretically possible number of racemates is larger than that of symmetric prochiral or meso compounds,1 the development of deracemization methods, which yield a single stereoisomer from a racemate is an important topic.1–3 Enantioconvergent processes are based on the transformation of a pair of enantiomers through opposite stereochemical pathways affecting retention and inversion of configuration. Depending on the stereochemical course of enzymatic and chemical reactions, three types of deracemization protocols were recently classified by Feringa et al.4 Two chemoenzymatic methods start with a biocatalytic kinetic resolution step, which yields a hetero- or homochiral 1:1 mixture of the formed product and nonconverted substrate enantiomer. The latter is subjected to a second (non-enzymatic) transformation with retention or inversion of configuration to yield a single stereoisomeric product. Although several one-pot, two-step protocols have been successfully demonstrated,5, 10c,d they typically rely on activated species, such as sulfonates,5a–d nitrate esters,5b or Mitsunobu intermediates,5e and negatively affect the overall atom economy of the process. The most elegant method relies on one (or two) enzyme(s), which mediate the transformation of both enantiomers through stereocomplementary pathways by retention and inversion. Since the requirements of such double selectivities are very difficult to meet, successful examples are rare: This approach has been applied to the hydrolysis of epoxides using two epoxide hydrolases showing opposite enantiopreference6 or a single enzyme that catalyzes the enantioconvergent hydrolysis of enantiomers with opposite regioselectivity.7


General
Competent cells One Shot® TOP10 and One Shot® BL21 Star™ (DE3) from Invitrogen were transformed according to the manufacturer´s protocol. Secondary alcohols 1b-7b were purchased from Sigma Aldrich, Acros and Alfa Aesar. 18 OH 2 for the preparation of 18 O-labeled buffer was from Rotem (>98%). NMR spectra were recorded on a Bruker spectrometer at 300 ( 1 H) and 75 ( 13 C) MHz. Chemical shifts (δ) are given in ppm and coupling constants (J) are given in Hz. Optical rotation values were determined on a Perkin Elmer Polarimter 341. GC-MS measurements were performed on an Agilent 7890A GC system, equipped with an Agilent 5975C mass-selective detector (EI 70 eV) and a HP-5-MS column (30 m x 0.25 mm x 0.25 µm film) using helium gas at a flow rate of 0.55 mL/min. GCmeasurements were conducted using an Agilent Technologies 7890A GC-FID system equipped with an Agilent Technologies 7683B autosampler and a Varian Chirasil Dex CB column (25 m x 0.32 mm x 0.25 µm film). HPLC-MS measurements were done on a Shimadzu Nexera instrument equipped with a Shimadzu LCMS-2020 MS-detector and a Machery Nagel EC 150/4 Nucleosil ® 120-5 C4 column (150 mm x 4 mm). Further details are shown below.

Cloning, expression and purification of PISA1
PISA1 was obtained according to previously published procedures. [1,2]

Expression and purification of Pseudomonas aeruginosa arylsulfatase (PAS)
E. coli BL21 (DE3) was transformed with PAS harboring a N-terminal strep-tag (pASK-IBA5plus vector with EcoRI and HindIII as the respective restriction sites). Cells were grown in LB-medium containing ampicillin (100 µg/mL) at 37 °C and 120 rpm until the culture reached an A 600 of 0.6. After cooling to 30 °C, expression was induced with anhydrotetracyclin (200 µg/L). After 6 h, cells were harvested at 4 °C and 8000 rpm for 10 min. The cell pellet was washed with NaCl (0.9%) and stored at -20° C. After resuspension in Tris-HCl buffer (100 mM, 150 mM NaCl, pH 8.0), cells were disrupted by sonication using a Sonics & Materials Vibra Cell CV26 (13 mm tip, 5 min, 40% amplitude, pulse 1 sec on, 2 sec off). After centrifugation (4 °C, 18000 rpm, 2x15 min), the supernatant was filtered through a MN 615 filter paper (Machery Nagel) and subjected to strep-tag purification on a 5 mL gravity flow column according to the protocol of the supplier (IBA BioTAGnology). The protein was eluted with 6 x 2.5 mL of EDTA-free elution buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 2.5 mM desthiobiotin). The combined elution fractions were afterwards dialyzed in 5 L of Tris-HCl (100 mM, pH 8.0) overnight and concentrated with a Vivaspin centrifugal concentrator (10,000 MWCO) to a final concentration of ~20 mg/mL. Aliquots of the concentrated enzyme solution were shock frozen in liquid nitrogen and stored at -20 °C. The yield was ca. 100 mg/L culture.

Determination of protein concentrations
Protein concentrations were determined by measuring the absorbance at 280 nm on an Eppendorf Biophotometer plus spectrophotometer. Purified protein solution (60 µL, diluted to fit into the linear range of the spectrophotometer) was directly measured in an UV permeable cuvette with buffer from the preceding dialysis used for blank determination and dilution of the sample. The concentration was calculated according to Lambert-Beer's law using an extinction coefficient obtained from the ProtParam tool from ExPASy (ε 280 = 102790 M -1 cm -1 ). [3] Enantioselectivity assays for PISA1 Enantioselectivity assays were conducted according to a known procedure [1] with slight modifications: 20 mg of substrate was used and quantities for all other reactants were adapted accordingly. Reaction were conducted with 1.77 nM PISA1 for substrates rac-1a, -2a, -3a, -5a and -6a, and with 7.08 nM PISA1 for rac-4a and -7a. Reaction times, enantiomeric excesses and E-values are found in the main paper.

Enantioselectivity assays for PAS
Enantioselectivity assays have been conducted as described above for PISA1. [1] Reactions were conducted with 3.54 nM PISA1 for substrates rac-1a-7a. Reaction times, enantiomeric excess and Evalues are displayed in the main text.

Determination of absolute configuration
Absolute configurations of products 1b-7b were determined after derivatization to the corresponding acetates 1c-7c as previously shown. [1,2]

Determination of enantiomeric excess of products
The enantiomeric excess of alcohols 1b-7b was determined via GC after derivatization to the corresponding acetates 1c-7c using an Agilent Technologies 7890A GC-FID system equipped with an Agilent Technologies 7683B autosampler and a Varian Chirasil Dex CB column (25 m x 0.32 mm x 0.25 µm film) as previously shown. [1] The following methods were used:

Enantioselectivity assays for the enantioconvergent process
Alkyl sulfate rac-1a-7a (5 mg) was dissolved in Tris-HCl (100 mM, pH 8.0) and an aliquot of PISA1 and PAS enzyme solution ( Table 2) was added to a total volume of 1 mL. The reaction mixture was shaken at 30 °C and 120 rpm for the respective reaction times ( Table 2 main paper).

Determination of enantiomeric excess
The residual 500 µL of the reaction mixture were extracted with ethyl acetate (750 µL) and the organic phase was dried over anhydrous sodium sulfate. The e nantiomeric excess of alcohols 1b-7b was determined after derivatization to the corresponding acetates 1c-7c as shown above.

OH 2 Labeling experiments
Enzymatic assays of alkyl sulfate rac-6a (2.5 mg, 11 µmol) were conducted in unlabeled and 18 O-labeled Tris-HCl (500 µL, 100 mM, pH 8.0) with PAS and PISA1. The reaction mixture was shaken at 30 °C and 120 rpm for 24 h. After extraction with ethyl acetate (500 µL), the organic phase was dried over anhydrous sodium sulfate. Product 6b was derivatized to the corresponding acetate 6c and analyzed on an