(1S,2S,3R)-3-Hydroxy-2-nitrocyclohexyl acetate could be produced through the following synthetic routes.

A. (1R,2r,3S)-2--1,3-diol (CAS NO.: ). A 1-L, round-bottomed flask, equipped with a magnetic stirrer, a thermometer, and an ice–ethanol bath is charged with 175 mL (0.455 mol) of an aqueous 25% solution of glutaric dialdehyde, 38 mL (0.708 mol) of nitromethane, and 600 mL of methanol. At 0–5°C 12 mL of aqueous 2 M sodium hydroxide is added gradually. The cooling bath is removed and the reaction mixture is stirred for 4 hr at room temperature. The resulting yellow solution is neutralized by adding 15 g of acidic cation-exchange resin and stirring for an additional 20 min. The resin is filtered off and washed with a small volume of methanol. The filtrate is evaporated to a semisolid residue using reduced pressure and a 35°C water bath. The residue is dissolved in 100 mL of absolute ethyl alcohol with heating and diluted by gradual addition of 250 mL of toluene. The resulting two-phase mixture is again evaporated, with azeotropic removal of water. The resulting residue is again taken up in 100 mL of hot ethyl alcohol and diluted with 250 mL of toluene. The almost colorless crystals are filtered and dried at high vacuum to yield 44–52 g (60–70%) of nitrodiol, mp 152–155°C (dec).

B. (1R,2r,3S)-3-Acetoxy-2-nitrocyclohexyl acetate. In a 1-L flask 52 g (0.323 mol) of the nitrodiol are suspended in 150 mL of acetic anhydride. Without cooling, 3–6 drops of concentrated sulfuric acid are added. After 1 hr 500 mL of ice/water is rapidly added and stirring is continued for 60 min. The resulting colorless crystals are filtered, washed with water, and air-dried. The product thus obtained, 75.6 g (95.6%) of colorless crystals, is pure by TLC (4 : 1 hexane/ethyl acetate) and NMR, and melts at 89–90°C.

C. (1S,2S,3R)-3-Hydroxy-2-nitrocyclohexyl acetate. A 500-mL flask equipped with a magnetic stirrer is charged with 10 g (41 mmol) of powdered nitrodiacetate and 300 mL of 0.2 M phosphate buffer of pH 7.0 prepared by dissolving 11 g of potassium dihydrogenphosphate and 3.3 g of potassium hydroxide in 300 mL of deionized (or distilled) water. To the stirred suspension is added 30 mg of purified PLE [Esterase (EC 3.1.1.1), suspended in 2.8 mL of 3.2 M ammonium sulfate buffer] and the mixture is stirred for 24 to 48 hr. The continuously measured pH drops to about 5.6 during the reaction and then remains almost constant, while the mixture has turned to a practically clear, pale-yellow solution. The solution is filtered through a paper filter and the filtrate is extracted 3 times with 100 mL of ether. The organic phase is dried over anhydrous magnesium sulfate and filtered. Removal of the solvent and of the acetic acid under reduced pressure leaves 7–8 g (85–95%) of colorless monoacetate as a crystalline solid. Recrystallization by dissolving in 100 mL of ether and adding 250 mL of pentane gives 5–6 g (60–70%) of pure (1S,2S,3R)-3-hydroxy-2-nitrocyclohexyl acetate, mp 90–91°C, [α]_D + 9.5° (CHCl₃, c 1.0). From the mother liquor, another 1–2 g (12–24%) of monoacetate, mp 89–90°C, can be obtained.