Figure S1. 1H NMR spectrum (500 MHz, in CDCl3) of trans-2-azidocyclohexanol (1)

Figure S2. 13C NMR spectrum (126 MHz, in CDCl3) of trans-2-azidocyclohexanol (1)

Figure S3. LC–MS analysis results for trans-2-azidocyclohexanol (1). Top: total absorbance (red) and total ion current (black) chromatograms. Bottom: ESI-MS spectrum recorded at 3.918 min, only fragmentation peaks visible

Figure S4. 1H NMR spectrum (500 MHz, in CDCl3) of trans-2-azidocyclohexyl 9H-fluoren-9-ylmethyl carbonate (2)

Figure S5. 1H NMR spectrum (300 MHz, in CDCl3) of trans-2-azidocyclohexyl 9H-fluoren-9-ylmethyl carbonate (2), with signal assignment

Figure S6. 13C NMR spectrum (126 MHz, in CDCl3) of trans-2-azidocyclohexyl 9H-fluoren-9-ylmethyl carbonate (2)

Figure S7. LC–MS analysis results for trans-2-azidocyclohexyl 9H-fluoren-9-ylmethyl carbonate (2). Top: total absorbance (red) and total ion current (black) chromatograms. Bottom: ESI-MS spectrum recorded at 9.109 min, only fragmentation peaks visible.

Figure S8. Figure S4. 1H NMR spectrum (500 MHz, in CDCl3) of trans-2-azidocyclohexyl acetate (3)

Figure S9. 13C NMR spectrum (126 MHz, in CDCl3) of trans-2-azidocyclohexyl acetate (3)

Figure S10. LC–MS analysis results for trans-2-azidocyclohexyl acetate (3). Top: total absorbance (red) and total ion current (black) chromatograms. Bottom: ESI-MS spectrum recorded at 6.193 min, only fragmentation peaks visible.

Figure S11. An example chromatogram recorded using method A (non-chiral) for a mixture of trans-2-azidocyclohexanol (1, tR = 4.08 min) and trans-2-azidocyclohexyl acetate (3, tR = 8.17 min)

Table S1: Development of the fmoc derivatization procedure. All reactions have been performer at room temperature After the indicated time, pyridine was evaporated and the dry residue was dissolved in acetonitrile and analyzed with RP-HPLC

Figure S12: An example chromatogram obtained after derivatization of racemic trans-2-azidocyclohexanol with Fmoc chloride in pyridine.

Figure S13: Peak areas for trans-2-azidocyclohexanol 1 (blue) and trans-2-azidocyclohexyl acetate 3 (green), recorded while following an initial enzymatic hydrolysis reaction in pH 7.4 phosphate buffer without any modifier.

Table S2: trans-2-azidocyclohexyl acetate (3) peak areas recorded for pH 7.4 phosophate buffer and various concentrations of methanol, acetonitrile and ethanol

Figure S14: Conversion and ee values obtained using 20% v/v of selected organic modifiers: acetonitrile, ethanol and THF

Table S3: Collected conversion and enantiomeric excess data for the studies on organic modifier and temperaturę influence. Typically, for each entry, two reactions have been conducted, with one conversion and ee measurement for each reaction, if otherwise, it is indicated by asterisks

Table S4: Collected conversion and enantiomeric excess results for the calcium influence study. For each entry, two reactions have been performed, one conversion measurement for each. For the highest and lowest Ca2+ concentrations, single ee measurements have been taken