P 1030 polarimeter

All reagents were purchased from commercial sources and used without further purification. Substrates which were not commercially available were synthesized according to known literature procedures (see Supporting Information for details). NMR spectra were performed on a Bruker AC-300 or Bruker Avance-400 (Bruker Corporation, Karlsruhe, Germany) using CDCl₃ as solvent and TMS as internal standard unless otherwise stated. Optical rotations were measured on a Jasco P-1030 Polarimeter with a 5 cm cell (c given in g/100 mL). Enantioselectivities were determined by HPLC analysis (Agilent 1100 Series HPLC) equipped with a G1315B diode array detector and a Quat Pump G1311A (Agilent Technologies, Palo Alto, CA, United States) equipped with the corresponding Daicel chiral column; the retention time of the major enantiomer is highlighted in bold. HRMS were measured using HPLC–HRMS (ESI) equipment (Agilent 1100 Series LC/MSD Trap SL, Agilent Technologies). Analytical TLC was performed on Merck silica gel plates and the spots were visualized with UV light at 254 nm (Merck Millipore, Billerica, MA, USA). Flash chromatography employed Merck silica gel 60 (0.040–0.063 mm) (Merck Millipore, Billerica, MA, USA).

¹H and ¹³C NMR spectra were recorded on a JEOL ECZ500 (500 MHz for ¹H NMR) or a JEOL ECZ400 (400 MHz for ¹H NMR) spectrometers. Chemical shifts are denoted in δ (ppm) relative to residual solvent peaks as internal standard (DMSO-d₆, ¹H δ 2.50, ¹³C δ 39.5). ESI-MS spectra were measured by a Thermo Scientific Exactive mass spectrometer or a SHIMADZU LCMS-2050 spectrometer. Optical rotations were recorded on a JASCO P-1030 polarimeter. High-performance liquid chromatography (HPLC) experiments were performed with a SHIMADZU HPLC system equipped with a LC-20AD intelligent pump. MS/MS analysis was performed with amaZon SL-NPC (Bruker Daltonics) using helium gas with an amplitude value 1.0 V. The chemical structures of enzyme substrates and enzymatic reaction products are analyzed by MS/MS (Supplementary data 1) and/or NMR (Supplementary data 2). All reagents were used as supplied unless otherwise noted.

Analytical TLC was performed on silica gel 60 F254 (Merck). Silica and octadecyl silica gel column chromatography was conducted using Biotage Sfär Silica High Capacity Duo (Biotage, Uppsala, Sweden) and Biotage Sfär C18 Duo eluted by Isolera (Biotage, Uppsala, Sweden). NMR spectra were recorded on a JEOL ECA-600 spectrometer and a Bruker AVANCE 600 spectrometer. ¹H and ¹³C NMR chemical shifts are given in parts per million (δ) relative to tetramethylsilane (δ_H 0.00) or residual solvent signals (δ_H 7.26, δ_C 77.0) as internal standards. Mass spectra were measured using JEOL JMS-700 and JMS-DX303 spectrometers. ECD spectra were measured on a J-1100DS spectrometer. Optical rotations were measured using a JASCO P-1030 polarimeter.

The ¹H-NMR spectra of the products were measured with a Varian Gemini-2000 (400 MHz) or a Bruker (500 MHz) spectrometer (Bruker, Billerica, MA, USA) in pyridine-d₅ at 80 °C. IR spectra were obtained with a JASCO FT/IR-620 spectrophotometer (JASCO, Tokyo, Japan). The CD spectra of 3,5-dimethylphenylcarbamates of cellulose were measured on a JASCO J-720 spectrophotometer using a quartz cell with a path length of 0.10 mm. The concentrations of samples in THF were 3.3–4.6 mg/5 mL. Optical rotation was measured with a JASCO P-1030 polarimeter. Thermal analysis of products was performed on a SEIKO SSC/5200TG instrument. The molecular weights (Mn) and its distribution (Mw/Mn) or DPs of the celluloses were estimated as the tris(3,5-dimethylphenylcarbamate)s by SEC on Shodex GPC system-21 at 40 °C using polystyrene standards and tetrahydrofuran (THF) as solvent. Two SEC columns, Shodex KF-806 and KF-803, were used in a series. For the samples with DP 40 and 52, the SEC on Waters 600 GPC system was used with three SEC columns, Styragel HR 0.5, HR 3 and HR 4. The HPLC analysis of racemates was carried out with a JASCO HPLC system equipped with a PU-980 pump and a JASCO OR-990 polarimetric detector or a JASCO CD-2095 detector.

The specific rotations were measured on a JASCO P-1030 polarimeter. UV and IR spectra were recorded on a Shimadzu UV-1800 spectrophotometer and a PerkinElmer Spectrum 100 spectrophotometer, respectively. NMR spectra were obtained on a Bruker AVANCE II 500 MHz NMR spectrometer in DMSO-d₆ supplemented with a trace amount of trifluoroacetic acid using the signals of the residual solvent protons (δ_H 2.49) and carbon atoms (δ_C 39.5) as internal standards for compounds 1–5, or in CDCl₃ using the signals of the residual solvent protons (δ_H 7.27) and carbon atoms (δ_C 77.0) as internal standards for other compounds. HRESITOFMS spectra were recorded on a Bruker micrOTOF focus mass spectrometer. An Agilent HP1200 system equipped with a diode array detector was used for analysis and purification.

The optical rotations were measured in MeOH on a JASCO P-1030 polarimeter (Loveland, CO, USA) in a 10 cm tube. The UV–Vis and ECD spectra were recorded on a JASCO J-815 spectrometer (Loveland, CO, USA) in MeOH using a 1 cm cell. Scan speed was set at 200 nm/min in continuous mode between 800 and 190 nm with 3 accumulations. NMR spectroscopic data were recorded on a Bruker Avance Neo 600 MHz NMR spectrometer equipped with a QCI 5 mm cryoprobe and a SampleJet automated sample changer (Bruker BioSpin, Rheinstetten, Germany). One-dimensional and two-dimensional NMR experiments (¹H, COSY, ROESY, and edited-HSQC) were recorded in DMSO-d₆. Chemical shifts are reported in parts per million (δ) and coupling constants (J) in Hz. The residual DMSO-d₆ signal (δ_H 2.50; δ_C 39.5) was used as internal standards for ¹H and ¹³C NMR, respectively.

Optical rotations were measured on a JASCO P-1030 polarimeter (Jasco, Tokyo, Japan). UV and IR spectra were obtained on a Jasco V-520 UV/Vis spectrophotometer and a Horiba FT-710 Fourier transform infrared spectrophotometer (Horiba, Kyoto, Japan), respectively. NMR experiments were measured on a Bruker Avance 500, 600 and 700 MHz spectrometers (Bruker, Billerica, MA, USA), with tetramethylsilane (TMS) as an internal standard. Positive ion HR-ESI-MS spectra were recorded using a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Column chromatography (CC) was performed on Diaion HP-20 (Mitsubishi Chemical Corp., Tokyo, Japan), silica gel 60 (230–400 mesh, Merck, Darmstadt, Germany), and octadecyl silica (ODS) gel (Cosmosil 75C₁₈-OPN (Nacalai Tesque, Kyoto, Japan; Φ = 35 mm, L = 350 mm), and TLC was performed on precoated silica gel plates 60 GF₂₅₄ (0.25 mm in thickness, Merck). HPLC was performed on ODS gel (Inertsil ODS-3, GL-science, 10 mm × 250 mm, flow rate 2.5 mL/min) with a mixture of H₂O, acetone and MeOH, and the eluate was monitored by refractive index and/or a UV detector. An amino column (Shodex Asahipak NH2P-50 4E (4.6 mm × 250 mm), CH₃CN-H₂O (3:1) 1 mL/min) together with a chiral detector (Jasco OR-2090plus) was used for HPLC analysis of sugars obtained after hydrolysis [15 (link)].