Av 700 mhz nmr spectrometer

Optical rotations were measured using a 341 Polarimeter (Perkin-Elmer, Inc., Norwalk, CT, USA). UV spectra were measured on a U-2900 spectrophotometer (Hitachi, Tokyo, Japan). IR spectra were recorded on an Affinity-1 FT-IR spectrometer (Shimadzu, Tokyo, Japan). Melting points were measured using an SGW^®X-5 micro melting point meter (Shanghai INESA Physico optiacal Instrument Co., Ltd., Shanghai, China). 1D and 2D NMR spectra were recorded on a Bruker AV-700 MHz NMR spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) with tetramethylsilane (TMS) as the internal standard. Mass spectrometric data were obtained on a quadrupole-time-of-flight mass spectrometer (Bruker Maxis 4G) for HRESIMS. Column chromatography was performed using silica gel (100–200 mesh, 300–400 mesh; Jiangyou Silica gel development, Inc., Yantai, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). HPLC was carried out while a reversed-phase column (Phenomenex Gemini C18, 250 mm × 4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA) with UV detection at 254 nm and 360 nm. Semi-preparative HPLC was performed on a Hitachi HPLC station (Hitachi-L2130) with a Diode Array Detector (Hitachi L-2455) using a Phenomenex ODS column (250 mm × 10.0 mm, 5 μm; Phenomenex, Torrance, CA, USA) with UV detection at 360 nm.

HR-ESI-MS was obtained on a Bruker maXis ESI-QTOF mass spectrometer (Bruker Daltonics, Bremen, Germany) in the positive-ion mode. LR-ESI-MS was recorded on a Bruker amaZon SL mass spectrometer (Bruker Daltonics, Bremen, Germany) in both the positive- and negative-ion modes. One- and two-dimensional NMR spectra were measured on a Bruker AV-700 MHz NMR spectrometer (Bruker Scientific Technology Co. Ltd., Karlsruhe, Germany). UV spectra were recorded on a UV-2600 UV-Vis spectrophotometer (SHIMADZU, Kyoto, Japan) and optical rotations determined on an MCP 500 modular circular polarimeter (Anton Paar GmbH, Seelze, Germany) with a 1.0 cm cell at 25 °C. Preparative HPLC was performed on a Waters 2535 pump equipped with a YMC C₁₈ reversed-phase column (250 × 10 mm i.d., 5 μm, Kyoto, Japan) and a 2998 photodiode array detector coupled with a 2424 evaporative light scattering detector (Waters Corporation, Milford, NY, USA). For column chromatography, silica gel (100–200 mesh, Qingdao Mar. Chem. Ind. Co. Ltd., Qingdao, China) and C₁₈ reversed-phase silica gel (ODS-A-HG 12 nm, 50 µm, YMC, Kyoto, Japan) were employed.

Recombinant ¹⁵N-labeled His-tagged FGF2-C77/95S (pQE30) was expressed in Escherichia coli W3110Z1 cells using M9 minimal medium with ¹⁵NH₄Cl as the sole nitrogen source. Purification was performed as described above. Purified FGF2-C77/95S was diluted in 25 mM HEPES buffer (pH 7.4), containing 150 mM KCl and 10% D₂O, to a final concentration of 160 µM. For the IP₃ and heparin disaccharide titration experiments, 500 µl of 80 µM FGF2-C77/95S were titrated with defined volumes of 80 µM FGF2-C77/95S and either 900 µM IP₃ (Sigma 74148, Sigma Aldrich, St. Louis, MO) or heparin disaccharide (Sigma H9267). The endpoint of the IP₃ titration was further titrated with defined volumes of 80 µM FGF2-C77/95S and 900 µM heparin disaccharide and vice versa. NMR spectra were recorded on a Bruker AV 700 MHz NMR spectrometer equipped with a 5 mm triple resonance cryo-probe at 300 K. For each titration step 2D ¹⁵N¹H-HSQC spectra were recorded with 1024 points in the ¹H dimension and 96 points in the ¹⁵N dimension and averaged over eight transients. Spectra were processed with TopSpin (Bruker, Billerica, MA) using CcpNmr Analysis software (Vranken et al., 2005 (link)). Signals were assigned using previously published data (accession code: 1BLA; (Moy et al., 1996 (link), 1995 (link))) Chemical shift differences were calculated using the equation ${\displaystyle \mathrm{\Delta }\delta =\sqrt{{\left(\delta H\right)}^2+{\left(0,15\ast \delta N\right)}^2}}$ .