Avance 3 hd 400 mhz spectrometer

All reagents were commercially available and used as supplied without further purification. Deuterated solvents were purchased from Cambridge Isotope Laboratory (Andover, MA). Compound 1^{39 (link)}, 2^{40 (link)}, 4^{39 (link)}, 5^{40 (link)} was prepared according to the published procedures. ¹H NMR, ³¹P{¹H} NMR spectra and 2D COSY NMR spectra were recorded on Bruker AVANCE III HD 400 MHz spectrometer and Bruker AVANCE III HD 600 MHz spectrometer. ¹H NMR chemical shifts are reported relative to residual solvent signals. Mass spectra were recorded on the Micromass Quattro II triple-quadrupole mass spectrometer using electrospray ionization, Thermo Scientific Q Exactive mass spectrometer using electrospray ionization and Agilent 6545 Q-TOF using electrospray ionization. The single crystals data were collected on a Nonius KappaCCD diffractometer equipped with Mo K-alpha radiation (λ = 0.71073 Å) and a BRUKER APEXII CCD.

The chemical structure of the dual-targeting dendrimer was characterized by ¹H NMR measurement on AVANCE III HD 400 MHz spectrometer (Bruker, USA) using D₂O as solvent. ¹H NMR (D₂O, 400 MHz, ppm): δ 2.25-2.40 (-CH₂CH₂CONH-); δ 2.45-2.55 (-CH₂CH₂N<); δ 2.68-2.74 (-NCH₂CH₂CO-); δ 2.95-3.20 (-CONHCH₂CH₂- and -CH₂CH₂NH₂); δ 3.32-3.75 (-CH₂CH₂O-); δ 6.79 (-COCH=CHCO-); δ 3.30 (-OCH₃). Size distribution and zeta potential of the dendrimer-based carriers were measured by Zetasizer Nano ZS nanoparticle size analyzer (Malvern Instruments Ltd., UK) at a concentration of 0.1 mg/mL in PBS. The samples were sonicated for 3 min and filtered with 200 nm filter membrane before detection. Morphological characterization of the dendrimer-based carriers was performed on a Ht-7700 transmission electron microscope (Hitachi, Japan) at acceleration voltage of 80 KV. TEM samples were made by dropping 2.5 μL samples (1 mg/mL) on carbon-coated grids and deposited for 30 min. Then the samples were negative stained by 1 wt% uranyl acetate for 25 s before test. Stability of the dual-functional dendrimer in water and PBS was detected by DLS within 72 h. Briefly, the samples were dissolved into deionized water and PBS at a concentration of 1 mg/mL. Then the particle size was detected by a Zetasizer Nano ZS nanoparticle size analyzer at 0 h, 12 h, 24 h, 48 h and 72 h.

Zeta potential was detected by Malvern Zetasizer Nano ZS (Zetasizer Nano ZS 90, Malvern Instrument, UK) at 25 °C. The morphological of hydrogel was recorded by scanning electron microscopy (SEM, ZEISS-SUPRA55, Germany). Rheology tests were performed on the rheometer (MCR 302, Aaton paar, Austria) to investigate mechanical properties and stability of NCTD Gel. In order to explore self-assembly mechanism of NCTD Gel, thermodynamic mechanism was recorded on a NANO ITC (TA, USA). UV–vis spectra were recorded on a UV–vis spectrophotometer (HITACHI UH5300, Japan) with the range of 200–400 nm. FT-IR spectra were recorded on a Fourier transform infrared spectrometer (NicoletiS10, Thermo, USA) with the range of 4000-400 cm⁻¹. CD spectra were obtained by using Chirascan V100 (Applied Photophysics, UK) with the range of 200–400 nm. ¹H-NMR (Avance IIIHD 400 MHz spectrometer, Bruker, America) spectra were used to ensure the formation mechanism between GA and NCTD. MD simulation was performed by GROMACS 2019.6 software to reveal the self-assembly mechanism of NCTD Gel.

Technical products of Isoprothiolane (IPT) and Phenazine-1-carboxylic acid (PCA) were provided by the College of Agriculture, Yangtze University. All chemicals and solvents were commercially obtained and used directly without further purification. Column chromatography was performed over pure silica gel 60 (200–300 mesh, Liang Chen Gui Yuan Co., Ltd., Anhui, China). Thin-layer chromatography (TLC) was performed on silica gel 60 GF254 (Qingdao Hai Yang Chemical Co., Ltd., Qingdao, China). The melting points of all target compounds were determined by using a WRR melting point apparatus (Shanghai Jingke Industrial Co., Ltd., Shanghai, China) and are uncorrected. ¹H and ¹³C NMR spectra were recorded in DMSO-d₆ or CDCl₃ as the solvent using an AVANCE III HD 400 MHz spectrometer (Bruker Co., Ltd., Fällanden, Switzerland). Tetramethylsilane was used as the internal standard. High-Resolution Mass Spectra (HRMS) were obtained using electrospray ionization (ESI) technique by collision-induced dissociation on a Thermo Scientific Q Exactive instrument (Thermo Fisher Scientific, Bremen, Germany). The samples were dissolved in HPLC methanol and filtered by a 0.2 μm membrane. Then, it was infused using a syringe pump with a flow rate of 10 μL/min, and analyzed in the positive mode within a mass range m/z 100–1500.

Nuclear magnetic resonance (NMR) data were acquired using a Bruker AVANCE III HD 400 MHz spectrometer (Bruker Corp., Billerica, MA, USA). A (S)-5-oxotetrahydrofuran-2-carboxylic acid sample was dissolved in CDCl₃ with 0.03% v/v tetramethylsilane (Sigma-Aldrich) and transferred to 5 mm NMR standard tubes. (S)-2HG was dissolved in double-distilled water. The (S)-2HG aqueous solution was analyzed inside a 5 mm NMR tube using a coaxial system (Wilmad^® coaxial insert, Wilmad-LabGlass, Vineland, NJ, USA), filled with D₂O with 0.75% sodium 3-(trimethylsilyl)propionate-2,2,3,3-d₄(TSP) (Sigma-Aldrich).
¹H and ¹³C-NMR spectra of (S)-5-oxotetrahydrofuran-2-carboxylic acid were obtained using standard acquisition parameters. The (S)-2HG proton spectrum was acquired using the noesypr1d pulse sequence for water signal suppression. Compounds were identified by comparing the obtained data with previously reported spectra (Bal & Gryff-Keller, 2002 (link)). DEPT-135, HMBC, and HSQC spectra were also acquired. Relative (S)-2HG purity was determined by the integration of all the signals of the ¹H-NMR spectrum, with exception of the ¹³C couplings and the TSP signal using a method similar to published chromatographic procedures (Pauli, Jaki & Lankin, 2007 (link)). The obtained data were processed and analyzed using Bruker TopSpin 3.2 software (Bruker Corp.) and used to confirm (S)-2HG synthesis.