Avance neo 500 mhz

Allylpalladium chloride dimer (98%) and 1,4-bis(diphenylphosphino)butane (dppb) (98%) were purchased from Aldrich. DMA (99+%) extra pure was purchased from Acros. KOPiv (95%) was purchased from Doug Discovery. These compounds were not purified before use. All reagents were weighed and handled in air. All reactions were carried out under an inert atmosphere with standard Schlenk techniques. ¹H, ¹⁹F and ¹³C NMR spectra were recorded on Bruker Avance III 400 MHz and Bruker Avance neo 500 MHz spectrometers. High-resolution mass spectra were measured on a Bruker MaXis 4G spectrometer. Melting points were determined with a Kofler hot bench system. Chromatography purifications were performed using a CombiFlash NextGen 300 with Buchi FlashPure cartridges containing 40 μm irregular silica.

Starting materials were purchased from
commercial suppliers and were used without further purification. Reactions
were monitored by LCMS (Agilent 1100 Series) equipped with an ESI-MS
detector (Waters Micromass ZQ 2000) or by TLC-MS (API, Advion Expression).
The phosphonamidate series were purified with preparative RP-HPLC
(VWR LaPrep P110) with single wavelength detection (254 nm), using
an ACE5 C8 column (5 μm, 100 Å, ϕ 21.2 mm L 250 mm)
and gradients of CH₃CN/H₂O as the mobile phase
at a 10 mL/min flow rate. NMR spectra of the synthetic intermediates
were recorded on a Varian Unity 400 MHz, Bruker Avance Neo 500 MHz,
or a Bruker Avance Neo 600 MHz spectrometer. The Bruker instruments
were equipped with TXO and TCI cryogenic probes. The chemical shifts
are reported using the residual solvent signal as an indirect reference
to TMS. Chemical shift titrations and 3D NOESY were acquired on the
Bruker Avance Neo 600 MHz spectrometer, whereas spectra for assignment
were obtained on the Bruker Avance HD 800 MHz spectrometer equipped
with a 3 mm TCI cryogenic probe. Purity analysis of the final phosphonamidate
inhibitors was performed using ¹H NMR, with the original
spectra being shown in the Supporting Information and the original NMR raw data files (FID) available open access
at Zenodo (DOI:10.5281/zenodo.4773990).

The NMR spectra of PI_14.6PS_34.8AmPEO_1.9, PI_35.1PS_14.8AmPEO_1.9 and EmPEO_1.9 were recorded using an AVANCE NEO 500 MHz (Bruker, Billerica, MA, USA), that of mPEO_1.9, PI_24.8PS_25.0AmPEO_1.9 and PI_26.1PS_67.3AmPEO_1.9 using an AVANCE NEO 400 MHz (Bruker) and for PI_6.8PS_17.3AmPEO_1.9 using an AVANCE III 400 MHz (Bruker). NMR measurements with number of scans = 64 were recorded, and the recycle delay D1 between transients was set to 30 s to ensure full relaxation to equilibrium magnetization and thus the acquisition of quantitative spectra, except for mPEO_1.9. All NMR spectra were recorded at 300 K using CDCl₃ as deuterated solvent, where all signals were referenced to CDCl₃ (δ = 7.3 ppm for ¹H and δ = 77.2 ppm for ¹³C relative to tetramethylsilane) [46 (link)]. The spectra were analyzed with the software MestReNova (version: 12.0.4-22023, Mestrelab Research, Santiago de Compostela, Spain).

The polyoxometalates used in this study, K₆[α-P₂W₁₈O₆₂]·14H₂O (abbreviated P₂W₁₈), K₁₂[α-H₂P₂W₁₂O₄₈]·16H₂O (P₂W₁₂), Na₁₂[α-P₂W₁₅O₅₆]·24H₂O (P₂W₁₅), and (NH₄)₁₄[NaP₅W₃₀O₁₁₀]·31H₂O (P₅W₃₀) (Figure 1, Table 2), were synthesized according to published procedures [54 ,55 ], and their purity was confirmed by infrared spectroscopy. Stock solutions of POTs were freshly prepared by dissolving the solid compound in water and keeping the solution at low temperature to avoid POT decomposition. The concentrations of the stock solutions were 10 mM and/or 1 mM for all POTs.
For solution stability studies, ³¹P-NMR spectroscopy was used. ³¹P-NMR spectra were recorded with a Bruker FT-NMR spectrometer Avance Neo 500 MHz (Bruker, Rheinstetten, Germany) at 25 °C and 202.53 MHz. Chemical shifts were measured relative to 85% H₃PO₄.

Optical rotations were
measured on a PerkinElmer 341-LC instrument. NMR spectra were acquired
on an Agilent MR-400-DD2 spectrometer equipped with a 5 mm OneNMR
probe and on a Bruker Avance NEO 500 MHz or Bruker Avance NEO 800
MHz instrument equipped with a 5 mm cryogenic TXO probe. The spectra
were processed using MestReNova 14.1 software and were referenced
to the residual solvent peak. LC-ESIMS data were acquired on a Waters
Micromass ZQ multimode ionization electrospray ionization (ESI) instrument
connected to an Agilent 1100 series gradient pump system and a C₈ column (Gemini), using a Milli-Q H₂O/MeCN (5:95
to 95:5, with 1% HCO₂H over 4 min) eluent mixture. HRESIMS
spectra were acquired with a Q-TOF LC/MS spectrometer with a lock
mass-ESI source (Stenhagen Analysis Lab AB, Gothenburg, Sweden), using
a 2.1 × 30 mm, 1.1 μm reverse phase^{49 (link)}-C₁₈ column and a H₂O/MeCN
gradient
(5:95 to 95:5, with 0.2% HCO₂H). TLC analyses were carried
out on Merck precoated silica gel 60 F₂₅₄ plates. Preparative
TLCs were performed on glass plates of 20 × 20 cm dimension,
precoated with silica gel 60F₂₅₄ having 0.25 to 1 mm thickness.
Column chromatography was performed on silica gel (40–63 μm
mesh), and gel filtration was performed on Sephadex LH-20.

Bruker AVANCE NEO 500MHz nuclear magnetic resonance spectrometer (with TMS as internal standard, DMSO-d6 and CDCl₃ as solvents); Agilent LC1200/MS Q-TOF6520 liquid mass spectrometer; Gemini E single crystal diffractometer and an X-4 precision microscope melting-point tester were used. Soybean aphids were raised in the laboratory. The column chromatography silica gel (200–300 mesh) and TLC plates used in this experiment were produced by Qingdao Ocean Chemical Co., Ltd. The reagents used were all commercially available analytical or chemically pure reagents. Unless otherwise specified, the solvents used were not treated with water.

Chitosan (the deacetylation degree ≥ 95%), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl), N-hydroxysuccinimide (NHS), Phloretic acid (PA), 2-(N-Morpholino)ethanesulfonic acid (MES), Sodium tripolyphosphate (TPP), and Sodium molybdate (Na₂MoO₄) and horseradish peroxidase (HRP) (>300 U/mg, lyophilized powder) were all purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) and used as received. 5-Fluorouracil (5-FU) was obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Nuclear magnetic resonance (NMR) spectrometer (AVANCE NEO, 500 MHz, Bruker) and Fourier transform infrared (FTIR, MAGNA550, Nicolet) was used for structural analysis. Nanoparticle size analyzer (Nano-ZS90, Malvern) was used to determine the size of the nanogels. UV-Vis spectrophotometer (T6, Persee) was used to determine the concentrations of 5-FU in the drug loading and release experiments.