Dd2 600 mhz spectrometer

3,4-Dimethoxycinnamic acid (DMCA, 15.6 mg, 0.08 mmol), helenalin (21.9 mg, 0.08 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (14.4 mg, 0.08 mmol) and 4-dimethyl amino pyridine (18.4 mg, 0.15 mmol) were dissolved in dry dichloromethane (5 mL) and stirred at room temperature. Loss of alcohol was monitored by thin layer chromatography (silica gel plates 60 F₂₅₄, Merck KGaA, Darmstadt, Germany with mobile phase ethyl acetate/hexane (4/1)). After 24 h, the solvent was evaporated under reduced pressure. The residue was dissolved in acetonitrile and purified by preparative HPLC (Jasco, Groß-Umstadt, Germany) with preparative reverse phase column Reprosil 100 C-18 (5 μm, 250 mm, 20 mm, Macherey-Nagel, Düren, Germany). The yield was 14.97 mg. NMR spectra were recorded on an Agilent DD2 600 MHz spectrometer and calibrated with the solvent (CDCl₃) peak as reference. NMR data are reported in Table S1, Supplementary Materials. The purity as determined by UHPLC with diode array detector (DAD) was >95% (Figure S1, Supplementary Materials).

Proteins (450 µL) in 20 mM PBS buffer were mixed with 50 µL of D₂O to prepare a 9:1 H₂O/D₂O NMR sample. All NMR data were acquired using a Agilent DD2 600 MHz spectrometer equipped with a z-axis pulsed-field gradient triple resonance (1H, 13C, 15N) cold probe at 298 K. ¹H NMR spectra were recorded using the double pulsed-field gradient spin echo water suppression (DPFGSE) pulse sequence, 64 scans, and a 1.5-s relaxation delay. The acquired spectral width was 12 kHz and 16k complex points were obtained with a total acquisition time of 2.5 min. The final figure was generated on MestreNova.

The samples were defrosted at room temperature, and 250 μL aliquots were mixed with 250 μL of 1.5 M phosphate buffer (pH 7.4) to minimize variation in pH. The samples were centrifuged at 13,000 rpm for 10 min at 4 °C to separate any precipitate. Then, the filter liquor was added with 4, 4-dimethyl-4-silapentane-1-sulfonic acid (DSS) (50 μL) and vortexed for 10 s. Afterwards, the mixed liquor was centrifuged for 2 min at 13,000 rpm. Finally, the 480 μL of total supernatant was injected into the nuclear magnetic tube. ¹H NMR was measured on a DD2 600 MHz spectrometer (Agilent, CA, USA) operating at a 599.83 MHz magnet frequency and equipped with a triple-resonance cryoprobe. Therein, 256 scans were collected with a spectral width of 7225.434 Hz at 25 °C; the recycle delay time was set as 0.01 s and the water signals were suppressed during relaxation time. The free induction decay (FID) was transported into the Chenomx NMR suit (version 8.0, Edmonton, Alberta, Canada) software, and the ¹H NMR spectra were manually phased and baseline-corrected. The DSS at 0.0 ppm was used as reference for chemical shifts.

The self-diffusion coefficients of D₂O in H₂O were measured using ²H PGSE Dbppste pulse sequence on Agilent DD2 600 MHz spectrometer (Santa Clara, California, USA) equipped with DOTY DSI-1372 multinuclear probe-head with a maximum magnetic field gradient of 30 T/m (Doty Scientific, Clemson Rd, Columbia, USA). The self-diffusion coefficients of H₂O in D₂O were measured using ¹H PGSE Dbppste⁷⁴ using Agilent DD2 800 MHz spectrometer. The exact temperatures were calibrated using ethylene glycol by analyzing the chemical shift difference between CH₂ (ethylene glycol) and OH groups according to the Bruker VT-Calibration Manual. Gradient calibration constants on both spectrometers were determined using a water sample obtained from Mili-Q using the Agilent gradient calibration procedure at 21 °C.
NMR samples were poured into 5 mm NMR sample tubes (0.01% D₂O in H₂O and 0.01% H₂O in D₂O). The spin-echo intensities were fitted according to the Stejskal-Tanner equation⁷⁵ . using MestReNova 14 software (https://mestrelab.com/software/mnova/).
The self-diffusion coefficient at T₀ = 25 °C, D₂₅, and the activation energy, E_a, were estimated according to the modified Arrhenius Eq. (11) implemented in Origin (version 9.9; www.originlab.com). $$\text{D}\left(\text{T}\right)={\text{D}}_{25}·\text{exp}\left(\frac{{\text{E}}_{\text{a}}}{\text{R}},·,\frac{T-T_0}{\text{T}·T_0}\right)$$

2D NMR spectra of parent peptide and hybrid 6 were recorded on Agilent DD2 600 MHz spectrometer. The compounds were dissolved in 9:1 DPBS buffer:D₂O at a concentration of around 10 mg/mL. 1D spectra were recorded with a double pulsed field gradient spin echo (DPFGSE) water signal suppression sequence. Sequences and parameters for 2D spectra were as follows: TOCSY DPFGSE water signal suppression—512 × 256 time domain complex points zero-filled to 2048 × 1024 complex points, and apodized by cosine square function in both dimensions; spectral width 6 kHz in both dimensions; number of scans 16 and mixing time 65 ms. ROESY DPFGSE water signal suppression—512 × 256 time domain complex points zero-filled to 2048 × 1024 complex points, and apodized by cosine square function in both dimensions; spectral width 6 kHz in both dimensions; number of scans 48 and mixing time 200 ms. 13C-1H HSQC—1442 × 400 time domain complex points zero-filled to 2048 × 1024 complex points, and apodized by cosine square function in both dimensions; spectral width 9.6 × 25.6 kHz in F2 and F1, respectively; number of was scans 8. COSY water signal suppression using presaturation—900 × 256 time domain complex points zero-filled to 2048 × 2048 complex points, and apodized by cosine square function on both dimensions; spectral width 6 kHz in both dimensions; number of scans 16.