Dd2 nmr spectrometer

NMR spectra were recorded with TMS as internal standard on an Agilent DD2 NMR spectrometer (500 and 125 MHz for ¹H and ¹³C NMR, respectively). HRESIMS and ESIMS spectra were obtained from a Micromass Q-TOF spectrometer. Optical rotations were measured on a JASCO P-1020 digital polarimeter. Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200–300 mesh), and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography (CC). TLC silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin-layer chromatography. Semipreparative HPLC was carried out on a Waters 1525 system using a semipreparative C18 (Kromasil, 5 μm, 10 × 250 mm) column equipped with a Waters 2996 photodiode array detector, at a flow rate of 2.0 mL/min.

All reagents were obtained commercially and used without further purification. BPA (¹⁰B) was supplied from Stella Pharma Corp. (Osaka, Japan). Synthesis of the intermediates was performed as previously described [23 (link),25 (link)] with minor modifications. The synthetic scheme is shown in Figure S1 and the details are described in the Supplementary Methods. Purified BS-631 was obtained as a brown oil and characterized using an EI-MS, a high-resolution mass spectra (HRMS)(JMS-700(2) mass spectrometer, JEOL Ltd., Tokyo, Japan), and ¹H and ¹³C-NMR (DD2 NMR Spectrometer, Agilent, CA, USA, 600 MHz). MS (EI) m/z: 247; EI-HRMS m/z: 247.1681 (chemical formula: C₁₄H₂₁N₃O, calculated m/z: 247.1685); ¹H NMR (600 MHz, CDCl₃): δ 7.76 (s, 1H), 6.18 (s, 1H), 6.04 (s, 1H), 3.44 (t, J = 5.0 Hz, 2H), 3.35 (q, J = 7.0 Hz, 2H), 3.29 (s, 3H), 3.20 (q, J = 7.0 Hz, 2H), 3.11 (t, J = 5.0 Hz, 2H), 1.17 (t, J = 5.0 Hz, 3H), and 1.16 (t, J = 5.0 Hz, 3H); ¹³C NMR (600 MHz, CDCl₃): δ 165.0, 162.2, 143.0, 127.1, 112.0, 107.4, 98.5, 47.5, 45.6, 45.6, 45.4, 41.8, 10.6, and 10.3.

¹H (500 MHz) and ¹³C (125 MHz) spectra were acquired in CDCl₃ or CD₃OD, on a 500 MHz Agilent DD2 NMR spectrometer operated using VnmrJ software version 4.2 rev. A, with reference to solvent signals (δ 7.26 ppm and 77.0 ppm for CDCl₃, or δ 3.31 ppm and 49.0 ppm for CD₃OD). Two-dimensional NMR spectra were recorded on the same instrument, and these included gCOSY, HSQCAD, and gHMBCAD NMR experiments. A Bruker 300 MHz Avance III NMR spectrometer operated with Bruker TopSpin software version 3.6.5, with reference to solvent signals (δ 7.26 ppm and 77.0 ppm for CDCl₃ or δ 3.31 ppm and 49.0 ppm for CD₃OD) was also used for the acquisition of rapid ¹H NMR experiments using a Bruker SampleCase^TM 24-slot autosampler. “Bruker TopSpin”, “MestReNova” and “ACD Spectrus” software was used for processing the NMR data.

The Faradaic efficiency of gas products was calculated as follows. $Faradaicefficiency\left(\%\right)=N\times F\times \nu \times r/\left(i\times V_m\right)$ where N represents the number of electrons transferred, F represents the Faradaic constant, v represents the gas flow rate at the cathodic outlet, r represents the concentration of product(s) in ppm, i represents the total current, and V_m represents the unit molar volume of product(s). The gas flow rate at the cathode outlet was measured via a bubble flow meter.
The liquid products of CO₂R or COR were analyzed by using ¹H NMR spectroscopy (600 MHz Agilent DD2 NMR Spectrometer) with suppressing water peak. Dimethyl sulfoxide (DMSO) was used as the reference standard, whereas deuterium oxide (D₂O) was used as the lock solvent. The NMR spectra collected at each current density were used to calculate the Faradaic efficiency toward liquid products of CO₂R or COR as follows. $Faradaicefficiency\left(\%\right)=N\times F\times n_{product}/Q$ where N represents the number of electrons transferred, F represents the Faradaic constant, n_product represents the total mole of product(s), and Q = i × t represents the total charge passing during the liquid product collection.

Assignments were made using 3D ¹³C,¹³C,¹H HMQC-NOESY-HMQC data (250 msec mixing time) recorded on a 900 MHz (21.1 T) Varian VNMRS NMR spectrometer equipped with a cryogenically cooled z-axis gradient probe (Rocky Mountain Regional 900 MHz Facility, University of Colorado, Denver). NOE-based assignments were validated and extended by recording 2D ¹³C,¹H methyl-TROSY HMQC spectra on cysteine point mutations (I190C, V768C, V866C, L103C, L176C, and L51C) in the absence and presence of MTSL nitroxide spin label (Toronto Research Chemicals). Additionally, Rad50 hinge region mutations (V156M, V157M, V160M, and L163M) were produced to help resolve a crowded region in the Rad50^NBD structure. The choice of the mutation to methionine was derived from a sequence alignment of Rad50, which showed the need for a hydrophobic residue at these positions, and the desire to put the newly labeled methyl group in an uncrowded region of the methyl-TROSY spectra. HMQC spectra on these mutations were recorded on a 600 MHz (14.1 T) Agilent DD2 NMR spectrometer equipped with a room temperature z-axis gradient probe. All NMR data were collected at 50 °C, and spectra were processed with NMRPipe^{43 (link)} and analyzed with CCPN analysis^{44 (link)}.

Optical rotations were measured using a P-1020 polarimeter (JASCO). UV spectrua were obtained with a DU 640 spectrophotometer (Beckman). ECD spectra were acquired on a JASCO J-815-150S CD spectrometer. IR spectra were obtained via a Nicolet-Nexus-470 spectrometer. NMR spectra were recorded on an Agilent DD2 NMR spectrometer (500 MHz). ESIMS and HRESIMS spectra were obtained by a Q-TOF (Micromass) and a LTQ Orbitrap XL (Thermo Scientific) spectrometer, respectively. Single-crystal analysis were performed on a Gemini A Ultra system using Cu Kα radiation (Aglient Technologies). A 1525 separation module (Waters) equipped with a C₁₈ (Kromasil, 5 μm, 10 × 250 mm) column was used for semi-preparative HPLC. ODS (Unicorn; 45–60 μm), Sephadex LH-20 (Amersham Biosciences), and silica gel (200–300 mesh; Qing Dao Hai Yang Chemical Group Co.) were applied for column chromatography. TLC (G60, F-254; Yan Tai Zi Fu Chemical Group Co.) was used in the compounds detection.

B16F10-bearing mice were intravenously injected with 3-BPA or 4-BPA mixed with fructose (3-BPA-Fru, 4-BPA-Fru, 1 mg/100 μL PBS (-), 2.2 w/v% Fru, pH 7.4), and sacrificed 10, 30, 60, or 120 min after administration. Samples of the plasma and the tissues of interest were excised, weighed, and ashed by nitric acid, followed by ICP-MS measurements of the boron amount. The accumulation rate was calculated as the % injected dose/g (%ID/g) of the quantified dose of boron in the administrated solution.
LAT1-positive T3M-4 xenograft mice were intravenously injected with 3-BPA-Fru, 4-BPA-Fru (1 mg/100 μL PBS (-), 2.2 w/v% Fru, pH 7.4), or fructose-free 3-BPA (1 mg/100 μL PBS (-)) and sacrificed 60 min later, followed by boron amount measurement in the same manner. The details of the preparation of 3-BPA-Fru and 4-BPA-Fru and measurement of ¹¹B-NMR (DD2 NMR Spectrometer, Agilent, Santa Clara, CA, USA, 600 MHz) to confirm the formation of the complex with fructose are described in Supplementary Methods.