The concentration of the final NMR sample was typically 1 mM dissolved in PBS (20 mM phosphate and 150 mM NaCl) with 10% (v/v) 2H2O. All NMR experiments were acquired at 25°C on a cryoprobe-equipped Varian VNMRS 600 MHz spectrometer. The backbone resonances were assigned using HNCA and HNCO spectra and side-chain resonances using 3D 15N-edited TOCSY, HCCH-TOCSY and 13C-edited NOESY spectra. Distance restraints for structure calculations were derived from aliphatic and aromatic 13C-edited and 15N-edited NOESY spectra as well as a 2D 1H-1H NOESY spectrum recorded at 20°C. All of the NMR data were processed using NMRPipe [31 ], and the spectra were visualized and assigned using CcpNmr Analysis version 2.2 [32 (link)]. Structure calculations were performed using Aria 2.3 [33 (link)] using spin diffusion correction throughout, network anchoring in the first three iterations, and 8000 cooling steps in both the cool1 and cool2 phases. Structures (100) were calculated in iteration 8, of which the ten lowest energy structures were further water refined. PSVS 1.4 [34 (link)] and CING [35 (link)] were used to validate the structures and NMR data. The chemical shifts and structural restraints were deposited with the Biological Magnetic Resonance Data Bank (BMRB) [36 (link)] under accession number 19464. The structure was deposited with the PDB under code 4C26.
Vnmrs 600 mhz spectrometer
Manufactured by Agilent Technologies
Sourced in United States
The VNMRS 600 MHz spectrometer is a high-resolution nuclear magnetic resonance (NMR) instrument designed for analytical and research applications. It operates at a frequency of 600 MHz, which provides enhanced sensitivity and resolution for the analysis of chemical structures and molecular interactions.
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Lab products found in correlation
Sephadex lh 20, by GE Healthcare (3 mentions)
Silica gel 60, by Merck Group (2 mentions)
Lc 8a pump, by Shimadzu (2 mentions)
P 2000 polarimeter, by Jasco (2 mentions)
Cosmosil 5c18 ar 2 column, by Nacalai Tesque (2 mentions)
Guanidine hydrochloride, by Thermo Fisher Scientific (1 mentions)
Lichrosolv, by Merck Group (1 mentions)
Uvasol, by Merck Group (1 mentions)
Hp 8452a, by Hewlett-Packard (1 mentions)
D8 discover, by Bruker (1 mentions)
Labram hr evolution, by Horiba (1 mentions)
Usb2000 miniature fiber optic spectrometer, by OceanOptics (1 mentions)
Leo 912ab microscope, by Zeiss (1 mentions)
Bx 51 w dp70 microscope, by Olympus (1 mentions)
Agilent 8453 uv vis spectrometer, by Agilent Technologies (1 mentions)
Nicolet is50, by Thermo Fisher Scientific (1 mentions)
Finnigan flash ea 1112, by Thermo Fisher Scientific (1 mentions)
Mercury plus, by Agilent Technologies (1 mentions)
Vnmrs 500 mhz spectrometer, by Agilent Technologies (1 mentions)
Masslynx v4, by Waters Corporation (1 mentions)
25 protocols using vnmrs 600 mhz spectrometer
1
NMR Structure Determination Protocol
2
Enzymatic Kinetics of Uridine Analogues
3
Spectroscopic analysis of organic compounds
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NMR spectra were recorded in 5 mm NMR tubes on a VNMRS 600 MHz spectrometer (600 MHz for 1H; 150 MHz for 13C, Varian, Inc., Palo Alto, CA, USA) in DMSO-d6 and CDCl3 as solvents. Chemical shifts are referenced to tetramethylsilane (TMS) as an internal standard. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra of all the described experiments were measured on a Nicolet 6700 FTIR (from ThermoNicolet Corp., Madison, WI, USA). Spectra were recorded with ATR mathematical corrections yielding a 1.0 cm−1 actual resolution and 40 measurements were averaged. Electronic absorption spectra were obtained on an HP 8452A diode array spectrophotometer (Hewlett Packard, Palo Alto, CA, USA). The solvents used (CHCl3 and DMF) were HPLC (CHCl3; LiChrosolv®, Merck, Darmstadt, Germany) or UV-spectroscopy grade (DMF; Uvasol®, Merck) and were used without further purification.
4
NMR Spectroscopic Characterization Protocol
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For NMR measurements, samples were dissolved in deuterochloroform (CDCl3). Spectra were measured on the Varian VNMRS 600 MHz spectrometer at 25 °C. One-dimensional (1-D) 1H and 13C spectra and 2-D 1H-1H COSY, 1H-13C HSQC, and 1H-13C HMBC spectra were acquired. Additionally, 1-D NOESY spectra have been recorded for the selected proton signals. Plots of the original spectra prepared using Spinworks v. 4.05 software are shown in the Supplemental Figure 1.
Obtained data are summarized below.
Obtained data are summarized below.
5
NMR Analysis of Isotopically Labeled Mid1p
6
Multimodal Characterization of Materials
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1H NMR and 13C NMR spectra were obtained with a Varian VNMRS (600 MHz) spectrometer. UV-vis spectra were recorded on a single beam Agilent 8453 UV-vis spectrometer (Agilent Technologies). Absorption spectra were obtained with a USB2000 miniature fiber-optic spectrometer (Ocean Optics). XRD spectra were recorded on a D8 discover (Bruker). Optical microscopic images were obtained with an Olympus BX 51 W/DP70 microscope. TEM images were collected using a Zeiss LEO912AB microscope. SEM images were collected with a NOVA NanoSEM 450 at an accelerating voltage of 15 kV. IR spectra were recorded on a Thermo Nicolet NEXUS 870 FT-IR with KBr plate. Raman spectra were obtained with a Raman microscope (785 nm laser, LabRAM HR Evolution, Horiba).
7
Chemical Conjugation Analysis of R9G10-GC
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FT-IR spectroscopy was used to confirm the chemical conjugation between the R9G10 and GC (Nicolet IS50, Thermo Fisher Scientific; Waltham, MA, USA). The GC or R9G10-GC samples were mixed with dry KBr in a fine powder form, and a disk was prepared through compression. Each disk was scanned over a wavenumber region of 400–2000 cm−1 (resolution, 4 cm−1; scan rate, 4 mm/s). 1H NMR spectra were recorded using a Varian VNMRS 600 MHz spectrometer. Samples were dissolved in D2O ([sample] = 10 mg/mL). The degree of substitution of R9G10 conjugated to GC was calculated from elemental analysis (Thermo Finnigan Flash EA 1112, Bremen, Germany).
8
NMR Characterization of U3G and U3DG
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Nuclear Magnetic Resonance spectra (NMR) of U3G and U3DG isolated from in vitro experiments with Sace_3599 and in vivo feeding experiments with S. erythraea and S. albus Gluc x pUWL-A-sace_3599 were measured using a Varian VNMR-S 600MHz spectrometer equipped with 3 mm triple resonance inverse and 3 mm dual broadband probes. Pulse sequences were taken from Varian pulse sequence library. Spectra were recorded in 150 µL DMSO-d6 at 35 °C. Variable temperature experiments were measured at 25, 35, 50, 65 and 80 °C in DMSO-d6. EXSY spectra were recorded using the NOESY pulse sequence with a mixing time of 3 s.
9
Synthesis and Characterization of Organic Compounds
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All reagents used in the synthesis were obtained commercially and were used without further purification, unless otherwise specified. The reactions were monitored using thin-layer chromatography (TLC) with TLC sheets ALUGRAM-SIL G/UV254 (Macherey Nagel, Germany). Purification by flash chromatography was performed using silica gel 60 Å (0.0040–0.063 mm, Merck, Darmstadt, Germany) with the indicated eluent. Melting points were determined using a Boetius hot-stage apparatus and are presented in uncorrected form. NMR spectra were recorded at room temperature (RT) on a Varian Mercury Plus (Varian) 400 MHz spectrometer operating at 400 MHz for 1H and 100 MHz for 13C and on a Varian VNMRS 600 MHz spectrometer (operating at 600 MHz for 1H and 150 MHz for 13C). Chemical shifts (δ in ppm) are given from the internal solvent, DMSO-d6.
10
NMR Spectroscopy Analysis of Sample
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We used a Varian VNMRS (600 MHz) spectrometer (Varian, Palo Alto, CA, USA) for the analysis. The purified sample was dissolved in acetone-d6/D2O at a ratio of 1:1.
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