Prior to the structural NMR analysis of the P-1 fraction, we prepared an asialo P-1 fraction in addition to the intact P-1 preparation. Sialic acid was released from the P-1 fraction by adding 5 mM HCl at 80 °C for 50 min under N2 gas. The reaction was terminated by adjusting the pH to 7.5 at 0 °C followed by desalting. 1H- and 13C-NMR and 2D NMR experiments (1H-13C HSQC, COSY, H2BC, HMBC, TOCSY and ROESY) were performed using JNM-α500 or JNM-ECA920 spectrometers (JEOL Ltd., Tokyo, Japan). The oligosaccharide preparation was permethylated following the method of Ciucanu and Kerek [14 (link)] prior to GC-MS analysis. Hydrolysis of the permethylated oligosaccharide was conducted with 2 M trifluoroacetic acid at 121 °C for 1 h. Reduction was performed by adding DMSO-NaBD4 at 40 °C for 1.5 h, and peracetylation was performed by adding acetic acid, 1-methyl imidazole and acetic anhydride successively. The GC-MS system consisted of a HP5890 gas chromatograph (Hewlett-Packard Co., Palo Alto, PA, USA), a JMS DX-303 mass spectrograph and a JMA DA5000 data module (JEOL).
Jnm α500
Manufactured by JEOL
Sourced in Japan
The JNM-α500 is a high-performance nuclear magnetic resonance (NMR) spectrometer manufactured by JEOL. It is designed to provide accurate and reliable analysis of chemical samples. The core function of the JNM-α500 is to detect and measure the magnetic properties of atomic nuclei within a sample, allowing for the identification and characterization of various chemical compounds.
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2 protocols using jnm α500
1
Structural Analysis of Asialo P-1 Fraction
2
Synthesis of Fluorescent Polymer-Conjugated Dendrimer
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Synthesis of FP-CDE CDE was prepared according to a previously described method, 23) (link) using a polyamidoamine dendrimer (ethylenediamine core, generation 2.0) (Sigma-Aldrich Japan, Tokyo, Japan) and GUG-β-CyD (Ensuiko Sugar Refining, Tokyo, Japan). The number of GUG-β-CyD molecules attached to the CDE was determined by comparing the peak areas of the anomeric proton of GUG-β-CyD and the ethylene protons of the dendrimer in the 1 H-NMR spectrum using an NMR spectrometer (JNM-α-500, JEOL, Tokyo, Japan). FP-COOH was prepared according to a previous method, 25) (link) using FA (Nacalai Tesque, Kyoto, Japan), N,N′dicyclohexylcarbodiimide (NHS), N-hydroxysuccinimide, and SUNBRIGHT PA-020HC (Yuka Sangyo, Tokyo, Japan). FP-COOH (50 mg), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (37.2 mg), and NHS (22.3 mg) were dissolved in borate buffer (0.2 M, pH 9, 1 mL) and stirred for 2 h. CDE (69.8 mg) was dissolved in borate buffer (0.2 M, pH 9, 1 mL) and stirred for 48 h at room temperature (r.t.) after addition of the solution containing activated FP-COOH. The product was dialyzed in H 2 O for 5 d (dialysis membrane; MWCO = 8000) and lyophilized to obtain FP-CDE.
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