Ascend avance 3 600 mhz

All experiments were performed on a Bruker Ascend Avance III 600 MHz system equipped with a broadband cryoprobe (Bruker). Experiments were conducted at pH 5.5 or 6.0 at 10 °C in 10% D₂O containing 50 mM sodium deuterated acetate buffer, 10 mM NaCl, and 100 μM 2,2,3,3-²H₄ (trimethylsilyl)propionic acid sodium salt (TMSP) as a chemical shift reference (0 ppm). Water signal suppression was achieved using an excitation-sculpting scheme. Thirty-two scans were collected for each spectrum to yield a 15K-point free induction decay.
To assess the anomerase activity of YhcH, 1 mM 2,3-EN was used as starting substrate in the presence of 20 μM NAD⁺ and 1.5 μM of the hydratase YjhC, which converts 2,3-EN to a mixture of 2,7-AN and α-Neu5Ac (6 ). Anomerases YhcH and NanM were tested at concentrations of 15 μM and 0.5 μM, respectively. We monitored the time course of appearance of shifts specific to H3 axial for both α- and β-Neu5Ac.
To study the anomerization of Neu5Ac catalyzed by divalent cations, 0.6 U/ml of C. perfringens sialidase A (Sigma Aldrich) was used to hydrolyze 1 mM 3’-sialyllactose to lactose and α-Neu5Ac. CoCl₂, NiCl₂, CaCl₂, MgCl₂ were tested at 5 mM and ZnCl₂ was tested at 1 mM. We monitored the time-dependent evolution of peaks corresponding to H3 axial of the alpha-anomer and H3 equatorial of beta-anomer of Neu5Ac.

The samples were prepared in PBS (pH 7.4 or 6.8) from a stock solution of olaparib (10 mM in DMSO-d₆) and NADH (10 mM in PBS pH 7.4 or 6.8). The final concentration of DMSO-d₆ was 5%. All spectra were acquired at 310 K on a Bruker (Kontich, Belgium) Ascend Avance III 600 MHz system equipped with a broadband cryoprobe. Water signal suppression was achieved using an excitation-sculpting scheme. A total of 32 scans were collected for each experiment sample.