28217-28-9Relevant academic research and scientific papers
Scope of the DMC mediated glycosylation of unprotected sugars with phenols in aqueous solution
Fairbanks, Antony J.,Qiu, Xin
, p. 7355 - 7365 (2020/10/13)
Activation of reducing sugars in aqueous solution using 2-chloro-1,3-dimethylimidazolinium chloride (DMC) and triethylamine in the presence of para-nitrophenol allows direct stereoselective conversion to the corresponding 1,2-Trans para-nitrophenyl glycosides without the need for any protecting groups. The reaction is applicable to sulfated and phosphorylated sugars, but not to ketoses or uronic acids or their derivatives. When applied to other phenols the product yield was found to depend on the pKa of the added phenol, and the process was less widely applicable to 2-Acetamido sugars. For 2-Acetamido substrates an alternative procedure in which the glycosyl oxazoline was pre-formed, the reaction mixture freeze-dried, and the crude product then reacted with an added phenol in a polar aprotic solvent system with microwave irradiation proved to be a useful simplification.
Mechanistic evaluation of MelA α-galactosidase from citrobacter freundii: A family 4 glycosyl hydrolase in which oxidation is rate-limiting
Chakladar, Saswati,Cheng, Lydia,Choi, Mary,Liu, James,Bennet, Andrew J.
experimental part, p. 4298 - 4308 (2012/03/22)
The MelA gene from Citrobacter freundii, which encodes a glycosyl hydrolase family 4 (GH4) α-galactosidase, has been cloned and expressed in Escherichia coli. The recombinant enzyme catalyzes the hydrolysis of phenyl α-galactosides via a redox elimination-addition mechanism involving oxidation of the hydroxyl group at C-3 and elimination of phenol across the C-1-C-2 bond to give an enzyme-bound glycal intermediate. For optimal activity, the MelA enzyme requires two cofactors, NAD+ and Mn2+, and the addition of a reducing agent, such as mercaptoethanol. To delineate the mechanism of action for this GH4 enzyme, we measured leaving group effects, and the derived βlg values on V and V/K are indistinguishable from zero (-0.01 ± 0.02 and 0.02 ± 0.04, respectively). Deuterium kinetic isotope effects (KIEs) were measured for the weakly activated substrate phenyl α-d-galactopyranoside in which isotopic substitution was incorporated at C-1, C-2, or C-3. KIEs of 1.06 ± 0.07, 0.91 ± 0.04, and 1.02 ± 0.06 were measured on V for the 1-2H, 2- 2H, and 3-2H isotopic substrates, respectively. The corresponding values on V/K were 1.13 ± 0.07, 1.74 ± 0.06, and 1.74 ± 0.05, respectively. To determine if the KIEs report on a single step or on a virtual transition state, we measured KIEs using doubly deuterated substrates. The measured DV/K KIEs for MelA-catalyzed hydrolysis of phenyl α-d-galactopyranoside on the dideuterated substrates, DV/K(3-D)/(2-D,3-D) and DV/K (2-D)/(2-D,3-D), are 1.71 ± 0.12 and 1.71 ± 0.13, respectively. In addition, the corresponding values on V, DV (3-D)/(2-D,3-D) and DV(2-D)/(2-D,3-D), are 0.91 ± 0.06 and 1.01 ± 0.06, respectively. These observations are consistent with oxidation at C-3, which occurs via the transfer of a hydride to the on-board NAD+, being concerted with proton removal at C-2 and the fact that this step is the first irreversible step for the MelA α-galactosidase-catalyzed reactions of aryl substrates. In addition, the rate-limiting step for Vmax must come after this irreversible step in the reaction mechanism.
