Artificial Glycosyl Phosphorylases

Abstract

alpha- and beta-Cyclodextrin 6(A),6(D)-diacids (1 and 2), beta-cyclodextrin-6-monoacid (14), beta-cyclodextrin 6(A),6(D)-di-O-sulfate (16) and beta-cyclodextrin-6-heptasulfate (19) were synthesised. Acids 1, 2 and 14 were made from perbenzylated alpha- or beta-cyclodextrin, by diisobutylaluminum hydride (DIBAL)-promoted debenzylation, oxidation and deprotection. Addition of molecular sieves was found to improve the debenzylation reaction. Sulfates 16 and 19 were made by sulfation of the appropriately partially protected derivatives and deprotection. Catalysis of 4-nitrophenyl glycoside cleavage by these cyclodextrin derivatives was studied. Compounds 1, 2 and 16 were found to catalyse the reaction, with the catalysis following Michaelis-Menten kinetics and depending first order on the phosphate concentration. In a phosphate buffer (0.5 M, 59 degrees C, pH 8.0), K(M) varied from 2-10 mM and the k(cat)/k(uncat) ratio from 80-1000 depending on the stereochemistry of the substrate and the catalyst, with 2 being the best catalyst and with the sulfated 16 also displaying catalytic ability. The monoacid 14 and the heptasulfate 19 were not catalytic.

Cyclodextrin derivatives that display Enzyme Catalysis

“…At low phosphate concentration (0.05 M) the rate acceleration is modest and the best catalysis was k cat /k uncat ＝35, which was found for hydrolysis of the 4-nitrophenyl b-D-glucopyranoside by 38 (45). On the other hand, the catalyzed rate increased linearly with the phosphate concentration and at phosphate 0.5 M, pH 8.0 the kcat/kuncat was as high as 989 (46). The catalysis is presumably caused by electrostatic stabilization of the transition state, since the carboxylate is required and the disulfate 39 also is a catalyst with a decent kcat/kuncat of 309.…”

Synthesis of Some Trifluoromethylated Cyclodextrin Derivatives and Analysis of Their Properties as Artificial Glycosidases and Oxidases

“…[45] (95 % yield) as a colorless foam. 1 [38][39][40] To a solution of compound 5 (7.56 g, 2.50 mmol) and 4-Å molecular sieves (53 g) in toluene (350 mL), stirred under N 2 for 1 h, was added DIBAL-H (1.5 ) (80 mL, 120 mmol) dropwise, and left stirring overnight. The reaction was monitored by TLC (EtOAc/pentane, 1:3) until no starting material (R f = 0.55) and no monool (R f = 0.26) were observed, and only the diol occurred (R f = 0.13).…”

“…The synthesis of 2 and 4 was carried out from the nonadecabenzylated diol 6 readily available from β-cyclodextrin in two steps (Scheme 1): [38][39][40] The β-cyclodextrin is perbenzylated with NaH and benzyl chloride giving the perbenzylated cyclodextrin 5 in 85 % yield. Selective debenzylation with DIBAL (0.2 ) and 4-Å molecular sieves at 25°C and 18 h gives the diol 6 in 81 % yield.…”

Substantial Spatial Flexibility and Hydrogen Bonding within the Catalysis Exerted by Cyclodextrin Artificial Glycosidases

“…These unprotected carboxylate CD chemzymes can effect reaction rate en- www.eurjoc.orghancements of up to 1000 times for hydrolysis of aryl glycosides, and their mechanism of catalysis is proposed to involve electrostatic stabilization of the transition state by the carboxylate groups, with subsequent nucleophilic substitution with phosphate. [12,13] Two carboxylate catalytic groups are needed for chemzyme catalysis to occur; cyanohydrin CDs suffice having just one catalytic entity (Figure 2).…”

“…[8,9] The inspiration for the design of the CD glycosidases came from the world of natural glycosidases which in their catalysis of glycosidic bond hydrolysis typically encompass two catalytically active carboxylate groups in their active site. [10,11] This acid/base-catalysis principle spawned the invention of firstly the carboxylate cyclodextrin glycosidases that were able to catalyze aryl glycoside bond hydrolysis reactions with rate enhancements of up to 1000 times (k cat / k uncat ), following the enzyme-characteristic Michaelis-Menten rate law pattern [12,13] (see below). By obeying MichaelisMenten kinetics, the chemzymes reveal themselves as enzyme-like mimics, displaying saturation kinetics, and not mere catalysts.…”