4-Deoxy-substrates for β-N-acetylhexosaminidases: How to make use of their loose specificity

Abstract

beta-N-Acetylhexosaminidases feature so-called wobbling specificity, which means that they cleave substrates both in gluco- and galacto- configurations, with the activity ratio depending on the enzyme source. Here we present the new finding that fungal beta-N-acetylhexosaminidases are able to hydrolyze and transfer 4-deoxy-N-acetylhexosaminides with high yields. This clearly demonstrates that the 4-hydroxy moiety at the substrate pyranose ring is not essential for substrate binding to the enzyme active site, which was also confirmed by molecular docking of the tested compounds into the model of the active site of beta-N-acetylhexosaminidase from Aspergillus oryzae. A set of four 4-deoxy-N-acetylhexosaminides was synthesized and screened against a panel of beta-N-acetylhexosaminidases (extracellular and intracellular) from various sources (fungal, human, animal, plant and bacterial) for hydrolysis. The results of this screening are reported here, as well as the structures of three novel 4'-deoxy-disaccharides prepared by transglycosylation reaction with high yields (52% total disaccharide fraction) using beta-N-acetylhexosaminidase from Talaromyces flavus.

Charged Hexosaminides as New Substrates for β‐N‐Acetylhexosaminidase‐Catalyzed Synthesis of Immunomodulatory Disaccharides

“…Sulfates 4 (gluco-) and 6 (galacto-) were prepared in a 5-step synthesis starting from the respective unprotected pnitrophenyl hexosaminide as already described in our previous work. [6] The preparation of phosphates 5 (gluco-) and 8 (galacto-) was the most challenging task and was complicated by the presence of the nitro moiety on the aromatic ring. Here, the optimal procedure was the phosphorylation of the respective partially protected p-nitrophenyl hexosaminide with POCl 3 .…”

“…The catalyst used in all the preparative reactions presented was the b-N-acetylhexosaminidase from Talaromyces flavus CCF 2686, which proved to be among the most efficient enzymes in the screening and, additionally, was known for its good synthetic capacity and tolerance to substrate modifications, as shown previously. [6,7,11] The carboxyl group was introduced into the disaccharidic compounds in two ways -via aldehyde 2 in combination with in situ quantitative oxidation of the product by NaClO 2 or directly via uronate 3. This was because, from our previous experience with galactoderivatives, [11] we (correctly) supposed uronate 3 to be a much poorer substrate than aldehyde 2, both in terms of the cleavage and transglycosylation.…”

“…(6): Compound 6 was synthesized analogously to compound 4; [5] it was obtained in a 30% overall yield. [6] (7; 200 mg, 0.469 mmol) and dry DIPEA in CH 2 Cl 2 (11 mL, 1:10 v/v). After 15 h of stirring at ambient temperature, another portion of DIPEA (5 mL) was added, followed by the addition of water (1 mL).…”

“…[3] Previously, we have demonstrated that fungal b-N-acetylhexosaminidases possess a wide tolerance to substrates bearing various modifications, including N-acyls, [4] sulfates, [5] and an absent C-4 hy-droxy. [6] Moreover, GlcNAc substrates with two unusual C À N linked aglycones, namely the azido group [7] and the aromatic triazole moiety, [8] were cleaved by some fungal b-N-acetylhexosaminidases; the azide even worked as an efficient glycosyl donor in transglycosylation reactions. [7] As for the biological and especially the immunomodulatory activity of glycosides and glycoconjugates, our previous studies described the ability of N-acetyld-hexosamine structures to stimulate the immune response: they can activate natural killer (NK) cells by binding to their activation receptors, such as NKR-P1 and CD69 proteins.…”

Efficient and Regioselective Synthesis of β-GalNAc/GlcNAc-Lactose by a Bifunctional Transglycosylating β- N -Acetylhexosaminidase from Bifidobacterium bifidum

“…However, the available enzyme sources for synthesis remain limited. Most of the reported ␤-N-acetylhexosaminidases with transglycosylation ability are obtained from fungi, especially those belonging to the genera Aspergillus (22)(23)(24)(26)(27)(28)(29), Penicillium (28,30), and Talaromyces (31,32), and only three are obtained from the bacteria Nocardia orientalis and Serratia marcescens YS-1 (33)(34)(35). Moreover, most ␤-N-acetylhexosaminidases with transglycosylation ability display low glycosyl transfer efficiency at 1% to 10% yield, as well as poor regioselectivity, resulting in isomer production with multiple glycosyl linkages that are difficult to isolate (33,35,36).…”