Inhibition of GlcNAc-Processing Glycosidases by C-6-Azido-NAG-Thiazoline and Its Derivatives

Abstract

NAG-thiazoline is a strong competitive inhibitor of GH20 β-N-acetylhexosaminidases and GH84 β-N-acetylglucosaminidases. Here, we focused on the design, synthesis and inhibition potency of a series of new derivatives of NAG-thiazoline modified at the C-6 position. Dimerization of NAG-thiazoline via C-6 attached triazole linkers prepared by click chemistry was employed to make use of multivalency in the inhibition. Novel compounds were tested as potential inhibitors of β-N-acetylhexosaminidases from Talaromyces flavus, Streptomyces plicatus (both GH20) and β-N-acetylglucosaminidases OPEN ACCESSMolecules 2014, 19 3472 from Bacteroides thetaiotaomicron and humans (both GH84). From the set of newly prepared NAG-thiazoline derivatives, only C-6-azido-NAG-thiazoline displayed inhibition activity towards these enzymes; C-6 triazole-substituted NAG-thiazolines lacked inhibition activity against the enzymes used. Docking of C-6-azido-NAG-thiazoline into the active site of the tested enzymes was performed. Moreover, a stability study with GlcNActhiazoline confirmed its decomposition at pH < 6 yielding 2-acetamido-2-deoxy-1-thio-α/β-D-glucopyranoses, which presumably dimerize oxidatively into S-S linked dimers; decomposition products of NAG-thiazoline are void of inhibitory activity.

Mechanism of Human Nucleocytoplasmic Hexosaminidase D

“…Notably, functionalized derivatives of NAG-thiazoline have been developed as potent and selective inhibitors of the lysosomal β-hexosaminidases HexA/B and OGA, which have proven to be useful tools for interrogating the biological role of these enzymes. 14 , 23 , 37 , 38 Unfortunately, Gal-NAG-thiazoline has limited use as a chemical tool for studying the physiological role played by HexD because it also inhibits the lysosomal β-hexosaminidases ( Table 4 ). These studies, however, lay important groundwork for the rational design of selective inhibitors of HexD.…”

How Site‐Directed Mutagenesis Boosted Selectivity of a Promiscuous Enzyme

“…Among GlcNAcases, many representatives also exhibit purely hydrolytic activity . Moreover, even in the very selective GlcNAcases, traces of GalNAcase activity may be found ( e. g ., in O ‐GlcNAcase, the GalNAcase/GlcNAcase ratio is 0.03). It is also noteworthy that although the main source of specificity of GalNAcase/GlcNAcase is encoded by the primary amino acid sequence of the β‐ N ‐acetylhexosaminidase, other factors, not yet fully understood, are undeniably involved, e. g ., the effect of posttranslational modifications or of heterologous expression in a different host …”

A divergent synthesis to generate targeted libraries of inhibitors for endo-N-acetylglucosaminidases

“…The sensitivity of the thiazoline ring of NAG-thiazoline limits the conditions to which this molecule can be subjected, which in turn circumscribes those chemical transformations that can be used to derivatize this compound. For example, in acidic conditions (pH < 6.0) thiazolines are protonated to form an easily hydrolyzed thiazolinium, 38 in oxidative conditions the sulfur can be oxidized, and in reductive or radical conditions the N=C double bond can be degraded. 37 Thus, mild protection and deprotection conditions are required.…”