Inhibition of glycosidases has great potential in the quest for highly potent and specific drugs to treat diseases such as diabetes, cancer, and viral infections. One of the most effective ways of designing such compounds is by mimicking the transition state. Here we describe the structural, kinetic, and thermodynamic dissection of binding of two glucoimidazole-derived compounds, which are among the most potent glycosidase inhibitors reported to date, with two family 1 beta-glycosidases. Provocatively, while inclusion of the phenethyl moiety improves binding by a factor of 20-80-fold, this does not appear to result from better noncovalent interactions with the enzyme; instead, improved affinity may be derived from significantly better entropic contributions to binding displayed by the phenethyl-substituted imidazole compound.
It was shown that retaining b-glucosidases and galactosidases of families 1 ± 3 feature a strong interaction between C(2)OH of the substrate and the catalytic nucleophile. An analogous interaction can hardly take place for retaining b-mannosidases. A structureÀactivity comparison between the inhibition of the b-glucosidase from Caldocellum saccharolyticum (family 1) and b-glucosidase from sweet almonds by the gluco-imidazoles 1 ± 6, and the inhibition of snail b-mannosidase by the corresponding manno-imidazoles 8 ± 13 does not show any significant difference, suggesting that also the mechanisms of action of these glycosidases do not differ significantly. For this comparison, we synthesized and tested the manno-imidazoles 9 ± 13, 28, 29, 32, 35, 40, 41, 43, 46, 47, and 50. Among these, the alkene 29 is the strongest known inhibitor of snail b-mannosidase (K i 6 nm, non-competitive); the aniline 35 is the strongest competitive inhibitor (K i 8 nm).Introduction. ± The strong inhibition of b-glucosidases by imidazoles of type 1 [1 ± 3] has been rationalized by the similarity of shape of the inhibitor and of the putative reactive intermediate, an oxycarbenium cation, and by the cooperative interaction of the imidazole with the catalytic nucleophile and acid [4]. A correlation between the inhibition constant and the pK value of the C(2)-and C(3)-acetamido imidazoles 7 and 14 ± 16, and by related azoles has established that substituents at C(3) lower the inhibitory activity [5]. The structureÀactivity relation (SAR) resulting from varying the C(2)-substituents has been studied in detail [6]. It was shown that the HOCH 2 group at C(2) in 2, and particularly the flexible hydrophobic PhCH 2 CH 2 group in 3 lead to an improved inhibition, with K i values as low as 0.1 nm (against Caldocellum saccharolyticum b-glucosidase) 1 ). The C(2)-substituents affect both the strength and the type of the inhibition (competitive or mixed, with a varying between 2.5 and 15).Legler and Withers evidenced that the C(2)OH group of b-glucosides and bgalactosides interacts with the catalytic nucleophile of the retaining b-glucosidases and b-galactosidases of families 1 [8], 2 [9], and 3 [10] [11]. 2-Deoxy-and 2-deoxy-2-fluorob-d-glucosides and -galactosides are cleaved much less readily than the parent substrates, the rate-determining step being deglycosylation of the enzyme. The transition state for this reaction is considered very similar to that of the enzyme glycosylation [9], and the most important interaction in the transition state was considered with the C(2)OH 2 ). That 2-deoxyglucosides are cleaved less readily than the parent substrates is surprising, as the OH group at C(2) is known to destabilize an
The gluco-configured analogue 15 of nagstatin (1) and the methyl ester 14 were synthesized via condensation of the thionolactams 17 or 18 with the b-amino ester 19. The silyl ethers 20 and 21 resulting from 17 were desilylated to 22 and 23; these alcohols were directly obtained by condensing 18 and 19. The attempted substitution of the C(8)ÀOH group of 22 by azide under Mitsunobu conditions led unexpectedly to the deoxygenated a-azido esters 24. The desired azide 25 was obtained by treating the manno-configured alcohol 23 with diphenyl phosphorazidate. The azide was transformed to the debenzylated acetamido ester 14 that was hydrolyzed to the nagstatin analogue 15. The imidazole-2-acetates 14 and 15 are nanomolar inhibitors of the Nacetyl-b-glucosaminidases from Jack beans and from bovine kidney, submicromolar to micromolar inhibitors of the b-glucosidase from Caldocellum saccharolyticum, and rather weak inhibitors of the snail b-mannosidase. In all cases, the ester was a stronger inhibitor than the corresponding acid. As expected from their glucoconfiguration, both imidazopyridines 14 and 15 are stronger inhibitors of the b-N-acetylglucosaminidase from bovine kidney than nagstatin.Introduction. ± Nagstatin (1), a strong inhibitor of several hexosaminidases [1 ± 4], is a N-acetylgalactosamine-derived tetrahydropyridoimidazole-2-acetic acid [5]. Its inhibitory activity is essentially associated with the imidazole ring and not with the carboxymethyl substituent [2], although substituents on the imidazole ring may strongly affect the inhibition of b (and a-)-glycosidases [6 ± 8]. In the preceding paper [9], we described the influence of the hydrophobic character of C(2)-methyl ester and carboxylic acid substituents on the inhibition of b-glycosidases. Imidazole-2-propionates 10 ± 13 are stronger inhibitors of the b-glucosidase from Caldocellum saccharolyticum and of the b-mannosidase from snail than imidazole-2-acetates 6 ± 9, and these are stronger than imidazole-2-carboxylates 2 ± 5. There is a parallel sequence of inhibitory activity for the methyl esters and the corresponding acids, with the esters being stronger inhibitors.
The acetylcholinesterase inhibitor (-)-huperzine A was synthesized from (S)-4-hydroxycyclohex-2-enone in 17 steps by a route that involved two cyclobutane fragmentations. The first of these employed a retro-aldol cleavage to generate the α-pyridone ring of huperzine A, and the second invoked a novel intramolecular aza-Prins reaction in tandem with stereocontrolled scission of a cyclobutylcarbinyl cation to create the aminobicyclo[3.3.1]nonene framework of the natural alkaloid.
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