L-Fucosidase

The substrate, fucosyl-GMj, was separated from its product, GMj, by chromatography on a LiChrosorb-NH2 column (4 mm X 250 mm, 5 /urn). The mobile phase was a 25 75 mixture of 5 mM potassium phosphate buffer (pH 5.5) and acetonitrile. Similarly, fucosyl-GDib was separated from GDib. except the concentration of phosphate buffer was 20 mM Gangliosides were detected by their absorbance at 195 nm. 

The enzyme preparation used was purified to apparent homogeneity from human liver by affinity chromatography on agarose-e-aminocaproylfucos-amine resin. 

This assay measures the ability of a-lV-acetylgalactosaminyltransferase to transfer the sugar moiety of UDP-yV-acetylgalactosamine to an oligosaccharide that bears a fluorescent label. There are two kinds of a-TV-acetylgalactosami-nyltransferase One transfers sugar to a protein, the other to an oligosaccharide. 

The standard reaction mixture contained in a final volume of 100 fxL 50 mM Tris-HCl (pH 7.0), 10 mM MnCl2, 0.5% Triton X-100, 1.0 mM UDP-GalNAc, 20 fiL of enzyme, and about 10 pmol of pyridylamino lacto-N-fucopentaose I. The mixture was incubated at 37°C for 2.5 hours, and the reaction was terminated by adding 3 volumes of ethanol. After centrifugation, the supernate was dried and reconstituted in 100 fiL of water. A 5 fiL aliquot was analyzed by HPLC. 

Microsomes were prepared from the antrum and corpus regions of rat stomach. The microsomal fraction from 1 g of wet tissue was suspended in 50 fiL of 0.25 M sucrose containing 20 m M Tris-HCl (pH 7.2). 

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The guanidino analogue 90 of the 7-membered cyclic urea system was prepared, enantiomerically pure, from D-mannitol. The derivative 90 selectively inhibits bovine kidney a-L-fucosidase at 2.8pM <00BMC307>. 

Instead of a triflate, the electrophile on the glycosyl acceptor can be an a,(3-unsaturated carbonyl group. This is the case reported in Fig. 25, in which a stereoselective Michael addition of the 1-thiosugar 56 to the a,(3-conjugated system of levoglucosenone 57, generated after deprotection a couple of L-fucopyranosyl-4-thiodisaccharides 61 and 62 presenting inhibitory activity on a-L-fucosidase.54... 

Selected from among their contributions were the first example of a synthetic furanoid iminoalditol, l,4-dideoxy-l,4-imino-D-mannitol (19, Scheme 7) 49 the first synthesis of the powerful L-fucosidase inhibitor l,5-dideoxy-l,5-imino-L-fucitol (20),50 the synthesis and structure confirmation of l,4-dideoxy-l,4-imino-D-arabinitol... 

The inverting GH 95 1,2-oc-L-fucosidase (EC 3.2.1.63) from Bifidobacterium bifi-dum in complex (PDB 2EAC) with 1-deoxy-L-fuconojirimycin (20) showed the inhibitor in C4 chair conformation, resembling L-fucose and the entrance to the active site was narrowed by the gate keeper loops.315 A calcium ion associated with the protein was not involved in the catalytic process but was considered to stabilize the enzyme. 

A. Lammerts van Bueren, A. Ardevol, J. Fayers-Kerr, B. Luo, Y. Zhang, M. Sollogoub, Y. Bleriot, C. Rovira, and G. J. Davies, Analysis of the reaction coordinate of a-L-fucosidases A combined structural and quantum mechanical approach,./. Am. Chem. Soc., 132 (2010) 1804-1806. 

M. Nagae, A. Tsuchiya, T. Katayama, K. Yamamoto, S. Wakatsuki, and R. Kato, Structural basis of the catalytic reaction mechanism of novel 1,2-a-L-fucosidase from Bifidobacterium bifidum, J. Biol. Chem., 282 (2007) 18497-18509. 

The inhibitory activity of compound (42) (Scheme 13) was examined during the hydrolysis of p-nitrophenyl a-L-fucopyranoside by a-L-fucosidases. As reported above, this compound showed mixed inhibition [32]. 

In the series of a-L-fucopyranosyl-thio-glycosides, the most powerful inhibitor of a-L-fucosidases was the a(l ->3) linked isomer obtained by deprotection of the disaccharide (65 b). It has been shown to be a competitive inhibitor (Kj= 0.65 mmol/1) [32]. 

Less active derivatives might therefore be desirable in some cases. In this context, Paulsen and co-workers synthesized a range of chain-extended as well as bicyclic analogues of 1-deoxyfuconojirimydn, amongst others the 6-C-ethyl (54) and 6-C-methyl derivatives as well as l-deoxy-6,7,8-a-tri-epi-castanosper-mine (55). All of these derivatives exhibited considerable inhibitory activities towards a-L-fucosidase and were slightly to appreciably less active than the parent compound [107]. In an attempt to design a simple synthesis of 1-deoxy-... 

An amidrazone (58) derived from 5-amino-5-deoxy-L-fuconolactam was found to inhibit a recombinant human a-L-fucosidase with a K -value of 820 nmol/1 [ 111 ]. A simple synthesis of 1,5-dideoxy-1,5-imino-D-arabinitol (59), previously prepared by Ganem et al. [49] as a potential maimosidase inhibitor, was applied to the affinity purification of a-L-fucosidase from bovine kidney by an improved method and the characterization of the enzyme thus obtained [112]. The relatively low affinity of this compound to the enzyme (Kj 2.2 pmol/1 at pH 7) compared to 1-deoxyfuconoJirimycin (51) turned out to be advantageous in terms of enzyme recovery and yield. Structurally related, suitably protected 5-amino-5-deoxy-D-arabinopyranose (60), was coupled with a N-acetyl-6-deoxy-6-thio-D-glucosaminide (61) to give a stable thioglycoside (62) [113]. 

A L-/wco-related analogue (71) of isofagomine [61], which was recently prepared from D-ribose by Ichikawa et al. following previously established chemistry [63,121], was found to be an a-L-fucosidase inhibitor with Kj 8 pmol/1. No inhibition was found with tetrazole derivatives such as (72) [ 122] obtained from L-gulonolactone. 

Fucosidase inhibiting l,4,5-trideoxy-l,4-imino-L-lyxitol (78) was prepared [124] from D-ribose via protected 5-amino-5-deoxy-L-lyxose (79) by a chemical route. This compound, as well as the 1-aminomethyl homologue (80), obtained by Kiliani-Fischer chain extension of (79), inhibited a-L-fucosidase with K around 2 pmol/1. l,4-Dideoxy-l,4-imino-D-iditol (81) [71] was found to be a moderate inhibitor of the enzyme. 

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