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Fucosidase inhibitors represent captivating targets for various biological applications. Iminosugars featuring a fluoroalkyl chain in the pseudoanomeric position were synthesized by nucleophilic addition of a perfluoroalkyl Grignard reagent to a nitrone. Reduction of the N–O bond of the hydroxylamine was achieved by treatment with an aqueous solution of sulfur dioxide. The pKa = 4.5 of the target pyrrolidine reflected a strong electronic effect of the fluoroalkyl chain. Inhibitory potencies of the fluorinated iminosugars and their corresponding hydroxylamines were evaluated against α-L-fucosidase at various pH values.

L-Fucose-containing glycoconjugates are essential for a myriad of physiological and pathological activities, such as inflammation, bacterial and viral infections, tumor metastasis, and genetic disorders. Fucosyltransferases and fucosidases, the main enzymes involved in the incorporation and cleavage of L-fucose residues, respectively, represent captivating targets for therapeutic treatment and diagnosis. We herein review the important breakthroughs in the development of fucosyltransferase and fucosidase inhibitors. To demonstrate how the synthesized small molecules interact with the target enzymes, i.e. delineation of the structure–activity relationship, we cover the reaction mechanisms and resolved X-ray crystal structures, discuss how this information guides the design of enzyme inhibitors, and explain how the molecules were optimized to achieve satisfying potency and selectivity.

Trichloroacetimidate method was proved to be effective for 5-thio- D -arabinopyranosylation and 5-thio- L -fucopyranosylation of N -acetyl- D -glucosaminides and D -galactosides. In these 5-thioglycosylation the neighboring group participation of 2- O -acetyl function was not decisive of anomeric stereoselectivity to give 1,2- cis pseudodisaccharides predominantly. Allyl 2- O -(5-thio-α- L -fucopyranosyl)-β- D -galactopyranoside showed potent inhibitory activity towards α- L -fucosidase (Ki=30μM).

The first synthesis of the novel azasugar α- l -homofuconojirimycin is reported. The biological activity of this compound is described and is shown to be more specific for α- l -fucosidase than compounds previously tested.

Poly-N-acetyllactosamine extension has been found in O-glycans in addition to N-glycans and glycosphingolipids (Fukuda, M. (1994) In Molecular Glycobiology, (ed. M. Fukuda and O. Hindsgaul), pp. 1–52, Oxford University Press, Oxford). However, the extent of poly-N-acetyllactosamine formation between O-glycans and N-glycans in a given glycoprotein has not been determined. Attempts were made in HL-60 cells to determine the amount of sialyl Lex structures in O-glycans and N-glycans attached to the major lysosomal membrane glycoproteins, lamp-1 and lamp-2. Lamp molecules were immunoprecipitated from 3H-glucosamine labeled HL-60 cells. Glycopeptides were prepared by pronase or trypsin digestion, and O-glycan containing glycopeptides were isolated by affinity chromatography using Jacalin-Agarose. The glycopeptides bound to Jacalin-Agarose were treated with alkaline borohydride, and the released O-glycans were fractionated by Bio-Gel P-4 filtration. Similarly, the glycopeptides unbound to Jacalin-Agarose, which represent N-glycans, were isolated by Sephadex G-50 gel filtration. Sequential glycosidase digestion of the O- and N-glycans, with or without pretreatment by fucosidase or neuraminidase, revealed the following features: (1) each lamp-1 and lamp-2 molecule contains about 2 and 1 poly-N-acetyllactosaminyl O-glycan chains, (2) about 65% of these poly-N-acetyllactosaminyl O-glycans contain sialyl Lex termini, NeuNAcα2→3Galβ1→4(Fucα1→3)GlcNAcβ1→R, and (3) N-glycans and O-glycans are modified in almost equal efficiency to express sialyl Lex structures. These results indicate that in lamp molecules both O-glycans and N-glycans contribute to the expression of sialyl Lex structures.

5-Thio- l -fucopyranose tetraacetate was synthesized in 11 steps from 5-O- trityl- d -arabino-furanose or d -arabinose diethyl dithioacetal by one-carbon elongation at C-5. Highly diastereo-selective addition of MeLi in ether to a 1,2-O- isopropylidene -β- d -arabino- pentodialdo-1,4-furanose derivative was achieved to give the corresponding 6-deoxy-β- d -altrofuranose isomer in good yield. A sulfur atom was introduced at C-5 of 6-deoxy- d -altrofuranose derivatives via substitution of a 5-tosylate with KSAc in HMPA with inversion of configuration, giving 5-thio- l -fucopyranose. A 3-O- substituted- l -fucose derivative was also prepared from 6-deoxy-β- d -altrofuranose derivatives. 5-Thio- d -arabinopyranose tetraacetate, the 5-demethyl analog of 5-thio- l -fucose, was also synthesized from 5-O- trityl- d -arabinofuranose in 5 steps. 5-Thio- d -arabinose showed weak inhibitory activity against α- l -fucosidase from bovine kidney ( K i = 0.77 mM ).

Four 5-thio-l-fucose-containing disaccharides having α(1→6), α(1→3), α(1→4)GlcNAc, and α(1→2)Gal linkages (compounds 1−4, respectively) were synthesized as potential α-l-fucosidase inhibitors. The glycosylation reactions using 2,3,4-tri-O-acetyl-5-thio-l-fucopyranosyl trichloroacetimidate as a glycosyl donor and BF3·OEt2 as a catalyst gave mainly α-linked disaccharides. Only α(1→2)-linked disaccharide 4 showed inhibitory activity (Ki = 0.21 mM) against Bacillus α-l-fucosidase which hydrolyzes the Fucα(1→2) linkage specifically. The results suggested that sequence specificity of an enzyme could be estimated from the inhibitory activities of the compounds 1−4. In contrast, every disaccharide showed inhibitory activity (Ki = 30−91 μM) against bovine epididymis α-l-fucosidase.

Five 5-thio-α- l -fucopyranosyl derivatives were prepared and examined as inhibitors of the α- l -fucosidase from bovine epididymis. The inhibitory activities of the fucosides strongly depend on the hydrophobicity of the aglycon. The best inhibition ( K i = 3.3 μM) was obtained with p -nitrophenyl 1,5-dithio-α- l -fucopyranoside.

A chemoenzymatic strategy has been developed for the synthesis of libraries of iminocyclitol derivatives for the discovery of new and selective fucosidase inhibitors.

A thermally induced intramolecular 1,3-dipolar cycloaddition of an azidoester and subsequent sodium cyanoborohydride reduction of the resulting bicyclic vinylogous urethane to give a bicyclic aminolactone allows access to an eight carbon homologue of α-homomannojirimycin which is a weak fucosidase inhibitor. Intermediates with both an α- and β-amino acid moiety are described and may be useful for incorporation of homopipecolic acids into novel peptide libraries.

The potential of synthetic thiasugars, the ring sulfur analogs of carbohydrate, as glycosidase inhibitor was discussed in terms of activity and specificity, based on our own studies. The syntheses of these analogs including di- and tri-saccharide derivatives were also outlined. 5-Thiofucose was the first thiapyran to show a Ki value of micromolar range against a glycosidase. The structure-activity relationship studies indicated that its fucosidase inhibition was due in part to a hydrophobic interaction between the ring sulfur and the enzyme. A sulfylimine compound having a thiafuran structure was created as a transition-state analog inhibitor of glucosidases and found to have a weak activity. The same thiafuran structure was later found by another group in a natural product, salacinol, which is a sulfonium derivative and is a strong glucosidase inhibitor. The structure-activity relationship study with synthetic analogs of salacinol indicated importance of the sulfonium ion and its internal counter anion, sulfate, for the inhibition activity.

Nitrone 3 analogue of 5-deoxy- l -lyxose was derived from d -ribose. Its reduction with aqueous SO2 gave the corresponding 4-amino-sugar 2 , a potent α- l -fucosidase inhibitor. 1,3-Dipolar cycloaddition of 3 with alkenes allowed the synthesis of acetohydroxamic acid and ethane–phosphonate derivatives. Hydroxy-ethane derivative 15 is a nanomolar α- l -fucosidase inhibitor.

Polyhydroxypiperidines and pyrrolidines (also called iminocyclitols or aza-sugars) have attracted increasing attention because of their inhibitory activity against various glycosidases. The glucose-type iminocyclitol deoxynojirimycin (DNJ), for example, has been used for the treatment of noninsulin-dependent diabetes (miglitol, targeting the intestinal disaccharidases). Recent studies further indicate that DNJ and its derivatives are effective against hepatitis B and C, as well as glycosphingolipid storage disorders (such as Gaucher disease). The efficacy of iminocyclitols is attributed to their mimicry of the transition state of enzymatic glycosidic cleavage. Of the members of the glycosidase family, a-fucosidase is involved in the hydrolytic degradation of numerous fucosecontaining glycoconjugates. The existence of this enzyme is associated with a variety of essential functions. For example, the abnormal accumulation of fuco-glycoconjugates, which results from the absence or deficiency of a-fucosidase, leads to the genetic neurovisceral storge disease fucosidosis. An aberrant distribution of intracellular and extracellular afucosidase is also found in cystic fibrosis 11] and colon–rectal cancer. 13] Though the physiological functions of a-fucosidase are not completely understood, potent fucosidase inhibitors may be used as probes for the study of fucosidases with regard to their functions and for the development of potential therapeutic agents. Recently we have established a novel combinatorial approach to five-membered iminocyclitols based on reductive amination and the Strecker reaction, and a potent inhibitor has been identified from the library. The process, that is, synthesis and isolation of individual compounds for testing, is however very slow. To further facilitate the discovery of new glycosidase inhibitors, we aimed to develop a new method for the rapid derivatization of an iminocyclitol core designed for a specific glycosidase family (for example, fuconojirimycin derivatives for fucosidases in this study) without protecting group manipulation and under such conditions that the product could be used directly for screening in situ without isolation. This concept was first demonstrated by the development of new HIV protease inhibitors. Herein we report the generation of a library based on fuconojirimycin (FNJ) in a microtiter plate, followed by the direct in situ evaluation of these reaction mixtures as fucosidase inhibitorse without product isolation. This approach has led to the discovery of the most potent inhibitors of a-fucosidases from bovine kidney and Corynebacterium sp. Previous work on the development of glycosidase inhibitors indicated the existence of an additional binding component in the inhibitor. 16–25] This study was thus aimed at generating an FNJ derivative 1 (Scheme 1) as a core structure to mimic the transition state of the fucose moiety. A baminomethyl group was attached to the C1-position for identification of a new binding component throught the subsequent in situ amide-bond formation and screening. Since 1-deoxy-FNJ has been shown to be a potent inhibitor against a-fucosidases (Ki= 9.8 nm for the a-fucosidase from human neutrophil and 6.2 nm from bovine epididymal)

The synthesis of the disaccharides α-L-Fucp-(1,2)- and α-L-Fucp-(1,3)-β-D-Galp(1)-4-methylumbelliferone as fucosidase substrates was accomplished by activation of the anomeric centre of 2,3,4-tri-O-acetyl- and 2,3,4,6-tetra-O-acetyl-α,β-L-fuco- and -D-galactopyranosyl propane-1,3-diyl phosphates with TMSOTf and with minimal protection of 4-methylumbelliferyl β-D-galactopyranoside.

The ribofuranoside (I) reacts with trimethylsilyl cyanide (II) to form the nitriles (III) and (IV).