Design and synthesis of biologically active carbaglycosylamines: From glycosidase inhibitors to pharmacological chaperones

For over 50 years, our group has been involved in synthetic studies on biologically active cyclitols including carbasugars. Among a variety of compounds synthesized, this review focuses on carbaglycosylamine glycosidase inhibitors, highlighting the following: (1) the naturally occurring N-linked carbaoligosaccharide α-amylase inhibitor acarbose and related compounds; (2) the novel synthetic β-glycosidase inhibitors, 1′-epi-acarviosin and its 6-hydroxy analogue as well as β-valienaminylceramide and its 4′-epimer; (3) the discovery of the β-glycosidase inhibitors with chaperone activity, N-octyl-β-valienamine (NOV) and its 4-epimer (NOEV); and (4) the recent development of the potential pharmacological chaperone N-alkyl-conduramine F-4 derivatives.


Introduction
In the early 1960s, Prof. Umezawa and coworkers 1) began to work on the total synthesis and chemical modification of the aminocyclitol antibiotic kanamycin, according to their proposed relative structure of the main component kanamycin A (1) (Fig. 1a). One of the authors (SO) was conducting joint research with Prof. Umezawa's group at Keio University, synthesizing the model compounds aminocyclohexyl glucosaminides. 2) The work was gradually extended to collaboration with Prof. Suami at the same university and the synthesis of aminocyclitols of biochemical interest, 3), 4) including the two main components of aminocyclitol antibiotics, streptamine (2) and 2-deoxystreptamine (3), leading to a practical synthesis of 3 from myo-inositol (4). 5) This synthesis was later applied to the preparation of specifically 14 C-labeled 3 6) to elucidate the biosynthetic pathway of the antibiotic neomycin. 7), 8) The findings were then utilized to develop the microbial production of novel neomycin analogs (hybrimycins) by replacement of 3 with various aminocyclitols. 9), 10) Since then, SO has been involved in the synthesis of new bioactive inositol derivatives, such as anhydro-and dianhydroinositols, 11) and highly oxygenated cyclohexane compounds, such as crotepoxide. 12) In 1970, the discovery of an antibiotic with potent trehalase inhibitory activity, validamycin A (5) (Fig. 1b), was reported by Takeda Chemical Co. Ltd. 13)-15) Structural studies demonstrated the presence of the active core N-linked carbadisaccharide validoxylamine A (7), composed of the aminocarbasugars validamine (9) 16) and its unsaturated derivative valienamine (11). 17) Carbasugars, originally referred to as pseudo-sugars, are carbocyclic analogues of carbohydrates (Fig. 1b, inset). 18) The ,,,trehalose-like structure of 7 is believed to mimic the transition state of the trehalase reaction. In addition to 5, validamycin B 14) (6) was isolated and found to have a core validoxylamine B (8) composed of hydroxyvalidamine 16) (10) instead of 9. Later, the aminocarbasugars 9, 10, and 11 were also isolated together with a new aminocarbasugar ,-glucosidase inhibitor valiolamine (12) from validamycin fermentation broth. 19) It is noteworthy that 3 years before the discovery of 5, some carbasugars were chemically synthesized by McCasland and co-workers, 20  Particularly, O-valienaminylceramide ((E)-24) and its 4B-epimer (E)-25 ( Fig. 2a) have been shown to be potent and specific inhibitors of gluco-and galactocerebrosidase, respectively. 31) During the course of the synthetic studies, the structure-activity relationships of 21 and (E)-24 were investigated and have led to the development of the potent glycosidase inhibitor N-alkyl-carbaglycosylamines including N-octyl-O-valienamine (NOV) (26) 32) and its 4-epimer (NOEV) (27) 33) (Fig. 2b). To facilitate the accessibility of N-alkyl-carbaglycosylamines, an improved and practical synthetic route to chiral carbaglycosylamines from myo-inositol (4) has been established. 34) Both NOV (26) and NOEV (27) have later been found to possess chaperone activity for mutant forms of their target glycosidases. Since then, it has become of interest to develop carbaglycosylamine-based pharmacological chaperones for lysosomal glycosidases associated with lysosomal storage disorders (LSDs). 35)-37) Recently, the N-alkyl-conduramine F-4 derivatives have been identified as potential pharmacological chaperones for GM1-gangliosidosis, with the N-cyclohexylmethyl derivative 28 (Fig. 2c) being the most promising among them. 38), 39) 2. Total synthesis of N-linked carbaoligosaccharide ,-amylase inhibitors 2.1. Preparation of validamine-and valienamine-derived carbaglycosyl acceptors and donors for the construction of N-linked carbaoligosaccharides. A practical preparative method for constructing N-linked carbaoligosaccharides is the ring opening of carbasugar acceptor epoxides with appropriate donor amines, and vice versa, as demonstrated in the total synthesis of antibiotic validamycins including 5 and 6. 30) All the carbasugar derivatives used in the study can be prepared from a single starting compound, namely the Diels-Alder endo-adduct 29 of furan and acrylic acid (Fig. 3a), 40) which is now obtainable in both chiral forms, (D)-29 and (!)-29, by chiral resolution of racemic 29 with (!)-and (D)-1-phenylethylamines, respectively 41) ; thus, all the carbasugar derivatives described are available in both chiral forms. For the sake of simplicity, except noted otherwise, only the D-series of carbasugar derivatives derived from (!) 29 are shown in Figs. 3b and 4. The Diels-Alder adduct 29 was treated with hydrogen peroxide or bromine to give the lactones 30 or 31, respectively (Fig. 3b)  both of which were converted into the corresponding 1,4-anhydro-5a-carba-,-glucopyranose derivatives 32 and 33 by reductive opening of the lactone ring with lithium aluminum hydride and subsequent acetylation. 42) The anhydro rings of 32 and 33 were then cleaved with HBr/AcOH giving rise to the dibromocarbaglucose 34 and tribromocarbaglucose 35, respectively, which are versatile intermediates for the synthesis of various carbasugar derivatives including the validamine-and valienamine-derived acceptors and donors.
The preparation of the validamine-derived acceptor and donor starts from 34 (Fig. 4a). 43)-46) Treatment of 34 with an excess of sodium benzoate resulted in benzoate for the primary bromide substitution (! 36) and subsequent elimination of the secondary bromide giving rise to the cyclohexene 37. Because epoxidation of 37 with m-chloroperbenzoic acid (MCPBA) gave a mixture of nearly equal amounts of ,and O-epoxides 38, and 38O, the conformationally flexible 37 was first converted to the more rigid 39 by deacylation followed by benzylidenation and acetylation. The MCPBA epoxidation of 39 then proceeded stereoselectively yielding exclusively the acceptor O-epoxide 40. Notably, after our synthesis of 40, the strong O-glucosidase inhibitor cyclophellitol 47) (Fig. 4a, inset), which is the deprotected form of 40, was discovered from the fermentation broth of Phellinus sp. When 34 was treated with an equivalent amount of sodium benzoate, the primary benzoate 36 was preferentially formed; subsequent S N 2 substitution of the secondary bromide with an azide anion then yielded 41. After diacylation and di-O-isopropylidenation (! 42), the azido group was reduced to give the protected validamine 43 as the donor amine.
The dibromocarbaglucose 34 is also the starting point for the preparation of the valienamine-derived donors (Fig. 4b). 44 44,43) which was reacted with an equivalent amount of bromine in CCl 4 at !15°C to provide the 1,4-addition products 45, and 45O in a ratio of 1:2; the ratio was reversed to 3:1 when the bromination was carried out at room temperature. Selective replacement of the primary bromide in 45, with benzoate was accompanied by the epimerization of the allylic secondary bromide yielding an inseparable mixture of 46, and 46O, azidation of which led to a separable 1:1.7 mixture of 47, and 47O. After deacylation and di-O-isopropylidenation, the resulting azides 48, and 48O were reduced with H 2 S or PPh 3 to afford the protected valienamine 49, and O-valienamine 49O, respectively, as the donor amines. The efficient synthesis of 49, was later achieved from the spiro-epoxide 50 prepared by selective epoxidation of 44 with MCPBA 48) ; thus, opening of the epoxide ring in 50 with hydrochloric acid and subsequent acetylation (! 51) followed by S N 2B substitution with azide anion gave selectively the ,-azide 52, which was then converted to 49, in a similar manner as 47,.
The valienamine-derived acceptors are prepared from the tribromocarbaglucose 35 (Fig. 4c). 45) After vicinal debromination of 35 with zinc powder, the alkene 53 thus obtained was subjected to allylic bromination using a slight excess of N-bromosuccinimide (NBS) in CCl 4 , resulting in the formation of an equilibrium mixture of the allyl bromides 54 and 55 together with the cyclohexadiene 56 and the tribromocyclohexene 57. It seemed that both 54 and 55 tend to undergo dehydrobromination (! 56) followed by addition of bromine generated in situ by the reaction of NBS and HBr (! 57). Therefore, the reaction was carried out with an excess of NBS to convert 53 exclusively to 57. The primary bromide in 57 was then replaced with benzoate, and the resulting monobenzoate 58 was treated with zinc powder yielding the 1,3-cyclohexadiene 59. Regioselective epoxidation with MCPBA, followed by column chromatography separation, afforded 60, and 60O in a ratio of 1:1.5 as the valienamine-derived acceptor epoxides.
The first effort was made to construct oligobiosamine (23) (Fig. 5), 49) which is the biologically active core of oligostatins (20) and contains hydroxyvalidamine (10). The coupling reaction of the validamine-derived acceptor epoxide 40 (racemic) and the donor amine, methyl 4amino-4,6-dideoxy-,-D-glucopyranoside 50) (61), was carried out in propan-2-ol at 120°C for 70 h. After de-O-benzylidenation followed by acetylation and column chromatography, all four products formed were isolated and their structures and absolute configurations were determined using 1 H NMR analysis and specific rotations. As expected, the epoxide opening proceeded mainly in a trans-diaxial fashion to give Two other active cores, acarviosin (21) and 6hydroxyacarviosin (22), are composed of the valienamine (11) moiety. Therefore, a similar coupling reaction was investigated with the valienaminederived acceptor 60O (Fig. 6a). 45) When an equimolar mixture of 60O (racemic) and 61 was reacted in propan-2-ol at 120°C in a sealed tube, the ring opening occurred exclusively at the allylic position C-1 giving rise to the trans-diaxial products (D, D)-65 and (L, D)-65 which, after deprotection, yielded the ,-methyl glycoside of the 2B-epimer of 21, methyl 2B-epiacarviosin (D, D)-66 (46%) and its diastereomer (L, D)-66 (31%), respectively. 51) The attempted transformation of (D, D)-66 to 21 by inversion of the 2B-hydroxy group was unsuccessful due to the neighboring group participation of the nucleophilic imino group.
Two alternative routes to 21 were, therefore, investigated starting from other N-linked carbadisaccharide cores synthesized: (1) deoxygenation of the 6-hydroxy group of 70 (Fig. 7a) 51) and (2) introduction of the C5B-C6B double bond in 64 by dehydration of the 6B-hydroxy group (Fig. 7b). 53), 54) The first route began with the coupling product (D, D)-68. After protection of the 2-and 3-hydroxy groups by isopropylidenation (! 73), the remaining 6-hydroxy group was converted to p-toluenesulfonate (! 74) and replaced with iodide (! 75). Without the protection of the 3-hydroxy group, the iodide for p-toluenesulfonate substitution led to the predominant formation of the 3,6-anhydro ring by intramolecular attack of the 3-hydroxy group. The iodide 75 was treated with lithium triethylborohydride followed by acetylation to give 76, which, after deacetylation, yielded the ,-methyl glycoside of 21, methyl acarviosin (77). For the second route, selective protection of the 6B-and 7B-hydroxy groups in 64 was first carried out using an equivalent amount of 2,2-dimethoxypropane to yield, after acetylation, the desired 6B,7B-and the isomer 4B,7B-O-isopropylidene derivatives 78 and 79, respectively, in a ratio of 1:3. Removal of the isopropylidene group of 78 (! 80) was followed by chlorination with sulfuryl chloride affording the dichloride 81. Because selective substitution of the primary chloride with acetate was accompanied by aziridine formation (! 82), the chloro group was reinstalled at C6B by opening of the aziridine ring with chloride (! 83). The C5B-C6B double bond was then introduced by treatment of 83 with DBU providing 76, which was deacetylated to give 77.

Total synthesis of acarbose and related compounds.
The total synthesis of N-linked carbatrisaccharide inhibitors, amylostatin-XG (15) 55) and adiposin-1 (17), 56) was first accomplished ( Next, the total synthesis of N-linked carbatetrasaccharide inhibitors, acarbose (14) and adiposin-2 (18), was achieved using the protected chiral valienamine D-49, (Fig. 9). 58 derivative 91 increased the yield of the transdiequatorial product affording the desired 94 and the trans-diaxial product 95 in 33% and 21% isolated yields, respectively, of which 94 was converted to 18 by acetolysis and deacetylation. It is noteworthy that the role of the 6-hydroxy group in favoring the trans-diequatorial opening of the 3,4-anhydro ring, possibly through hydrogen bond formation to the epoxide oxygen, needs further investigation.
Although the coupling reaction is not fully optimized, the successful synthesis of N-linked carbaoligosaccharides would allow for the development of novel N-linked carbaoligosaccharide glycosidase inhibitors.
All four analogues were found to be slightly less active than 77 but were still potent inhibitors. Surprisingly, changing the conformation of the sugar moiety from 4 C 1 to 1 C 4 by 3,6-anhydro formation did not have any significant detrimental effect on the activity.
To gain further insight into the structural requirement of the sugar moiety, the 1,6-anhydride of 6-hydroxyacarviosin (22), namely 116a, and its several derivatives 116b-h were synthesized (Fig. 10b) and their inhibitory activities were compared with that of 77 (Table 1b) and deprotection afforded the 2-amino derivative 116c, whereas the reduction followed by N-acetylation and deprotection yielded the 2-acetamido derivative 116d. The mono-and dideoxy derivatives (116g and 116h, respectively) were synthesized by coupling of D-49, with the 2-thioether 110, prepared by S N 2 displacement of the 2-trifluoromesylate in 107 by p-toluenethiolate, and subsequent desulfurization of the product 115 with Raney-nickel followed by deprotection. The ratio of the two products 116g and 116h was 1.4:1; the dideoxy derivative 116h was presumed to be formed through the intermediate episulfide.
As in the case of the 3,6-anhydro derivative 104, the 1 C 4 conformation of the 1,6-anhydro sugar moiety did not have any negative effect on the inhibitory activity. The 1,6-anhydride 116a, its 2-epimer 116f, and its 2-azido and 2-fluoro derivatives (116b and 116e, respectively) showed similar activities to 77, but the 2-amino derivative 116c and, particularly, the 2-acetamide derivative 116d were significantly less active. The most interesting finding was the increased activity of the deoxy derivatives: both mono-and dideoxy-derivatives (116g and 116h, respectively) were nearly 10 times more active than 77.
The X-ray crystallographic analysis of the acarbose (14) and glucoamylase complex was later published by Aleshin et al., 64) revealing: (1) an extensive hydrogen bond network between the valienamine moiety and the surrounding amino acids; (2) a salt bridge between the imino linkage and the putative catalytic Glu; and (3) the hydrophobic interactions between the hydrophobic faces of the sugar moieties and the Tyr/Trp at the binding site. The strong interaction of the valienamine moiety with the enzyme active site probably contributes to the potent activity of the synthetic analogues of 77 except 116c and 116d. The poor activity of these two analogues may be attributed to the disruption of the salt bridge interaction by the amino/acetamido group being 1,3-diaxially oriented to the imino linkage. The hydrophobic amino acids present in the sugar binding site may also explain the higher activity of the deoxy analogues 116g and 116h than 77. These results seemed to clearly suggest that the sugar moiety of 77 may be replaced by simple alkyl groups without impairing the inhibitory potency. Kameda et al. 65) reported similar findings based on their semi-synthetic studies on valienamine-containing ,-glycosidase inhibitors, in which several N-alkyl and N-aralkyl valienamines were synthesized from valienamine (11)   microbial degradation of validamycin A (5), and the findings were used to guide the development of voglibose 66) (Fig. 10b, inset), which is an N-substituted derivative of valiolamine (12) and a practical alternative to 14 for the treatment of type 2 diabetes mellitus.

Synthesis of novel carbaglycosylaminebased O-glycosidase inhibitors
Several valienamine-type carbaglycosylamine derivatives available in our laboratory were exploited to develop new transition-state analogue inhibitors of O-glycosidases.

Synthesis of O-valienaminylceramide and 4-epi-O-valienaminylceramide.
Glucocerebrosidase and galactocerebrosidase are the lysosomal O-glucosidase and O-galactosidase, respectively, and involved in glycolipid metabolism through hydrolysis of glucosyl-and galactosylceramide, respectively. It was reasoned that replacing the carbohydrate moiety of glucosylceramide and galactosylceramide with an appropriate valienamine derivative may provide a transition-state analogue inhibitor of the respective glycosylceramidase. 31) The ring opening of sphingosine-derived aziridines with proper carbaglycosylamines was found to be effective for the synthesis of N-carbaglycosylceramides. The protected aziridine (E)-129 was prepared from the known azidosphingosine (E)-126 69)-71) in three steps (Fig. 12a): (1)  selective benzoylation of the primary hydroxy group (! (E)-127); (2) protection of the residual hydroxy group as a tert-butyldimethylsilyl ether followed by debenzoylation (! (E)-128); and (3) aziridine formation by reduction of the azide with triphenylphosphine and subsequent protection of the aziridine with a 2,4-dinitrophenyl group (! (E)-129). The aziridine product (E)-129 was formed in 39% yield together with the amino alcohol (E)-130 (45%), which was readily converted to (E)-129 (86%) by iodination (! 131) followed by reductive cyclization. The isomer (Z)-129 was similarly prepared from the (Z)-126. The 4-epi-O-valienamine donor 136 was prepared from the precursor of the O-valienamine donor D-47O (Fig. 12b) by epimerization of the 4-hydroxy group, after a series of protecting group manipulations including deisopropylidenation (! 132), benzylidenation and subsequent methoxymethylation (! 133), and debenzylidenation followed by selective protection of the primary hydroxy group with tetrabutyldimethyl-silyl ether (! 134 ! 135). Epimerization at the 4 position was then achieved by oxidation of the 4 hydroxy group of 135 with pyridinium chlorochlomate, followed by reduction with diisopropylaluminum hydride, which also reduced the azido group, to give 136.
Their inhibitory activities were tested against mouse liver glucocerebrosidase and galactocerebrosidase ( Table 3). As expected, (E)-24 and (E)-25 were potent and selective inhibitors of glucocerebrosidase and galactocerebrosidase, respectively. Notably, their isomers (Z)-24 and (Z)-25 were also found to be potent and selective inhibitors. Both glucocerebrosidase and galactocerebrosidase appear to have a rather broad specificity for the ceramide moiety.   that the ceramide moiety of (E)-24 and (E)-25 can be replaced by simple alkyl chains without compromising their activities. Thus, several N-alkyl derivatives of O-valienamine were first prepared by Nacylation of the protected O-valienamine D-49O with n-alkanoyl chlorides (! 139a-e) followed by lithium aluminum hydride reduction (! 140a-e) and deprotection (! 141a-e) (Fig. 13a) and evaluated for their inhibitory activity against mouse liver glucocerebrosidase (Table 4a). 32) All the N-alkyl derivatives, except for the shortest alkyl chain derivative 141a, showed similar or better activity than (E)-24. The  potency seemed to depend on the length of the Nalkyl chain: the potency increased rapidly from C 4 (141a) to C 6 (141b) and to C 8 (26) and then decreased gradually for C 10 (141c), C 14 (141d) and C 18 (141e). The most potent C 8 derivative, NOV (26), exhibited 10-fold greater activity than (E)-24. Accordingly, the C 8 derivative of 4-epi-O-valienamine, NOEV (27), was similarly synthesized from the protected 4-epi-O-valienamine 136 and n-octanoyl chloride (Fig. 13b) and tested for its inhibitory activity against mouse liver galactocerebrosidase as well as bovine liver O-galactosidase (Table 4b). 33) Of note, NOEV (27) was found to be only a moderate inhibitor of galactocerebrosidase but rather a potent inhibitor of O-galactosidase. Moreover, it was later discovered that NOEV is a potent inhibitor of human lysosomal O-galactosidase responsible for the hydrolysis of the terminal O-galactose residue from GM1ganglioside. 35) The potent O-galactosidase inhibitory activity of (E)-25 and NOEV (27) spawned an increased interest in the N-substituted 4-epi-O-valienamine and, therefore, a more straightforward synthesis of a suitable 4-epi-O-valienamine donor has been developed starting from the tribromocarbaglucose L-35 derived from the Diels-Alder adduct (D)-29 (Fig. 14). 37),72),73) Treatment of 35 with sodium methoxide gave after acetylation the epoxide 142, which was subjected to acid hydrolysis followed by acetylation providing the dibromocarbaaltrose 143. Following the same sequence of reactions described for the preparation of the valienamine-donors 49,,O from 45,,O (Fig. 4b), the carbaaltrose derivative 143 was converted to the 4-epi-O-valienamine donor 149O and its ,-isomer 149, via the conjugated alkene 144, the 1,4-conjugate addition products 145,,O, the primary benzoates 146,,O, the allyl azides 147, and 147O (at a ratio of ca. 1:1; 50%), and the di-O-isopropylidene derivatives 148, and 148O. Furthermore, after protecting group manipulation of 144 (deacetylation followed by isopropylidenation and acetylation), the resulting 150 was similarly converted to a mixture of the allyl bromides 151,,O and then to a mixture of 152,,O, which then reacted with alkylamines, providing selectively O-amine 153. 73) The alkylamination of 152O likely proceeded via a cyclic acetoxonium ion. 71) After deprotection, NOEV (27) and 154a-c thus obtained were evaluated for their inhibitory activity (Table 5). Both N-decyl and N-dodecyl derivatives (154b and 154c, respectively) were found to be more potent O-galactosidase inhibitor than NOEV (27). Notably, the most potent inhibitor 154c was also a potent inhibitor of O-glucosidase. Pharmacological chaperone therapy 76) is an emerging approach to treat LSDs using smallmolecule ligands that specifically bind and stabilize mutant enzymes, thereby facilitating proper folding and thus improving lysosomal trafficking and activities. 77),78) Pharmacological chaperone therapy is particularly advantageous for the treatment of LSDs affecting the central nervous system, because of the potential of small-molecule ligands to cross the blood-brain barrier. As such, reversible compet-itive inhibitors are obvious candidates for the development of specific pharmacological chaperones. In 1999 the imino sugar 1-deoxygalactonojirimycin (migalastat), an ,-galactosidase inhibitor, was shown to function as a pharmacological chaperone for Fabry disease, which is caused by a deficiency in ,galactosidase A activity and subsequent accumulation of the substrate globotriosylceramide. 79) Its hydrochloride has been approved for the oral treatment of some variants of Fabry disease in several countries including the European Union, U.S.A., and Japan. 80)

Discovery of chaperone activities of NOV and NOEV.
Gaucher disease and GM1gangliosidosis are LSDs caused by deficiencies of lysosomal glucocerebrosidase and O-galactosidase, respectively, and characterized by the intracellular accumulation of the corresponding substrates, glucosylceramide and GM1-ganglioside. 81) The selective inhibitors of these enzymes are potential candidates for the development of specific pharmacological chaperones: the glucocerebrosidase inhibitor NOV (26) (see Fig. 2b) for Gaucher disease and the human O-galactosidase inhibitor NOEV (27) (see Fig. 2b) for GM1-gangliosidosis.
The increased activity of the mutant enzyme due to the addition of 26, namely chaperone activity of 26, was evaluated in fibroblasts expressing F213I mutant glucocerebrosidase, which is a common Table 5. Inhibitory activity of NOEV (27) (Table 6a). 82),83) After incubation with 30 µM 26, the maximum enhancement of enzyme activity (96-fold compared to without 26) was observed together with lysosomal localization of the mutant enzyme and intracellular clearance of the substrate glucosylceramide. 82) This finding was rather surprising, considering that the IC 50 of 26 for wild-type human glucocerebrosidase was found to be 3 µM. Of note, a similar enhancement of activity was reproduced by 3 µM of the hydrochloride of 26 probably due to its increased water solubility. 83) In addition to the F213I mutant, the hydrochloride of 26 (3 and 30 µM) was effective against other mutant glucocerebrosidases, including N188S, G202R, and N370S (each 92-fold in an ex vivo enzyme assay), but not against the D409H and L444P mutants. The potent human O-galactosidase inhibitor 27 (IC 50 F 0.2 µM) was evaluated for its chaperone activity in fibroblasts expressing R201C mutant Ogalactosidase causing juvenile GM1-gangliosidosis (Table 6b). 35) Incubation with 0.2 µM 27 resulted in the maximum enzyme activity (5.1-fold compared to without 27) as well as intracellular reduction of the substrate GM1-ganglioside. The enhancement of activity was also observed in fibroblasts expressing other mutant O-galactosidases including R201H (4.5fold), R457Q (2.4-fold), W273L (2.2-fold), and Y83H (2.0-fold). An animal study was also performed using GM1-gangliosidosis model mice expressing human R201C mutant O-galactosidase. The oral administration of 27 enhanced the mutant enzyme activity and decreased GM1-ganglioside levels in neuronal cells in the brain.
Computational analysis of the interaction of 26 84) and 27 85) with their target enzymes indicated that their binding free energies were higher at pH 5 (lysosome) than at pH 7 (ER) because of the reduced number of hydrogen bonds caused by protonation of functional residues in the enzyme active site. Thus, both 26 and 27 bind to the respective mutant enzymes stabilizing them in the ER and dissociate from them thus restoring their activity in the lysosome, which is consistent with the observed chaperone activity of 26 and 27.
4.3. Practical synthesis of NOV and NOEV from quercitols. The initial synthesis of NOV (26) and NOEV (27) began with chiral resolution of the racemic Diels-Alder adduct of furan and acrylic acid 29 into (!)-29 by crystallization with (D)-1-phenylethylamine (Fig. 3a), which is rather cumbersome and time-consuming and, moreover, is not atomeconomical because the other enantiomer (D)-29 is not used. In order to facilitate the advancement of carbaglycosylamine-based glycosidase inhibitors as well as pharmacological chaperones, a more practical synthesis from readily available chiral compounds was investigated.
The first step in the practical synthesis of 26 from 155 involves selective oxidation of the axial hydroxy group in 155, which was achieved by means of bio-oxidation (Fig. 15b). 88) Thus, an aqueous solution of 155 was incubated with Gluconobacter sp. at ambient temperature for 1 day to give, after purification by cation-and anion-exchange chromatography, (!)-2-deoxy-scyllo-inosose (157) 90) in practically quantitative yield. Subsequent acetylation of 157 in acidic conditions was accompanied by O-elimination yielding the ,,O-unsaturated ketone 158 in 83% yield. exo-Methylenation was then carried out using acidic Nysted reagent to afford the conjugated alkene 44 but in lower yield (25%). Due to the base sensitivity of 158, conventional Wittig olefination was not applicable. From this point, the synthesis was able to follow the initial synthesis of 26: 44 ! 49 (Fig. 4b) and 49 ! 26 (Fig. 13a). Here, a more straightforward approach 73) was used: after 1,4-dibromination of 44 to 45,,O, the primary bromide was replaced with acetate and the resulting mixture of allyl bromides 159,,O was reacted with n-octylamine yielding 26 as a sole product in 31% yield. The reaction of 159O likely proceeded via a cyclic acetoxonium ion.
For the practical synthesis of 27 from 156, isopropylidenation of 156 was first carried out to give the diisopropylidene derivative 160 (Fig. 15c). 89) The residual hydroxy group was subsequently oxidized with SO 3 (27) have shown promising chaperone activity, yet their potent inhibitory activity might be counterproductive. Therefore, special considerations are necessary to maximize the stabilizing effect and to minimize the inhibitory activity. Mena-Barragán et al. 92) have devised sp 2 -iminosugar inhibitors bearing pH-sensitive hydrophobic tails by incorporating an orthoester linker; thus, they are strong inhibitors of human glucocerebrosidase or ,-galactosidase at pH 7 (in the ER) but become non/less-inhibitory at pH 5 (in the lysosome) due to the loss of the hydrophobic tails, which are necessary for the inhibitory activity, by hydrolysis of the acid-labile orthoester linker.
Suzuki et al. 93) reported a comparative crystallographic analysis of human O-galactosidase in complex with 27 and sp 2 -iminosugar-type inhibitors, revealing the major interactions of 27 with the enzyme: (1) the hydrogen bond interactions involving the 2-, 3-and 4-hydroxy groups are crucial for the specific binding to the enzyme; (2) the salt bridge between the exocyclic nitrogen and the catalytic Glu is also crucial for the binding and specificity; and (3) the hydrogen bond and hydrophobic interactions involving the 6-hydroxy and n-octyl groups, respectively, are important for the stabilization of the complex. Based on these findings, in order to reduce the inhibitory activity of 27 while maintaining a certain affinity to the enzyme for the chaperone activity, modification of the interactions that stabilize the complex appeared to be a rational approach. Therefore, the 6-deoxy and dehydroxymethyl derivatives of 27 (170 and 175, respectively) were first prepared to eliminate the hydrogen bond interaction involving the 6-hydroxy group.
The 6-deoxy derivative 170 was synthesized from a mixture of the 1,4-dibromides 164,,O (Fig. 16a). 89) Selective reduction of the primary bromide with NaBH 4 yielded the mono bromides 168, and 168O in 68% and 23% yields, respectively. The ,-bromide 168, was then debenzoylated to give 169 (51%), which was subjected to S N 2 replacement of the ,-bromide by n-octylamine and subsequent de-O-isopropylidenation to afford 170 in 90% yield. The dehydroxymethyl derivative 175, on the other hand, was synthesized from the di-O-isopropylidene derivative 160 (Fig. 16b). 38) Mesylation of the hydroxy group (! 171) followed by E2 elimination of the mesylate using DBU furnished the cyclohexenetetrol (known as conduritol F) derivative 172 in 76% yield. After selective removal of the trans-O-isopropylidene group with weak acid, the diol 173 thus obtained was treated with either Martin sulfurane or Mitsunobu reagent to give the ,-epoxide 174 as the sole product in 69% and 59% yields, respectively. The epoxide ring opening with n-octylamine proceeded regio-and stereo-selectively to yield, after acidic removal of the isoproprylidene group, the hydrochloride of 175 (N-octyl-conduramine F-4) quantitatively.
The hydrochlorides of 27, 170, and 175 were evaluated for their inhibitory activity against human wild-type O-galactosidase, and their chaperone activity was assessed using fibroblasts expressing R201C mutant O-galactosidase (Table 7). 38) Both 170, and 175 indeed showed much lower inhibitory activity than 27; the IC 50 values of 170 and 175 were 27and 71-fold higher than that of 27, respectively. The higher IC 50 values of 175 than 170 may be attributed to the 5-methyl group in 170, which might be involved in a hydrophobic interaction with the enzyme. Notably, the chaperone activities of 170 and 175 were slightly higher than that of 27.
Next, several N-alkyl derivatives of conduramine F-4 were prepared similarly to the N-octyl derivative 175 using the corresponding alkylamines (Table 8). 38) Because the strength of hydrophobic interactions depends on the size and shape of the hydrophobic molecules, different types of alkylamines were selected including various linear and branched amines.
Linear alkyl groups shorter or longer than C 8 led to slightly more active inhibitors than 175; meanwhile, the chaperone activity increased gradual-ly with the length of the alkyl chain up to C 8 (Table 8a). It should be noted that the C 10 derivative showed high cytotoxicity to fibroblasts at 20 µM, probably due to its cationic surfactant character; therefore, linear alkyl groups longer than C 10 were not investigated.
The binding affinity was significantly impaired when the linear alkyl group was replaced by either hydroxypropyl, 1-ethylpropyl, or cyclohexyl group (Table 8b). The hydrophilic hydroxypropyl group obviously did not form the hydrophobic interaction necessary for binding. On the other hand, both the 1-ethylpropyl and cyclohexyl groups are attached to the nitrogen atom through their secondary carbons; thus, they create more steric hindrance around the nitrogen, which may interfere with the formation of the salt bridge between the NH and the catalytic Glu. Indeed, reduction of the steric hindrance by insertion of a CH 2 group between the nitrogen and the secondary carbon, namely the N-2-ethylbutyl and N-cyclohexylmethyl derivatives 176 and 28, respectively, restored the binding affinity (Table 8c). Of note, replacement of the terminal 3-pentyl group in 176 with an isopropyl group and the terminal cyclohexyl group in 28 with a phenyl group resulted in weaker inhibitory and chaperone activities, which  were improved to some degree by insertion of an additional CH 2 group. Among the N-alkyl derivatives synthesized, the N-cyclohexylmethyl-conduramine F-4 (28) showed the best activity profile for a potential pharmacological chaperone with moderate inhibitory activity (IC 50 F 60 µM) and the highest chaperone activity (8.5-fold activity enhancement). In addition, the N-2-ethylbutyl derivative 176 had the second-best activity profile with IC 50 of 15 µM and 7.4-fold activity enhancement. Further in vivo studies will be needed to confirm the promising chaperone activity of 28 as well as 176.

Prospects
It is now well appreciated that carbohydrates are involved in cellular communication and interaction essential for physiological and pathological events. Despite the important role carbohydrates play, the molecular basis of carbohydrate-mediated recognition processes remains poorly understood. This is in part caused by their structural complexity and diversity, but it is also because their structures are, unlike those of nucleic acids and proteins, generated in a non-template driven manner by various glycosyltransferases and glycosidases. In this context, carbohydrate mimetics that selectively interfere with these carbohydrate-processing enzymes as well as carbohydrate-binding proteins are useful as molecular probes to understand the structurefunction relationships of carbohydrates. Among the carbohydrate mimetics, carbasugars and their derivatives are of immense value because of their ability to mimic the oxocarbenium ion-like transition state of glycosidase-catalyzed hydrolysis in addition to the substrates of glycosyltransferases and the ligands of carbohydrate-binding proteins.
In the course of the total synthesis of naturally occurring N-linked carbaoligosaccharides, including the amylase inhibitor acarbose and related compounds, a general strategy has been established to link carbasugars to carbohydrates via an imino linkage by epoxide ring opening of either carbasugar epoxides with amino carbohydrates or carbohydrate epoxides with carbaglycosylamines. In addition, the synthesis of chiral carbasugars has been substantially improved with the ready availability of quercitols through the bioconversion of myo-inositol in conjunction with the well-established carbasugar synthesis from the Diels-Alder endo-adducts of furan and acrylic acid. This practical approach greatly facilitates the availability of carbasugars and their derivatives of interest, including carbaglycosylamines and N-linked carbaoligosaccharides, for their applications in glycobiology.
Several carbohydrate-processing enzymes and carbohydrate-binding proteins have been recognized as potential targets for therapeutic interventions. Therefore, carbasugars and their derivatives are also useful for the development of therapeutics against these targets. We were pleased to find the chaperone activity of the carbaglycosylamine-based transition-state analogue inhibitors NOV and NOEV; moreover, chemical modification of NOEV has led to the identification of N-cyclohexylmethylconduramine F-4 as a potential pharmacological chaperone for GM1-gangliosidosis related human lysosomal O-galactosidase.
Given their ready availability through synthesis, carbasugars and their derivatives now hold great promise for applications in glycobiology and therapeutics.

Acknowledgements
Our synthetic journey towards carbaglycosylamine glycosidase inhibitors described in this review would not have been possible without the hard work and dedication of students as well as the valuable suggestions of colleagues and collaborators, whose names appear on the cited references. SO is particularly grateful to have had such outstanding students in his laboratory at Keio University.
When we started our synthetic journey, we had no idea it would lead to the development of potential pharmacological chaperones. The outcome of research is indeed unpredictable, but for this reason research is interesting and worth conducting. It is important to focus and execute your research with care and confidence and see what it leads to. Enjoy your journey!