Conformationally-Locked C-Glycosides: Tuning Aglycone Interactions for Optimal Cheperone Behaviour in Gaucher Fibroblasts

Organic & Biomolecular Chemistry RSCPublishing ARTICLE This journal is © The Royal Society of Chemistry 2015 Org. Biomol. Chem., 2015, 00, 1-3 | 1 Cite this: DOI: 10.1039/x0xx00000x Received 00th February 2015, Accepted 00th February 2015 DOI: 10.1039/x0xx00000x www.rsc.org/ Conformationally-Locked C-Glycosides: Tuning Aglycone Interactions for Optimal Cheperone Behaviour in Gaucher Fibroblasts C. D. Navo,a F. Corzana,a E. M. Sánchez-Fernández,b J. H. Busto,a A. Avenoza,a E. Namba,c K. Higaki,c C. Ortiz Mellet,b* J. M. García Fernándezd* and J. M. Peregrinaa* A series of conformationally locked C-glycosides based on the 3-aminopyrano[3,2-b]pyrrol2(1H)-one (APP) scaffold has been synthesized. The key step involved a totally stereocontrolled C-Michael addition of a serine-equivalent C-nucleophile to tri-O-benzyl-2nitroD -galactal. Stereoselective transformations of the corresponding Michael adduct allowed us the synthesis of compounds with monoor diantennated aglycone moieties and different topologies. In vitro screening showed highly selective inhibition of bovine liver βglucosidase/-galactosidase and specific inhibition of human -glucocerebrosidase among lysosomal glycosidases for compounds bearing palmitoyl chains in the aglycone, with a marked dependence of the inhibition potency upon their number and location. Molecular dynamics simulations highlighted the paramount importance of an optimal orientation of the hydrophobic substituent to warrant efficient non-glycone interactions, which are critical for the binding affinity. The results further provide a rational for the strong decrease of the inhibiton potency of APP compounds on going from neutral to acidic pH. The best candidate was found to behave as a chaperone in Gacuehr fibroblasts with homozygous N370S and F213I mutations, with enzyme activity enhancements similar to those encountered for the reference compound Ambroxol®. Introduction Glycosidases are enzymes that catalyze the hydrolysis of glycosidic bonds, thereby playing a key role in biochemical routes that are essential for life such as the processing of glycoproteins and the catabolism of polysaccharides and glycoconjugates. Deregulation of any of these enzymes generally translates into significant biological and pathological consequences, which has fuelled research in compounds interfering with their activity as potential drug candidates. 1 The carbohydrate mimics (glycomimetics) of the iminosugar type, such as the piperidine derivative 1-deoxynojirimicyn (DNJ) or the indolizidine analogue castanospermine (CS, Figure 1), represent by far the most studied family of molecules towards these channels. 2 Their polyhydroxylated structure emulates that of the natural monosaccharides, enabling them to compete with the substrate for the glycone pocket in the active site of glycosidases, behaving as competitive inhibitors..However, classical iminosugars cannot fully reproduce the structure of the putative glycoside substrates due to the instability of aminoacetal functional groups, which often translates into poor selectivity. Even though rigidification of the azaheterocyclic core in bicyclic derivatives can be exploited to improve the discrimination capacity between isoenzymes, the absence of a defined anomeric configuration generally results in simultaneous inhibition of glycosidases acting on anomeric substrates, i.e. - and -glucosidases in the case of DNJ and CS, which represents a serious handicap for clinical applications. 3 The urgent need for more specific glycosidase inhibitors has fuelled research on synthetic glycomimetics targeting medically relevant enzymes. 4,5. In principle, molecular prototypes mirroring the full structure of glycosides, that is incorporating both glyconeand aglycone-like portions and benefiting from a defined pseudoanomeric configuration, are better suited for that purpose. 6 For example, sp 2 -iminosugar cores, 7 in which the endocyclic nitrogen is part of a pseudoamide functionality, are compatible with the incorporation of axially-oriented pseudoanomeric substituents through stable O-, Sor N- ARTICLE Organic & Biomolecular Chemistry 2 | Org. Biomol. Chem., 2015, 00, 1-3 This journal is © The Royal Society of Chemistry 2015 glycosidic linkages (Figure 1), which has been exploited in the design of specific -glucosidase inhibitors exhibiting anticancer ad antileishmanial activities. 8 N-alkylated bicyclic isoureas derived from aminocyclitol scaffolds (e.g., benzo[d]oxazolimine derivatives, Figure 1) behaved instead as very selective inhibitors of human lysosomal -glucosidase, exhibiting strong pharmacological chaperone potential for the treatment of Gaucher disease. 9 Interestingly, some examples of glycosidase inhibitors keeping the pyranose ring as the glycone constituent have also been reported, which presents advantages in term of synthetic procedures. An example is thiamet G (Figure 1), a fused pyranose-thiazoline derivative, that acts as a potent inhibitor of O-linked 2-acetamino-2-deoxy-βD - glucopyranoside hydrolysis currently in preclinical evaluation as a potential therapeutic for the treatment of Alzheimer’s disease. 10 The conformationally-locked bicyclic N-glycoside derivatives of the pyranose-sulfanyl-1,3-oxazoline family (PSO, Figure 1) exhibit β-glucosidase inhibitory activity that is strongly dependent on the nature of the psudoaglycone Ssubstituent, some representatives behaving as pharmacological chaperones for Gaucher disease. 11 Figure 1. Some iminosugars and bicyclic derivatives glycosidase inhibitors. The pyrano[3,2-b]pyrrole framework (Figure 1) has also been suggested as a suitable scaffold for the preparation of glycosidase inhibitors. 12 The lack of effective synthetic methods compatible with the incorporation of aglycone-type susbtituents has burdened optimization of the affinity and selectivity towards biomedically relevant enzyme targets, however. 13 The development of efficient approaches towards this framework through synthetic routes compatible with structurally diversity-oriented strategy is therefore highly wanted. The possibility of accessing conformationallyrestricted multiantennated derivatives with C-glycoside structure is particularly appealing 14 given that many medically relevant glycosidases, such as those that are dysfunctional in glycosphingolipid-related metabolic disorders, act on substrates with branched aglycone segments. 15 With this background, we have now envisioned the synthesis of 3a mino-6,7-dihydroxy-3,5bis(hydroxymethyl)hexahydro p yrano[3,2-b] p yrrol-2(1H)-one derivatives (APP, Figure 1) as a new family of non-iminosugartype glycosidase inhibitors. The disubstitution pattern at the exocyclic quaternary anomeric carbon, which bears simultaneously a hydroxymethyl and an amino group susceptible of chemical modification, offer broad opportunities for the incorporation of non-glycone moieties. The optimization of the synthesis of a key synthetic precursor for APP synthesis, the subsequent transformation into a broad battery of monoand diantennated APP derivatives and the assessment of the affinity and selectivity of the final compounds against a panel of commercial and human glycosidase enzymes is presented. Results and discussion Recently, a totally stereocontrolled C-Michael addition of a serine-equivalent C-nucleophile to tri-O-benzyl-2-nitroD - galactal (nitrogalactal) was used as the key step to synthesize several pyrano[3,2-b]pyrrole structures, e.g. compound 1 , following reduction of the nitro group in the corresponding Michael adduct. 16 Interestingly, spirocycle 1 can be regarded as a protected form of the target APP derivatives (Scheme 1). To explore this channel, an optimized preparation of 1 in gram scale was a prerequisite. This goal was conveniently achieved by using platinized Raney Ni under an atmosphere of hydrogen 17 in the reduction step. The transient amino group reacted spontaneously with the methyl ester group to close the corresponding five-membered lactam ring with retention of the configuration at both stereocenters. Scheme 1. Retrosynthetic analysis of APP derivatives. Compound 1 was next used as a precursor for the synthesis of a battery of APP derivatives. In a first step, concomitant hydrolysis of the acetal and cyclic carbamate groups, by Journal Name ARTICLE This journal is © The Royal Society of Chemistry 2012 Org. Biomol. Chem., 2015, 00, 1-3 | 3 treatment with 4 M aqueous HCl at 40 °C for 12 h and liberation of the resulting ammonium hydrochloride salt by the action of propylene oxide in ethanol at reflux, provided aminoalcohol 2 in excellent yield (96%). Acylation of 2 with palmitoyl chloride in the presence of trimethylamine at room temperature for 12 h afforded the diantennated derivative 3 (70% yield), which was subsequently de-O-benzylated by hydrogenolysis in acidified methanol, using palladium on carbon as a catalyst ,to give the unprotected APP 4 in only three steps and 62% overall yield (Scheme 2). Scheme 2. Synthesis of target compounds 4 , 6 , 8 and 11 incorporating palmitoyl subtituents. Compound 3 was also selectively de-O-acylated with sodium methoxide in methanol at pH 9 to give the corresponding alcohol 5 , which after debenzylation following the above procedure afforded the corresponding APP derivative 6 , bearing a single-chain N-palmitoyl aglycone moiety (Scheme 2). The homologous O-palmitoyl monantennated adduct 8 , keeping a free amino group in the aglycone, was obtained from the pivotal precursor 1 after acid hydrolysis, in situ regioselective O-acylation with palmitoyl chloride, neutralization with propylene oxide in ethanol and hydrogenolysis of the tribenzyl ether 7 (62% overall yield; Scheme 2). The possibility of conducting chemoselective acylation of the masked hydroxy and amino groups in 1 is further compatible with the incorporation of different substituent at each position. Thus, palmitoylation of the previously reported N-acetylated compound 9 16 followed by hydrogenolysis of the resulting ester 10 led to the new APP derivative 11 (Scheme 2). Finally, compounds 13 - 15 , featuring O-acetyl and a free amino groups, free hydroxyl and amino groups, or free hydroxy and N-acetyl groups in the aglycone segment, respectively were prepared as controls to assess the influence of the presence of the long hydrophobic segments in the glycosidase inhibitory properties of the PAA (Scheme 3). ARTICLE Organic & Biomolecular Chemistry 4 | Org. Biomol. Chem., 2015, 00, 1-3 This journal is © The Royal Society of Chemistry 2015 Scheme 3. Synthesis of target compounds 13 - 15 without large alkyl chains. Compounds 4 , 6 , 8 , 11 , 13 , 14 and 15 were first evaluated against a panel of commercially available glycosidase enzymes including -glucosidase (baker yeast), isomaltase (baker yeast), amyloglucosidase (A. niger), -glucosidase/-galactosidase (bovine liver), -galactosidase (E. coli), -galactosidase (green coffee), -glucosidase (almonds), -mannosidase (Jack bean), -mannosidase (H. pomatia) and -N-acetylglucosaminidase (human placenta, bovine kidney and Jack bean). Inhibition constants (K i ,  M ) were determined from the slope of Lineweaver-Burk plots (see Figure 2 for a representative example) and are collected in Table 1. The data confirm that the presence of a hydrophobic segment in the aglycone ( 4 , 6 , 8 and 11 ) is necessary to elicit a significant activity, isomaltase (K i , 13-80  M ) and -glucosidase/-galactosidase (K i , 5.8-86  M ) being the more sensitive enzymes to APP inhibition. The presence of a bulky substituent at the nitrogen atom ( 4 and 6 ) is detrimental for isomaltase inhibition, whereas it has the opposite effect in the case of the mammalian -glucosidase/- galactosidase enzyme. Compound 8 , keeping a free amino group in the aglycone, additionally behaves as a  M inhibitor of -glucosidase (K i , 27  M ) and -galactosidase (K i , 67  M ) Table 1. Glycosidase inhibitory activities (K i ,  M ) for APP derivatives against commercial glycosidases. [a] [a] Inhibition, when detected, was competitive in all cases. No inhibition was detected for any of the compounds at concentrations up to 2 mM against amyloglucosidase (A. niger),  -glucosidase (almonds),  -galactosidase (E. coli),  -mannosidase (Jack bean),  -mannosidase (H. pomatia) and  -N-acetylglucosaminidase (human placenta, bovine kidney and Jack bean). [b] Determined from the slope of Lineweaver_Burk Plots and Double Reciprocal Analysis. n.i. not inhibition detected at concentrations up to 2 mM. Inhibition of bovine liver β-glucosidase/-galactosidase has been often used as a preliminary parameter to select candidates as pharmacological chaperones for mutant forms of human βglucocerebrosidase (GCase) associated with Gaucher disease. 18 Although the predictive character of the data must be taken with care, 19 the significant inhibitory potential and high selectivity encountered for some of the new APP compounds in Table 1 against the commercial enzyme warranted further evaluation in this sense. The concentrations of the di- ( 4 ) and monoantennated derivatives ( 6 , 8 and 11 ) given 50% inhibition of human GCase are collected in Table 2. The corresponding data for the non-glycomimetic type pharmacological chaperone Ambroxol® (ABX), currently in preclinical studies for the treatment of Gaucher disease, 20 are also included for comparative purposes. Enzymes 4 6 8 11 13 14 15  -Glucosidase (Baker yeast) 225±25 124±13 27±3 244±28 n.i. 532±58 n.i. Isomaltase (Baker yeast) 80±9 34±4 13±1 19±2 97±11 283±31 n.i.  -Glucosidase/  -Galactosidase (Bovine liver) 10±1 [b] 5.8±0.6 [b] 86±9 9.2±8 [b] n.i. n.i. n.i.  -Galactosidase (Green coffee) 657±72 515±56 67±7 n.i. n.i. n.i. n.i. Journal Name ARTICLE This journal is © The Royal Society of Chemistry 2012 Org. Biomol. Chem., 2015, 00, 1-3 | 5 Figure 2. Lineweaver-Burk plot for Ki determination (5.8  M ) of APP 6 against  -galactosidase (bovine liver) (pH 7.3). Table 2. IC 50 values (M) against human GCase for APP derivatives 4 , 6 , 8 and 11 . Compound GCase pH 7 GCase pH 5 41±1 470±20 23±1 272±10 49±2 606±10 67±5 707±20 7.6±0.5 90±5 The inhibition potency against GCase followed a slightly different trend as compared to that encountered for the commercial mammalian β-glucosidase/-galactosidase. The presence of the O-acyl substituent in the aglycone seems to be detrimental for the human enzyme. Thus, compound 6 (IC 50 23 M at pH 7), missing this group, is an about 2to 3-fold stronger inhibitor than the three other APP derivatives assayed (IC 50 41-67 M at pH 7), though it is still 3-fold weaker than the reference compound ABX (7.6 M). No inhibition of other lysosomal enzymes, such as -glucosidase, -galactosidase, - galactosidase, and -hexosaminidase, was observed. Most interestingly, an about one-order-of-magnitude decrease in the GCase inhibition strength was observed at pH 5, a favorable feature for chaperone candidates. 21 The ensemble of data in Tables 1 and 2 highlight the paramount importance of non-glycone interactions in the affinity and selectivity towards glycosidases. They also underline the need for implementing strategies allowing structural modifications with a relatively low synthetic cost to optimize such interactions. The superiority of compound 6 as GCase inhibitor within the APP series is remarkable. To get further structural information, we performed 50 ns MD simulations 22 on 6 in complex with the human enzyme (pdb code: 2WCG). 23 The results support that the pyranoid glycone moiety is engaged in hydrogen bond networking in a similar manner to that previously encountered for other glycomimetic-type competitive inhibitors (Figure 3). Indeed, although the putative substrate of GCase is D-glucosylceramide, it has been shown that D-galacto configured glycomimetics do also fit well in the catalytic site. Noticeably, the calculations predict that the exocyclic amide nitrogen is involved in a hydrogen bond with the catalytic glutamic acid nucleophile Glu235. Protonation of this residue may be responsible for the large difference observed in the inhibitory potency of APP compounds at neutral and acidic pH. Moreover, this hydrogen bond orients the aliphatic N-substituent towards the hydrophobic region at the entrance of the active site of the enzyme, reinforcing the stability of the complex. Figure 3. Representative frame obtained from the 50 ns MD simulation on human GCase:APP 6 complex showing the hydrogen bonds between the glycone unit of compound 6 and the residues in the binding site (left panel). The side chain of compound APP 6 is engaged in hydrophobic contacts with the surface of the enzyme (right panel). The hydrophobic residues of the enzyme are shown in orange. ARTICLE Organic & Biomolecular Chemistry 6 | Org. Biomol. Chem., 2015, 00, 1-3 This journal is © The Royal Society of Chemistry 2015 All the four amphiphilic APP compounds were further assayed for their chaperoning capabilities using healthy and Gaucher fibroblasts from patients having the N370S/N370S, F213I/F213I or L444P/L444P GCase mutations. These variants are associated to the non-neuronopathic (type 1) and neuronopathic (types 2 and 3) phenotypes of Gaucher disease, respectively. 24 Only the first one, with the highest prevalence and located in the catalytic domain of GCase, is responsive to the enzyme replacement or substrate reduction therapies currently available, The F213I/F213I mutation is also located in the catalytic domain of the enzyme, while the L444P/L444P mutation is located in a noncatalytic domain, which makes it less prone to rescuing by pharmacological chaperones. 25 The cells were cultured for 5 days in the absence and in the presence of 2 or 20 M concentrations of the APP compounds, then lysed and the GCase activity determined using 4methylumbelliferyl -D-glucopyranoside as substrate. Enzyme activity variations relative to the control in the absence of any compound were thus monitored. Statistically significant activity enhancements were observed only for the N370S/N370S and F213I/F213I GCase mutants after treatment with compound 6 at 20 M concentration, with relative increases of 1.3and 1.5folds, respectively, which approach those achieved with the reference compound ABX (1.5and 1.8-fold, respectively). None of the APP derivatives did exhibit toxic effect on any of the normal or mutant cell lines assayed for 5 days incubation. Conclusions In summary, we have devised an efficient methodology for the synthesis of conformationally locked C-glycosides based on the 3-amino-3-hydroxymethylpyrano[3,2-b]pyrrol-2(1H)-one (APP) scaffold compatible with the incorporation of mono and multibranched aglycone moieties. Evaluation against a variety of glycosidase enzymes showed that amphiphilic derivatives bearing long hydrophobic substituents behaved as M competitive inhibitors of bovine liver -glucosidase/- galactosidase. Most interestingly, they also behaved as selective inhibitors of human lysosomal -glucosidase (- glucocerebrosidase) at neutral pH, with a remarkable decrease in the inhibitory potency upon acidification (pH 5). The pH dependency of glucocerebrosidase binding makes this type of compounds good candidates as pharmacological chaperones for Gaucher disease. Indeed, compound 6 , having a palmitoylamido segment in the aglycone, was able to increase the activity of N370S/N370S and F213I/F213I glucocerebrosidase mutants in fibroblasts of Gaucher patients with an efficiency similar to that of the reference compound Ambroxol®. MD simulations support the existence of a hydrogen bond involving the amide proton of the chaperone and the carboxylate group of the catalytic glutamic acid residue Glu235 thta favorably orients the palmitoyl chain towards a hydrophobic pocket in theenzyme. Since the protonation state of Glu235 changes in the pH 7-5, this structural feature offer further interesting opportunities for pH-dependent chaperone design. Research in that direction is currently sought in our laboratories. Experimental General Information Solvents were purified according to standard procedures. All reactions were followed by thin layer chromatography (TLC) where practical, using silica gel 60 F254 fluorescence treated silica gel plates, which were visualized under UV light (250 nm). Column chromatography was performed using silica gel 60 (230−400 mesh). 1 H and 13 C NMR spectra were recorded on a 400 MHz spectrometer using CDCl 3 , CD 3 OD, or D 2 O as the solvent; chemical shifts are reported in parts per million on the δ scale, and coupling constants are reported in Hertz. All of the resolved signals in the 1 H NMR spectra were assigned on the basis of coupling constants and ge-COSY and ge-HSQC experiments performed on the 400 MHz spectrometer. The results of these experiments were processed with MestReC and MestreNova software. Melting points were determined on a melting-point apparatus and are uncorrected. Optical rotations were measured on a polarimeter from solutions in 1.0 dm cells of capacity 1.0 or 0.3 mL. Electrospray mass spectra were recorded on a micrOTOF spectrometer; accurate mass measurements were achieved by using sodium formate as an external reference. Copies of NMR spectra for all new compounds are provided in the Supporting Information. Preparation of (3R,3aS,5R,6R,7R,7aS,7′R,7′aS)-6,7-dibenzyloxy5-(benzyloxymethyl)-7′-methoxy-7′,7′adimethylspiro[1,3a,5,6,7,7a-hexahydropyran[3,2-b]pyrrol-3,3′- 2H-oxazolo[4,3-b]oxazol]- 2,5′-dione 1 Raney Ni (2.00 g) was suspended in H 2 O (12 mL), and hexachloroplatinic acid (50 mg) and sodium hydroxide 20% (400 μL) were added under stirring. The mixture was heated at 50 °C. After the mixture was stirred for 2.5 h, sodium hydroxide 40% (6 mL) was added, keeping the stirring and the heating. After 1.5 h of stirring, a white cloud in the top of the flask appeared, which was removed by decantation; the resulting solution was then washed with warm water (3 × 15 mL) and ethanol (3 × 15 mL). The catalyst obtained was suspended in ethanol (10 mL) and prehydrogenated for 10 min. An ethanol/ethyl acetate solution (5:2, 7 mL) of Michael adduct (200 mg, 0.28 mmol) was added, and the mixture was stirred under molecular hydrogen at room temperature and atmospheric pressure for 5 h. The crude product was filtered, and the liquid phase was concentrated and purified by silica gel column chromatography (ethyl acetate/hexane, 7:3), to afford compound 1 (105 mg, 0.16 mmol, 57%) as a colorless oil. Physical data are agree with those reported in the literature. ref Preparation of (3R,3aS,5R,6R,7R,7aS)-3-Amino-6,7bis(benzyloxy)-5-(benzyloxymethyl)-3-(hydroxymethyl)-2-oxo1,3a,5,6,7,7a-hexahydropyrano[3,2-b]pyrrol 2 Journal Name ARTICLE This journal is © The Royal Society of Chemistry 2012 Org. Biomol. Chem., 2015, 00, 1-3 | 7 Compound 1 (105 mg, 0.163 mmol) was dissolved in THF (10 ml). A 4 M solution of HCl (3.6 mL) was then added and the mixture was heated up to 40 ºC for 12 h under stirring. The crude obtained after concentration was dissolved in absolute ethanol (5 mL). Propylene oxide (5 mL) was added and the mixture was heated to reflux for 2 h under stirring. Concentration and purification of the crude product by silica gel column chromatography (dichloromethane/methanol, 15:1) afforded compound 2 (60 mg, 0.116 mmol, 71%) as a colourless oil. [𝛼] 𝐷 20 = +64.8 (c 1.0 in CHCl 3 ). HRMS ESI+ (m/z) = 519.2495 [M+H] + ; calculated for C 30 H 35 N 2 O 6+ = 519.2490. 1 H NMR (400 MHz, CDCl 3 )  = 3.52-3.61 (m, 2H, H 7 , BnOCH a H b ), 3.67 (d, 1H, J = 12.0 Hz, CH c H d OH), 3.713.83 (m, 2H, CH c H d OH, BnOCH a H b ), 4.03-4.15 (m, 3H, H 5 , H 6 , H 7a ), 4.24 (d, 1H, J = 4.8 Hz, H 3a ), 4.48-4.59 (m, 4H, PhCH 2 O, PhCH e H f O, PhCH g H h O), 4.68 (d, 1H, J = 11.6 Hz, PhCH e H f O, 4.84 (d, 1H, J = 11.6 Hz, PhCH g H h O), 6.84 (s, 1H, NH), 7.20-7.40 (m, 15H, Ph). 13 C NMR (100 MHz, CDCl 3 )  = 56.2 (C 7a ), 62.3 (C 3 ), 65.1 (CH 2 OH), 67.2 (BnOCH 2 ), 71.9 (PhCH e H f O), 72.5 (C 6 ), 73.6 (PhCH 2 O), 74.5 (PhCH g H h O), 76.2 (C 5 ), 76.7 (C 3a ), 81.3 (C 7 ), 127.8, 127.9, 128.0, 128.3, 128.5, 128.6, 128.8, 137.6, 137.9, 138.1 (Ph), 177.8 (CO). Preparation of [(3R,3aS,5R,6R,7R,7aS)-6,7-Bis(benzyloxy)-5- (benzyloxymethyl)-2-oxo-3-palmitamido-1,3a,5,6,7,7ahexahydropyrano[3,2-b]pyrrol-3-yl]methyl palmitate 3 Compound 2 (45 mg, 0.086 mmol) was dissolved in dichloromethane (2 mL) and palmitoyl chloride (80 L, 0.26 mmol) was added. Et 3 N (50 L, 0.36 mmol) was then added and the mixture was stirred at room temperature for 12 h. A 0.5 M solution of HCl (2 mL) was added. The organic layer was separated and the aqueous one was washed with dichloromethane (3 × 2 mL). The organic phases were collected and dried with anhydrous Na 2 SO 4 . Concentration and purification of the crude product by silica gel column chromatography (hexane/ethyl acetate, 65:35) afforded compound 3 (60 mg, 0.06 mmol, 70%) as a colourless oil. [𝛼] 𝐷 20 = +34.5 (c 1.0 in CHCl 3 ). HRMS ESI+ (m/z) = 995.7071 [M+H] + ; calculated for C 62 H 95 N 2 O 8+ = 995.7083. 1 H NMR (400 MHz, CDCl 3 )  = 0.88 (t, 6H, J = 6.5 Hz, CH 3 ), 1.19-1.35 (m, 48H, (CH 2 ) 12 CH 3 ), 1.51-1.64 (m, 4H, CH 2 (CH 2 ) 12 CH 3 ), 2.18 (t, 2H, J = 7.6 Hz, NHCOCH 2 ), 2.24 (t, 2H, J = 7.6 Hz, OCOCH 2 ), 3.52-3.60 (m, 2H, H 7 , BnOCH a H b ), 3.63-3.70 (m, 1H, BnOCH a H b ), 4.03-4.10 (m, 1H, H 5 ), 4.11-4.15 (m, 1H, H 6 ), 4.29-4.36 (m, 2H, H7a, CH c H d OCO), 4.42-4.54 (m, 4H, CH c H d OCO, PhCH 2 O, PhCH e H f O), 4.58 (d, 1H, J = 11.6 Hz, PhCH g H h O), 4.66-4.72 (m, 2H, H 3a , PhCH e H f O), 4.88 (d, 1H, J = 11.6 Hz, PhCH g H h O), 6.06 (NH), 6.32 (NH), 7.22-7.40 (m, 15H, Ph). 13 C NMR (100 MHz, CDCl 3 )  = 14.2 (CH 3 ), 22.8, 24.9, 25.3, 29.3, 29.5, 29.6, 29.7, 29.8, 32.1 ((CH 2 ) 13 CH 3 ), 34.2 (OCOCH 2 ), 36.2 (NHCOCH 2 ), 53.5 (C 3 ), 56.0 (C 7a ), 63.1 (CH 2 OCO), 67.8 (BnOCH 2 ), 71.5 (PhCH e H f O), 71.7 (C 6 ), 73.6 (PhCH 2 O), 74.4 (PhCH g H h O), 75.9 (C 3a ), 76.5 (C 5 ), 81.4 (C 7 ), 127.7, 127.8, 127.9, 128.2, 128.4, 128.6, 128.8, 137.6, 138.1, 138.4 (Ph), 172.4 (OCO), 173.2 (NHCO), 173.7 (NHCO). Preparation of [(3R,3aS,5R,6R,7R,7aR)-6,7-Dihydroxy-5- (hydroxymethyl)-2-oxo-3-palmitamido-1,3a,5,6,7,7ahexahydropyrano[3,2-b]pyrrol-3-yl]methyl palmitate 4 A hydrogenolysis of a methanol solution (5 mL) of the compound 3 (37 mg, 0.037 mmol) was held under ambient pressure and temperature, using Pd-C (35 mg) and HCl conc. (3 drops) as catalyst. The reaction was performed for 12 h and the catalyst was filtered over diatomaceous earth. The liquid phase was concentred affording compound 4 (25 mg, 0,034 mmol, 93%) as a yellow oil. [𝛼] 𝐷 20 = +20.6 (c 0.99 in methanol). HRMS ESI+ (m/z) = 725.5669 [M+H] + ; calculated for C 41 H 77 N 2 O 8+ = 725.5674. 1 H NMR (400 MHz, CD 3 OD)  = 0.89 (t, 6H, J = 6.5 Hz, CH 3 ), 1.24-1.35 (m, 48H, (CH 2 ) 12 CH 3 ), 1.54-1.67 (m, 4H, CH 2 (CH 2 ) 12 CH 3 ), 2.23 (t, 2H, J = 6.7 Hz, NHCOCH 2 ), 2.36 (t, 2H, J = 7.3 Hz, OCOCH 2 ), 3.70-3.78 (m, 3H, CH 2 OH, H 7 ), 3.79-3.85 (m,1H, H 5 ), 3.91-4.02 (m, 2H, H 6 , H 7a ), 4.31 (d, 1H, J = 10.7 Hz, CH a H b OCO), 4.45-4.52 (m, 1H, CH a H b OCO), 4.83 (m, 1H, H 3a ). 13 C NMR (100 MHz, CD 3 OD)  = 14.4 (CH 3 ), 23.7, 25.8, 26.8, 30.2, 30.4, 30.5, 30.6, 30.7, 30.8, 33.0 ((CH 2 ) 13 CH 3 ), 34.9 (OCOCH 2 ), 36.7 (NHCOCH 2 ), 56.9 (C 7a ), 61.6 (CH 2 OH), 62.6 (CH 2 OCO), 63.1 (C 3 ), 69.4 (C 6 ), 73.3 (C 7 ), 76.0 (C 3a ), 78.8 (C 5 ), 174.7 (OCO), 175.7 (NHCO), 176.0 (NHCO). Preparation of N-[(3R,3aS,5R,6R,7R,7aS)-6,7-Bis(benzyloxy)-5- (benzyloxymethyl)-3-hydroxymethyl-2-oxo -1,3a,5,6,7,7ahexahydropyrano[3,2-b]pyrrol-3-yl]palmitamide 5 A methanol solution of sodium methoxide 0.5 M (2 mL) was added to a methanol solution (4 mL) of the compound 3 (76 mg, 0.076 mmol). The mixture was stirred for 1 h and sulfonic acid resine Dowex ® was then added. The liquid phase was filtered, concentrated and the residue was purified by silica gel column chromatography (hexane/ethyl acetate, 15:85) to afford compound 5 (40 mg, 0.053 mmol, 70%) as a colourless oil. [𝛼] 𝐷 20 = +66.0 (c 1.0 in CHCl 3 ). HRMS ESI+ (m/z) = 757.4788 [M+H] + ; calculated for C 46 H 65 N 2 O 7+ = 757.4786. 1 H NMR (400 MHz, CDCl 3 )  = 0.88 (t, 3H, J = 6.7 Hz, CH 3 ), 1.22-1.33 (m, 24H, (CH 2 ) 12 CH 3 ), 1.55-1.65 (m, 2H, CH 2 (CH 2 ) 12 CH 3 ), 2.20 (t, 2H, J = 7.6 Hz, NHCOCH 2 ), 3.48 (dd, 1H, J = 10.3, 4.7 Hz, BnOCH a H b ), 3.56 (d, 1H, J = 8.1 Hz, H 7 ) , 3.67-3.75 (m, 2H, BnOCH a H b , CH c H d OH), 3.98 (d, 1H, J = 12.0 Hz, CH c H d OH), 4.09 (d, 1H, J = 4.9 Hz, H 6 ), 4.11-4.18 (m, 1H, H 5 ), 4.38 (t, 1H, J = 7.2 Hz, H 7a ), 4.47 (d, 1H, J =11.9 Hz, PhCH e H f O), 4.49 (s, 2H, PhCH 2 O), 4.55 (d, 1H, J = 11.8 Hz, PhCH g H h O), 4.61 (d, 1H, J = 6.3, H 3a ) 4.68 (d, 1H, J = 11.8 Hz, PhCH e H f O), 4.87 (d, 1H, J = 11.8 Hz, PhCH g H h O), 6.22 (NH), 6.56 (NH), 7.20-7.39 (m, 15H, Ph). 13 C NMR (100 MHz, CDCl 3 )  = 14.1 (CH 3 ), 22.7, 25.3, 29.2, 29.4, 29.5, 29.7, 32.0 ((CH 2 ) 13 CH 3 ), 35.8 (NHCOCH 2 ), 56.3 (C 7a ), 63.5 (C 3 ), 64.0 (CH 2 OH), 67.4 (BnOCH 2 ), 71.5 (C 6 ), 71.6 (PhCH e H f O), 73.5 (PhCH 2 O), 74.2 (PhCH g H h O), 76.7 (C 5 , C 3a ), 81.4 (C 7 ), 127.6, 127.8, 127.9, 128.2, 128.4, 128.5, 128.7, 137.5, 137.8, 138.0 (Ph), 173.5 (NHCO), 173.9 (NHCO). Preparation of N-[(3R,3aS,5R,6R,7R,7aR)-6,7-Dihydroxy-3,5bis(hydroxymethyl)-2-oxo -1,3a,5,6,7,7a-hexahydropyrano[3,2b]pyrrol-3-yl]palmitamide 6 A hydrogenolysis of a methanol solution (5 mL) of the compound 5 (35 mg, 0.046 mmol) was held under ambient pressure and temperature, using Pd-C (35 mg) and HCl conc. (3 drops) as catalyst. The reaction was performed for 12 h and the catalyst was filtered over diatomaceous earth. The liquid phase ARTICLE Organic & Biomolecular Chemistry 8 | Org. Biomol. Chem., 2015, 00, 1-3 This journal is © The Royal Society of Chemistry 2015 was concentred affording compound 6 (22 mg, 0,045 mmol, 98%) as a yellow oil. [𝛼] 𝐷 20 = +15.1 (c 1.0 in methanol). HRMS ESI+ (m/z) = 487.3386 [M+H] + ; calculated for C 25 H 47 N 2 O 7+ = 487.3378. 1 H NMR (400 MHz, CD 3 OD)  = 0.83 (t, 3H, J = 5.8 Hz, CH 3 ), 1.13-1.36 (m, 24H, (CH 2 ) 12 CH 3 ), 1.49-1.62 (m, 2H, CH 2 (CH 2 ) 12 CH 3 ), 2.38 (t, 2H, J = 6.7 Hz, NHCOCH 2 ), 3.654.02 (m, 6H, CH 2 OH, H 5 , H 6 , H 7 , H 7a ), 4.39 (d, 1H, J = 11.3 Hz, C 3 CH a H b OH), 4.48 (d, 1H, J = 11.7 Hz, C 3 CH a H b OH), 4.74 (d, 1H, J = 5.3 Hz, H 3a ). 13 C NMR (100 MHz, CD 3 OD)  = 14.3 (CH 3 ), 23.5, 25.5, 30.0, 30.3, 30.4, 30.5, 30.6, 32.9 ((CH 2 ) 13 CH 3 ), 34.4 (NHCOCH 2 ), 55.8 (C 7a ), 61.6 (C 3 CH 2 OH), 61.9 (CH 2 OH), 63.0 (C 3 ), 69.2 (C 6 ), 73.3 (C 7 ), 74.6 (C 3a ), 79.0 (C 5 ), 174.1 (2 NHCO). Preparation of [(3R,3aS,5R,6R,7R,7aS)-3-Amino-6,7bis(benzyloxy)-5-(benzyloxymethyl)-2-oxo-1,3a,5,6,7,7ahexahydropyrano[3,2-b]pyrrol-3-yl]methyl palmitate 7 Compound 1 (82 mg, 0.127 mmol) was dissolved in THF (8 mL). A 4 M solution of HCl (2.8 mL) was then added and the mixture was heated up to 40 ºC for 12 h under stirring. The crude obtained after concentration was dissolved in TFA (4 mL) and palmitoyl chloride was added (385 L, 1.27 mmol). The mixture was stirred at room temperature for 15 min and absolute ethanol (10 mL) was added to quench the reaction. After concentration, the crude was dissolved in absolute ethanol (5 mL). Propylene oxide (5 mL) was added and the mixture was heated to reflux for 2 h under stirring. Concentration and purification of the crude product by silica gel column chromatography (hexane/ethyl acetate, 65:35) afforded compound 7 (64 mg, 0.085 mmol, 67%) as a colourless oil. [𝛼] 𝐷 20 = +52.9 (c 1.0 in CHCl 3 ). HRMS ESI+ (m/z) = 757.4788 [M+H] + ; calculated for C 46 H 65 N 2 O 7+ = 757.4786. 1 H NMR (400 MHz, CDCl 3 )  = 0.88 (t, 3H, J = 6.7 Hz, CH 3 ), 1.20-1.36 (m, 24H, (CH 2 ) 12 CH 3 ), 1.54-1.64 (m, 2H, CH 2 (CH 2 ) 12 CH 3 ), 2.27 (t, 2H, J = 7.8 Hz, OCOCH 2 ), 3.55 (d, 1H, J = 5.3 Hz, H 7 ), 3.70 (d, 1H, J = 5.2 Hz, BnOCH 2 ) , 4.05-4.12 (m, 2H, H 3a , H 5 ), 4.144.20 (m, 2H, H 7a , H 6 ), 4.47 (d, 1H, J = 2.7 Hz, CH 2 OCO), 4.494.61 (m, 4H, PhCH a H b O, PhCH c H d O, PhCH 2 O), 4.69 (d, 1H, J = 11.8 Hz, PhCH a H b O), 4.81 (d, 1H, J = 11.5 Hz, PhCH c H d O), 6.57 (NH), 7.24-7.38 (m, 15H, Ph). 13 C NMR (100 MHz, CDCl 3 )  = 14.2 (CH 3 ), 22.8, 26.1, 29.3, 29.5, 29.6, 29.7, 29.8, 32.0 ((CH 2 ) 13 CH 3 ), 28.2 (NHCOCH 2 ), 57.3 (C 7a ), 67.2 (BnOCH 2 ), 68.3 (CH 2 OCO), 72.0 (PhCH a H b O), 73.0 (C 3a ), 73.5 (PhCH 2 O), 74.2 (PhCH c H d O), 74.6 (C 6 ), 76.2 (C 5 ), 78.7 (C 3 ), 80.1 (C 7 ), 127.8, 127.9, 128.2, 128.5, 128.6, 128.7, 137.7, 138.1, 138.2 (Ph), 170.9 (OCO), 175.5 (NHCO). Preparation of [(3R,3aS,5R,6R,7R,7aR)-3-Amino-6,7bis(hydroxy)-5-(hydroxymethyl)-2-oxo-1,3a,5,6,7,7ahexahydropyrano[3,2-b]pyrrol-3-yl]methyl palmitate 8 A hydrogenolysis of a methanol solution (5 mL) of the compound 7 (50 mg, 0.065 mmol) was held under ambient pressure and temperature, using Pd-C (50 mg) and HCl conc. (3 drops) as catalyst. The reaction was performed for 12 h and the catalyst was filtered over diatomaceous earth. The liquid phase was concentred affording compound 8 (30 mg, 0,062 mmol, 94 %) as a yellow oil. [𝛼] 𝐷 20 = -3.9 (c 1.0 in methanol). HRMS ESI+ (m/z) = 487.3388 [M+H] + ; calculated for C 25 H 47 N 2 O 7+ = 487.3378. 1 H NMR (400 MHz, CD 3 OD)  = 0.89 (t, 3H, J = 6.4 Hz, CH 3 ), 1.22-1.39 (m, 24H, (CH 2 ) 12 CH 3 ), 1.58-1.67 (m, 2H, CH 2 (CH 2 ) 12 CH 3 ), 2.43 (t, 2H, J = 7.4 Hz, OCOCH 2 ), 3.70-3.78 (m, 3H, CH 2 OH, H 7 ), 3.79-3.85 (m, 1H, H 5 ), 3.93 (t, 1H, J = 7.6 Hz, H 7a ), 3.99 (s, 1H, H 6 ), 4.44 (d, 1H, J = 12.1 Hz, CH a H b OCO), 4.53 (d, 1H, J = 11.9 Hz, CH a H b OCO), 4.77 (d, 1H, J = 7.2 Hz, H 3a ). 13 C NMR (100 MHz, CD 3 OD)  = 14.4 (CH 3 ), 23.7, 25.7, 30.1, 30.4, 30.6, 30.7, 30.8, 33.0 ((CH 2 ) 13 CH 3 ), 34.5 (OCOCH 2 ), 55.9 (C 7a ), 61.7 (CH 2 OCO), 62.0 (CH 2 OH), 63.1 (C 3 ), 69.3 (C 6 ), 73.5 (C 7 ), 74.8 (C 3a ), 79.2 (C 5 ), 170.3 (OCO), 174.1 (NHCO). Preparation of [(3R,3aS,5R,6R,7R,7aS)-3-Acetamido-6,7bis(benzyloxy)-5-(benzyloxymethyl)-2-oxo-1,3a,5,6,7,7ahexahydropyrano[3,2-b]pyrrol-3-yl]methyl palmitate 10 Compound 9 (60 mg, 0.107 mmol) was dissolved in dichloromethane (2 mL) and palmitoyl chloride (70 L, 0.23 mmol) was added. Et 3 N (44 L, 0.32 mmol) was then added and the mixture was stirred at room temperature for 12 h. A 0.5 M solution of HCl (2 mL) was added. The organic layer was separated and the aqueous one was washed with dichloromethane (3 × 2 mL). The organic phases were collected and dried with anhydrous Na 2 SO 4 . Concentration and purification of the crude product by silica gel column chromatography (hexane/ethyl acetate, 35:65) afforded compound 10 (63 mg, 0.079 mmol, 74%) as a colourless oil. [𝛼] 𝐷 20 = +41.0 (c 1.0 in CHCl 3 ). HRMS ESI+ (m/z) = 799.4853 [M+H] + ; calculated for C 48 H 67 N 2 O 8+ = 799.4892. 1 H NMR (400 MHz, CDCl 3 )  = 0.88 (t, 3H, J = 6.7 Hz, CH 3 ), 1.21-1.30 (m, 24H, (CH 2 ) 12 CH 3 ), 1.51-1.58 (m, 2H, CH 2 (CH 2 ) 12 CH 3 ), 1.98 (s, 3H, NHAc), 2.23 (t, 2H, J = 7.5 Hz, OCOCH 2 ), 3.53-3.59 (m, 2H, BnOCH a H b , H 7 ), 3.66 (dd, 1H, J = 9.5, 6.9 Hz, BnOCH a H b ), 4.07 (dd, 1H, J = 11.1, 6.3 Hz, H 5 ), 4.13 (d, 1H, J = 4.2 Hz, H 6 ), 4.27-4.34 (m, 2H, H 7a , CH c H d OCO), 4.42-4.52 (m, 4H, CH c H d OCO, PhCH 2 O, PhCH e H f O), 4.58 (d, 1H, J = 11.6, PhCH g H h O), 4.66-4.72 (m, 2H, PhCH e H f O, H 3a ), 4.87 (d, 1H, J = 11.6 Hz, PhCH g H h O), 6.07-6.18 (m, 1H, NHAc), 6.38 (s, 1H, NHCO), 7.22-7.40 (m, 15H, Ph). 13 C NMR (100 MHz, CDCl 3 )  = 14.1 (CH 3 ), 23.1 (NHCOCH 3 ), 22.7, 24.8, 29.2, 29.3, 29.4, 29.5, 29.7, 31.9, 34.1 (CO(CH 2 ) 14 CH 3 ), 55.9 (C 7a ), 63.0 (CH 2 OCO), 63.2 (C 3 ) 67.7 (BnOCH 2 ), 71.4 (PhCH e H f O), 71.6 (C 6 ), 73.5 (PhCH 2 O), 74.3 (PhCH g H h O), 75.7 (C 3a ), 76.4 (C 5 ), 81.2 (C 7 ), 127.7, 127.8, 127.9, 128.2, 128.3, 128.5, 128.7, 137.5, 138.0, 138.2 (Ph), 170.0 (OCO), 172.3 (NHCOCH 3 ), 173.6 (NHCO). Preparation of [(3R,3aS,5R,6R,7R,7aR)-3-Acetamido-6,7bis(hydroxy)-5-(hydroxymethyl)-2-oxo-1,3a,5,6,7,7ahexahydropyrano[3,2-b]pyrrol-3-yl]methyl palmitate 11 A hydrogenolysis of a methanol solution (5 mL) of the compound 10 (63 mg, 0.079 mmol) is held under ambient pressure and temperature, using Pd-C (60 mg) and HCl conc. (3 drops) as catalyst. The reaction is performed for 12 h and the catalyst is filtered over diatomaceous earth. The liquid phase is concentred affording the compound 11 (35 mg, 0,066 mmol, 84 %) as a yellow oil. [𝛼] 𝐷 20 = +22.7 (c 1.0 in H 2 O). HRMS ESI+ (m/z) = 529.3493 [M+H] + ; calculated for C 27 H 49 N 2 O 8+ = 529.3483. 1 H NMR (400 MHz, CD 3 OD)  = 0.88 (t, 3H, J = 6.4 Hz, CH 3 ), 1.19-1.37 (m, 24H, (CH 2 ) 12 CH 3 ), 1.53-1.66 (m, 2H, CH 2 (CH 2 ) 12 CH 3 ), 2.26-2.39 (m, 2H, OCOCH 2 ), 3.68-4.03 (m, 6H, CH 2 OH, H 5 , H 6 , H 7 , H 7a ), 4.25-4.35 (m, 1H, CH a H b OCO), 4.46-4.54 (m, 1H, CH a H b OCO), 4.83 (m, 1H, H 3a ). 13 C NMR (100 MHz, CD 3 OD)  = 14.3 (CH 3 ), 23.6, 25.8, 25.9, 30.1, 30.2, 30.3, 30.4, 30.5, 30.6, 30.7, 32.9 ((CH 2 ) 13 CH 3 ), 34.7 (OCOCH 2 ), 56.9 (C 7a ), 61.6 (CH 2 OH), 62.2 (CH 2 OCO), 64.9 Journal Name ARTICLE This journal is © The Royal Society of Chemistry 2012 Org. Biomol. Chem., 2015, 00, 1-3 | 9 (C 3 ), 69.3 (C 6 ), 73.1 (C 7 ), 75.9 (C 3a ), 78.7 (C 5 ), 171.4 (OCO), 174.6 (NHCO), 176.0 (NHCO). Preparation of [(3R,3aS,5R,6R,7R,7aS)-3-Amino-6,7bis(benzyloxy)-5-(benzyloxymethyl)-2-oxo-1,3a,5,6,7,7ahexahydropyrano[3,2-b]pyrrol-3-yl]methyl acetate 12 Compound 1 (73 mg, 0.113 mmol) was dissolved in THF (7 mL). A 4 M solution of HCl (2.5 mL) was then added and the mixture was heated up to 40 ºC for 12 h under stirring. The crude obtained after concentration was dissolved in TFA (4 mL) and acetyl chloride was added (80 L, 1.13 mmol). The mixture was stirred at room temperature for 15 min and absolute ethanol (10 mL) was added to quench the reaction. After concentration, the crude was dissolved in absolute ethanol (5 mL). Propylene oxide (5 mL) was added and the mixture was heated to reflux for 2 h under stirring. Concentration and purification of the crude product by silica gel column chromatography using ethyl acetate as eluent afforded compound 12 (27 mg, 0.048 mmol, 42%) as a colourless oil. [𝛼] 𝐷 20 = +76.7 (c 1.0 in CHCl 3 ). HRMS ESI+ (m/z) = 561.2590 [M+H] + ; calculated for C 32 H 37 N 2 O 7+ = 561.2595. 1 H NMR (400 MHz, CDCl 3 )  = 1.99 (s, 3H, CH 3 ), 3.54 (s, 1H, H 7 ), 3.67-3.73 (m, 2H, BnOCH 2 ), 4.06-4.12 (m, 2H, H 3a , H 5 ), 4.15-4.20 (m, 2H, H 6 , H 7a ), 4.41-4.49 (m, 2H, CH 2 OAc), 4.49-4.61 (m, 4H, PhCH a H b O, PhCH c H d O, PhCH 2 O), 4.70 (d, 1H, J = 11.8 Hz, PhCH a H b O), 4.82 (d, 1H, J = 11.4 Hz, PhCH c H d O), 6.51 (s, 1H, NH), 7.24-7.39 (m, 15H, Ph). 13 C NMR (100 MHz, CDCl 3 )  = 14.0 (CH 3 ), 57.4 (C 7a ), 67.2 (BnOCH 2 ), 68.6 (CH 2 OAc), 71.9 (PhCH a H b O), 72.9 (C 3a ), 73.5 (PhCH 2 O), 74.3 (PhCH c H d O), 74.6 (C 6 ), 76.2 (C 5 ), 78.8 (C 3 ), 80.2 (C 7 ), 127.8, 127.9, 128.3, 128.5, 128.6, 128.8, 137.7, 138.1, 138.2 (Ph), 167.8 (COCH 3 ), 175.5 (NHCO). Preparation of [(3R,3aS,5R,6R,7R,7aR)-3-Amino-6,7bis(hydroxy)-5-(hydroxymethyl)-2-oxo-1,3a,5,6,7,7ahexahydropyrano[3,2-b]pyrrol-3-yl]methyl acetate 13 A hydrogenolysis of a methanol solution (5 mL) of the compound 12 (27 mg, 0.048 mmol) was held under ambient pressure and temperature, using Pd-C (25 mg) and HCl conc. (3 drops) as catalyst. The reaction was performed for 12 h, the catalyst was then filtered over diatomaceous earth and the liquid phase is concentred. The residue was dissolved in H 2 O (5 mL) and extracted with ethyl acetate (2 × 5 mL). The combined aqueous phases were concentrated and the residue was dissolved in H 2 O (2 mL) and eluted through a reverse-phase Sep-pak C18 cartridge affording, after evaporation of water, compound 13 (13 mg, 0,044 mmol, 94%) as a yellow oil. [𝛼] 𝐷 20 = +42.3 (c 1.0 in H 2 O). HRMS ESI+ (m/z) = 291.1188 [M+H] + ; calculated for C 11 H 19 N 2 O 7+ = 291.1187. 1 H NMR (400 MHz, D 2 O)  = 2.17 (s, 3H, CH 3 ), 3.69-3.84 (m, 2H, CH 2 OH), 3.844.08 (m, 4H, H 5 , H 6 , H 7 , H 7a ), 4.51 (d, 1H, J = 12.3 Hz, CH a H b OAc), 4.66 (d, 1H, J = 12.3 Hz, CH a H b OAc), 4.96 (d, 1H, J = 8.1 Hz, H 3a ). 13 C NMR (100 MHz, D 2 O)  = 20.1 (CH 3 ), 53.1 (C 7a ), 61.1 (CH 2 OAc), 61.4 (CH 2 OH), 61.8 (C 3 ), 67.4 (C 6 ), 71.8 (C 7 ), 74.3 (C 3a ), 77.4 (C 5 ), 169.7 (COAc), 172.6 (NHCO). Preparation of (3R,3aS,5R,6R,7R,7aR)-3-Amino-6,7-dihydroxy3,5-bis(hydroxymethyl)- 1,3a,5,6,7,7a-hexahydropyrano[3,2b]pyrrol-2-one 14 A hydrogenolysis of a methanol solution (5 mL) of the compound 2 (35 mg, 0.067 mmol) was held under ambient pressure and temperature, using Pd-C (35 mg) and HCl conc. (3 drops) as catalyst. The reaction was performed for 12 h, the catalyst was then filtered over diatomaceous earth and the liquid phase was concentred. The residue was dissolved in H 2 O (5 mL) and extracted with ethyl acetate (2 × 5 mL). The combined aqueous phases were concentrated affording compound 14 (16 mg, 0,064 mmol, 96 %) as a yellow oil. [𝛼] 𝐷 20 = +32.6 (c 1.0 in H 2 O). HRMS ESI+ (m/z) = 249.1087 [M+H] + ; calculated for C 9 H 17 N 2 O 6+ = 249.1081. 1 H NMR (400 MHz, D 2 O)  = 3.71-3.84 (m, 2H, CH 2 OH), 3.90-4.16 (m, 6H, H 5 , H 6 , H 7 , H 7a , C 3 CH 2 OH), 4.96 (d, 1H, J = 5.6 Hz, H 3a ). 13 C NMR (100 MHz, D 2 O)  = 53.0 (C 7a ), 59.0 (C 3 CH 2 OH), 61.4 (CH 2 OH), 63.6 (C 3 ), 67.4 (C 6 ), 71.8 (C 7 ), 73.8 (C 3a ), 77.3 (C 5 ), 170.7 (NHCO). Inhibition Studies with Commercial Enzymes . Inhibition constant (K i ) values were determined by spectrophotometrically measuring the residual hydrolytic activities of the glycosidases against the respective p-nitrophenyl αor βD -glycopyranoside, or o-nitrophenyl βD -galactopyranoside (for β-galactosidases), in the presence of compounds 4 , 6 , 8 , 11 , 13 , 14 and 15 . Each essay was performed in phosphate buffer at the optimal pH of each enzyme. The reactions were initiated by addition of enzyme to a solution of the substrate in the absence or presence of various concentrations of inhibitor. The mixture was incubated for 10-30 min at 37 °C, and the reaction was quenched by addition of 1 M Na 2 CO 3 . Reaction times were appropiate to obtain 10 − 20% conversion of the substrate in order to achieve linear rates. The absorbance of the resulting mixture was determined at 405. Approximate values of K i were determined using a fixed concentration of substrate (around the K m value for the different glycosidases) and various concentrations of inhibitor. Full K i determinations and enzyme inhibition mode were determined from the slope of Lineweaver − Burk plots and double reciprocal analysis. Lysosomal enzyme activity assay . Lysosomal enzyme activities in cell lysates were determined as described previously. 26 Briefly, cells were scraped in ice-cold 0.1% Triton X-100 in water. After centrifugation (6,000 rpm for 15 min at 4 ºC) to remove insoluble materials, protein concentrations were determined using Protein Assay Rapid Kit (Wako, Tokyo, Japan). The lysates were incubated at 37 ºC with the corresponding 4-methylumbelliferyl -D-glycopyranoside solution in 0.1 M citrate buffer (pH 4). The librated 4methylumbelliferone was measured with a fluorescence plate reader (exitation 340 nm; emission 460 nm; Infinite F500, TECAN Japan, Kawasaki, Japan). For enzyme inhibition assay, cell lysates from normal skin fibroblasts were mixed with the 4methylumbelliferyl -D-glycopyranoside substrates in the absence or presence of increasing concentrations of the tested derivatives. Cell culture and GCase activity enhancement assay. Human skin fibroblasts from a healthy and three Gaucher disease patients (with N370S/ N370S, F213I/ F213I and L444P/L444P mutations) were maintained in our laboratory with DMEM supplemented with 10% FBS as the culture medium. For enzyme activity enhancement assay, cells were cultured in the presence of different concentrations of the candidates or DMSO alone (as a control) for 5 days and harvested by scraping. 26 Cytotoxicity of the compounds was monitored by measuring the lactate dehydrogenase activities in the cultured supernatants (LDH assay kit, Wako, Tokyo, Japan).