Synthesis and Evaluation of Novel Iminosugars Prepared from Natural Amino Acids

Cyclopropanated iminosugars have a locked conformation that may enhance the inhibitory activity and selectivity against different glycosidases. We show the synthesis of new cyclopropane-containing piperidines bearing five stereogenic centers from natural amino acids l-serine and l-alanine. Those prepared from the latter amino acid may mimic l-fucose, a natural-occurring monosaccharide involved in many molecular recognition events. Final compounds prepared from l-serine bear S configurations on the C5 position. The synthesis involved a stereoselective cyclopropanation reaction of an α,β-unsaturated piperidone, which was prepared through a ring-closing metathesis. The final compounds were tested as possible inhibitors of different glycosidases. The results, although, in general, with low inhibition activity, showed selectivity, depending on the compound and enzyme, and in some cases, an unexpected activity enhancement was observed.


Introduction
Natural or synthetic polyhydroxylated piperidines are iminosugars able to act as biomimetics of their corresponding pyranose analogs. For instance, nojirimycin, its epimers, and their deoxyanalogs have been used as lead molecules to design glycosidase and glycosyl transferase inhibitors and modulators [1][2][3]. Some iminosugars such as miglitol (Glyset ® ) [4], migalastat (Galafold ® ) [5], and miglustat (Zavesca ® ) [6] are commercially available, and others are actually in different clinical phases ( Figure 1). The interaction with glycosidases is generally attributed to a structural similarity to diverse conformational oxocarbenium transition states formed during the hydrolysis of carbohydrates [7]. There are important variations in these transition states for different glycosidases [8,9]. Thus, there is a great interest in designing conformationally restricted inhibitors in order to achieve selective inhibition and adequate metabolic stability.
Our group engaged in the synthesis of novel piperidine iminosugars fused to a cyclopropane ring, resulting in structures with a locked conformation [10,11]. The cyclopropane renders a twist-like conformation to the piperidine ring that is found, as preferred for interactions with certain glycosidases [12,13]. We expect these compounds to be starting points for the finding of products of pharmacological interest ( Figure 2). The possibility of variations in the substitution pattern of the cyclopropane allows different configurations that could direct their selectivity to different glycosidases. Although the development of synthetic routes to iminosugars has received much attention in the synthetic community [14,15], the preferred chiral pools are carbohydrates, which are transformed using reductive aminations [16,17], or other transformation strategies [18]. In our case, we developed synthetic approaches from natural amino acids, which have less precedents [19,20].

Introduction
Natural or synthetic polyhydroxylated piperidines are iminosugars able to act as biomimetics of their corresponding pyranose analogs. For instance, nojirimycin, its epimers, and their deoxyanalogs have been used as lead molecules to design glycosidase and glycosyl transferase inhibitors and modulators [1][2][3]. Some iminosugars such as miglitol (Glyset ® ) [4], migalastat (Galafold ® ) [5], and miglustat (Zavesca ® ) [6] are commercially available, and others are actually in different clinical phases ( Figure 1). The interaction with glycosidases is generally attributed to a structural similarity to diverse conformational oxocarbenium transition states formed during the hydrolysis of carbohydrates [7]. There are important variations in these transition states for different glycosidases [8,9]. Thus, there is a great interest in designing conformationally restricted inhibitors in order to achieve selective inhibition and adequate metabolic stability.  Our group engaged in the synthesis of novel piperidine iminosugars fused to a cyclopropane ring, resulting in structures with a locked conformation [10,11]. The cyclopropane renders a twist-like conformation to the piperidine ring that is found, as preferred for interactions with certain glycosidases [12,13]. We expect these compounds to be starting points for the finding of products of pharmacological interest ( Figure 2). The possibility of variations in the substitution pattern of the cyclopropane allows different configurations that could direct their selectivity to different glycosidases. Although the development of synthetic routes to iminosugars has received much attention in the synthetic community [14,15], the preferred chiral pools are carbohydrates, which are transformed using reductive aminations [16,17], or other transformation strategies [18]. In our case, we developed synthetic approaches from natural amino acids, which have less precedents [19,20]. Other asymmetric or biocatalyzed approaches have been used [21]. In our previous work, starting from natural amino acid L-serine, we synthesized iminosugars bearing the R configuration on the carbon adjacent to nitrogen, which was supposed to mimic C5 in natural carbohydrates and iminosugars. The present work is focused on new compounds prepared both from L-alanine and L-serine. In the case of L-alanine, the derived compounds could mimic 6-dehydroxylated sugars as fucose. Fucose-containing glycans, such as in blood groups and Lewis oligosaccharides and related ones, are critical for a wide range of cell events [22]. These include cell-cell adhesion, immune response, viral and bacterial infection, and tumor progression. We prepared new bicyclic iminosugars that include the cyclopropane motif fused with a piperidine that may mimic the L-fucose ring and evaluated them against fucosidase and other glycosidases. In addition, we prepared cyclopropane-containing piperidine iminosugars starting from L-serine but now with an S configuration at the carbon that mimics C5. A preliminary glycosidase inhibition evaluation is shown. The synthesis implies building final compounds with five stereogenic centers [23][24][25].

Chemistry
The synthesis of the novel iminosugars started from L-serine or L-alanine as the chiral pool. Both amino acids were protected using di-tert-butyl dicarbonate (Boc2O) [26]. Without further purification, these latter intermediates were submitted to coupling with N,Odimethylhydroxylamine and, for the L-serine derivative, protection with tert-butyldimethylsilyl of the hydroxy group. Thus, intermediates 1a (78%) and 1b (76%) were obtained in good yields. Treatment with a base and allyl bromide, followed by a reaction with vinylmagnesium bromide at −30 °C, gave the precursors of the ring-closing metathesis reaction (RCM). For the RCM, a second-generation Grubbs' catalyst (Grubbs' Catalyst ® M204, 3 mol %) was used, affording the α,β-unsaturated ketones 2a and 2b in 76% and 60% yield, respectively (Scheme 1) [27]. These compounds reacted with tert-butyl 2-(tetrahydro-1λ 4 -thiophen-1-ylidene)acetate to give a mixture (as seen in 1 H-NMR) of cyclopropane exo:endo isomers 3a (77%) and 4a (5%) from 2a in a 15:1 ratio, while 3b (71%) and 4b (18%) from 2b in a 4:1 exo:endo ratio [28]. These isomers were isolated and separately characterized. 1 H-NMR at 90 °C in DMSO-d6 determined that compounds 3b and 4b were The present work is focused on new compounds prepared both from L-alanine and L-serine. In the case of L-alanine, the derived compounds could mimic 6-dehydroxylated sugars as fucose. Fucose-containing glycans, such as in blood groups and Lewis oligosaccharides and related ones, are critical for a wide range of cell events [22]. These include cell-cell adhesion, immune response, viral and bacterial infection, and tumor progression. We prepared new bicyclic iminosugars that include the cyclopropane motif fused with a piperidine that may mimic the L-fucose ring and evaluated them against fucosidase and other glycosidases. In addition, we prepared cyclopropane-containing piperidine iminosugars starting from L-serine but now with an S configuration at the carbon that mimics C5. A preliminary glycosidase inhibition evaluation is shown. The synthesis implies building final compounds with five stereogenic centers [23][24][25].

Chemistry
The synthesis of the novel iminosugars started from L-serine or L-alanine as the chiral pool. Both amino acids were protected using di-tert-butyl dicarbonate (Boc 2 O) [26]. Without further purification, these latter intermediates were submitted to coupling with N,O-dimethylhydroxylamine and, for the L-serine derivative, protection with tert-butyldi methylsilyl of the hydroxy group. Thus, intermediates 1a (78%) and 1b (76%) were obtained in good yields. Treatment with a base and allyl bromide, followed by a reaction with vinylmagnesium bromide at −30 • C, gave the precursors of the ring-closing metathesis reaction (RCM). For the RCM, a second-generation Grubbs' catalyst (Grubbs' Catalyst ® M204, 3 mol %) was used, affording the α,β-unsaturated ketones 2a and 2b in 76% and 60% yield, respectively (Scheme 1) [27]. These compounds reacted with tert-butyl 2-(tetrahydro-1λ 4 -thiophen-1-ylidene)acetate to give a mixture (as seen in 1 H-NMR) of cyclopropane exo:endo isomers 3a (77%) and 4a (5%) from 2a in a 15:1 ratio, while 3b (71%) and 4b (18%) from 2b in a 4:1 exo:endo ratio [28]. These isomers were isolated and separately characterized. 1 H-NMR at 90 • C in DMSO-d 6 determined that compounds 3b and 4b were obtained as a mixture of conformers due to the slow rotation of the Boc group in a 55:45 ratio in both cases ( Figure 3). obtained as a mixture of conformers due to the slow rotation of the Boc group in a 55:45 ratio in both cases ( Figure 3).  The cyclopropanation reaction has two steps, ylide addition to the double bond and ring closure. It is known that the sulfur ylide attack is nonselective, and the exo:endo selectivity is determined in the second step [29][30][31]. The result depends on many different issues, such as temperature, reagent concentration, and the presence of a base. For instance, in the cyclopropanation reaction of 2a, the 15:1 exo:endo ratio was observed when carrying out the reaction at 0 °C and a low concentration of ylide (0.15 M), while a higher temperature and ylide concentration promoted higher ratios in favor of an exo isomer. In the case of the cyclopropanation of compound 2b with an ylide concentration of 2M and   The cyclopropanation reaction has two steps, ylide addition to the double bond and ring closure. It is known that the sulfur ylide attack is nonselective, and the exo:endo selectivity is determined in the second step [29][30][31]. The result depends on many different issues, such as temperature, reagent concentration, and the presence of a base. For instance, in the cyclopropanation reaction of 2a, the 15:1 exo:endo ratio was observed when carrying out the reaction at 0 °C and a low concentration of ylide (0.15 M), while a higher temperature and ylide concentration promoted higher ratios in favor of an exo isomer. In the case of the cyclopropanation of compound 2b with an ylide concentration of 2M and The cyclopropanation reaction has two steps, ylide addition to the double bond and ring closure. It is known that the sulfur ylide attack is nonselective, and the exo:endo selectivity is determined in the second step [29][30][31]. The result depends on many different issues, such as temperature, reagent concentration, and the presence of a base. For instance, in the cyclopropanation reaction of 2a, the 15:1 exo:endo ratio was observed when carrying out the reaction at 0 • C and a low concentration of ylide (0.15 M), while a higher temperature and ylide concentration promoted higher ratios in favor of an exo isomer. In the case of the cyclopropanation of compound 2b with an ylide concentration of 2M and temperature of 20 • C, the ratio of exo/endo isomers can increase up to 20:1, as seen by NMR. Similar behavior has already been reported [32]. With the intermediates 3 and 4 in hand, the final products were prepared by a reduction of the ketone and further hydrolysis or reduction of the ester group. Thus, compound 3a was treated with NaBH 4 to give isomers 5a and 5b in a 3:2 ratio (Scheme 2). These alcohols were isolated in 55% (5a) and 19% (5b) yields and separately treated with diisobutylaluminium hydride (DIBAL-H), giving the corresponding intermediates in 55% and 67% yields, respectively. A final treatment with trifluoroacetic acid (TFA) afforded compounds 6a (85%) and 6b (83%) after a final elution through a basic DOWEX resin. This two-step reduction allowed the separation of the isomers 5a and 5b. When using stronger conditions to perform the reduction in one step from 3a, a mixture of diols was obtained but could not be separated. On the other hand, isomer 4a reacted with DIBAL-H to give only a product in 32% yield, which, after hydrolysis with TFA and further elution through a basic DOWEX resin, afforded free amine 6c (81%). temperature of 20 °C, the ratio of exo/endo isomers can increase up to 20:1, as seen by NMR. Similar behavior has already been reported [32]. With the intermediates 3 and 4 in hand, the final products were prepared by a reduction of the ketone and further hydrolysis or reduction of the ester group. Thus, compound 3a was treated with NaBH4 to give isomers 5a and 5b in a 3:2 ratio (Scheme 2). These alcohols were isolated in 55% (5a) and 19% (5b) yields and separately treated with diisobutylaluminium hydride (DIBAL-H), giving the corresponding intermediates in 55% and 67% yields, respectively. A final treatment with trifluoroacetic acid (TFA) afforded compounds 6a (85%) and 6b (83%) after a final elution through a basic DOWEX resin. This two-step reduction allowed the separation of the isomers 5a and 5b. When using stronger conditions to perform the reduction in one step from 3a, a mixture of diols was obtained but could not be separated. On the other hand, isomer 4a reacted with DIBAL-H to give only a product in 32% yield, which, after hydrolysis with TFA and further elution through a basic DOWEX resin, afforded free amine 6c (81%). Scheme 2. Reagents and conditions: (a) NaBH4, EtOH, 0 °C to r.t., 55% (5a) and 19% (5b). (b) DIBAL-H, DCM, 0 °C to r.t., 55% (from 5a), 67% (from 5b), and 32% (from 4a). (c) Trifluoroacetic acid (TFA), MeOH, r.t., 85% (6a), 83% (6b), and 81% (6c).
Compound 3b diastereoselectively reacted with NaBH4, resulting in 7 as the only reaction product. 4b reacted, giving a separable mixture of compounds 8 and 9. The selectivity of this reduction is governed by the configuration of the cyclopropane ring but not by the location of the bulky tert-butyldimthylsilyl (OTBS) group; as in the case of 4b, the hydride reacts by the face of the OTBS group. The Felkin-Anh model corroborates these results: on the exo isomer 3b, the tert-butyl ester group gets far from the carbonyl, but the OTBS is the one making the steric hindrance to determine the stereochemistry of the reaction. On the other hand, the endo isomer 4b has both bulky groups near the carbonyl, staying close to the ester. Moreover, CH2OTBS can rotate to get farther from carbonyl, while the tert-butyl ester cannot ( Figure 4). Deprotection of the hydroxyl group of compound 7 using tetrabutylammonium fluoride trihydrate (TBAF.3H2O) and further treatment with trifluoroacetic acid (TFA) resulted in the final product 10a as a trifluoroacetate salt (Scheme 3) [33]. On the other hand, the reduction of the tert-butyl ester in 7 with DIBAL-H gave the N-Boc and OTBS protected intermediate, which was treated as previous to give the corresponding trifluoroacetate salt. After treatment of this salt with a basic DOWEX resin, the final compound 11a was Compound 3b diastereoselectively reacted with NaBH 4 , resulting in 7 as the only reaction product. 4b reacted, giving a separable mixture of compounds 8 and 9. The selectivity of this reduction is governed by the configuration of the cyclopropane ring but not by the location of the bulky tert-butyldimthylsilyl (OTBS) group; as in the case of 4b, the hydride reacts by the face of the OTBS group. The Felkin-Anh model corroborates these results: on the exo isomer 3b, the tert-butyl ester group gets far from the carbonyl, but the OTBS is the one making the steric hindrance to determine the stereochemistry of the reaction. On the other hand, the endo isomer 4b has both bulky groups near the carbonyl, staying close to the ester. Moreover, CH 2 OTBS can rotate to get farther from carbonyl, while the tert-butyl ester cannot ( Figure 4). temperature of 20 °C, the ratio of exo/endo isomers can increase up to 20:1, as seen by NMR. Similar behavior has already been reported [32]. With the intermediates 3 and 4 in hand, the final products were prepared by a reduction of the ketone and further hydrolysis or reduction of the ester group. Thus, compound 3a was treated with NaBH4 to give isomers 5a and 5b in a 3:2 ratio (Scheme 2). These alcohols were isolated in 55% (5a) and 19% (5b) yields and separately treated with diisobutylaluminium hydride (DIBAL-H), giving the corresponding intermediates in 55% and 67% yields, respectively. A final treatment with trifluoroacetic acid (TFA) afforded compounds 6a (85%) and 6b (83%) after a final elution through a basic DOWEX resin. This two-step reduction allowed the separation of the isomers 5a and 5b. When using stronger conditions to perform the reduction in one step from 3a, a mixture of diols was obtained but could not be separated. On the other hand, isomer 4a reacted with DIBAL-H to give only a product in 32% yield, which, after hydrolysis with TFA and further elution through a basic DOWEX resin, afforded free amine 6c (81%). Scheme 2. Reagents and conditions: (a) NaBH4, EtOH, 0 °C to r.t., 55% (5a) and 19% (5b). (b) DIBAL-H, DCM, 0 °C to r.t., 55% (from 5a), 67% (from 5b), and 32% (from 4a). (c) Trifluoroacetic acid (TFA), MeOH, r.t., 85% (6a), 83% (6b), and 81% (6c).
Compound 3b diastereoselectively reacted with NaBH4, resulting in 7 as the only reaction product. 4b reacted, giving a separable mixture of compounds 8 and 9. The selectivity of this reduction is governed by the configuration of the cyclopropane ring but not by the location of the bulky tert-butyldimthylsilyl (OTBS) group; as in the case of 4b, the hydride reacts by the face of the OTBS group. The Felkin-Anh model corroborates these results: on the exo isomer 3b, the tert-butyl ester group gets far from the carbonyl, but the OTBS is the one making the steric hindrance to determine the stereochemistry of the reaction. On the other hand, the endo isomer 4b has both bulky groups near the carbonyl, staying close to the ester. Moreover, CH2OTBS can rotate to get farther from carbonyl, while the tert-butyl ester cannot ( Figure 4). Deprotection of the hydroxyl group of compound 7 using tetrabutylammonium fluoride trihydrate (TBAF.3H2O) and further treatment with trifluoroacetic acid (TFA) resulted in the final product 10a as a trifluoroacetate salt (Scheme 3) [33]. On the other hand, the reduction of the tert-butyl ester in 7 with DIBAL-H gave the N-Boc and OTBS protected intermediate, which was treated as previous to give the corresponding trifluoroacetate salt. After treatment of this salt with a basic DOWEX resin, the final compound 11a was Deprotection of the hydroxyl group of compound 7 using tetrabutylammonium fluoride trihydrate (TBAF.3H 2 O) and further treatment with trifluoroacetic acid (TFA) resulted in the final product 10a as a trifluoroacetate salt (Scheme 3) [33]. On the other hand, the reduction of the tert-butyl ester in 7 with DIBAL-H gave the N-Boc and OTBS protected intermediate, which was treated as previous to give the corresponding trifluoroacetate salt. After treatment of this salt with a basic DOWEX resin, the final compound 11a was obtained. Compound 8 was deprotected to give compound 10b as a trifluoroacetate salt. Finally, compound 9 was treated similarly to afford final compound 11b.

Modeling and NOE Experiments
The stereochemistry of all the synthesized products was assigned by means of NO-ESY experiments and coupling constant calculations. All compounds were first modeled on Chimera 1.13.1, using ANTECHAMBER for the computing charges [34]. With these models, we could calculate the relevant dihedral angles and predict the expected NOE effects, which were checked with those experimentally obtained. Figure 5 shows the models and main NOE effects of compounds 5a and b and compound 6c derived from L-alanine. H5 in compound 5a shows a NOE interaction with H4 and H7. On the other hand, the other reduction isomer, 5b, gave a NOE signal between H5 and H6 and an intense effect between H5 and the methyl group. The final product 6c obtained from the endo isomer, after the cyclopropanation reaction, gave analog signals as 5b.

Modeling and NOE Experiments
The stereochemistry of all the synthesized products was assigned by means of NOESY experiments and coupling constant calculations. All compounds were first modeled on Chimera 1.13.1, using ANTECHAMBER for the computing charges [34]. With these models, we could calculate the relevant dihedral angles and predict the expected NOE effects, which were checked with those experimentally obtained. Figure 5 shows the models and main NOE effects of compounds 5a and b and compound 6c derived from L-alanine. H5 in compound 5a shows a NOE interaction with H4 and H7. On the other hand, the other reduction isomer, 5b, gave a NOE signal between H5 and H6 and an intense effect between H5 and the methyl group. The final product 6c obtained from the endo isomer, after the cyclopropanation reaction, gave analog signals as 5b.
The models and NOE interactions for compounds 7 and 11b are shown in Figure 6. Compound 7 was assigned with the NOE effects observed between H7 and H5, H5 and H4, and between H7 and H4. The constant couplings measured in 1 H-NMR also agreed with the modeled angles. On the other hand, compound 11b only showed one NOE signal between H5 and H6 ( Figure 6). The measured coupling constant agreed with the calculated ones from models using the Carplus equation (see Supporting Information Table S1). The models and NOE interactions for compounds 7 and 11b are shown in Figure 6. Compound 7 was assigned with the NOE effects observed between H7 and H5, H5 and H4, and between H7 and H4. The constant couplings measured in 1 H-NMR also agreed with the modeled angles. On the other hand, compound 11b only showed one NOE signal between H5 and H6 ( Figure 6). The measured coupling constant agreed with the calculated ones from models using the Carplus equation (see Supporting Information Table  S1).

Enzymatic Assays
Glycosidase activities were assessed in 80-μL reaction volumes in Eppendorf vials. Buffer composition and enzyme concentrations were adjusted depending on the enzyme: 20-mM Na2HPO4 at pH 7.3 for β-glucosidase from Almonds (3 μg/mL) and β-galactosidase from Escherichia coli (1 μg/mL), 20-mM Na2HPO4 at pH 6.8 for α-glucosidase from Bacillus stearothermophilus (1 μg/mL) and α-galactosidase from Green coffee (20 μM), 20-mM NaH2PO4 at pH 5.5 for α-and β-mannosidase from Jack beans and Helix pomatia, respectively (7 μM and 2 μM, respectively), 0.1-M NaOAc at pH 4.0 with 1 mg/mL of BSA (bovine serum albumin) for α-L-fucosidase from Homo sapiens (2 μM), and 50-mM NaOAc at pH 5.0 for neuraminidase from Vibrio cholerae (6 μM). The inhibitors were tested at 1-, 5-, and 25-mM final concentrations in the assays. Each mixture of enzyme and inhibitor  The models and NOE interactions for compounds 7 and 11b are shown in Figure 6. Compound 7 was assigned with the NOE effects observed between H7 and H5, H5 and H4, and between H7 and H4. The constant couplings measured in 1 H-NMR also agreed with the modeled angles. On the other hand, compound 11b only showed one NOE signal between H5 and H6 ( Figure 6). The measured coupling constant agreed with the calculated ones from models using the Carplus equation (see Supporting Information Table  S1).

Enzymatic Assays
Glycosidase activities were assessed in 80-µL reaction volumes in Eppendorf vials. Buffer composition and enzyme concentrations were adjusted depending on the enzyme: 20-mM Na 2 HPO 4 at pH 7.3 for β-glucosidase from Almonds (3 µg/mL) and βgalactosidase from Escherichia coli (1 µg/mL), 20-mM Na 2 HPO 4 at pH 6.8 for α-glucosidase from Bacillus stearothermophilus (1 µg/mL) and α-galactosidase from Green coffee (20 µM), 20-mM NaH 2 PO 4 at pH 5.5 for αand β-mannosidase from Jack beans and Helix pomatia, respectively (7 µM and 2 µM, respectively), 0.1-M NaOAc at pH 4.0 with 1 mg/mL of BSA (bovine serum albumin) for α-L-fucosidase from Homo sapiens (2 µM), and 50-mM NaOAc at pH 5.0 for neuraminidase from Vibrio cholerae (6 µM). The inhibitors were tested at 1-, 5-, and 25-mM final concentrations in the assays. Each mixture of enzyme and inhibitor was homogenized and preincubated for 10 min at 37 • C or 40 • C (α-L-fucosidase). Each reaction was initiated and brought to a final volume of 80 µL by the addition of an aliquot of the corresponding p-nitrophenyl glycoside substrate to obtain the following final concentrations in the reaction mixtures: p-nitrophenyl αand β-D-glucopyranoside (1 mM), p-nitrophenyl αand β-D-galactopyranoside (0.5 mM), p-nitrophenyl αand β-D-mannopyranoside (1 mM), p-nitrophenyl α-L-fucopyranoside (1 mM), or p-nitrophenyl neuraminic acid (1 mM). After 10 min of incubation time at the same temperature, each reaction was quenched with 400 µL of 1.0-M Na 2 CO 3 , and the absorbance at 405 nm was measured. Assays were repeated twice, and the data was averaged. The enzymatic activity was calculated by the ratio in the absorbance measured at 405 nm of the phenoxide released in the enzymatic reaction. The final compounds were screened at 1, 5, and 25 mM. The assays at 1 mM of synthesized compounds did not give clear results. Thus, the activity detected at 5 mM is shown in Table 1. None of the synthesized compounds showed activity against α-glucosidase, α-mannosidase, or neuraminidase at the concentrations used. In the case of products 11a and 6a, no activity was observed against any of the enzymes. Interestingly, products 6c and 10b inhibited only one enzyme, β-glucosidase, decreasing their activity to 43% and 39% at 5 mM, respectively. The results at 25 mM were 20% and 13% of the residual activity, respectively. Product 11b inhibited the activity of β-galactosidase to 25% at 5 mM but without an inhibition increase at 25 mM.
On the other hand, some assays showed an enhancement in the enzyme activity. Thus, compound 6b increased the activity of β-mannosidase and α-L-fucosidase up to 148% and 142% at 5 mM, respectively. This increase raised up to 240% at 25 mM. Compound 10a activated α-galactosidase and β-mannosidase up to around 155% at 5 mM.
Regarding the inhibitory results, we can conclude that these compounds are very weak inhibitors only against certain enzymes far from the inhibition values of well-known iminosugars such as deoxinojirimycin [35] or castanospermine analogs [36]. However, the activation observed in certain cases deserves some comments, as there are few precedents of this behavior [37,38], including our previous results with similar compounds [10]. This activation does not have a clear explanation. The possibility that the compounds could work as efficient transglycosidation acceptors and, thus, accelerate the nitrophenol release was checked following the enzymatic reaction in a NMR tube and recorded spectra each 5 min. However, no potential transglycosilation product was detected (see the Supporting Information). Other cases of glycosidase activity enhancements were described; thus, some glycosidases were found to activate when using multivalent iminosugars [37]. Other reports explained the activation mechanism by the introduction of a small molecule in the active site, locking the reactive form of the glycosidase [38], or through an allosterictype interaction that changed the conformation of the enzyme into the active one. These observations need further research to explain this behavior.

Conclusions
We described the synthesis of bicyclic piperidine-based iminosugars from natural amino acids L-alanine and L-serine. The procedure involves the preparation of enantiomerically pure α,β-unsaturated ketones in four steps and high yields from the natural amino acids. These intermediates, upon a stereoselective cyclopropanation reaction and further straightforward transformations, give the final products, which contain five stereogenic centers. The synthetic methodology used allows the obtention of different configurations at some of the asymmetric carbons, which, in this project, is interesting, because selectivity towards different enzymes could be achieved. The behavior of the products against different glycosidases showed that inhibition was generally low but selective towards one or two enzymes. The activation of the target enzymes was observed in some cases.

Supplementary Materials:
The following are available online, 1D and 2D NMR spectra of all new compounds (Figures S1-S72). Figure S41: Transglycosidation monitorization by 1 H-NMR). Table S1: Measured constant coupling from products derived from L-serine.
Author Contributions: Conceptualization of the work, J.P.-C. and G.D.; methodology, all authors; synthetic work, A.P.; biological assays, A.P. and F.J.C.; writing-original draft preparation, A.P. and J.P.-C.; writing-review and editing, all authors; and funding acquisition, J.P.-C. and F.J.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Spanish MINECO, grant numbers RTI2018-095588-B-I00 and RTI2018-094751-B-C22 (co-funded by the European Regional Development Fund/European Social Fund, "Investing in your future"), FUSP-CEU (PC17/17), and CIBERES, an initiative from the Spanish Institute of Health Carlos III.