Stereoselective Synthesis of a 4- -Glucoside of Valienamine, Inhibition, and X-Ray Structural Studies Involving a Bacterial Glycoside Hydrolase-Like Enzyme of The GH13 Family


 Glycoside hydrolases (GH) are a large family of hydrolytic enzymes found in all domains of life. As such, they control a plethora of normal and pathogenic biological functions. Thus, understanding selective inhibition of GH enzymes at the atomic level can lead to the identification of new classes of therapeutics. In these studies, we identified a 4-⍺-glucoside of valienamine (8) as an inhibitor (IC50 of 484 ± 51 µM) of Streptomyces coelicolor (Sco) GlgE1-V279S which belongs to the GH13 Carbohydrate Active EnZyme (CAZy) family. The synthetic route to 8 and a closely related 4-⍺-glucoside of validamine (7) was achieved starting from readily available D-maltose. A key step in the synthesis was a chelation-controlled addition of vinylmagnesium bromide to a maltose-derived enone intermediate. X-ray structures of both 7 and 8 in complex with Sco GlgE1-V279S were solved to resolutions of 1.75 and 1.78 Å, respectively. Structural analysis revealed the valienamine derivative 8 binds the enzyme in an E2 conformation for the cyclohexene fragment. Also, the cyclohexene fragment shows a new hydrogen-bonding contact from the pseudo-diaxial C(3)-OH to the catalytic nucleophile Asp 394 at the enzyme active site. Asp 394, in fact, forms a bidentate interaction with both the C(3)-OH and C(7)-OH of the inhibitor. In contrast, compound 7 disrupts the catalytic sidechain interaction network of Sco GlgE1-V279S via steric interactions resulting in a conformation change in Asp 394. These findings will have implications for the design other aminocarbasugar-based GH13-inhibitors and will be useful for identifying more potent and selective inhibitors.


Introduction
Glycoside hydrolase (GH) enzymes are hydrolases involved in a large array of biological phenomena. [1][2][3] Inhibiting the GH activity alters the glycosylation or catabolism of glycans, making the GH inhibitors highly potent drugs. [4][5][6][7] An important class of GH inhibitors in glycobiology are the carbasugars, wherein the endocyclic ring oxygen is replaced by a methylene group or an sp 2 hybridized carbon. 8,9 Some of these pseudosaccharides may mimic the transition state (TS) of the catalyzed reaction while others may mimic the Michaelis complex. 4 The exact nature of GH inhibition by carbasugars remains complex as to whether they are actual TS inhibitors or merely tight binders. 10-13 1-Aminocarbasugars are thought to mimic charge development in the TS by incorporating a basic nitrogen atom in place of C(1)-O. 14 One example of this compound class is Acarbose (1), an inhibitor of the GH13 enzyme human pancreatic αamylase and a treatment for type-II diabetes mellitus (T2D), Figure 1, 15,16 while aminocarbasugars related to validamine (2a) and valienamine (6) 17,18 show inhibitory activity against α-GHs such as α-amylase, rice α-glucosidase and yeast α-glucosidase. 19,20 IC 50 values for compounds 2 -6 against α-amylase are in the range of 10 -2 mol/L and against yeast α-glucosidase is between 10 -5 -10 -4 mol/L. 19 Voglibose (5), a derivative of 2a, has also found use as an α-glucosidase inhibitor and treatment for T2D. 20 The inhibitory activities of 1 and related compounds has been commonly ascribed to the ability to mimic a glucopyranosyl half-chair conformation intermediate of glycosidase catalysis 12,15,21 and form favorable coulombic interactions. 22 For valienamine-base carbasugars, an initial Michaelis complex in the 2 H 3 against various α-glucosidases like rat intestinal maltase, sucrase, glucoamylase, isomaltase and trehalase suggested that 2a, 6, 7 and 8 are highly potent (IC 50 = 10 -5 to 10 -2 M). 17,18 However, relying on natural products and enzymatic methods to obtain 7 and 8 is very challenging due to the availability of biological sources, complex puri cations, and complex spectroscopic characterization of closely related regioisomers. In order to overcome these di culties, the development of an e cient synthetic strategy for the aminocarbasugars 7 and 8 was developed. In 1999, Kapferer et al. reported a practical synthetic route to the formation of valienamine (6) that involved a stereoselective addition of vinyl magnesium bromide to give separable dienes followed by a ring closing metathesis to give a cyclohexene adduct which could be transformed to the desired 6. 34 In this work, we applied this method to the regiospeci c synthesis of aminocarbasugars 7 and 8. Scheme 1 illustrates the retrosynthetic route for the desired target inhibitors. Compounds 7 and 8 were then prepared in fourteen steps using commercially available D-(+)-maltose as the starting material.

General methods
All chemicals and solvents were purchased from Fisher Scienti c, Acros Organics, Alfa Aesar or Sigma-Aldrich. Solvents were dried by through solvent puri cation system by passing through activated alumina and copper catalyst columns. All reactions were carried out at room temperature under a nitrogen atmosphere using a nitrogen balloon unless mentioned otherwise. Reactions were monitored by TLC (silica gel, f 254 ) under UV light or by charring (5% H 2 SO 4 -MeOH) and the puri cation was performed by column chromatography on silica gel (230-400 mesh) using the solvent system speci ed, solvents were used without puri cation for chromatography. 1 H NMR was recorded on Bruker Avance III 600 MHz spectrometer using CDCl 3 and D 2 O as an internal reference. 13 C were recorded on Bruker Avance III 600 MHz spectrometer using CDCl 3 and D 2 O as internal reference. High resolution mass spectrometry was recorded on TOF MS-ES+ instrument. Low resolution mass spectrometry was recorded on ESquire-LC-MS.

Metathesis procedure
A solution of dialkene in dichloromethane was degassed by passing nitrogen gas through it for 20 minutes. After that, 1 st generation Grubb's catalyst (10 mol%) was added to the solution and the reaction was kept under nitrogen atmosphere using nitrogen balloon for 7 days or until the catalyst turned dark brown or black. Then, everything was evaporated under reduced pressure and puri ed using ash column chromatography on silica gel. 1,2,3,6,2' ,3' ,4' ,6'-octa-O-acetyl-D-maltose (21) 65 D-(+)-Maltose monohydrate (2.00 g, 5.84 mmol) was suspended in dry pyridine (7.20 mL, 89.0 mmol) and acetic anhydride (6.28 mL, 66.8 mmol). A catalytic amount of 4-(dimethylamino)pyridine was added to the solution. The solution was stirred at ambient temperature for 16 h. The reaction mixture was diluted with ethyl acetate and washed successively with 1 N HCl (20 mL) and saturated aq. NaHCO 3 (40 mL).
The resulting organic phase was dried with anhydrous Na 2 SO 4 , ltered and the ltrate was concentrated under reduced pressure to give product 21: yield 95% (3.50 g); silica gel TLC R f = 0.5 (50% ethyl acetate : hexane). All the NMR values match with the reported data. 65 4-methylphenyl 1-thio-β-D-maltopyranopyranoside (22) 66 Peracetylated maltose 21 (3.45 g, 5.08 mmol) was dissolved in dichloromethane. 4-Methylthiophenol (1.26 g, 10.2 mmol) and boron tri uoride diethyl etherate (0.75 mL, 6.10 mmol) were added to the solution at 0 °C. The resulting solution was stirred at ambient temperature under nitrogen atmosphere. The reaction was monitored by TLC and appeared to be complete after 4 h. The reaction mixture was diluted with dichloromethane (20 mL) and successively washed with saturated aq. NaHCO 3 (25 mL), brine (25 mL), and water (15 mL). The resulting organic phase was dried (anhydrous Na 2 SO 4 ), ltered and the ltrate was concentrated under reduced pressure. The product was dried overnight and deacetylated by dissolving in dry methanol followed by addition of a catalytic amount of sodium metal until the solution reached pH 9. The reaction was monitored for completion using TLC. The reaction was neutralized by adding Amberlite IRA-118H H + resin until the pH reached 7. Then, the resin was ltered away and the ltrate was concentrated under reduced pressure to afford the 4-methylthiophenyl maltoside 22: yield 90% (3.10 g). All the NMR values match with the reported data. 66 4-methylthiophenyl 2,3,6-Tri-O-benzyl-4-O-(2',3',4',6'-tetra-O-benzyl-α-D-glucopyranosyl)-β-Dglucopyranoside (23) 67 A solution of 22 (2.95 g, 9.04 mmol) in dry N,N-dimethylformamide (50 mL) was cooled to 0 °C. The solution was treated drop-wise with a suspension of sodium hydride (60% dispersion in mineral oil) (4.55 g, 114.0 mmol) in dry N,N-dimethylformamide. Benzyl bromide (11.6 mL, 95.3 mmol) was added dropwise over 15 min. and the solution stirred at room temperature for 16 h. The reaction was poured over ice and extracted with ethyl acetate (50 mL). The combined organic layers were washed with brine. The resulting organic phase was dried (anhydrous Na 2 SO 4 ), ltered and the ltrate was concentrated under reduced pressure to obtain a product. The product was puri ed by silica gel ash column chromatography. The product fractions were combined, concentrated, and dried in vacuum to afford a yellow oily product 23: yield 85% (2.50 g); silica gel TLC R f = 0.61 (25% ethyl acetate : hexane). All the NMR values match with the reported data. 67 2,3,6-Tri-O-benzyl-4-O-(2',3',4',6'-tetra-O-benzyl-α-D-glucopyranosyl)-α/β-D-glucopyranoside (24) 44 N-bromosuccinimide (0.90 g, 5.08 mmol) was added to a solution of 6 (2.47 g, 2.45 mmol) in 9:1 acetonewater (50 mL) and stirred at room temperature for 45 min. The solvent was evaporated at room temperature until turbid. A solution of the residue in ethyl acetate (100 mL) was washed successively with saturated aqueous NaHCO 3 , (3 X 50 mL) and water (3 X 50 mL). The solution was dried with anhydrous Na 2 SO 4 and evaporated. The product was puri ed by silica gel ash column chromatography.
The resulting mixture was slowly warmed to room temperature and stirred for 1 h. In a separate ask, sodium hydride suspension in 60% mineral oil (0.01 g, 0.39 mmol) was added to a solution of benzyl alcohol (41.0 µL, 0.39 mmol) in dry tetrahydrofuran (2 mL) at 0 °C. After 1 hr, the solution of sodium benzyloxide was added to the generated isocyanate and the reaction progress was followed by TLC. After 24 h. volatiles are removed under reduced pressure. The reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (10 mL X 4). The organic layer was separated washed with brine (10 mL X 2), dried over anhydrous Na 2 SO 4 . The solvent was evaporated under reduced pressure and puri cation was performed by ash column chromatography on silica gel with 1:5 ethyl acetate-hexane to afford product as colorless liquid 30: yield 60% (0.13 g); silica gel TLC R f = 0.43 (30% ethyl acetate : 3.47 (dd, J = 10.6, 1.9 Hz, 1H, H-6b'). 13  L-chiro-Inisitol-1-amino-1,5,6-trideoxy-4-O-(α-D-glucopyranosyl)-5-(hydroxymethyl) or 4-α-glycoside derivative of validamine (7) Compound 30 (40.0 mg, 0.04 mmol) was dissolved in 10 mL of methanol and 20% Pd(OH) 2 /C on carbon (20.0 mg) was added. The mixture was stirred for 16 h. under hydrogen (1 atm.). The catalyst was ltered away through a plug of Celite® 545 and washed with methanol (5mL). The combined ltrate and washings were concentrated to dryness and the residue was passed through reverse phase C-18 silica gel (washed with water) to afford 7 as a white solid: yield quantitative (5.00 mg); 1 27,52 In the various reactions, the compound 8 concentration was varied from 0 to 1000 µM, except in the positive control that lacked inhibitor, and the negative control that lacked enzyme. All of the reactions were performed in triplicate using a Synergy H4 plate reader (Bio Tek) to measure the absorbance at 360 nm. The percent enzymatic activity was calculated by using the equation (V/V 0 ) × 100, where V and V 0 denote the rates of the inhibited and uninhibited enzyme (positive control), respectively. The percent enzymatic activity vs inhibitor concentration was plotted using Prism 8 and the equation y = 100/ (1+( IC 50 /x) Hillslope ) was used to determine the IC 50 . Using this assay, 100 µM concentration of both 7 and 8 were tested separately against Sco GlgE1-V279S. Dose-response experiments for 8 were performed as described above while varying the concentration of 8.

Crystallization of Sco GlgE1-V279S complexes
Prior to the experiment, 20 mM aqueous solutions of 7 and 8 were prepared. The protein-compound mixture was prepared by mixing the Sco GlgE1-V279S with each compound separately to a nal concentration as 8 mg/mL 51 of Sco GlgE1-V279S and 10 mM of the respective compound. The hanging drop vapor diffusion method was used for the crystallization and each crystallization drop consisted with 2 µL of Sco GlgE1-V279S/compound mixture and 2 µL of the well solution (0.2 mM Sodium citrate pH 7 and 10 % PEG 3,350). 45 The crystallization drop was equilibrated against 100 µL of the well solution and crystals were formed within 3 days. Following crystal formation, an additional 4 µL of the appropriate 20 mM compound stock was added to the drop and incubated for an additional 12 hours. Crystals were cryoprotected by adding 4 µL of 50% PEG 2000 to the crystallization drop, and crystals were ash-cooled using liquid nitrogen in preparation for the cryo-crystallographic experiments.

X-ray Diffraction experiments
The LS-CAT beamline at Advanced Photon Source of Argonne National Labs, IL was used to perform the X-ray diffraction experiments. The X-ray diffraction data corresponding to the Sco GlgE1-V279S/7 and Sco GlgE1-V279S/8 complexes were indexed and reduced, using DIALS, iMos m, and Scala in the CCP4 suite. The re nements were carried out by using PHENIX, and the previously published Sco GlgE1-V279S in complex with maltose-C-phosphonate (RCSB accession number 4U31) was used for difference Fourier analysis to calculate phases and difference maps prior to manual and computational re nement. 68 The visualization and manual re nements of the structures were carried out using COOT. 69 The nal occupancy of each ligand was changed manually and then re ned in PHENIX.

Results And Discussion
Before synthesizing the target pseudodisaccharides, we synthesized valienamine (6) from commercially available D-(+)-glucose (9) to obtain spectroscopic diastereoselectivity metrics for the intermediates afforded by the stereoselective addition of vinylmagnesium bromide to ketone 15, Scheme 2. The spectral analysis of the intermediates 16A, 16B, 17A, and 17B proved critical in analyzing the desired diastereomer of the maltosyl analogues. Compound 15 was obtained starting from 9 in seven steps that involved Wittig ole nation and Moffatt oxidation (Scheme S1 in Supporting Information). 27,35,36 The key step in the synthesis is the diastereoselective addition of vinylmagnesium bromide to enone 15 to afford separable diastereomers 16A and 16B (9:1). The high selectivity for the conformer 16A over the conformer 16B can be explained by Cram's-chelate TS model. 37,38 According to previous reports, the torsional strain in enone 15 implies a staggered conformation in the TS where the nucleophile attacks preferentially from the si face, Scheme 2. 34,37,39 Moreover, the down eld shift of the C(6)-OH signal of 16A relative to the chemical shift of C(6)-OH of 16B indicated a strong intramolecular H-bond (sixmembered ring) to C(4)-OBn in 16A and a ve-membered H-bond to C(9)-OBn in 16B, Figure 3.
Although, there is little to distinguish the relative stereochemistry of 16A and 16B, after the intramolecular ring closing metathesis (RCM), Kapferer et al. assigned the absolute stereochemistry of the carbasaccharides 17A and 17B by comparing the spectroscopic data and Nuclear Overhauser Effect (NOE) experiments. 34 Finally, 17A was converted to valienamine (6) in three steps involving Ichikawa rearrangement, (Scheme S1 in Supporting Information). 40 The spectral data of all the intermediates were found nearly identical to the previously reported data (Supporting Information). 34,41 After successfully synthesizing valienamine (6), we synthesized the pseudodisaccharides 7 and 8 starting from D-(+)-maltose, Scheme 3, and the intermediates of Scheme 2 were used to assign the diastereomers. D-(+)-maltose (20) was peracetylated with acetic anhydride followed by the treatment with p-thiocresol and boron-tri uoride diethyl etherate to generate a peracetylated β-thiomaltoside. 42 Subsequently, deacetylation under Zemplén conditions afforded glycoside 22 followed by benzylation to afford perbenzylated thiomaltosid e (23) in 85% yield. 43 We treated thiomaltoside 23 with Nbromosuccinimide (NBS) in acetone:water (9:1) to obtain the reducing sugar 24. 44 Wittig methylenation of 24 using methyltriphenylphosphonium bromide in the presence of n-butyl lithium afforded alkene 25 in 55% yield. 45 After that, the secondary alcohol 25 was oxidized under Moffatt oxidation conditions to afford ketone 26 in 90% yield. 36 Compound 26 was subjected to the stereoselective addition using vinylmagnesium bromide in dry THF to afford diastereomeric dienes 27A and 27B in a 4:1 ratio, respectively (Scheme 3). Hz) values for 27A are smaller than the J(9a, 9b = 9.2 Hz) values for 27B. Indeed, a similar pattern is noticed in the glucosyl dienes suggesting that 27A and 27B possess the same conformation as 16A J(9a, 9b = 8.7 Hz) and 16B J(9a, 9b = 9.3 Hz) respectively. However, the exact stereochemistry of the diastereomers 27A and 27B could not be con dently con rmed using only the 1 H NMR chemical shift and J values.
Complete structure elucidation of the desired epimer was achieved by further subjecting the dienes 27A and 27B to RCM using Grubb's 1st generation catalyst, Scheme 4. The reaction was optimized by using various conditions to improve the yield, Table 2. The highest yield (25%) was obtained using Grubb's rstgeneration catalyst under DCM re ux conditions.  34 The C-atoms carrying an equatorial substituent, especially oxy groups, resonate at a higher eld than those with axial substituents, justifying the 13 C NMR chemical shifts for C(4) of 28A (73.10 ppm) and 28B (75.07 ppm) ( Table 3). 46 A similar trend in the 13 C NMR chemical shift values was also observed in the glucosyl diastereomers The synthesis continued with the protection of the tertiary alcohol of 28A with trichloroacetyl isocyanate in dichloromethane (DCM) at 0 °C, followed by hydrolysis with K 2 CO 3 in aqueous MeOH giving an 85% yield of the carbamate 29. Compound 29 was subjected to an Ichikawa rearrangement 40 that proved useful in the synthesis of valienamine (6). 34 Here, the dehydration of 29 with tri uoroacetic anhydride (TFAA) and Et 3 N at -20 °C formed an intermediate allylic cyanate that underwent a spontaneous [3,3]sigmatropic rearrangement to afford the isocyanate. The isocyanate was treated in situ with 60% NaH in mineral oil and benzyl alcohol solution to yield protected maltosyl valienamine analog 30 in 60% overall yield, Scheme 5.

Kozikowski et al. reported that hydrogenation of substituted cyclohexenes yields two diastereomers
where the major isomer has the hydroxy methylene at the equatorial position. 47 However, hydrogenation of 30 using 20% Pd(OH) 2 /C afforded a single isomer 7 in a quantitative yield. We compared the 1 H NMR chemical shift values of the obtained compound with reported validamine (2a) 48 and epi-validamine (2b) 49 to determine the stereochemistry at C(5), Table 4. Although the proton chemical shift value of 7 at C(5) is similar to 2b, no sound conclusion can be derived due to difference in the chemical environment. Therefore, we compared the C(6) protons of 7 with the C(6) protons of 2a and 2b. For epi-validamine, the Ha-(6) and Hb-(6) appear at different chemical shifts (1.78 ppm and 2.11 ppm) whereas for validamine (2a), both the C(6) protons appear as a multiplet (1.74-1.59 ppm). After the spectral analysis of 7, we observed a similarity in the chemical shifts and peak splitting pattern of the C(6) protons (1.71-1.55 ppm) to validamine (2a), thereby, con rming that the hydroxy methylene in target compound 7 is at the equatorial position. Finally, the benzyl carbamate 30 was subjected to deprotection under Birch reduction conditions to afford target compound 8.
Inhibition studies were carried out using 7 and 8 separately against Sco GlgE1-V279S. The maltosyltransferase activity of Sco GlgE1-V279S using M1P was determined by coupling with purine nucleoside phosphorylase (PNP), using the previously published method. 27 Enzyme activity of Sco GlgE1-V279S decreased by 32 % at 100 µM concentration of 8, but 7 failed to exhibit any inhibition of Sco GlgE1-V279S. Similarly, a 1 mM concentration of 7 also failed to inhibit the enzyme. Therefore, only compound 8 was used for dose-dependence studies at concentrations between 0 and 1000 µM. Using the data plotted in Figure 4, the IC 50 of compound 8 was determined to be 484 ± 51 µM. Unfortunately, lack of 8 solubility beyond 1 mM prevents inhibitor saturation of Sco GlgE1-V279S and a more accurate IC 50 determination of the compound. However, as the two highest inhibitor concentrations clearly exhibit enzyme activity below 50 % when normalized with the rate of the uninhibited enzyme, the calculated IC 50 for compound 8 is reasonably accurate.
The X-ray crystal structures of Sco GlgE1-V279S/7 and Sco GlgE1-V279S/8 complexes were pursued to gain insight regarding the lack of e cacy of 7 and evaluate the role of ring attening in affording the inhibitory activity of 8. The results of the X-ray diffraction experiment afforded re nement of the Sco GlgE1-V279S/7 and Sco GlgE1-V279S/8 complex structures to resolutions of 1.75 Å and 1.78 Å, respectively. The evidence from the 2F O -F C composite omit maps ( Figure 5) demonstrate the presence of each compound in the M1P binding site. In addition to the strong density observed for both 7 and 8 in the respective structures, the GlgE1-V279S/7 complex also exhibits a well-ordered spherical density located 3.2 Å from the exocyclic amine moiety of 7 that we have modeled as a chloride ion. Based on the proximity of the C(1) ammonium moiety to Glu 423 in both structures, the presence of the chloride ion in the Sco GlgE1-V279S/7 complex structure, and the pH of the crystallization condition (pH 7.5), we expect the "amine" moiety of 7 (and 8) to harbor a positively charged ammonium moiety and the following discussion will re ect that assumption.
The compounds bound within the M1P binding sites in each structure provide important information about enzyme-compound interactions and the geometry of the compounds within the active site. In both enzyme-compound structures, the interactions of the glucose moiety bound within the -2 subsite are the same as that observed in previously published Sco GlgE1-V279S/inhibitor complex structures. [50][51][52][53] However, interactions between the carbocycle moieties and the amino acid residues forming the -1 subsite differ signi cantly from each other. In the Sco GlgE1-V279S/7 complex, the interactions are similar to those observed in the substrate-bound (M1P and maltose) and substrate-mimic (MCP) complexes because the saturated carbocycle in 7 maintains the expected chair conformation.
Speci cally, the C(2)-OH and C(3)-OH functional groups form a bidentate hydrogen-bonded interaction with Asp 480, which is a unique feature to the GH13 family of proteins and has been observed previously in GlgE ( Figure 6A). 54,55 In addition, the C(2)-OH forms a hydrogen bond with the sidechain nitrogen atom/NH1 of Arg 392.
An unexpected difference between the Sco GlgE1-V279S/7 complex structure and other complexes of disaccharide-like compounds is the rotation of the Asp 394 side chain away from the substrate analog.
As this residue is known to form a hydrogen bond with the C6-OH group on incoming saccharides to allow positioning for nucleophilic attack on the anomeric carbon, it was anticipated to form the same hydrogen bond with 7. However, the superimposed active sites of Sco GlgE1-V279S/7 and Sco GlgE1/Maltose (PDB:3ZT5) 50 complexes ( Figure 6B) clearly highlight χ2 dihedral rotation in Asp 394. This is exhibited unambiguously by the 2F O -F C composite omit map density contoured at 1.5σ (Figure 7).
The reason for this movement of Asp 394 appears to derive from steric hindrance with atom C(1) of 7, apparently due to the ionic attraction between the C(1) ammonium moiety and the Glu 423 side chain.
Since the crystals were grown at pH 7.5, the Asp side chains would be expected to be in the carboxylate form and harbor a negative charge, it is likely that has this charge attraction has shifted the carbocycle closer to Glu 423 than in other Sco GlgE1-V279S complexes. Inspection of the interaction network in this area illustrates that the side chains of Trp 281 and Tyr 251 form hydrogen bonds with each of the opposing aspartate side chains. Importantly, the Arg 392 side chain is positioned near the two aspartates and forms hydrogen bonds of 2.7 and 3.5 Å with Asp 394 and Asp 320, respectively. In apparent contrast to those stabilizing interactions, the 2.4 Å distance between O(D1) atoms of Asp 320 and Asp 394 represent the only possibly destabilizing interaction in this region. However, the very short distance between those atoms can be rationalized if either of the two side chains is protonated and the O(D1) atoms in those residues are using that hydrogen in a low barrier hydrogen bond. It is suspected that the steric hindrance producing this energetically unfavorable repositioning of Asp 394 signi cantly limits the inhibitory activity of compound 7.
When inspecting the Sco GlgE1-V279S/8 complex, the amino acid residues forming the -1 subsite are consistent with previous Sco GlgE1-V279S complexes and do not undergo the structural change observed in the compound 7 complex. This is due to the expected structural differences observed compound 8 resulting from the presence of the sp 2 hybridized carbons at positions 5 and 6 in the carbocycle. The ring exhibits a attened conformation that differs signi cantly from the chair conformation in 7 (Figure 8).
This changes the hydroxyls at positions C(2) and C (3)  have a hydroxyl at this position that interacts directly with the enzyme nucleophile. 50,52 Although speculative, such a bidentate interaction could be envisioned to stabilize a 3 H 4 or B 2,5 -TSs in the enzyme catalyzed reaction. We note some studies have suggested that reactions catalyzed by this enzyme class proceed through a 4 C 1 → 4 H 3 -TS → 1 S 3 conformational itinerary leading to a 4 C 1 enzyme-bound intermediate. 56,57 On the other hand, kinetic isotope effect studies have suggested a 2,5 B TS could be at play for some GH13 enzymes. 58 Others have noted the same data could be interpreted as a 4 C 1 → 3 S 1 → 3 H 4 -TS. 59 While more recently it was proposed S. cerevisiae α-glucosidase passes through a 4 H 3 TS. 60 Further support of the 3 H 4 or B 2,5 -TSs hypothesis is derived from inspection of other GH13 enzymes in complex with acarbose, which is a tetrasaccharide possessing an N-glycosidic linkage that functions as an inhibitor of a-amylases. Inspection of available GH13 enzymes in complex with acarbose or acarbosederived oligosaccharides (accession codes: 4B9Z, 4E2O, 6GXV, and 6LGE) show that the N-glucosyl moieties in these structures maintain a chair conformation with C(2)-OH and C(3)-OH in equatorial positions. This difference is likely due to the enzyme/acarbose complex resembling the Michaelis complex rather than mimicking a reaction intermediate as we propose for compound 8. [61][62][63][64] The ammonium moieties in both compounds form polar interactions with the side chain of the General acid/base, Glu 423. This interaction is consistent with the role of Glu 423 in protonating the OH leaving group during the rst catalytic step and is likely stabilizing the complex due to the charge complementary between Glu 423 and the ammonium moieties. The difference in polar bond distances is a function of the ring attening in 8 that both affords closer approach of the ammonium to Glu 423 while avoiding steric hindrance with Asp 394 ( Figure 6A and Figure 8). This shorter polar bond distance and lack of movement in Asp 394 may be the major factor de ning the differences in a nity observed for 7 and 8. Additionally, in the Sco GlgE1-V279S/7 complex, the well-ordered chloride ion appears to form ionic interactions with the nitrogen atom of 7 and the side chain nitrogen atom of Lys 355. However, these interactions are rather long so likely contribute little to the binding a nity of 7.
The active site comparison of both structures (Figure 9) indicates an upward shift in the carbocycle ring of compound 8. Also, we observed some minor shifts in the side chains of Lys 355, Arg 392, Asp 394 and Glu 423, but these are within coordinate error. The superposition shown in Figure 6 also highlights the signi cant rotation of the χ2 dihedral observed in the Sco GlgE1-V279S/7 complex. When measuring the χ1 dihedral angle (between Cα and Cβ atoms) of Asp 394 in GlgE1-V279S/8 and Sco GlgE1-V279S/7, we identi ed a 119.4° dihedral difference. Due to this bond rotation, Asp 394 in Sco GlgE1-V279S shifts away from the substrate binding site and does not form any direct interaction with 7 ( Figure 6A). In addition to the speculated low-barrier hydrogen bond with Asp 320, the carbonyl group of Asp 394 was further stabilized by formation of hydrogen bonds with the side chain nitrogen atom/NH1 of Arg 392 and the indole of Trp 281.
The previous studies relating to the synthesis and characterization of zwitterionic pyrrolidinephosphonates showed that such compounds are reasonable mimics of the TSs formed during the enzymatic reaction. Out of those zwitterionic pyrrolidine-phosphonates, the methyl phosphonate pyrrolidine (MPP) is the most potent inhibitor against Sco GlgE1-V279S exhibiting an equilibrium dissociation constant (K i ) of 45 ± 4 μM. 52 The comparison of the active sites ( Figure 10 Further consideration of the Sco GlgE1-V279S/8 and Sco GlgE1-V279S/MPP complexes suggest valuable information to design future inhibitors for Sco GlgE-V279S and the homologous Mtb GlgE. With this assessment, it seems that endocyclic amines are preferable to α-exocyclic amines because the approach of the positively charged group toward the Glu 423 side chain does not encroach upon the volume of space occupied by the Asp 394 side chain and therefore would not promote protein structural changes that are detrimental to inhibitor binding. Additionally, while GlgE can accommodate the axial hydroxyl moieties at positions 2 and 3, it seems that only the axial C(3)-OH group in 8 strongly supports binding. Speci cally, the bene t of an axial C(3)-OH is that it interacts directly with the Asp 394 side chain affording a bidentate interaction between the inhibitor and the enzyme nucleophile. In contrast, the axial C(2)-OH in 8 does not form any notable interactions with the enzyme. Indeed, the presence of an equatorial C(2)-OH group is bene cial for binding since it forms two hydrogen bonded interactions with amino acid residues in the -1 subsite. Further, in the -1 subsite of all the compound bound structures of Sco GlgE1-V279S, we observe some volume that can be targeted with additional functional groups. Therefore, we can extend the length of the new inhibitors to access those unoccupied areas and form additional interactions with the conserved amino acid residues present in those areas.

Conclusions
The results obtained in these studies relate to both synthesis and structural evaluation of aminocarbocycles as inhibitors of GH13 enzymes. Herein, we have analyzed the diastereoselectivity of the Grignard reaction using the Cram's-chelate TS model. It is noted that the selectivity follows the rule for 4-α-glucoside series. However, there is an erosion of stereoselectivity in the 4-α-glucoside series (4:1) with respect to the gluco series (9:1). We were successful in identifying the desired isomer 27A after subjecting the obtained dienes (27A and 27B) to RCM and comparing their spectral data with the alkenes 17A and 17B. The success of the synthetic route was highly dependent on the development of suitable experimental conditions due to highly sensitive nature of the Grubb's RCM. Also, we have successfully synthesized and determined the stereochemistry of the target 1-aminocarbasugars 7 and 8. In the case of 7, the stereoselectivity of the hydrogenation was improved relative to the gluco series. Of these two compounds, 8 exhibited inhibitory activity of the enzyme at a modest IC 50 value of 484 ± 51 µM.
Compound 8 formed each of the expected interactions in the -2 subsite as these are consistent with previously published Sco GlgE1-V279S structures. The origins of the inhibitory ability of 8 derives from the attened ring system, coulombic interactions with conserved active site residues, and the ability to adopt a E 2 conformation in the cyclohexene fragment. This causes the 3-OH to form a new hydrogen bond with the catalytic nucleophile while also maintaining an electrostatic interaction between the exocyclic amine and the general acid/general base Glu 423. We note an E 2 conformation of 8 bears similarity to either 3 S 1 , 3 H 4 or even B 2,5 conformations that are possible for the conformational literary of natural substrates. However, this conformation could also be a result of the favorable Glu 423ammonium electrostatic interaction which would be absent in a 2 H 3 conformation and the low interconversion barrier for 2 H 3 to 3 H 2 . Overall, the inclusion of the exocyclic amine was not as bene cial to binding as expected. While the short polar interactions between the amine and Glu 423 are likely stabilizing complex formation, this bene t is negated in the complex with compound 7 due to unexpected protein structural changes induced by steric hindrance. In conclusion, we have reported an effective regiospeci c method to prepare the 4-α-glucosides of 1-aminocarbasugars using a readily available starting material. This structural and inhibitory information provides valuable insights into the atomic level understanding of valienamine-based GH inhibitor interactions and reveals new avenues for the design of improved inhibitors based on this important scaffold. Figure 1 Aminocarbasugar-based GH inhibitors and targets 7 and 8.