Exploration of Strategies for Mechanism‐Based Inhibitor Design for Family GH99 endo‐α‐1,2‐Mannanases

Abstract endo‐α‐1,2‐Mannosidases and ‐mannanases, members of glycoside hydrolase family 99 (GH99), cleave α‐Glc/Man‐1,3‐α‐Man‐OR structures within mammalian N‐linked glycans and fungal α‐mannan, respectively. They are proposed to act through a two‐step mechanism involving a 1,2‐anhydrosugar “epoxide” intermediate incorporating two conserved catalytic carboxylates. In the first step, one carboxylate acts as a general base to deprotonate the 2‐hydroxy group adjacent to the fissile glycosidic bond, and the other provides general acid assistance to the departure of the aglycon. We report herein the synthesis of two inhibitors designed to interact with either the general base (α‐mannosyl‐1,3‐(2‐aminodeoxymannojirimycin), Man2NH2DMJ) or the general acid (α‐mannosyl‐1,3‐mannoimidazole, ManManIm). Modest affinities were observed for an endo‐α‐1,2‐mannanase from Bacteroides thetaiotaomicron. Structural studies revealed that Man2NH2DMJ binds like other iminosugar inhibitors, which suggests that the poor inhibition shown by this compound is not a result of a failure to achieve the expected interaction with the general base, but rather the reduction in basicity of the endocyclic nitrogen caused by introduction of a vicinal, protonated amine at C2. ManManIm binds with the imidazole headgroup distorted downwards, a result of an unfavourable interaction with a conserved active site tyrosine. This study has identified important limitations associated with mechanism‐inspired inhibitor design for GH99 enzymes.


Introduction
Glycoside hydrolases of the carbohydrate-activee nzyme( see www.cazy.org;w ww.cazypedia.org) [1,2] family GH99 are endoacting mannosidases that cleave a-mannosidel inkages within mammalian high mannose N-glycans (endo-a-1,2-mannosidases) [3][4][5][6][7] and fungal a-mannans (endo-a-1,2-mannanases,F igure 1A). [8,9] Inhibitor design for these enzymesi sd riven by their potential use to understand glycoproteinb iosynthesis and maturation in the secretory pathway,a nd to manipulate fungal mannan degradation processes in the human gut microbiota. Structural and mechanistic studies of family GH99 enzymes suggest that they utilise an unusual mechanism involv-ing neighbouring group participationb yt he substrate 2-hydroxy to form a1 ,2-anhydrosugari ntermediate. [10] In this proposed mechanism,aconserved active site residue acts as a general base to deprotonatet he 2-OH group,t hereby facilitating its nucleophilic attack on C1 ( Figure 1A). This process has little biological precedent( for ar elatedp roposal see Ref. [11]), but occurs in the base-promoted solvolysis of a-mannosides. [12] Efforts to develop inhibitors of GH99 enzymes have relied upon appending 1,3-linked a-glucosyl (to target mammalian endo-a-1,2-mannosidases) or 1,3-linked a-mannosyl (to target bacterial endo-a-1,2-mannanases) groups to various sugarshapedh eterocycles. Spiro and co-workersr eportedt he discoveryo fa-glucosyl-1,3-deoxymannojirimycin (GlcDMJ) as an effective inhibitor of the mammalian enzyme, [13,14] and followon studies by Fleet and co-workersr evealed a-mannosyl-1,3deoxymannojirimycin (ManDMJ) to be as lightly weaker inhibitor for this enzyme ( Figure 1B). [15] The potencyo fG lcDMJ was subsequently exceededb ya-glucosyl-1,3-isofagomine (GlcIFG). [10,16] Equivalent results have been noted for bacterial GH99 enzymes, whichl ed to the development of a-mannosyl-1,3-isofagomine (ManIFG;d issociationc onstant, K D = 0.14 mm for Bacteroidest hetaiotaomicron GH99). [8] Furthermore, reintroductiono ft he "missing" 2-OH of 1,3-isofagomine( IFG) into ManIFG gave a-mannosyl-1,3-noeuromycin( ManNOE), which was shown to be five-fold more potent towards the B. thetaiotaomicron GH99 enzyme (K D = 0.03 mm). [17] These compounds bind in ag round-state 4 C 1 conformation, as seen in complexes of inactive enzyme with substrate and thus proposed for the conformation of substrate within the Michaelis complex (Figure 1A), which suggests that potent inhibition of GH99e nzymes can be achieved simply by mimicry of the chargei nt he transition state. [17] Separately,S piro and co-workerss howedt hat the neutral compound GlcGlucal ( Figure 1C)w as am odesti nhibitor of mammalian GH99 (rat Golgi preparation, IC 50 = 2.3 mm;f or GlcDMJ IC 50 = 1.7 mm); [14,18] the equivalent molecule targeting bacterialG H99, ManGlucal, wasa lso al igand with mildly potent affinity (K D = 15 mm for BtGH99). [17] Computational freeenergy landscapea nalysiso ft he preferredc onformation of dglucal suggested that the inhibition of the glucal-based inhibitors arises from mimicry of the proposed 4 E conformation of the transition state or the proposed 4 H 5 conformation of the 1,2-anhydro sugar intermediate, but with no contribution from charge mimicry owing to the neutraln ature of this compound. [17] We report here our efforts to explore two new inhibitor design strategies for the inhibition of GH99 enzymes. Considering the role of the basic residue implicated in the 1,2-anhydrosugar mechanism of GH99 enzymes,w es peculated that introductiono fa na mino group into the structure of ManDMJt o give Man-2NH 2 DMJ (1; Figure 1E)c ould promote the formation of af avourable ionic interaction upon inhibitor binding (Figure 1D). Separately,t he glycoimidazole class of inhibitors were developedf ollowing the discovery of the naturalp roduct nagstatin, [19] and are believed to derive their potencyf rom their ability to mimic the shape of the oxocarbenium-like transition state as well as from the ability of the imidazole glycosidic nitrogent oe ngage in ah ydrogen bond with an appropriately situated carboxylate residuei nt he active site ( Figure 1D). [20] For the present work, this would requiret he synthesis of Man-ManIm (2;F igure 1E). Thus, we report herein on the synthesis of these two target inhibitors, the structural characterisation of their binding modes and measurement of their bindingc onstants.

Results and Discussion
Synthesis of Man2NH 2 DMJ and ManManIm Man2NH 2 DMJ (1)w as prepared by substitution of known tosylate 3 [21] with sodium azide in DMF to afford azide 4 (Scheme 1). Coupling of azide 4 with trichloroacetimidate 5 [22] under the agency of TfOH afforded the disaccharide 6 in a yield of 83 %. The deprotection of 6 was achieved in astepwise manner,a sa ttempts to perform ag lobal deprotection that involved simultaneous removal of benzyloxycarbonyl (Cbz), benzylidene and benzyle thers as well as the reduction of the azide was unsuccessful. Deacetylation of 6 (NaOMe/MeOH) and then hydrolysis of the benzaldehyde acetal (TFA/H 2 O) afforded triol 7.T he azide group was reduced with dithiothreitol( DTT)/ pyridine buffer to afford amine 8.R emoval of the Cbz and benzylg roups then proceeded smoothly by using H 2 and Pearlman'sc atalyst to afford 1.
ManManIm (2)w as synthesised throughasequence involving the preparation of the protected mannoimidazole alcohol 22,f ollowed by elaboration to the disaccharide (Scheme2). The known alcohol 9 [23] was treated with 2-naphthylmethyl bromide (NapBr)/NaHi nD MF to afford 10.H ydrolysis of the thioglycoside with N-iodosuccinimide (NIS) in H 2 O/acetone gave the hemiacetal 11,w hich was oxidised to the lactone 12 under Albright-Goldman conditions. [24] For the conversion of the lactone 12 to the lactam 17 we followed the protocol developedb yO verkleeft et al., [25] which involved aminolysis to the acyclic amide 13,A lbright-Goldmano xidation (!14)a nd ring closure promoted by ammonia/MeOH (!15). Reduction of the hemiaminals 15 with NaCNBH 3 afforded a2:1 mixture of the d-manno and l-gulo lactams, from which the d-manno lactam 17 was isolated in ay ield of 38 %. Conversion of the lactam to the thionolactam 18 was achieved by using Lawesson's reagent and pyridine in toluene. Annulation of the imidazole ring was achieved by following the general approach of Vasella and co-workers. [26] Reaction of the thionolactam 18 with aminoacetaldehyde dimethyl acetal afforded the amidine 19,a nd imidazole ring formation was achievedb yc atalysis with TsOH to provide am ixture of d-gluco and d-manno imida-zoles in a2:1 ratio, from whicht he d-manno imidazole 21 was isolated in ay ield of 32 %o ver two steps. The naphthylmethyl group was removedu nder the agency of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)a nd CH 2 Cl 2 /H 2 Ot oa fford the alcohol 22.
Coupling of 22 with trichloroacetimidate 5 [22] catalysed by TfOH afforded the disaccharide 23 in ay ield of 47 %. Deprotection was achieved in two steps under conditions chosen to avoid epimerisation at C2. Treatment of 23 with K 2 CO 3 /MeOH afforded the alcohol 24,a nd hydrogenation with Pearlman's catalystafforded 2.

Bindingaffinities and 3D structures
Isothermal titrationc alorimetry (ITC) was used to assess the binding of 1 and 2 to ab acterial endomannosidase. Titration of BtGH99 with Man2NH 2 DMJ (1)r evealed binding with K D = 97.7 AE 4.9 mm (Figure 2), whereas no binding with ManManIm (2)w as evident by ITC. Placed in context, 1 has ap oorer binding affinity towards BtGH99 than GlcDMJ( K D = 24 mm); [10] the equivalent data is not available for ManDMJ, but as this enzymep refers to bind Man-configured substrates, the difference would be expected to be even greater.
Three-dimensionals tructures were obtained for 1 and 2 bound to the endo-a-1,2-mannanase BxGH99 from Bacteroides xylanisolvens,which is closely related to BtGH99but more amenable to complex formation. These complexes diffracted to a resolution of 1.1 and 1.3 ,r espectively ( Table 1). Occupancy of the active site for the complexwith 1 was essentially complete, whereas that with 2,with prolonged soaking,was estimated to be 80 %, likely ac onsequence of the poor affinity of the compound for the enzyme. As predicted, both compounds bound in the À2/À1s ubsites of the enzyme (sub-site nomenclature from Ref. [27]) and will be discussed in turn.
Structural analysis of the BxGH99-1 complex ( Figure 3A)r evealed the piperidine ring in a 4 C 1 conformation, which matches that seen for complexes of the wild-type enzyme with GlcDMJ and isofagomine-based inhibitors [8,10,17] as well as that of ad isabled mutant with substrate. [8] The 2-amino group is situated appropriatelyt oi nteract with the E333 residue, that which is proposed to act as ag eneral base/acid through deprotonation of the 2-hydroxy group. Overlayo ft his complex with that of BxGH99-GlcDMJ reported previously [10] revealed that the positioning and conformationso ft he rings in the À1 and À2s ub-sites are essentially identical, and that no amino acid residues undergo significant movement ( Figure 3C). In particular, the E333···O2 and E333···N2 distances are 2.54 and 2.59 ,r espectively.T he poor binding affinity of 1 compared with GlcDMJt herefore does not result from incorrect binding of the inhibitor,a nd must insteadr eflect af ailure to fully capitalise on the proposed interactions. It is widely acknowledged that iminosugars such as DMJ (and thus GlcDMJ) achieve inhibition through binding to glycosidases in their protonated  form; [28] this is supported by first-principles consideration of the basicity of these inhibitors and the relevant pK a valueso f the catalytic residues,a sw ell as by studies of the pH dependence of inhibition. In the case of 1,t here are two basic nitrogen residues.However,f or vicinal diamines, protonation at one nitrogen has ap rofound effect on the pK a value at the second nitrogen;i na cyclics ystemst his effect has been estimated to be DpK a = 3.6 units for NH 3 + andN R 3 + . [29] Moreover, in cyclic systemst here are stereoelectronic and conformational contributions, notable examples for various diamines (pK a1 ,p K a2 )i nclude piperazine (9.8, 5.7), [29] cis-1,3-diaminocyclohexane (10.3, 8.3) [30] and trans-1,3-diaminocyclohexane (10.4, 8.5). [30] Finally, vicinal hydroxy groups can also perturb amine pK a values;i n Man2NH 2 DMJ, O4 is antiperiplanarw ith respect to the endocyclic nitrogen and would be expected to reduce its basicity by around1.3 pK a units. [30] Collectively,this analysis would suggest that N2 is protonated by the general acid E333, and that it is unlikely that the dicationi sf ormed, and therefore Man2NH 2 DMJ fails to appropriately mimic an oxocarbeniumlike transition state. Ar elated example of this phenomenon was reported in which introduction of as econd amine vicinal to ap re-existing one in apramycin resulted in ad ramatic loss of binding to ab acterial ribosomeo fa pproximately 100fold. [31] Additionally,t he proposed binding mode of 1 shown in Figure 1D highlights the factt hat the 2-amino group has additional hydrogen substituents that may cause an energy penalty upon binding of the inhibitor.
Structural analysis of the BxGH99-2 complex revealed the piperidine ring of the mannoimidazole moiety to be in an unusual 2 H 3 /E 3 conformation ( Figure 3B). [32] Overlay of the complex with that of BxGH99-GlcDMJ [10] revealed that although the À2s ugar residues occupy similarp ositions, the mannoimidazole headgroup is atypically positioned such that the heterocycle projects downward into the active site, below the plane of the piperidine ring of the GlcDMJ complex ( Figure 3D). In this case the E336···N (imidazole ring) distance is 2.65 ,s imilar to that seen in relatedg lycoimidazole complexes. [33] In the originalf ormulation by Heightman and Vasella, b-equatorial glycosidases were proposed to performp rotonation from the side, in what was termed "lateral protonation",w ith the acid either on the same side as the endocyclic oxygen (syn)o ro pposed to it (anti). [20] In as ubsequentp ublication Nerinckx et al. formalised this concept by dividing the space aroundt he À1 sugar into anti and syn hemispherest hrough ap lane defined by the glycosidico xygen, C1 and H1 of the sugar residue. [34] Analysiso fc omplexes of various anti-protonating glycosidases revealed that the acid/base or acid residues responsible for protonating the leaving group are in fact not universally located lateral to the mean plane of the sugar,b ut are more commonly positioned above or below it, so as to better protonate the leaving group oxygen. However,t his does not prevent glycoimidazoles binding in normal orientationsa nd engaging in hydrogen-bonding interactions with the imidazole nitrogen. For example, in the case of the retaining GH116 b-glucosidase from Thermoanaerobacterium xylanolyticum,t he acid/base is positioned above the mean plane of the sugar,b ut an ormal orientation and conformation of glucoimidazole was observed. [35] Mannoimidazole also binds in the normalf ashiont o an inverting GH47 a-mannosidase from Caulibacter sp. in which the acid is below the mean plane of the inhibitor,b ut insteadt he inhibitor establishes an interaction with another conserved active site carboxylic acid that lies lateralt othe imidazole. [36] BxGH99 is an anti-protonatinge nzyme with its general acid/base Glu336 positioned below the plane of the ring to facilitate classical anti protonation of the axial glycosidic oxygen (O5-C1-O1 angle is approximately6 0 8). The distorted mode of binding of the mannoimidazolem oiety of 2 seemst o be ac onsequence of the imidazole binding to maximise this interaction with the acid/base. Close examination of the active site of BxGH99 revealed that if the ManIm moiety were to be shifted up to the same position as that of the piperidine of GlcDMJ, as teric interaction would result with Tyr252, ac onserved residue. In fact, the distance between the imidazole C= Cb ond and Tyr252 Ce is only 3.2 ,w hichc ausest he wwPDB validation software [37] to report H/H steric clashes in this region. In fact, at ernary complex of GlcDMJ and a-1,2-mannobiose highlighted the fact that the active site of the enzyme involves as harp bend in the À1a nd + 1s ub-sites.T he failure of 2 to bind in at ypical positioni nt he À1s ub-site is thus likely a result of af ailure to accommodate the imidazole ring owing to the location of Tyr252.

Conclusions
We have reported here the design and synthesis of two "mechanism-based" inhibitors of family GH99 endomannanases. Al-thoughM an2NH 2 DMJ (1)b ound to the bacterial endomannanase BxGH99 in the expected manner,i ts affinity for BtGH99 did not exceed that seen for GlcDMJ. Thisa ppears to be a result of the perturbing effect of the 2-amino substituent, which reducest he basicity of the endocyclic nitrogen and its ability to be protonated in the active site and therebyr esemble the oxocarbenium-liket ransition state. On the other hand, the binding of ManManIm (2)t oBtGH99 could not be detect- ed by ITC and, consistent with this, the X-ray structure of 2 complexed with BxGH99 displayed incomplete occupancy.T he poor binding of this inhibitor appears to be ac onsequence of an inability of the active site of BxGH99 to accommodate the annulated imidazole ring because of an interaction with ac onserved Tyra ctive-site residue. This study provides important insights that will inform future strategies for the development of mechanism-inspired and transition-state mimicking inhibitors of GH99 enzymes.

Experimental Section
General: 1 Ha nd 13  . Melting points were obtained by using aR eichert-Jung hot-stage apparatus. TLC analysis was performed with aluminium-backed Merck Silica Gel 60 F254 sheets, detection was achieved by using UV light, 5% H 2 SO 4 in MeOH or ceric ammonium molybdate ("Hanessian's stain") with charring as necessary.F lash chromatography was performed by using Geduran silica gel according to the method of Still et al. [38] Dry CH 2 Cl 2 ,T HF and Et 2 O were obtained from ad ry solvent apparatus (Glass Contour of SG Water,N ashua). [39] DMF and DMSO were dried over 4 molecular sieves.  3,4,6-Tri-O-benzyl-a-d-mannopyranosyl-(1!3)-2-azido-N-benzyloxycarbonyl-1,2,5-trideoxy-1,5-imino-d-mannitol (7):Asolution of sodium methoxide in methanol (0.1 m,1 0 mL, 1 mmol) was added to 6 (60 mg, 0.068 mmol) in methanol (0.5 mL) and the mixture was stirred for 1hand then concentrated under reduced pressure to give an alcohol, which was used without purification. TFA/ H 2 O( 9:1, 100 mL) was added to the crude alcohol and the mixture was stirred for 30 min, concentrated and azeotroped with toluene  (10):Adry solution of the alcohol 9 [23] (167 mg, 0.30 mmol) in DMF (5 mL) was cooled to 0 8C. The solution was charged with NaH (60 %d ispersion in mineral oil, 36 mg, 0.9 mmol) and the mixture stirred for 30 min. 2-Bromomethylnaphthalene (79.6 mg, 0.36 mmol) was added and the mixture stirred overnight. The mixture was diluted with Et 2 O( 20 mL), poured into ice/water and washed with water (3 20 mL) and brine (1 20 mL). The organic extracts were dried (MgSO 4 ), the solvent was removed under reduced pressure and the resulting residue was subjected to flash chromatography (EtOAc/pet. ether,1 5:85) to give the protected thioglycoside 10 (179.3 mg, 86 %) as ac olourless oil.  (12):As olution of the hemiacetal 11 (742 mg, 1.26 mmol) in acetic anhydride (6.1 mL) and dry DMSO (6.6 mL) was stirred under N 2 for 22 h. The mixture was diluted with EtOAc (20 mL), quenched with ice and washed with water (3 20 mL) and brine (1 20 mL). The organic extracts were dried (MgSO 4 )a nd the solvent was evaporated. Azeotropic toluene was used to remove any residual AcOH to afford the crude lactone 12 (823 mg), which was used directly in the next step. Ap ortion of 12 obtained from as eparate experiment was purified by flash chromatography (EtOAc/pet. ether, 1 :9) to yield analytically pure 12 as ac olourless oil. Ad ry-ice/acetone cold finger cooling trap was used to condense ammonia (50 mL) into as olution of the crude lactone 12 (823 mg) in dry THF (30 mL) at À78 8C. The solution was allowed to reflux at 0 8Cf or 4h.T he mixture was then evaporated to dryness to afford the crude amide 13 (771 mg), which was used directly in the next step. Ap ortion obtained from an independent experiment was pu- (3S,4S,5S,6R/S)-3,5-Bis(benzyloxy)-6-(benzyloxymethyl)-6-hydroxy-4-(2-naphthylmethoxy)piperidin-2-one (15):Asolution of the crude amide 13 (771 mg) in acetic anhydride (6.1 mL) and dry DMSO (6.6 mL) was stirred under N 2 for 21 h. The reaction mixture was diluted with EtOAc (20 mL), quenched with ice and washed with water (3 20 mL) and brine (1 20 mL). The organic extracts were dried (MgSO 4 )a nd the solvent was evaporated to affordt he keto-amide 14 as aw hite solid. Ad ry-ice/acetone cold finger was used to condense ammonia (20 mL) into as olution of the crude keto-amide in dry methanol (30 mL) at 0 8C. The solution was allowed to warm to room temperature and stirred under N 2 for 16 h. The solvent was removed under reduced pressure and the resulting residue was subjected to flash chromatography (EtOAc/pet. ether,1 :1) to give as eparable mixture of the hydroxy-lactams 15 (669 mg, 88 %o ver four steps; d-manno/l-gulo 2.2:1). 1 HNMR (500 MHz, CDCl 3 ), partial spectrum of the mixture of diastereomers: d The organic extracts were dried (MgSO 4 )a nd the solvent removed under reduced pressure to affordt he amidines 19 as ac olourless residue. p-Toluenesulfonic acid monohydrate (0.14 g, 0.74 mmol) was added to as olution of the crude amidines in toluene (9.5 mL) and the mixture was stirred at 60 8Co vernight. The mixture was then diluted with DCM (20 mL) and washed with NaHCO 3 (2 20 mL) and brine (1 20 mL). The organic extracts were dried (MgSO 4 ), the solvent was removed under reduced pressure and the residue was subjected to flash chromatography (EtOAc/pet. 0.11mmol) was again added and the mixture stirred for 3days when TLC analysis (EtOAc/pet. ether,8:2) indicated complete consumption of the starting material. The mixture was then diluted with DCM (20 mL), washed with water (3 20 mL) and aq. sat. NaHCO 3 (3 20 mL), dried (MgSO 4 ), filtered and concentrated. The crude product was purified by flash chromatography (EtOAc/pet. ether,8 0:20 to 100:0) to afford the alcohol 22 (11.7 mg, 67 %) as a yellow oil.
Crystallisation and data collection: BxGH99 protein [10] was crystallised by using the vapour diffusion hanging drop method in 3 m sodium acetate at pH 7.4. Crystals were grown at 19 8Ci na2 4-well plate with 500 mLo fr eservoir solution in each well and sealed with vacuum grease. The droplet was created by mixing 1 mLo fBxGH99 solution (34 mg mL À1 in 25 mm HEPES buffer,p H7.0, 100 mm NaCl) with 1 mLo ft he crystallant solution. Crystals were fished from the droplet by using an ylon cryoloop, without cryoprotection. Data were collected at Diamond Light Source beamline i04 using X-rays with awavelength of 0.979 .
Structure solution and refinement:I mages containing diffraction patterns were indexed and integrated by using DIALS [42] through xia2. [43] The hkl index of each data set was then matched to ap revious solution in Aimless. [44] Refinement was performed by using Refmac5 [45] and real-space model building in Coot. [46] Model geometry and agreement with electron density were validated in Coot and Edstats. [47] The quality of the carbohydrates and nitrogen heterocycles were verified by using Privateer. [32] The modelling and refinement processes were aided by using ccp4i2 interface. [48]