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

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), Man2NH2 DMJ) 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 Man2NH2 DMJ 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‐active enzyme (see www.cazy.org; www.cazypedia.org)1, 2 family GH99 are endo‐acting mannosidases that cleave α‐mannoside linkages within mammalian high mannose N‐glycans (endo‐α‐1,2‐mannosidases)3, 4, 5, 6, 7 and fungal α‐mannans (endo‐α‐1,2‐mannanases, Figure 1 A).8, 9 Inhibitor design for these enzymes is driven by their potential use to understand glycoprotein biosynthesis and maturation in the secretory pathway, and 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 involving neighbouring group participation by the substrate 2‐hydroxy to form a 1,2‐anhydrosugar intermediate.10 In this proposed mechanism, a conserved active site residue acts as a general base to deprotonate the 2‐OH group, thereby facilitating its nucleophilic attack on C1 (Figure 1 A). This process has little biological precedent (for a related proposal see Ref. 11), but occurs in the base‐promoted solvolysis of α‐mannosides.12

Figure 1

(A) Proposed mechanism for family GH99 enzymes retaining endomannosidases/endomannanases. Only the first half of the catalytic cycle is shown. (B) Saturated basic heterocyclic inhibitors for GH99 enzymes mimicking the ground state conformation. (C) Neutral glycal inhibitors for GH99 enzymes mimicking the transition state. (D) Two inhibitor design concepts explored herein. (E) Structures of Man2NH2DMJ (1) and ManManIm (2).

Efforts to develop inhibitors of GH99 enzymes have relied upon appending 1,3‐linked α‐glucosyl (to target mammalian endo‐α‐1,2‐mannosidases) or 1,3‐linked α‐mannosyl (to target bacterial endo‐α‐1,2‐mannanases) groups to various sugar‐shaped heterocycles. Spiro and co‐workers reported the discovery of α‐glucosyl‐1,3‐deoxymannojirimycin (GlcDMJ) as an effective inhibitor of the mammalian enzyme,13, 14 and follow‐on studies by Fleet and co‐workers revealed α‐mannosyl‐1,3‐deoxymannojirimycin (ManDMJ) to be a slightly weaker inhibitor for this enzyme (Figure 1 B).15 The potency of GlcDMJ was subsequently exceeded by α‐glucosyl‐1,3‐isofagomine (GlcIFG).10, 16 Equivalent results have been noted for bacterial GH99 enzymes, which led to the development of α‐mannosyl‐1,3‐isofagomine (ManIFG; dissociation constant, K D=0.14 μm for Bacteroides thetaiotaomicron GH99).8 Furthermore, reintroduction of the “missing” 2‐OH of 1,3‐isofagomine (IFG) into ManIFG gave α‐mannosyl‐1,3‐noeuromycin (ManNOE), which was shown to be five‐fold more potent towards the B. thetaiotaomicron GH99 enzyme (K D=0.03 μm).17 These compounds bind in a ground‐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 1 A), which suggests that potent inhibition of GH99 enzymes can be achieved simply by mimicry of the charge in the transition state.17

Separately, Spiro and co‐workers showed that the neutral compound GlcGlucal (Figure 1 C) was a modest inhibitor of mammalian GH99 (rat Golgi preparation, IC50=2.3 μm; for GlcDMJ IC50=1.7 μm);14, 18 the equivalent molecule targeting bacterial GH99, ManGlucal, was also a ligand with mildly potent affinity (K D=15 μm for BtGH99).17 Computational free‐energy landscape analysis of the preferred conformation of d‐glucal 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 neutral nature 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, we speculated that introduction of an amino group into the structure of ManDMJ to give Man‐2NH2DMJ (1; Figure 1 E) could promote the formation of a favourable ionic interaction upon inhibitor binding (Figure 1 D). Separately, the glycoimidazole class of inhibitors were developed following the discovery of the natural product nagstatin,19 and are believed to derive their potency from their ability to mimic the shape of the oxocarbenium‐like transition state as well as from the ability of the imidazole glycosidic nitrogen to engage in a hydrogen bond with an appropriately situated carboxylate residue in the active site (Figure 1 D).20 For the present work, this would require the synthesis of ManManIm (2; Figure 1 E). Thus, we report herein on the synthesis of these two target inhibitors, the structural characterisation of their binding modes and measurement of their binding constants.

Results and Discussion

Synthesis of Man2NH2DMJ and ManManIm

Man2NH2DMJ (1) was 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 a stepwise manner, as attempts to perform a global deprotection that involved simultaneous removal of benzyloxycarbonyl (Cbz), benzylidene and benzyl ethers as well as the reduction of the azide was unsuccessful. Deacetylation of 6 (NaOMe/MeOH) and then hydrolysis of the benzaldehyde acetal (TFA/H2O) afforded triol 7. The azide group was reduced with dithiothreitol (DTT)/pyridine buffer to afford amine 8. Removal of the Cbz and benzyl groups then proceeded smoothly by using H2 and Pearlman's catalyst to afford 1.

Scheme 1

Reagents and conditions: a) NaN3, DMF, reflux, 74 %; b) TfOH, CH2Cl2, −30 to 0 °C, 87 %; c) i. NaOMe, MeOH, ii. 9:1 TFA/H2O, 83 %; d) DTT, pyr, pH 9.2 NaHCO3/Na2CO3, 80 %; e) H2, Pd(OH)2/C, aq. HCl, 2:2:1 EtOAc/MeOH/H2O, 70 %.

ManManIm (2) was synthesised through a sequence involving the preparation of the protected mannoimidazole alcohol 22, followed by elaboration to the disaccharide (Scheme 2). The known alcohol 9 23 was treated with 2‐naphthylmethyl bromide (NapBr)/NaH in DMF to afford 10. Hydrolysis of the thioglycoside with N‐iodosuccinimide (NIS) in H2O/acetone gave the hemiacetal 11, which 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 developed by Overkleeft et al.,25 which involved aminolysis to the acyclic amide 13, Albright–Goldman oxidation (→14) and ring closure promoted by ammonia/MeOH (→15). Reduction of the hemiaminals 15 with NaCNBH3 afforded a 2:1 mixture of the d‐manno and l‐gulo lactams, from which the d‐manno lactam 17 was isolated in a yield 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, and imidazole ring formation was achieved by catalysis with TsOH to provide a mixture of d‐gluco and d‐manno imidazoles in a 2:1 ratio, from which the d‐manno imidazole 21 was isolated in a yield of 32 % over two steps. The naphthylmethyl group was removed under the agency of 2,3‐dichloro‐5,6‐dicyano‐1,4‐benzoquinone (DDQ) and CH2Cl2/H2O to afford the alcohol 22.

Scheme 2

A) Preparation of imidazole alcohol 22. Reagents and conditions: a) NapBr, NaH, DMF, 86 %; b) NIS, H2O, acetone, 0 °C, 99 %; c) DMSO, Ac2O; d) NH3, THF, reflux; e) DMSO, Ac2O; f) NH3, MeOH, 88 % over steps c–f; g) HCO2H, NaBH3(CN), 38 % d‐manno, 33 % l‐gulo; h) Lawesson's reagent, pyridine, 4 Å molecular sieves, toluene, 93 %; i) H2NCH2CH(OMe)2; j) TsOH⋅H2O, toluene, 60 °C, yields over steps i and j: 42 % d‐gluco, 32 % d‐manno; k) DDQ, CH2Cl2/H2O, 67 %. B) Synthesis of ManManIm (2). Reagents and conditions: l) TfOH, 4 Å molecular sieves, toluene, −20 °C, 47 %; m) K2CO3/MeOH, 46 %; n) H2 (34 bar), Pd(OH)2/C, AcOH, EtOAc, MeOH, H2O, 48 %.

Coupling of 22 with trichloroacetimidate 5 22 catalysed by TfOH afforded the disaccharide 23 in a yield of 47 %. Deprotection was achieved in two steps under conditions chosen to avoid epimerisation at C2. Treatment of 23 with K2CO3/MeOH afforded the alcohol 24, and hydrogenation with Pearlman's catalyst afforded 2.

Binding affinities and 3D structures

Isothermal titration calorimetry (ITC) was used to assess the binding of 1 and 2 to a bacterial endomannosidase. Titration of BtGH99 with Man2NH2DMJ (1) revealed binding with K D=97.7±4.9 μm (Figure 2), whereas no binding with ManManIm (2) was evident by ITC. Placed in context, 1 has a poorer binding affinity towards BtGH99 than GlcDMJ (K D=24 μm);10 the equivalent data is not available for ManDMJ, but as this enzyme prefers to bind Man‐configured substrates, the difference would be expected to be even greater.

Figure 2

Isothermal titration calorimetry thermogram showing the binding of Man2NH2DMJ (1) to Bacteroides thetaiotaomicron endo‐α‐1,2‐mannanase (BtGH99). DP=differential power. Binding parameters K D=97.7±4.9 μm, N=1 (fixed) and ΔH=−5.9±0.1 kcal mol−1.

Three‐dimensional structures were obtained for 1 and 2 bound to the endo‐α‐1,2‐mannanase BxGH99 from Bacteroides xylanisolvens, which is closely related to BtGH99 but more amenable to complex formation. These complexes diffracted to a resolution of 1.1 and 1.3 Å, respectively (Table 1). Occupancy of the active site for the complex with 1 was essentially complete, whereas that with 2, with prolonged soaking, was estimated to be 80 %, likely a consequence of the poor affinity of the compound for the enzyme. As predicted, both compounds bound in the −2/−1 subsites of the enzyme (sub‐site nomenclature from Ref. 27) and will be discussed in turn.

Table 1

Data collection and refinement statistics for the complexes of BxGH99 with 1 and 2.

	BxGH99 complexedwith aminoDMJ (1)	BxGH99 complexedwith ManManIm (2)
Data collection
Space group	I4	I4
Cell dimensions
a [Å]	108.1	108.6
b [Å]	108.1	108.6
c [Å]	67.5	67.8
α [°]	90	90
β [°]	90	90
γ [°]	90	90
resolution [Å]	76.44–1.13 (1.15–1.13)[a]	76.81–1.30 (1.32–1.30)[a]
R merge	0.069 (1.501)	0.054 (1.224)
R pim	0.026 (0.735)	0.020 (0.610)
CC(1/2)	0.999 (0.400)	(0.999) 0.486
I/σI	10.2 (1.0)	14.0 (0.9)
completeness [%]	99.1 (86.0)	99.5 (92.7)
redundancy	7.5 (4.8)	7.5 (4.6)
Refinement
resolution [Å]	76.44–1.13	76.81–1.30
no. reflectionsall/free	143544/7133	96144/4810
R work/R free	0.122/0.144	0.134/0.162
no. atoms
protein	3188	3146
ligand/ion	22	25
water	467	427
B factors [Å2]
protein	17.2	20.5
ligand/ion	20.3	22.4
water	35.1	36.7
r.m.s. deviations
bond lengths [Å]	0.0101	0.011
bond angles [°]	1.495	1.497
PDB ID	6FAM	6FAR

[a] Values in parentheses are for the highest‐resolution shell.

Structural analysis of the BxGH99–1 complex (Figure 3 A) revealed 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 inhibitors8, 10, 17 as well as that of a disabled mutant with substrate.8 The 2‐amino group is situated appropriately to interact with the E333 residue, that which is proposed to act as a general base/acid through deprotonation of the 2‐hydroxy group. Overlay of this complex with that of BxGH99–GlcDMJ reported previously10 revealed that the positioning and conformations of the rings in the −1 and −2 sub‐sites are essentially identical, and that no amino acid residues undergo significant movement (Figure 3 C). In particular, the E333⋅⋅⋅O2 and E333⋅⋅⋅N2 distances are 2.54 and 2.59 Å, respectively. The poor binding affinity of 1 compared with GlcDMJ therefore does not result from incorrect binding of the inhibitor, and must instead reflect a failure 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 values of the catalytic residues, as well as by studies of the pH dependence of inhibition. In the case of 1, there are two basic nitrogen residues. However, for vicinal diamines, protonation at one nitrogen has a profound effect on the pK a value at the second nitrogen; in acyclic systems this effect has been estimated to be ΔpK a=3.6 units for NH3 + and NR3 +.29 Moreover, in cyclic systems there are stereoelectronic and conformational contributions, notable examples for various diamines (pK a1, pK a2) include 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; in Man2NH2DMJ, O4 is antiperiplanar with respect to the endocyclic nitrogen and would be expected to reduce its basicity by around 1.3 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 dication is formed, and therefore Man2NH2DMJ fails to appropriately mimic an oxocarbenium‐like transition state. A related example of this phenomenon was reported in which introduction of a second amine vicinal to a pre‐existing one in apramycin resulted in a dramatic loss of binding to a bacterial ribosome of approximately 100‐fold.31 Additionally, the proposed binding mode of 1 shown in Figure 1 D highlights the fact that the 2‐amino group has additional hydrogen substituents that may cause an energy penalty upon binding of the inhibitor.

Figure 3

Three‐dimensional structures of BtGH99 complexed with A) Man2NH2DMJ (1) and B) ManManIm (2). Electron density maps are maximum likelihood/σ A weight F o−F c difference syntheses contoured at 0.5 and 0.3 e Å−3 for panels A and B, respectively, visible before refining the structure model with the ligand added. (C) Overlay of Man2NH2DMJ (1) with GlcDMJ (PDB code 4FAM). (D) Overlay of ManManIm (2) with GlcDMJ (PDB code 4FAR).

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 3 B).32 Overlay of the complex with that of BxGH99–GlcDMJ10 revealed that although the −2 sugar residues occupy similar positions, 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 3 D). In this case the E336⋅⋅⋅N (imidazole ring) distance is 2.65 Å, similar to that seen in related glycoimidazole complexes.33 In the original formulation by Heightman and Vasella, β‐equatorial glycosidases were proposed to perform protonation from the side, in what was termed “lateral protonation”, with the acid either on the same side as the endocyclic oxygen (syn) or opposed to it (anti).20 In a subsequent publication Nerinckx et al. formalised this concept by dividing the space around the −1 sugar into anti and syn hemispheres through a plane defined by the glycosidic oxygen, C1 and H1 of the sugar residue.34 Analysis of complexes 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, but are more commonly positioned above or below it, so as to better protonate the leaving group oxygen. However, this does not prevent glycoimidazoles binding in normal orientations and engaging in hydrogen‐bonding interactions with the imidazole nitrogen. For example, in the case of the retaining GH116 β‐glucosidase from Thermoanaerobacterium xylanolyticum, the acid/base is positioned above the mean plane of the sugar, but a normal orientation and conformation of glucoimidazole was observed.35 Mannoimidazole also binds in the normal fashion to an inverting GH47 α‐mannosidase from Caulibacter sp. in which the acid is below the mean plane of the inhibitor, but instead the inhibitor establishes an interaction with another conserved active site carboxylic acid that lies lateral to the imidazole.36 BxGH99 is an anti‐protonating enzyme 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 approximately 60°). The distorted mode of binding of the mannoimidazole moiety of 2 seems to be a consequence 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, a steric interaction would result with Tyr252, a conserved residue. In fact, the distance between the imidazole C=C bond and Tyr252 Cϵ is only 3.2 Å, which causes the wwPDB validation software37 to report H/H steric clashes in this region. In fact, a ternary complex of GlcDMJ and α‐1,2‐mannobiose highlighted the fact that the active site of the enzyme involves a sharp bend in the −1 and +1 sub‐sites. The failure of 2 to bind in a typical position in the −1 sub‐site is thus likely a result of a failure 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. Although Man2NH2DMJ (1) bound to the bacterial endomannanase BxGH99 in the expected manner, its affinity for BtGH99 did not exceed that seen for GlcDMJ. This appears to be a result of the perturbing effect of the 2‐amino substituent, which reduces the basicity of the endocyclic nitrogen and its ability to be protonated in the active site and thereby resemble the oxocarbenium‐like transition state. On the other hand, the binding of ManManIm (2) to BtGH99 could not be detected by ITC and, consistent with this, the X‐ray structure of 2 complexed with BxGH99 displayed incomplete occupancy. The poor binding of this inhibitor appears to be a consequence of an inability of the active site of BxGH99 to accommodate the annulated imidazole ring because of an interaction with a conserved Tyr active‐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: 1H and 13C NMR spectra were recorded by using 400, 500 or 600 MHz Varian INOVA spectrometers. All signals were referenced to TMS (δ=0.00 ppm) or solvent peaks (CDCl3: δ=7.26 ppm for 1H and 77.16 ppm for 13C; D2O: δ=4.80 ppm for 1H and TMS: δ=0.00 ppm for 13C; [D4]MeOH: δ=3.49 ppm for 1H and δ=49.0 ppm for 13C). Melting points were obtained by using a Reichert‐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 % H2SO4 in MeOH or ceric ammonium molybdate (“Hanessian's stain”) with charring as necessary. Flash chromatography was performed by using Geduran silica gel according to the method of Still et al.38 Dry CH2Cl2, THF and Et2O were obtained from a dry solvent apparatus (Glass Contour of SG Water, Nashua).39 DMF and DMSO were dried over 4 Å molecular sieves.

2‐Azido‐4,6‐: Sodium azide (57.8 mg, 0.890 mmol) was added to a solution of 4,6‐O‐[(R)‐benzylidene]‐N‐benzyloxycarbonyl‐1,5‐dideoxy‐2‐O‐(p‐toluenesulfony1)‐d‐glucitol21 (3; 120 mg, 0.222 mmol) in DMF (1 mL). The suspension was heated at reflux for 18 h, poured into ice, extracted into EtOAc (3×20 mL), washed with brine (2×20 mL), dried over anhydrous MgSO4 and evaporated to dryness. Column chromatography (AcOEt/pet. ether 40‐60, 1:5) gave the azide 4 (67.7 mg, 74 %) as a white solid. [α] =−21.9 (c=1.12 in CHCl3); 1H NMR (CDCl3, 500 MHz): δ=2.74 (s, 1 H; NH), 2.82 (dd, J=1.6, 14.5 Hz, 1 H; 1‐Ha), 3.06 (td, J=4.6, 10.2 Hz, 1 H; 5‐H), 3.74 (dd, J=3.8, 9.2 Hz, 1 H; 3‐H), 3.79–3.93 (m, 2 H; 2,4‐H), 4.31 (dd, J=3.0, 14.5 Hz, 1 H; 1‐Hb) 4.46 (t, J=11 Hz, 1 H; 6‐Ha), 4.66 (dd, J=4.6, 11.6 Hz, 1 H; 6‐Hb), 5.01 (d, J=3.1 Hz, 2 H; CH2), 5.48 ppm (s, 1 H; CH); 13C NMR (CDCl3, 125 MHz): δ=48.1, 55.8, 60.1, 67.8, 69.2, 73.6, 78.2 (7 C; C1–C6, CH2), 101.8 (1 C; CH), 126.3, 128.3, 128.4, 128.5, 128.7, 129.4, 136.0, 137.3 (12 C; Ph), 155.0 ppm (1 C; C=O); HRMS (ESI, +ve): m/z calcd for C21H22N4O5: 411.1663 [M+H]+; found: 411.1664.

2‐: TfOH (0.043 μL, 0.0049 mmol) was added to a mixture of acceptor 4 (20 mg, 0.049 mmol) and 2‐O‐acetyl‐3,4,6‐tri‐O‐benzyl‐α‐d‐mannopyranosyl trichloroacetimidate (5;22 37 mg, 0.058) in CH2Cl2 over 4 Å sieves at −30 °C, The mixture was stirred for 30 min, warmed to 0 °C and quenched with Et3N (7 μL, 0.05 mmol) and then concentrated under reduced pressure. Flash chromatography (EtOAc/pet. ether, 25:75) gave the disaccharide 6 (37.4 mg, 87 %) as a colourless oil. [α] =−4.2 (c=0.89 in CHCl3); 1H NMR (CDCl3, 500 MHz): δ=2.80 (dd, J 1,1=14.4, J 1,2=0.9 Hz, 1 H; 1‐Ha), 3.15 (dt, J=10.1, 4.6 Hz, 1 H; 5‐H), 3.70–4.00 (m, 6 H; 3,4,4′,5′‐H, 6“‐Ha, 6′‐Hb), 4.03 (dd, J=9.3, 3.4 Hz, 1 H; 3′‐H), 4.17–4.20 (m, 1 H; 2‐H), 4.28 (dd, J=14.5, 2.2 Hz, 1 H; 1‐Hb), 4.47–4.52 (m, 3 H; 3×CH 2Ph), 4.60–4.64 (m, 2 H; 6‐Ha, CH 2Ph), 4.69 (d, J=11 Hz, 1 H; CH 2Ph), 4.76 (dd, J=11.6, 4.5 Hz, 1 H; 6‐Hb), 4.86 (d, J=11 Hz, 1 H; CH 2Ph), 5.12 (d, J=3.6 Hz, 2 H; CH2), 5.28 (d, J=1.6 Hz, 1 H; 1′‐H), 5.59 (dd, J=3.3, 1.8 Hz, 1 H; 2′‐H), 5.64 (s, 1 H; CH), 7.17–7.46 ppm (m, 25 H; Ph); 13C NMR (CDCl3, 125 MHz): δ=48.3 (1 C; C‐1), 56.3 (1 C; C‐5), 60.0, 72.7, 74.4, 77.8 (4 C; C‐3,4,4′,5), 67.7 (1 C; CH2), 68.5 (1 C; C‐2′), 69.1 (1 C; C‐6), 69.3 (1 C; C‐6′), 72.2, 73.6, 75.1 (3 C; CH2Ph), 78.1 (1 C; C‐2), 78.2 (1 C; C‐3′), 99.5 (1 C; C‐1′), 100.90 (1 C; CH), 100.92, 126.0, 127.77, 127.79, 127.83, 127.9, 128.0, 128.2, 128.28, 128.29, 128.41, 128.44, 128.5, 128.7, 128.9 ppm (30 C; Ph); HRMS (ESI, +ve): m/z calcd for C50H52N4O11: 907.3525 [M+Na]+; found: 907.3544.

3,4,6‐Tri‐: A solution of sodium methoxide in methanol (0.1 m, 10 μL, 1 μmol) was added to 6 (60 mg, 0.068 mmol) in methanol (0.5 mL) and the mixture was stirred for 1 h and then concentrated under reduced pressure to give an alcohol, which was used without purification. TFA/H2O (9:1, 100 μL) was added to the crude alcohol and the mixture was stirred for 30 min, concentrated and azeotroped with toluene (3×10 mL). Flash chromatography (EtOAc/pet. ether, 9:1) gave the triol 7 (42.5 mg, 83 %,). [α] =44.6 (c=1.03 in MeOH); 1H NMR (500 MHz, CD3OD): δ=2.67–4.20 (13 H; 1‐Ha–6‐Hb, 2′‐H–6′‐Hb), 4.43–4.46 (m, 2 H; 2×CH 2Ph), 4.52 (d, J=12.0 Hz, 1 H; CH 2Ph), 4.70 (d, J=12.7 Hz, 1 H; CH 2Ph), 4.72 (d, J=11.2 Hz, 1 H; CH 2Ph), 4.89 (d, J=2.1 Hz, 1 H; 1′‐H), 5.12 (s, 2 H; CH2), 5.15 (app. s, 1 H; 1′‐H), 7.03–7.42 ppm (m, 20 H; 4×Ph); 13C NMR (CDCl3, 125 MHz): δ=59.5, 68.0, 68.9, 69.0, 71.9, 72.5, 73.5, 74.2, 74.9, 79.5 (13 C; C‐1,2,3,4,5,6,1′,2′,3′,4′,5′,6′, CH2) 127.8, 127.9, 128.0, 128.1, 128.16, 128.19, 128.4, 128.5, 128.6, 128.7, 137.9, 138.0, 138.3 (24 C; Ph), 156.5 ppm (1 C; C=O); HRMS (ESI, +ve): m/z calcd for C41H46N4O10: 755.3287 [M+H]+; found: 755.3300.

3,4,6‐Tri‐: DTT (51 mg, 0.331 mmol) was added to a solution of azide 7 (25 mg, 0.0331 mmol) in pyridine (1 mL) and NaHCO3/H2CO3 buffer (0.625 mL, pH 9.16). The mixture was stirred at room temperature for 4 h, concentrated and azeotroped with toluene (5×10 mL). Flash chromatography (EtOAc/MeOH/H2O, 94:4:2) gave the amine 8 (80 %, 19.2 mg). 1H NMR (500 MHz, CD3OD): δ=2.89 (t, J=12.4 Hz, 1 H; 2‐H), 3.21–4.13 (13 C; m, 1‐Ha, 1‐Hb, 3,5‐H, 6‐Ha, 6‐Hb, 1′–6b′‐H), 4.36 (t, J=7.8 Hz, 1 H; 4‐H),4.46–4.54 (m, 2 H; 2×CH 2Ph), 4.58 (d, J=12.0 Hz, 1 H; CH 2Ph), 4.66 (d, J=11.8 Hz, 1 H; CH 2Ph), 4.77–4.81 (m, 2 H; 2×CH 2Ph), 4.98 (d, J=2.5 Hz, 1 H; 1′‐H), 5.15 (s, 2 H; CH2), 7.16–7.47 ppm (m, 20 H; Ph); 13C NMR (CDCl3, 125 MHz): δ=46.8, 59.9, 65.6, 68.5, 69.4, 70.4, 72.6, 73.7, 74.4, 75.4, 75.7, 78.1, 80.1, 100.8 (16 C; C‐1–6, C1′–6′, 4×CH2), 128.81, 128.84, 129.2, 129.28, 128.30, 129.3, 129.4, 129.5, 138.0, 139.3, 139.5, 139.6 ppm (24 C; Ph); HRMS (ESI, +ve): m/z calcd for C41H48N2O10: 729.3385 [M+H]+; found: 729.3398.

α‐: The triol 8 (19.2 mg, 0.0264 mmol) in EtOAc/MeOH/H2O (2:2:1, 3 mL) and 10 % HCl in methanol (0.3 mL) was treated with Pd(OH)2/C (50 mg) and H2 (20 atm, 18 h). The suspension was filtered, concentrated and purified with cation and anion resin (eluted with aqueous NH3) to give ManNH2DMJ (1; 70 %, 6.02 mg) as a colourless oil. [α] =17.2 (c=0.08 in H2O); 1H NMR (500 MHz, D2O): δ=2.78–2.84 ( m, 1 H; 5‐H), 3.09 (dd, J 1a,1b=14.0, J 1a,2=2.1 Hz, 1 H; 1‐Ha), 3.25 (dd, J 1a,1b=14.0, J 1a,2=3.2 Hz, 1 H; 1‐Hb), 3.62–3.95 (m, 9 H; 2,3,4,4′,5′‐H, 6‐Ha, 6′‐Ha, 6‐Hb, 6′‐Hb), 3.98 (dd, J 3′,4′=9.2, J 2′,3′=4.3 Hz, 1 H; 3′‐H), 4.09 (dd, J 2′,3′=3.3, J 1′,2′=1.8 Hz, 1 H; 2′‐H), 5.24 ppm (d, J 1′,2′=1.6 Hz, 1 H; 1′‐H); 13C NMR (125 MHz, D2O): δ=44.5, 50.4, 60.0, 60.8, 61.0, 66.6, 67.3, 69.7, 70.1, 73.7, 77.3, 101.6 ppm; HRMS (ESI, +ve): m/z calcd for C12H24N2O8: 325.1605 [M+H]+; found: 325.1606.

4‐Methylphenyl 2,4,6‐tri‐: A dry solution of the alcohol 9 23 (167 mg, 0.30 mmol) in DMF (5 mL) was cooled to 0 °C. The solution was charged with NaH (60 % dispersion 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 Et2O (20 mL), poured into ice/water and washed with water (3×20 mL) and brine (1×20 mL). The organic extracts were dried (MgSO4), the solvent was removed under reduced pressure and the resulting residue was subjected to flash chromatography (EtOAc/pet. ether, 15:85) to give the protected thioglycoside 10 (179.3 mg, 86 %) as a colourless oil. [α] =+65 (c=0.69 in CHCl3); 1H NMR (500 MHz, CDCl3): δ=2.28 (s, 3 H; TolMe), 3.78 (dd, J 5,6a=1.8, J 6a,6b=10.9 Hz, 1 H; 6‐Ha), 3.87 (dd, J 5,6b=5.2, J 6a,6b=10.9 Hz, 1 H; 6‐Hb), 3.97 (dd, J 2,3=3.0, J 3,4=9.3 Hz, 1 H; 3‐H), 4.04 (dd, J 1,2=3.0, J 2,3=1.8 Hz, 1 H; 2‐H), 4.11 (m, 1 H; 4‐H), 4.33 (ddd, J 4,5=9.8, J 5,6a=5.1, J 5,6b=1.6 Hz, 1 H; 5‐H), 4.49 (d, J=11.9 Hz, 1 H; CH 2Ph), 4.57–4.67 (m, 3 H; 3×CH 2Ph), 4.74 (m, 3 H; CH 2Ph, 2×CH 2Nap), 4.96 (d, J=10.9 Hz, 1 H; CH 2Ph), 5.58 (d, J 1,2=1.5 Hz, 1 H; 1‐H), 7.02 (app. d, J=7.9 Hz, 2 H; Tol), 7.21–7.37 (m, 17 H; 3×Ph, Tol), 7.44–7.47 (m, 3 H; Nap), 7.74–7.83 ppm (m, 4 H; Nap); 13C NMR (125 MHz, CDCl3): δ=21.2 (1 C; TolMe), 69.3 (1 C; C‐6), 71.9 (1 C; CH2Ph), 72.2 (1 C; CH2Nap), 72.8 (1 C; C‐5), 73.3 (1 C; CH2Ph), 75.1 (1 C; C‐4), 75.2 (1 C; CH2Ph), 76.3 (1 C; C‐2), 80.3 (1 C; C‐3), 86.1 (1 C; C‐1), 125.9–126.5 (4 C; Nap), 127.5–128.4 (18 C; 3×Ph, Nap), 129.8 (2 C; Tol), 132.3 (2 C; Tol), 133.4, 135.8, 137.6, 138.0, 138.5, 138.6 ppm (6 C; Cq); HRMS (ESI, +ve): m/z calcd for C45H44O5S: 719.2802 [M+Na]+; found: 719.2809.

2,4,6‐Tri‐ : N‐Iodosuccinimide (216 mg, 0.961 mmol) was added to a solution of the thioglycoside 10 (447 mg, 0.641 mmol) in acetone (1 % aq., 10 mL) at 0 °C and left to stir for 2.5 h. The solution was quenched with aq. Na2S2O3 (0.5 m, 10 mL), diluted with EtOAc (20 mL) and washed with aq. Na2S2O3 (0.5 m, 3×20 mL), NaHCO3 (2×20 mL) and brine (1×20 mL). The organic extracts were dried (MgSO4), the solvent was removed under reduced pressure and the resulting residue was subjected to flash chromatography (EtOAc/pet. ether/Et3N, 30:69.5:0.5) to afford the hemiacetals 11 (344 mg, 91 %; α/β 3.3:1) as a white powder. α anomer: 1H NMR (500 MHz, CDCl3): δ=3.69 (dd, J 5,6a=6.6, J 6a,6b=10.5 Hz, 1 H; 6‐Ha), 3.74 (dd, J 5,6b=2.0, J 6a,6b=10.4 Hz, 1 H; 6‐Hb), 3.83 (dd, J 1,2=2.0, J 2,3=2.8 Hz, 1 H; 2‐H), 3.91 (t, J 3,4=J 4,5=9.6 Hz, 1 H; 4‐H), 4.05 (dd, J 2,3=3.0, J 3,4=9.4 Hz, 1 H; 3‐H), 4.10 (ddd, J 4,5=8.7, J 5,6a=5.8, J 5,6b=1.9 Hz, 1 H; 5‐H), 4.51–4.59 (m, 3 H; 3×CH 2Ph), 4.74–4.76 (m, 4 H; 2×CH 2Ph, 2×CH 2Nap), 4.94 (d, J=11.0 Hz, 1 H; CH 2Ph), 5.27 (d, J 1,2=1.8 Hz, 1 H; 1‐H), 7.18–7.41 (m, 17 H; 3×Ph), 7.45–7.47 (m, 3 H; Nap), 7.72–7.83 ppm (m, 4 H; Nap); 13C NMR (125 MHz, CDCl3): δ=69.7 (1 C; C‐6), 71.4 (1 C; C‐5), 72.2 (1 C; CH2Nap), 72.7 (1 C; CH2Ph), 73.3 (1 C; CH2Ph), 75.1 (1 C; CH2Ph), 75.1 (1 C; C‐2), 75.3 (1 C; C‐4), 79.8 (1 C; C‐3), 92.6 (1 C; C‐1), 125.8–126.3 (4 C; Nap), 127.6–128.5 (18 C; 3×Ph, Nap), 133.0, 133.4, 136.1, 138.0, 138.5 ppm (6 C; Cq); HRMS (ESI, +ve): m/z calcd for C38H38O6: 608.3007 [M+NH4]+; found: 608.3007.

2,4,6‐Tri‐: A solution of the hemiacetal 11 (742 mg, 1.26 mmol) in acetic anhydride (6.1 mL) and dry DMSO (6.6 mL) was stirred under N2 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 (MgSO4) and 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. A portion of 12 obtained from a separate experiment was purified by flash chromatography (EtOAc/pet. ether, 1:9) to yield analytically pure 12 as a colourless oil. [α] =+4.05 (c=0.44 in CHCl3); 1H NMR (500 MHz, CDCl3): δ=3.61 (m, 2 H; 6‐Ha, 6‐Hb), 3.80 (dd, J 2,3=1.5, J 3,4=7.2 Hz, 1 H; 3‐H), 4.09 (dd, J 1,2=2.6, J 2,3=1.6 Hz, 1 H; 2‐H), 4.23 (m, 2 H; 5‐H, 4‐H), 4.38 (d, J=2.6 Hz, 1 H; CH 2Ph), 4.48 (app. d, 2 H; 2×CH 2Ph), 4.56 (d, J=11.8 Hz, 1 H; CH 2Ph), 4.77 (d, J=12.5 Hz, 1 H; CH 2Ph), 4.94 (d, J=12.5 Hz, 1 H; CH 2Ph), 5.06 (m, 2 H; 2×CH 2Nap), 6.96–7.45 (m, 18 H; 3×Ph, Nap), 7.69–7.78 ppm (m, 4 H; Nap); 13C NMR (125 MHz, CDCl3): δ=69.0 (1 C; C‐6), 71.6 (1 C; C‐4), 72.8 (1 C; CH2Ph), 72.9 (1 C; CH2Nap), 73.3 (1 C; CH2Ph), 75.5 (1 C; CH2Ph), 75.8 (1 C; C‐3), 76.5 (1 C; C‐2), 78.4 (1 C; C‐5), 125.9–126.1 (3 C; Nap), 126.9 (1 C; Nap), 127.6–128.9 (18 C; 3×Ph, Nap), 132.9, 133.0, 135.0, 136.7, 137.3, 137.6 (6 C; Cq), 169.3 ppm (1 C; C=O); HRMS (ESI, +ve): m/z calcd for C38H36O6: 606.2850 [M+NH4]+; found: 606.2853.

2,4,6‐Tri‐: A dry‐ice/acetone cold finger cooling trap was used to condense ammonia (50 mL) into a solution of the crude lactone 12 (823 mg) in dry THF (30 mL) at −78 °C. The solution was allowed to reflux at 0 °C for 4 h. The mixture was then evaporated to dryness to afford the crude amide 13 (771 mg), which was used directly in the next step. A portion obtained from an independent experiment was purified by flash chromatography (EtOAc/pet. ether, 3:2) to yield analytically pure 13 as a yellow solid. M.p. 120 °C; [α] =+7.21 (c=0.41 in CHCl3); 1H NMR (500 MHz, CDCl3): δ=3.20 (d, J 5,OH=6.2 Hz, 1 H; OH), 3.61 (m, 2 H; 6‐Ha, 6‐Hb), 3.87 (dd, J 3,4=5.9, J 4,5=7.3 Hz, 1 H; 4‐H), 3.98 (m, 1 H; 5‐H), 4.13 (dd, J 2,3=3.5, J 3,4=5.8 Hz, 1 H; 3‐H), 4.33 (d, J 2,3=3.5 Hz, 1 H; 2‐H), 4.43–4.60 (m, 6 H; 6×CH 2Ph), 4.82 (s, 2 H; 2×CH 2Nap), 5.50 (br s, 1 H; NH), 6.54 (br s, 1 H; NH), 7.11–7.27 (m, 15 H; 3×Ph), 7.38–7.43 (m, 3 H; Nap), 7.68–7.76 ppm (m, 4 H; Nap); 13C NMR (125 MHz, CDCl3): δ=71.1 (1 C; C‐5), 71.4 (1 C; C‐6), 72.9 (1 C; CH2Ph), 73.6 (1 C; CH2Ph), 74.6 (1 C; CH2Ph), 75.0 (1 C; CH2Nap), 79.1 (1 C; C‐4), 80.2 (1 C; C‐2), 81.6 (1 C; C‐3), 126.0–126.3 (3 C; Nap), 126.9 (1 C; Nap), 127.8–128.7 (18 C; 3×Ph, Nap), 133.1, 133.4, 135.7, 137.2, 138.2, 138.4 (6 C; Cq), 173.4 ppm (1 C; C=O); HRMS (ESI, +ve): m/z calcd for C38H39NO6: 606.2844 [M+H]+; found: 606.2850 ppm.

(3: A solution of the crude amide 13 (771 mg) in acetic anhydride (6.1 mL) and dry DMSO (6.6 mL) was stirred under N2 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 (MgSO4) and the solvent was evaporated to afford the keto‐amide 14 as a white solid. A dry‐ice/acetone cold finger was used to condense ammonia (20 mL) into a solution of the crude keto‐amide in dry methanol (30 mL) at 0 °C. The solution was allowed to warm to room temperature and stirred under N2 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 a separable mixture of the hydroxy‐lactams 15 (669 mg, 88 % over four steps; d‐manno/l‐gulo 2.2:1). 1H NMR (500 MHz, CDCl3), partial spectrum of the mixture of diastereomers: δ=3.38 (d, J=9.8 Hz, 1 H; CH 2(C6) d‐manno), 3.43 (d, J=9.6 Hz, 1 H; CH 2(C6) l‐gulo), 3.47 (d, J=9.8 Hz, 1 H; CH 2(C6) d‐manno), 3.57 (d, J=9.6 Hz, 1 H; CH 2(C6) l‐gulo), 3.72 (br s, 1 H; OH), 4.22 (d, J 3,4=3.0 Hz, 1 H; 3‐H d‐manno), 4.26 (d, J 3,4=3.1 Hz, 1 H; 3‐H l‐gulo), 4.98 (d, J=12.5 Hz, 1 H; CH 2Ph d‐manno), 5.10 (d, J=12.3 Hz, 1 H; CH 2Ph l‐gulo), 6.33 (br s, 1 H; NH l‐gulo), 6.22 ppm (br s, 1 H; NH d‐manno); 13C NMR (125 MHz, CDCl3): δ=74.0 (1 C; CH2(C6) d‐manno), 74.5 (1 C; C‐3 d‐manno), 169.6 (1 C; C=O d‐manno), 170.2 ppm (1 C; C=O l‐gulo); HRMS (ESI, +ve): m/z calcd for C38H37NO6: 604.2694 [M+H]+; found: 606.2698 ppm.

(3: Sodium cyanoborohydride (90.4 mg, 1.44 mmol) was added to a solution of the hydroxy‐lactams 15 (86.9 mg, 0.144 mmol) and formic acid (0.52 mL) in dry acetonitrile (3 mL) and the mixture stirred under N2 for 20 h. Sodium cyanoborohydride (90.4 mg, 1.44 mmol) was added and the reaction mixture was stirred for a further 24 h when TLC analysis (EtOAc/pet. ether, 1:3) indicated complete consumption of the starting material. The mixture was diluted with EtOAc (20 mL) and washed with aq. sat. NaHCO3 (3×20 mL) and brine (1×20 mL). The aqueous extracts were treated with sodium hypochlorite prior to disposal. The organic extracts were dried (MgSO4), the solvent was removed under reduced pressure and the resulting residue was subjected to flash chromatography (EtOAc/pet. ether, 1:1) to afford the l‐gulo lactam 16 (28.2 mg, 33 %) and the d‐manno lactam 17 (32.5 mg, 38 %), both as colourless oils.

Characterisation for 16: [α] =−57 (c=0.535 in CHCl3); 1H NMR (400 MHz, CDCl3): δ=3.36 (dd, J 6,6a=4.27, J 6a,6b=9.11 Hz, 1 H; CH 2(C6)), 3.46 (m, 2 H; 6‐H, CH 2(C6)), 3.57 (m, 1 H; 3‐H), 3.91 (dd, J 3,4=3.1, J 4,5=4.4 Hz, 1 H; 4‐H), 3.95 (m, 1 H; 6‐H), 4.08–4.19 (m, 3 H; 2×CH 2Ph, 5‐H), 4.40 (m, 2 H; 2×CH 2Ph), 4.66 (d, J=12.4 Hz, 1 H; CH 2Ph), 4.71 (d, J=12.3 Hz, 1 H; CH 2Nap), 4.93 (d, J=12.3 Hz, 1 H; CH 2Nap), 5.10 (d, J=12.4 Hz, 1 H; CH 2Ph), 5.83 (br s, 1 H; NH), 6.84 (app. d, J=7.05 Hz, 2 H; Ph), 7.07–7.45 (m, 16 H; Ph, Nap), 7.62 (s, 1 H; Nap), 7.72–7.79 ppm (m, 3 H; Nap); 13C NMR (100 MHz, CDCl3): δ=52.8 (1 C; C‐6), 70.3 (1 C; CH2(C6)), 72.5 (1 C; CH2Nap), 73.6 (1 C; CH2Ph), 73.6 (1 C; CH2Ph), 73.7 (1 C; CH2Ph), 74.2 (1 C; C‐5), 74.3 (1 C; C‐3), 74.8 (1 C; C‐4), 126.0–126.3 (3 C; Nap), 126.8 (1 C; Nap), 127.8–128.6 (18 C; 3×Ph, Nap), 133.2, 133.3, 135.6, 137.0, 137.6, 138.4 (6 C; Cq), 171.3 ppm (1 C; C=O); HRMS (ESI, +ve): m/z calcd for C38H37NO5: 588.2749 [M+H]+; found: 588.2747.

Characterisation for 17: [α] =−9.49 (c=0.715 in CHCl3); 1H NMR (400 MHz, CDCl3): δ=3.41 (m, 1 H; CH 2(C6)), 3.54 (m, 2 H; 6‐H, CH 2(C6)), 3.66 (t, J 4,5=J 5,6=5.2 Hz, 1 H; 5‐H), 3.98 (dd, J 3,4=2.9, J 4,5=5.0 Hz, 1 H; 4‐H), 4.18 (d, J 3,4=2.9 Hz, 1 H; 3‐H), 4.38 (d, J=11.6 Hz, 1 H; CH 2Ph), 4.42–4.49 (m, 2 H; 2×CH 2Ph), 4.55 (d, J=11.6 Hz, 1 H; CH 2Ph), 4.69 (d, J=12.1 Hz, 1 H; CH 2Ph), 4.74 (d, J=12.2 Hz, 1 H; CH 2Nap), 4.88 (d, J=12.2 Hz, 1 H; CH 2Nap), 5.06 (d, J=12.2 Hz, 1 H; CH 2Ph), 5.91 (br s, 1 H; NH), 7.08–7.49 (m, 18 H; 3×Ph, Nap), 7.72–7.84 ppm (m, 4 H; Nap); 13C NMR (100 MHz, CDCl3): δ=55.5 (1 C; C‐6), 71.5 (1 C; CH2(C6)), 72.9 (1 C; CH2Nap), 72.9 (1 C; CH2Ph), 73.4 (1 C; CH2Ph), 73.5 (1 C; CH2Ph), 75.0 (1 C; C‐5), 75.2 (1 C; C‐3), 77.8 (1 C; C‐4), 126.1–126.3 (3 C; Nap), 127.0 (1 C; Nap), 127.8–128.6 (18 C; 3×Ph, Nap), 133.2, 133.3, 135.5, 137.5, 138.1 (6 C; Cq), 169.6 ppm (1 C; C=O); HRMS (ESI, +ve): m/z calcd for C38H37NO5: 588.2744 [M+H]+; found: 588.2747.

(3: Lawesson's reagent (202 mg, 0.50 mmol) was added to a mixture containing the mannonolactam 17 (98 mg, 0.167 mmol), pyridine (6.7 μL, 0.083 mmol), freshly activated 4 Å molecular sieves and distilled toluene (6 mL) and the mixture was stirred for 20 h. The mixture was then filtered, stirred with MeOH (1.68 mL) for 2 h and the solvent removed under reduced pressure. The residue obtained was subjected to flash chromatography (EtOAc/pet. ether, 20:80) to afford the thionolactam 18 (94 mg, 93 %) as a white solid. M.p. 147 °C; [α] =−52 (c=0.215 in CHCl3); 1H NMR (400 MHz, CDCl3): δ=3.43 (m, 1 H; CH 2(C6)), 3.56 (m, 2 H; 6‐H, CH 2(C6)), 3.83 (apt. t, 1 H; 5‐H), 3.91 (dd, J 3,4=2.6, J 4,5=7.2 Hz, 1 H; 4‐H), 4.42 (d, J 3,4=2.5 Hz, 1 H; 3‐H), 4.44–4.52 (m, 3 H; 3×CH 2Ph), 4.68–4.73 (m, 2 H; CH 2Nap, CH 2Ph), 4.79 (d, J=12.1 Hz, 1 H; CH 2Nap), 4.83 (d, J=12.0 Hz, 1 H; CH 2Ph), 5.08 (d, J=12.1 Hz, 1 H; CH 2Ph), 7.14–7.52 (m, 18 H; 3×Ph, Nap), 7.73–7.85 (m, 4 H; Nap), 8.13 ppm (br s, 1 H; NH); 13C NMR (100 MHz, CDCl3): δ=59.8 (1 C; C‐6), 70.6 (1 C; CH2(C6)), 72.5 (1 C; CH2Nap), 73.2 (1 C; CH2Ph), 73.5 (1 C; CH2Ph), 73.7 (1 C; CH2Ph), 74.2 (1 C; C‐5), 78.3 (1 C; C‐4), 79.8 (1 C; C‐3), 125.9–126.3 (3 C; Nap), 126.8 (1 C; Nap), 127.8–128.7 (18 C; 3×Ph, Nap), 133.1, 133.3, 135.4, 137.3, 137.6, 138.0 (6 C; Cq), 200.0 ppm (1 C; C=O); HRMS (ESI, +ve): m/z calcd for C38H37NO4S: 604.2516 [M+H]+; found: 604.2524 [].

(5: Thionolactam 18 (256 mg, 0.424 mmol) was dissolved in aminoacetaldehyde dimethyl acetal (0.69 mL, 6.33 mmol) and the mixture stirred under N2 for 18 h. The mixture was diluted with Et2O (20 mL) and washed with H2O (2×20 mL) and brine (1×20 mL). The organic extracts were dried (MgSO4) and the solvent removed under reduced pressure to afford the amidines 19 as a colourless residue. p‐Toluenesulfonic acid monohydrate (0.14 g, 0.74 mmol) was added to a solution of the crude amidines in toluene (9.5 mL) and the mixture was stirred at 60 °C overnight. The mixture was then diluted with DCM (20 mL) and washed with NaHCO3 (2×20 mL) and brine (1×20 mL). The organic extracts were dried (MgSO4), the solvent was removed under reduced pressure and the residue was subjected to flash chromatography (EtOAc/pet. ether, 1:1) to afford the glucoimidazole 20 (110 mg, 42 % over two steps) as a colourless oil and the mannoimidazole 21 (83.3 mg, 32 % over two steps) as a yellow oil.

Characterisation for 20: [α] =+52 (c=0.315 in CHCl3; lit.:39 +52 (in CHCl3)); 1H NMR (600 MHz, CDCl3): δ=3.75 (dd, J 5,5a=5.0, J 5a,5b=10.3 Hz, 1 H; CH 2(C5)), 3.87 (m, 2 H; 6‐H, CH 2(C5)), 4.13 (dd, J 6,7=7.5, J 7,8=5.8 Hz, 1 H; 7‐H), 4.18 (m, 1 H; 5‐H), 4.45 (app. d, 2 H; 2×CH 2Ph), 4.51 (d, J=11.2 Hz, 1 H; CH 2Ph), 4.78 (d, J 7,8=5.8 Hz, 1 H; 8‐H), 4.84 (d, J=11.6 Hz, 1 H; CH 2Ph), 4.86 (d, J=11.2 Hz, 1 H; CH 2Ph), 4.89 (d, J=11.5 Hz, 1 H; CH 2Nap), 4.97 (d, J=11.5 Hz, 1 H; CH 2Ph), 5.19 (d, J=11.5 Hz, 1 H; CH 2Nap), 7.04 (s, 1 H; 2‐H), 7.12 (s, 1 H; 3‐H), 7.14–7.48 (m, 18 H; 3×Ph, Nap), 7.68–7.83 ppm (m, 4 H; Nap); 13C NMR (125 MHz, CDCl3): δ=58.3 (1 C; C‐5), 68.5 (1 C; CH2(C5)), 72.9 (1 C; CH2Nap), 73.4 (1 C; CH2Ph), 74.3 (1 C; CH2Ph), 74.4 (1 C; CH2Ph), 74.5 (1 C; C‐8), 76.2 (1 C; C‐6), 82.2 (1 C; C‐7), 117.4 (1 C; C‐2), 126.1–126.9 (3 C; Nap), 127.7 (1 C; Nap), 127.8–128.6 (18 C; 3×Ph, Nap), 129.5 (1 C; C‐3), 133.2, 133.4, 135.5, 137.4, 137.7, 138.4 (6 C; Cq), 144.2 ppm (Cq, imidazole).

Characterisation for 21: [α] =−24 (c=0.24 in CHCl3: lit.:39 −20 (in CHCl3)); 1H NMR (600 MHz, CDCl3): δ=3.57 (dd, J 5,5a=7.1, J 5a,5b=10.1 Hz, 1 H; CH 2(C5)), 3.71 (dd, J 5,5a=3.4, J 5a,5b=10.1 Hz, 1 H; CH 2(C5)), 3.84 (dd, J 6,7=9.3, J 7,8=3.1 Hz, 1 H; 7‐H), 4.06 (m, 1 H; 5‐H), 4.25 (dd, J 5,6=9.3, J 6,7=7.2 Hz, 1 H; 6‐H), 4.39 (m, 2 H; 2×CH 2Ph), 4.56–4.66 (m, 3 H; 2×CH 2Ph, CH 2Nap), 4.69 (d, J=12.2 Hz, 1 H; CH 2Nap), 4.74 (d, J=12.0 Hz, 1 H; CH 2Ph), 4.78 (d, J 7,8=3.0 Hz, 1 H; 8‐H), 4.96 (d, J=11.2 Hz, 1 H; CH 2Ph), 6.98 (s, 1 H; 3‐H), 7.09 (s, 1 H; 2‐H), 7.17–7.39 (m, 18 H; 3×Ph, Nap), 7.62–7.74 ppm (m, 4 H; Nap); 13C NMR (125 MHz, CDCl3): δ=60.0 (1 C; C‐5), 68.3 (1 C; C8), 70.6 (1 C; CH2Nap), 71.2 (1 C; CH2(C5)), 71.8 (1 C; CH2Ph), 73.3 (1 C; CH2Ph), 74.3 (1 C; C‐6), 75.0 (1 C; CH2Ph), 80.2 (1 C; C‐3), 119.5 (1 C; C‐2), 125.2–126.9 (3 C; Nap), 126.7 (1 C; Nap), 128.6–127.7 (18 C; 3×Ph, Nap), 129.4 (1 C; C‐3), 133.2, 133.3, 135.4, 137.6, 138.2, 138.3 (6 C; Cq), 143.0 ppm (Cq, imidazole).

(5: DDQ (25.2 mg, 0.111 mmol) was added to a solution of the mannoimidazole 21 (22.6 mg, 0.037 mmol) in DCM/H2O (9:1, 1 mL) and the reaction mixture was stirred at room temperature overnight. DDQ (25 mg, 0.11 mmol) was again added and the mixture stirred for 3 days 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. NaHCO3 (3×20 mL), dried (MgSO4), filtered and concentrated. The crude product was purified by flash chromatography (EtOAc/pet. ether, 80:20 to 100:0) to afford the alcohol 22 (11.7 mg, 67 %) as a yellow oil. [α] =−35 (c=0.585 in CHCl3; lit.:40 −6 (in CHCl3)); 1H NMR (500 MHz, CDCl3): δ=3.64 (dd, J 5,5a=5.9, J 5a,5b=10.2 Hz, 1 H; CH 2(C5)), 3.78 (dd, J 5,5a=2.5, J 5a,5b=10.2 Hz, 1 H; CH 2(C5)), 4.03 (m, 3 H; 7‐H, 6‐H, 5‐H), 4.42 (app. s, 2 H; 2×CH 2Ph), 4.54 (d, J=11.2 Hz, 1 H; CH 2Ph), 4.65 (d, J=11.6 Hz, 1 H; CH 2Ph), 4.70 (d, J 7,8=3.3 Hz, 1 H; 8‐H), 4.85 (d, J=11.6 Hz, 1 H; CH 2Ph), 4.90 (d, J=11.2 Hz, 1 H; CH 2Ph), 7.05 (s, 1 H; 3‐H), 7.13 (s, 1 H; 2‐H), 7.19–7.28 ppm (m, 15 H; 3×Ph); 13C NMR (125 MHz, CDCl3): δ=59.1 (1 C; C‐5), 70.2 (1 C; CH2(C5)), 71.2 (2 C; C‐8, CH2Ph), 72.4 (1 C; C‐6), 73.2 (1 C; CH2Ph), 74.6 (1 C; CH2Ph), 75.3 (1 C; C‐7), 118.9 (1 C; C‐2), 127.7–128.5 (15 C; 3×Ph), 129.6 (1 C; C‐3), 137.5, 137.7, 137.8 (3 C; Cq), 142.3 ppm (Cq, imidazole).

(5: A mixture of the alcohol 22 (13.8 mg, 0.029 mmol), 2‐O‐acetyl‐3,4,6‐tri‐O‐benzyl‐α‐d‐mannopyranosyl trichloroacetimidate (5;22 32.5 mg, 0.051 mmol) and freshly activated 4 Å molecular sieves in toluene (1.5 mL) was stirred at room temperature for 30 min. Triflic acid (1 μL, 0.011 mmol) was added to the mixture at −20 °C and the mixture was stirred for 1 h, then at 0 °C for 20 min, and at room temperature for another 20 min, quenched with pyridine (1 drop) and filtered through a pad of Celite. The solvent was removed under reduced pressure and the resulting residue was subjected to flash chromatography (EtOAc/pet. ether/ Et3N 80:19:1) to recover alcohol 26 (6.4 mg) and afford the disaccharide 23 (12.9 mg, 47 %) as a colourless oil. [α] =+7.2 (c=0.175 in CHCl3); 1H NMR (600 MHz, CDCl3): δ=2.11 (s, 3 H; Ac), 3.49 (dd, J 5′,5a′=1.7, J 5a“,5b′=10.9 Hz, 1 H; CH 2(C5′)), 3.55 (dd, J 5,5a=6.7, J 5a,5b=10.2 Hz, 1 H; CH 2(C5)), 3.63 (dd, J 5′,5b′=3.5, J 5a”,5b′=10.8 Hz, 1 H; CH 2(C5′)), 3.67 (dd, J 5,5b=3.2, J 5a,5b=10.2 Hz, 1 H; CH 2(C5)), 3.87 (m, 1 H; 5′‐H), 3.93 (t, J 3′,4′=J 4′,5′=9.5 Hz, 1 H; 4′‐H), 4.01 (dd, J 2′,3′=3.3, J 3′,4′=9.5 Hz, 1 H; 3′‐H), 4.07 (dd, J 6,7=9.5, J 7,8=3.1 Hz, 1 H; 7‐H), 4.13 (1 H, m, 5‐H), 4.29 (dd, J 5,6=7.1, J 6,7=9.5 Hz, 1 H; 6‐H), 4.41 (m, 2 H; 2×CH 2Ph), 4.46 (d, J=10.9 Hz, 1 H; CH 2Ph), 4.51 (d, J=11.3 Hz, 1 H; CH 2Ph), 4.54 (d, J=12.0 Hz, 1 H; CH 2Ph), 4.57 (d, J=11.3 Hz, 1 H; CH 2Ph), 4.64 (app. d, 3 H, 3×CH 2Ph), 4.81 (d, J 2,3=3.1 Hz, 1 H; 2‐H), 4.84 (m, 2 H; 2×CH 2Ph), 5.19 (d, J 1′,2′=1.6 Hz, 1 H; 1′‐H), 5.48 (dd, J 1′,2′=1.6, J 2′,3′=3.3 Hz, 1 H; 2′‐H), 7.07 (s, 1 H; 3‐H), 7.14 (s, 1 H; 2‐H), 7.08–7.34 ppm (m, 30 H; 6×Ph); 13C NMR (125 MHz, CDCl3): δ=21.2 (1 C; Me), 60.0 (1 C; C‐5), 68.5 (1 C; C‐6′), 69.1 (1 C; C‐2′), 70.3 (1 C; CH2Ph), 70.8 (1 C; CH2(C5)), 70.9 (1 C; C‐8), 72.1 (1 C; CH2Ph), 72.4 (1 C; C‐5′), 73.4 (1 C; CH2Ph), 73.7 (1 C; CH2Ph), 74.2 (1 C; C‐4′), 74.4 (1 C; C‐6), 75.1 (2 C; CH2Ph), 78.2 (1 C; C‐3′), 80.3 (1 C; C‐7), 100.1 (1 C; C‐1′), 119.4 (1 C; C‐2), 127.6–128.7 (30 C; 6×Ph), 129.5 (1 C; C‐3), 137.6, 137.7, 137.9, 138.1, 138.2, 138.8 (6 C; Cq), 142.6 (Cq, imidazole), 170.4 ppm (1 C; C=O); HRMS (ESI, +ve): m/z calcd for C58H60N2O10: 945.4321 [M+H]+; found: 945.4322.

(5: K2CO3 (1 mg, 0.007 mmol) was added to a solution of the acetate 23 (13.1 mg, 0.014 mmol) in dry methanol (0.3 mL) and the resulting suspension was stirred at room temperature for 6.5 h. The reaction mixture was quenched with acetic acid (5 μL, 0.087 mmol), the solvent was removed under reduced pressure and the resulting residue was subjected to flash chromatography (EtOAc/pet. ether/Et3N 50:49.5:0.5) to afford the alcohol 24 (5.8 mg, 46 %) as a colourless oil. [α] =+13 (c=0.305 in CHCl3); 1H NMR (500 MHz, CDCl3): δ=2.40 (d, J 2′,OH=2.5 Hz, 1 H; OH), 3.49 (dd, J 5′,6a′=1.8, J 6a“,6b′=10.8 Hz, 1 H; 6′‐Ha), 3.58 (m, 2 H; CH 2(C5), 6′‐Hb), 3.70 (dd, J 5,5a=3.2, J 5a,5b=10.1 Hz, 1 H; CH 2(C5)), 3.87 (m, 1 H; 5′‐H), 3.91 (m, 2 H; 4′,3′‐H), 4.03 (m, 1 H; 2′‐H), 4.08 (dd, J 6,7=9.6, J 7,8=3.1 Hz, 1 H; 7‐H), 4.13 (1 H, m, 5‐H), 4.28 (dd, J 5,6=7.3, J 6,7=9.6 Hz, 1 H; 6‐H), 4.40–4.53 (m, 5 H; 5×CH 2Ph), 4.57–4.68 (m, 5 H; 5×CH 2Ph), 4.79 (m, 2 H; 2×CH 2Ph), 4.85 (d, J 7,8=3.1 Hz, 1 H; 8‐H), 5.23 (d, J 1′,2′=1.5 Hz, 1 H; 1′‐H), 7.08 (s, 1 H; 3‐H), 7.14 (s, 1 H; 2‐H), 7.11–7.35 ppm (m, 30 H; 6×Ph); 13C NMR (125 MHz, CDCl3): δ=60.0 (1 C; C‐5), 68.6 (1 C; C‐6′), 69.0 (1 C; C‐2′), 70.3 (1 C; CH2Ph), 70.7 (1 C; C‐8), 71.1 (1 C; CH2(C5)), 72.0 (1 C; C‐5′), 72.4 (1 C; CH2Ph), 73.4 (1 C; CH2Ph), 73.7 (1 C; CH2Ph), 74.3 (2 C; C‐6,3′), 75.1 (2 C; CH2Ph), 80.1 (1 C; C‐4′), 80.4 (1 C; C‐7), 101.8 (1 C; C‐1′), 119.3 (1 C; C‐2), 127.6–128.7 (30 C; 6×Ph), 129.6 (1 C; C‐3), 137.6, 137.8, 138.1, 138.3, 138.7 (6 C; Cq), 142.7 ppm (Cq, imidazole); HRMS (ESI, +ve): m/z calcd for C56H58N2O9: 903.4215 [M+H]+; found: 903.4214.

(5: Pd(OH)2/C (20 %, 24 mg) was added to a solution of the deacetylated disaccharide 24 (12.6 mg, 0.014 mol) in EtOAc/MeOH/H2O (5:17:3, 1.50 mL) and AcOH (0.34 mL). The reaction vessel was filled with H2 (34 bar) and agitated for 4 days. At this point TLC analysis (EtOAc/MeOH/H2O, 7:3:2) indicated complete conversion to a single species along with baseline by‐products. The suspension was filtered through a pad of Celite, the solvent was evaporated and the resulting residue was subjected to flash chromatography (EtOAc/MeOH/H2O, 5:2:1) to afford ManManIm (2; 2.4 mg, 48 %) as a colourless residue. [α] =+13 (c=0.12 in H2O); 1H NMR (500 MHz, D2O): δ=3.57 (t, J 3′,4′=J 4′,5′=9.8 Hz, 1 H; 4′‐H), 3.66 (dd, J 5′,6a′=6.3, J 6a“,6b′=12.1 Hz, 1 H; 6′‐Ha), 3.77 (m, 1 H; 5′‐H), 3.83 (m, 2 H; 3′‐H, 6′‐Hb), 3.91 (m, 1 H; 5‐H), 3.95 (dd, J 5,5a=3.3, J 5a,5b=12.7 Hz, 1 H; CH 2(C5)), 3.99 (dd, J 6,7=10.2, J 7,8=3.7 Hz, 1 H; 7‐H), 4.02 (dd, J 1′,2′=3.4, J 2′,3′=1.7 Hz, 1 H; 2′‐H), 4.13 (dd, J 5,5b=2.6, J 5a,5b=12.7 Hz, 1 H; CH 2(C5)), 4.27 (dd, J 5,6=8.6, J 6,7=10.2 Hz, 1 H; 6‐H), 4.97 (d, J 7,8=3.7 Hz, 1 H; 8‐H), 5.23 (d, J 1′,2′=1.6 Hz, 1 H; 1′‐H), 7.01 (s, 1 H; 3‐H), 7.20 ppm (s, 1 H; 2‐H); 13C NMR (125 MHz, D2O): δ=59.3 (1 C; CH2(C5)), 60.9 (1 C; C‐5,6′), 63.5 (1 C; C‐8), 63.9 (1 C; C‐6), 66.7 (1 C; C‐4′), 69.9 (1 C; C‐2′), 70.3 (2 C; C‐4,3′), 73.5 (1 C; C‐5′), 78.1 (1 C; C‐7), 102.1 (1 C; C‐1′), 118.3 (1 C; C‐2), 128.7 (1 C; C‐3), 144.7 ppm (Cq, imidazole); HRMS (ESI, +ve): m/z calcd for C14H22N2O9: 363.1398 [M+H]+; found: 363.1398.

Isothermal titration calorimetry (ITC): The binding affinity of Man2NH2DMJ (1) to BtGH99 was determined by using a Microcal iTC200 calorimeter (GE Healthcare/Malvern Instruments). The assay was carried out at 25 °C with 18×2 μL injections of the inhibitor (6 mm) titrated into the ITC cell containing 117 μm BtGH99. Owing to the low affinity of the ligand, which prevented the observation of a sigmoidal binding isotherm, N was fixed at 1.41 An initial ITC experiment was conducted by using 1 m inhibitor in the syringe and 52 μm protein with 24×1.5 μL injections. The dissociation constant (K D), change in enthalpy (ΔH) and measurement uncertainty were calculated by using the MicroCal PEAQ‐ITC Analysis Software (Malvern Instruments).

Crystallisation and data collection: BxGH99 protein10 was crystallised by using the vapour diffusion hanging drop method in 3 m sodium acetate at pH 7.4. Crystals were grown at 19 °C in a 24‐well plate with 500 μL of reservoir solution in each well and sealed with vacuum grease. The droplet was created by mixing 1 μL of BxGH99 solution (34 mg mL−1 in 25 mm HEPES buffer, pH 7.0, 100 mm NaCl) with 1 μL of the crystallant solution. Crystals were fished from the droplet by using a nylon cryoloop, without cryoprotection. Data were collected at Diamond Light Source beamline i04 using X‐rays with a wavelength of 0.979 Å.

Structure solution and refinement: Images containing diffraction patterns were indexed and integrated by using DIALS42 through xia2.43 The hkl index of each data set was then matched to a previous solution in Aimless.44 Refinement was performed by using Refmac545 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

Conflict of interest

The authors declare no conflict of interest.

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Supplementary

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