Combined inhibitor free-energy landscape and structural analysis reports on the mannosidase conformational coordinate

Article abstract of DOI:10.1002/anie.201308334

Mannosidases catalyze the hydrolysis of a diverse range of polysaccharides and glycoconjugates, and the various sequence-based mannosidase families have evolved ingenious strategies to overcome the stereoelectronic challenges of mannoside chemistry. Using a combination of computational chemistry, inhibitor design and synthesis, and X-ray crystallography of inhibitor/enzyme complexes, it is demonstrated that mannoimidazole-type inhibitors are energetically poised to report faithfully on mannosidase transition-state conformation, and provide direct evidence for the conformational itinerary used by diverse mannosidases, including β-mannanases from families GH26 and GH113. Isofagomine-type inhibitors are poor mimics of transition-state conformation, owing to the high energy barriers that must be crossed to attain mechanistically relevant conformations, however, these sugar-shaped heterocycles allow the acquisition of ternary complexes that span the active site, thus providing valuable insight into active-site residues involved in substrate recognition. Shipshape inhibitors: Quantum mechanical calculations of the free-energy landscape (see figure) of the glycosidase transition-state mimics isofagomine and mannoimidazole reveals that only the latter is energetically poised to report upon the mannosidase transition-state conformation. X-ray structures of β-mannanases from different families reveal they both adopt a boat conformation, thus allowing unification of the enzymatic conformational itinerary of a range of diverse α- and β-mannosidases.

Full text of DOI:10.1002/anie.201308334

Supporting Information
ꢀ
Wiley-VCH 2014
69451 Weinheim, Germany
Combined Inhibitor Free-Energy Landscape and Structural Analysis
Reports on the Mannosidase Conformational Coordinate**
Rohan J. Williams, Javier Iglesias-Fernꢀndez, Judith Stepper, Adam Jackson,
Andrew J. Thompson, Elisabeth C. Lowe, Jonathan M. White, Harry J. Gilbert, Carme Rovira,*
Gideon J. Davies,* and Spencer J. Williams*
anie_201308334_sm_miscellaneous_information.pdf

Table of Contents
Figure S1: X-ray structures of ManIFG bound to GH26 and GH113 -mannanases. ...S3
Figure S2: GH26 and GH113 β-mannanases in complex with Man-IFG and Man-MIm
(including a single ternary complex with Man-IFG and Man-Man). ...............................S4
Figure S3: Conformational free energy landscape (Mercator projection) of isolated
isofagomine 1 annotated with the conformations of isofagomine-type inhibitors that
have been observed on-enzyme for mannosidases and glucosidases.................................S5
Figure S4: Remodelled conformations for DmGManII GH38-bound mannoimidazole.S6
Figure S5: Plot of atomic charge for protonated mannoimidazole 2 at C1......................S7
Figure S6: C5-O5-C1-C2 dihedral angle for each relevant conformation of protonated
mannoimidazole 2. .................................................................................................................S8
Figure S7. Computed TS index (TSi) corresponding to the four relevant protonated
mannoimidazole 2 conformations.........................................................................................S9
Figure S8: X-ray structure for D-gluco isomer of 15........................................................S10
Figure S9: HPLC purification of 17...................................................................................S11
Table S1: X-ray data and structure refinement statistics................................................S12
Table S2: Calculated values of qC1, the C5-O5-C1-C2 dihedral angle, Grel and TSi for
the four relevant canonical conformations........................................................................S13
Table S3: Crystal data and structure refinement for (5R,6R,7S,8S)-7,8-bis(benzyloxy)-5-
[
(benzyloxy)methyl]-6-hydroxy-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine. ..................S14
Computational Chemistry...................................................................................................S15
Cloning, gene expression and protein purification. ..........................................................S15
Collection of structural data, processing and structure solution.....................................S16
Calculation of transition state inhibition index (TSi). ......................................................S17
Re-refinement of GH38 mannoimidazole complex...........................................................S18
Small molecule crystallographic methods. ........................................................................S18
Crystallographic data..........................................................................................................S18
Synthetic overview. ..............................................................................................................S20
General synthetic methods..................................................................................................S20
4
5
5
,5’-O-Benzylidene-3-O-p-toluoyl-N-benzyloxycarbonyl-isofagomine (6)......................S21
’-O-Benzyl-3-O-p-toluoyl-N-benzyloxycarbonyl-isofagomine (7). ................................S21
’-O-Benzyl-4-O-(2,3-di-O-benzyl-4,6-O-benzylidene--D-mannopyranosyl)-3-O-p-
toluoyl-N-benzyloxycarbonyl-isofagomine (8)..................................................................S22
(3R,4R,5R)-3-Hydroxy-5-(hydroxymethyl)-4-(-D-mannopyranosyloxy)piperidine
(ManIFG; 3). ......................................................................................................................S23
4
4
-Methylphenyl 2,3,6-tri-O-benzyl-1-thio--D-glucopyranoside (9)................................S24
-Methylphenyl 2,3,6-tri-O-benzyl-4-O-(4-methoxybenzyl)-1-thio--D-glucopyranoside
(10). ....................................................................................................................................S24
2
2
2
,3,6-Tri-O-benzyl-4-O-(4-methoxybenzyl)-D-glucopyranose.........................................S25
,3,6-Tri-O-benzyl-4-O-(4-methoxybenzyl)-D-gluconolactone (11). ...............................S27
,3,6-Tri-O-benzyl-4-O-(4-methoxybenzyl)-D-gluconamide............................................S27
(
3R,4S,5S,6R)-3,4,-Bis(benzyloxy)-6-[(benzyloxy)methyl]-5-(4-
methoxybenzyloxy)piperidin-2-one...................................................................................S27

(
3R,4S,5S,6S)-3,4-Bis(benzyloxy)-6-[(benzyloxy)methyl]-5-(4-
methoxybenzyloxy)piperidin-2-thione (13). ......................................................................S28
Glycoimidazole formation..................................................................................................S29
i)
tetrahydroimidazo[1,2-a]pyridine (15)...........................................................................S29
ii) (5R,6R,7S,8S)-7,8-Bis(benzyloxy)-5-[(benzyloxy)methyl]-6-hydroxy-5,6,7,8-
(5R,6R,7S,8R)-7,8-Bis(benzyloxy)-5-[(benzyloxy)methyl]-6-hydroxy-5,6,7,8-
tetrahydroimidazo[1,2-a]pyridine...................................................................................S29
Glycosylation of 15. ...........................................................................................................S30
i) (5R,6R,7S,8R)-6-(2,3-Di-O-benzyl-4,6-O-benzylidene--D-mannopyranosyloxy)-7,8-
bis(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine
(17).
.
.......................................................................................................................................S30
ii) (5R,6R,7S,8R)-6-(2,3-Di-O-benzyl-4,6-O-benzylidene--D-mannopyranosyloxy)-7,8-
bis(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine. ......S31
(
5R,6R,7S,8R)-7,8-Dihydroxy-5-[(hydroxy)methyl]-6-(-D-mannopyranosyloxy)-5,6,7,8-
tetrahydroimidazo[1,2-a]pyridine-7,8-diol (ManMIm; 4). ................................................S31
References.............................................................................................................................S33
NMR Spectra........................................................................................................................S35
4
5
5
,5’-O-Benzylidene-3-O-p-toluoyl-N-benzyloxycarbonyl-isofagomine (6)......................S35
’-O-Benzyl-3-O-p-toluoyl-N-benzyloxycarbonyl-isofagomine (7) .................................S36
’-O-Benzyl-4-O-(2,3-di-O-benzyl-4,6-O-benzylidene--D-mannopyranosyl)-3-O-p-
toluoyl-N-benzyloxycarbonyl-isofagomine (8)..................................................................S38
(3R,4R,5R)-3-Hydroxy-5-(hydroxymethyl)-4-(-D-mannopyranosyloxy)piperidine
(ManIFG; 3) .......................................................................................................................S41
4
2
2
-Methylphenyl 2,3,6-tri-O-benzyl-1-thio--D-glucopyranoside (9)................................S43
,3,6-Tri-O-benzyl-4-O-(4-methoxybenzyl)-D-gluconolactone (11) ................................S46
,3,6-Tri-O-benzyl-4-O-(4-methoxybenzyl)-D-gluconamide............................................S48
(
3R,4S,5S,6S)-3,4,-Bis(benzyloxy)-6-[(benzyloxy)methyl]-5-(4-
methoxybenzyloxy)piperidin-2-one (12) ...........................................................................S50
3R,4S,5S,6R)-3,4,-Bis(benzyloxy)-6-[(benzyloxy)methyl]-5-(4-
methoxybenzyloxy)piperidin-2-one...................................................................................S52
3R,4S,5S,6S)-3,4,-Bis(benzyloxy)-6-[(benzyloxy)methyl]-5-(4-
methoxybenzyloxy)piperidin-2-thione (13) .......................................................................S54
5R,6R,7S,8R)-7,8-Bis(benzyloxy)-5-[(benzyloxy)methyl]-6-hydroxy-5,6,7,8-
tetrahydroimidazo[1,2-a]pyridine (15)...............................................................................S56
5R,6R,7S,8S)-7,8-Bis(benzyloxy)-5-[(benzyloxy)methyl]-6-hydroxy-5,6,7,8-
tetrahydroimidazo[1,2-a]pyridine ......................................................................................S58
5R,6R,7S,8R)-6-(2,3-Di-O-benzyl-4,6-O-benzylidene--D-mannopyranosyloxy)-7,8-
bis(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine...........S60
5R,6R,7S,8R)-6-(2,3-Di-O-benzyl-4,6-O-benzylidene--D-mannopyranosyloxy)-7,8-
bis(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine (17) ...S62
(
(
(
(
(
(
(
5R,6R,7S,8R)-7,8-Dihydroxy-5-[(hydroxy)methyl]-6-(-D-mannopyranosyloxy)-5,6,7,8-
tetrahydroimidazo[1,2-a]pyridine-7,8-diol (ManMIm; 4) .................................................S64
S2

Figure S1: X-ray structures of ManIFG bound to GH26 and GH113 -
mannanases.
Binary complex of ManIFG 3 bound to (a) CjMan26C and (b) GH113 AaManA.
A o c
Depicted electron density is a REFMAC maximum-likelihood/σ weighted 2F −F
3
synthesis contoured at 0.41 electrons per Å .
S3

Figure S2: GH26 and GH113 β-mannanases in complex with Man-IFG and Man-
MIm (including a single ternary complex with Man-IFG and Man-Man).
Figures show identical views to those depicted in Figures 2 and S1, with identical
REFMAC maximum-likelihood/σ
.0σ (blue density). Additionally, in order to confirm binding, unbiased F
with phases calculated prior to the incorporation of any ligand in refinement are also
shown (green density). Difference maps are REFMAC maximum-likelihood/σ
A
weighted 2F
o
−F
c
syntheses, each contoured at
1
o
−F maps
c
A
weighted F
o
−F
c
syntheses contoured at 3.0σ.
S4

Figure S3: Conformational free energy landscape (Mercator projection) of
isolated isofagomine 1 annotated with the conformations of isofagomine-type
inhibitors that have been observed on-enzyme for mannosidases and
glucosidases.
1
: 3 bound to GH26 CjMan26C (this work, PDB code 4CD4); 2: 3 bound to GH113
AaManA (this work, PDB code 4CD6); 3: 1 bound to GH1 Thermotoga maritima b-
[
1]
glucosidase (PDB code 1OIF); 5: 1 bound to GH3 Aspergillus aculeatus b-
[
2]
glucosidase (PDB code 4IIC); 6: -Glc-1,4-isofagomine bound to GH10
Cellulomonas fimi xylanase/cellulase (PDB code 3CUF); 7: -Glc-1,4-isofagomine
[
3]
bound to GH5 Rhodococcus sp. endoglycoceramidase II (PDB code 2OYK); 8: 1
[
4]
bound to GH30 Homo sapiens acid -glucosidase (PDB code 3GXF); 9: -Glc-1,4-
isofagomine bound to GH6 Mycobacterium tuberculosis cellulase (PDB code
[
5]
1
UP2); 10: -Glc-1,4-isofagomine bound to GH5 Bacillus agaradhaerans Cel5A
[
6]
(
PDB code 1OCQ); 11: -Glc-1,4-isofagomine bound to GH6 Humicola insolens
[
7]
Cel6A (PDB code 1OCN); 12,13: -Glc-1,3-isofagomine bound to BxGH99 (PDB
[
8]
–1
code 4AD2, 4AD4); FEL contoured at 1 kcal mol .
S5

Figure S4: Remodelled conformations for DmGManII GH38-bound
mannoimidazole.
4
[9]
(
a) “Side” and (b) “front” view of the published E and remodelled near-B2,5
conformations. Electron density shown is a 2F
o
-F
c
c
synthesis contoured at about 1σ
3
(
0.4 electrons/Å in blue) with the difference F
o
-F
map contoured at approximately
3
2
.3σ (0.15 / -0.15 electrons/Å in green/red respectively). Phases used for maps are
calculated from the deposited coordinates, prior to the incorporation of the second
modelled conformation in refinement.
S6

0
0
0
.3
.2
.1
0
3H4
B2,5
2,5B
-
0.1
0.2
4H3
-
conformation
Figure S5: Plot of atomic charge for protonated mannoimidazole 2 at C1.
ESP charge computed from the electronic density.
S7

6
4
2
0
2
4
3H4
B2,5
2,5B
4H3
-
-
conformation
Figure S6: C5-O5-C1-C2 dihedral angle for each relevant conformation of
protonated mannoimidazole 2.
S8

Figure S7. Computed TS index (TSi) corresponding to the four relevant
protonated mannoimidazole 2 conformations.
S9

Figure S8: X-ray structure for D-gluco isomer of 15.
Plot shows thermal ellipsoid plot (at 20% probability level) for one of the two
independent molecules of (5R,6R,7S,8S)-7,8-bis(benzyloxy)-5-[(benzyloxy)methyl]-
6-hydroxy-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine.
S10

DAD1 A, Sig=220,16 Ref=600,100 (R:\111114\RW.D)
mAU
400
300
200
100
0
1
2
14
16
16
16
18
18
18
20
20
20
22
22
22
24
24
24
26 min
26 min
26 min
DAD1 A, Sig=220,16 Ref=600,100 (R:\111116\RW-7-38-F1.D)
mAU
400
300
200
100
0
12
14
DAD1 A, Sig=220,16 Ref=600,100 (R:\111116\RW-7-38-F2.D)
mAU
400
300
200
100
0
12
14
Figure S9: HPLC purification of 17.
a) Analytical HPLC trace of anomeric mixture of glycosylation prior to HPLC
purification; b) analytical HPLC trace of 17 after preparative HPLC purification; c)
analytical HPLC trace of (5R,6R,7S,8R)-6-(2,3-di-O-benzyl-4,6-O-benzylidene--D-
mannopyranosyloxy)-7,8-bis(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-
tetrahydroimidazo[1,2-a]pyridine minor product after preparative HPLC purification.
t
Minor impurities eluting at approximately R = 15 min are attributed to low levels of
hydrolysis of the benzylidene acetal under the acidic conditions employed. The
impurities were not detectable by NMR.
S11

Table S1: X-ray data and structure refinement statistics.
CjMan26C-
ManIFG
CjMan26C-
ManMIm
AaManA-ManIFG AaManA-ManIFG AaManA-
mannobiose ManMIm
Space Group
Resolution (Å)
P6
1
22
P6
1
22
P2
1
2
1
2
1
1
2
1
2
1
1 1 1
P2 P2 2 2
[a]
[a]
1.20 (1.22)
1.10 (1.12)
0.097 (2.17)
11.7 (1.0)
1.64 (1.69)
1.65 (1.68)
0.156 (0.63)
6.7 (1.8)
1.48 (1.52)
0.062 (0.68)
14.1 (2.7)
R
merge
0.062 (0.694)
21.9 (0.8)
0.066 (0.61)
12.7 (2.5)
Mean (I/σI)
Completeness
88.8 (20.8)
100.0 (100.0)
98.0 (98.1)
97.9 (78.3)
98.8 (98.2)
(%)
R
cryst/Rfree (%)
11.1/13.4
0.01
14.2/16.4
0.01
14.4/20.8
0.01
15.1/19.6
0.01
15.2/20.1
0.01
r.m.s.d bonds (Å)
r.m.s.d angles (°)
PDB code
1.52
1.30
1.36
1.41
1.43
4CD4
4CD5
4CD6
4CD7
4CD8
[
a] Values in parentheses denote highest resolution shell.
S12

Table S2: Calculated values of qC1, the C5-O5-C1-C2 dihedral angle, Grel and
TSi for the four relevant canonical conformations.
Values of the different properties of interest, along with its score (in grey) and the
resulting preactivation index (TSi), associated to each canonical conformation. ESP
charges are given in electrons, the dihedral angle in degrees and free energy (∆Grel
)
-
1
in kcal mol .
q
C1
dihed. angle
5.29
∆Grel
0.00
TSi
0
.20
00.00
0.12
3
4
H
4
67
1
0.00
100.00
1.36
-
-1.65
H
3
53
53
56
0
.00
82.74
0.89
76.52
5.78
0.07
2
,5
B
59.25
100.00
1.41
0.00
0.10
5.21
B
2,5
70.69
88.16
9.97
S13

Table S3: Crystal data and structure refinement for (5R,6R,7S,8S)-7,8-
bis(benzyloxy)-5-[(benzyloxy)methyl]-6-hydroxy-5,6,7,8-tetrahydroimidazo[1,2-
a]pyridine.
Identification code
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
CCDC 924306
C H N O
29 30 2 4
470.55
130.0(1) K
1.5418 Å
Triclinic
P 1
Unit cell dimensions
a = 6.2702(2) Å
b = 13.9470(4) Å
c = 14.0503(4) Å
= 104.692(2)°.
= 90.398(2)°.
 = 93.268(2)°.
3
Volume
Z
1186.31(6) Å
2
3
Density (calculated)
1.317 Mg/m
-1
Absorption coefficient
F(000)
0.706 mm
500
3
Crystal size
Theta range for data collection
Index ranges
Reflections collected
Independent reflections
Completeness to theta = 74.08∞
Absorption correction
Max. and min. transmission
0.55 x 0.13 x 0.08 mm
3.25 to 74.08°.
-7<=h<=7, -17<=k<=17, -16<=l<=17
18067
8830 [R(int) = 0.0276]
98.5 %
Semi-empirical from equivalents
1.00000 and 0.84722
2
Refinement method
Full-matrix least-squares on F
Data / restraints / parameters
8830 / 3 / 639
2
Goodness-of-fit on F
1.055
Final R indices [I>2sigma(I)]
R indices (all data)
Absolute structure parameter
R1 = 0.0301, wR2 = 0.0767
R1 = 0.0311, wR2 = 0.0777
0.01(9)
-
3
Largest diff. peak and hole
0.170 and -0.209 e.Å
S14

Computational Chemistry. Quantum mechanical calculations were performed using
Density Functional Theory-based molecular dynamics (MD), according to the Car-Parrinello
[
10]
method.
The system analyzed consists of a single protonated mannoimidazole unit (27
atoms) enclosed in an isolated orthorhombic box of size 14.0 Å  14.0 Å  13.0 Å. The
Kohn-Sham orbitals were expanded in a plane wave (PW) basis set with a kinetic energy
cutoff of 70 Ry. Ab initio pseudopotentials generated within the Troullier-Martins scheme
[
11]
were employed.
The Perdew, Burke and Ernzerhoff generalized gradient-corrected
[
12]
approximation (PBE) was selected in view of its good performance in previous work on
[13]
[14]
[15]
isolated sugars, glycosidases and glycosyltransferases. A fictitious electron mass of
[
16]
8
50 au and a time step of 0.12 fs were used. The metadynamics algorithm was used to
explore the conformational free energy landscape of mannoimidazole, taking as collective
[17]
variables two of the puckering coordinates of Cremer and Pople (θ, φ), in the spirit of the
[
18]
pioneering work by Dowd, French and Reilly. Initially, the height of these Gaussian terms
-1
was set at 0.19 kcal·mol and once the free-energy surface was fully explored it was reduced
-1
to 0.13 kcal·mol , to ensure sufficient accuracy for the reconstruction of the free energy
surface. The width of the Gaussian terms was set to 0.10 Å according to the oscillations of the
selected collective variables observed in a free dynamics. At the beginning of the
metadynamics simulation, a new Gaussian-like potential was added every 400 MD steps,
which was increased up to 1000 MD steps towards the end of the simulation. The simulation
was stopped after having added 2490 Gaussians. In terms of simulation time this corresponds
6
to ≈ 1,5 x 10 MD steps (≈ 180 ps). The FEL of isofagomine was computed using the same
setup as for mannoimizadole. The isofagomine molecule was considered as the neutral
+
species to avoid spurious interactions of the hydroxymethyl and the NH
2
that take place in
the absence of the enzyme environment and heavily affect the shape of the FEL. In the case
of mannoimidazole, the planarity of the imidazole ring avoids such interactions and the FEL
is not affected by protonation of imidazole nitrogen.
Cloning, gene expression and protein purification. The C. japonicus
[19]
mannobiohydrolase gene (man26C) was cloned and expressed as described earlier. The A.
acidocaldarius mannanase gene (man113A) was optimized for expression in Escherichia coli
and synthesized by GenScript, then cloned into pET28a (Novagen) and expressed in E. coli
2
+
BL21 (DE3). AaManA-6x-His was purified using a Ni agarose column (His TrapTM FF,
Amersham Biosciences) and then further purified by gel filtration using a HiLoad 16/60
Superdex 200 prep grade column and eluted with 50 mM HEPES, pH 7.5, 150 mM NaCl.
S15

Collection of structural data, processing and structure solution. Pure CjMan26C
[19]
was crystallized as described before, with 10 mM ManIFG or 10 mM ManMIm present in
the protein solution. Purified AaManA was crystallized in 0.1 M sodium acetate pH 4.6 and
4
% PEG 4000. Crystals of the ManIFG, ManIFG+mannobiose and ManMIm complexes were
obtained by soaking P2 AaManA crystals for 10 min in mother liquor supplemented with
0mM ManIFG, 10 mM ManIFG and 10 mM mannobiose, or 10 mM ManMIm, respectively.
X-ray data for both mannanase complexes were collected at the Diamond Light Source and
1 1 1
2 2
1
[
13c]
[20]
[21]
processed using XIA2
implementations of XDS or MOSFLM. Both structures were
[
22]
solved by molecular replacement using the CCP4 implementation
of the MOLREP
The E338A CjMan26C-mannobiose (PDB 2VX7) and the native A.
acidocaldarius AaManA (PDB 3CIV) were used as search models. The structures, each with
[
23]
program.
[
24]
one molecule in the asymmetric unit, were refined using numerous cycles of REFMAC
[
25]
and manual corrections using COOT. Data and structure quality statistics are in Table 1,
Further details on structure solution and refinement are included in the PDB headers and the
[
26]
Supporting Information. Structural figures were drawn with CCP4MG.
Diamond Light Source is thanked for provision of beamline facilities.
The staff at the
o
Enzyme kinetics. The activity of CjMan26C (5 nM) was determined at 37 C using
mannotriose as the substrate at 500 M, which is <<K
were carried out in the absence or presence of inhibitor at concentrations ranging from 50 to
00 nM. The reaction product mannose was determined continuously, using the
mannose/glucose/fructose detection kit supplied by Megazyme International, in 50 mM Na-
HEPES buffer containing 2 mM MgCl . The activity of AaManA-6x-His at 20 nM was
M
estimated at 5 mM. The reactions
5
2
M
determined using 600 M of the substrate mannotetraose (<<K of 7 mM) in 50 mM
o
potassium phosphate/12 mM citrate buffer pH 6.5 at 50 C. The inhibitor, when incorporated
into the assay, was at 3 mM for ManMIm and from 0.1 to 2 mM for ManIFG. In this
discontinuous assay the rate of substrate depletion was measured using Dionex high
[27]
performance anion-exchange chromatography as described previously. The reaction rates
for both enzymes gave a direct read out of kcat/K in the presence of a range of [I] for
CjMan26C (single [I] for AaManA-6x-His), enabling K to be calculated from equation (1):
M
I
௩బ
ଵ
= ௄
 [I] +1
(1)
௩౟
౅
S16

where v
0
and v
i
are the rates of the reaction in the absence and presence of inhibitor,
the fractional decrease in rate thus yields the
respectively. Under conditions where [S]>> K
M
[
28]
I
K for a competitive inhibitor.
Calculation of transition state inhibition index (TSi). For configurationally-matched
inhibitors, design features that mimic the transition state of glycosidase-catalyzed hydrolysis
reactions include: a) a suitably positioned positive charge that can interact with the catalytic
carboxylate groups; and b) chemical modifications that enforce planarity of the 'carbohydrate'
ring. The charge development at the anomeric carbon (qC1) and the value of the dihedral
angle (C2-C1-O5-C5) of protonated mannoimidazole 1 were calculated in order to assess
which conformations are the most 'pre-activated' for inhibition. A set of approximately 100
structures (approx 25 structures for each one of the conformations of interest) were selected
from the metadynamics simulation and submitted to geometry optimization. The optimized
structures were clustered to each one of the four possible transition state conformations
[
17]
according to the values of their θ and φ puckering coordinates. Atomic charges (ESP), C2-
C1-O5-C5 dihedral angle and free energy were extracted for each structure within the group
and average values were computed. Plots of atomic charge and dihedral angle for each
conformation are available as supporting information (Figure S5 and S6).
Both parameters (qC1 and dihedral angle) were combined, along with the relative free
energy (∆Grel), into an index that reflects the likelihood for a given conformation of the
inhibitor to be adopted on-enzyme. This was done by assigning for each conformation, j, a
[13a]
i
score for each parameter, x , using the equations (2) and (3):
min
(
(
2)
3)
xi, j  xi, j
score x
i, j 
100 for x q
i C1

max
i, j
min
 xi, j
x
max
x
 xi, j
100 for x Grel, Dihed.angle
min
i C2C1O5C5
i, j
max
i, j
score x
i, j 

x
 xi, j
The values of the parameters and the corresponding scores are given in Table S2. Since the
score for each parameter is normalized, they can be directly compared. The transition state
j
index, TSi , is defined as the average of the scores for the n parameters (n = 3) for a given
conformation j (equation (4)):
TSi score x / n
j

i, j

i
(4)
The values are plotted in Figure S7.
S17

Re-refinement of GH38 mannoimidazole complex. The counter-intuitive observation
4
of a E conformation for mannoimidazole in the deposited DmGManII GH38 complex (PDB:
[
9]
3
D4Y) led us to inspect the coordinates. It is clearly apparent from the density that although
4
the deposited E conformation is a major conformer, that an unmodelled second conformer
exists, reflected in positive F
o
-F
c
difference density “above” the imidazole ring
-F difference density on the
(
Supplementary Figure 1, coloured green) and negative F
o
c
imidazole ring, as modelled. In order to investigate the nature and relative occupancy of such
an additional mannoimidazole conformer, we refined the original deposited data (structure
factors that accompany PDB 3D4Y) both against dual occupancy ligands inserted into both
the MIm-bound model (3D4Y) and, in order to remove any possible bias, against an earlier
entirely ligand-free model (PDB 3BUB). Manual modelling was carried out using the real-
[
29]
space refinement options of COOT, followed by several refinement rounds of maximum
[
30]
likelihood refinement with REFMAC. The second conformer indeed refines to a near B2,5
conformation for the mannoimidazole, however, it is difficult to judge the relative
occupancies of the two conformations. Manual manipulation of the occupancies of the two
respective conformers, such that they yield the same refined average temperature factor
2
(
about 24 Å ) post-refinement, suggests an approximate relative occupancy of 0.65:0.35
envelope:boat) for structures refined using either starting model.
(
Small molecule crystallographic methods. Crystals of (5R,6R,7S,8S)-7,8-
bis(benzyloxy)-5-[(benzyloxy)methyl]-6-hydroxy-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine
were slowly grown in ethyl acetate and mounted in low temperature oil then flash cooled to
1
30 K using an Oxford low temperature device. Intensity data were collected at 130 K with
an Oxford SuperNova X-ray diffractometer with CCD detector using Cu-K radiation ( =
[
22]
1
.54184 Å). Data were reduced and corrected for absorption. The structures were solved
[
31]
by direct methods and difference Fourier synthesis using the SHELX suite of programs as
[
32]
implemented within the WINGX software. Thermal ellipsoid plots were generated using
the program ORTEP-3 (Figure S8).
Crystallographic data. Crystal data for (5R,6R,7S,8S)-7,8-bis(benzyloxy)-5-
[
(benzyloxy)methyl]-6-hydroxy-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine: C29
30 2 4
H N O M =
4
70.55, T = 130.0(1) K, = 1.54180, triclinic, space group P1 a = 6.2702(2) b = 13.9470(4),
3
-3
-1
c = 14.0503(4) Å, V = 1186.31(6) Å , Z = 2, D = 1.317 Mg M (Cu-K) 0.706 mm ,
c
F(000) = 500, crystal size 0.55 x 0.13 x 0.08 mm. 18067 reflections measured, 8830
S18

2
independent reflections (Rint = 0.0276), the final R was 0.0301 [I > 2(I)] and wR(F ) (all
data) was 0.0777. This data can be obtained free of charge from the Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif under the code
CCDC 924306.
S19

Synthetic overview. ManIFG 3 was prepared by a -selective mannosylation using Crich's
[33]
pre-activation protocol
on the protected acceptor alcohol 7, followed by global
deprotection (Scheme 1A). The synthesis of ManMIm 4 commenced through annulation of
the imidazole ring onto the amidine 8 utilizing the approach developed by Vasella and co-
[
34]
workers (Scheme 1B). Cyclization of 14 using TsOH resulted in simultaneous removal of
the PMB group and afforded a mixture of D-gluco- and D-manno-configured alcohols whose
3
assignment on the basis of JH,H coupling constants was ambiguous. Fortunately, the D-gluco-
diastereomer of 15 was crystalline, allowing its characterization by X-ray crystallography
(
Figure S7). Efforts to -mannosylate the D-manno-alcohol 15 utilizing donor 16 under
Crich’s pre-activation protocol were unsuccessful. The weakly -selective mannosylation
[
35]
conditions reported by Shin and co-workers
using the same donor 16 afforded pseudo-
disaccharide 17 in 40% yield, with a modest -stereoselectivity of 2:1. Purification of 17 was
achieved by HPLC (Figure S9). Global deprotection afforded 4, in 17 linear steps (from D-
glucose).
General synthetic methods. Thin layer chromatography (TLC) was performed using
aluminium-backed plates of Silica Gel 60 F . Detection was by visualisation in UV light
2
54
and/or by charring in a mixture of 5% H
or solution of ceric ammonium molybdate (made from 1:5:10:90
Ce(SO)
2 4
SO /MeOH, 10% (w/v) phosphomolybic acid/EtOH
a
1
13
4
4 6 7 2 2 4 2
/(NH ) Mo O24·4H O/H SO /H O). H, C and 2-D NMR spectra were collected on
1
13
a nominal 500 MHz instrument (499.7 MHz for H and 125.8 MHz for C) at 25.0 ºC.
1
Spectra are referenced to the following solvent peaks: CDCl
3
( 7.27 ppm for H; 77.16 ppm
1
3
1
13
A,B
for C) or d
4
-methanol ( 3.34 ppm for H; 49.0 ppm for C). Superscript
specifying
carbohydrate rings starting from the non-reducing end for pseudo-disaccharides. Flash
[
36]
chromatography was performed according to the method of Still et al. with Silica Gel 60.
Pyridine was distilled over KOH. Acetonitrile was distilled over P . Toluene was distilled
over K CO . Dimethylformamide and dimethylsulfoxide were dried using 4 Å molecular
sieves. CH Cl was dried using a solvent purification apparatus (Glass Contour, USA) as
described by Pangborn et al.
rotary evaporator. [] values are given in deg.dm cm g . Melting points were obtained
using a hot stage melting point apparatus and are corrected. High resolution mass spectra
HRMS) were acquired on a Finnegan FT-ICR-MS.
2
O
5
2
3
2
2
[
37]
Solvents were evaporated under reduced pressure using a
-1
3
-1
D
(
S20

4
,5’-O-Benzylidene-3-O-p-toluoyl-N-benzyloxycarbonyl-isofagomine
(6).
4-
Dimethylaminopyridine (0.0130 g, 0.109 mmol) was added to a
Ph
O
O
O
NCbz
solution of 4,5’-O-benzylidene-N-benzyloxycarbonyl-isofagomine
Tol
[
38]
5
(0.202 g, 0.546 mmol), p-toluoyl chloride (0.144 ml, 1.09
mmol), distilled pyridine (0.44 ml, 5.46 mmol) and dry dichloromethane (5.0 ml) under N
After 3 h TLC analysis (1:1 EtOAc/pet. spirits with 1% Et N) indicated complete
consumption of starting material. The mixture was diluted with dichloromethane and washed
with sat. aq. NaHCO (3 × 5 ml) and brine. The organic extracts were dried (MgSO ), the
solvent evaporated under reduced pressure and the resulting residue purified by flash
chromatography (30:70 EtOAc/pet. spirits with 1% Et N) to afford the protected carbamate 6
0.234 g, 88%) as a colourless oil that crystallised. A small portion was recrystallised from
O
2
.
3
3
4
3
(
2
4
EtOAc/pet. spirits, 149-150 °C; []D -47 (c 0.9, CHCl );  (499.7 MHz, CDCl ) 2.10-2.19
3
H
3
(
1H, m, H5), 2.39 (3H, s, CH
3
), 2.50-2.61, 2.86-2.97 (2H, 2m, H2ax, H6ax), 3.67 (1H, dd, J5,5’
1
4
0.5, J5’,5’ 11.0 Hz, CH (C5)), 3.87 (1H, J3,4 9.5, J4,5 10.0 Hz, H4), 4.00-4.11 (1H, m, H6eq),
2
.15-4.22 (1H, m, CH
2
(C5)), 4.60-4.74 (1H, m, H2eq), 5.11-5.29 (3H, m, H3,PhCH
2
), 5.61
(125.8
(
1H, s, PhCH), 7.22 (2H, app d, Ar), 7.29-7.45 (10H, m, Ar), 7.93 (2H, app d, Ar); 
MHz, CDCl ) 21.77 (1C, CH
1.84 (4C, C3,4,5’,PhCH ), 101.58 (1C, PhCH), 126.12, 127.27, 128.12, 128.28, 128.68,
28.97, 129.17, 129.87, 136.39, 137.77, 143.95 (18C, Ar), 155.11 (1C, NC=O), 165.72 (1C,
C
3
3
), 37.61 (1C, C5), 43.37, 46.20 (2C, C2,6), 67.79, 68.33, 69.86,
8
1
2
+
+
OC=O); HRMS m/z (ESI ) 510.1887 (C29
6
H29NNaO [M + Na] requires 510.1887).
5
’-O-Benzyl-3-O-p-toluoyl-N-benzyloxycarbonyl-isofagomine (7). Trifluoroacetic acid
BnO
(0.027 ml, 0.368 mmol) was added to a mixture of protected
carbamate 6 (0.036 g, 0.074 mmol), triethylsilane (0.058 ml, 0.368
mmol), freshly-activated 4 Å molecular sieves and dry
HO
O
NCbz
Tol
O
2
dichloromethane (0.50 ml). After 5.5 h under N TLC analysis (40:60 EtOAc/toluene with
1
% Et N) indicated that significant amounts of starting material remained and further
3
trifluoroacetic acid (0.027 ml, 0.368 mmol) and triethylsilane (0.058 ml, 0.368 mmol) were
added. After 20 h TLC analysis (40:60 EtOAc/toluene with 1% Et N) indicated complete
3
consumption of starting material and the mixture was filtered through Celite, diluted with
dichloromethane and washed with sat. aq. NaHCO
were dried (MgSO ), the solvent removed under reduced pressure and the resulting residue
subjected to flash chromatography (15:85 EtOAc/toluene with 1% Et N) to afford the alcohol
(0.034 g, 93%) as a colourless oil. []D +10 (c 0.77, CHCl );  (499.7 MHz, CDCl )
3
(3 × 5 ml) and brine. The organic extracts
4
3
2
3
7
3
H
3
1
.92-1.99 (1H, m, H5), 2.42 (3H, s, CH
3
), 2.77-2.89 (1H, m, H6ax), 2.91 (1H, J2,2 13.0, J2,3
S21

1
0.0 Hz, H2ax), 3.06 (1H, bs, 4-OH), 3.61 (1H, dd, J5,5’ 5.0, J5’,5’ 9.5 Hz, CH
2
(C5)), 3.65-3.77
(
1H, m, CH
2
(C5)), 3.81-3.94, 4.14-4.25 (2H, 2m, H4,6eq), 4.41-4.49 (1H, m, H2eq), 4.52 (2H,
s, PhCH
2
2
), 4.92-4.99 (1H, m, H3), 5.09-5.23 (2H, m, PhCH ), 7.24-7.38 (12H, m, Ar), 7.93
(
2H, app d, Ar);  (125.8 MHz, CDCl ) 21.84 (1C, CH ), 42.20 (1C, C5), 44.71, 45.51 (2C,
C
3
3
2
C2,6), 67.63, 69.56, 73.34, 73.60, 73.68 (5C, C3,4,5’, 2 × PhCH ), 127.01, 127.75, 127.91,
128.08, 128.21, 128.58, 128.66, 129.27, 129.95, 136.59, 137.95, 144.22 (18C, Ar), 155.25
+
+
(
1C, NC=O), 166.49 (1C, OC=O); HRMS m/z (ESI ) 512.2044 (C29
6
H31NNaO [M + Na]
requires 512.2044).
5
’-O-Benzyl-4-O-(2,3-di-O-benzyl-4,6-O-benzylidene--D-mannopyranosyl)-3-O-p-
BnO
toluoyl-N-benzyloxycarbonyl-isofagomine (8).
Triflic anhydride (0.059 ml, 0.351 mmol) was
added to a solution of 4-methylphenyl 2,3-di-O-
BnO
O
O O
NCbz
O
OBn
Ph
O
Tol
O
[
39]
benzyl-4,6-O-benzylidene-1-thio--D-mannopyranoside
diphenylsulfoxide (0.142 g, 0.703 mmol) and 2,4,6-tri-tert-butylpyrimidine (0.187 g, 0.753
mmol) in dry dichloromethane (4 ml) at -65 °C under N . After 10 min a solution of alcohol 7
0.090 g, 0.183 mmol) in dry dichloromethane (2 ml) was cannulated into the sulfoxide
16 (0.140 g, 0.251 mmol),
2
(
solution. The mixture was stirred at -65 °C for 1 h then was slowly warmed to -40 °C. This
temperature was maintained for 30 min and then the mixture was allowed to slowly warm to
rt. TLC analysis (30:70 EtOAc/pet. spirits with 1% Et
3
N) indicated consumption of acceptor
at approximately -40 °C. The reaction was quenched with sat. aq. NaHCO , diluted with
(3 × 5 ml) and brine. The organic extracts
7
3
dichloromethane and washed with sat. aq. NaHCO
3
were dried (MgSO ), the solvent removed under reduced pressure and the resulting residue
4
subjected to flash chromatography (25:75 EtOAc/pet. spirits with 1% Et
3
N) to afford the
2
D
4
pseudo-disaccharide 8 (0.101 g, 60%) as a colourless oil. []
-37 (c 0.62, CHCl ); 
3
H
B
(
499.7 MHz, (CD
3
)
2
SO, 60.0 °C) 2.03-2.10 (1H, m, H5 ), 2.40 (3H, s, CH
3
), 3.19 (1H, ddd,
A
B
J4,5 10.0, J5,6 10.0, J5,6 4.5 Hz, H5 ), 3.37 (1H, dd, J 8.0, J6,6 13.5 Hz, H6 ), 3.41 (1H,
5,6
ax
B
A
B
bdd, J2,2 13.5, J2,3 7.0 Hz, H2ax ), 3.53-3.60 (3H, m, H6 , 2 × CH
2
(C5) ), 3.67 (1H, dd, J2,3
A
B
A
3
.0, J3,4 10.0 Hz, H3 ), 3.76-3.81 (1H, m, H6eq ), 3.78 (1H, dd, J6,6 10.0 Hz, H6 ), 3.89 (1H,
A
B
A
dd, H4 ), 3.92 (1H, bdd, J2,3 4.5 Hz, H2eq ), 3.97 (1H, app d, H2 ), 4.01 (1H, dd, J3,4 7.0, J4,5
B
7
.0 Hz, H4 ), 4.41-4.46 (2H, m, 2 × PhCH
2
), 4.63 (2H, s, 2 × PhCH
2
), 4.64 (1H, J 11.5 Hz,
A
B
PhCH
2
), 4.74 (1H, J 11.5 Hz, PhCH
2
), 4.77 (1H, app s, H1 ), 4.96 (1H, ddd, H3 ), 5.06-5.13
(
2H, m, PhCH ), 5.58 (1H, s, PhCH), 7.22-7.40 (27H, m, Ar), 7.86 (2H, app d, Ar);  (125.8
2
C
B
MHz, CDCl
3
, 25.0 °C) 21.84 (1C, CH
3
), 29.84 (1C, C5 ), 41.05, 42.98, 43.26, 44.82 (2C,
S22

B
(
(
(
C2,6) ), 66.00, 67.48, 67.73, 68.44, 68.58, 70.52, 72.71, 73.52, 74.85, 76.58, 78.22, 78.68
A
B
2
12C, (C2,3,4,5,6) , (C3,4,5’) , 4 × PhCH ), 101.50 (JC,H 161.1 Hz), 102.45 (JC,H 155.8 Hz)
A
2C, C1 , PhCH), 126.21, 127.43, 127.67, 127.74, 127.94, 127.98, 128.13, 128.28, 128.31,
1
1
28.49, 128.57, 128.62, 128.99, 129.23, 129.84, 137.73, 137.98, 138.53, 143.97 (36C, Ar),
+
55.75 (1C, NC=O), 165.41 (1C, OC=O); HRMS m/z (ESI ) 942.3823 (C56
H57NNaO11 [M +
+
Na] requires 942.3824).
(
3R,4R,5R)-3-Hydroxy-5-(hydroxymethyl)-4-(-D-mannopyranosyloxy)piperidine
(
ManIFG; 3). NaOMe (1.0 M, 0.060 ml) was added to a
solution of the protected pseudo-disaccharide 8 (0.029 g,
.030 mmol) in methanol (2.0 ml) and dichloromethane
HO
HO
HO
HO
O
O
OH
HO
NH
0
(
3
1.0 ml). After 15 h TLC analysis (30:70 EtOAc/pet. spirits with 1% Et N) indicated
+
complete consumption of starting material. Amberlite IR-120 resin (H form) was added to
neutralise the solution and, following filtration, the solvent was removed under reduced
pressure. Pd(OH)
AcOH/H O/THF (4:2:1, 1.75 ml). The reaction vessel was filled with H
for 16 h. At this point TLC analysis (5:95 MeOH/H O upon NH -neutralised TLC plate)
2
(30 mg, 20% w/w) was added to a solution of the crude filtrate in
2
2
(6 bar) and agitated
2
3
indicated formation of a highly polar product. The suspension was filtered through Celite
then was concentrated under reduced pressure. The residue was purified by ion-exchange
-
+
chromatography [i) Dowex 1X-8 (OH form), eluted with water; ii) Dowex-50W-X2 (H
form), eluted with water then 6 M aq. NH ] followed by C18 reversed-phase chromatography
3
2
D
4
(
5:95 MeOH/H O) to afford 3 (0.070 g, 80%) as a colourless glass. [] -24 (c 0.36, H O);
2
2
B
B

H
2
(499.7 MHz, D O) 1.86 (1H, m, H5 ), 2.50 (1H, app bt, J2,2 12.5 Hz, H2ax ), 2.57 (1H,
B
B
app bt, J 13.5 Hz, H6ax ), 3.18 (1H, dd, J5,6 4.0, J6,6 12.5 Hz, H6eq ), 3.27 (1H, dd, J2,2 12.5,
B
A
J
2,3 5.0 Hz, H2eq ), 3.44 (1H, ddd, J4,5 9.0, J5,6 2.0, J5,6 6.5 Hz, H5 ), 3.57-3.61 (2H, m,
A
A
A
B
B
B
H3 ,4 ), 3.66-3.79 (5H, m, H6 ,3 ,4 , 2 × CH (C5) ), 3.94 (1H, dd, J 2.0, J6,6 12.0 Hz,
2
5,6
A
A
A
H6 ), 4.11 (1H, app d, J2,3 3.5 Hz, H2 ), 4.75 (1H, app s, H1 ); 
C
2
(125.8 MHz, D O) 42.91
B
B
(
(
[
1C, C5 ), 45.86, 48.30 (2C, (C2,6) ), 59.61, 60.85, 66.61, 70.05, 70.36, 72.80, 76.29, 83.53
A
B
A
+
8C, (C2,3,4,5,6) , (C3,4,5’) ), 100.47 (1C, C1 ); HRMS m/z (ESI ) 310.1507 (C12H24NO
8
+
M + H] requires 310.1496).
S23

4
-Methylphenyl 2,3,6-tri-O-benzyl-1-thio--D-glucopyranoside (9). Trifluoroacetic acid
(
3.34 ml, 45.1 mmol) was slowly added to a solution of 4-
methylphenyl 2,3-di-O-benzyl-4,6-O-benzylidene-1-thio--D-
glucopyranoside (5.00 g, 9.01 mmol) and triethylsilane (7.20 ml,
(50 ml) at 0 ºC under N . After 90 min TLC analysis (30:70
BnO
O
HO
STol
BnO
BnO
[40]
4
5.1 mmol) in CH
2
Cl
2
2
EtOAc/pet. spirits) of the mixture showed complete consumption of the starting material. The
mixture was diluted with CH Cl and quenched with ice then was washed with sat. aq.
NaHCO (× 3) and brine. The organic extracts were dried (MgSO ), the solvent removed
under reduced pressure and the resulting residue subjected to flash chromatography (20:80
2
2
3
4
2
D
4
EtOAc/pet. spirits) to afford the alcohol 9 (4.47 g, 89%) as a colourless oil; []
-32 (c 1.0,
[
41]
CHCl
3
lit. -30); 
H
(499.7 MHz, CDCl
3
) 2.31 (3H, s, CH
3
), 2.54 (1H, d, J4,OH 2.5 Hz, OH),
3
.45 (1H, dd, J1,2 9.5, J 9.0 Hz, H2), 3.46 (1H, m, H5), 3.50 (1H, dd, J 9.0 Hz, H3), 3.64
2,3 3,4
(
1H, ddd, H4), 3.73-3.80 (2H, m, H6,6), 4.54 (1H, d, J 12.0 Hz, PhCH ), 4.58 (1H, d, J 12.0
2
Hz, PhCH ), 4.62 (1H, d, H1), 4.73 (1H, d, J 12.0 Hz, PhCH ), 4.77 (1H, d, J 12.0 Hz,
2
2
PhCH
2
), 4.90 (1H, d, J 11.5 Hz, PhCH
2
2
), 4.92 (1H, d, J 10.5 Hz, PhCH ), 7.04 (2H, app d,
Ar), 7.26-7.27 (17H, m, Ar); 
C
(125.8 MHz, CDCl
3
) 21.25 (1C, CH
3
), 70.61, 71.94, 73.82,
Ph), 127.85, 128.03,
7
5.49, 75.64, 78.18, 80.63, 86.36, 88.13 (9C, C1,2,3,4,5,6, 3 × CH
2
1
28.07, 128.10, 128.42, 128.56, 128.58, 128.75, 129.82, 129.94, 132.78, 137.92, 138.20,
38.64 (24C, Ar).
1
4
-Methylphenyl 2,3,6-tri-O-benzyl-4-O-(4-methoxybenzyl)-1-thio--D-glucopyranoside
(
10). Sodium hydride (60% dispersion in mineral oil, 0.832 g, 20.8
STol mmol) was added to alcohol 9 (5.79 g, 10.4 mmol) in dry DMF (40
ml) under N . After ten minutes para-methoxybenzyl chloride (2.12
BnO
PMBO
BnO
O
BnO
2
ml, 15.6 mmol) was added dropwise at 0 ºC. The mixture was slowly allowed to attain room
temperature and left to stand overnight. At this time TLC analysis (10:60:30
EtOAc/toluene/pet. spirits) showed complete consumption of the starting material. The
reaction was diluted with EtOAc then was quenched with ice and washed with H
brine. The organic extracts were dried (MgSO ), the solvent removed under reduced pressure
and the resulting residue subjected to flash chromatography (5:60:35 EtOAc/toluene/pet.
2
O (× 2) and
4
2
4
spirits with 1% Et
.5 (c 0.7, CHCl ); 
dd, J1,2 9.5, J2,3 9.0 Hz, H2), 3.60 (1H, dd, J3,4 9.0, J4,5 9.5 Hz, H4), 3.67 (1H, dd, H3), 3.70
3
N) to afford the thioglycoside 10 (6.62 g, 94%) as a colourless oil; []
D
-
5
3
H
(499.7 MHz, CDCl ) 2.30 (3H, s, ArCH ), 3.45 (1H, m, H5), 3.47 (1H,
3
3
3
(1H, dd, J5,6 4.5, J6,6 10.5 Hz, H6), 3.76 (1H, dd, J5,6 2.0 Hz, H6), 3.77 (3H, s, OCH ), 4.51
S24

(
1H, d, J 11.0 Hz, ArCH
2.0 Hz, ArCH ), 4.71 (1H, d, J 10.5 Hz, ArCH
d, J 10.5 Hz, ArCH ), 4.89 (2H, d, J 11.0 Hz, 2 × ArCH
app d, Ar), 7.10 (2H, app d, Ar), 7.23-7.49 (17H, m, Ar);  (125.8 MHz, CDCl ) 21.24 (1C,
2
), 4.53 (1H, d, J 11.5 Hz, ArCH
), 4.73 (1H, d, J 10.5 Hz, ArCH
), 6.79 (2H, app d, Ar), 7.02 (2H,
2
), 4.59 (1H, d, H1), 4.60 (1H, d, J
1
2
2
2
), 4.86 (1H,
2
2
C
3
ArCH
3
), 55.42 (1C, CH
3
OAr), 69.21, 71.72, 73.56, 74.84, 75.51, 75.92, 77.69, 79.25, 80.96.
Ar), 113.98, 127.66, 127.82, 127.89, 127.97,
8
1
1
6.94, 87.81 (11 C, C1,2,3,4,5,6, 5 × CH
2
28.37, 128.48, 128.54, 128.55, 128.59, 129.55, 129.79, 129.92, 130.38, 132.83, 137.81,
+
38.26, 138.52, 138.65, 159.46 (30 C, Ar); HRMS m/z (ESI ) 699.2748 (C42
H44NaO
6
S [M +
+
Na] requires 699.2751).
2
,3,6-Tri-O-benzyl-4-O-(4-methoxybenzyl)-D-glucopyranose. N-Iodosuccinimide (1.15 g,
.13 mmol) was added to a solution of the thioglycoside 10 (2.32 g,
.42 mmol) in acetone (1% aq., 60 ml) at 0 ºC. After 30 min TLC
analysis (30:70 EtOAc/pet. spirits) of the mixture showed complete
and
(50 ml), NaHCO
), the solvent removed
5
BnO
PMBO
BnO
O
3
OH
BnO
consumption of the starting material. The solution was quenched with 0.5 M aq. Na
diluted with EtOAc (100 ml) before being washed with 0.5 M aq. Na
3 × 50 ml) and brine (50 ml). The organic extracts were dried (MgSO
2 2 3
S O
2
S
2
O
3
3
(
4
under reduced pressure and the resulting residue recrystallised from EtOAc/pet. spirits to
afford 2,3,6-tri-O-benzyl-4-O-(4-methoxybenzyl)-D-glucopyranose (1.48 g, 78%) as a white
H 3
powder in a mixture of anomers ( ratio 25:1); -anomer signals:  (499.7 MHz, CDCl )
3
.57 (1H, dd, J 3.5, J 10.0 Hz, H2), 3.61 (1H, dd, J 9.5, J 10.0 Hz, H4), 3.64 (1H, dd,
1
,2
2,3
3,4
4,5
J
5,6 2.5, J6,6 11.0 Hz, H6), 3.70 (1H, dd, J5,6 4.0 Hz, H6), 3.79 (3H, s, OCH
3
), 3.95 (1H, dd,
H3), 4.02 (1H, ddd, H5), 4.43 (1H, d, J 10.5 Hz, ArCH
2
2
), 4.50 (1H, d, J 12.0 Hz, ArCH ),
4
.61 (1H, d, J 12.0 Hz, ArCH ), 4.69 (1H, d, J 12.0 Hz, ArCH ), 4.74 (1H, d, J 10.5 Hz,
2
2
ArCH
2
), 4.77 (1H, d, J 12.0 Hz, ArCH
2
2
), 4.86 (1H, d, J 11.0 Hz, ArCH ), 4.95 (1H, d, J 10.5
2
Hz, ArCH ), 5.23 (1H, d, H1), 6.80 (2H, app d, Ar), 7.05 (2H, app d, Ar), 7.26-7.36 (15H, m,
Ar).
(
3R,4S,5S,6S)-3,4,-Bis(benzyloxy)-6-[(benzyloxy)methyl]-5-(4-
methoxybenzyloxy)piperidin-2-one (12). Acetic anhydride (8.30
ml) and dry dimethylsulfoxide (13.9 ml) were added to 2,3,6-tri-O-
benzyl-4-O-(4-methoxybenzyl)-D-glucopyranose (2.90 g, 5.09
BnO
PMBO
BnO
6
N1 H
O
OBn
2
mmol) and the solution was stirred under N for 18 h. At this time
TLC analysis (30:70 EtOAc/pet. spirits) showed complete consumption of the starting
material. The reaction was diluted with EtOAc then was quenched with ice and washed with
S25

2 4
H O (× 5) and brine. The organic extracts were dried (MgSO ) and evaporated. Azeotropic
evaporation using toluene was performed to remove residual AcOH. The crude residue was
employed directly in the next step. A dry-ice/acetone cold finger cooling trap was employed
2
to condense ammonia (6 ml) into a solution of crude lactone 11 in dry Et O (65 ml) at -78 °C.
The solution was then allowed to reflux at 0 °C for 2 h. The solution was evaporated to
dryness and the crude residue was employed directly in the next step. Acetic anhydride (8.30
ml) and dry dimethylsulfoxide (13.9 ml) were added to the crude 2,3,6-tri-O-benzyl-4-O-(4-
2
methoxybenzyl)-D-gluconamide and the mixture was stirred under N for 18 h. At this time
TLC analysis (70:30 EtOAc/pet. spirits) showed complete consumption of the starting
material. The reaction was diluted with EtOAc then was quenched with ice and washed with
H
2
O (× 4) and brine. The organic extract was dried (MgSO
residue was dissolved in dry acetonitrile (70 ml) and formic acid (18.8 mL) then sodium
cyanoborohydride (1.64 g, 26.2 mmol) were added. After 2 h stirring under N TLC analysis
40:60 EtOAc/toluene) of the solution indicated complete consumption of the starting
material. The reaction was diluted with EtOAc then was washed with sat. aq. NaHCO (× 3)
and brine. The aqueous extract was treated with aq. sodium hypochlorite prior to disposal.
The organic extracts were dried (MgSO ), the solvent removed under reduced pressure and
the resulting residue was subjected to flash chromatography (30:70 EtOAc/pet. spirits with
% Et N) to afford the lactam 12 (2.13 g, 74% over four steps) as a colourless oil that
crystallised upon standing. A small portion was recrystallised from EtOAc/pet. spirits, mp
4
) and evaporated. The crude
2
(
3
4
1
3
2
D
2
1
9
2
00-102 °C; []
.0 Hz, CH (C6)), 3.50 (1H, dd, J4,5 8.5, J5,6 9.0 Hz, H5), 3.56 (1H, m, H6), 3.58 (1H, dd, J6,6’
.5 Hz, CH (C6)), 3.81 (3H, s, OCH ), 3.90 (1H, dd, J 8.0 Hz, H4), 3.99 (1H, d, H3), 4.42
+96 (c 1.0, CHCl
3
); 
H 3
(499.7 MHz, CDCl ) 3.22 (1H, dd, J6,6’ 8.0, J6’,6’
2
2
3
3,4
(
1H, d, J 10.5 Hz, ArCH
2
), 4.44 (1H, d, J 12.0 Hz, ArCH
2
2
), 4.47 (1H, d, J 11.5 Hz, ArCH ),
4
.74 (1H, d, J 11.0 Hz, ArCH ), 4.76 (1H, d, J 10.5 Hz, ArCH ), 4.77 (1H, d, J 11.0 Hz,
2
2
ArCH
2
), 4.86 (1H, d, J 11.0 Hz, ArCH
2
), 5.17 (1H, d, J 11.5 Hz, ArCH
(125.8 MHz, CDCl
3.93, 55.39, 70.12, 73.44, 74.33, 74.71, 74.80, 76.78, 78.91, 82.50 (10C, C3,4,5,6,6’, 4 ×
2
), 5.87 (1H, broad s,
NH), 6.82 (2H, m, Ar), 7.10 (2H, m, Ar), 7.28-7.43 (15H, m, Ar); 
C
3
)
5
ArCH
2
, OCH
3
), 113.98, 127.88, 127.98, 128.07, 128.11, 128.46, 128.51, 128.65, 129.83,
+
1
29.95, 137.44, 137.99, 138.23, 159.60 (24C, Ar), 170.64 (1C, C2); HRMS m/z (ESI )
+
5
6
90.2509 (C35H37NNaO [M + Na] requires 590.2513).
Intermediates in this sequence were purified and characterised in parallel experiments as
described below:
S26

2
,3,6-Tri-O-benzyl-4-O-(4-methoxybenzyl)-D-gluconolactone (11). A portion of the
lactone 11, resulting from a parallel experiment, was purified by
BnO
O
PMBO
flash chromatography (10:90 EtOAc/ pet. spirits) to afford a
BnO
O
2
2
OBn
colourless oil; []
.67 (1H, dd, J5,6 3.5, J6,6 11.0 Hz, H6), 3.72 (1H, dd, J5,6 2.0 Hz, H6), 3.79 (3H, s, OCH
.91 (dd, J2,3 6.5, J3,4 6.5 Hz, H3), 3.95 (dd, J4,5 8.0 Hz, H4), 4.13 (1H, d, H2), 4.44 (1H, m,
), 4.49 (1H, d, J 12.0 Hz, ArCH ), 4.58 (1H, d, J 11.5 Hz,
), 4.61 (1H, d, J 11.5 Hz, ArCH ), 4.65 (1H, d, J 12.0 Hz, ArCH ), 4.74 (1H, d, J 11.5
Hz, ArCH ), 4.99 (1H, d, J 11.5 Hz, ArCH ), 4.82 (2H, m, Ar), 7.10 (2H, m, Ar), 7.26-7.40
15H, m, Ar); (125.8 MHz, CDCl ) 55.39, 68.41, 73.70, 73.84, 73.86, 75.79, 77.55, 78.35,
1.17 (10C, C2,3,4,5,6, 4 × ArCH , OCH ), 113.98, 127.94, 128.04, 128.10, 128.22, 128.52,
28.57, 128.60, 129.74, 129.84, 137.10, 137.73, 137.77, 159.59 (24C, Ar), 169.48 (1C,
+76 (c 1.2, CHCl
H 3
);  (499.7 MHz, CDCl )
D
3
3
3
3
),
H5), 4.46 (1H, d, J 11.0 Hz, ArCH
2
2
ArCH
2
2
2
2
2
(
C
3
8
1
2
3
+
+
7
C=O); HRMS m/z (ESI ) 591.2351 (C35H36NaO [M + Na] requires 591.2353).
2
,3,6-Tri-O-benzyl-4-O-(4-methoxybenzyl)-D-gluconamide. A portion of 2,3,6-tri-O-
benzyl-4-O-(4-methoxybenzyl)-D-gluconamide, resulting from a
BnO
OH
PMBO
NH2
parallel experiment, was purified by flash chromatography (60:40
BnO
BnO
23
O
EtOAc/ pet. spirits) to afford a colourless oil; []
(499.7 MHz, CDCl
1H, J5,6 3.0 Hz, H6), 3.80 (3H, s, OCH ), 3.85 (1H, dd, J 6.0, J4,5 8.0 Hz, H4), 3.91 (1H,
D
+24 (c 1.1,
CHCl
3
); 
H
3
) 2.85 (1H, bs, OH), 3.58 (1H, J5,6 5.0, J6,6 9.5 Hz, H6), 3.65
(
3
3,4
m, H5), 4.07 (1H, dd, J2,3 3.0 Hz, H3), 4.26 (1H, d, H2), 4.45 (1H, d, J 10.5 Hz, ArCH
2
), 4.51
(
1H, d, J 11.5 Hz, ArCH
2
), 4.57 (1H, d, J 12.0 Hz, ArCH
2
), 4.59 (1H, d, J 11.0 Hz, ArCH ),
2
4
.60 (1H, d, J 12.5 Hz, ArCH ), 4.64 (1H, d, J 11.0 Hz, ArCH ), 4.65 (1H, d, J 11.0 Hz,
2
2
ArCH
2
), 4.71 (1H, d, J 11.0 Hz, ArCH
2
), 4.59 (1H, bs, NH), 6.60 (1H, bs, NH), 6.82 (2H, m,
Ar), 7.15 (2H, m, Ar), 7.28-7.36 (15H, m, Ar);
C
(125.8 MHz, CDCl
3
) 55.41, 71.27, 71.51,
7
1
1
6
3.56, 73.90, 73.92, 75.32, 77.31, 79.80, 80.74 (10C, C2,3,4,5,6, 4 × ArCH , OCH ), 113.90,
2
3
27.84, 127.99, 128.01, 128.39, 128.45, 128.47, 128.49, 128.54, 128.57, 128.77, 129.90,
+
30.45, 136.94, 137.93, 138.26, 159.40 (24C, Ar), 174.16 (1C, C=O); HRMS m/z (ESI )
+
08.2615 (C H NNaO [M + Na] requires 608.2619).
3
5
39
7
(
3R,4S,5S,6R)-3,4,-Bis(benzyloxy)-6-[(benzyloxy)methyl]-5-(4-
methoxybenzyloxy)piperidin-2-one. A portion of (3R,4S,5S,6R)-
6
N1 H
PMBO
BnO
3,4,-Bis(benzyloxy)-6-[(benzyloxy)methyl]-5-(4-
methoxybenzyloxy)piperidin-2-one resulting from a parallel
experiment, was purified by flash chromatography (30:70 EtOAc/
O
OBn
BnO
S27

pet. Spirits with 1% Et
lactam 12 (0.103 g, 74%) was also isolated in this experiment. []
499.7 MHz, CDCl (C6)), 3.61 (1H, dd, J6,6’ 9.0 Hz,
CH (C6)), 3.68 (1H, dd, J4,5 5.0, J5,6 3.5 Hz, H5), 3.75 (1H, m, H6), 3.81 (3H, s, OCH ), 3.92
1H, dd, J3,4 6.0 Hz, H4), 4.00 (1H, d, H3), 4.43 (1H, d, J 11.5 Hz, ArCH ), 4.47 (1H, d, J
2.0 Hz, ArCH ), 4.52 (1H, d, J 12.0 Hz, ArCH ), 4.56 (1H, d, J 11.5 Hz, ArCH ), 4.57 (1H,
d, J 11.5 Hz, ArCH ), 4.65 (1H, d, J 11.5 Hz, ArCH ), 4.75 (1H, d, J 11.5 Hz, ArCH ), 5.11
1H, d, J 11.5 Hz, ArCH ), 5.88 (1H, broad s, NH), 6.82 (2H, m, Ar), 7.12 (2H, m, Ar), 7.25-
.43 (15H, m, Ar);  (125.8 MHz, CDCl ) 52.21, 55.42, 69.87, 71.73, 73.34, 73.63, 74.30,
3
N) to afford a colourless oil (0.013 g, 10%). The diastereomeric
2
D
5
+22 (c 0.2, CHCl
3
); 
H
(
3 2
) 3.54 (1H, dd, J6,6’ 3.5, J6’,6’ 9.0 Hz, CH
2
3
(
2
1
2
2
2
2
2
2
(
2
7
7
1
1
C
3
4.98, 78.35, 79.02 (10C, C3,4,5,6,6’, 4 × ArCH
2 3
, OCH ), 113.98, 127.89, 127.93, 127.99,
28.02, 128.07, 128.49, 128.50, 128.57, 128.66, 129.64, 129.76, 137.62, 138.01, 138.15,
+
+
6
59.58 (24C, Ar), 170.68 (1C, C2); HRMS m/z (ESI ) 590.2511 (C35H37NNaO [M + Na]
requires 590.2513).
(
3R,4S,5S,6S)-3,4-Bis(benzyloxy)-6-[(benzyloxy)methyl]-5-(4-
methoxybenzyloxy)piperidin-2-thione (13). Lawesson’s reagent
0.40 g, 0.99 mmol) was added to a mixture containing the lactam
12 (0.28 g, 0.493 mmol), pyridine (20 l, 0.246 mmol), freshly
activated 4 Å molecular sieves and dry toluene (20 ml). The reaction mixture was stirred for
0 h when TLC analysis (40:60 EtOAc/toluene) indicated complete consumption of the
BnO
PMBO
BnO
6
N1 H
(
S
OBn
2
starting material. The mixture was filtered, stirred with MeOH (5 ml) for 2 h and the solvent
removed under reduced pressure. The residue was subjected to flash chromatography (1:1
2
3
EtOAc/pet. spirits) to afford the thiolactam 13 (0.284 g, 99%) as a colourless oil; []
D
+123
(C6)), 3.53
(C6)), 3.78 (3H, s, OCH ),
.84 (1H, m, H6), 3.89 (1H, dd, J3,4 4.5 Hz, H4), 4.28 (1H, d, J 11.0 Hz, ArCH ), 4.44 (1H, d,
J 12.0 Hz, ArCH ), 4.45 (1H, d, H3), 4.46 (1H, d, J 11.5 Hz, ArCH ), 4.47 (1H, d, J 12.0 Hz,
ArCH ), 4.52 (1H, d, J 11.0 Hz, ArCH ), 4.66 (1H, d, J 12.0 Hz, ArCH ), 4.73 (1H, d, J 12.5
Hz, ArCH ), 5.01 (1H, d, J 11.5 Hz, ArCH ), 6.79-6.81 (2H, m, Ar), 7.05-7.07 (2H, m, Ar),
(c 0.9, CHCl
3
H 3 2
);  (499.7 MHz, CDCl ) 3.34 (1H, dd, J6,6’ 7.5, J6’,6’ 9.5 Hz, CH
(
1H, dd, J4,5 4.5, J5,6 9.0 Hz, H5), 3.61 (1H, dd, J6,6’ 3.5 Hz, CH
2
3
3
2
2
2
2
2
2
2
2
7
6
1
1
.26-7.42 (15H, m, Ar), 8.10 (1H, broad s, NH); 
C
(125.8 MHz, (CDCl
3
) 55.44, 56.06,
, OCH ),
8.44, 72.54, 72.67, 72.74, 73.54, 78.03, 81.57, 82.63 (10C, C3,4,5,6,6’, 4 × ArCH
2
3
13.97, 128.02, 128.13, 128.25, 128.33, 128.46, 128.53, 128.60, 128.73, 128.89, 128.95,
+
37.25, 137.58, 137.67, 159.61 (24C, Ar), 200.58 (1C, C2); HRMS m/z (ESI ) 606.2284
+
(
C
35
H
37NNaO
5
S [M + Na] requires 606.2284).
S28

Glycoimidazole formation. Thiolactam 13 (0.144 g, 0.247 mmol) was dissolved in
aminoacetaldehyde dimethyl acetal (0.40 ml, 3.71 mmol) and stirred under N for 18 h. At
2
this time TLC analysis (30:70 EtOAc/pet. spirits) indicated complete consumption of the
starting material. The solution was evaporated to dryness and p-toluenesulfonic acid (0.085 g,
0
.494 mmol) was added to a solution of the crude amidine 14 in toluene (1.1 ml) and water
0.05 ml). The solution was stirred at 50 °C for 5 d. At this time the reaction was diluted with
EtOAc then was washed with NaHCO (× 3) and brine. The organic extracts were dried
MgSO ), the solvent removed under reduced pressure and the residue was subjected to flash
chromatography (40:60 EtOAc /pet. spirits) to afford the glycoimidazoles 15 and
5R,6R,7S,8S)-7,8-bis(benzyloxy)-5-[(benzyloxy)methyl]-6-hydroxy-5,6,7,8-
tetrahydroimidazo[1,2-a]pyridine (0.077 g, 65% over two steps) as separable diastereomers
D-Man/D-Glc ratio 4:3).
(
3
(
4
(
(
i)
(5R,6R,7S,8R)-7,8-Bis(benzyloxy)-5-[(benzyloxy)methyl]-6-hydroxy-5,6,7,8-
2
4
tetrahydroimidazo[1,2-a]pyridine (15). []D -61 (c 0.8, CHCl3);
(499.7 MHz, CDCl ) 3.06 (1H, broad s, 6-OH), 3.66 (1H, dd,
OBn
3
BnO
HO
BnO
4
N

H
3
6
2
5
8
N1
7
J
6,7 10.0, J7,8 3.0 Hz, H7), 3.79 (1H, dd, J5,5’ 7.0, J5’,5’ 10.0 Hz,
(C5)), 4.08 (1H, ddd, J5,6 8.0 Hz, H5), 4.41 (1H, dd,
), 4.57 (1H, d, J 12.0 Hz, PhCH ), 4.61 (1H, d, J 12.0 Hz,
), 4.65 (1H, d, J 12.0 Hz, PhCH ), 4.68 (1H, d, J 12.5 Hz, PhCH ), 4.75 (1H, d, J 12.5
Hz, PhCH ), 4.85 (1H, d, H8), 7.10 (1H, d, J2,3 1.0 Hz, H3), 7.27-7.42 (16H, m, H2, Ph); 
125.8 MHz, CDCl ) 60.13, 65.81, 67.15, 70.65, 71.29, 71.57, 73.57, 78.94 (8C, C5,5’,6,7,8,
2 2
CH (C5)), 4.05 (1H, dd, J5,5’ 2.5 Hz, CH
H6), 4.45 (1H, d, J 11.5 Hz, PhCH
2
2
PhCH
2
2
2
2
C
(
3
3
1
× PhCH ), 119.55 (1C, C3), 127.76, 127.78, 127.90, 127.99, 128.14, 128.23, 128.33,
2
28.43, 128.62, 128.69, 129.16, 137.34, 137.66, 138.07 (19C, C2, Ph), 142.75 (1C, C8’);
+
+
HRMS m/z (ESI ) 471.2278 (C29
31 2 4
H N O [M + H] requires 471.2278).
ii) (5R,6R,7S,8S)-7,8-Bis(benzyloxy)-5-[(benzyloxy)methyl]-6-hydroxy-5,6,7,8-
2
D
4
tetrahydroimidazo[1,2-a]pyridine. mp 92-93 °C; []
0.9, CHCl );  (499.7 MHz, CDCl ) 3.69 (broad d, J6,OH 6.0 Hz, 6-
OH), 3.74 (dd, J5,5’ 7.5, J5’,5’ 10.5 Hz, CH (C5)), 3.86 (dd, J5,5’ 4.0
+57 (c
BnO
HO
BnO
4
3
N
6
2
5
3
H
3
8
N1
7
OBn
2
2
Hz, CH (C5)), 4.04 (1H, dd, J6,7 6.5, J7,8 5.0 Hz, H7), 4.09 (1H, broad ddd, J5,6 6.5 Hz, H6),
4
.36 (1H, ddd, H5), 4.51 (1H, d, J 12.0 Hz, PhCH ), 4.55 (1H, d, J 12.0 Hz, PhCH ), 4.57
2
2
(
1H, d, J 11.5 Hz, PhCH
1.5 Hz, PhCH ), 5.10 (1H, d, J 12.0 Hz, PhCH
H2), 7.22-7.39 (15H, m, Ar); 
2
), 4.77 (1H, d, J 11.5 Hz, PhCH
2
), 4.78 (1H, d, H8), 4.84 (1H, d, J
1
2
2
), 7.15 (1H, d, J2,3 1.0 Hz, H3), 7.19 (1H, d,
C
(125.8 MHz, CDCl
S29
3
) 61.07, 68.12, 71.12, 72.38, 72.69,