A 1-acetamido derivative of 6-epi-valienamine: An inhibitor of a diverse group of β-N-acetylglucosaminidases

Article abstract of DOI:10.1039/b709681j

The synthesis of an analogue of 6-epi-valienamine bearing an acetamido group and its characterisation as an inhibitor of β-N- acetylglucosaminidases are described. The compound is a good inhibitor of both human O-GlcNAcase and human β-hexosaminidase, as well as two bacterial β-N-acetylglucosaminidases. A 3-D structure of the complex of Bacteroides thetaiotaomicron BtGH84 with the inhibitor shows the unsaturated ring is surprisingly distorted away from its favoured solution phase conformation and reveals potential for improved inhibitor potency. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b709681j

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A 1-acetamido derivative of 6-epi-valienamine: an inhibitor of a diverse group
of b-N-acetylglucosaminidases
Adrian Scaffidi,a Keith A. Stubbs,*b Rebecca J. Dennis,c Edward J. Taylor,c Gideon J. Davies,c
David J. Vocadlob and Robert V. Sticka
Received 27th June 2007, Accepted 16th July 2007
First published as an Advance Article on the web 10th August 2007
DOI: 10.1039/b709681j
The synthesis of an analogue of 6-epi-valienamine bearing an acetamido group and its characterisation
as an inhibitor of b-N-acetylglucosaminidases are described. The compound is a good inhibitor of both
human O-GlcNAcase and human b-hexosaminidase, as well as two bacterial b-N-acetylglucos-
aminidases. A 3-D structure of the complex of Bacteroides thetaiotaomicron BtGH84 with the inhibitor
shows the unsaturated ring is surprisingly distorted away from its favoured solution phase
conformation and reveals potential for improved inhibitor potency.
[2,1-d]-D2ꢀ-thiazoline (NAG-thiazoline) 1 (Fig. 1) has been found
Introduction
to be a potent inhibitor of family 20 hexosaminidases2,3 and,
more recently, family 84 O-GlcNAcases.5 Its poor inhibitory
2-Acetamido-2-deoxy-b-D-glycopyranosides are naturally occur-
ring glycosides found within many of the glycoconjugates and
potency toward family 3 is consistent with this family of enzy-
oligosaccharides that are present in an extremely wide variety of
organisms, ranging from microbes to humans. Unsurprisingly,
given their abundance in nature, there are several classes of
enzymes comprising both hydrolases and lyases that have evolved
to cleave glycosidic linkages involving this saccharide residue.
Glycoside hydrolases are particularly abundant and have been
mes. O-(2-Acetamido-2-deoxy-D-glucopyranosylidene)amino-N-
phenylcarbamate (PUGNAc) 2,12 the tetrahydroimidazopyridines,
with the most important being 8-acetamido-5,6,7,8-tetrahydro-
6,7-dihydroxy-5-(hydroxymethyl)imidazo[1,2-a]pyridine-2-acetic
acid, gluco-nagstatin 3,13 and the triazoles, all prepared by Vasella
et al., have all been shown to potently inhibit family 3 b-N-acetyl-
glucosaminidases,14 family 20 human b-hexosaminidases9,13 and
family 84 O-GlcNAcases.6,15,16,17 Some imino- and azasugars,
as well as being good inhibitors of glucosidases, have also
been found to be inhibitors of b-N-acetylglucosaminidases. The
N-acetylamino analogues 4 and 5 of 1-deoxynojirimycin and
isofagomine, respectively, are potent inhibitors of family 20 b-
hexosaminidases.18,19
classified into many different families on the basis of structural
and primary sequence similarities.† Members of each family
have similar three-dimensional structures and use similar cat-
alytic mechanisms. Interestingly though, just two main enzyme-
catalyzed hydrolytic mechanisms are known for cleaving the b-
glycosidic linkage of 2-acetamido-2-deoxy-b-D-glycopyranosides.
Examples of these two mechanistic classes include the exo-b-N-
acetylglucosaminidases from family 3, and those from families 20
and 84 of the glycoside hydrolases, all of which are functionally
related in that they catalyze the release of terminal 2-acetamido-2-
deoxy-b-D-glycopyranose residues from glycoconjugates.1–4 exo-b-
N-Acetylglucosaminidases from family 3 use a catalytic mech-
anism involving the formation and breakdown of a covalent
glycosyl–enzyme intermediate.4 The enzymes from families 201,2
and, more recently, 845–7 of the glycoside hydrolases have been
shown to differ in that they use a catalytic mechanism involving
assistance from the 2-acetamido group of the substrate.
As a result of the biological importance of these b-N-acetyl-
glucosaminidases, the design of small molecule inhibitors2,5,8–11
has received considerable attention.10 2ꢀ-Methyl-a-D-glucopyrano-
aChemistry M313, School of Biomedical, Biomolecular and Chemical
Sciences, The University of Western Australia, 35 Stirling Hwy, Crawley,
WA, Australia 6009; Fax: +61 8 6488 1005; Tel: +61 8 6488 320
bDepartment of Chemistry, Simon Fraser University, 8888 University Drive,
Burnaby, BC, Canada V5A 1S6. E-mail: kstubbs@sfu.ca; Tel: +1 604-291-
3530
cYork Structural Biology Laboratory, Department of Chemistry, The Uni-
versity of York, Heslington, York, United Kingdom YO10 5YW
† For the classification of glycoside hydrolases, see the CAZY Database
(available at http://afmb.cnrs-mrs.fr/CAZY/).
Fig. 1 Compounds referred to in this study, as well as one of the likely
transition states (4H3) for an a-glucosidase.
This journal is
The Royal Society of Chemistry 2007
Org. Biomol. Chem., 2007, 5, 3013–3019 | 3013
©

View Article Online
To expand the repertoire of inhibitors of b-N-acetylgluco-
saminidases, we thought to modify the known inhibitor, epi-
valienamine 6, which is the C-6 epimer of valienamine.20 Va-
lienamine is an essential core unit in many kinds of pseudo-
oligosaccharidic a-glucosidase inhibitors, such as acarbose,21
acarviosin,21 adiposin-222 and validoxylamine A.23 The valien-
amine core itself is also an inhibitor of some a-glucosidases.19
A
rationale for the potency of valienamine against a-glucosidases
is that it is thought to mimic both the charge and the ring
distortion of the transition state. The 1H2 (2H3 conformation using
the numbering as according to D-glucopyranose) conformation‡
exhibited by valienamine resembles in some aspects both the 4H3
and 2,5B conformations for the D-glucopyranose transition state,
found for related hydrolases,24,25 which are the two likely transition
state conformations for a-glucosidases (Fig. 1). The potency of
valienamine stems from the crucially important double bond.
“Dihydro” acarbose, a compound where the double bond has
been saturated, is only a modestly good glucoamylase inhibitor
(low micromolar Ki) compared to the very potent (picomolar Ki)
acarbose which retains the double bond.26,27
Scheme 1 (a) i. HCl, MeOH, ii. PhCH(OMe)2, camphor-10-sulfonic acid,
DMF; (b) BnBr, NaH, THF; (c) AcOH–H2O (4 : 1); (d) i. TsCl, pyridine,
ii. NaI, DMF; (e) NaH, BnBr, DMF; (f) i. Et3N·BH3, AlCl3, CH2Cl2,
ii. I2, PPh3, imidazole, PhMe, iii. NaH, BnBr, DMF.
On the other hand, little is known about the inhibitory potency
of 6-epi-valienamine 6.28 Some N-alkyl analogues have been syn-
thesised and have been found to be potent b-D-glucocerebrosidase
inhibitors,29 and one, N-octyl-6-epi-valienamine, has potential as
a therapeutic for Gaucher disease.30 We felt that owing to the
success of 6-epi-valienamine analogues as potent inhibitors of
b-glucocerebrosidases, an analogue of 6-epi-valienamine, where
the hydroxyl group at C-1 was replaced with an acetamido
group, as in 7, could yield a useful inhibitor scaffold for b-
N-acetylglucosaminidases. We therefore engaged in a rational
synthesis of such a putative 6-epi-valienamine-based inhibitor.
Scheme 2 (a) i. Hg(CF3COO)2, acetone–H2O (4 : 1), ii. MsCl, Et3N,
CH2Cl2; (b) Mg, BnOCH2Cl, HgCl2, THF.
and resulting in its axial addition. To circumvent this problem
associated with the presence of the 2-acetamido moiety, we
envisaged that a 2-azido group could instead be used.
Returning to the diol 9, we obtained 15 after using hydrazine hy-
drate; treatment of 15 with trifluoromethanesulfonyl azide then af-
forded the azide 16 in good yield (Scheme 3). Iodination of 16 gave
17, which upon benzylation and elimination furnished the alkene
18 in good yield. Alkene 18 was then treated with mercury(II)
trifluoroacetate to provide smoothly the intermediate carbocycle,
which was dehydrated to form the enone 19. Gratifyingly, as we
had predicted, treatment of the azide 19 with benzyloxymethyl-
magnesium chloride gave the desired alkene 20. Continuing on,
alkene 20 was next treated with triphenylphosphine to reduce the
azide to the corresponding amine, which was acetylated in situ
to provide 21 (Scheme 4). Treatment of 21 with sodium azide
and pure tetrakis(triphenylphosphine)palladium(0)34 yielded the
azide 22. At this point, the required exchange of protecting groups
went smoothly to give 23. The azide 23 was then subjected
to triphenylphosphine to give the presumed intermediate amine
which was treated in situ with tert-butyl dicarbonate to furnish
the Boc-protected amine (not shown). Deacetylation followed by
removal of the protecting group gave the desired compound 7 as
its hydrochloride salt 24.
We evaluated the inhibitor 7 against family 3 b-N-acetyl-
glucosaminidase, NagZ from Vibrio cholerae (Ki = 50 lM) and
the family 20 human placental b-hexosaminidase (Ki = 34 lM).
Also, family 84 human O-GlcNAcase (Ki = 6.2 lM) and a close
bacterial homologue BtGH846 (Ki = 26 lM) were assayed with
this inhibitor. It is interesting that despite the close structural
relationship of 6-epi-valienamine 6 and the 1-acetamido analogue
24, compound 24 is clearly much more potent. Indeed, Ogawa et al.
saw no inhibition of b-glucocerebrosidase with 6-epi-valienamine
Results and discussion
The methyl glycoside 8 was efficiently prepared in three steps from
N-acetyl-D-glucosamine (Scheme 1).31 Removal of the benzylidene
ring gave the diol 9. Selective iodination of the liberated primary
hydroxyl group via a tosylate intermediate furnished the iodide
10. Following benzylation of the free hydroxyl group of 10,
elimination of hydrogen iodide afforded the alkene 11.32 The
alkene 11 could also be prepared via a reductive ring-opening
of the benzylidene ring of 8, followed by sequential iodination
and elimination (Scheme 1). Treatment of the alkene 11 with
mercury(II) trifluoroacetate under Ferrier reaction conditions33
smoothly provided the presumed intermediate carbocycle (not
shown), which was dehydrated to form the enone 12 (Scheme 2).
Addition of the required exocyclic carbon to 12 is well precedented
and we chose to use benzyloxymethylmagnesium chloride to
facilitate the conversion. Interestingly, performing the Grignard
reaction using 12 did not proceed to give the desired alcohol 13
but resulted in a Michael addition to give a ketone, assigned
the structure 14. We propose that the amide moiety is first
deprotonated, directing the Grignard reagent to the wrong carbon
‡ The ring conformations referred to in this paper are based on the IUPAC
naming system for each individual compound. Since valienamine and its
analogues discussed here bind in a manner that mimics the pyranose ring
of the substrate, a more intuitive (although incorrect) numbering system
using the glucopyranose ring system is given in brackets for convenient
comparison.
3014 | Org. Biomol. Chem., 2007, 5, 3013–3019
This journal is
The Royal Society of Chemistry 2007
©

View Article Online
Scheme 3 (a) N2H4·H2O, H2O; (b) TfN3, CuSO4·5H2O, MeOH, CH2Cl2; (c) i. TsCl, pyridine, ii. NaI, DMF; (d) NaH, BnBr, DMF; (e) i. Hg(CF3COO)2,
acetone–H2O (4 : 1), ii. MsCl, Et3N, CH2Cl2; (f) i. Mg, BnOCH2Cl, HgCl2, THF.
Asn339). Surprisingly, the unsaturated ring of 7 is distorted away
from its favoured solution phase 1H6 (2H3) conformation toward a
2S6 (1S3) conformation in which the amine moiety is pseudo-axial
˚
and makes a 2.7 A hydrogen bond to the catalytic acid, Asp243.
Such a conformation does not resemble the predicted transition
state geometry but rather mimics those observed for the Michaelis
complexes of retaining enzymes active on b-glycosides of the gluco-
series (for example, ref. 35,36) and reflects in-line geometry for
nucleophilic substitution at the anomeric centre: the O–C2–N2
(O–C1–N1) angle is 167◦. Such distortion appears critical for
establishing a powerful electrostatic interaction between the amine
7 and Asp 243.
Scheme
4
(a) i. PPh3, THF, H2O, ii. Ac2O, DMAP, pyridine;
(b) Pd(PPh3)4, NaN3, THF, H2O; (c) i. FeCl3, CH2Cl2, ii. Ac2O, DMAP,
pyridine; (d) i. PPh3, THF, H2O, ii. Boc2O, KOH, MeOH, iii. 6 M HCl,
MeOH.
6,29 which differs from the results described here using the
1-acetamido analogue. It is difficult to ascertain the basis for
these differences.
Conclusions
We have devised a route that enables the rapid synthesis of an
inhibitor of b-N-acetylglucosaminidases from a diverse range
of glycoside hydrolase families. Also, this inhibitor scaffold has
the potential to be used to create more potent and potentially
selective inhibitors by elaborating the functional groups present
within the inhibitor. The structural analysis of BtGH84 in complex
with 7 given here should facilitate this development. Notably,
although partial distortion away from the expected half-chair
must come with a significant energetic penalty, the additional
electrostatic interaction this permits clearly mitigates these costs.
Furthermore, the pseudo-axial C2–N2 geometry suggests that
augmentation of the inhibitor at the amine position should
generate even more powerful inhibitors in which aglycone binding
energy may be captured. Such inhibitors could be useful molecules
for studying the cellular role of O-GlcNAc since they may have
useful pharmacological properties.
Therefore, to gain a more detailed understanding of the
molecular basis for these apparent differences, we evaluated the
interactions between7 andafamily84 b-N-acetylglucosaminidase.
The 3-D structure of BtGH84 (whose active centre residues are
invariant with the human enzyme) was solved in complex with 7 by
˚
X-ray crystallography at a resolution of approximately 2 A. To our
knowledge this is the first time that a crystal structure involving any
6-epi-valienamine analogue has been reported. The 3-D structure
revealed clear and unambiguous electron density for 7 bound in
the “−1” subsite of BtGH84 (Fig. 2). The acetamide 7 makes
similar interactions to those previously observed for thiazoline
complexes of the enzyme: notably a tight network of hydrogen
bonds (O6 to NZ of Lys166 and the main chain carbonyl of
Gly135, O5 and O in the hydroxymethyl group to Asp344 and
the acetyl NH and carbonyl group to OD2 of Asp242 and ND2 of
Experimental
General
Experimental procedures have been given previously.37 Elemental
analyses of all synthesized compounds used in enzyme assays were
performed at the Simon Fraser University.
Methyl 2-acetamido-3-O-benzyl-2,6-dideoxy-6-iodo-a-D-gluco-
pyranoside 10. (i) The acetal 8 (18.5 g) was heated in AcOH–
H2O (4 : 1, 200 mL) (70 ◦C, 1 h). The solution was concentrated
to leave a powder that was washed with toluene, followed by
hexanes, to afford a colourless powder (15.5 g). This material,
presumably methyl 2-acetamido-3-O-benzyl-2-deoxy-a-D-gluco-
pyranoside 9,31 was used in the next step without any further
purification.
Fig. 2 Observed electron density for 7 binding to BtGH84 shown in
divergent (wall-eyed) stereo; 7 is shown in grey and with observed 2Fobs
F
−
calc electron density at 0.4 electrons A−3. Interacting amino-acids discussed
˚
in the text are shown and are coloured green.
This journal is The Royal Society of Chemistry 2007
Org. Biomol. Chem., 2007, 5, 3013–3019 | 3015
©

View Article Online
(ii) 4-Toluenesulfonyl chloride (7.6 g, 40 mmol) was added to
the material from (i) (6.5 g, 20 mmol) and Et3N (10 mL) in CH2Cl2
(125 mL) at 0 ◦C and the solution was kept at room temperature
(3 h) before being quenched with H2O. A usual workup (CH2Cl2)
yielded a yellow residue. Sodium iodide (15 g, 0.10 mol) was added
to the residue in N,N-dimethylformamide (DMF, 100 mL) and the
solution was stirred at 100 ◦C (3 h). Concentration of the reaction
mixture followed by a usual workup (CH2Cl2) gave a brown residue
that was subjected to flash chromatography (EtOAc–petrol, 1 : 1)
to return the iodide 10 (5.8 g, 67%) as a colourless solid. Rf 0.5
(EtOAc–hexane, 7 : 3); mp 150–152 ◦C; [a]D20 +74.2; dH (600 MHz)
7.37–7.28 (m, 5H, Ph), 5.59 (d, 1H, J2,NH 9.5 Hz, NH), 4.70, 4.67
(ABq, 2H, J 11.7 Hz, CH2Ph), 4.65 (d, 1H, J1,2 3.7 Hz, H1), 4.27
(ddd, 1H, J2,3 10.4 Hz, H2), 3.56 (dd, 1H, J5,6 2.4 Hz, J6,6 10.7 Hz,
H6), 3.55 (dd, 1H, J3,4 8.7 Hz, H3), 3.48 (dd, 1H, J4,5 9.3 Hz,
H4), 3.43–3.40 (m, 1H, H5), 3.40 (s, 3H, OCH3), 3.30 (dd, 1H,
J5,6 7.1 Hz, H6), 1.92 (s, 3H, COCH3); dC (150.9 MHz) 169.94
(COCH3), 138.05–127.98 (Ph), 98.66 (C1), 79.90, 73.91, 70.53,
52.01 (4C, C2, C3, C4, C5), 73.92 (CH2Ph), 55.30 (OCH3), 23.34
(COCH3), 6.73 (C6); m/z (FAB) 436.0600, (M + H)+ requires
436.0621.
78.93, 49.52 (3C, C1, C5, C6), 73.82, 73.57 (2C, CH2Ph), 23.15
(COCH3); m/z (FAB) 366.1696, (M + H)+ requires 366.1705.
N-[(1S,2R,3S,5S)-2,3-Dibenzyloxy-6-benzyloxymethyl-4-oxo-
cyclohex-1-yl]acetamide 14. Magnesium (180 mg, 7.4 mmol),
benzyl chloromethyl ether (0.90 mL, 6.7 mmol) and HgCl2 (25 mg)
in THF (10 mL) were stirred at 0 ◦C (1.5 h). The enone 12 (100 mg,
0.30 mmol) in THF (5 mL) was added to the mixture at −78 ◦C
and the mixture was stirred (2 h) before being quenched with
saturated NaHCO3 solution. The suspension was filtered through
Celite, followed by a usual workup (EtOAc), to give a pale yellow
oil that was subjected to flash chromatography (EtOAc–petrol, 3 :
2) to yield 14 (90 mg, 67%) as colourless plates. Rf 0.35 (EtOAc–
hexane, 7 : 3); mp 146–148 ◦C; [a]D20 +14.3; dH (600 MHz) 7.35–7.23
(m, 15H, Ph), 6.12 (d, 1H, J1,NH 8.3 Hz, NH), 4.71, 4.49 (ABq, 2H,
J 11.2 Hz, CH2Ph), 4.65, 4.61 (ABq, 2H, J 11.9 Hz, CH2Ph),
4.52–4.47 (m, 1H, H1), 4.47, 4.36 (ABq, 2H, J 11.8 Hz, CH2Ph),
3.95 (d, 1H, J2,3 5.6 Hz, H3), 3.90 (dd, 1H, J1,2 5.9 Hz, H2), 3.47
(dd, 1H, J7,7 9.7 Hz, J6,7 5.3 Hz, H7), 3.40 (dd, 1H, J6,7 5.6 Hz,
H7), 2.69–2.64 (m, 1H, H6), 2.55–2.52 (m, 2H, H5, H5), 1.75
(s, 3H, COCH3); dC (150.9 MHz) 205.94 (C4), 169.68 (COCH3),
137.88–127.49 (Ph), 84.07, 78.82, 49.51 (3C, C1, C2, C3), 73.20,
73.03, 72.69, 70.61 (4C, C7, CH2Ph), 38.46 (C5), 36.86 (C6), 23.16
(COCH3); m/z (FAB) 488.2441, (M + H)+ requires 488.2437
Methyl 2-acetamido-3,4-di-O-benzyl-2,6-dideoxy-a-D-xylohex-
5-enoside 11. Sodium hydride (60% dispersion in mineral oil,
500 mg, 12.5 mmol) was added to 10 (1.0 g, 2.3 mmol) and BnBr
(0.30 mL, 2.5 mmol) in DMF (20 mL) and the mixture was stirred
(1 h). The reaction was monitored by 1H NMR spectroscopy
before being quenched with MeOH, concentrated and subjected
to a usual workup (EtOAc), followed by flash chromatography
(EtOAc–hexane, 1 : 1), to return 11 (700 mg, 77%)◦as a colourless
solid. Rf 0.25 (EtOAc–hexane, 1 : 1); mp 148–150 C; [a]2D0 +67.9;
dH (600 MHz) 7.38–7.26 (m, 10H, Ph), 5.44 (d, 1H, J2,NH 9.3 Hz,
NH), 4.85–4.84, 4.74–4.73 (2 m, 2H, H6, H6), 4.84, 4.63 (ABq,
2H, J 11.9 Hz, CH2Ph), 4.78, 4.71 (ABq, 2H, J 11.2 Hz, CH2Ph),
4.74 (d, 1H, J1,2 3.3 Hz, H1), 4.33 (ddd, 1H, J2,3 9.2 Hz, H2), 4.00
(ddd, 1H, J3,4 8.4 Hz, J4,6 ≈ J4,6 1.5 Hz, H4), 3.64 (dd, 1H, H3),
3.40 (s, 3H, OCH3), 1.84 (s, 3H, COCH3); dC (150.9 MHz) 169.79
(COCH3), 153.67 (C5), 138.28–127.79 (Ph), 99.50 (C1), 97.57 (C6),
79.62, 78.50, 51.69 (3C, C2, C3, C4), 74.34, 73.83 (2C, CH2Ph),
55.53 (OCH3), 23.35 (COCH3); m/z (FAB) 398.1956, (M + H)+
requires 398.1967.
Methyl 2-amino-3-O-benzyl-2-deoxy-a-D-glucopyranoside 15.
The amide 9 (13.0 g) was dissolved in N2H4·H2O (200 mL) and
the solution was refluxed (30 h). The solution was concentrated,
followed by flash chromatography (MeOH–CHCl3, 1 : 19), to yield
15 (10.0 g, 88%) as a gum. Rf 0.7 (MeOH–CHCl3, 1 : 4); [a]D20
+62.2; dH (600 MHz) 7.33–7.25 (m, 5H, Ph), 4.91, 4.72 (ABq, 2H,
J 11.7 Hz, CH2Ph), 4.67 (d, 1H, J1,2 3.6 Hz, H1), 3.77 (dd, 1H, J6,6
11.9 Hz, J5,6 3.8 Hz, H6), 3.73 (dd, 1H, J5,6 3.1 Hz, H6), 3.60–3.52
(m, 2H, H4, H5), 3.40 (dd, 1H, J2,3 9.9 Hz, J3,4 8.5 Hz, H3), 3.33
(s, 3H, OCH3), 2.69 (dd, 1H, H2); dC (150.9 MHz) 138.58–127.77
(Ph), 100.34 (C1), 83.57, 71.66, 71.11 (3C, C3, C4, C5), 75.14
(CH2Ph), 61.83 (C6), 55.26, 55.03 (2C, C2, OCH3); m/z (FAB)
284.1497, (M + H)+ requires 284.1498.
Methyl 2-azido-3-O-benzyl-2-deoxy-a-D-glucopyranoside 16.
A freshly prepared solution of trifluoromethanesulfonyl azide
(TfN3) in CH2Cl2 (0.50 M, 100 mL, 50 mmol)38 was added
dropwise to 15 (8.8 g, 31 mmol), 4-(dimethylamino)pyridine
(DMAP, 4.6 g, 38 mmol) and CuSO4·5H2O (200 mg) in MeOH
(100 mL) and the solution was stirred at room temperature
(3 h). Toluene (50 mL) was added to the solution before being
concentrated to approximately 50 mL. Flash chromatography
(EtOAc–petrol, 1 : 1) yielded 16 (9.5 g, 99%) as a colourless gum.
Rf 0.6 (EtOAc–hexane, 7 : 3); [a]2D0 +95.5; dH (600 MHz) 7.41–7.31
(m, 5H, Ph), 4.79 (d, 1H, J1,2 3.5 Hz, H1), 4.96, 4.74 (ABq, 2H,
J 11.3 Hz, CH2Ph), 3.83–3.76, 3.66–3.60 (2 m, 5H, H3, H4, H5,
H6, H6), 3.43 (s, 3H, OCH3), 3.34 (dd, 1H, J2,3 10.1 Hz, H2); dC
(150.9 MHz) 137.86–128.01 (Ph), 98.79 (C1), 80.09, 71.00, 70.82,
63.16 (4C, C2, C3, C4, C5), 75.01 (CH2Ph), 62.11 (C6), 55.22
(OCH3); m/z (FAB) 308.1236, (M + H)+ requires 308.1246.
N-[(1S,5S,6R)-5,6-Dibenzyloxy-4-oxocyclohex-2-en-1-yl]acet-
amide 12. Mercury(II) trifluoroacetate (5.0 mg, 17 lmol) was
added to 11 (200 mg, 0.5 mmol) in dioxane–aqueous H2SO4
(5 mM) (2 : 1, 8 mL) and the mixture was heated at 80 ◦C
(3 h). The mixture was cooled and subjected to a usual workup
(CH2Cl2) to leave a colourless powder that was dissolved in
CH2Cl2 (10 mL) and treated with methanesulfonyl chloride (MsCl,
0.20 mL, 2.6 mmol) and Et3N (0.70 mL, 5.3 mmol). The mixture
was quenched with H2O, followed by a usual workup (CH2Cl2) to
afford a brown residue that was subjected to flash chromatography
(EtOAc–petrol, 7 : 3) to furnish 12 (70 mg, 38%) as flakes. Rf 0.5
◦
(EtOAc); mp 164–166 C; [a]2D0 +118.5; dH (600 MHz) 7.40–7.27
(m, 10H, Ph), 6.72 (dd, 1H, J2,3 10.2 Hz, J1,2 3.3 Hz, H2), 6.06 (dd,
1H, J1,3 2.1 Hz, H3), 5.58 (d, 1H, J1,NH 7.8 Hz, NH), 4.82–4.77 (m,
1H, H1), 4.92, 4.66 (ABq, 2H, J 11.4 Hz, CH2Ph), 4.79, 4.66 (ABq,
2H, J 11.7 Hz, CH2Ph), 4.03 (d, 1H, J5,6 7.9 Hz, H5), 3.80 (dd,
1H, J1,6 7.7 Hz, H6), 1.83 (s, 3H, COCH3); dC (150.9 MHz) 196.07
(C4), 169.67 (COCH3), 146.90 (C2), 137.57–127.99 (Ph), 82.31,
Methyl 2-azido-3-O-benzyl-2,6-dideoxy-6-iodo-a-D-glucopyra-
noside 17. 4-Toluenesulfonyl chloride (8.0 g, 42 mmol) was added
to◦16 (9.5 g, 31 mmol) in pyridine–CH2Cl2 (1 : 3, 100 mL) at
0 C. The solution was stirred at room temperature (3 h) before
being quenched with MeOH. A usual workup (CHCl3) gave a
3016 | Org. Biomol. Chem., 2007, 5, 3013–3019
This journal is
The Royal Society of Chemistry 2007
©

View Article Online
yellow residue. Sodium iodide (22.0 g, 147 mmol) was added to
the residue in DMF (100 mL) and the solution was stirred at 100 ◦C
(2 h). Concentration of the reaction mixture followed by a usual
workup (CHCl3) gave a brown residue that was subjected to flash
chromatography (EtOAc–petrol, 3 : 17) to yield 17 (10.5 g, 82%)
as an oil. Rf 0.6 (EtOAc–hexane, 7 : 3); [a]2D0 +62.5; dH (600 MHz)
7.40–7.33 (m, 5H, Ph), 4.97, 4.67 (ABq, 2H, J 11.3 Hz, CH2Ph),
4.82 (d, 1H, J1,2 3.6 Hz, H1), 3.78 (dd, 1H, J2,3 10.1 Hz, J3,4 8.3 Hz,
H3), 3.53 (dd, 1H, J6,6 10.7 Hz, J5,6 2.3 Hz, H6), 3.45 (s, 3H,
OCH3), 3.44–3.37 (m, 2H, H4, H5), 3.37 (dd, 1H, H2), 3.30 (dd,
1H, J5,6 6.4 Hz, H6), 2.23 (d, 1H, J4,OH 3.0 Hz, OH); dC (150.9 MHz)
137.72–128.11 (Ph), 98.74 (C1), 79.75, 74.18, 70.02, 63.27 (4C, C2,
C3, C4, C5), 75.07 (CH2Ph), 55.55 (OCH3), 6.65 (C6); m/z (FAB)
420.0445, (M + H)+ requires 420.0420.
followed by a usual workup (EtOAc), to give a pale yellow oil that
was subjected to flash chromatography (EtOAc–toluene, 1 : 19) to
yield 20 (260 mg, 76%) as an oil. Rf 0.4 (EtOAc–toluene, 1 : 9);
[a]D20 +10.0; mmax (film)/cm−1 2100 (N3); dH (600 MHz) 7.38–7.28 (m,
15H, Ph), 5.79 (dd, 1H, J2,3 10.2 Hz, J2,4 2.4 Hz, H2), 5.58 (dd, 1H,
J3,4 2.4 Hz, H3), 4.85, 4.82 (ABq, 2H, J 10.5 Hz, CH2Ph), 4.84,
4.73 (ABq, 2H, J 10.7 Hz, CH2Ph), 4.61, 4.57 (ABq, 2H, J 11.0 Hz,
CH2Ph), 4.06 (ddd, 1H, J4,5 6.9 Hz, H4), 3.83 (dd, 1H, J5,6 9.8 Hz,
H5), 3.79 (d, 1H, H6), 3.74, 3.62 (ABq, 2H, J 9.2 Hz, H7, H7); dC
(150.9 MHz) 138.39–124.34 (C2, C3, Ph), 83.51, 80.56, 63.08 (3C,
C4, C5, C6), 75.50, 75.06, 73.70, 73.45 (4C, C7, CH2Ph), 75.38
(C1); m/z (FAB) 472.2232, (M + H)+ requires 472.2236.
(1R,4S,5R,6S)-4-Acetamido-5,6-dibenzyloxy-1-benzyloxymeth-
yl-cyclohex-2-en-1-yl acetate 21. Triphenylphosphine (70 mg,
0.30 mmol) was added to 20 (100 mg, 0.20 mmol) in THF–H2O
(3 : 1, 4 mL) and the solution was heated at 50 ◦C (4 h). The
reaction was subjected to a usual workup (EtOAc) to afford a
yellow gum. Acetic anhydride (0.20 mL, 2.0 mmol) and DMAP
(5 mg) were then added to the yellow gum in pyridine (5 mL) and
the solution was heated at 60 ◦C (3 h) before being quenched with
MeOH, followed by a usual workup (EtOAc), to leave a gum that
was subjected to flash chromatography (EtOAc–petrol, 1 : 1) to
yield 21 (90 mg, 80%) as a colourless gum. Rf 0.5 (EtOAc–hexane,
7 : 3); [a]2D0 +75.3; dH (600 MHz) 7.36–7.25 (m, 15H, Ph), 5.90 (dd,
1H, J2,3 10.3 Hz, J2,4 2.3 Hz, H2), 5.62 (dd, 1H, J3,4 3.2 Hz, H3),
4.96 (d, 1H, J4,NH 8.9 Hz, NH), 4.83, 4.73 (ABq, 2H, J 11.3 Hz,
CH2Ph), 4.74, 4.62 (ABq, 2H, J 12.0 Hz, CH2Ph), 4.70–4.65 (m,
H4), 4.56, 4.50 (ABq, 2H, J 11.4 Hz, CH2Ph), 4.40 (d, 1H, J5,6
8.8 Hz, H6), 4.07, 3.78 (ABq, 2H, J 9.3 Hz, H7, H7), 3.87 (dd,
1H, J4,5 6.5 Hz, H5), 1.87, 1.56 (2s, 6H, COCH3); dC (150.9 MHz)
169.71, 169.08 (2C, COCH3), 138.46–127.63 (Ph), 132.05, 127.20
(C2, C3), 83.36 (C1), 80.86, 79.66, 50.79 (3C, C4, C5, C6), 75.32,
73.78, 73.36, 70.30 (4C, C7, CH2Ph), 23.10, 21.98 (2C, COCH3);
m/z (FAB) 530.2529, (M + H)+ requires 530.2542.
Methyl 2-azido-3,4-di-O-benzyl-2,6-dideoxy-a-D-xylohex-5-
enoside 18. Sodium hydride (60% dispersion in mineral oil, 4.5 g,
110 mmol) was added to BnBr (5.0 mL, 42 mmol) and 17 (9.5 g,
23 mmol) in DMF (100 mL) and the mixture was stirred (6 h).
1
The reaction was monitored by H NMR spectroscopy before
being quenched with MeOH, concentrated and subjected to a
usual workup (CH2Cl2). Flash chromatography of the residue
(EtOAc–petrol, 1 : 19) then returned 18 (6.6 g, 76%) as an oil
that solidified on standing. Rf 0.5 (EtOAc–hexane, 1 : 4); [a]D20
+62.5; dH (600 MHz) 7.40–7.28 (m, 10H, Ph), 4.94–4.93 (m, 1H,
H6), 4.94, 4.83 (ABq, 2H, J 10.7 Hz, CH2Ph), 4.84 (d, 1H, J1,2
3.5 Hz, H1), 4.82, 4.77 (ABq, 2H, J 11.2 Hz, CH2Ph), 4.80–4.78,
4.00–3.95, 3.53–3.50 (3 m, 4H, H2, H3, H4, H6), 3.47 (s, 3H,
OCH3); dC (150.9 MHz) 153.16 (C5), 137.72–127.79 (Ph), 99.26
(C1), 97.54 (C6), 80.14, 79.29, 63.13 (3C, C2, C3, C4), 75.52,
74.40 (2C, CH2Ph), 55.48 (OCH3); m/z (FAB) 382.1747, (M +
H)+ requires 382.1767.
(4S,5R,6S)-4-Azido-5,6-dibenzyloxy-cyclohex-2-enone
19.
Mercury(II) trifluoroacetate (60 mg, 0.10 mmol) was added to
18 (700 mg, 1.80 mmol) in acetone–H2O (4 : 1, 10 mL) and the
suspension was stirred (12 h). Volatile solvents were removed and
the resulting aqueous mixture was subjected to a usual workup
(EtOAc) to give a colourless gum. Methanesulfonyl chloride
(0.50 mL, 6.4 mmol) and Et3N (1.1 mL, 7.9 mmol) were added to
the gum in CH2Cl2 (20 mL) at 0 ◦C. The solution was then left at
room temperature (2 h), followed by a usual workup (CH2Cl2),
to leave a gum that was subjected to flash chromatography
(EtOAc–petrol, 1 : 9) to return 19 (395 mg, 64%) as a gum. Rf 0.8
(EtOAc–hexane, 2 : 3); [a]2D0 +7.8; dH (500 MHz) 7.45–7.30 (m,
10H, Ph), 6.64 (dd, 1H, J2,3 10.3 Hz, J2,4 2.2 Hz, H2), 6.10 (dd, 1H,
J3,4 2.6 Hz, H3), 5.11, 4.74 (ABq, 2H, J 11.3 Hz, CH2Ph), 4.98,
4.80 (ABq, 2H, J 10.6 Hz, CH2Ph), 4.37 (ddd, 1H, J4,5 8.7 Hz,
H4), 4.11 (d, 1H, J5,6 10.5 Hz, H6); dC (125.8 MHz) 196.78 (C1),
144.94 (C3), 137.42–127.97 (C2, Ph), 84.17, 83.39, 62.86 (3C, C4,
C5, C6), 75.66, 74.62 (2C, CH2Ph); m/z (FAB) 350.1533, (M +
H)+ requires 350.1505.
N-[(1S,2R,5R,6R)-2-Azido-5,6-dibenzyloxy-4-benzyloxymethyl-
34
cyclohex-3-en-1-yl]acetamide 22. Freshly prepared Pd(PPh3)4
(12 mg, 8.9 lmol) was added to NaN3 (75 mg, 1.2 mmol) and 21
(100 mg, 0.20 mmol) in deoxygenated THF–H2O (2 : 1, 3 mL)
and the mixture was refluxed (6 h). A usual workup (EtOAc)
returned a brown gum that was subjected to flash chromatography
(EtOAc–petrol, 3 : 7) to afford an orange solid that was washed
with Et2O to furnish 22 (55 mg, 55%) as a pale yellow powder.
◦
Rf 0.6 (EtOAc–hexane, 1 : 1); mp 116–118 C; [a]2D0 −192.2; dH
(600 MHz) 7.37–7.18 (m, 15H, Ph), 6.47 (d, 1H, J1,NH 8.3 Hz,
NH), 5.90 (d, 1H, J2,3 3.8 Hz, H3), 4.75, 4.60 (ABq, 2H, J 12.0 Hz,
CH2Ph), 4.56, 4.54 (ABq, 2H, J 11.0 Hz, CH2Ph), 4.55, 4.41
(ABq, 2H, J 11.7 Hz, CH2Ph), 4.47–4.43 (m, 1H, H1), 4.27 (d,
1H, J7,7 12.9 Hz, H7), 4.09 (d, 1H, J5,6 3.3 Hz, H5), 3.98 (d, 1H,
H7), 3.89 (dd, 1H, J1,6 4.8 Hz, H6), 3.86 (m, 1H, H2), 1.78 (s, 3H,
COCH3); dC (150.9 MHz) 169.59 (COCH3), 137.86–127.74 (C4,
Ph), 122.16 (C3), 74.10, 72.93, 57.79, 49.43 (4C, C1, C2, C5, C6),
73.57, 72.05, 71.63, 70.38 (4C, C7, CH2Ph), 23.26 (COCH3); m/z
(FAB) 513.2487, (M + H)+ requires 513.2502.
(1R,4S,5R,6S)-4-Azido-5,6-dibenzyloxy-1-benzyloxymethyl-
cyclohex-2-en-1-ol 20. Magnesium (105 mg, 4.30 mmol), benzyl
chloromethyl ether (0.50 mL, 3.7 mmol) and HgCl2 (14 mg) in
dry THF (5 mL) were stirred at 0 ◦C (1.5 h). The en◦one 19
(250 mg, 0.70 mmol) in THF (5 mL) was added at −78 C and
the mixture was stirred (2 h) before being quenched with saturated
NaHCO3 solution. The suspension was filtered through Celite,
N-[(1S,2R,5R,6R)-2-Azido-5,6-diacetoxy-4-acetoxymethyl-cyclo-
hex-3-en-1-yl]acetamide 23. Anhydrous iron(III) chloride
(115 mg, 0.70 mmol) was added to 22 (30 mg, 0.06 mmol) in
dry CH2Cl2 (3 mL) at 0 ◦C. The mixture was stirred at room
This journal is
The Royal Society of Chemistry 2007
Org. Biomol. Chem., 2007, 5, 3013–3019 | 3017
©

View Article Online
temperature (3 h) before being quenched with H2O (0.1 mL).
The mixture was concentrated and co-evaporated with toluene to
leave a brown residue. Acetic anhydride (0.20 mL, 2.0 mmol) and
DMAP (5 mg) were then added to the brown residue in pyridine
(5 mL) and the solution was heated at 50 ◦C (1 h) before being
quenched with MeOH, followed by a usual workup (CH2Cl2),
to leave a gum. Flash chromatography (EtOAc–petrol, 3 : 2)
yielded pale yellow plates that were washed with Et2O to return
23 (9.0 mg, 42%) as colourless plates. Rf 0.4 (EtOAc–hexane, 7 :
3); mp 147–149 ◦C; mmax (film)/cm−1 2095 (N3); dH (600 MHz) 5.85
(s, 1H, H3), 5.83–5.80 (m, 1H, H5), 5.70 (d, 1H, J1,NH 9.4 Hz,
NH), 5.17 (dd, 1H, J1,6 11.2 Hz, J5,6 7.8 Hz, H6), 4.68 (d, 1H, J7,7
13.3 Hz, H7), 4.45–4.35 (m, 2H, H1, H7), 4.12–4.08 (m, 1H, H2),
2.08, 2.06, 2.05, 2.00 (4s, 12H, COCH3); dC (150.9 MHz) 171.24,
170.22, 170.20, 169.57 (4C, COCH3), 134.17 (C4), 126.34 (C3),
72.05, 69.92, 60.73, 52.81 (4C, C1, C2, C5, C6), 62.41 (C7), 23.12,
20.61, 20.56, 20.50 (4C, COCH3); m/z (FAB) 369.1418, (M + H)+
requires 369.1410.
NaCl, 0.1% BSA, pH 4.25 for b-hexosaminidase B. The progress
of the reaction at the end of 30 minutes for stopped assays
was determined by measuring the amount of 4-nitrophenolate
liberated as determined by UV measurements at 400 nm using a 96-
well plate (Sarstedt) and 96-well plate (Molecular Devices) reader.
Reaction velocities for continuous based assays were determined
by monitoring the liberation of 4-nitrophenolate using linear re-
gression of the linear region of the reaction progress curve between
the first and third minute. O-GlcNAcase and b-hexosaminidase B
were used in the inhibition assays at a concentration (lg lL−1) of
0.0406 and 0.012, respectively, using 4-nitrophenyl-2-acetamido-2-
deoxy-b-D-glucopyranoside as the substrate at a concentration of
0.50 mM. The inhibitor was tested at seven concentrations ranging
from three times to one-third Ki. Ki values were determined by
linear regression of data from Dixon plots.
Crystallization and structure solution of BtGH846
N-Terminally His6-tagged BtGH84 was produced via isopropyl-
1-thio-b-D-galactopyranoside (IPTG) induction (overnight) of
pGH84-containing Escherichia coli BL21 (DE3; Novagen) cul-
tures grown at 37 ◦C to an OD600 = 0.6. BtGH84 was purified
from cell free extracts via Ni-affinity chromatography followed by
gel filtration. Purified protein was concentrated to 5 mg mL−1 and
exchanged into 20 mM HEPES (4-(2-hydroxyethyl)piperazine-
N -[(1S,2R,5R,6R)-2-Amino-5,6-dihydroxy-4-hydroxymethyl-
cyclohex-3-en-1-yl]acetamide hydrochloride 24. Triphenylphos-
phine (20 mg, 76 lmol) was added to 23 (20 mg, 54 lmol) in
THF–H2O (2 : 1, 3 mL) and the solution was heated at 50 ◦C
(5 h). The mixture was concentrated, the residue was dissolved
in MeOH (2 mL), followed by the addition of Boc2O (25 mg,
0.10 mmol) and KOH (20 mg, 0.40 mmol). The solution was stirred
at room temperature (1 h) before being concentrated and subjected
to flash chromatography (MeOH–EtOAc, 1 : 4) to furnish a gum.
This gum was treated with 6 M HCl in MeOH (2 mL) to afford 24
1-ethanesulfonic acid) (pH 7.5) using
a 10 kDa cut-off
concentrator. BtGH84 crystals were grown using hanging
drop vapour diffusion as for the native structure6 with N-
[(1S,2R,5R,6R)-2-amino-5,6-dihydroxy-4-hydroxymethyl-cyclo-
hex-3-en-1-yl)acetamide hydrochloride 24 included at a concen-
tration of 10 mM. X-Ray data were collected from a single crystal
at 100 K at the European Synchrotron Radiation Facility on ID
14–2 with 30% v/v glycerol used as the cryo-protectant. All X-
ray data were processed with MOSFLM from the CCP4 suite39
(Table 1). Structure solution was carried out using the molecular
replacement technique as implemented in the CCP439 version
of MOLREP using BtGH84 (PDB code 2CHO) as the search
model. COOT40 was used to make manual corrections to the model
with refinement using REFMAC.41 Solvent molecules were added
using COOT and checked manually. Structural figures were
◦
(10 mg, 73%) as a colourless solid. Mp >230 C; [a]2D0 −59.6; dH
(600 MHz, D2O) 5.28–5.25 (m, 1H, H3), 4.35–4.32 (m, 1H, H5),
4.30–4.18 (m, 2H, H7, H7), 4.12 (dd, 1H, J1,2 9.8 Hz, J1,6 11.0 Hz,
H1), 4.05–4.02 (m, 1H, H2), 3.72 (dd, 1H, J5,6 8.0 Hz, H6), 2.10 (s,
3H, COCH3); dC (150.9 MHz, D2O) 175.62 (COCH3), 143.68 (C4),
117.29 (C3), 73.00, 71.76, 57.83, 52.67 (4C, C1, C2, C5, C6), 60.84
(C7), 22.30 (COCH3); m/z (FAB) 217.1189, (M − Cl)+ requires
217.1188. Anal. calcd for C9H17ClN2O4: C, 42.78; H, 6.78. Found:
C, 42.52; H, 6.59%.
Kinetic analysis of b-N-acetylglucosaminidases
O-GlcNAcase and Vibrio cholerae NagZ were overexpressed and
purified just prior to use.5,14 Human placental b-hexosaminidase
B was purchased from Sigma (lot 043K3783). Assays involving
human b-hexosaminidase and B BtGH84 were carried out in
triplicate at 37 ◦C for 30 minutes by using a stopped assay
procedure in which the enzymatic reactions (50 lL) were quenched
by the addition of a four-fold excess (200 lL) of quenching buffer
(200 mM glycine, pH 10.75). Assays using NagZ and human
O-GlcNAcase were carried out using continuous based assays
in which the enzyma◦tic reactions (80 lL) were conducted at
Table 1 X-Ray data quality and structure refinement statistics for the
complex BtGH84 with 24
BtGH84 with 24a
Data collection
˚
Resolution/A
Rmerge
I/rI
20–1.95 (2.06–1.95)
0.086 (0.53)
8.3 (1.6)
97 (94)
2.5 (2.4)
Completeness (%)
Redundancy
◦
25 C (NagZ) and 37 C (human O-GlcNAcase) as the enzymes
Refinement
were considerably more stable at these temperatures. Assays were
initiated by the careful addition, via pipette, of enzyme, and in all
cases the final pH of the resulting quenched solution was greater
than 10. Time-dependent assays of NagZ, b-hexosaminidase B,
O-GlcNAcase and BtGH84 revealed that the enzymes were stable
and rates were linear in their respective buffers over the period of
the assay: 50 mM NaH2PO4, 100 mM NaCl, 0.1% BSA, pH 6.5
for O-GlcNAcase and BtGH84, and 50 mM citrate, 100 mM
˚
Resolution/A
work/Rfree
20–1.95
0.20 (0.24)
R
Root mean square deviations
˚
Bond lengths/A
0.014
1.4
Bond angles/◦
a Highest resolution shell in parentheses.
3018 | Org. Biomol. Chem., 2007, 5, 3013–3019
This journal is
The Royal Society of Chemistry 2007
©

View Article Online
drawn with MOLSCRIPT42/BOBSCRIPT.43 Coordinates have
been deposited with the Protein Data Bank, PDB code 2JIW.
17 H. C. Dorfmueller, V. S. Borodkin, M. Schimpl, S. M. Shepherd, N. A.
Shpiro and D. M. F. van Aalten, J. Am. Chem. Soc., 2006, 128, 16484.
18 E. Shitara, Y. Nishimura, F. Kojima and T. Takeuchi, Bioorg. Med.
Chem., 1999, 7, 1241.
19 E. Kappes and G. Legler, J. Carbohydr. Chem., 1989, 8, 371.
20 X. Chen, Y. Fan, Y. Zheng and Y. Shen, Chem. Rev., 2003, 103, 1955.
21 B. Junge, F. R. Heiker, J. Kurz, L. Muller, D. D. Schmidt and C.
Wunsche, Carbohydr. Res., 1984, 128, 235.
22 S. Namiki, K. Kangouir, T. Nagata, N. Hara, K. Sugita and S. Omura,
J. Antibiot., 1982, 35, 1234.
23 H. M. Salleh and J. F. Honek, FEBS Lett., 1990, 262, 359.
24 Y. Tanaka, W. Tao, J. S. Blanchard and E. J. Hehre, J. Biol. Chem.,
1994, 269, 32306.
Acknowledgements
DJV is a scholar of the MSFHR and a Canada Research Chair
in Chemical Glycobiology. GJD is a Royal Society–Wolfson Re-
search Merit Award recipient. We thank the Natural Sciences and
Engineering Research Council of Canada, and the Biotechnology
and Biochemical Sciences Research Council for Funding.
25 G. J. Davies, V. M. Ducros, A. Varrot and D. L. Zechel, Biochem. Soc.
Trans., 2003, 31, 523.
26 B. Svensson and M. R. Sierks, Carbohydr. Res., 1992, 227, 29.
27 B. W. Sigurskjold, C. R. Berland and B. Svensson, Biochemistry, 1994,
33, 10191.
28 F. Nicotra, L. Panza, F. Ronchetti and G. Russo, Gazz. Chim. Ital.,
1989, 119, 577.
29 S. Ogawa, M. Ashiura, C. Uchida, S. Watanabe, C. Yamazaki, K.
Yamagishi and J. Inokuchi, Bioorg. Med. Chem. Lett., 1996, 6, 929.
30 H. Lin, Y. Sugimoto, Y. Ohsaki, H. Ninomiya, A. Oka, M. Taniguchi,
H. Ida, Y. Eto, S. Ogawa, Y. Matsuzaki, M. Sawa, T. Inoue, K. Higaki,
E. Nanba, K. Ohno and Y. Suzuki, Biochim. Biophys. Acta, 2004, 1689,
219.
References
1 I. Tews, A. Perrakis, A. Oppenheim, Z. Dauter, K. S. Wilson and C. E.
Vorgias, Nat. Struct. Biol., 1996, 3, 638.
2 S. Knapp, D. Vocadlo, Z. N. Gao, B. Kirk, J. P. Lou and S. G. Withers,
J. Am. Chem. Soc., 1996, 118, 6804.
3 B. L. Mark, D. J. Vocadlo, S. Knapp, B. L. Triggs-Raine, S. G. Withers
and M. N. James, J. Biol. Chem., 2001, 276, 10330.
4 D. J. Vocadlo, C. Mayer, S. He and S. G. Withers, Biochemistry, 2000,
39, 117.
5 M. S. Macauley, G. E. Whitworth, A. W. Debowski, D. Chin and D. J.
Vocadlo, J. Biol. Chem., 2005, 280, 25313.
31 S. S. Rana, J. J. Barlow and K. L. Matta, Carbohydr. Res., 1983, 113,
6 R. J. Dennis, E. J. Taylor, M. S. Macauley, K. A. Stubbs, J. P.
Turkenburg, S. J. Hart, G. N. Black, D. J. Vocadlo and G. J. Davies,
Nat. Struct. Mol. Biol., 2006, 13, 365.
257.
32 J. K. Fairweather, M. J. McDonough, R. V. Stick and D. M. G. Tilbrook,
Aust. J. Chem., 2004, 57, 197.
7 F. V. Rao, H. C. Dorfmueller, F. Villa, M. Allwood, I. M. Eggleston
and D. M. van Aalten, EMBO J., 2006, 25, 1569.
8 G. Legler, E. Lullau, E. Kappes and F. Kastenholz, Biochim. Biophys.
Acta, 1991, 1080, 89.
9 M. Horsch, L. Hoesch, A. Vasella and D. M. Rast, Eur. J. Biochem.,
1991, 197, 815.
10 J. Liu, A. R. Shikhman, M. K. Lotz and C.-H. Wong, Chem. Biol.,
2001, 8, 701.
11 K. A. Stubbs, N. Zhang and D. J. Vocadlo, Org. Biomol. Chem., 2006,
4, 839.
12 D. Beer, J. L. Maloisel, D. M. Rast and A. Vasella, Helv. Chim. Acta,
1990, 73, 1918.
13 M. Terinek and A. Vasella, Helv. Chim. Acta, 2005, 88, 10.
14 K. A. Stubbs, M. Balcewich, B. L. Mark and D. J. Vocadlo, J. Biol.
Chem., 2007, 282, 21382.
15 D. L. Dong and G. W. Hart, J. Biol. Chem., 1994, 269, 19321.
16 B. Shanmugasundaram, A. W. Debowski, R. J. Dennis, G. J. Davies,
D. J. Vocadlo and A. Vasella, Chem. Commun., 2006, 4372.
33 F. Chretien and Y. Chapleur, J. Chem. Soc., Chem. Commun., 1984,
1268.
34 J. E. Backvall, J. E. Nystrom and R. E. Nordberg, J. Am. Chem. Soc.,
1985, 107, 3676.
35 G. J. Davies, L. Mackenzie, A. Varrot, M. Dauter, M. A. Brzozowski,
M. Schulein and S. G. Withers, Biochemistry, 1998, 37, 11707.
36 V. A. Money, N. L. Smith, A. Scaffidi, R. V. Stick, H. J. Gilbert and
G. J. Davies, Angew. Chem., Int. Ed., 2006, 45, 5136.
37 A. Scaffidi, B. W. Skelton, R. V. Stick and A. H. White, Aust. J. Chem.,
2006, 59, 426.
38 Q. Liu and Y. Tor, Org. Lett., 2003, 5, 2571.
39 S. Bailey, Acta Crystallogr., Sect. D: Biol. Crystallogr., 1994, 50, 760.
40 P. Emsley and K. Cowtan, Acta Crystallogr., Sect. D: Biol. Crystallogr.,
2004, 60, 2126.
41 G. N. Murshudov, A. A. Vagin and E. J. Dodson, Acta Crystallogr.,
Sect. D: Biol. Crystallogr., 1997, 53, 240.
42 P. J. Kraulis, J. Appl. Crystallogr., 1991, 24, 946.
43 R. M. Esnouf, J. Mol. Graphics Modell., 1997, 15, 132.
This journal is
The Royal Society of Chemistry 2007
Org. Biomol. Chem., 2007, 5, 3013–3019 | 3019
©