The synthesis and biological evaluation of some carbocyclic analogues of PUGNAc

Article abstract of DOI:10.1016/j.carres.2008.08.012

The synthesis of some analogues of O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenylcarbamate, PUGNAc, an inhibitor of β-N-acetylglucosaminidases, is described. The analogues were tested against a range of β-N-acetylglucosaminidases to establish any biological activity. As well, the analogues were tested as inhibitors of a uridine diphosphate-N-acetyl-d-glucosamine: polypeptidyl transferase, OGT, a critical protein involved in the post-translational modification of nuclear and cytosolic proteins by N-acetyl-d-glucosamine.

Full text of DOI:10.1016/j.carres.2008.08.012

Carbohydrate Research 343 (2008) 2744–2753
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
The synthesis and biological evaluation of some carbocyclic
analogues of PUGNAc
b
a
Adrian Scaffidi a, Keith A. Stubbs a,b, , David J. Vocadlo , Robert V. Stick
*
a Chemistry M313, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
b Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 June 2008
Received in revised form 4 August 2008
Accepted 9 August 2008
Available online 15 August 2008
The synthesis of some analogues of O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenylcar-
bamate, PUGNAc, an inhibitor of b-N-acetylglucosaminidases, is described. The analogues were tested
against a range of b-N-acetylglucosaminidases to establish any biological activity. As well, the analogues
were tested as inhibitors of a uridine diphosphate-N-acetyl-
OGT, a critical protein involved in the post-translational modification of nuclear and cytosolic proteins
by N-acetyl- -glucosamine.
D-glucosamine: polypeptidyl transferase,
D
Keywords:
GlcNAc
Ó 2008 Elsevier Ltd. All rights reserved.
Glycosidases
Inhibitors
Glycosyltransferases
1. Introduction
in the development of insulin resistance and gluconeogenesis,
respectively.
The post-translational modification of nuclear and cytoplasmic
proteins with 2-acetamido-2-deoxy- -glucopyranose (GlcNAc), to
generate 2-acetamido-2-deoxy-b- -glucopyranosides, is found in
To maintain the appropriate levels of cellular O-GlcNAc, two dif-
ferent carbohydrate-processing enzymes are critical. The enzyme
catalyzing the installation of O-GlcNAc is a known glycosyltrans-
ferase termed OGT (E.C. 2.4.1.94); ,17,18 this enzyme has been found
to be essential at the single cell level.19 The enzyme responsible for
catalyzing the cleavage of O-GlcNAc from modified proteins and
returning them to their unmodified state is the glycoside hydrolase
known as O-GlcNAcase (E.C. 3.2.1.52)20,21 (Fig. 1). As a result of the
biological importance of these two enzymes, the design of small
molecules to inhibit their activity is receiving considerable attention.
Inhibitors for O-GlcNAcase are known22–27 but in the case of OGT,
very few inhibitors are currently available to study the physiological
role of this enzyme in terms of the O-GlcNAc post-translational mod-
D
D
a wide variety of organisms, ranging from microbes to humans.1
This monosaccharide, attached to serine or threonine residues
(O-GlcNAc), is found on many cellular proteins having a wide range
of vital functions, including those involved in transcription,2–5 pro-
teosomal degradation6 and cellular signalling;7 O-GlcNAc is also
found on many structural proteins.8–10 It has also been observed
that the O-GlcNAc modification is dynamic, with cycles of addition
and removal occurring several times during the lifetime of a pro-
tein.11 A reciprocal relationship has also been noted between glo-
bal levels of cellular O-GlcNAc and O-phosphorylation,12 and
residues that bear the O-GlcNAc modification are, at least in some
cases, also known to be phosphorylated.13,14 Not surprisingly, ow-
ing to the abundance of O-GlcNAc on intracellular proteins, this
post-translational modification has been implicated in the etiology
of several disease states. For example, recent studies have sug-
gested that elevation of O-GlcNAc levels in adipocytes7,15 as well
as on TORC2 (Crtc2), the transducer of regulated cyclic adenosine
monophosphate response element-binding protein16 are involved
ification. Alloxan 1 (Fig. 2) inhibits OGT with an IC50 of 100 l
M,28 but
this inhibitor causes several other cellular effects limiting its use.
Walker and co-workers have developed a high-throughput screen
of small molecule libraries29 and identified compounds 2–4, all of
which have moderate inhibitory potency (IC50 ꢀ50
lM) towards
OGT.29 Hanover and co-workers recently developed a C-linked com-
pound 5 that mimics the donor-sugar substrate of OGT, UDP-GlcNAc
6. Unfortunately, 5 was found to be a poor inhibitor of the enzyme.30
On the other hand, more success has been realized with inhib-
itors of O-GlcNAcase. 20-Methyl- 20-
a-D-glucopyrano-[2,1-d]-D
thiazoline (NAG-thiazoline) 7 (Fig. 3) has been found to be a potent
* Corresponding author at present address: Chemistry M313, School of Biomed-
ical, Biomolecular and Chemical Sciences, The University of Western Australia, 35
Stirling Highway, Crawley, WA 6009, Australia. Tel.: +61 64881755.
E-mail address: kstubbs@cyllene.uwa.edu.au (K. A. Stubbs).
OGT is more correctly known as uridine diphosphate-N-acetyl-D-glucosamine:
polypeptidyl transferase.
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.08.012

A. Scaffidi et al. / Carbohydrate Research 343 (2008) 2744–2753
2745
Figure 1. The O-GlcNAc modification of intracellular proteins is dynamic. OGT catalyzes the attachment of N-acetyl-
target proteins, and O-GlcNAcase catalyzes the hydrolysis.
D-glucosamine (GlcNAc) to serine/threonine residues in
Figure 2. Some previously reported inhibitors of OGT.
inhibitor of O-GlcNAcase,22 with analogues of this compound, such
as 8–10, showing selectivity for O-GlcNAcase over other mechanis-
tically similar enzymes found in humans.22,27 O-(2-Acetamido-2-
Recently, we prepared compound 1536 based on the known gly-
coside hydrolase inhibitor, epivalienamine, the C-6 epimer of
valienamine,37 and found it had good potency against O-GlcNAcase
deoxy-
D
-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc)
(Ki = 6 lM). As a result of this work, and with the aid of a co-crystal
11,31 the tetrahydroimidazopyridines, with the most important
structure of 15 in complex with a bacterial homologue of O-GlcNA-
case,36 we thought to increase the potency of 15 by mimicking cer-
tain elements of PUGNAc. As well, we felt that some of these
compounds might be effective inhibitors of OGT because they
share some structural elements common to the donor-sugar sub-
strate as well as potentially mimicking the oxocarbenium ion-like
transition state, that glycosyltransferases are thought to stabilize.38
Thus, the compounds of interest to us were the carbamate 16, the
ethyl ester 17 and the acid 18.
being 8-acetamido-5,6,7,8-tetrahydro-6,7-dihydroxy-5-(hydroxy-
methyl)imidazo[1,2-a]pyridine-2-acetic
acid,
gluco-nagstatin
12,32 prepared by Vasella et al., as well as GlcNAcstatin 1326 pre-
pared by the van Aalten group, have also been shown to be potent
inhibitors of O-GlcNAcase.20,22,23,26 Three structural features of
PUGNAc are associated with its binding affinity to O-GlcNAcase.
Firstly, its sp2 hybridized carbon is thought to mimic some part
of the transition state of the enzyme-catalyzed reaction.à Secondly,
the phenyl ring is thought to bestow significant binding energy
owing to the fact that 2-acetamido-2-deoxy-D-glucohydroximino-
1,5-lactone (LOGNAc) 1431 is a significantly weaker inhibitor.48
Finally, the oxime nitrogen is involved in favourable hydrogen bond-
ing to the catalytic acid residue in the active site.§ 35
2. Results and discussion
The alkene 19, prepared previously from 2-acetamido-2-deoxy-
D
-glucopyranose,36 was subjected to a Ferrier transformation39 to
give the alcohol 20 in 53% yield (Scheme 1). A somewhat ambitious
Grignard reaction was conducted on the alcohol 20, using excess
reagent, to furnish the diol 21, which was subsequently oxidised
to the ketone 22 with the Dess–Martin periodinane.40 At this stage
it was decided to investigate the possibility of eliminating water
à
Recently,
a report suggested that PUGNAc may be a poor transition state
analogue.33
§
As the three-dimensional crystal structure of a true eukaryotic O-GlcNAcase is yet
to be determined, this piece of data was obtained from a sequence-related bacterial
homologue.35

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A. Scaffidi et al. / Carbohydrate Research 343 (2008) 2744–2753
Figure 3. Some previously reported inhibitors of O-GlcNAcase, and compounds prepared in this study.
from across C4 and C5 of 22, a critical procedure in the overall syn-
thesis of the desired targets.
the benzyl ethers from 31, followed by acetylation, afforded the tri-
acetate 32. Subsequent deacetylation of 32 with sodium ethoxide (in
order to avoid transesterification of the ethyl ester) yielded the triol
17. On the other hand, to gain access to the acid 18, the sodium eth-
oxide was replaced with sodium hydroxide to afford 18 in good
yield.
Initial attempts at the proposed dehydration involved treating
22 with trifluoromethanesulfonic anhydride and pyridine; how-
ever, only the oxazole 23 was produced. In a second attempt, the
ketone 22 was treated with chloroform saturated with hydrogen
chloride; however, only the enone 24 was isolated. As a last at-
tempt, treatment of the ketone 22 with sulfuryl chloride at 0 °C re-
sulted in the elimination of water to give the desired enone 25 in
69% yield. Having now established that dehydration across C4
and C5 was possible, we proceeded with the synthetic route. The
ketone 22 was converted into the oxime 26 in 86% yield (Scheme
2); the assignment of stereochemistry of this molecule was based
on previous work by Abell and co-workers.41 Treatment of the
oxime 26 with phenyl isocyanate furnished the phenylcarbamate
27, which was dehydrated to afford the alkene 28. Removal of
the benzyl ethers was effected using ferric chloride.42 Acetylation
to give the triacetate 29 and subsequent deacetylation aided isola-
tion of the desired carbamate 16. Attention was then turned to the
ester 17 and the acid 18.
With the ketone 22 in hand, the alkene 30 was prepared using a
Wittig olefination involving ethyl (triphenylphosphoranylid-
ene)acetate,43 with the stereochemistry once again tentatively as-
signed (Scheme 3). It has been shown that in order to minimize
dipole–dipole interactions during the Wittig olefination, ylides
containing stabilizing electron-withdrawing substituents, such as
esters, react with gluco- and galacto-configured substrates to give
predominately Z-isomers while manno-configured substrates re-
sult in E-isomers.44,45 With this in mind, an E configuration was as-
signed for the alkene 30 in order to avoid repulsion between the
ester and acetamido moieties.– The diene 31 was prepared from
the alkene 30 via the established dehydration protocol. Removal of
At this point, we evaluated the prepared compounds 16, 17 and
18 against O-GlcNAcase. Unfortunately, none of the compounds
inhibited the enzyme significantly (Table 1). Thus, it was decided
to evaluate the compounds against other known b-N-acetylglucos-
aminidases. The enzymes chosen for evaluation were BtGH84,34
which is a close bacterial homologue of O-GlcNAcase; human pla-
cental b-hexosaminidase, which is an enzyme that has a catalytic
mechanism similar to that of O-GlcNAcase but acts on various
glycosphingolipids; and finally NagZ, a b-N-acetylglucosaminidase
involved in the peptidoglycan recycling pathway used by Gram-
negative bacteria. Again, no significant inhibition was observed
with any of these enzymes, with the exception of NagZ, which
showed high micromolar inhibition with the carbamate 16 and
the acid 18 (Table 1). These results seem to lend credence to what
is observed in the co-crystal structural of 15 with BtGH84:36 the
important structural motif of 15, and potentially the reason for
its good potency against O-GlcNAcase, is the amino group, in that
it establishes powerful electrostatic interactions with an active site
residue. Any changes that remove this interaction (e.g., incorporat-
ing a phenylcarbamate moiety to mimic PUGNAc) seem to reduce
greatly the affinity of the resultant molecule.
We next evaluated the three synthetic compounds as inhibitors
of OGT, using recombinant OGT18 and monitoring the O-GlcNAc
modification of recombinantly expressed nuclear pore protein,
p62, via Western blot.8 To confirm that the p62 glycosylation assay
was viable, various controls were performed (Fig. 4). The results
clearly indicate that only in the presence of all of the reagents
was p62 O-GlcNAc modified, and that the assay appeared approx-
–
Note the priority changes between the pyranose and cyclohexane ring.

A. Scaffidi et al. / Carbohydrate Research 343 (2008) 2744–2753
2747
O
BnO
BnO
OH
BnO
BnO
BnO
O
a
b
OCH3
BnO
OH
BnO
OH
AcHN
19
AcHN
AcHN
20
21
OH
BnO
OH
BnO
BnO
BnO
c
d
6
BnO
O
BnO
1
O
AcHN
N
23
22
BnO
OH
BnO
e
O
AcHN
24
BnO
BnO
BnO
f
O
AcHN
25
Scheme 1. Reagents and conditions: (a) HgSO4, H2SO4, dioxane, H2O, 53%; (b) Mg, BnOCH2Cl, HgCl2, THF, 53%; (c) Dess–Martin periodinane, CH2Cl2, 90%; (d) Tf2O, pyridine,
CH2Cl2, 58%; (e) HCl, CHCl3, 89%; (f) SO2Cl2, pyridine, CH2Cl2, 69%.
OH
OH
BnO
BnO
BnO
BnO
a
b
c
22
OH
OC(O)NHPh
N
N
BnO
BnO
AcHN
26
AcHN
27
BnO
AcO
BnO
AcO
AcO
d
e
16
OC(O)NHPh
OC(O)NHPh
N
N
BnO
AcHN
AcHN
29
28
Scheme 2. Reagents and conditions: (a) NH2OHꢁHCl, NaOAc, CH3OH, 86%; (b) PhNCO, Et3N, THF, 69%; (c) SO2Cl2, pyridine, CH2Cl2, 86%; (d) (i) FeCl3, CH2Cl2; (ii) Ac2O, 48% over
two steps; (e) NaOCH3, CH3OH, 95%.

2748
A. Scaffidi et al. / Carbohydrate Research 343 (2008) 2744–2753
BnO
OH
BnO
BnO
BnO
BnO
a
b
22
CO2Et
CO2Et
BnO
AcHN
AcHN
31
30
AcO
AcO
AcO
c
d
e
17
18
CO2Et
AcHN
32
Scheme 3. Reagents and conditions: (a) Ph3PCHCO2Et, CH2Cl2, 78%; (b) SO2Cl2, pyridine, CH2Cl2, 67%; (c) (i) FeCl3, CH2Cl2; (ii) Ac2O, 81% over two steps (d) NaOEt, EtOH, 79%;
(e) NaOH, EtOH, H2O, 75%.
cules 16–18. Based on the kinetic results obtained against O-
GlcNAcase with the synthetic molecules, it seems that modification
of the inhibitor by incorporating an sp2 hybridized carbon (as
found in PUGNAc) results in poorer inhibition of the enzyme. As
well, removal of the amine functionality from the epivalienamine
scaffold also results in poor binding of the compounds.
Table 1
Inhibition constants determined for 16, 17 and 18 against O-GlcNAcase and other b-
N-acetylglucosaminidases
Enzyme
Compound Ki (mM)
16
17
18
Human O-GlcNAcase
Bacterial O-GlcNAcase
b-Hexosaminidase
NagZ
>5
>5
>2.2
0.95
>5
>5
>5
3.1
>5
>5
>5
0.75
4. Experimental
4.1. General methods
1H and 13C NMR spectra were obtained on a BrukerAV600
(600 MHz for 1H and 150.9 MHz for 13C) spectrometer. Unless sta-
ted otherwise, deuterated chloroform (CDCl3) was used as the sol-
vent with CHCl3 (dH 7.26) or CDCl3 (dC 77.0) being employed as
internal standards. NMR spectra run in D2O used internal CH3OH
(dH 3.34, dC 49.0) as the standard. Melting points were determined
on a Reichert hot stage melting point apparatus. Optical rotations
were performed with a Perkin–Elmer 141 Polarimeter in a micro-
cell (1 mL, 10 cm path length) in CHCl3 at room temperature, un-
less stated otherwise. Mass spectra were recorded with a VG-
Autospec spectrometer using the fast atom bombardment (FAB)
imately linear over the 5h reaction time. We next turned our atten-
tion to observing the effect of 15, 16, 17 and 18 on OGT activity. Co-
incubation of p62 with UDP-GlcNAc, OGT, and each of the synthetic
compounds (1 lM–1 mM concentrations) failed to show any
detectable differences in the intensity of the band corresponding
to glycosylated p62 when compared to the control (Fig. 5).
3. Conclusion
We have presented an extension of some of the chemistry of the
known inhibitor of O-GlcNAcase, 15, in the synthesis of the mole-
Figure 4. Western blot analysis showing assay validation. Controls to establish the O-GlcNAc modification assay of recombinant nuclear pore protein p62 by OGT monitored
using Western blot with CTD110.6 as the primary antibody to detect O-GlcNAc modified proteins. From left to right; glycosylation assay performed without p62, glycosylation
assay performed without UDP-GlcNAc, and glycosylation assay with all reagents to establish the approximate linearity of the assay. Bands at positions corresponding to
proteins other than p62 stem from lack of specificity of CTD110.6 binding when used with the unoptimized conditions for this assay.

A. Scaffidi et al. / Carbohydrate Research 343 (2008) 2744–2753
2749
except DMF and MeCN were distilled before use and dried accord-
ing to the methods of Burfield and Smithers.46 ‘Usual workup’ re-
fers to dilution with water, repeated extraction into an organic
solvent, sequential washing of the combined extracts with hydro-
chloric acid (1 M, where appropriate), saturated aqueous sodium
bicarbonate and brine solutions, followed by drying over anhy-
drous magnesium sulfate, filtration and evaporation of the solvent
by means of a rotary evaporator at reduced pressure. All buffer
salts used in this study were obtained from Bioshop. Milli-Q
(18.2 M
minidase (lot 043K3783) and 4-nitrophenyl 2-acetamido-2-
deoxy-b- -glucopyranoside were purchased from Sigma. The
X
cmꢂ1) water was used to prepare all buffers. b-Hexosa-
D
mouse anti-O-GlcNAc monoclonal IgM antibody (mAb CTD110.6)
was purchased from Covance.
4.2. N-[(1S,2R,3S,6S)-2,3-Dibenzyloxy-6-hydroxy-4-
oxocyclohexyl]acetamide (20)
Mercury(II) sulfate (200 mg, 0.70 mmol) was added to 19 (3.6 g,
12 mmol) in dioxane–aqueous H2SO4 (5 mM) (2:1, 120 mL), and
the mixture was heated at 80 °C (3 h). The mixture was cooled
and subjected to a usual workup (CH2Cl2) to yield a colourless
powder that was washed with Et2O to afford 20 (2.4 g, 53%); mp
226–228 °C; [
a]
D ꢂ50.0 (c 1.0, CH3OH); 1H NMR (600 MHz, CD3OD)
1.95 (s, 3H, COCH3), 2.47 (dd, 1H, J5,5 = 14.2, J5,6 = 3.5 Hz, H-5), 2.85
(dd, 1H, J5,6 = 2.5 Hz, H-5), 3.87 (dd, 1H, J1,2 = 10.0, J2,3 = 9.4 Hz, H-
2), 4.07–4.11 (m, 1H, H-6), 4.32 (d, 1H, H-3), 4.36–4.40 (m, 1H,
H-1), 4.51, 4.88 (ABq, 2H, J = 11.4 Hz, CH2Ph), 4.61, 4.80 (ABq, 2H,
J = 11.1 Hz, CH2Ph), 7.23–7.32 (m, 10H, Ph), 7.39 (d, 1H,
J1,NH = 7.2 Hz, N–H); 13C NMR (150.9 MHz, CD3OD) 21.24 (COCH3),
45.12 (C-5), 54.42, 67.17, 80.15, 86.44 (C-1, C-2, C-3, C-6), 73.17,
74.32 (2C, CH2Ph), 127.00–138.58 (Ph), 171.78 (COCH3), 205.55
(C-4); HR-MS m/z (FAB) 384.1819; [M+H]+ requires 384.1811.
4.3. N-[(1S,2R,3S,4R,6S)-2,3-Benzyloxy-4-benzyloxymethyl-4,6-
dihydroxycyclohexyl]acetamide (21)
Magnesium (500 mg, 21 mmol), benzyl chloromethyl ether
(2.5 mL, 16 mmol) and HgCl2 (70 mg, 0.30 mmol) in THF (25 mL)
were stirred at 0 °C (1.5 h). The ketone 20 (300 mg, 0.8 mmol) in
THF (20 mL) was added at ꢂ78 °C, and the mixture was stirred at
0 °C (1 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) to yield 21 as colourless needles
(210 mg, 53%); mp 110–112 °C; [a]
+29.3 (c 1.0, CDCl3); 1H NMR
D
(600 MHz, CDCl3) 1.87 (s, 3H, COCH3), 1.97 (dd, 1H, J5,5 = 15.3,
J5,6 = 3.1 Hz, H-5), 2.02 (dd, 1H J5,6 = 3.0 Hz, H-5), 3.21, 3.44 (Abq,
2H, J7,7 = 8.7 Hz, H-7), 3.82 (d, 1H, J2,3 = 9.3 Hz, H-3), 3.84 (dd, 1H,
J1,2 = 10.3 Hz, H-2), 3.93–3.95 (m, 1H, H-6), 4.08–4.12 (m, 1 H, H-
1), 4.42, 4.47 (ABq, 2H, J = 11.9 Hz, CH2Ph), 4.55, 4.89 (ABq, 2H,
J = 10.8, CH2Ph), 4.68, 4.83 (ABq, 2H, J = 11.4 Hz, CH2Ph), 6.18 (d,
1H, J1,NH = 9.3 Hz, N–H), 7.22–7.35 (m, 15H, Ph); 13C NMR
(150.9 MHz, CDCl3) 23.23 (COCH3), 35.15 (C-5), 54.71, 69.98,
78.85, 81.50 (C-1, C-2, C-3, C-6), 72.99, 73.08, 75.24, 75.39 (4C,
C-7, CH2Ph), 76.65 (C-4), 127.47–138.25 (Ph), 169.95 (COCH3);
HR-MS m/z (FAB) 506.2569; [M+H]+ requires 506.2543.
Figure 5. Inhibition assay of OGT versus 15, 16, 17 and 18. The extent of the O-
GlcNAc modification of p62 was monitored at 0 and 5 h with various concentrations
(1 lM, 10 lM, 100 lM and 1 mM) of (A) 15, (B) 16, (C) 17 and (D) 18. The O-GlcNAc
modification of p62 was monitored using Western blot with CTD110.6 as the
primary antibody to monitor O-GlcNAc modification of p62 in this assay. Bands at
positions corresponding to proteins other than p62 stem from lack of specificity of
CTD110.6 binding when used with the unoptimized conditions for this assay.
technique, with 3-nitrobenzyl alcohol as a matrix, unless otherwise
stated. Elemental analyses of all synthesized compounds used in
enzyme assays were performed at the Simon Fraser University.
Flash chromatography was performed on BDH silica gel or Geduran
Silica Gel 60 with the specified solvents. Thin-layer chromato-
graphy (TLC) was effected on Merck Silica Gel 60 F254 alumi-
num-backed plates that were stained by heating (>200 °C) with
5% sulfuric acid in EtOH. Percentage yields for chemical reactions
as described are quoted only for those compounds that were puri-
fied by recrystallization or by column chromatography, and the
purity was assessed by TLC or 1H NMR spectroscopy. All solvents
4.4. N-[(1R,2R,3S,4R)-2,3-Dibenzyloxy-4-benzyloxymethyl-4-
hydroxy-6-oxocyclohexyl]acetamide (22)
Dess–Martin periodinane (360 mg, 0.85 mmol) was added to 21
(200 mg, 0.40 mmol) in CH2Cl2 (10 mL), and the solution was kept
(1 h). The reaction mixture was diluted with CH2Cl2, washed with
aqueous Na2S2O3 and then saturated with aqueous NaHCO3, and
then dried over MgSO4. Filtration followed by evaporation returned

2750
A. Scaffidi et al. / Carbohydrate Research 343 (2008) 2744–2753
a colourless solid that was washed with Et2O to return 22 as a col-
ourless powder (180 mg, 90%); mp 156–158 °C; [
J = 11.2 Hz, CH2Ph), 4.74, 4.88 (ABq, 2H, J = 11.0 Hz, CH2Ph), 6.10–
6.15 (m, 1H, H-3), 6.73 (d, 1H, J1,NH = 8.6 Hz, N–H), 7.27–7.40 (m,
15H, Ph); 13C NMR (75.5 MHz, CD3CN) 23.07 (COCH3), 60.40,
80.73, 84.07 (C-1, C-5, C-6), 69.53 (C-7), 73.43, 75.62, 76.23 (3C,
CH2Ph), 124.23 (C-3), 128.71–139.40 (Ph), 161.34 (C-4), 171.10
(COCH3), 194.69 (C-2), HR-MS m/z (FAB) 486.2287; [M+H]+ re-
quires 486.2280.
a]
ꢂ9.8 (c 1.0,
D
CH3CN); 1H NMR (600 MHz, CD3CN) 1.90 (s, 3H, COCH3), 2.35,
2.95 (ABq, 2H, J5/7,5/7 = 14.6 Hz, H-5/7), 3.22, 3.65 (ABq, 2H,
J7/5,7/5 = 8.8 Hz, H-7/5), 3.84 (dd, 1H, J1,2 = 10.3, J2,3 = 9.5 Hz, H-2),
4.05 (m, 1H, H-3), 4.50, 4.54 (ABq, 2H, J = 12.0 Hz, CH2Ph), 4.65,
4.75 (ABq, 2H, J = 11.0 Hz, CH2Ph), 4.66, 4.88 (ABq, 2H,
J = 11.0 Hz, CH2Ph), 4.73–4.76 (m, 1H, H-1), 6.60 (d, 1H,
J1,NH = 9.0 Hz, N–H), 7.25–7.38 (m, 15 H, Ph); 13C NMR
(150.9 MHz, CD3CN) 23.05 (COCH3), 47.83 (C-5), 61.95, 82.68,
83.08 (C-1, C-2, C-3), 73.92, 74.00, 76.04, 76.15 (4C, C-7, CH2Ph),
75.16 (C-4), 128.55–139.72 (Ph), 170.92 (COCH3), 203.68 (C-6);
HR-MS m/z (FAB) 504.2423; [M+H]+ requires 504.2386.
4.8. N-[(E)-(1S,2R,3S,4R)-2,3-Dibenzyloxy-4-benzyloxymethyl-
4-hydroxy-6-(hydroxyamino)cyclohexyl]acetamide (26)
Hydroxylamine hydrochloride (20 mg, 0.30 mmol) was added
to 22 (45 mg, 0.10 mmol) and NaOAcꢁ3H2O (60 mg, 0.40 mmol)
in DMF–MeOH (5 mL, 1:19), and the solution was kept overnight.
The mixture was concentrated and subjected to a usual workup
(CH2Cl2), followed by flash chromatography (EtOAc–petrol, 4:1),
to afford a colourless residue that was crystallized to yield 26 as
4.5. (4R,5S,6R)-4,5-Dibenzyloxy-6-benzyloxymethyl-2-methyl-
4,5,6,7-tetrahydrobenzoxazol-6-ol (23)
Trifluoromethanesulfonic anhydride (140 mg, 0.50 mmol) in
CH2Cl2 (1 mL) was added dropwise to 22 (50 mg, 0.10 mmol) in
pyridine–CH2Cl2 (5 mL, 1:9) at 0 °C, and the solution was kept
(1 h). The reaction mixture was quenched with CH3OH (2 mL)
and subjected to a usual workup (CH2Cl2), followed by flash chro-
matography (EtOAc–petrol, 3:7), to yield 23 as a gum (28 mg, 58%);
a colourless powder (40 mg, 86%); mp 128–130 °C (Et2O); [a]
D
+3.7 (c 1.0, CH3CN); 1H NMR (600 MHz, CD3CN) 1.88 (s, 3H,
COCH3), 2.18, 3.26 (ABq, 2H, J5/7,5/7 = 15.1 Hz, H-5/7), 3.05 (br s,
1H, OH), 3.30, 3.59 (ABq, 2H, J7/5,7/5 = 8.9 Hz, H-7/5), 3.74 (dd, 1H,
J1,2 = 9.3, J2,3 = 8.8 Hz, H-2), 3.77 (d, 1H, H-3), 4.48, 4.53 (ABq, 2H,
J = 12.0 Hz, CH2Ph), 4.60, 4.83 (ABq, 2H, J = 11.1 Hz, CH2Ph), 4.67,
4.74 (ABq, 2 H, J = 11.0 Hz, CH2Ph), 4.67–4.72 (m, 1H, H-1), 6.63
(d, 1H, J1,NH = 9.5 Hz, N–H), 7.27–7.37 (m, 15H, Ph), 8.80 (br s,
1H, NOH); 13C NMR (150.9 MHz, CD3CN) 23.30 (COCH3), 31.17
(C-5), 54.69, 82.69, 83.54 (C-1, C-2, C-3), 73.93, 74.45, 75.93,
75.98 (4C, C-7, CH2Ph), 74.89 (C-4), 128.49–139.84 (Ph), 154.36
(C-6), 170.54 (COCH3); HR-MS m/z (FAB) 519.2503; [M+H]+ re-
quires 519.2495.
[a
]
D
ꢂ27.6 (c 1.0, CH3CN); 1H NMR (600 MHz, CD3CN) 2.38 (s, 3H,
CH3), 2.65, 3.04 (ABq, 2H, J8,8 = 16.7 Hz, H-8), 3.25 (br s, 1H, OH),
3.44, 3.67 (ABq, 2H, J7,7 = 9.3 Hz, H-7), 3.86 (d, 1H, J4,5 = 5.3 Hz, H-
5), 4.49, 4.54 (ABq, 2H, J = 12.0 Hz, CH2Ph), 4.55–4.57 (m, 1H, H-
4), 4.67, 4.82 (ABq, 2H, J = 11.3 Hz, CH2Ph), 4.77, 4.95 (ABq, 2H,
J = 11.6 Hz, CH2Ph), 7.26–7.38 (m, 15H, Ph); 13C NMR (150.9 MHz,
CD3CN) 14.20 (CH3), 30.96 (C-7), 72.93, 73.95, 74.48, 75.33 (4C,
C-8, CH2Ph), 75.78, 82.06 (C-4, C-5), 76.25 (C-6), 128.43–139.91
(Ph), 132.92, 146.44 (C-1, C-3), 162.06 (C-2); HR-MS m/z (FAB)
486.2267; [M+H]+ requires 486.2280.
4.9. O-[(2S,3R,4S,5R)-2-Acetamido-3,4-dibenzyloxy-5-
benzyloxymethyl-5-hydroxycyclohexylidene]amino N-
Phenylcarbamate (27)
4.6. N-[(3S,4R)-3-Benzyloxy-4-benzyloxymethyl-4-hydroxy-6-
oxocyclohexenyl]acetamide (24)
Phenyl isocyanate (0.20 mL, 1.7 mmol) was added to 26
(100 mg, 0.20 mmol) and Et3N (0.5 mL) in THF (10 mL), and the
solution was kept (3 h). Concentration of the mixture followed by
flash chromatography (EtOAc–petrol, 2:3 to 1:1) gave 27 as a col-
Hydrogen chloride gas was bubbled through CHCl3 until the
mixture was saturated. One drop of this solution was added to
the ketone 22 (50 mg, 0.10 mmol) in CHCl3 (5 mL), and the solution
was kept (2 h). Concentration of the mixture followed by flash
chromatography ((EtOAc–petrol, 3:7) yielded 24 as an oil (35 mg,
ourless powder (85 mg, 69%); mp 187–189 °C; [a] +23.3 (c 1.0,
D
CH3CN); 1H NMR (600 MHz, CD3CN) 1.94 (s, 3H, COCH3), 2.45
(dd, 1H, J7,7 = 14.9, J7,OH = 1.5 Hz, H-7), 3.24 (d, 1H, OH), 3.28, 3.60
(ABq, 2H, J6,6 = 8.9 Hz, H-6), 3.34 (d, 1H, H-7), 3.80–3.84 (m, 2H,
H-3, H-4), 4.48, 4.53 (ABq, 2H, J = 12.0 Hz, CH2Ph), 4.57, 4.82
(ABq, 2H, J = 11.0 Hz, CH2Ph), 4.66, 4.75 (ABq, 2H, J = 11.0 Hz,
CH2Ph), 4.86–4.91 (m, 1H, H-2), 6.87 (d, 1H, J2,NH = 9.4 Hz, N–H),
7.08–7.48 (m, 20H, Ph), 8.39 (s, 1H, N–H); 13C NMR (150.9 MHz,
CD3CN) 23.35 (COCH3), 33.78 (C-6), 54.99, 82.47, 83.57 (C-2, C-3,
C-4), 73.98, 74.09, 76.16, 76.30 (4C, C-7, CH2Ph), 75.17 (C-5),
120.64–139.63 (Ph), 153.01 (C-1), 162.14 (CNHPh), 170.01
(COCH3); HR-MS m/z (FAB) 638.2841; [M+H]+ requires 638.2866.
89%); [a]
+87.2 (c 1.0, CDCl3); 1H NMR (600 MHz) 2.12 (s, 3H,
D
COCH3), 2.69, 2.84 (ABq, 2H, J5/7,5/7 = 17.0 Hz, H-5/7), 3.20, 3.56
(ABq, 2H, J7/5,7/5 = 9.0 Hz, H-7/5), 4.40, 4.46 (ABq, 2H, J = 11.9 Hz,
CH2Ph), 4.55, 4.82 (ABq, 2H, J = 11.4 Hz, CH2Ph), 4.62 (d, 1H,
J2,3 = 2.8 Hz, H-3), 7.23–7.36 (m, 10H, Ph), 7.68 (d, 1H, H-2), 7.83
(br s, 1H NH); 13C NMR (150.9 MHz) 24.50 (COCH3), 43.56 (C-5),
72.15, 72.72, 73.21 (3C, C-7, CH2Ph), 73.17 (C-3), 75.30 (C-4),
122.71 (C-2), 127.66–137.51 (Ph), 131.75 (C-1), 168.92 (COCH3),
192.18 (C-6); HR-MS m/z (FAB) 396.1841; [M+H]+ requires
396.1811.
4.10. O-[(4R,5R,6S)-6-Acetamido-4,5-dibenzyloxy-3-
benzyloxymethylcyclohex-2-enylidene]amino
N-Phenylcarbamate (28)
4.7. N-[(1R,5R,6R)-5,6-Dibenzyloxy-4-benzyloxymethyl-2-oxo-
cyclohex-3-enyl]acetamide (25)
Sulfuryl chloride (15 mg, 0.10 mmol) in CH2Cl2 (1 mL) was
added dropwise to 22 (30 mg, 0.06 mmol) and pyridine (0.3 mL)
in CH2Cl2 (5 mL) at 0 °C (1 h). The reaction mixture was diluted
with CH2Cl2, washed with 2 M citric acid solution and then satu-
rated with aqueous NaHCO3, followed by drying over MgSO4. Fil-
tration followed by evaporation and flash chromatography
Sulfuryl chloride (25 mg, 0.20 mmol) in CH2Cl2 (1 mL) was
added dropwise to 27 (55 mg, 0.10 mmol) and pyridine (0.5 mL)
in CH2Cl2 (6 mL) at ꢂ70 °C, and the mixture was kept (1 h). The
reaction mixture was diluted with CH2Cl2, washed with 2 M citric
acid and then saturated with aqueous NaHCO3 solution, followed
by drying over MgSO4. Filtration followed by evaporation and flash
chromatography (EtOAc–petrol, 2:3) afforded a powder that was
washed with EtOAc–Et2O (1:1) to leave 28 as a colourless powder
(EtOAc–petrol, 1:1) yielded 25 as a gum (20 mg, 69%); [
a]
ꢂ15.2
D
(c 1.0, CH3CN); 1H NMR (300 MHz, CD3CN) 1.90 (s, 3H, COCH3),
4.00 (dd, 1H, J1,6 = 11.1, J5,6 = 8.4 Hz, H-6), 4.15–4.35 (m, 2H, H7),
4.50–4.60 (m, 4 H, H-1, H-5, CH2Ph), 4.68, 4.76 (ABq, 2H,
(46 mg, 86%); mp 182–184 °C; [
a]
ꢂ52.4 (c 1.0, CH3CN); 1H NMR
D
(600 MHz, CD3CN) 1.85 (s, 3H, COCH3), 4.02 (dd, 1H, J4,5 = 6.4,

A. Scaffidi et al. / Carbohydrate Research 343 (2008) 2744–2753
2751
J5,6 = 4.6 Hz, H-5), 4.17–4.23 (m, 2H, H-7), 4.24 (d, 1H, H-6), 4.51,
4.55 (ABq, 2H, J = 11.8 Hz, CH2Ph), 4.59, 4.70 (ABq, 2H,
J = 11.1 Hz, CH2Ph), 4.66, 4.70 (ABq, 2H, J = 11.4 Hz, CH2Ph), 5.03
(d, 1H, H-4), 6.93 (s, 1H, H-2), 7.08–7.50 (m, 20H, Ph); 13C NMR
(150.9 MHz) 23.33 (COCH3), 50.67, 76.71, 77.89 (C-4, C-5, C-6),
70.80, 73.63, 74.20, 75.01 (4C, C-7, CH2Ph), 113.70 (C-2), 120.84–
139.25 (Ph), 151.28, 153.29, 156.45 (C-1, C-3, CNHPh), 171.51
(COCH3); HR-MS m/z (FAB) 620.2731; [M+H]+ requires 620.2761.
1H, H-20), 4.60, 4.81 (ABq, 2H, J = 12.5 Hz, CH2Ph), 4.60, 4.84
(ABq, 2H, J = 10.9 Hz, CH2Ph), 5.47 (br s, 1H, N–H), 5.77–5.79 (m,
1H, H-2), 7.22–7.38 (m, 15H, Ph); 13C NMR (150.9 MHz) 14.06
(CH2CH3), 23.08 (COCH3), 33.98 (C-60), 56.15, 80.92, 81.55 (C-20,
C-30, C-40), 59.96 (CH2CH3), 73.27 (C-70), 73.40, 74.36, 75.37 (3C,
CH2Ph), 74.44 (C-50), 115.99 (C-2), 127.40–138.11 (Ph), 152.92
(C-10), 166.30 (C-1), 169.91 (COCH3); HR-MS m/z (FAB) 574.2843;
[M+H]+ requires 574.2805.
4.11. O-[(4R,5R,6S)-6-Acetamido-4,5-diacetoxy-3-
acetoxymethylcyclohex-2-enylidene]amino
N-Phenylcarbamate (29)
4.14. (E)-{Ethyl [(40R,50R,60S)-60-acetamido-40,50-dibenzyloxy-30-
benzyloxymethylcyclohex-20-enylidene]acetate} (31)
Sulfuryl chloride (10 mg, 0.07 mmol) in CH2Cl2 (1 mL) was
added dropwise to 30 (20 mg, 0.03 mmol) and pyridine (0.2 mL)
in CH2Cl2 (3 mL) at ꢂ70 °C, and the solution was kept (1 h). The
reaction mixture was diluted with CH2Cl2, washed with 2 M citric
acid solution and then saturated aqueous NaHCO3 solution, fol-
lowed by drying over MgSO4. Filtration followed by evaporation
and flash chromatography (EtOAc–petrol, 3:7) afforded 31 as a
Anhydrous ferric chloride (300 mg, 1.9 mmol) was added to 28
(110 mg, 0.20 mmol) in dry CH2Cl2 at 0 °C. The mixture was stirred
at room temperature (2 h) before being treated with Ac2O (1.0 mL).
The mixture was kept (0.5 h) before being quenched with satu-
rated NaHCO3 solution, followed by a usual workup (CH2Cl2), to
leave a solid. Flash chromatography (EtOAc–petrol, 3:2) yielded a
powder that was washed with EtOAc–Et2O (1:1) to furnish 29 as
powder (13 mg, 67%); mp 157–159 °C, [
a]
D
ꢂ31.1 (c 1.0, CH3CN);
a colourless powder (40 mg, 48%); mp 166–168 °C, [
a
]
ꢂ52.6 (c
1H NMR (600 MHz, CD3CN) 1.24 (t, 3H, J = 7.1 Hz, CH2CH3), 1.84
D
1.0, CH3CN); 1H NMR (600 MHz, CD3CN) 1.95, 2.00, 2.05, 2.07 (4s,
12H, COCH3), 4.66–4.70 (m, 1H, H-7), 4.76–4.80 (m, 1H, H-7),
5.12 (dd, 1H, J5,6 = 9.7, J6,NH = 8.5 Hz, H-6), 5.28 (dd, 1H,
J4,5 = 7.2 Hz, H-5), 5.82–5.85 (m, 1H, H-4), 6.75 (d, 1H, N–H),
6.99–7.01 (m, 1H, H-2), 7.11–7.52 (m, 5H, Ph), 8.38 (s, 1H, N–H);
13C NMR (150.9 MHz, CD3CN) 20.87, 20.91, 23.07 (COCH3), 50.60,
70.18, 72.76 (C-4, C-5, C-6), 63.44 (C-7), 116.28 (C-2), 120.61,
125.01, 130.01, 138.68 (Ph), 146.42, 152.35, 155.04 (C-1, C-3,
CNHPh), 170.79, 170.82, 170.92, 170.99 (4C, COCH3); HR-MS m/z
(FAB) 476.1664; [M+H]+ requires 476.1669.
(s, 3H, COCH3), 3.84 (dd, 1H, J5 ,6 = 7.5, J4 ,5 = .4 Hz, H-50), 4.14 (q,
2H, CH2CH3) 4.12–4.15 (m, 1H, H-70), 4.23–4.27 (m, 2H, H-40,H-
70), 4.47, 4.53 (ABq, 2H, J = 11.7 Hz, CH2Ph), 4.63, 4.73 (ABq, 2H,
J = 11.1 Hz, CH2Ph), 4.67, 4.69 (ABq, 2H, J = 11.4 Hz, CH2Ph), 4.83
0
0
0
0
(ddd, 1H, J6 ,NH = 9.0, J2 ,6 = 1.4 Hz, H-60), 5.77–5.78 (m, 1H, H-2),
6.75 (d, 1H, N–H), 7.28–7.38 (m, 15H, Ph), 7.56–7.57 (m, 1H, H-
20); 13C NMR (150.9 MHz, CD3CN) 14.50 (CH2CH3), 23.29 (COCH3),
52.92, 77.68, 79.15 (C-40, C-50, C-60), 60.92 (CH2CH3), 71.09, 73.05,
74.06, 74.69 (4C, C-70, CH2Ph), 116.92 (C-2), 122.05 (C-20),
128.63–139.41 (Ph), 144.76, 149.78 (C-10, C-30), 166.83 (C-1),
170.40 (COCH3); HR-MS m/z (FAB) 556.2642; [M+H]+ requires
556.2699.
0
0
0
4.12. O-[(4R,5R,6S)-6-Acetamido-4,5-dihydroxy-3-
hydroxymethylcyclohex-2-enylidene]amino
N-Phenylcarbamate (16)
4.15. (E)-{Ethyl [(40R,50R,60S)-60-acetamido-40,50-diacetoxy-30-
acetoxymethylcyclohex-20-enylidene]acetate} (32)
A small piece of Na was added to CH3OH (2 mL) and the result-
ing solution was added to 29 (20 mg, 0.05 mmol) and the solution
was kept at room temperature (1 h). Concentration of the mixture
followed by flash chromatography (CH3OH–EtOAc, 1:9) afforded
16 as a colourless powder (14 mg, 95%); mp 170–172 °C; 1H NMR
(600 MHz, D2O) 2.10 (s, 3H, COCH3), 3.76 (dd, 1H, J5,6 = 10.8,
J4,5 = 8.0 Hz, H-5), 4.34–4.45 (m, 3H, H-4, H-7), 4.82 (d, 1H, H-6),
6.85 (br s, 1H, H-2), 7.16–7.42 (m, 5H, Ph); 13C NMR (150.9 MHz,
D2O) 23.55 (COCH3), 54.06, 73.26, 75.94 (C-4, C-5, C-6), 62.52 (C-
7), 111.90 (C-2), 122.02, 126.35, 130.72, 138.13 (Ph), 155.81,
157.35, 158.71 (C-1, C-3, CNHPh), 175.93 (COCH3); HR-MS m/z
(FAB) 350.1364; [M+H]+ requires 350.1352. Anal. Calcd for
C16H19N3O6: C, 55.01; H, 5.48. Found: C, 55.22; H, 5.38.
Anhydrous ferric chloride (300 mg, 1.9 mmol) was added to 31
(100 mg, 0.20 mmol) in dry CH2Cl2 at 0 °C. The mixture was stirred
at room temperature (2 h) before being treated with Ac2O (1.0 mL).
The mixture was kept (0.5 h) before being quenched with satu-
rated NaHCO3 solution, followed by a usual workup (CH2Cl2), to
leave a gum. Flash chromatography (EtOAc–petrol, 1:1) yielded
32 as a glass (60 mg, 81%); [
a]
ꢂ27.4 (c 1.0, CH3CN); 1H NMR
D
(600 MHz, CD3CN) 1.26 (t, 3H, J = 7.2 Hz, CH2CH3), 1.94, 1.97,
2.02, 2.04 (4s, 12H, COCH3), 4.16 (q, 2H, CH2CH3), 4.60, 4.74
(ABq, 2H, J7 ,7 = 14.2 Hz, H-70), 4.93–4.97 (m, 1H, H-60), 5.09 (dd,
0
0
1H, J5 ,6 = 10.8, J4 ,5 = 7.8 Hz, H-50), 5.77–5.79 (m, 1H, H-2), 5.81–
0
0
0
0
5.84 (m, 1H, H-40), 6.56 (d, 1H, J6 ,NH = 9.1 Hz, N–H), 7.65–7.67
0
(m, 1H, H-20); 13C NMR (150.9 MHz, CD3CN) 14.52 (CH2CH3),
20.91, 20.94, 23.00 (4C, COCH3), 52.21, 71.18, 73.56 (C-40, C-50, C-
60), 61.25, 63.84 (C-70, CH2CH3), 117.04 (C-2), 124.60 (C-20),
140.09, 148.01 (C-10, C-30), 166.57 (C-1), 170.91, 171.04, 171.08,
171.12 (COCH3); HR-MS m/z (FAB) 412.1627; [M+H]+ requires
412.1634.
4.13. (E)-{Ethyl [(20S,30R,40S,50R)-20-acetamido-30,40-
dibenzyloxy-50-benzyloxymethyl-50-hydroxy-
cyclohexylidene]acetate} (30)
Ethyl (triphenylphosphoranylidene)acetate (300 mg, 0.90 mmol)
was added to 22 (90 mg, 0.20 mmol) in CH2Cl2, and the solution
was kept (3 h). The reaction mixture was diluted with CH2Cl2,
washed with 1 M HCl and then saturated aqueous NaHCO3 solution
followed by drying over MgSO4. Filtration followed by evapora-
tion and flash chromatography (EtOAc–petrol, 2:3) yielded 30 as
4.16. (E)-{Ethyl 2-[(40R,50R,60S)-60-acetamido-40,50-dihydroxy-30-
hydroxymethylcyclohex-20-enylidene]acetate} (17)
A small piece of Na was added to EtOH (2 mL) and the resulting
solution was added to 32 (20 mg, 0.05 mmol) and the solution was
kept at room temperature (1 h). Concentration of the mixture fol-
lowed by flash chromatography (EtOH–EtOAc, 3:17–3:10) afforded
17 as a colourless powder (11 mg, 79%); mp 158–160 °C; 1H NMR
(600 MHz, D2O) 1.30 (t, 3H, J = 7.2 Hz, CH2CH3), 2.17 (s, 3H, COCH3),
a powder (80 mg, 78%); mp 179–181 °C; [
a] +68.5 (c 1.0, CDCl3);
D
1H NMR (600 MHz) 1.23 (t, 3H, J = 7.1 Hz, CH2CH3), 1.84 (s, 3H,
COCH3), 2.50, 3.85 (ABq, 2 H, J6 /7 ,6 /7 = 15.0 Hz, H-60/70), 3.26,
0
0
0
0
3.49 (ABq, 2H, J7 /6 ,7 /6 = 8.7 Hz, H-70/60), 3.67 (dd, 1H,
0
0
0
0
J2 ,3 ꢃ J3 ,4 = 9.0 Hz, H-30), 3.94 (d, 1H, H-40), 4.05–4.15 (m, 2H,
CH2CH3), 4.40, 4.50 (ABq, 2H, J = 11.9 Hz, CH2Ph), 4.51–4.56 (m,
0
0
0
0
3.67 (dd, 1H, J5 ,6 = 11.2, J4 ,5 = 8.2 Hz, H-50), 4.20–4.25 (m, 2H,
0
0
0
0

2752
A. Scaffidi et al. / Carbohydrate Research 343 (2008) 2744–2753
CH2CH3), 4.32–4.40 (m, 2H, H-70), 4.43–4.45 (m, 1H, H-40), 4.66 (dd,
use.47,48 Concentrations of NagZ and human O-GlcNAcase used in
the inhibition assays were 0.07
Lꢂ1, using pNP-GlcNAc at a
concentration of 0.5 mM. All inhibitors were tested at several con-
centrations, ranging from one-third to three times Ki where
possible.
1H, J2 ,6 = 1.8 Hz, H-60), 5.73–5.74 (m, 1H, H-2), 7.39–7.41 (m, 1H,
H-20); 13C NMR (150.9 MHz, D2O) 14.06 (CH2CH3), 22.66 (COCH3),
54.95, 72.82, 75.05 (C-40, C-50, C-60), 61.86, 62.12 (C-70, CH2CH3),
113.61, 119.59 (C-2, C-20), 148.84, 149.72 (C-10, C-30), 168.93,
175.46 (C-1, COCH3); HR-MS m/z (FAB) 286.1305; [M+H]+ requires
286.1291; Anal. Calcd for C13H19NO6: C, 54.73; H, 6.71. Found: C,
54.58; H, 6.58.
lg l
0
0
4.18.2. Activity of 15, 16, 17, 18 against OGT analyzed by
Western blot
An aliquot of OGT18 (20
l
L, 5.9 mg/mL) was combined with re-
combinant nuclear pore protein p628 (40
L, 2.3 mg/L) in the pres-
ence of UDP-GlcNAc (5 L, 500 M) in PBS buffer containing MgCl2
(1.25 L, 12.5 mM), 2-mercaptoethanol (2 L, 1 mM) and the syn-
4.17. (E)-[(40R,50R,60S)-60-Acetamido-40,50-dihydroxy-30-
l
hydroxymethylcyclohex-20-enylidene]acetic acid (18)
l
l
l
l
Sodium hydroxide (50 mg, 1.2 mmol) was added to 32 (30 mg,
0.07 mmol) in EtOH–H2O (1:1, 2 mL), and the solution was kept
(1 h). The reaction mixture was neutralized with AcOH and con-
centrated to leave a pale yellow residue that was subjected to flash
chromatography (MeOH–EtOAc–AcOH, 2:8:2) to yield 18 as a col-
ourless powder (14 mg, 75%); mp 145–147 °C; 1H NMR (600 MHz,
thetic compounds 15–18 (various concentrations), and the mixture
was incubated at room temperature; total volume of the assay was
100
lL. A small aliquot (10
lL) was removed and diluted with SDS/
PAGE loading buffer (10
l
L) before being heated at 96 °C for 5 min.
Aliquots were loaded onto 10% TrisꢁHCl polyacrylamide gels. After
electrophoresis, the samples were electroblotted to nitrocellulose
0
0
0
0
D2O) 2.04 (s, 3H, COCH3), 3.51 (dd, 1H, J5 ,6 = 10.9, J4 ,5 = 8.1 Hz, H-
membrane (0.45 lm, Bio-Rad). Transfer was confirmed by visual
50), 4.13, 4.24 (ABq, 2H, J7 ,7 = 14.7 Hz, H-70), 4.30 (d, 1H, H-40), 4.48
(d, 1H, H-60), 5.65 (s, 1H, H-2), 6.85 (s, 1H, H-20); 13C NMR
(150.9 MHz, D2O) 22.63 (COCH3), 54.25, 72.68, 75.22 (C-40, C-50,
C-60), 62.09 (C-70), 121.56, 122.12 (C-2, C-20), 139.34, 143.19 (C-
10, C-30), 175.42, 181.58 (C1, COCH3); HR-MS m/z (FAB) 257.0895;
[M]+ requires 257.0899; Anal. Calcd for C11H15NO6: C, 51.36; H,
5.88. Found: C, 51.43; H, 5.71.
inspection of the transferred pre-stained markers (Dual Colour Pre-
cision Plus Protein Standards, Bio-Rad). The membrane was treated
with a blocking buffer, 5% BSA in PBS pH 7.4 containing 0.1% Tween
20, for 1 h at room temperature with rocking. The blocking buffer
was decanted and a solution of blocking buffer containing mouse
anti-O-GlcNAc monoclonal IgM antibody (mAb CTD110.6,
Covance) (1:2500) was added and rocked for 1 h. After blocking,
the solution was decanted, the membrane was washed two times
for 5 min and three times for 15 min with washing buffer (PBS,
pH 7.4, containing 0.1% Tween 20). A second antibody, goat anti-
mouse-IgG–HRP conjugate (1:10000) in blocking buffer, was added
and the membrane rocked for 1 h at room temperature. After
blocking, the solution was decanted, and the membrane was
washed two times for 5 min and three times for 15 min with
washing buffer. Detection of membrane-bound goat anti-mouse
IgM–HRP conjugate was accomplished by chemiluminescence
using the SuperSignal West Pico chemiluminescent detection kit
(Pierce) and film (Kodak Biomax MR).
0
0
4.18. Kinetic analysis of b-hexosaminidase and BtGH84
All Michaelis–Menten kinetic experiments were conducted
using concentrations of one-fifth to five times Km and incubated
at 37 °C for 30 min. All assays were conducted in triplicate by using
a stopped-based assay procedure, where the enzymatic reactions
(50
quenching buffer (200 mM glycine, pH 10.75). Enzyme addition
was via pipette (5 L) and in all cases the final pH of the quenched
lL) were quenched with the addition of a fourfold excess of
l
solution was greater than 10. Buffer systems for b-hexosaminidase
and BtGH84 were 50 mM citrate–100 mM NaCl pH 4.5, and 50 mM
NaH2PO4–100 mM NaCl pH 7.4, respectively; time dependent as-
says of b-hexosaminidase and BtGH84 showed that the enzymes
were stable in their respective buffers over the period of the assays.
The reactions, quenched after 30 min, were monitored by deter-
mining the extent of liberated 4-nitrophenolate by UV measure-
ments at 400 nm, using a 96-well plate (Stastedt) and 96-well
plate reader (Molecular Devices). Human b-hexosaminidase was
purchased from Sigma and BtGH84 was overexpressed and purified
prior to use.34 Concentrations of b-hexosaminidase and BtGH84
Acknowledgements
The authors thank Hau-Wing Chan for providing the recombi-
nant p62 protein and recombinant human OGT as well as Professor
Gideon J. Davies for providing the BtGH84 construct. D.J.V. is a
scholar of the Michael Smith Foundation of Health Research and
a Canada Research Chair in Chemical Glycobiology. This research
was supported by the Canadian Institutes of Health Research by
a grant to D.J.V.
used in inhibition assays were 0.04 lg l lg l ,
Lꢂ1 and 0.07 Lꢂ1
respectively, using pNP-GlcNAc at a concentration of 0.5 mM. All
inhibitors were tested at several concentrations, ranging from
one-third to three times Ki for those inhibitors for which Ki values
were determined.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2008.08.012.
4.18.1. Kinetic analysis of NagZ and human O-GlcNAcase
All Michaelis–Menten kinetic experiments were conducted
using concentrations of one-fifth to five times Km at 37 °C. All as-
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