A divergent synthesis of 2-acyl derivatives of PUGNAc yields selective inhibitors of O-GlcNAcase

Article abstract of DOI:10.1039/b516273d

A divergent route facilitating the rapid synthesis of a series of O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenylcarbamate (PUGNAc)-based inhibitors, bearing different N-acyl groups has been developed. All compounds of this series are inhibito

Full text of DOI:10.1039/b516273d

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www.rsc.org/obc | Organic & Biomolecular Chemistry
A divergent synthesis of 2-acyl derivatives of PUGNAc yields selective
inhibitors of O-GlcNAcase†
Keith A. Stubbs, Nelson Zhang and David J. Vocadlo*
Received 16th November 2005, Accepted 13th December 2005
First published as an Advance Article on the web 18th January 2006
DOI: 10.1039/b516273d
A divergent route facilitating the rapid synthesis of a series of O-(2-acetamido-2-deoxy-D-glucopyrano-
sylidene)amino N-phenylcarbamate (PUGNAc)-based inhibitors, bearing different N-acyl groups has
been developed. All compounds of this series are inhibitors of both human O-GlcNAcase and human
b-hexosaminidase, yet some effectively exploit differences between the active site architectures of these
two human enzymes which render them selective for O-GlcNAcase. Such inhibitors may be valuable
tools in dissecting the role of the O-GlcNAc post-translational modification at the cellular and
organismal level since these compounds may have different pharmacokinetic properties when
compared to other inhibitors of b-N-acetyl-glucosaminidases.
As a result of the biological importance of these b-N-acetyl-
Introduction
glucosaminidases, the design of small molecule inhibitors17–21 has
Humans have three genes encoding for enzymes that re-
received considerable attention both as tools for establishing their
role in biological processes22 as well as in potential therapeutic
applications.19,23 A major challenge in developing inhibitors for
blocking the function of mammalian glycosidases, including O-
GlcNAcase, is the large number of functionally related enzymes
present in tissues of higher eukaryotes. Accordingly, the use of
non-selective inhibitors in studying the cellular and organismal
physiological role of one particular enzyme is complicated because
complex phenotypes arise from the concomitant inhibition of such
functionally related enzymes.
Many inhibitors of b-N-acetyl-glucosaminidases are known,
but two of the most potent and best characterized are NAG-
thiazoline 1 and PUGNAc 2. 2ꢀ-Methyl-a-D-glucopyrano-[2,1-d]-
D2ꢀ-thiazoline (NAG-thiazoline) 1, has been found to be a potent
inhibitor of family 20 hexosaminidases,20,24 and more recently,
the family 84 O-GlcNAcases.21 The potent inhibition of these
enzymes by NAG-thiazoline 1 is consistent with these enzymes
sharing a common catalytic mechanism involving anchimeric
assistance from the 2-acetamido group (Fig. 1). Support for such
a catalytic mechanism operating for the family 20 b-N-acetyl-
glucosaminidases is supported by both kinetic and structural
studies.20,24 Macauley et al. have shown that the family 84 b-N-
acetyl-glucosaminidases also use anchimeric assistance through
kinetic analyses of human O-GlcNAcase.21 The potent inhibition
by NAG-thiazoline 1 is presumably due its similarity to the
oxazoline or oxazolinium ion intermediate or a closely derived
transition state. Despite its potency, a downside to using NAG-
thiazoline 1 in a complex biological context, is that it lacks
selectivity and therefore perturbs multiple cellular processes.
move N-acetyl-D-glucosamine residues from glycoconjugates.
The first of these, O-glycoprotein 2-acetamido-2-deoxy-b-D-
glucopyranosidase (O-GlcNAcase), is a member of family 84‡
of the glycoside hydrolases.1,2 This enzyme acts to hydrolyse O-
GlcNAc off serine and threonine residues of modified proteins.3
The post-translational modification of nucleocytoplasmic proteins
with 2-acetamido-2-deoxy-b-D-glucopyranoside (O-GlcNAc) is
found in every tissue of multi-cellular eukaryotes that have been
investigated to date.4 O-GlcNAc mediates the function of many
cellular proteins including, for example, those involved in cellu-
lar signaling,5 proteasomal degradation,6 and transcription.7–10
Consistent with the presence of O-GlcNAc on many intracellular
proteins, this enzyme appears to have a role in the etiology of
several diseases including cancer,11 Alzheimers,12–14 and type II
diabetes.5,15
HEXA and HEXB are the two other genes that encode
enzymes catalyzing the hydrolytic cleavage of terminal N-acetyl-
D-glucosamine from glycoconjugates. The two gene products of
HEXA and HEXB yield predominantly two dimeric isozymes.
The heterodimeric isozyme, hexosaminidase A, is composed of an
a- and a b-subunit. The heterodimeric isozyme, hexosaminidase
B, is composed of two b-subunits. Both of these enzymes are
classified as members of family 20 of the glycoside hydrolases and
are normally localised within lysosomes. Dysfunction of either
of these enzymes results in the accumulation of gangliosides and
other glycoconjugates within lysosomes causing the inheritable
neurodegenerative disorders known as Tay-Sachs and Sandhoff
diseases.16
O-(2-Acetamido-2-deoxy-D-glucopyranosylidene)amino
N-
phenylcarbamate (PUGNAc)18,28 2 is another compound that
suffers from the same problem of lack of selectivity, yet has
enjoyed use as an inhibitor of both human O-GlcNAcase3,26
and the family 20 human b-hexosaminidases.25 This molecule,
developed by Vasella and coworkers, was found to be a potent
competitive inhibitor of the b-N-acetyl-glucosaminidases from
Department of Chemistry, Simon Fraser University, 8888 University Drive,
Burnaby, BC, Canada V5A 1S6. E-mail: dvocadlo@sfu.ca; Tel: +1 604-
291-3530
† Electronic supplementary information (ESI) available: NMR spectra.
See DOI: 10.1039/b516273d
‡ For the classification of glycoside hydrolases, see refs 1 and 2 and the
CAZY database (available at http://afmb.cnrs-mrs.fr/CAZY/).
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Fig. 1 The catalytic mechanism of family 20 and 84 b-N-acetyl-glucosaminidases. The mechanism involves anchimeric assistance from the 2-acetamido
group to form an oxazoline intermediate.
Canavalia ensiformis, Mucor rouxii, and the b-hexosaminidase
from bovine kidney.18 One rationale for the potency of PUGNAc
2 toward both the family 84 O-GlcNAcases and the family
20 b-hexosaminidases is the sp2 hybridisation of the anomeric
carbon. This sp2 hybridisation is thought to mimic the geometry
of the dissociative transition states of most glycosidases and
is accordingly a common design feature of many glycosidase
inhibitors.27 Indeed, the inhibitory potency of PUGNAc 2 has
prompted the suggestion that it is a transition state analogue,18
although this assertion waits to be rigourously substantiated.
To generate more useful probes of O-GlcNAcase and circum-
vent the problems associated with the concomitant inhibition
of the lysosomal b-hexosaminidases, Macauley et al. recently
prepared a series of thiazoline inhibitors 3a–f.21 Several of these
compounds proved both potent and highly selective for human O-
GlcNAcase over human b-hexosaminidase. As well, some of these
inhibitors were shown to rapidly block O-GlcNAcase function in
cultured cells resulting in rapid and marked increases in cellular
O-GlcNAc levels. Accordingly, these molecules are superior
candidates for studying the cellular and physiological processes
involving the O-GlcNAc post-translational modification.21 Based
on these observations we envisaged that making modifications in
the pendant N-acyl chain of PUGNAc 2, might yield an additional
set of potent and selective inhibitors for O-GlcNAcase based on
a different inhibitor scaffold. Such inhibitors may have different
pharmacokinetic properties and be valuable tools in dissecting
the role of the O-GlcNAc post-translational modification at the
cellular and organismal level.
Results and discussion
Both previous syntheses of PUGNAc 2 described in the
literature28,29 use N-acetyl-D-glucosamine as a starting material.
To prepare the series of compounds we desired, we envisaged
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Scheme 1 (a) Boc2O, Et3N, THF; (b) (NH4)2CO3, THF, MeOH; (c) NH2OH.HCl, C5H5N, MeOH; (d) DBU, NCS, CH2Cl2.
that a divergent synthesis would facilitate their rapid preparation.
Accordingly, N-acetyl-D-glucosamine is not a viable starting
material since we aimed to generate a panel of compounds with
various N-acyl groups. Despite this limitation, we expected that the
general synthetic route of Mohan and Vasella,29 using N-acetyl-
D-glucosamine to prepare 2, could be adapted to our needs since
it is a much improved and convenient approach when compared
to the first report outlining the synthesis of PUGNAc 2.28
by flash chromatography (Scheme 2). At this stage of the synthesis
we expected that even though 13 contains two carbamate groups,
the phenyl carbamate would be more stable to acidic conditions
unlike the Boc-carbamate. Indeed, the Boc protecting group
is smoothly removed using anhydrous trifluoroacetic acid in
dichloromethane. Treating the resulting crude amine salt 14 with
the appropriate acyl chloride yields amides 15a–g in good overall
yields. Detailed analyses of 15a and 15b support the identities
of this entire N-acylated series of compounds. Subsequent de-O-
acetylation of 15a with saturated ammonia in methanol affords
PUGNAc 2 in good yield (Scheme 3). 16a was obtained from 15b
in a similar fashion. To highlight the ease of these conversions,
the common intermediate, 14 can be treated with a range of acyl
chlorides to provide 15c–g as crude products. Immediate de-O-
acetylation of these crude intermediates readily furnishes the triols
16b–f, in good yield.
Due to the nature of the synthesis described by Mohan and
Vasella, which primarily involves base-catalyzed reactions we felt
that the tert-butoxycarbonyl group (Boc) would be stable through-
out the synthesis. We also expected that this group could be easily
removed in the presence of all the other functionalities at the end of
the sequence. This common intermediate obtained at a late stage
in the synthesis should be amenable to rapid diversification.
Starting from the readily accessible hydrochloride 4,30 the
Boc group was introduced to protect the amine moiety to
give the tetraacetate 531(Scheme 1). With the tetraacetate 5 in
hand, selective de-O-acetylation using (NH4)2CO3 afforded the
hemiacetal 6 in excellent yield. Treating this hemiacetal 6 with
NH2OH.HCl yields the crude E and Z oximes 7 in good yield.
The next reaction, an oxidative ring closure, has been reported by
Mohan and Vasella to be quite temperamental. When oxidising
the oxime 8 using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and
N-chlorosuccinimide (NCS) careful control of the temperature of
the reaction mixture during the addition of the NCS was critical to
avoid formation of the undesired 1,4-lactone oxime 9.29 Consistent
with these observations several trial reactions with 8 show that
the temperature is indeed critical to successfully produce the 1,5-
lactone oxime 10 over the undesired 1,4-lactone oxime 9. Indeed,
at slightly elevated temperatures (−20 ◦C) only the 1,4-lactone
oxime 9 is obtained.
Treating the oxime 7 with DBU/NCS under the literature
conditions29 provided us with a mixture of the desired 1,5-lactone
11 and the unwanted 1,4-lactone oxime 12 in a 4 : 1 ratio. We found,
however, that having NCS already in solution with 7 and only then
adding DBU to the mixture furnished exclusively 11, albeit in a
somewhat lower yield. One complication encountered here is that
succinimide, a by-product of these reactions, is difficult to separate
from 11. Furthermore, spectral analysis of 11 is complicated due
to broadening of the signals obtained in the 1H NMR spectrum.
Nonetheless, treating impure hydroximolactone 11 with phenyl
isocyanate yields pure carbamate 13 in good yield after purification
Inhibition studies
It has been previously established that PUGNAc 2 is a po-
tent competitive inhibitor of both O-GlcNAcase3,26 and b-
hexosaminidase.18,25 For the human enzymes the respective KI
values are 46 nM and 36 nM.3,21 As described above, selective
inhibitors of these enzymes are of interest and we therefore
evaluated the selectivity of putative inhibitors 16a–f. Using pNP-
GlcNAc 17 as a substrate we found that these compounds are
indeed inhibitors of both human O-GlcNAcase and human b-
hexosaminidase (Table 1).
Analysis of the inhibition of human b-hexosaminidase reveals
that increasing the chain length of the N-acyl group results in a
marked decrease in the potency of these inhibitors (Table 1). The
inclusion of only one methylene group (compound 16a) results in a
33-fold increase in the KI value for b-hexosaminidase as compared
to the parent PUGNAc 2. Additional increases in chain length lead
to still greater increases in KI values. Furthermore, as we hoped,
O-GlcNAcase tolerates increases in chain length better than b-
hexosaminidase (Table 1).
Interestingly, for these two human enzymes, direct comparison
of the KI values of the previously prepared NAG-thiazoline
derivatives, 3a–f,21 reveals that the modified thiazolines are better
inhibitors than the corresponding PUGNAc-based compounds,
16a–f. These observations point to the importance of the position
of the acyl group of the inhibitor and potentially, the hybridisation
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Scheme 2 (a) PhNCO, Et3N, THF; (b) CF3COOH, CH2Cl2; (c) RC(O)Cl, C5H5N.
Scheme 3 (a) NH3, MeOH.
Table 1 Inhibition constants and selectivities of inhibitors for O-GlcNAcase and b-hexosaminidase
Selectivity ratio
(b-hexosaminidase KI/O-GlcNAcase KI)
Compound
O-GlcNAcase KI/lM
b-hexosaminidase KI/lM
2
0.0463,21
1.2
2.4
40
210
9
190
0.03621
1.2
26
1
1
11
6
16a
16b
16c
16d
16e
16f
220
> 900
20
≥5
2
> 1200
≥6
of the anomeric carbon. The thiazolines, 1 and 3a–f, with a sp3
hybridised anomeric carbon, comprise a bicyclic scaffold that
restricts movement of the acyl chain as compared to the acyl chain
on the corresponding PUGNAc derivatives, 2 and 16a–f, that have
a sp2 hybridised anomeric carbon. As well, the precise positioning
of the side chain within the active sites must vary between
these two sets of compounds. Together, these two factors must
contribute to both the overall somewhat poorer inhibition and
lesser selectivity of the PUGNAc-derived compounds compared
to the thiazoline derivatives. Consistent with these observations is
that streptozotocin (STZ) 18, a poor inhibitor of O-GlcNAcase
(KI = 1.5 mM),21,32,33 also has a bulky, freely rotating acyl
chain and shows moderate selectivity for O-GlcNAcase over b-
hexosaminidase.21,32,33
These differences between the thiazoline-based compounds, 1
and 3a–f, and the PUGNAc derivatives, 2 and 16a–f, may stem
from the former compounds emulating the presumed bicyclic-
like transition state involved in the catalytic mechanism of
O-GlcNAcase21 and b-hexosaminidase.20,24,34,35 In contrast, the
position of the N-acyl group of the PUGNAc analogues and
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STZ may resemble that of the natural substrate N-acetyl-D-
glucopyranoside. The different hybridisation of the anomeric
carbons of the PUGNAc and thiazoline derivatives may also
contribute to the precise positioning of these N-acyl groups.
Further crystallographic and kinetic studies defining the molecular
basis accounting for these differences between NAG-thiazoline 1
and PUGNAc 2 would be of considerable interest.
NaCl, 0.1% BSA, pH 4.25. The progress of the reaction at the end
of 30 min was determined by measuring the extent of 4-nitrophenol
liberated as determined by UV measurements at 400 nm using a 96-
well plate (Sarstedt) and 96-well plate reader (Molecular Devices).
Human placental b-hexosaminidase was purchased from Sigma
(lot043K3783), and O-GlcNAcase was overexpressed and purified
freshly, prior to use.39 O-GlcNAcase and b-hexosaminidase were
used in the inhibition assays at a concentration (lg lL−1) of
0.0406 and 0.012, respectively using substrate 17 at a concentration
of 0.5 mM. All inhibitors were tested at seven concentrations
ranging from three times to 1/3 KI, with the exception of the
assay of inhibitor 16d with b-hexosaminidase, where such high
concentrations of inhibitor could not be reached because of its
high KI value. KI values were determined by linear regression of
data from Dixon plots.
Conclusions
We have devised a divergent route that enables the rapid synthesis
of a series of PUGNAc-based inhibitors bearing different N-
acyl groups. All of the prepared compounds were inhibitors of
both human O-GlcNAcase and human b-hexosaminidase. These
compounds, however, exploit differences in active site architectures
between these two enzymes, which results in them being selective
for O-GlcNAcase. This observation is fairly consistent with what
has been reported for a set of thiazoline analogues.21 Despite
the somewhat surprising lower selectivity of these PUGNAc ana-
logues as compared to the thiazoline derivatives, these compounds
may have different pharmacokinetic properties owing to their dif-
ferent scaffold. Accordingly, these PUGNAc derivatives may prove
to be valuable tools for dissecting the role of the O-GlcNAc post-
translational modification at the cellular and organismal level.
Indeed, elaboration of other glycosidase inhibitors has yielded
clinically useful compounds targeting entirely different enzymes.36
As well, using the strategy outlined here, systematic elaboration of
other b-N-acetyl-glucosaminidase inhibitor scaffolds37,38 may also
yield selective inhibitors for human O-GlcNAcase.
(E)- and (Z)-3,4,6-Tri-O-acetyl-2-tert-butoxycarbonamido-2-
deoxy-D-glucose oxime (7). Hydroxylamine hydrochloride (3.2 g,
46 mmol) was added to the hemiacetal 630 (12 g, 31 mmol) and
pyridine (6.3 mL, 77 mmol) in MeOH (200 mL) and the resulting
solution stirred at reflux (2 h). The solution was concentrated and
co-evaporated with toluene (2 × 20 mL). The residue was taken
up in EtOAc and washed with water (2 × 50 mL), brine (50 mL),
dried (MgSO4), filtered and concentrated to give the presumed
oxime 7 (9.5 g). The residue was used without further purification.
3,4,6-Tri-O-acetyl-2-tert-butoxycarbonamido-2-deoxy-D-gluco-
nohydroximo-1,5-lactone (11). (a) 1,8-Diazabicyclo[5.4.0]undec-
7-ene (0.98 mL, 6.6 mmol) was added to the crude oxime 7 (2.5 g,
5.9 mmol) in CH2Cl2 (60 mL) at −45 ◦C and the mixture stirred
(5 min). N-chlorosuccinimide (0.87 g, 6.5 mmol) was then added
to the solution in such a way that the temperature did not go
above −40 ◦C and the resulting mixture was allowed to stir for
30 min at this temperature and then was allowed to warm to room
temperature over two h. The mixture was quenched with water and
diluted with EtOAc (100 mL). The organic layer was separated
and washed with water (2 × 50 mL), brine (1 × 50 mL), dried
(MgSO4) filtered and concentrated. Flash chromatography of the
resultant residue (EtOAc/hexanes 2 : 3) gave the title compound
11 as a colourless oil (1.7 g, 68%). The 1H and 13C NMR spectra
appeared to show the compound of interest but was contaminated
with the 1,4-lactone oxime 12 and succinimide.
(b) 1,8-Diazabicyclo[5.4.0]undec-7-ene (3.0 mL, 20 mmol) was
added to the oxime 7 (7.7 g, 18 mmol) and N-chlorosuccinimide
(2.7 g, 20 mmol) in CH2Cl2 (110 mL) at −45 ◦C in such a way that
the temperature did not go above −40 ◦C and the resulting mixture
was allowed to stir for 30 min at this temperature and then was
allowed to warm to room temperature over two h. The mixture
subsequently treated as in (a) to give the title compound 11 (4 g,
52%§). Rf 0.16 (EtOAc/hexane 1 : 1); dH (500 MHz, CD3OD):
9.12 (br s, 1H, NOH), 5.33–5.18 (m, 3H, H3, H4, NH), 4.60–4.52
(m, 1H, H2), 4.33 (dd, J = 3.5, 12.5, 1H, H6), 4.33 (dd, J = 2.0,
1H, H6), 4.19 (ddd, J = 9.5, 1H, H5), 2.08 (s, 3H, CH3), 2.03 (s,
3H, CH3), 2.02 (s, 3H, CH3), 1.40 (s, 9H, C(CH3)3). dC (125 MHz,
Experimental
General
All solvents were dried prior to use. Synthetic reactions were
monitored by TLC using Merck Kieselgel 60 F254 aluminium-
backed sheets. Compounds were detected by charring with a 10%
concentrated sulfuric acid in ethanol solution and heating. Flash
chromatography under a positive pressure was performed with
Merck Kieselgel 60 (230–400 mesh) using specified eluants. 1H and
13C NMR spectra were recorded on a Bruker AMX400 at 400 MHz
(100 MHz for 13C) or a Varian AS500 Unity Innova spectrometer
at 500 MHz (125 MHz for 13C) (chemical shifts quoted relative to
CDCl3 or CD3OD where appropriate). Elemental analyses of all
synthesized compounds used in enzyme assays were performed at
the Simon Fraser University or the University of British Columbia
Analytical Facility.
Kinetic analysis of O-GlcNAcase and b-hexosaminidase
All assays were carried out in triplicate at 37 ◦C for 30 min by
using a stopped assay procedure in which the enzymatic reactions
(50 lL) were quenched by the addition of a 4-fold excess (200 lL)
of quenching buffer (200 mM glycine, pH 10.75). Assays were
initiated by the careful addition, via pipette, of enzyme (5 lL),
and in all cases the final pH of the resulting quenched solution
was greater than 10. Time-dependent assay of O-GlcNAcase
and b-hexosaminidase revealed that enzymes were stable in their
respective buffers over the period of the assay: 50 mM NaH2PO4,
100 mM NaCl, 0.1% BSA, pH 6.5 and 50 mM citrate, 100 mM
=
CD3OD): 171.5, 170.5, 170.3, 169.0 (4C, C O), 150.7 (C1), 76.5,
72.4, 67.3 (3C, C3, C4, C5), 61.5 (C6), 50.8 (C2), 29.6 (C(CH3)3),
28.2 (C(CH3)3), 20.7, 20.6, 20.5 (3C, CH3).
§ The quoted yield does not include the 5% (w/w) of succinimide in the
1H and 13C NMR spectra.
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O-(3,4,6-Tri-O-acetyl-2-tert-butoxycarbonamido-2-deoxy-D-
glucopyranosylidene)amino N-phenylcarbamate (13). Phenyl
isocyanate (0.5 mL, 3.7 mmol) was added to the lactone 11 (1.3 g,
3.1 mmol) and Et3N (1.3 mL, 9.3 mmol) in THF (50 mL) and
the solution stirred (r.t., 3 h). Concentration followed by flash
chromatography of the resultant residue (EtOAc/hexanes 1:4)
yielded the carbamate 13 as a colourless oil (1.2 g, 71%). Rf
0.65 (EtOAc/hexane 1:1); dH (500 MHz, CDCl3): 7.83 (br s, 1H,
PhNH), 7.42 (m, 2H, Ar), 7.32 (m, 2H, Ar), 7.11 (m, 1H, Ar),
5.38–5.30 (m, 2H, H3, H4), 5.18 (br s, 1H, NH), 4.62 (m, 1H, H2),
4.45–4.40 (m, 2H, H5, H6), 4.31 (dd, J = 3.5, 13.5, 1H, H6), 2.12
(s, 3H, CH3), 2.09 (s, 3H, CH3), 2.07 (s, 3H, CH3), 1.44 (s, 9H,
C(CH3)3). dC (125 MHz, CDCl3): 171.1, 170.3, 170.0, 169.1 (4C,
O-(2-Acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl-
carbamate (2). (32%) Gave 1H and 13C NMR spectra consistent
with that found in the literature.27 Rf 0.15 (MeOH/EtOAc 1 : 19).
O-(2-Deoxy-2-propamido-D-glucopyranosylidene)amino N-phenyl-
carbamate (16a). (32%) Rf 0.11 (MeOH/EtOAc 1 : 19); dH
(500 MHz, CD3OD): 7.41 (m, 2H, Ar), 7.26 (m, 2H, Ar), 7.03
(m, 1H, Ar), 4.54 (m, 1H, H2), 3.96–3.93 (m, 2H, H5, H6), 3.83
(dd, J = 4.0, 12.5, 1H, H6), 3.75–3.71 (m, 2H, H3, H4), 2.31 (q,
J = 7.5, CH2), 1.16 (t, 3H, CH3); dC (125 MHz, CD3OD): 177.5
=
(C O), 159.5 (CONHPh), 154.7 (C1), 139.3, 129.9, 124.8, 120.3
(4C, Ar), 84.1, 74.4, 69.9 (3C, C3, C4, C5), 61.8 (C6), 52.9 (C2),
30.2 (CH2), 10.2 (CH3); Anal. calcd for C16H21N3O7.H2O: C, 50.00;
H, 5.77; N, 10.93. Found: C, 50.02; H, 5.78; N, 10.76%.
=
C O), 155.0 (CONHPh), 151.4 (C1), 136.8, 129.1, 124.2, 119.4
(4C, Ar), 77.2, 71.0, 67.2 (3C, C3, C4, C5), 61.2 (C6), 51.1 (C2),
30.6 (C(CH3)3), 28.2 (C(CH3)3), (20.7, 20.6, 20.5 (3C, CH3); Anal.
calcd for C24H31N3O11: C, 53.63; H, 5.81; N, 7.82. Found: C, 53.72;
H, 5.78; N, 7.78%.
O-(2-Deoxy-2-butamido-D-glucopyranosylidene)amino N-phenyl-
carbamate (16b). (26%) Rf 0.26 (MeOH/EtOAc 1 : 19); dH
(500 MHz, CD3OD): 7.41 (m, 2H, Ar), 7.27 (m, 2H, Ar), 7.03
(m, 1H, Ar), 4.54 (m, 1H, H2), 3.94–3.92 (m, 2H, H5, H6), 3.83
(dd, J = 4.5, 13.0, 1H, H6), 3.74–3.70 (m, 2H, H3, H4), 2.26 (t, J =
6.0, COCH2), 1.66 (m, 2H, CH2CH3), 0.96 (t, J = 7.0, 3H, CH3);
General procedure for the preparation of the O-(3,4,6-tri-O-
acetyl-2-acylamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl-
carbamates (15a–g). Trifluoroacetic acid (13 mmol) was added
to the carbamate 13 (1 mmol) in CH2Cl2 (10 mL) at 0 ◦C and
the solution stirred (2 h). Pyridine (200 mmol) was then slowly
added to the solution and the resulting mixture left to stand (0 ◦C,
10 min). The appropriate acyl chloride (3 mmol) was then added
at 0 ◦C and the solution allowed to stand at 4 ◦C overnight.
Concentration of the mixture gave a yellowish residue which was
dissolved in EtOAc (30 mL) and washed with (2 × 20 mL), brine
(1 × 20 mL), dried (MgSO4) filtered and concentrated. For the
presumed intermediate tri-O-acetates 15c–g these were carried
through without further purification. Flash chromatography of
the residues presumably 15a and 15b (EtOAc/hexanes 1 : 1) gave
the desired acyl derivatives 15a and 15b in yields of 48% and 42%
respectively.
=
dC (100 MHz, CD3OD): 176.7 (C O), 159.5 (CONHPh), 154.7
(C1), 139.3, 130.0, 124.8, 120.3 (4C, Ar), 84.1, 74.5, 69.9 (3C, C3,
C4, C5), 61.8 (C6), 52.9 (C2), 39.1 (CH2), 20.3 (CH2), 14.1 (CH3);
Anal. calcd for C17H23N3O7: C, 53.54; H, 6.08; N, 11.02. Found:
C, 53.52; H, 6.09; N, 10.90%.
O-(2-Deoxy-2-valeramido-D-glucopyranosylidene)amino N-phenyl-
carbamate (16c). (23%) Rf 0.43 (MeOH/EtOAc 1 : 19); dH
(400 MHz, CD3OD): 7.45 (m, 2H, Ar), 7.30 (m, 2H, Ar), 7.03
(m, 1H, Ar), 4.58 (m, 1H, H2), 3.99–3.94 (m, 2H, H5, H6), 3.87
(dd, J = 4.0, 12.4, 1H, H6), 3.79–3.75 (m, 2H, H3, H4), 2.33 (t, J =
7.2, COCH2), 1.64 (m, 2H, CH2CH2), 1.40 (m, 2H, CH2CH2), 0.92
=
(t, J = 7.2, 3H, CH3); dC (100 MHz, CD3OD): 176.8 (C O), 159.5
(CONHPh), 154.7 (C1), 139.3, 130.0, 124.8, 120.4 (4C, Ar), 84.1,
74.3, 69.9 (3C, C3, C4, C5), 61.8 (C6), 52.9 (C2), 37.0 (CH2), 29.0
(CH2), 23.5 (CH2), 14.2 (CH3); Anal. calcd for C18H25N3O7.2H2O:
C, 50.11; H, 6.34; N, 9.79. Found: C, 50.25; H, 6.04; N, 9.94%.
O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranosyli-
1
dene)amino N-phenylcarbamate (15a). Gave H and 13C NMR
spectra consistent with that found in the literature.27 (48%) Rf 0.2
(EtOAc/hexane 7 : 3).
O-(2-Deoxy-2-hexamido-D-glucopyranosylidene)amino N-phenyl-
carbamate 16d. (26%) Rf 0.48 (MeOH/EtOAc 1 : 19); dH
(500 MHz, CD3OD): 7.43 (m, 2H, Ar), 7.28 (m, 2H, Ar), 7.05
(m, 1H, Ar), 4.55 (m, 1H, H2), 3.97–3.92 (m, 2H, H5, H6), 3.85
(dd, J = 4.0, 12.5, 1H, H6), 3.77–3.72 (m, 2H, H3, H4), 2.30 (t,
J = 7.5, COCH2), 1.66 (m, 2H, COCH2CH2), 1.36–1.27 (m, 4H,
CH2CH2), 0.87 (t, J = 6.5, 3H, CH3); dC (125 MHz, CD3OD):
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-propamido-D-glucopyranosyli-
dene)amino N-phenylcarbamate (15b). (42%) Rf 0.11 (EtOAc/
hexane 1 : 1); dH (500 MHz, CDCl3): 7.71 (br s, 1H, PhNH), 7.41
(m, 2H, Ar), 7.32 (m, 2H, Ar), 7.11 (m, 1H, Ar), 6.59 (d, J = 7.5,
1H, NH), 5.40 (dd, J = 9.0, 1H, H3), 5.35 (dd, J = 9.0, 1H, H4),
4.89 (dd, 1H, H2), 4.53 (ddd, J = 3.0, 4.0, 1H, H5), 4.43 (dd, J =
13.0, 1H, H6), 4.32 (dd, 1H, H6), 2.22 (q, J = 7.5, CH2), 2.13 (s,
3H, COCH3), 2.07 (s, 3H, COCH3), 2.05 (s, 3H, COCH3), 1.14 (t,
3H, CH3). dC (125 MHz, CDCl3): 174.2, 170.4, 170.2, 169.1 (4C,
=
176.8 (C O), 159.5 (CONHPh), 154.7 (C1), 139.3, 130.0, 124.8,
120.4 (4C, Ar), 84.1, 74.3, 69.9 (3C, C3, C4, C5), 61.8 (C6), 52.9
(C2), 37.2 (CH2), 32.6 (CH2), 26.6 (CH2), 23.5 (CH2), 14.3 (CH3);
Anal. calcd for C19H27N3O7: C, 55.74; H, 6.65; N, 10.26. Found:
C, 55.55; H, 6.86; N, 9.99%.
=
C O), 155.1 (CONHPh), 151.7 (C1), 136.8, 129.2, 124.3, 119.2
(4C, Ar), 77.2, 71.2, 67.1 (3C, C3, C4, C5), 61.2 (C6), 49.6 (C2),
29.4 (CH2), 20.7, 20.6, 20.5 (3C, COCH3), 9.7 (CH3).
O-(2-Deoxy-2-isobutamido-D-glucopyranosylidene)amino N-phenyl-
carbamate (16e). (29%) Rf 0.22 (MeOH/EtOAc 1 : 19); dH
(500 MHz, CD3OD): 7.42 (m, 2H, Ar), 7.27 (m, 2H, Ar), 7.03 (m,
1H, Ar), 4.52 (m, 1H, H2), 3.96–3.92 (m, 2H, H5, H6), 3.83 (dd,
J = 4.5, 12.5, 1H, H6), 3.77–3.71 (m, 2H, H3, H4), 2.54 (m, J = 7.0,
CH), 1.16 (d, 3H, CH3), 1.14 (d, 3H, CH3); dC (125 MHz, CD3OD):
General procedure for the preparation of the O-(2-acylamido-2-
deoxy-D-glucopyranosylidene)amino N-phenylcarbamates 2, 16a–
f. A saturated solution of ammonia in MeOH (2 mL) was
added to the carbamate (0.3 mmol) in MeOH (10 mL) and the
solution left to stand (r.t., 2 h). Concentration followed by flash
chromatography of the residue (MeOH/EtOAc 3 : 97) gave the
desired triols 2, 16a–f in yields ranging from 21% to 32%.
=
180.6 (C O), 159.4 (CONHPh), 154.7 (C1), 139.3, 130.1, 124.8,
120.4 (4C, Ar), 84.1, 74.3, 69.9 (3C, C3, C4, C5), 61.8 (C6), 52.8
844 | Org. Biomol. Chem., 2006, 4, 839–845
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(C2), 36.4 (CH), 19.9, 19.8 (2C, CH3); Anal. calcd for C17H23N3O7:
C, 53.54; H, 6.08; N, 11.02. Found: C, 53.17; H, 6.18; N, 10.87%.
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O-(2-Deoxy-2-isovaleramido-D-glucopyranosylidene)amino N-
phenylcarbamate (16f). (23%) Rf 0.47 (MeOH/EtOAc 1 : 19);
dH (400 MHz, CD3OD): 7.44 (m, 2H, Ar), 7.30 (m, 2H, Ar), 7.06
(m, 1H, Ar), 4.59 (m, 1H, H2), 3.98–3.95 (m, 2H, H5, H6), 3.86
(dd, J = 4.4, 12.8, 1H, H6), 3.77–3.74 (m, 2H, H3, H4), 2.17 (d,
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=
dC (100 MHz, CD3OD): 176.0 (C O), 159.5 (CONHPh), 154.7
(C1), 139.3, 130.0, 124.8, 120.4 (4C, Ar), 84.1, 74.5, 69.9 (3C, C3,
C4, C5), 61.8 (C6), 52.9 (C2), 46.5 (CH2), 27.5 (CH(CH3)2), 23.0,
22.9 (2C, CH3); Anal. calcd for C18H25N3O7: C, 54.68; H 6.37; N,
10.63. Found: C, 54.33; H, 6.48; N, 10.56%.
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Acknowledgements
We thank Natural Science and Engineering Research Council
and The Canadian Protein Engineering Networks of Centres
of Excellence for funding. D.J.V. is supported by the Canada
Research Chairs Program as a Tier II Canada Research Chair
in Chemical Glycobiology. We also thank B.M. Pinto for access
to a UV–Visible spectrophotometer.
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